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UNIVERSIDAD NACIONAL AUTNOMA DE MXICO

FACULTAD DE MEDICINA VETERINARIA Y ZOOTECNIA SECRETARA DE EDUCACIN CONTINUA Y TECNOLOGA DEPARTAMENTO REPRODUCCIN

VII CURSO INTERNACIONAL DE REPRODUCCIN EN EQUINOS

MEMORIAS
Del 1 al 3 de Julio de 2009

DIRECTORIO

UNIVERSIDAD NACIONAL AUTNOMA DE MXICO


Dr. Jos Narro Robles RECTOR Dr. Sergio M. Alcocer Martnez de Castro SECRETARIO GENERAL Mtro. Juan Jos Prez Castaeda SECRETARIO ADMINISTRATIVO Dra. Rosaura Ruiz Gutirrez SECRETARIA DE DESARROLLO INSTITUCIONAL MC. Ramiro Jess Sandoval SECRETARIO DE SERVICIOS A LA COMUNIDAD Lic. Luis Ral Gonzlez Prez ABOGADO GENERAL Enrique Balp Daz DIRECTOR GENERAL DE COMUNICACIN SOCIAL

FACULTAD DE MEDICINA VETERINARIA Y ZOOTECNIA


Dr. Francisco Trigo Tavera DIRECTOR Dra. Ma. Elena Trujillo Ortega SECRETARIA GENERAL MCV. Germn Valero Elizondo SECRETARIO DE EDUCACIN CONTINUA Y TECNOLOGA MVZ. Patricia R. Daz Gemez JEFA DEL DEPTO. DE EDUCACIN CONTINUA Dr. Joel Hernndez Ceron JEFE DEL DEPTO. DE REPRODUCCIN

CONFERENCIANTES
Daniel Guillaume Juan Salazar Patrick McCue Mariano Hernndez Gil Gabriela Quijano Stephanie Retteg Nuria de Buen Alain Martnez Guillermo Gonzlez

COORDINADORES
COORDINADORA ACADMICA Myriam Boeta Acosta COORDINADORAS ADMINISTRATIVAS DIVISIN DE EDUCACIN CONTINUA Patricia Meja Gutirrez Mariana Figueroa Gmez EDITORAS Patricia Daz Gemez Mariana Figueroa Gmez Patricia Mejia Gutirrez

La reproduccin parcial o total de los trabajos no podr efectuarse sin la previa autorizacin por escrito del autor y citando estas memorias como referencia. La informacin contenida, as como estilo y ortografa en cada uno de los escritos es responsabilidad de los autores.

MEMORIAS PATROCINADAS POR:

ESTACIONALIDAD EN LA YEGUA
Salazar Ortiz, J.a , Nagy, P b. y Guillaume, D.b Colegio de Postgraduados, Campus Crdoba. Carretera Federal Crdoba-Veracruz Km. 348, Congregacin Manuel Len 94946 Amatln de los Reyes, Veracruz, Mxico. Apdo. Postal 143 Col. Centro 94500 Crdoba, Veracruz, Mxicoa. Equipe de Neurobiologie et Matrise des Fonctions Saisonnire. INRA, UMR85 Physiologie de la Reproduction et des Comportements, CNRS, Universit de Tours, Haras Nationaux, F-37380 Nouzilly, France b.

INTRODUCCIN
Los animales han desarrollado estrategias de reproduccin estacional que aseguren que las cras nazcan en el momento apropiado del ao. En los caballos, como en muchas otras especies, el ritmo circanual de reproduccin es regulado por las variaciones del fotoperiodo. Esta seal ambiental se traduce en una seal endocrina en la glndula pineal, secretando melatonina durante la fase oscura del da. En la yegua, los das decrecientes se asocian a una disminucin de la secrecin de gonadotropinas y de la actividad ovulatoria. El mecanismo por el que las gonadotropinas y probablemente la secrecin de GnRH, disminuyen durante el perodo de inactividad ovulatoria estacional no es bien conocido en la yegua. Es posible que la ausencia de la actividad cclica sea resultado de la falta de seales estimulantes, por ejem. das largos, condiciones climticas y alimenticias favorables, la presencia del semental, que estimule la secrecin GnRH y de gonadotropinas como durante la estacin de reproduccin. La inactividad reproductiva estacional puede ser consecuencia de una inhibicin directamente inducida entonces por los das cortos, clima adverso y nutricin pobre. En la yegua, el ritmo de reproduccin circanual endgeno parece ser sincronizado, por las variaciones estacionales del fotoperiodo, al invierno y al verano. La yegua es polistrica estacional, con el inicio de la estacin de reproduccin en primavera ligada al aumento en de la duracin del da, la temperatura y la disponibilidad de alimento. La estacin de reproduccin sucede entonces de abril a septiembre en el hemisferio Norte (Hughes et al., 1975). Arbitrariamente, se han establecido como la fecha oficial de nacimiento para los potros el 1 de enero en el hemisferio norte, y un periodo de reproduccin oficial de febrero a junio; como resultado, la edad oficial es la misma para todos los potros que nacen durante la estacin (Ginther, 1992). Esto genera una presin econmica, para que los criadores de caballos reproduzcan sus yeguas lo ms temprano en el ao y tener una ventaja de edad fisiolgica sobre los potros nacidos ms tarde. Los potros que nacen al inicio del ao presentan ventajas y se realizan mejor que los potros que nacen despus (Langlois y Blouin 1996). Esta normatividad ha estimulado la investigacin para entender los mecanismos de la estacionalidad reproductiva y desarrollar mtodos para la adelantar el inicio, cada vez ms temprano, de la estacin de reproduccin. Se ha demostrado que el fotoperiodo artificial, simulando das largos, puede utilizarse para avanzar la fecha de la primera ovulacin (Palmer y Guillaume, 1992). Estos estudios han aumentado la comprensin de los mecanismos y las limitaciones de este regulador importante en la funcin reproductiva estacional y se han desarrollado mtodos ms eficaces usando luz artificial (Guillaume et al., 2000). A pesar de la efectividad de la fotoestimulacin, para inducir la actividad ovulatoria cclica, este mtodo requiere que se inici en diciembre y existe adems una variacin en el intervalo de inicio de la foto estimulacin a la primera ovulacin. La mayora de las yeguas, en las zonas templadas, presenta el periodo de reposo sexual de octubre a mayo. Este periodo de inactividad ovulatoria es ms frecuente y ms largo en las yeguas de 2 a 3 aos y en yeguas adultas

que han amamantado un potrillo durante el verano, en comparacin con las yeguas adultas no lactantes. En este ltimo tipo de yeguas, la fase anovulatoria es muy corta o inexistente y la condicin corporal generalmente mejor que en los dos primeros tipos. Estas fluctuaciones de condicin corporal podran ser una de las causas de variacin de la duracin de la inactividad ovulatoria y de respuesta a la fotoestimulacin.

RITMO ENDGENO DE REPRODUCCIN CIRCANUAL


El patrn de reproduccin estacional es resultado de un ritmo endgeno circanual regulado por factores como el fotoperiodo, la temperatura, la nutricin y la condicin corporal. En la oveja, Karsch et al. (1989) aportaron evidencias de la existencia de un ciclo de reproduccin circanual. En ovejas ovariectomizadas, recibiendo implantes de estrgenos y mantenidas en condiciones constantes de fotoperiodo por 5 aos, mantuvieron cambios circanuales de secrecin de LH. Sin embargo, los perodos de niveles elevados de LH, comunes de la estacin de reproduccin, difieren entre los individuos, reflejando la ausencia de factores ambientales de sincronizacin. En caballos, la informacin disponible, indica que un ritmo de reproduccin endgeno tambin existe. Las yeguas mantenidas en condiciones fotoperiodo de das largos constantes (16 h), iniciando en el solsticio del verano, entran en inactividad ovulatoria estacional y las yeguas mantenidas bajo fotoperiodo de das cortos (8.5 h luz) iniciando en el solsticio de invierno reanudan la actividad reproductiva (Kooistra y Ginther, 1975; Palmer y Driancourt, 1981; Palmer et al., 1982). Asimismo, la extirpacin de la glndula pineal o del ganglio superior cervical no da lugar a la desaparicin de la actividad reproductiva estacional, pero el ritmo reproductivo de estos animales no es ms sincronizado por los cambios del fotoperiodo (Sharp et al., 1979; Grubaugh et al., 1982).

FACTORES EXTERNOS QUE ARTICULAN EL RITMO ANUAL DE REPRODUCCIN


Fotoperiodo En los equinos, el fotoperiodo es el factor externo ms importante que influye el ritmo endgeno de reproduccin circanual (Ginther, 1992). La exposicin adicional, de las yeguas en inactividad ovulatoria, a la luz artificial durante el invierno y al inicio de la primavera estimula la actividad ovrica y es de uso general para avanzar el inicio de la poca de reproduccin. Sin embargo, la respuesta correcta a este fotoperiodo artificial estimulante est sujeta a condiciones especficas y la actividad reproductiva no es siempre seguida a la exposicin a das largos. La capacidad de influenciar el ritmo reproductivo circanual depende de varios factores: 1. del estado de refractariedad a los cambios fotoperiodicos, 2. de la historia fotoperiodica y 3. la existencia de una fase fotosensible durante la noche. Cuando las yeguas se mantienen bajo condiciones de das constantes de iluminacin (largos o cortos), reanudaran su ritmo de reproduccin circanual a pesar de las condiciones de iluminacin estimulantes o inhibitorias, (Kooistra y Ginther 1975; Palmer et al., 1982; Scraba y Ginther, 1985). Esta condicin es conocida como refractariedad y se puede definir como la incapacidad de continuar respondiendo al fotoperiodo de ese momento. Por esta razn, las yeguas mantenidas bajo 16L: 8D iniciando en invierno (Palmer et al 1982; Kooistra) o en verano (Ginther, 1975; Scraba y Ginther, 1985), retornaran a la inactividad ovulatoria invernal. El desarrollo de la fotorefractariedad puede ser prolongado o ser prevenido cambiando el fotoperiodo en intervalos regulares. En ovejas, la fase fotorefractariedad se puede retrasar por la exposicin diaria a luz; la cual disminuye de 16 a 12 h de luz y entonces de 12 a 8 h de luz. (Malpaux et al., 1988). Esto permite que el sistema reproductivo mantenga una sensibilidad constante a la luz. En carneros, la actividad reproductiva permanente era si los fotoperiodos inhibitorios y

estimulantes son aplicados con alternancias rpidos, observadas de 1 mes ms (Pelletier y Almeida, 1987). Un experimento similar tambin se fue realizado en yeguas ovariectomizadas (Palmer y Guillaume, 1992). Los animales fueron mantenidos bajo fotoperiodo natural o bajo alteracin rpida de ciclos de da largo y corto por 25 meses (70LD: 35SD y 35LD: 35SD). La disminucin estacional de la concentracin de LH observada en las yeguas testigo durante el invierno fue atenuada en los grupos bajo alternacin fotoperiodica rpida. Sin embargo, en yeguas ovrio intactas (Guillaume comunicacin personal) y en machos ponis (Cox y Skidmore, 1991), la ocurrencia de la inactividad ovulatoria estacional no fue eliminada por este horario de iluminacin. La historia fotoperiodica determina entonces la respuesta a un fotoperiodo especfico. En ovejas, la exposicin a la luz diaria de 13 h es estimulante si sigue un fotoperiodo de luz de 16 h pero es inhibitoria cuando es precedida por un periodo de 10 h luz (Robinson y Karsch, 1987). No se ha realizado ningn estudio en yeguas. La actividad ovrica se puede inducir en yeguas en inactividad ovulatoria aplicando un 1flash de luz aproximadamente 9.5 h y de 2 h de duracin. La actividad ovrica se puede inducir en yeguas en inactividad ovulatoria estacional aplicando un flash de luz de 1 a 2 h aproximadamente 9.5-10 h despus del inicio sbito de la oscuridad (Palmer y Driancourt, 1981; Palmer et al., 1982; Malinowski et al., 1985). Estas observaciones indican que, similar a otras especies estacionales, las yeguas tambin tienen una fase fotosensible durante el perodo de oscuridad. La presencia o la ausencia de la luz a 9.5 h despus del inicio de la oscuridad ms que la duracin total de luz y de la oscuridad son importantes para la respuesta. La exposicin a 16L: 8D, 14.5L: 9.5D, 8L: 9.5D: 1L: 5.5D o bien 4L: 9.5D: 1L: 9.5D produce un respuesta ovrica similar, aun cuando las yeguas expuestas a el protocolo de iluminacin (4L: 9.5D: 1L: 9.5D) reciben solamente 5 h de luz por da (Palmer y Driancourt, 1981; Palmer et al., 1982). Bajo condiciones naturales de fotoperiodo, hay un cambio gradual entre la fase de luz y la fase de oscuridad del da, la intensidad de la oscuridad puede variar considerablemente y la longitud de cada fase cambia gradualmente. Es probable que, bajo condiciones naturales de iluminacin, la percepcin de la salida y la puesta del sol sea diferente de las situaciones experimentales antedichas y el lugar de la fase fotosensible durante el ritmo circadiano puede variar. En los caballos, se ha estudiado tambin la influencia del fotoperiodo sobre la reproduccin bajo latitudes templadas como bajo latitudes tropicales. Las fluctuaciones regulares de temperatura y disponibilidad en alimento se correlacionan estrechamente con los cambios del fotoperiodo en las regiones templadas, pero de manera menos pronunciada en las regiones tropicales. Con todo, es el fotoperiodo que determina el momento de la reproduccin; as pues, en Venezuela (10 de latitud N, lo que representa aprox. 1 o 2 h de diferencia entre das cortos y das largos) o en Mxico, Quintero et al. (1995) y Boeta et al. (2005) ponen de evidencia, en la yegua, una inactividad ovulatoria, durante los das cortos y una actividad ovulatoria durante los das largos. Temperatura Para los animales que viven en latitudes templadas o ms extremas, el verano es un perodo de abundancia, con temperaturas moderadas y comida en cantidades importantes. Al contrario el invierno, puede ser hostil, con temperaturas bajas y una calidad y/o una disponibilidad de comida disminuida. Los organismos que viven en tal medio ambiente se enfrentan al doble reto de aprovechar de un verano de abundancia, y sobrevivir a las dificultades de invierno. En la oveja, la temperatura medioambiental no puede influir el aspecto estacional de la reproduccin, como fue demostrado por Wodzicka-Tomaszewska et al (1967). Estos autores encontraron que a pesar de cambios dramticos de la temperatura, el ritmo anual de reproduccin persista en ovejas bajo fotoperiodo constante (12 h luz y 12 h de oscuridad al da) (Wodzicka-Tomaszewska et al, 1967). Sin embargo, la temperatura puede modificar el inicio de la temporada de reproduccin (Lees, 1966). As pues, se puso

de manifiesto que ovejas, mantenidas bajo temperaturas bajas durante el verano, comenzaron su temporada de reproduccin ms pronto que las mantenidas bajo temperaturas normales (Dutt y Bush, 1955; Godley et al, 1966). Tambin, Lees (1966) encontr una correlacin positiva entre la temperatura media en julio y la fecha de inicio de la temporada de reproduccin en ovejas de raza Clun. En la yegua, un estudio retrospectivo sobre 10 prximos aos de los registros de un establo de yeguas Pura-sangre a una latitud de 35 S, sobre el momento del principio de la actividad ovulatoria, mostr una variacin significativa entre los aos: el inicio de la actividad ovulatoria se correlaciona estrechamente a la temperatura medioambiental mxima (r= 0,56; P= 0,09) y mnima (r= -0,67; P<0,01). Este estudio puso de manifiesto tambin que las temperaturas registradas en las semanas que siguen la ovulacin tendieran a aumentar. La temperatura puede ser entonces un zeitgeber adicional para el momento de la primera ovulacin en la yegua (Guerin and Wang, 1994). Palmer (1979) propuso que la fase sensible a la luz (9,5 a 10 horas despus del crepsculo) que se sita habitualmente cuando la temperatura es mnima era una seal importante para el inicio de la actividad ovulatoria de la yegua. Datos de campo de yeguas Pura-Sangre, en el Reino Unido fueron que la fase de transicin hacia la actividad ovulatoria en la primavera puede ser retrasada por las bajas temperaturas (Allen, 1987). Sin embargo, el inicio de la poca de reproduccin se estimula utilizando la fotoestimulacin en condiciones de invierno muy marcadas (Cooper and Wert, 1975). As, parece que bajo condiciones similares de fotoperiodo, de nutricin y de manejo, la temperatura un tiene una implicacin en la sincronizacin del ritmo reproductivo circanual. Nutricin y Condicin Corporal El efecto de la nutricin y la condicin corporal en la reproduccin estacional han sido descritos por varios autores. van Niekerk y van Heerden (1972) demostraron que las yeguas que reciben un suplemento ovulan ms pronto una vez que la inactividad ovulatoria ha terminado, que la yeguas testigo sin suplementacin y tambin observaron que el perodo anovulatorio es ms corto en las yeguas que ganan peso durante el inicio de la primavera (Ginther, 1974). McDaniel et al. (1979) reportaron un efecto aditivo de la suplementacin alimenticia y del fotoperiodo artificial en el inicio de la actividad reproductiva. Henneke et al. (1984) observaron que el intervalo promedio a la primera ovulacin fue ms largo en yeguas con una nota de condicin corporal de menos 5.0 (escala de 1 a 9) comparado con las yeguas con condicin sobre 5.0. La interaccin entre el consumo de energa y la condicin corporal en el funcionamiento reproductivo de yeguas no gestantes fue evaluado por Kubiak et al. (1987). Un alto consumo de energa acorta el intervalo a la primera ovulacin en yeguas en transicin hacia la actividad ovulatoria con un nivel bajo de reservas corporales pero no beneficia a las yeguas en condicin corporal moderada o yeguas gordas. Las yeguas con 15% ms de grasa corporal tienen un intervalo ms corto a la primera ovulacin comparado con aquellas con un 15% ms bajo de de grasas corporal. Esto resultados sugieren, para condiciones practicas, que las yeguas no lactantes pueden mantenerse durante la estacin de reproduccin sobre 15% de grasas corporal y una nota de condicin corporal sobre 5.0 y mantenerlas en un balance energtico positivo para obtener un inicio temprano de la primera ovulacin. La condicin corporal pobre no slo afecta el tiempo de la fase de transicin y de la primera ovulacin anual, tambin disminuye la eficacia de la inyeccin de un extracto pituitario equino para la induccin de ovulacin (Bour et al., 1985). No slo el consumo de energa, sino tambin la calidad de la protena en la racin, afectan el inicio de poca de reproduccin. Los animales que reciben protena de alta calidad en la dieta, presentan un incremento de la secrecin de FSH y ovulan aproximadamente 3-6 semanas ms temprano que las yeguas alimentadas con protena de baja calidad (Niekerk van y van Niekerk, 1997). Un efecto estimulante, del forraje pastoreado, en el tiempo de la

primera ovulacin tambin se ha reportado. La primera ovulacin del ao ocurre ms tarde en yeguas pura sangre que son mantenidas encerradas en las caballera durante la noche y llevadas a las praderas por 4-6 h/da mientras que las yeguas que fueron alimentadas en la caballeras durante invierno ovularon en sincrona despus de que fueron puestas en libertad en primavera a exuberantes pastizales (Allen, 1987). Carnevale y Ginther (1997) demostraron el efecto benfico de pastorear al inicio de la actividad ovulatoria. Las yeguas de inactividad ovulatoria pastoreadas en hierba verde de buena calidad a partir a principios mayo ovularon ms pronto que las yeguas mantenidas en pastos secos y alimentados con heno. Es posible que el inicio temprano de la inactividad ovulatoria observado en yeguas jvenes (Ginther, 1992) y la alta incidencia de la inactividad ovulatoria en yeguas lactantes (Palmer y Driancourt, 1983) estn tambin relacionadas con factores nutricionales. Experimentos recientes, en nuestro laboratorio, demuestran claramente el papel de la nutricin y de la condicin corporal en inicio de la inactividad ovulatoria en yeguas (Guillaume, et al., 2002). En estos estudios (Salazar Ortiz, 2006), el porcentaje de yeguas alimentadas a mantenimiento que presenta una actividad ininterrumpida durante el ao (60%) es superior a los reportado por Diekman et al (2002), pero nuestros resultados son similares a los observados por Fitzgerald y McMnaus (2000) y Gentry et al (2002). En estos estudios, como en el nuestro, dnde las condiciones de alimentacin son uniformes a lo largo de las estaciones, la variabilidad de respuesta de un animal ante otros factores medioambientales y la repetibilidad de esta respuesta para un mismo animal, sugieren que la actividad ovulatoria pueda ser influida por factores genticos. Es probable que el conocimiento de la implicacin de del leptina mejoren nuestra comprensin de la interaccin nutricin y la estacionalidad reproductiva. En efecto, la concentracin media de leptina en suero ms importante fue observada en potrancas exhibiendo actividad ovulatoria continua y estas son ms bajas de aquellas exhibiendo actividad ovulatoria estacional. En las potrancas (1 ao de edad) en inactividad ovulatoria todo el ao, las concentraciones de leptina fueron ms bajas que en las potrancas con ciclicidad prolongada o continua, pero tuvieron concentraciones de leptina ms elevadas que las potrancas con la actividad ovulatoria estacional (ebulj-Kadunc et al., 2009). Igualmente se han constatado variaciones estacionales de la concentracin de esta hormona en la yegua (Buff et al., 2007).

CONTROL NEUROENDOCRINO DE LA REPRODUCCIN ESTACIONAL


Funcin de la melatonina La melatonina ha sido el tema de muchos estudios en caballos y hay evidencias fuertes que esta hormona es uno de los elementos dominantes en el control de la reproduccin estacional. La funcin de la melatonina en otras especies es ms all del alcance de este documento y ha sido ampliamente repasados (Malpaux et al., 1999). Se ha demostrado que la glndula pineal est implicada en el mecanismo de control de la reproduccin estacional y traduce la seal fotoperiodica registrada por el ojo a seales endocrinas. qEn yeguas, las concentraciones elevadas de melatonina se asocian fuertemente a la fase oscura. La secrecin de melatonina aumenta al principio de la fase oscura y disminuye rpidamente al final de la noche. Una exposicin corta a la luz durante la fase oscura da lugar a una disminucin inmediata de la concentracin de melatonina y es seguida por una vuelta a las concentraciones de melatonina de la fase oscuro- cuando la exposicin luz es terminados (Guillaume y Palmer, 1991; Palmer y Guillaume, 1992). Durante horas oscuras, la secrecin de melatonina es estimulada por el norepinefrina secretado por las neuronas sinpticas postganglionar del superior del cervical del ganglio. En el caballo, este concepto es apoyado por la observacin que el isoproterenol, un agonista uno- adrenrgico, estimula la melatonina secrecin (Sharp et al., 1980). Las

concentraciones medias de melatonina en plasma son ms altas durante otoo e invierno que durante el primavera y el verano y la actividad HIOMT, una de las enzimas esenciales para la sntesis del melatonina, es ms alta durante la estacin anovulatoria y disminuye perceptiblemente 2-3 meses antes del inicio de las estacin de reproduccin (Wesson et al., 1979). La primera evidencia directa de un implicacin funcional de la melatonina fue proporcionada por Grubaugh et al. (1982) usando yeguas pinealectomizadas bajo fotoperiodo de das largos. En estas yeguas, el inicio de la actividad reproductiva no fue avanzado por fotoperiodo artificial y en el invierno las yeguas pinealectomizadas ranudaron la actividad ovrica cclica perceptiblemente ms adelante que las yeguas testigo durante la segunda estacin de reproduccin despus de la pinealectomia. El papel de la melatonina en la transferencia de la seal fotoperiodica fue demostrado ms profundamente por Guillaume y Palmer (1991) quienes demostraron que la melatonina exgena administrada 4 h antes del inicio de noches cortas (14.5L: 9.5D) previenen el efecto estimulante de los das largos. Similarmente, yeguas en el inactividad ovulatoria estacional bajo fotoperiodo artificial (14.5L: 9.5D) no responden al fotoperiodo estimulante cuando la melatonina se administra cada 2 h durante un perodo de 12 h que incluya al periodo oscuro de 9.5 h (Palmer y Guillaume, 1992). En estas yeguas, el inicio de la oscuridad, marcado por un aumento en la secrecin del melatonina, y el inicio del da, evidente por una disminucin de la secrecin de melatonina, fue enmascarado por los niveles altos de melatonina exgena. No slo la administracin diaria de melatonina, sino tambin el uso constante, a largo plazo de melatonina, influencian la reproduccin estacional en caballos. Implantes insertados cerca del da ms corto del ao suprimen el efecto estimulante de los das largos pero no previenen la ocurrencia de la actividad ovrica cclica. Los implantes de melatonina insertados cerca del solsticio de verano avanzaron la estacin ovulatoria el ao siguiente en yeguas adultas, (Guillaume et al., 1995). La explicacin de esta ltima observacin es una secuencia de una percepcin temprana de los das cortos, causada por las altas concentraciones continuas de melatonina, seguidas por un inicio temprano del estado refractario a los das cortos. Bajo este estado refractario, el ciclo reproductivo anual es avanzado en su fase y se manifiesta en la ocurrencia temprana de la estacin ovulatoria el ao siguiente. Estas observaciones apoyan el concepto que el fotoperiodo y el ritmo de la secrecin de melatonina articulan el ritmo circanual endgeno pero no influencian la actividad reproductiva directamente. Aunque, el efecto del fotoperiodo est bien documentado, el sitio de la accin de la melatonina no se ha estudiado en caballos. De estudios en otras especies, se sabe que la melatonina no influencia directamente la secrecin GnRH, pero acta a travs de una red compleja de interneuronas que implican un nmero diverso de neurotransmisores y sus sitios de accin parecen estar situados en el hipotlamo (Malpaux et al., 1999). En los caballos, la afinidad especfica de la melatonina fue encontrada en la pars tuberalis, en la eminencia media y en los ncleos supraquiasmticos (Stankov et al., 1991). Las concentraciones de melatonina no parecen siempre reflejar las condiciones reales de iluminacin. Bajo fotoperiodo natural, el aumento de la concentraciones de melatonina circulante en la noche es a veces difcil de distinguir al principio de la fase oscura y puede incluso estar ausente durante perodo enteros de oscuridad (Fitzgerald y Schmidt, 1995). Es incierto si estas yeguas carecen de un ritmo circadiano o si los mtodos disponibles para medir concentraciones de melatonina en sangre perifrica son inadecuados para medir los cambios muy leves de la secrecin de melatonina entre el da y la noche. En ovejas, se ha demostrado que las concentraciones de melatonina son varias veces ms elevadas en el tercer ventrculo que en la sangre perifrica y se postula que en caballos, como en las ovejas, las concentraciones de melatonina circulante son un reflejo dbil de la secrecin de melatonina (Skinner y Malpaux, 1999; Tricoire et al., 2002). Asimismo, nosotros

hemos puesto en evidencia que la concentracin media de melatonina no se correlaciona con la manifestacin o la duracin de la fase de inactividad ovulatoria. Nuestros resultados confirman que los niveles plasmticos nocturnos de melatonina no estn relacionados a la duracin de la inactividad ovulatoria (Salazar Ortiz, 2006). Pero, sigue siendo probable que los niveles plasmticos de melatonina estn modificados dbilmente por el nivel de alimentacin o el estado corporal de la yegua (Salazar Ortiz, 2006).

EL PAPEL DE LOS NEUROTRANSMISORES


Semejantemente a otros criadores estacionales, los datos disponibles en yeguas sugiere que la ausencia de la actividad reproductiva durante inactividad ovulatoria estacional sea el resultado de una inhibicin de la secrecin de GnRH inducida por varios sistemas neuronales inhibitorios dentro del hipotlamo. Estos sistemas neuronales median el efecto de factores internos y externos tales como el ritmo, el fotoperiodo, la nutricin, y la temperatura endgenos, cada uno quizs acta con una va en parte independiente. Fue sugerido recientemente que un cambio interaccin existe entre estos sistemas inhibitorios y que sus contribuciones relativas a la inhibicin de la actividad reproductiva varan durante el perodo de inactividad ovulatoria estacional (Kao et al., 1992; Bertrand et al., 1998). En caballos, dos acercamientos experimentales se han utilizado para estudiar la implicacin de los neurotransmisores en el control de la secrecin de gonadotropinas y/o del GnRH. El papel de neurotransmisores ha sido examinado determinando el efecto agudo de agonistas y antagonistas en la secrecin de gonadotropinas (Fitzgerald y Mellbye, 1988; Irvine et al., 1994; Aurich et al., 2000). La administracin de antagonistas y agonistas por perodos prolongados para estudiar el efecto sobre actividad ovrica y de la poca de la primera ovulacin en la primavera (Besognet et al., 1996, 1997; Muehl y Cruz, 2000, Brendemuehl y Cross, 2000).

OPIOIDES
En yeguas, en inactividad ovulatoria estacional, la administracin de un antagonista de opioides, la naloxona, da lugar a un aumento inmediato de la secrecin LH (Aurich et al., 1994). Irvine et al., (1994) reportaron que el aumento inducido por la inyeccin de naloxona en la secrecin de gonadotropinas es dependiente de la dosis y la curva de respuesta es acampanada. Esto puede explicar en parte porque Sharp et al. (1985) usando dosis elevadas de naloxona (2 mg/ kg PV) no observaron un efecto de los opioides en la secrecin LH en yeguas en inactividad ovulatoria. Adems, la inhibicin por opioides es aumentada durante la estacin anovulatoria y la ocurrencia de la actividad ovulatoria cclica de algunas yeguas, durante la estacin de inactividad ovulatoria, es asociada a una reducida inhibicin opioidenergica del eje hipotlamo-pituitaria (Turner et al., 1995; Davison et al., 1998).

LAS CATECOLAMINAS: EL PAPEL DE LA DOPAMINA


Adems del sistema opioide, las neuronas catecolaminergicas han sido implicadas en la inhibicin estacional de la secrecin GnRH. La xylazina, un agonista alfa-adrenergico, aumenta la frecuencia de pulsos de FSH y LH en yeguas en inactividad ovulatoria estacional. La ausencia de aumento por inyeccin de xylazina en yeguas cclicas y yeguas expuestas a fotoestimulacin por 27, das indica que la actividad, de este sistema catecolaminergico inhibitorio, disminuye hacia la estacin de reproduccin (Fitzgerald y Mellbye, 1988). La funcin de la dopamina en la estacionalidad ha sido tema de estudios extensos en varias especies. En ovejas, la dopamina regula la retroalimentacin negativa estacional del estradiol durante la inactividad ovulatoria estacional (Havern et al., 1994). La secrecin de gonadotropinas durante la inactividad ovulatoria puede ser inhibida en parte por las neuronas dopaminergicas que actan directamente en las neuronas secretoras de GnRH (Pau et al., 1982; Kao et al., 1992; Le Corre y Chemineau, 1993; Havern et al., 1994). La inyeccin

sistmica de un antagonista de dopamina, a ovejas estrgeno-implantadas ovario-intactas y ovejas ovariectomizadas o a carneros, estimula la frecuencia de pulsos de LH durante la estacin de inactividad ovulatoria pero no durante la estacin de reproduccin (Meyer y Goodman, 1985; Meyer y Goodman, 1986; Le Corre y Chemineau, 1993; Tortonese y Lincoln, 1994). Similarmente, la secrecin de gonadotropinas es aumentada cuando los implantes con antagonista de dopamina son colocados en la eminencia media, si las vas de la dopamina son destruidas por la intervencin quirrgica o qumica o despus de la obstruccin de la sntesis de la dopamina en la eminencia media (Thiery et al., 1989; Havern et al., 1991; Bertrand et al., 1998; Vigui et al., 1998). En la yegua, la concentracin de dopamina en el fluido cerebroespinal (CSF) es ms elevada durante la fase anovulatoria estacional que durante la estacin de reproduccin (Melrose et al., 1990) y la inyeccin a largo plazo con un antagonista D2 de la dopamina (sulpiride, perphenazine, domperidone) induce la actividad ovrica cclica en yeguas en inactividad ovulatoria estacional (Besognet et al., 1996, 1997; Bennett-Wimbush et al., 1998). Sin embargo, el efecto del antagonista de dopamina en el inicio de la actividad reproductiva es variable entre experimentos y entre yeguas; esto plantea algunas preguntas sobre el papel relativo de la dopamina en la regulacin de la reproduccin estacional. En un estudio, fue observado que la administracin del antagonista D2 de la dopamina por 28 das dos veces/da, aumenta el desarrollo folicular y avanza el inicio de la actividad ovulatoria cclica en las yeguas que haban estado previamente en fotoperiodo estimulatorio por 28 d, pero no acelera la actividad reproductiva en las yeguas no fotoestimuladas y previamente consideradas en inactividad ovulatoria profunda (Nagy et al., 1999, Donadeu y Thompson, 2002). Estas ltimas observaciones indican que los antagonistas D2 de la dopamina inducen solamente la ovulacin cuando las yeguas han entrado en fase de transicin de inactividad ovulatoria profunda a actividad ovrica cclica y pueden explicar la variabilidad grande en intervalo del inicio del tratamiento a la primera ovulacin entre las yeguas y los estudios, y la falla del tratamiento en yeguas mantenidas bajo condiciones ambientales desfavorables (Nequin et al., 1993; Besognet et al., 1996, 1997; Bennett-Wimbush et al., 1998; Daels et al., 2000) aunque resultados recientes demuestran lo contrario (Mari et al., 2009). El mecanismo de accin de la dopamina en el control de la reproduccin estacional no es muy claro en la yegua. Algunos resultados, sobre la secrecin de gonadotropinas, despus de la administracin de un antagonista de la dopamina interrogan sobre el sitio de la accin central/hipotalmico de la dopamina (Nequin et al., 1993; Daels et al., 2000). La inyeccin de antagonistas D2 de la dopamina en yeguas durante la inactividad ovulatoria estacional no indujo cambios agudos en la secrecin LH y FSH (Nequin et al., 1993; Aurich et al., 2000). La administracin de un antagonista de la dopamina, a largo plazo, puede influenciar la secrecin de FSH y los niveles bajos de LH y FSH pueden ser perceptiblemente ms altos durante un periodo de 7 das precediendo el primer pico ovulatorio de LH en yeguas (Besognet et al., 1997). Sin embargo, Brendemuehl y Cross (2000) no encontraron ningn efecto sobre la secrecin de FSH despus de que el tratamiento con domperidone pero los niveles de LH y de estrgenos fueron aumentados perceptiblemente por 28 das de tratamiento. Las concentraciones medias FSH en plasma 30 das antes de la ovulacin no fueron diferentes en las yeguas tratadas con perphenazine y las yeguas no tratadas (Bennett-Wimbush et al., 1998). Finalmente, los parmetros de secrecin de FSH durante perodos de 24h muestreo intensivo permanecieron sin cambios en yeguas en inactividad ovulatoria estacional en yeguas tratadas con un antagonista de dopamina, el sulpiride (Daels et al., 2000). En el carnero, el sulpiride solamente tiene efectos menores en la secrecin de la LH, pero realza perceptiblemente el efecto estimulante de la naloxona en fotoperiodo inhibitorio (Tortonese, 1999). Esto sugiere una interaccin activa entre los sistemas dopaminergico y opioidergico

en el control de la reproduccin estacional e indica que el efecto de la dopamina, sobre la secrecin de GnRH, pueda no ser inhibitorio sino algo permisivo. Las interacciones dopaminergicas y opioidergicas tambin se han observado en la rata, pero los resultados no han podido demostrar esta interaccin en el caballo (Callahan et al., 1996; Aurich et al., 2000). Los resultados controversiales, sobre el papel de la dopamina en el control de la secrecin de gonadotropinas en yeguas, sugieren que los antagonistas de la dopamina pueden actuar en diversos sitios del eje reproductivo, y posiblemente en el ovario (Mari et al., 2009). La administracin de antagonista de la dopamina aumenta importantemente la secrecin de prolactina. La prolactina puede ejercer su efecto sobre el ovario aumentando el nmero de receptores de gonadotropinas y as regular su efecto en las gonadotropinas circulantes, como se ha demostrado en el hmster y la rata prepuber (Advis y Ojeda, 1978; Advis et al., 1981; Klemcke et al., 1984). Mientras que los receptores de prolactina se han localizado en las clulas de la granulosa en cerdos y en hamsters Oxberry y Greenwald, (1982; Bevers et al., 1988). No hay informacin de la presencia de los receptores de prolactina en el ovario equino. Sin embargo, la administracin del prolactina recombinante porcina adelanta la fecha de la primera ovulacin en yeguas en inactividad ovulatoria estacional (Thompson et al., 1997). La disminucin de la secrecin prolactina inducida por la inyeccin de bromocryptina no tiene un efecto en el desarrollo folicular, la esteroidogenesis y la ovulacin en el periodo postparto en yeguas (Neuschaefer et al., 1991) pero parece retrasar el crecimiento de los folculos de tamao preovulatorio sin afectar el intervalo a la primera ovulacin en yeguas en inactividad ovulatoria estacional (Bennett-Wimbush et al., 1998). Los datos disponibles sugieren que los antagonistas de la dopamina aceleran el inicio de la actividad ovrica cclica en yeguas transicin vernal, y el tratamiento puede ser eficaz igualmente durante la inactividad ovulatoria estacional profunda as como en el desarrollo folicular (Kelley et al., 2006; Mari et al., 2009). El inicio del perodo de transicin y la primera ovulacin son adelantados con la inyeccin de sulpiride pero no por el domperidone en todas la yeguas (Mari et al., 2009). El dimetro del folculo ms grande es afectado por la poca y aumentado significativamente por la inyeccin de sulpiride. Aunque un efecto principal del sulpiride en la concentracin plasmtica de LH en plasma no es observado, la poca es significativa. El intervalo a la primera ovulacin es ms corto por la inyeccin de sulpiride (36.9 d) comparado con la inyeccin de domperidone (74.7 d) o las yeguas no tratadas (81.4) (Mari et al., 2009). Esto sugiere, que la dopamina, similarmente a otros neurotransmisores, ejerce una inhibicin tnica a nivel hipotlamo en el control de la reproduccin estacional en yeguas.

AMINOCIDOS NEURO-EXCITATORIOS Y SEROTONINA


El cese de la actividad reproductiva durante la inactividad ovulatoria estacional podra ser en parte a la disminucin de la actividad neuronal. En un estudio, el N-metl-DL-aspartico (NMA), un agonista de los receptores para los aminocidos neuro-excitatorios, aumenta la secrecin de gonadotropinas en yeguas intactas como en las ovariectomizadas durante la inactividad ovulatoria estacional (Fitzgerald, 1996). Sin embargo, una funcin en la regulacin de la reproduccin estacional no se podra demostrar, puesto que los aumentos inducidos por NMA son similares en yeguas en inactividad ovulatoria estacional y yeguas que exhiben actividad ovrica cclica durante de la estacin de inactividad ovulatoria (Fitzgerald y Davis, 1997). El efecto de la serotonina en la inhibicin esteroide-independiente de la secrecin de GnRH en la oveja en inactividad ovulatoria estacional, no es conocido en la yegua (Meyer y Goodman, 1986; Le Corre y Chemineau, 1993).

FUNCIN DE LAS HORMONAS DE TIROIDEAS


En aves y en varias especies de mamferos, la funcin de las hormonas tiroideas en el control de la reproduccin estacional ha sido bien establecida (Nicholls et al., 1988; Moenter et al., 1991). En ovejas, la tiroidectomia durante el perodo de inactividad ovulatoria estacional bloquea la transicin de la estacin de reproduccin hacia la inactividad ovulatoria estacional. El efecto del tiroidectomia se puede prevenir completamente por la administracin de tiroxina durante la estacin de reproduccin (Webster et al., 1991). En yeguas, la tiroidectoma no altera el inicio de la inactividad ovulatoria estacional (Porter et al., 1995) pero los niveles plasmticos de tiroides son perceptiblemente ms bajo en las yeguas en inactividad ovulatoria estacional comparadas con aquellas que continan exhibiendo ciclos estrales durante la estacin anovulatoria (Fitzgerald y Davison, 1998; Huszenicza et al., 2000). Se sugiere que los niveles T3 y T 4 y la actividad reproductiva son regulados por mecanismos hipotalmicos de control similar. Comparado con las ovejas, las hormonas tiroides no parecen desempear un papel importante en el control de la reproduccin estacional en yeguas. Sin embrago, adems de presentar variaciones estacionales de las hormonas tiroideas, mas importantes en verano que en invierno, estas pueden controlar, en parte, la secrecin de leptina (Buff et al., 2007).

CONCLUSIN
La nutricin parece ser el factor principal que determina la duracin del periodo de inactividad ovulatoria. La entrada en reposo sexual dependera de la condicin corporal de las yeguas antes del otoo-invierno y de las variaciones de algunas hormonas relacionadas con la nutricin. La comprensin de la regulacin estacional de la reproduccin en yeguas ha aumentado perceptiblemente y estos aciertos conducirn indudablemente para mejorar las estrategias de tratamiento para la induccin de la actividad ovrica cclica al inicio de la primavera. En el futuro tambin tendremos una comprensin mejor de los mecanismos que controlan el desarrollo de la inactividad ovulatoria estacional.

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HORMONE THERAPY: BASIC AND ADVANCED


Patrick M. McCue, DVM, PhD, Diplomate ACT Equine Reproduction Laboratory College of Veterinary Medicine and Biomedical Sciences Colorado State University Ft. Collins, CO 80523

The goal of this summary is to present an update the hormones currently available for use in equine reproduction.

A. GnRH and GnRH Agonists


Gonadotropin Releasing Hormone (GnRH), also known as Luteinizing Hormone Releasing Hormone (LHRH) Native or natural GnRH is a small (10 amino acid) peptide hormone produced in the hypothalamus. Pulses of GnRH released from the hypothalamus stimulate production of gonadotropins (LH and FSH) from the anterior pituitary. In the horse, there is limited clinical use for exogenous natural GnRH. Administration of native GnRH is not effective in inducing ovulation in mares due to the relatively short half-life and low potency of the hormone. Low doses of GnRH may be used to test pituitary function or responsiveness. GnRH has been used in some stallions with low libido or poor semen characteristics (low sperm numbers and/or low motility) in an effort to enhance reproductive performance. Doses administered to stallions range from 50 g every 1-2 hours subcutaneously via a minipump to 500 g once to twice daily by intramuscular to subcutaneous injection. GnRh has also been administered as a 500 g bolus intramuscularly 1-2 hours prior to breeding for selected stallions with low libido, with (limited) anecdotal evidence of a positive response reported by owners and/or stallion managers. There is little scientific data on the efficacy of GnRH in improving sperm quality in stallions. The hormone is available from Bachem California (LHRH; product number: H-4005). GnRH Agonists OvuplantTM (Ft. Dodge Animal Health) is the trade name for the drug deslorelin acetate, which is a potent, synthetic agonist of GnRH. The drug is administered as a subcutaneous implant. The drug is very effective in inducing ovulation when administered to mares in estrus that have a follicle larger than 35 mm in diameter. Approximately 85 to 95 percent of mares will ovulate within 48 hours after treatment. The average interval from treatment to ovulation is 42 hours. OvuplantTM is used as an alternative to human chorionic gonadotropin (hCG) for induction of ovulation. Administration of multiple or repeated doses of hCG to a mare during a breeding season may result in a decrease in effectiveness. The most common use of OvuplantTM in a breeding program is the induction of a timed ovulation, such as when mares are being bred a single time in a live cover program, or with cooled-transported semen or frozen semen. For example, in a shipped semen program, OvuplantTM may be administered to a mare in the morning after a semen shipment is ordered. Semen shipped by overnight courier would arrive the following morning and the mare would be inseminated. The mare would ovulate later that evening and the interval from insemination to ovulation would be less than 24 hours. Induction of ovulation with OvuplantTM may also be advantageous in certain older mares that are prone to prolonged periods of uterine inflammation after they are bred. Mating or inseminating these mares only one time may limit the degree of post-breeding

inflammation, decrease the amount of medical treatments required to clean up the uterus after breeding and possibly enhance pregnancy rates. OvuplantTM was approved for use in the United States for the induction of ovulation in mares in 1998. After OvuplantTM was used extensively in the United States during the 1999 and 2000 breeding seasons, several reports suggested that the interovulatory interval may be prolonged in some individual mares treated to induce ovulation that did not become pregnant. Clinical studies subsequently confirmed these early reports. The cause of the prolonged interovulatory intervals was determined to be a temporary suppression of pituitary function in treated mares. It was also determined that mares in embryo transfer programs that received prostaglandins after an embryo recovery attempt 7 to 8 days after an ovulation induced by OvuplantTM were most prone to the prolonged interovulatory intervals. Subsequent studies have indicated that removal of the OvuplantTM implant approximately 48 hours after administration prevents the suppression of pituitary function and the adverse effect on follicle development. In order to facilitate easy administration and subsequent removal, it is currently recommended that the OvuplantTM implant be administered just beneath the skin of the vulva following infusion of a local anesthetic. The implant can be removed 48 hours later after ovulation has occurred by gently squeezing the implant out through the original insertion site. OvuplantTM is currently off the market in the United States, but is available in Canada, Australia and Europe. Injectable versions of deslorelin acetate are available in the United States from compounding pharmaceutical companies. Most products are provided at a concentration of 1.5 mg/ml and a 1.0 ml intramuscular dose is recommended. Reports indicate that the compounded products result in a similar interval from treatment to ovulation and similar ovulation rates as the implant (OvuplantTM). Compounded injectable products are not specifically approved for use in the horse. Injectable deslorelin is used primarily to induce ovulation in mares that do not ovulate in response to hCG.Since deslorelin is a small molecule (9 amino acids), it is considered unlikely that mares will develop antibodies against it. It has been administered to mares over multiple estrous cycles without a decrease in efficacy. Alteration of reproductive function in mares using GnRH has been accomplished by active immunization and down-regulation of gonadotropin secretion. It has been reported that immunization of colts and fillies with a GnRH vaccine resulted in a decrease in fertility. A follow-up study indicated that ovarian activity and estrous behavior of fillies vaccinated against GnRH was suppressed for 25-30 weeks. The effect was reversible as all vaccinated horses eventually returned to estrus and ovulated. In addition, three of the four treated fillies became pregnant the following year. Vaccination against GnRH is potentially safe, effective, and eventually reversible. However, the duration of suppression of reproductive activity may not be as predictable or controllable as with other treatments. An anti-GnRH vaccine (EquityTM, Pfizer, Inc.) has been approved for use in horses in Australia. Potent agonists of GnRH have been demonstrated to cause down-regulation of pituitary gonadotropin secretion and short-term alterations in ovarian function in mares. Implants containing GnRH agonists have been used to produce controlled, reversible longterm suppression of reproductive function in dogs and cattle. Little to no information is available on the efficacy of sustained-release GnRH implants on long-term suppression of ovarian follicular activity and estrous behavior in mares.

B. Pituitary Gonadotropins
Human Chorionic Gonadotropin (hCG) Human chorionic gonadotropin (hCG) is a glycoprotein hormone used primarily to induce ovulation in mares. It is also used diagnostically in a stimulation test to detect the presence or absence of testicular tissue in geldings exhibiting stallion-like behavior. The biological action of this human-origin hormone is due to its inherent luteinizing hormone (LH) type activity. In mares, a surge of natural or endogenous LH from the anterior pituitary during estrus causes final maturation and ovulation of the dominant follicle. Exogenous hCG essentially mimics the role of endogenous LH. Induction of ovulation is advantageous if a mare is in a timed breeding, shipped semen, frozen semen or embryo transfer program. In addition, in mares with a history of accumulating fluid in the uterus following mating or insemination, it may be beneficial to induce ovulation and limit the number of times the mare has to be bred. During a natural estrous cycle, mares are typically in heat for 4-7 days. A traditional breeding program may entail mating a mare every other day while she is showing heat. Such a strategy would result in an average of 2-3 breedings per cycle. This may not be an issue if the mare is young and reproductively healthy and if the stallion is located on the same farm and semen is not limited. Administration of an ovulation-inducing agent such as hCG is common for mares enrolled in a shipped semen program in which the stallion owner is likely to only provide semen once during a given estrous cycle. Use of hCG may also be very beneficial when breeding mares with frozen semen, since a majority of mares will ovulate within a relatively consistent time period. Consequently, a mare can be inseminated with one dose of frozenthawed semen within a few hours of when she is predicted to ovulate and a second dose after ovulation has been confirmed. Human chorionic gonadotropin is generally administered when a mare is in estrus, a follicle >35 mm is present and edema is visible in the uterus on ultrasound. Ovulation will usually occur an average of 36 hours after hCG administration. A wide dose range of hCG has been used successfully for induction of ovulation and the drug may be given intravenously or as an intramuscular injection. A common dosage is 2,500 units administered intravenously. The hormone is very effective in inducing ovulation in young mares that have not received the hormone previously and in middle-aged to older mares receiving the hormone for the first time or two within a single breeding season. However, the efficacy may be somewhat reduced if hCG is given to a mare repeatedly during the same breeding season (i.e. the mare may not ovulate in the predicted time period). Colts or geldings with an uncertain medical or surgical history suspected of being cryptorchid may be administered hCG as part of a diagnostic test to determine if testicular tissue is present. The hCG stimulation test is performed by collecting a blood sample immediately prior to administration of 10,000 units of hCG intravenously and collecting a second blood sample one to two hours later. The samples are then submitted to a diagnostic laboratory for evaluation of testosterone levels. True geldings will have low testosterone levels in both samples. Intact normal stallions will have high levels of testosterone in the first sample and even higher levels in the second sample. Cryptorchid horses usually have moderately low levels in the first sample and increased testosterone levels in the second

blood sample. The LH-like activity of hCG causes an increase in testosterone production from Leydig cells of the testes, if they are present.

Human chorionic gonadotropin is listed as a controlled drug in some countries/states.


Recombinant equine FSH (reFSH) Recombinant equine luteinizing hormone (reLH) has recently been reported to be successful in inducing ovulation in cycling mares. Current indications are that 750 ug of reLH is sufficient to induce ovulation in a majority of mares in estrus with follicles > 35 mm in diameter. Equine Follicle Stimulating Hormone (eFSH)

Induction of multiple ovulations has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Unfortunately, techniques successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone (pFSH) and equine chorionic gonadotropin (eCG) have little effect in the mare). The most effective and consistent therapy to induce multiple ovulations in mares has been administration of equine pituitary gonadotropins. Equine pituitary extract (EPE) has been used for many years to induce multiple ovulations in mares. However, EPE is not commercially available.
Recently a highly purified equine FSH product (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) has become available commercially. The original or standard protocol for use of eFSH involved initiating treatment 5-7 days after ovulation and administering a luteolytic dose of prostaglandins the day after treatment was started. Mares were typically treated with 12.5 mg eFSH twice daily for 7 to 8 days before a cohort of follicles > 35 mm in diameter were present. Treatment with eFSH was then discontinued and hCG was given to induce ovulation. A subsequent study suggested that follicular development would continue if mares were allowed to coast after several days of eFSH treatment once follicles achieved 32 mm in diameter. HCG was administered approximately 36 hours after the last dose of eFSH. Ovulation rates utilizing the coasting protocol were noted to be similar to the standard protocol and potentially one day of eFSH treatment cold be eliminated. A more recent study looked at the efficacy of eFSH treatment after mares were allowed to spontaneously develop a cohort of follicles during the common growth phase. Treatment with eFSH was initiated when a cohort of follicles reached a diameter of 25-30 mm in diameter. Mares were treated twice daily until follicles reached 32 mm in diameter. The mares were then allowed to coast for 36 hours before receiving hCG. The most recent research is looking at the effect of a fixed 3-day treatment period initiated when follicles reach 25-30 mm in diameter. The goal is to push development of multiple follicles through the window of follicle deviation and allow a cohort of follicles to continue to develop with minimal assistance. Ultimately, it is likely that eFSH therapy will have to be tailored to the individual needs of a specific mare. In general, mares treated with eFSH average 3 - 4 ovulations and 1.5 2.0 embryos collected per cycle. Unfortunately, some mares treated with eFSH develop multiple large follicles that do not ovulate in response to hCG. Superovulation treatment is more effective in younger mares than in older mares.

Equine FSH has also been used in transitional mares to stimulate follicular development and advance the first ovulation of the year. Ten mares in spring transition with follicles > 25 mm in diameter were administered 12 mg of eFSH intramuscularly twice daily. Ten additional transitional mares served as untreated controls. Mares were administered hCG (2,500 units) once one or more follicles were > 35 mm in diameter. Administration of eFSH followed by hCG resulted in ovulation in 80 % of mares treated. The interval from onset of treatment to ovulation was 7.6 days, while untreated control mares ovulated in 34.9 days. In addition, multiple ovulations were induced in 40 % of mares treated, whereas none of the control mares had multiple ovulations. It was concluded that treatment of transitional mares with eFSH might be helpful in hastening the first ovulation of the year. Close monitoring for multiple ovulations and twins would be essential.

C. Gonadal Steroids
The primary gonadal steroids used in broodmare practice are progesterone and estradiol. Progesterone is a product of the ovarian corpus luteum and the placenta of the pregnant mare. The primary clinical uses of progesterone are suppression of estrus and maintenance of pregnancy. Products available include natural progesterone (P4) and one synthetic progestin (Regumate). Other synthetic progestins have not been shown to have biologic activity in the horse. Estradiol (E2) and other estrogens have limited use in equine practice. Estradiol cypionate (ECP) is used to induce estrus in ovariectomized jump mares. Compounded products containing a combination of progesterone and estradiol are used for suppression of estrus and/or estrous synchronization. Natural Progesterone (P4) Natural progesterone is available in an oil base (50 mg/ml) that must be administered intramuscularly once daily to be effective. A dose of 100-150 mg (i.e. 2-3 mls) is required to maintain blood progesterone levels high enough to suppress estrus and 200 mg progesterone per day is required to maintain pregnancy. Unfortunately, local tissue swelling and pain may occur following a prolonged course of intramuscular administration. Consequently, progesterone-in-oil is not routinely used to block estrus in performance mares or to maintain pregnancy. Altrenogest (Regumate) The most common clinical uses of the synthetic progestin Regumate (Intervet, Inc.) are management of the transition period, suppression of estrous behavior, estrous synchronization, treatment of luteal insufficiency, management of high risk pregnancies, and management of twins. Follicular development is initiated in the late winter or spring in response to increased day length. Mares develop waves of follicles that grow and regress without ovulating during this transition between winter anestrus and the physiologic breeding season. Mares may exhibit irregular or often prolonged periods of sexual receptivity or heat during the transition. In an attempt to manage mares during the late transition period, Regumate may be administered once daily for 14-18 days, when follicles are >30 mm. The dose most commonly used is 1 ml/110 lbs or approximately 10 mls orally once daily. Regumate has little effect on follicular activity in mares in deep winter anestrus or early in the transition period. Mares may occasionally be difficult to train or manage or may perform inadequately during the estrous phase of their reproductive cycle. It may be advantageous to suppress the expression of estrous behavior in these individual mares during the training or performance

sessions. Regumate is the hormone used most commonly for suppressing heat. Treatment must begin a minimum of 2 to 3 days prior to the event. Mares ovulate at approximately 21-day intervals during the physiologic breeding season. It may be desirable to synchronize the ovulations of 2 or more mares for the purpose of embryo transfer or scheduled breedings. Regumate is administered once daily for 10 to 14 days. Prostaglandins may be administered on the last day of Regumate treatment. Progesterone production by the corpus luteum is required for maintenance of pregnancy during the first 2-3 months of gestation. Inadequate production of progesterone by the corpus luteum has been proposed to be a contributing factor to early embryonic loss in mares. Progesterone insufficiency is a controversial topic. Low serum concentrations of progesterone may be due to a primary luteal insufficiency or secondary to failure of maternal recognition of pregnancy and subsequent luteolysis. Progesterone levels above 4.0 ng/ml are generally considered adequate to maintain pregnancy. Mares with concentrations below 4.0 may be at increased risk of pregnancy loss. Therapy consists of progesterone supplementation and is usually initiated either 1 to 2 days after ovulation is detected or after ultrasonographic pregnancy diagnosis (i.e. day 14) and is continued until day 100 to 120 of pregnancy, at which time production of progesterone by the placenta is adequate to maintain pregnancy. Supplemental progesterone administration may be used to support problem pregnancies or pregnancies at risk due to endotoxemia, colic or other medical problems. In these instances, altrenogest is commonly given at twice the normal dose (i.e. 20 mls) during the time the mare is at high risk. Treatment can be continued for the duration of the pregnancy or tapered down gradually and discontinued. Regumate is usually administered at twice the standard dose for the management of high-risk pregnancies. Additional potential treatments may include flunixin meglumine, antibiotics or other medications.

In addition, Regumate may be given following manual reduction of a twin pregnancy to help with the maintenance of the remaining embryo.
Long-Acting Progesterone A compounded long-acting product containing natural progesterone that may be administered once every 7 days to maintain pregnancy in mares is available in some countries. Preliminary studies indicate that administration of 10 mls of long-acting progesterone (150 mg/ml) is effective at maintaining pregnancy in mares in the absence of endogenous progesterone. Some anecdotal reports indicate that some mares will have a significant inflammatory response at the injection site(s). Progesterone Implants Implants used to promote weight gain and feed efficiency in cattle that contain progesterone and estradiol (Synovex) have been administered to horses in an attempt to suppress heat. A dose of 8 Synovex pellets contains 200 mg of progesterone and 20 mg of estradiol benzoate. The implant is designed to release product over a period of 100-150 days in cattle. Assuming a constant rate of degradation (which is unlikely), these implants would release 1.3 2.0 mg of progesterone daily. A controlled clinical trial showed that administration of 8, 32 or 64 Synovex pellets did not prevent ovarian follicular development or suppression of estrus when mares were exposed to a stallion. In a follow-up study, 8 Synovex pellets were administered subcutaneously to each of 6 geldings. Blood samples were collected every 5 days for 45 days and subsequently assayed for progesterone levels. Progesterone remained undetectable in blood throughout the evaluation period. The low amount of progesterone in the implants and the designed slow release rate are responsible for the lack of efficacy. Consequently, the use of Synovex pellets for suppression of estrus in performance mares is

not supported or recommended. In addition, Synovex pellets are only licensed for use in cattle and are not approved for use in the horse. Other progestins Other synthetic progestins, such as medroxyprogesterone acetate (Depo-Provera), hydroxyprogesterone caproate (Hyproval), norgestomet (Synchro-Mate B) and megesterol acetate (Ovaban) have been administered to mares in an attempt to block estrus or maintain pregnancy. The main purported advantage of these implant or depot hormone preparations is that they may be administered periodically instead of daily. Unfortunately, there are no synthetic progestins other than altrenogest that are effective in suppression of estrus or maintenance of pregnancy in mares. The reason for the lack of efficacy of these compounds is failure to bind adequately to the equine progesterone receptor. Consequently, although these products may appear to be convenient, they should not be used in horses due to lack of efficacy.

D. Miscellaneous Hormones
Prostaglandins The most common clinical uses of prostaglandins in equine reproduction are short-cycling, estrous synchronization, treatment of persistent corpora lutea, termination of pregnancy, and evacuation of uterine fluid. The equine estrous cycle is 21 days in duration, and consists of an estrous (heat) phase of 4-6 days and a diestrus (nonsexually receptive) phase of 14-15 days. The interval between ovulations may be shortened by interrupting the diestrus or luteal phase of the cycle. The corpus luteum that forms from the follicle after ovulation can be lysed or destroyed by prostaglandins beginning 5 days after ovulation. This may be advantageous in a timed breeding program, after having missed a breeding or after a mismating. The most common dosages of prostaglandins used in clinical practice are: Lutalyse, 10 mg or Estrumate, 250 ug, intramuscularly, once. There is a higher incidence of side effects (i.e. sweating and/or abdominal cramping) with Lutalyse. It may be desirable to synchronize the ovulations of 2 or more mares for the purpose of scheduled breedings or embryo transfer. Estrous synchronization can be accomplished by administration of two doses of prostaglandins (Lutalyse or Estrumate) given 14 days apart. The corpus luteum that forms after ovulation is usually functional for 14-15 days in the nonpregnant mare. Luteolysis, or destruction of the corpus luteum, occurs as a result of prostaglandin release from the endometrium. Occasionally, however, a mare may fail to spontaneously regress her corpus luteum at the normal time. The most common causes of a persistent corpus luteum are inadequate prostaglandin release at days 13 to 15, ovulations late in diestrus resulting in corpora lutea that are immature (< 5 days old) at the time of prostaglandin release, embryonic loss after the time of maternal recognition of pregnancy and chronic uterine infections resulting in destruction of the endometrium, resulting in diminished prostaglandin release. If untreated, the corpus luteum may persist for 2 to 3 months. Elective termination of a pregnancy or a potential pregnancy may be indicated following an unplanned mating of a mare, breeding a mare with the wrong stallion, following purchase of a mare that was thought to be open or during other situations. Termination of an early pregnancy prior to endometrial cup formation (<35 days gestation) will generally allow the mare to return to estrus and ovulate within 5-10 days, provided the corpus luteum is lysed. Pregnancy termination using a technique other than prostaglandin administration after the time of maternal recognition of pregnancy (approximately 12-14 days postovulation) may

result in persistence of the corpus luteum. A single administration of prostaglandins (Lutalyse or Estrumate) is usually sufficient for complete luteolysis and termination of pregnancy from day 5 postovulation until formation of the secondary corpora lutea. The mare should be re-evaluated 5-10 days later to confirm that the pregnancy is gone. The presence of a small volume of ultrasonographically clear fluid within the lumen of the uterus is a normal clinical finding during estrus. Some mares will accumulate relatively large quantities of fluid during estrus. Reproductively healthy mares will usually reabsorb or expel all of the uterine fluid present during estrus. Uterine fluid present during diestrus may be associated with inflammation or infection and is considered to be potentially pathologic. Older mares may not be able to physically clear fluid from their uterus and fluid retention may be associated with subfertility. Mares with purulent endometritis or pyometra may also accumulate large quantities of fluid with echogenic particles detectable on ultrasonography. Prostaglandins may be given to stimulate uterine contractions and expel fluid through the cervix. The effect lasts 2 to 4 hrs. Several recent studies have determined that administration of the prostaglandin analog cloprostenol (Estrumate) to mares either the day ovulation is detected or for 1-2 days after ovulation will affect function of the developing corpus luteum. Complete luteolysis does not occur, but progesterone concentrations are significantly lower for the first 5-7 days of the diestrous period. Pregnancy rates have been reported to be lower in mares that received prostaglandins in the immediate postovulation period. In contrast, treatment with prostaglandins prior to ovulation does not affect CL function. Oxytocin does not have any adverse effects on corpus luteum development when administered either prior to or after ovulation. Consequently, prostaglandins or oxytocin may be used to evacuate uterine fluid prior to ovulation, but oxytocin appears to be a better choice for treatment of uterine fluid after ovulation is detected. Oxytocin The three most common clinical uses of oxytocin include evacuation of uterine fluid, induction of labor and management of retained placentas. The presence of a small volume of ultrasonographically clear fluid within the lumen of the uterus is a normal clinical finding during estrus. Some mares will accumulate relatively large quantities of fluid during estrus. Reproductively healthy mares will usually reabsorb or expel all of the uterine fluid present during estrus. Uterine fluid present during diestrus may be associated with inflammation or infection and is considered to be potentially pathologic. Older mares may not be able to physically clear fluid from their uterus and fluid retention may be associated with subfertility. Mares with purulent endometritis or pyometra may also accumulate large quantities of fluid with echogenic particles detectable on ultrasonography. Oxytocin may be given to stimulate uterine contractions and expel fluid through the cervix. The dose most commonly used is 20 IU, intravenously or intramuscularly, one to three times daily. Elective induction of labor in the mare for nonmedical reasons is generally not recommended. The stringent guidelines for induction of parturition in the mare that should be followed are a gestation length of at least 330 days, significant udder development and engorgement of the teats with colostrum and relaxation of the sacrosciatic ligaments and vulva. Additional criteria that may be used are softening of the mare's cervix as detected during a digital vaginal exam and milk calcium levels of > 200 ppm. Caution should be exercised when electing to induce labor as fetal maturity is critical to survival and early termination of a pregnancy, even at an apparently appropriate gestation length, can result in delivery of a dysmature foal. Oxytocin administration is a reliable

technique for induction of labor in the mare. Several dosage and administration regimens are available and delivery of the fetus usually occurs within 30 to 60 minutes following initiation of treatment. One commonly used dose regimen involves administration of 5.0 IU (0.25 mls of a 20 IU/ml product), intravenously, followed by 10 IU 15 minutes later. The mare will usually rupture her chorioallantoic membrane 8 to 10 minutes after the second dose of oxytocin. Retention of the fetal membranes is one of the most common postpartum problems in the mare. The placenta of the mare is considered to be pathologically retained at 3 hours postpartum. The entire chorioallantoic membrane or just the tip of the nonpregnant horn may be retained in the uterus. The incidence of retained placentas increases in mares with dystocia, prolonged gestation, hydrops, cesarean surgeries or induced labor. Complications associated with retained fetal membranes include metritis, laminitis, septicemia and death. Oxytocin may be given every hour beginning 2-3 hours after foaling. The dose of oxytocin recommended is 20 IU, intravenously or intramuscularly, repeated as needed. Additional treatments for retained placenta may include uterine lavage, intrauterine antibiotics, parenteral antibiotics, nonsteroidal anti-inflammatory drugs, and tetanus toxoid. Domperidone Domperidone, marketed under the name Equidone, is a medication in a class known as dopamine antagonists. Dopamine is a brain neurotransmitter that modulates or suppresses production of the hormone prolactin from the pituitary. Domperidone binds to the dopamine receptor and prevents the inhibition of prolactin secretion. Clinical uses of domperidone in equine reproduction include treatment of fescue toxicosis, stimulation of lactation in agalactic mares, induction of lactation in nurse mares and induction of follicular development in transitional mares. Fescue toxicosis is a clinical syndrome that may occur when pregnant mares ingest tall fescue grass infested with a specific endophytic fungus. Clinical signs associated with fescue toxicosis include lack of milk production, prolonged gestation, thickened fetal membranes, premature separation of the placenta or red bag, retained fetal membranes, abortion, dystocia, reduced fertility and increased neonatal mortality. These effects are due to production of a toxin by the fungus (called ergovaline) which binds to dopamine receptors and suppresses pituitary prolactin secretion. Management of fescue toxicity may include removal of late-term pregnant mares from affected pastures for the last 2-3 months of gestation and/or administration of domperidone beginning 10-15 days prior to the expected due date. It is recommended that domperidone therapy be continued until 5-10 days after foaling. Treatment with domperidone will prevent or lessen the severity of adverse clinical signs commonly associated with fescue toxicosis. Failure of udder development and lactation occasionally occurs in late-term or foaling mares not grazing on fescue pastures. Lack of milk production or agalactia is most common in young mares giving birth to their first foal. Failure of the mammary gland to produce colostrum at the end of pregnancy may result in failure of passive transfer of antibodies to the newborn foal. In addition, decreased milk production following foaling will result in inadequate nutrition for the growing foal. Lactation can often be stimulated in mares with poor milk production by administration of domperidone twice daily for 2-4 days and then once daily for the next 6-8 days. Domperidone therapy may be initiated prior to foaling if limited mammary development is noted as a mare approaches her due date.

Domperidone has also been used to induce lactation in non-foaling mares to be used for adoption of orphaned foals. Mares that are cycling and have had at least one foal previously are more likely to respond to treatment regimens designed to induce lactation. Orphaned foals have been successfully fostered to and raised to weaning age by mares induced to lactate. Continued therapy with domperidone for several days after adoption of a foal by a foster mare may be beneficial in maintaining or increasing milk production. Finally, domperidone has been used to stimulate follicular development and advance the first ovulation of the year in spring transition mares. Treatment of transitional mares with domperidone has been effective in promoting follicular growth in some clinical trials but not in others. Comments regarding clinical response to treatment from owners and veterinarians have also been mixed. It has been noted that domperidone therapy is less effective in inducing follicular development if environmental temperatures are low (i.e. colder climates or mares maintained outdoors) and more effective if mares are under lights. A current working hypothesis is that dopamine antagonists (i.e. domperidone and sulpiride) increase the number of gonadotropin receptors on the ovary, which would theoretically make the ovary more responsive to endogenous gonadotropins. The most commonly recommended protocol is to begin treatment with domperidone after mares have been under a stimulatory artificial photoperiod for at least 2 weeks. Prostaglandin E1 Topical application of prostaglandin E1 (Misoprostol) has been used in attempts to promote cervical relaxation in mares. Misoprostol (Cytotec; Pfizer, Inc.) is a human product currently approved in the USA only for reducing the risk of gastric ulcers caused by the chronic use of NSAIDs. Off-label use in obstetrics and gynecology include induction of labor, prevention of postpartum hemorrhage and induction of abortion. Little published scientific data exists on the effectiveness of misoprostol to induce cervical relaxation in mares. The classic use would be for an older maiden mare that has a very tight cervix while in estrus and will likely pool fluid in the uterus following breeding. Misoprostol cervical cream is available as a compounded product through various veterinary compounding pharmacies.

E. Alternative Therapies
Use of Glass Balls to Suppress Estrus A recent study reported that insertion of glass balls (marbles) into the uterus of mares resulted in prolonged suppression of estrus or heat. In the study, test mares were initially evaluated by teasing and ultrasonography to determine if they normally showed heat, grew a follicle and ovulated. Sterilized glass balls were inserted into the uterus of mares in estrus. Mares were subsequently examined by teasing, ultrasonography and hormone analysis. Sixty percent of the mares returned to heat on schedule. Four of the 10 mares (40 %) in which a large glass ball was placed in the uterus failed to return to estrus. The cause of the estrous suppression was a persistence of the corpus luteum or pseudopregnancy. The mechanism is likely to be a change in the secretory pattern of prostaglandins from the uterine lining or endometrium. A pulsatile release of prostaglandins from the uterus normally occurs 1315 days after ovulation in the nonpregnant mare. Presence of the glass balls may have altered or eliminated the normal pattern of pulsatile secretion.

Mares in this study were examined through the remainder of the season and then the glass ball was removed. Mares were bred the next season and no adverse effect on pregnancy rate was noted. Herbal Therapy Herbal supplements may have a label claim for calming or modifying undesirable behavior (i.e. Valerian root) or altering ovarian function (i.e. Chaste tree berries) in horses. In most instances the efficacy of the products have not been scientifically tested and the active ingredient(s) may not be known or standardized. In addition, American Horse Show Association regulations specifically forbid the use of herbal or natural products to affect performance of a horse. Surgery A last resort for behavioral modification is surgical removal of the ovaries or ovariectomy. This is analogous to castration of a colt in that it is a permanent, irreversible procedure that results in loss of any future reproductive potential. Consequently, owners should be advised to consider other modes of therapy prior to electing surgery. Ovariectomy would remove the source of hormones responsible for expression of estrus (estradiol) and the cause of periodic abdominal discomfort in some mares (ovulation). However, ovary removal would not be effective if the cause of the adverse or obnoxious behavior is something other than ovarian hormones. A common, but incorrect, supposition is that the behavior of a mare treated with exogenous progesterone would be similar to the behavior exhibited if the mare had her ovaries removed. Exposure of a mare to one hormone is not equivalent to removal or absence of another hormone. A more accurate comparison would be to evaluate the temperament and behavior of the mare during the winter anestrous period when ovarian function and hormone production are minimal.

THE PROBLEM MARE: DIAGNOSIS AND MANAGEMENT


Patrick M. McCue, DVM, PhD, Diplomate American College of Theriogenologists Equine Reproduction Laboratory Colorado State University 3194 Rampart Road Fort Collins, Colorado 80523, USA

INTRODUCTION
Reproductive problems are commonly encountered in a busy broodmare practice. A problem mare may be defined as 1) a mare that is not pregnant after being bred to a fertile stallion over 3 estrous cycles, 2) a mare that cannot successfully carry a foal to term, 3) a mare with known reproductive pathology, or 4) a mare with behavioral issues related to reproduction. Problem mares offer a significant challenge and therefore a significant opportunity for veterinarians. The following guidelines are offered for successful interaction with problem mares: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Provide maximal individualized attention to each problem mare Maintain a focus on every problem mare, every day (i.e. situations may change daily) Be detail oriented in your approach to diagnostics and therapeutics Be committed to succeed and be persistent in that quest Be flexible and change diagnostic and/or therapeutic course if indicated Be willing to consult with colleagues Provide frequent updates to owners Be optimistic in the face of despair However, be realistic with the prognosis for success based on an accurate diagnosis and the availability of expertise, therapeutic options and economic resources Know when to quit and/or when to refer

Occurrence of many problems can be predicted based on mare age (i.e. endometrosis in older mares), breed (i.e. poor perineal conformation in Thoroughbreds), parity (i.e. poor cervical function in older maiden mares) or additional factors. However, other reproductive abnormalities are more difficult or may be impossible to predict in advance. Ideally, all open or barren mares should be examined at the end of a breeding season while they are still cycling. The goals would be to diagnose the cause(s) of reduced fertility, implement a treatment plan, and assess the therapeutic response. It would be ideal to identify and resolve the reproductive problem prior to the off season. The other times mares are examined are a) at the beginning of the breeding season, and b) during the middle of the breeding season when problems are first noted. In our clinical practice, the most frequently diagnosed reproductive abnormalities are persistent post-mating endometritis, bacterial endometritis, and anovulatory or hemorrhagic follicles. A list of the more common and less common reproductive problems in non-pregnant mares is presented in Table 1. An accurate diagnosis of a problem is a prerequisite for development of a rational treatment plan and institution of an optimal management strategy (Ricketts and Troedsson, 2007). A systematic and thorough diagnostic plan should be devised for each problem mare. The diagnostic work-up should include, but not necessarily be limited to the following:

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Reproductive history and results of previous examinations or procedures Physical examination of the mare Teasing behavior Evaluation of perineal conformation Palpation of the reproductive tract per rectum Ultrasonography of the reproductive tract per rectum Speculum examination of the vaginal vault and external cervical os Manual or digital examination of the cervix Uterine culture (via swab, small volume uterine lavage, or biopsy) Endometrial cytology Endometrial biopsy Additional diagnostic tests (if indicated) a) Endoscopy of the uterine lumen b) Endocrine testing c) Karyotype d) Laparoscopy e) Oviductal patency test f) Test breed

REPRODUCTIVE HISTORY
Diagnostic workup of a problem mare should include a complete reproductive history (Greenhoff and Kenney, 1975; Shideler, 1993). The history should include age, breed, current reproductive status (maiden, barren, pregnant or foaling), number of cycles bred during the last season, date of last breeding, breeding technique used (artificial insemination, natural cover or pasture breeding), date of last foal, number of previous foals and any previous history of abnormal estrous cycles, uterine infections, embryonic loss or abortion. If possible, one should try to obtain reproduction records and results of previous diagnostic tests. In addition, information on stallion(s) used during previous unsuccessful breedings should be reviewed and notation made regarding other mares with reproductive issues on the farm or ranch. An accurate reproductive history can not only help identify current cause(s) of infertility, a history may help predict the occurrence of problems in the future. Williamson and colleagues (1989) reported that a reproductive history was more sensitive and specific than a uterine biopsy in predicting whether a mare would be resistant or susceptible to uterine infection.

PHYSICAL EXAMINATION
A general physical examination should be performed in addition to the reproductive examination to assess whether the mare is capable of carrying a foal to term. The evaluation should include, but is not limited to, examination of the oral cavity, eyes, and respiratory, cardiac and musculoskeletal systems. In addition, diet and body condition should be evaluated. Broodmares do not have to be fit for athletic competition, but they should not be severely overweight or underweight. It may be prudent to screen older mares for presence or absence of Equine Cushings disease, since this condition may be associated with reproductive abnormalities (McCue, 2002).

ESTRUS DETECTION
The mare should be teased with a stallion that exhibits good libido in order to evaluate estrous cycle stage. Adequate time should be taken to allow shy or nervous mares to express behavioral estrus. Maiden mares may not show heat well and foaling mares may not show

heat unless the foal is restrained and safely away from the stallion. It is generally more effective, and certainly more time consuming, to tease mares individually than to tease a group of mares in a paddock. Keys to successful teasing are patience, persistence and knowledge of the behavioral characteristics of each mare.

PERINEAL CONFORMATION
The external genitalia (vulva) should be evaluated for conformation and muscular tone. The vulva is the first physical barrier for prevention of contamination of the reproductive tract by pathogenic organisms. The optimal perineal conformation would consist of a vulva in a nearly vertical position (i.e. a slope of <10 degrees), with approximately two-thirds of the vulva below the pelvic floor (Pascoe, 1979). A formulation termed the Caslick Index, computed by multiplying the length of the vulva between the dorsal commissure and pelvic floor by the angle of declination of the vulva, has been used to predict reproduction problems related to perineal conformation (Pascoe, 1979). Poor perineal conformation has been reported to be a heritable trait in Thoroughbred mares (Pascoe, 1979). Trauma to the vulva during foaling and a decrease in muscular tone with age may lead to a compromise of the vulvar seal and predisposition for pneumovagina or windsucking. Sloping of the vulva secondary to recession of the anus and/or poor muscular tone to the labia of the vulva may predispose the mare to an ascending infection of the uterus (Caslick, 1937).

PALPATION
The goal of palpation is to identify significant features of the reproductive tract, determine stage of the estrous cycle and identify potential problems. Palpation is often performed in conjunction with transrectal ultrasonography. However, manual palpation can identify features of the tract that cannot be detected by ultrasonography, including tone in the uterus and cervix, softness of an ovarian follicle, and sensitivity of the ovary to touch.

ULTRASONOGRAPHY
Ultrasound is now the principle diagnostic tool used in modern broodmare practice (Ginther, 1986; Rantanen and McKinnon, 1998). Advantages of ultrasound are visualization of structures in the reproductive tract that cannot be discerned on palpation per rectum, early diagnosis of pregnancy, diagnosis and management of twins, and evaluation of potential ovarian or uterine pathology. Examples of pathology that can easily be detected by ultrasonography and difficult to detect by manual palpation are hemorrhagic follicles and uterine fluid.

VAGINAL SPECULUM EXAMINATION


A speculum examination is performed to evaluate the vaginal vault and the external os of the cervix, determine the stage of the estrous cycle, and detect pathologic conditions such as urine pooling, cervical discharge and trauma. The appearance of the external cervical os varies with season, stage of the estrous cycle, pregnancy status and presence of infection. Mares with endometritis may have a hyperemic cervix and/or a purulent discharge from the external cervical os. Urovagina or urine pooling is recognized by an accumulation of cloudy yellow fluid in the anterior vagina. Pooling of urine is most common in older mares in poor body condition with poor perineal conformation and is often most readily observed when affected mares are in estrus. A persistent hymen may be encountered during speculum examination of maiden mares. The hymen is located at the junction of the vestibule and vagina, approximately 6 to

8 cm inside the vulva. The hymen may be complete, occluding the entire vestibular-vaginal opening resulting in an accumulation of mucous and cellular debris cranial to the hymen, or the hymen may be a partial membrane. Vaginal varicose veins may be 0.5 cm or larger in diameter and are located in the region of the vestibular-vaginal junction. Spontaneous rupture of a vaginal varicose vein is the most likely issue when blood is noted emanating from the vulva of a mare.

MANUAL EXAMINATION OF THE CERVIX


After the speculum examination is completed, the cervix may be examined manually for tone, patency, and the presence of abnormalities. This examination is commonly performed during the process of collecting a culture swab from the uterus. Detection of a closed, tight cervix in a mare during estrus is abnormal, and is often associated with retention of fluid in the uterus after breeding. Failure of cervical relaxation is one factor attributed to reduced fertility of older maiden mares (Pycock, 1993; Pycock, 2006). Cervical lacerations that occur during foaling may cause a reduction in fertility. Digital examination for cervical competency should be performed in diestrus if possible. Prognosis for fertility is favorable after surgical repair (Embertson and Henderson, 2007).

ENDOMETRIAL CULTURE
Uterine infection can be suspected in a mare that exhibits an abnormally short estrous cycle, has a vaginal or cervical discharge, an inflamed cervix on speculum examination, and/or fluid in the uterus during diestrus. Infectious endometritis is a significant cause of reproductive failure in broodmares. Samples may be obtained via use of a double guarded culture swab (Blanchard et al., 1981) or a low volume uterine lavage (Ball et al., 1988; LeBlanc et al., 2007). In some instances a uterine biopsy may be collected for culture (Nielsen, 2005). It is critical that the pregnancy status of a mare be determined by ultrasonography prior to performing an invasive procedure such as collection of a uterine swab or biopsy. In the laboratory, the sample is applied to culture media plates and incubated. Bacterial growth is usually evident within 24 to 48 hours, while yeast often requires several days to grow. Antimicrobial susceptibility tests may be performed on microorganisms cultured. This may especially important for organisms for which the antibiotic susceptibility pattern is not predictable, such as gram negative bacteria and yeast. Antimicrobial therapy may be initiated when a uterine infection is first suspected, but therapy should be adjusted according to results of culture and susceptibility tests to determine the optimal treatment. The bacterium reported to be most commonly isolated during routine cultures of the uterus of mares in some studies is Streptococcus equi zooepidemicus (McCue et al., 1991; Ricketts et al., 1993; Riddle et al., 2007). However, other studies have reported that E. coli is the primary bacteria recovered from the uterus of problem mares (LeBlanc et al., 2007).

ENDOMETRIAL CYTOLOGY
Cytologic evaluation of the uterus involves the collection and interpretation of cells lining the uterus (endometrium) and within the uterine lumen. Cytology is often used in conjunction with culture and biopsy in the diagnosis of endometritis. Advantages of endometrial cytology include the ease of sample collection, low cost, and rapid availability of results, which may be critical when a prompt decision is required regarding breeding (Couto and Hughes, 1984; Wingfield-Digby, 1978).

Samples for endometrial cytology may be collected using a guarded swab (Brook, 1993) or cytology brush (Bourke et al., 1997), and directly applied to a clean a clean glass slide, air dried or fixed, and then stained (i.e. Diff Quick). Alternatively, a cytology sample may be obtained from the pellet following centrifugation of effluent from a low-volume uterine lavage (LeBlanc et al., 2007). Finally, sample for endometrial cytology can be obtained by smearing an endometrial biopsy on a glass slide (Nielsen, 2005). The presence and relative number of normal uterine epithelial cells, inflammatory cells (PMNs), bacteria, fungi and other debris is evaluated under high, dry magnification (400x). Presence of more than 1 to 2 neutrophils per high power field (hpf) is an indication that active inflammation is present. Higher numbers of neutrophils (i.e. > 5/hpf) indicate more severe inflammation. Riddle and colleagues (2007) recently reported on the relationship between uterine cytology results, culture results, and pregnancy rates in Thoroughbred mares. They evaluated results of 1,758 pairs of culture/cytology specimens and associated fertility. Endometrial cytology identified twice as many mares with endometritis as endometrial culture. Cytologic evidence of inflammation was not always associated with infection. The pregnancy rate was 60 % per cycle if the cytology was negative and no bacteria were isolated. Pregnancy rates decreased if either the cytology was positive or bacteria were isolated and were correlated with the severity of inflammation. Lowest pregnancy rates (23 %) were recorded in mares with severe inflammation only or with concurrent positive culture (18 %). Mares infected with -hemolytic Streptococcus were more likely to have severe inflammation as determined by cytology score than mares with E. coli or Pseudomonas infections (Riddle et al., 2007).

ENDOMETRIAL BIOPSY
The endometrial biopsy technique involves collection of a small sample of the endometrium for histological evaluation. It is primarily used as an aid in the diagnosis of uterine disease and as a prognostic indicator of the ability of a mare to carry a foal to term. It has been reported that collection of a single biopsy sample from one site is generally representative of the entire endometrium (Kenney, 1978), although collection of multiple samples may be advantageous in some mares. Endometrial biopsy samples are examined for the presence of inflammatory and degenerative changes. Acute inflammation is recognized by the presence of PMNs in endometrial tissue, while chronic endometritis is characterized by an accumulation of mononuclear cells (i.e. lymphocytes) in endometrial tissue. Inflammation is considered to be a potentially treatable condition. Endometrial glandular degeneration is most often recognized by a deposition of collagen in the form of fibrosis or scar tissue around the endometrial glands, and represents a permanent, untreatable condition. Development of degenerative changes in the endometrium is also part of the normal aging process. Endometrial biopsies are commonly classified on a I-III grading scale based on histology characteristics (Kenney and Doig, 1986). Grade I endometrium is essentially normal, with minimal inflammation or fibrosis. Grade III endometrium includes severe inflammatory and/or fibrotic changes. Grade II is a broad category encompassing all pathologic levels between Grades I and III. It is possible for an improvement in endometrial grade if the inflammation present is reduced or eliminated with proper treatment. Biopsy scores are sometimes used to provide a prognosis for a mare to carry a foal to term (Kenney, 1978). However, it must be emphasized that many additional factors, such as stallion and broodmare management, also play critical roles in reproductive success.

ENDOSCOPY OF THE UTERINE LUMEN (HYSTEROSCOPY)


The technique of direct visualization of the interior of the uterus, most commonly performed using an endoscope, is termed hysteroscopy. The endoscope may be used to detect intrauterine adhesions, cysts, retained endometrial cups, focal lesions, and foreign bodies (Bracher et al., 1992). A majority of common pathologic conditions, such as inflammation, infection and fibrosis, can be diagnosed with a combination of ultrasound, cytology, culture and biopsy. Uterine adhesions, however, are difficult to diagnose with any procedure other than endoscopy of the uterine lumen.

ENDOCRINOLOGY
Analysis of reproductive hormones in the non-pregnant mare is most commonly performed to evaluate corpus luteum function and to diagnose ovarian abnormalities. Progesterone concentrations > 1 ng/ml indicate the presence of an active corpus luteum. Testosterone and inhibin are occasionally measured to confirm or rule out the presence of an ovarian granulosa cell tumor. Approximately 50-60 % of mares with ovarian granulosa cell tumors have elevated concentrations of testosterone, while almost 90 % of affected mares have an elevation in plasma inhibin levels (McCue et al., 2006). The only laboratory currently assaying levels of inhibin in equine blood samples is the Clinical Endocrinology Laboratory, University of California, Davis.

KARYOTYPE
Analysis of the chromosome number and structure, termed karyotyping, may be an important diagnostic test in the evaluation of primary infertility in the mare (Hughes and Trommershausen-Smith, 1977). The most commonly reported chromosomal abnormality of the horse is XO gonadal dysgenesis, a condition in which one of the sex chromosomes has been deleted, resulting in a 63, X karyotype. Horses with XO gonadal dysgenesis develop as phenotypic females due to the absence of a Y sex chromosome. Affected mares are often small in size for their age and breed, have small, inactive ovaries and are infertile. Several other chromosomal abnormalities have been reported in the horse, but are rarely encountered. Chromosome analysis on equine blood or tissue samples is currently only performed at the Molecular Cytogenetics Laboratory, Maxwell H. Gluck Equine Research Center, University of Kentucky, and the Molecular Cytogenetics Laboratory, Department of Veterinary Anatomy and Public Health, Texas A&M University.

LAPAROSCOPY
Direct visualization of the reproductive organs within the abdominal cavity may be performed using laparoscopy. In addition, samples of tissue (i.e. an ovarian biopsy) may be collected for histological evaluation and medications may be topically applied to the surface of the reproductive tract (i.e. PGE2 onto the oviduct) using the laparoscope (Allen et al., 2006).

OVIDUCTAL PATENCY TEST


Evaluation of the equine oviduct is not easy. Mares may develop oviductal conditions that can adversely affect fertility, such as blockage and salpingitis (Saltiel et al., 1986). Fortunately, such conditions are considered to be rare in the horse. Techniques to determine if an oviduct is patent include deposition of starch granules (Allen et al., 1979) or fluorescent microspheres (Ley et al., 1998) onto the ovarian surface and subsequent recovery of the test particles from the uterus via lavage. In addition, flushing of the oviduct has been used as a combination diagnostic and therapeutic procedure in sub-fertile mares with no identified factors that would cause their reproductive failure (Bennett et al., 2002).

TEST BREED
Occasionally, routine diagnostic tests are not sufficient to determine the cause of reduced reproductive performance in a mare. Consequently, in some instances mares may be bred to a stallion of known high fertility and either an embryo collection procedure is performed 7 to 8 days after ovulation or an ultrasound examination is performed 14 to 16 days after ovulation to evaluate pregnancy status.

THERAPEUTIC PLAN FOR PROBLEM MARES


One should develop a logical therapeutic plan for problem mares, with specific treatment strategies dependent on the results of diagnostic tests. It is also advisable to have a back-up plan (or two) in case the first plan is unsuccessful. The primary goals are identify and eliminate pathogenic organisms from the uterus prior to breeding, limit the number of breedings to one per cycle, breed as close to ovulation as possible, and facilitate physical clearance of fluid, spermatozoa and inflammatory debris after breeding (LeBlanc, 2008; Pycock 2000; Troedsson, 2006). A therapeutic plan for a mare with persistent post-mating endometritis is outlined below. This plan would assume that the uterus of the mare had been cultured and determined to be free of pathogenic organisms. Therapy would be implemented during the next cycle once a mare had been determined to accumulate fluid in the uterus after breeding. Additional procedures listed at the end may be implemented depending on clinical circumstances. 1. 2. 3. Monitor the estrous cycle closely by teasing or ultrasonography. Lavage uterus with lactated Ringers solution and administer oxytocin when mare is in estrus if more than a trace of clear (i.e. non-echogenic) fluid is noted in the uterus Optimize breeding management; consult with owner to select the most fertile stallion that is acceptable; if more than one option for semen is available, breed with fresh semen if possible; select cooled semen second; frozen semen as a last resort; if the same stallion has been used over multiple cycles without success, change stallions Administer hCG or a GnRH agonist to elicit a timed ovulation Breed once (only) as close to ovulation as possible; breeding a second time may not be advantageous and may be detrimental Breed by artificial insemination (if allowed by the breed registry) and practice strict hygiene Lavage the uterus 4 to 6 hours after breeding with sterile saline or lactated Ringers solution Administer oxytocin and/or prostaglandins as needed to enhance uterine clearance post-breeding Confirm by ultrasonography that ovulation actually occurred Monitor the uterus by ultrasonography daily for several days after breeding for evidence of fluid accumulation Lavage and administer oxytocin as needed to clear uterine fluid up to 1 to 2 days after ovulation is deteced Additional considerations (if indicated): a) Administer intrauterine antibiotics pre-breeding and/or post-breeding b) Systemic antibiotics c) Administer supplemental progesterone/progestin therapy d) Caslicks vulvoplasty e) Other therapies (such as PGE1, acupuncture, corticosteroids, plasma, and alternative uterine infusions/lavages, including DMSO, hypertonic

4. 5. 6. 7. 8. 9. 10. 11. 11.

saline, vinegar, acetylcysteine)

povidone-iodine,

hydrogen

peroxide,

kerosene,

What do you do if your therapy is unsuccessful? Consider the possibility of multiple pathologic processes contributing to the problem(s); re-evaluate your original diagnostic results and therapeutic plan; consider alternative or non-traditional therapies; consider consultation with a specialist or consider referral; and finally consider advanced assisted reproductive techniques, such as oocyte transfer or intracytoplasmic sperm injection if the owner wants to pursue a pregnancy.

REFERENCES
Allen WE, Kessy BM, Noakes DE. 1979;105:364-366. Evaluation of uterine tube function in pony mares. Vet Rec

Allen WR, Wilsher S, Morris L, Crowhurst JS, Hillyer MH, Neal HN. Laparoscopic application of PGE2 to re-establish oviductal patency and fertility in infertile mares: a preliminary study. Equine Vet J 2006:38:454-459. Ball BA, Shin SJ, Patten VH, Lein DH, Woods GL. Use of a volume uterine flush for microbiologic and cytologic examination of the mares endometrium. Theriogenology 1988;29:1269-1283. Bennett S, Griffin R,Rhoads W. Surgical evaluation of oviductal disease and patency in the mare. Proc Amer Assoc Equine Pract 2002;48:347-349. Blanchard TL, Cummings MR, Garcia MC, Hurtgen JP, Kenney RMe. Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares. Theriogenology 1981;16:541-552.. Bourke M, Mills JN, Barnes AL. Collection of endometrial cells in the mare. Aust Vet J 1997;75:755758. Bracher V, Mathias S, Allen WR. Videoendoscopic evaluation of the mares uterus: II. Findings in subfertile mares. Equine Vet J 1992;24:279-284. Brook D. Uterine cytology. In: Equine Reproduction, McKinnon AO (Ed). Williams and Wilkins, Philadelphia, 1993, pp. 246-254. Caslick EA. The vulva and the vulvo-vaginal orifice and its relationship to genital health of the Thoroughbred mare. Cornell Vet 1937;27:178-187. Cuoto MA, Hughes JP. Technique and interpretation of cervical and endometrial cytology in the mare. J Equine Vet Sci 1984;4:265-273. Embertson RM, Henderson CE. Cervical tears. In: Current Therapy in Equine Reproduction Samper JC, Pycock JF, McKinnon AO (Eds.). Saunders Elsevier, St. Louis, 2007, pp 130-133. Ginther OJ. Ultrasonic imaging and reproductive events in the mare. Equiservices, Cross Plains, WI, 1986. Greenhoff GR, Kenney RM. Evaluation of the reproductive status of the non-pregnant mare. J Am Vet Med Assoc 1975;167:449-458. Hughes JP, Trommershausen-Smith A. Infertility in the horse associated with chromosomal abnormalities. Australian Vet J 1977;53:253-257. Kenney RM. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. JAVMA 1978;172:241-262. Kenny RM, Doig PA. Equine endometrial biopsy. In: Current Therapy in Theriogenology 2, Morrow DA (Ed.),. WB Saunders, Philadelphia, 1986, pp 723-729. LeBlanc MM. When to refer an infertile mare to a theriogenologist. Theriogenology 2008;70:421-429. LeBlanc MM, Magsig J, Stromberg AJ. Use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares. Theriogenology 2007;68:403-412.

Ley WB, Bowen JM, Pursewell BJ, Dascanio JJ, Parker NA, Bailey TL, DiGrassie WA. Modified technique to evaluate uterine tubal patency in the mare. Proc Am Assoc Equine Pract 1998;44:5659. Liu IKM, Lantz KC, Schlafke S, Bowers JM, Enders AC. Clinical observations of oviductal masses in the mare. Proc Amer Assoc Equine Pract 1991;36:41-45. McCue PM. Equine Cushings disease. Vet Clin Equine 2002;18:533-543. McCue, PM, Hughes JP, Jang SS, Biberstein EL. Antimicrobial susceptibility patterns for aerobic bacterial isolates from the uterus of mares. California Veterinarian. 1991;45(1): 25-28. McCue PM, Roser JF, Munro CJ, Liu IKM, Lasley BL. Granulosa cell tumors of the equine ovary. Vet Clinics North America Equine Practice 2006;22:799-817. Nielsen JM. Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. Theriogenology 2005;64:510-518. Pascoe RR. Observations on the length and angle of declination of the vulva and its relation to fertility in the mare. J Reprod Fert Suppl 1979;27:299-305. Pycock JF. Cervical function and uterine fluid accumulation in mares. Equine Vet J 1993;25:191. Pycock JF. Breeding management of the problem mare. In: Equine Breeding Management and Artificial Insemination Samper JC Ed.) . WB Saunders, Philadelphia, 2000, pp 195-228. Pycock JF. How to maximize the chances of breeding successfully from the older maiden mare. Proc Amer Assoc Equine Pract 2006;52:245-249. Rantanen NW, McKinnon AO. Equine diagnostic ultrasonography. Williams and Wilkins, Baltimore, 1998. Ricketts S, Troedsson MHT. Fertility expectations and management for optimal fertility. In: Current Therapy in Equine Reproduction, Samper JC, Pycock JF, McKinnonAO (Eds.). Saunders Elsevier, St. Louis, Missouri, 2007, pp 53-69. Ricketts SW, Young A, Medici EB. Uterine and clitoral cultures. In: Equine Reproduction, McKinnon AO (Ed.),. Williams and Wilkins, Philadelphia, 1993, pp 234-245. Riddle WT, LeBlanc MM, Stromberg AJ. Relationships between uterine culture, cytology and pregnancy rates in a Thoroughbred practice. Theriogenology 2007;68:395-402. Saltiel A, Paramo R, Murcia C, Tolosa J. 1986;47:594-597. Pathologic findings in the oviducts of mares. AJVR

Shideler RK. History. In: Equine Reproduction, McKinnon AO (Ed.): Williams and Wilkins, Philadelphia, 1993, pp. 196-198. Troedsson MHT. Breeding-induced endometritis in mares. Vet Clinics North America: Equine Practice 2006;22:705-712. Williamson P, Munyua SJM, Penhale J. Endometritis in the mare: a comparison between reproductive history and uterine biopsy as techniques for predicting susceptibility of mares to uterine infection. Theriogenology 1989;32:351-357. Wingfield-Digby NJ. The techniques and clinical application of endometrial cytology in mares. Equine Vet J 1978;10:167-170.

Table 1. Reproductive problems in non-pregnant mares.

Common Reproductive Problems Behavior Adverse behavior when in estrus Silent heat Ovary Hemorrhagic follicles Ovulation failure Persistent corpus luteum Premature luteolysis (endometritis) Oviduct Pera-ovarian cysts Uterus Persistent mating-induced endometritis Bacterial endometritis Uterine cysts Endometriosis Cervix Failure of cervical relaxation Cervical lacerations Vagina/Vestibule Urovagina Vericose veins Imperforate hymen Perineum Poor conformation Inadequate vulva tone Perineal lacerations Melanoma (grey mares) Miscellaneous Cushings disease

Less Common Reproductive Problems Behavior Persistent estrus Stallion-like behavior Ovary Ovarian tumors Failure of follicular development

Oviduct Oviductal blockage Salpingitis Uterus Fungal endometritis Pyometra Persistent endometrial cups Tumor (i.e. leiomyoma) Foreign body Cervix Cervical adhesions Tumor (i.e. leiomyoma) Vagina/Vestibule Lacerations Adhesions Vaginitis Perineum Squamous cell carcinoma Coital exanthema (EHV-3)

Miscellaneous Chromosomal abnormalities Mastitis Inappropriate lactation

NUTRICIN DEL EQUINO REPRODUCTOR


Mariano Hernndez-Gil

RESUMEN
El objetivo de la presente revisin es exponer y discutir las bases tericas y los mtodos actuales y vlidos para estimar requerimientos energticos y proteicos de equinos reproductores. Los equinos evolucionaron en condiciones de pastoreo y adaptaron su actividad reproductiva a los eventos ambientales determinados por el fotoperiodo y disponibilidad de alimento. En la actualidad los equinos son explotados con fines de trabajo y esparcimiento manteniendo la produccin de ejemplares de razas creadas para diferentes actividades zootcnicas; aunque en muchas ocasiones esto signifique alterar los ritmos naturales de los animales. La reproduccin eficiente de los equinos se debe apoyar entonces en la provisin de nutrientes en cantidad y calidad suficiente de acuerdo a la etapa de su fisiologa reproductiva en que se encuentren. Los sistemas de alimentacin mas fuertes disponibles en el mundo cuentan con modelos y mtodos apropiados para calcular los requerimientos nutricionales del equino reproductor con una precisin bastante aceptable y adaptable a diferentes condiciones. Las necesidades nutricionales para asegurar fertilidad en el equino no van ms all de lo que se propone para mantenimiento, aunque la calidad de los nutrientes si debe asegurarse. Las hembras gestantes tienen elevaciones discretas en su requerimiento hacia el final de la gestacin, mientras que al principio de la lactancia los requerimientos pueden representar el doble del mantenimiento. En el caso del garan, la mayora de los casos de infertilidad son atribuidles a factores distintos a la nutricin. Adems del plano energtico y proteico, la nutricin mineral y vitamnica de los equinos debe tambin ser considerada al momento de disear estrategias de alimentacin

INTRODUCCIN
El estudio de la biologa de la reproduccin del equino puede hacerse a diferentes niveles de agregacin, partiendo del individuo mismo hacia un nivel molecular llegando a factores de crecimiento y utilizacin de sustratos en clulas gonadales o hacia un nivel ms universal llegando al movimiento de la tierra, el fotoperiodo y la disponibilidad de alimento. Cualquiera que sea el sentido, siempre ser factible hallar explicaciones al comportamiento reproductivo de los equinos en estado natural y as controlarlo en condiciones de explotacin. Ciertamente, para el xito en la produccin equina ha sido necesario desarrollar mtodos que aseguran su reproduccin en condiciones artificiales. Sin duda, lo primero que debe asegurarse es un buen estado nutricional, lo que se logra mediante la provisin del alimento con la proporcin ideal de nutrientes. Esto es importante no solo para cubrir las demandas del animal en cada etapa fisiolgica, sino tambin por el inters en que los recursos se usen eficientemente tanto por razones ambientales, como por razones econmicas; sobre todo en equinos, cuyo nico producto vendible es un potro que prometa buen desempeo a futuro. Ello efectivamente lo conceden los cromosomas transmitidos gracias al trabajo de ovarios y testculos pero se limita o arruina si no hay aporte y utilizacin adecuados de nutrientes. Para asegurar el aporte de nutrientes, se ha requerido medir el gasto de energa y reciclaje de nitrgeno durante procesos fisiolgicos como produccin de hormonas, actividad
Mariano Hernndez Gil. MVZ CEq MC. Programa DS-WHW-UNAM. Centro de Enseanza, Investigacin y Extensin en Ganadera Tropical. Facultad de Medicina Veterinaria y Zootecnia, UNAM. Rancho El Clarn: Km 4.5 Carretera Federal Martnez de la Torre-Tlapacoyan. Tlapacoyan, Veracruz, Mxico. devitohgo@gmail.com

ovrica, ovulacin, fertilizacin, gestacin, lactacin, crecimiento, etc. Esto ya se ha hecho en varios trabajos, por lo que se dispone de sistemas de alimentacin para calcular requerimientos nutricionales y valor nutritivo de alimentos en los equinos. Para garantizar la utilizacin de nutrientes, al momento estimar requerimientos y formular una dieta deben considerarse las caractersticas de los ingredientes, as como la manera en que interactan con el tracto gastrointestinal para rendir los sustratos que cubren tales necesidades especiales, mientras se procuran condiciones que aseguren disponibilidad de oxgeno suficiente a nivel tisular. Los sistemas de alimentacin con bases ms slidas son el americano, francs, alemn, holands y escandinavo. Sus modelos han sido desarrollados a partir de datos generados en experimentos con condiciones, animales y alimentos distintos pero lgicamente comparables para alcanzar cierto grado de conciliacin. Esto trae ventajas al trabajar en Mxico, un pas importante en asuntos equinos no solo por su poblacin que lo coloca en tercer lugar a nivel mundial, sino tambin porque aqu se tienen casi todas las disciplinas ecuestres ms conocidas del mundo. Por tanto, la variedad de razas equinas que se cran es tan amplia como los climas, condiciones de explotacin y recursos de alimentacin. Puesto que cada raza ha sido desarrollada por siglos en su pas de origen en condiciones particulares de fotoperiodo, clima, suelo y alimentos, es sensato conducir la alimentacin estratgicamente para alcanzar eficiencia reproductiva, productividad y desempeo. Con tal objeto, la presente revisin expone y discute los mtodos actuales y vlidos para estimar requerimientos energticos y proteicos de equinos destinados a la produccin de ejemplares exitosos en la actividad y propsitos para los cuales han sido seleccionados.

NUTRICIN Y REPRODUCCIN EN ESTADO NATURAL


La historia natural coloca a los equinos como una especie excepcionalmente adaptada. Este xito puede atribuirse a dos aspectos que la evolucin lig: la nutricin y la reproduccin. En efecto, la eficiencia reproductiva ha sido parte esencial de la supervivencia de los equinos; originalmente con fines de preservacin, ahora aprovechada por el ser humano con fines productivos. Mas lo que hizo posible que el Hyracotherium evolucionara hasta convertirse en el Equus que actualmente se conoce, fue su capacidad de adaptarse al tipo y cantidad de alimento por los cambios climticos que sucedieron desde el Eoceno hasta el Pleistoceno. Los quidos evolucionaron para adaptarse a los pastizales que cubran los espacios restantes tras la desaparicin de los bosques debido a los cambios climticos.1,2 Desarrollaron cambios anatmicos a nivel digestivo para lograr una supervivencia en el corto plazo y adaptaciones en su biologa reproductiva para asegurar su estancia en el largo plazo. La estacionalidad del equino, especialmente notable en las hembras, demuestra tal adaptacin para ajustarse a la disponibilidad de nutrientes durante el ao. La Figura 1 ilustra lo que sucede al trazar fluctuaciones en fotoperiodo, crecimiento forrajero, actividad reproductiva y necesidad de nutrientes en estado natural a lo largo de dos ciclos anuales en el hemisferio boreal. El solsticio de invierno, hacia el 23 de diciembre determina el momento en que el sol en el cielo se encuentra a su mayor distancia angular al otro extremo del plano ecuatorial, es decir cuando el hemisferio norte de la tierra ha llegado al da ms corto para comenzar a volver y llegar a su da ms largo hacia el 20 de junio cuando comienza el verano. La naturaleza ha decidido que el momento ideal para la fertilidad de la yegua corresponda justo a esos das cercanos al solsticio de verano,3 de manera que el parto, once meses y das ms tarde, se presente hacia la primavera del siguiente ao, cuando las

condiciones sern ideales para que la yegua disponga de alimento en cantidad y calidad suficientes para producir leche de la mejor calidad y la cra se desarrolle correctamente. Corresponde adems, que cuando los cambios en el fotoperiodo dan la seal para que inicie la actividad de los rganos involucrados en la reproduccin en el interior de la yegua, ya en su exterior tambin se ha dado la seal para que las condiciones ambientales sean propicias para el crecimiento de pastos y la activacin de su metabolismo para hacer disponibles nutrientes. Los pastos tienen un metabolismo tal como cualquier organismo vivo. Captan, convierten, depositan y reciclan molculas necesarias para su funcin y estructura.4 La forma en que estas molculas se encuentran cuando el equino las ingiere, determina el grado en que quedarn cubiertas sus necesidades nutricionales generales y especficas.5 El principio de la primavera determina la activacin del ciclo de la mayora de las especies forrajeras y con ello la disponibilidad de nutrientes para los equinos. Las necesidades de la yegua reproductora ciclando no son tan altas en cantidades absolutas de energa, protena y materia seca, pero s lo son en calidad de nutrientes, lo cual est asegurado con las caractersticas moleculares de los forrajes a este estado fenolgico. Durante este momento, el garan tiene una participacin importante en la poca reproductiva, cuando la yegua est receptiva, justo tambin cuando la calidad del forraje es ptima, asegurando su fertilidad y efectividad. Si la yegua es servida por un garan y hay fertilizacin, iniciar entonces una gestacin. En este momento del ao se aseguran nutrientes durante una de las etapas ms crticas de la gestacin pues un desequilibrio puede llevar a la reabsorcin embrionaria.6 Ya hacia el otoo, la menor disponibilidad de alimento con su concentracin de nutrientes coincide con el momento de menor requerimiento durante los primeros siete meses de gestacin. Adems, la disponibilidad de forraje durante el verano permiti a la yegua crear reservas suficientes para pasar el invierno y llegar a la siguiente primavera que trae forraje con las primeras lluvias justo cuando las necesidades de la yegua incrementan debido al aumento en la deposicin de energa y protena en el feto, para parir hacia el ltimo tercio de la primavera. La yegua tiene sus necesidades nutricionales mayores durante la primera mitad de la lactacin justo en el verano, cuando dispone de nutrientes en calidad debido al contenido de solubles en los pastos que los hacen ms suculentos, asegurando el aporte de aminocidos y azcares necesarios para los propios de la leche. Es tambin durante este momento rico en nutrientes cuando la yegua es capaz de mostrar un celo frtil que en aquellos tiempos de competencia entre herbvoros por el recurso forrajero y de amenaza constante de predadores relegados de los bosques, aseguraron la preservacin de la especie equina.1 En la segunda mitad de la lactacin, cuando ya el potro ha comenzado a ingerir forraje, el final del verano y principio de otoo con sus condiciones de humedad y radiacin solar aseguran la disponibilidad de nutrientes en cantidad y calidad para sostener a la yegua y al potro creciendo. Ya entrado el otoo, cuando el potro ha crecido y depende menos de la yegua las

reservas corporales en ambos son suficientes para pasar el invierno y llegar a la primavera siguiente para compensar su crecimiento con la disponibilidad de alimento, reiniciando el ciclo. Comprendiendo este ciclo, parece entonces que lo ms natural es adaptar la crianza de los equinos a lo que sucede en condiciones naturales, comenzando por lo que respecta a la provisin de nutrientes. En los casos en que las exigencias de la actividad zootcnica para la cual sea explotada cierta raza equina demande alterar lo que sucede en condiciones naturales por lo que actividad reproductiva y, por tanto, las necesidades nutricionales no coinciden con el resto de los eventos a lo largo del ao (Figura 2), ser necesario investigar en el gasto energtico y proteico que representa al equino cada una de las fases del ciclo reproductivo y a partir de ello fijar los mtodos que faciliten la estimacin de sus requerimientos nutricionales y planear las prcticas encaminadas a cubrir deficiencias.

REQUERIMIENTOS NUTRICIONALES
El trmino requerimientos nutricionales implica la cantidad de nutrientes necesarios para cubrir el gasto metablico de un organismo para mantener sus procesos basales y el costo adicional que le representan estados especiales como actividad reproductiva, gestacin, lactacin y crecimiento.7 Los requerimientos se estiman como valores y se cubren asignando nutrientes en cantidad absoluta o porcentaje de inclusin, dependiendo de los ingredientes elegidos y su valor nutricional. As entonces, es posible aportar al animal los nutrientes para alcanzar el nivel de desempeo permitido por su potencial gentico. 8, 9, 10 La primera forma de energa de un alimento es la energa bruta. Cuando se descuenta la fraccin de energa perdida en heces se obtiene el estimado de energa digestible (ED). Una parte de esta ED se pierde con el metano de la fermentacin y con la orina restando entonces la fraccin de energa disponible al metabolismo llamada energa metabolizable (EM). Finalmente, parte de la EM se pierde en el llamado incremento calrico y la parte disponible o energa neta (EN) es lo que el organismo utiliza para cubrir sus requerimientos de mantenimiento y para los propsitos extra. Aunque lo ideal es conocer el requerimiento de energa neta para cada proceso dentro del equino, esto aun no es posible; por tal motivo, lo ms conveniente por ahora es trabajar a nivel requerimientos de Energa Digestible (ED). Los requerimientos de protena suelen estimarse como porcentaje de protena cruda (PC). Esto conduce a errores pues la calidad de la protena no es similar entre alimentos, mucho menos su valor nutricio determinado por el proceso de digestin. De manera entonces que, ahora que se cuenta con informacin, lo ideal es trabajar con protena cruda digestible (PCD), debido sobre todo a las variaciones en concentracin de las diferentes fracciones nutrientes y su digestibilidad en los distintos forrajes y concentrados utilizados en Mxico.

LA YEGUA REPRODUCTORA
Mantenimiento Los requerimientos de mantenimiento describen la cantidad de nutrientes necesaria para conservar una ganancia de peso igual a cero considerando el gasto que imponen procesos como: metabolismo basal, ciclos bioqumicos, reciclaje de nitrgeno y fosfolpidos, funciones de secrecin y excrecin, motilidad intestinal, circulacin sangunea, tono muscular, desplazamiento mnimo necesario, bsqueda y cosecha de alimento, masticacin, incremento calrico y termorregulacin.9 Aunque no siempre se precisa, es conveniente incluir en mantenimiento el gasto que representa la funcin de los rganos involucrados en la biologa reproductiva en cualquiera de sus estadios previos a la concepcin. As pues, los requerimientos de reproduccin corresponden a la fraccin de nutrientes en cantidad y calidad tales que aseguren la funcin reproductiva, el desempeo eficiente y la fertilidad. Precisar cantidades de nutrientes para cada evento de la funcin reproductiva no es posible, pues la informacin no es aun disponible; sin embargo, el tipo de nutrientes para promover eficiencia a diferentes niveles del proceso reproductivo han sido ya documentados. Los mecanismos involucrados en como el nivel de nutricin y las reservas corporales influyen en el ciclo estral y crecimiento folicular no estn del todo definidas en equinos.11 De manera general, se propone que la nutricin de la yegua reproductora debe enfocarse en cubrir adecuadamente sus requerimientos de mantenimiento, asegurndolo con alimentos de buena calidad. El xito, medido por tiempo a primera ovulacin y tasa de concepcin, est relacionado principalmente con el consumo de energa y el mantenimiento de la condicin corporal.11,12,13 Las yeguas que mantienen o aumentan su condicin corporal durante la poca reproductiva muestran mayores tasas de concepcin, 14 lo que ahora se explica con la relacin directa entre cantidad de tejido adiposo y niveles circulantes de leptinas.15,16 El inicio de la actividad reproductiva ha sido relacionado con la disponibilidad de alimento y las condiciones climticas al inicio de la primavera, sobre todo por el efecto que tales condiciones pueden tener sobre el crecimiento de los forrajes y la necesidad de energa en las hembras para termorregular.17 El consumo de energa est bien relacionado con la actividad ovrica. De hecho, un aporte excesivo de energa favorece las ovulaciones dobles.18 El pastoreo durante la primavera ha mostrado efecto positivo sobre el tiempo a la primera ovulacin en yeguas anstricas.19 As mismo, est demostrado el efecto benfico de la complementacin energtica para acortar el tiempo anovulatorio del invierno.20, 21 Cuando el gasto energtico supera el consumo energtico, esto es balance energtico negativo, la actividad ovrica est comprometida. Esto se ha propuesto porque en diversas especies se ha observado que ante balance energtico negativo hay reduccin en la cantidad de folculos llegando a estadios finales, as como disminucin en niveles sricos de gonadotropinas, niveles circulantes de insulina, IGF-I, leptina y glucosa. 22 Por el contrario, cuando el consumo de energa es de 10-15% por arriba del mantenimiento hay una ganancia de peso que resulta en aumento de la condicin corporal y efecto benfico en la reproduccin. De hecho, se propone que los ingredientes grasos que aporten una buena cantidad de colesterol favorecen la fertilidad por la provisin de precursores de hormonas esteroides. 23 Energa Digestible El nutriente ms limitante para los equinos es la energa. Esto significa que la formulacin de raciones debe comenzar por estimar el requerimiento de mantenimiento de energa y no por estimar su potencial de consumo de materia seca. El requerimiento de energa digestible

(ED) depende de la talla del animal. Por tal motivo, el NRC24,25 se apoya en una ecuacin que calcula la cantidad de megacaloras (MCal) de ED por da, despus de sumar la constante 1.4 al producto de multiplicar el factor 0.03 por el peso vivo (PV). As entonces: ED (MCal/da) = 1.4 + 0.03 PV (1)

Sin embargo, esta ecuacin est limitada a animales de hasta 600 kg de PV. Ms all de ese peso se debe utilizar la ecuacin que el mismo NRC 24,25 presenta: ED (MCal da-1) = 1.82 + 0.0383 PV 0.000015 PV2 (2)

Tabla 1. Comparacin entre requerimiento estimado de energa digestible para mantenimiento con tres ecuaciones diferentes y para tres pesos distintos. Peso vivo (Kg) Ecuacin (1) Requerimiento de ED (Mcal da-1) (2) (3) (4) 180 6.80 ----6.88 7.00 420 14.00 ----12.99 13.30 860 ----22.4 23.6 22.8

El Sistema Francs (INRA) se apoya en varios trabajos26, 27 y estima los requerimientos de energa digestible para mantenimiento al multiplicar la constante 0.586 por PV elevado a la potencia 0.75. Elevar PV0.75 resulta en lo que se conoce como peso metablico (PM), el cual es un exponente de la masa corporal que otorga proporcionalidad en estimaciones de ndices de ciertos procesos28 (Webster, 1989) y es por tanto aplicable en equinos igual como en cualquier especie. El producto de multiplicar PV0.75 por 0.586 se divide entre la constante 4.185 que es la cantidad de joules en una calora. La ecuacin queda entonces como: ED (MCal da-1) = 0.586 PV 0.75 / 4.185 (3)

La ecuacin (3) es factible de utilizarse con caballos de cualquier talla y resuelve problemas que pudieran surgir por pesos mayores a 600 Kg.29, 30 El Sistema Alemn tambin basa sus clculos en el PM, aunque utiliza una constante mayor a la del Sistema Francs: ED (MCal da-1)= 0.6 PV 0.75 / 4.185 (4)

En la Tabla 1 se confrontan las estimaciones de requerimiento de ED para caballos de 180, 420 y 860 kilogramo de PV con las cuatro ecuaciones mostradas. Los resultados, similares entre s, demuestran homogeneidad en teora nutricional equina entre de un sistema a otro. Ajuste de ED por tasa metablica Aunque el gasto energtico de mantenimiento es proporcional al peso, existen variaciones individuales asociadas con el temperamento del animal. Los aumentos en necesidades por tales razones van de 5 a 20 % y se deben a diferencias en tasa metablica tisular, tono muscular y actividad fsica espontnea. 31,32 Es muy probable que el efecto de la raza est ligado al efecto de temperamento. Caballos de razas ligeras muestran gastos energticos mayores hasta en 20 % que los ejemplares de razas pesadas o an los ponies.32,33

Es sabido que los ejemplares Pura Sangre Ingls suelen mostrar una tasa metablica mayor; lo cual dificulta mantenerlos en un buen estado nutricional. Considerando diferencias en tasa metablica, Vermorel et al.34 presentan factores para ajustar necesidades de mantenimiento (Tabla 2). En cada caso, el factor se multiplica por lo calculado como de mantenimiento. El punto est en definir el tipo de caballo. No ser difcil con un PSI, pero s para ciertas razas, lneas o cruzamientos cuyo tipo no est definido. Por ejemplo, un Cuarto de Milla de carreras, tendr una tasa metablica mayor que el de alguna lnea seleccionada para trabajo de ganado o conformacin. Los caballos pesados o sangre fra son fcilmente identificables. Percheron, Clydesdale y Shire son comunes en Mxico, aunque recientemente se han introducido ejemplares de razas pesadas, pero han comenzado a ser cruzadas con animales de tipo ligero o sangre caliente, conduciendo a aberraciones complicadas de definir. Tabla 2. Factores de ajuste de requerimientos de ED para mantenimiento en equinos dependiendo del tipo y nivel de actividad. Nivel de actividad Pesado En reposo En trabajo
Adaptado de Vermorel et al., 1984 34

Tipo de equino Silla 1.05 1.10 Pura sangre 1.10 1.15

1.00 1.05

Existen otros factores que afectan el requerimiento de ED para mantenimiento. El incremento en el nivel de alimentacin eleva el gasto debido al incremento en el metabolismo a nivel visceral y heptico.35 Un aspecto importante a considerar es el tipo de alimento y, en consecuencia, el sustrato encaminado a cubrir los requerimientos de cada rubro. La energa perdida como calor por el trabajo de digestin puede variar ampliamente en caballos dependiendo de la composicin de la dieta. Las dietas que se digieren eminentemente por fermentacin en el intestino grueso resultan ms termognicas que aquellas cuya digestin se da mayormente en el intestino delgado.36 Al respecto, se menciona que el requerimiento de energa digestible se incrementa de un 15 a 25 % cuando la dieta se compone exclusivamente de forraje. Entonces, un equino de 400 Kg cuya necesidad diaria de ED es de 13.4 MCal, requiere de 15.41 a 16.75 MCal al ser alimentado exclusivamente con forraje. Se propone que el sustrato utilizado para cubrir el gasto energtico afecta la eficiencia en el uso de la energa. Se ha demostrado que los quidos recibiendo grasas en sustitucin de carbohidratos muestran una reduccin en el requerimiento de ED para mantenimiento.35 Lo anterior guarda relacin con la composicin corporal y la manera en que esta afecta la eficiencia de termorregulacin. Cuando el animal es magro, hay una prdida excesiva de calor corporal en condiciones fras, lo que incrementa el requerimiento de energa.36 As mismo, una proporcin excesiva de grasa corporal resulta en aumentos en el requerimiento de energa digestible en caballos ubicados en climas templados o clidos.37 Los caballos con un contenido excesivo de grasa requieren ms energa para disipar el calor que aquellos con una condicin magra, particularmente en climas templados y clidos.36 El clima entonces interviene de manera importante y debe tomarse en cuenta que temperaturas extremas significan una demanda extra de energa. Aunque, el rango aceptable es de 15 a 30 C, las variaciones por clima no estn bien definidas. El gasto energtico de mantenimiento es alrededor de un 10 % mayor en verano que en invierno en regiones templadas.35

Protena Cruda Digestible Los requerimientos de PCD para mantenimiento son ligeros, salvo en caso de baja condicin corporal. La estimacin se apoya en una razn protena:energa que debe guardarse sobre todo ante incrementos en el requerimiento energtico. El NRC24,25 estima que la necesidad de mantenimiento de un equino equivale a 2.8 g de PCD por Kg de peso metablico (PM). Lo que equivale a 19.19 g por MCal de ED.38 El sistema Alemn estima que los requerimientos de PCD para mantenimiento son de de 3 g por kilogramo de PM o 20.93 g de PCD por MCal de ED.39 En Francia se utiliza un sistema de evaluacin que considera la digestin prececal y cecoclica de la protena, calculando que el requerimiento de PCD para mantenimiento es de 2.4 g de PCD por kg de PM.40 La Tabla 3, muestra los factores para calcular el requerimiento diario de PCD para mantenimiento con base en peso metablico, lo que en un rango de peso vivo de 50 a 1200 kilogramos promediara a 19.19 (+ 1.10) g de PCD por MCal de ED. 38

Tabla 3. Requerimiento de protena cruda digestible para mantenimiento en equinos de acuerdo con tres sistemas de alimentacin. PCD = 2.8 g * PV 0.75 PCD = 2.4 g * PV PCD = 3.0 g * PV
0.75 0.75

NRC INRA Sistema Alemn

Respecto al equilibrio de aminocidos, tanto el INRA como el NRC sugieren que este no es de importancia significativa en caballos en mantenimiento. La lisina y la treonina son los aminocidos ms limitantes. El requerimiento de lisina recomendado por el NRC24,25 es de 0.035 g de lisina por gramo de protena cruda, lo que corresponde 0.05 g de lisina por kilogramo de peso vivo. En general, los requerimientos de aminocidos de caballos adultos en mantenimiento y trabajo se cubren bajo condiciones de alimentacin normal. Lo extra debe calcularse por separado y adicionarse al requerimiento de mantenimiento.39 Las protenas son el mayor componente de msculos, sangre y otros tejidos. Proveen de aminocidos y nitrgeno para crecimiento y reparacin tisular; representando un componente estructural y regulador de procesos metablicos en todas las clulas. En quidos, la protena de la dieta repara tejidos degradados y compensa prdidas por sudoracin.7 Las necesidades estn en funcin de la tasa de reciclaje de protena en los diferentes tejidos del animal y el equilibrio de protena muscular; esta ltima dependiente de la demanda de aminocidos por el resto de los tejidos y la disponibilidad de aminocidos en sangre.40 La efectividad de la dieta para cubrir la demandas de este metabolismo depende mucho de la cantidad de compuestos nitrogenados que rinda para su absorcin desde el tracto digestivo. La nutricin equina incluye alimentos variables en calidad y cantidad proteica. La protena debe ser digerida hasta aminocidos en intestino delgado para ser absorbidos. Por tanto, el sitio de digestin es importante 7,41 sobre todo porque el equilibrio de protena resulta positivo solo durante la fase de absorcin de aminocidos. Fuera de este periodo el requerimiento por el tracto digestivo, hgado, enzimas y hormonas es mayor y los msculos tienen que proveer aminocidos. La sangre cuenta para el 1 % de los aminocidos libres, aunque la variacin en calidad de protena de la dieta se refleja en la variacin de proporciones de aminocidos en sangre.41,42 En mantenimiento se sintetizan 15 g diarios de protena por Kg de PM. El caballo sintetiza 3 a 5 veces ms protena que su consumo de aminocidos, por lo que la mayora de los aminocidos provienen de la degradacin de protena corporal. 40

Los requerimientos de protena para yeguas no lactando durante la poca reproductiva y la gestacin temprana no difieren de los de mantenimiento. Sin embargo, se ha demostrado que las concentraciones de protena en la dieta influyen en los valores de progesterona circulantes, con efectos en la fertilidad.24, 25 As mismo, se reporta que la calidad de la protena de la dieta tiene efecto sobre la activacin de la funcin ovrica, sobre todo porque en yeguas consumiendo protena de buena calidad se observa un incremento en la secrecin se FSH, adelantando el tiempo a la primera ovulacin.11

GESTACIN
La gestacin es una etapa esencial en la produccin equina. La hembra debe parir un potro sano y en la temporada apropiada de acuerdo a la funcin zootcnica para la cual se cra. La nutricin debe ser idnea no solo para asegurar el sano crecimiento del feto, sino tambin para sostener las altas demandas al inicio de la lactacin y promover la fertilidad dentro del primer mes posparto, asegurando un parto para el ao siguiente.43 El potro inicia su crecimiento desde el vientre materno.44 Cualquier evento que facilite su crecimiento como el aporte de nutrientes en calidad y cantidad suficiente, incrementa la posibilidad de que se trate de un caballo fuera del promedio. El aporte de nutrientes no solo es necesario en calidad y cantidad, para que estos nutrientes sean aprovechados debe haber una cantidad de oxgeno en el ambiente y ultimadamente en la sangre de la yegua suficiente para que se lleve en perfecto orden el metabolismo placentario y fetal. Los cuales sobreviven preferencialmente con glucosa, un sustrato que el mismo feto no es capaz de producir. A partir de ello es sencillo predecir que cualquier falla en tales requerimientos o procesos, compromete el futuro producto. Aunque se sugiere que el desarrollo embrionario y crecimiento fetal no requieren nutrientes adicionales durante los primeros siete meses de gestacin, es siempre benfico proporcionar una dieta bien equilibrada durante este tiempo. 44 Los mayores requerimientos se relacionan principalmente con el aumento en la deposicin a nivel tisular en el feto hacia los ltimos cuatro meses de gestacin, cuyo crecimiento se acelera de 0.08 a 0.33 kg/da.43 De acuerdo con Martin-Rosset et al.43 durante los ltimos cuatro meses de gestacin la ganancia de peso, energa y protena del feto cuenta para 80, 90 y 90 por ciento de la ganancia total, respectivamente. La placenta y el tero crecen y cambian en composicin qumica,45, 46 pues estos tejidos son muy activos metablicamente al ser responsables de la transferencia de substancias al feto y de la sntesis de nutrientes, hormonas y factores de crecimiento.47 Por tanto, la demanda de substratos, oxgeno y subsecuentes requerimientos por estos tejidos es considerable.48 Un aspecto importante es que el feto es altamente dependiente del aporte de glucosa (85 %)49 ya que es capaz de producirla por s mismo.43 Energa Digestible El NRC24,25 calcula los requerimientos diarios de energa digestible de la yegua a partir del noveno mes de gestacin de acuerdo con el siguiente modelo: EDG = EDM x F Donde EDG, energa digestible para gestacin, resulta de multiplicar el requerimiento de energa digestible de mantenimiento (EDM) por el factor F que corresponde a: 1.11 en el noveno, 1.13 en el dcimo y 1.20 en el onceavo mes de gestacin. Los sistemas: francs, alemn, holands y escandinavo basan sus clculos en ecuaciones que consideran la ganancia de peso y la composicin bioqumica del feto a distintos tiempos de gestacin. Sus estimaciones no difieren ampliamente de lo estimado por el NRC (Tabla 4). Lo destacable es que los cuatro sistemas europeos consideran aumentos a partir del octavo mes de gestacin.

Tabla 4. Factores para calcular requerimientos de energa digestible para gestacin propuestos por cinco diferentes sistemas de alimentacin de equinos. Porcentaje por arriba de mantenimiento Mes gestacin 8 9 10 11 NRC 0.11 0.13 0.20 INRA 0.13 0.19 0.19 0.26 Alemn 0.10 0.10 0.24 0.26 Holands 0.05 0.10 0.10 0.15 Escandinavo 0.07 0.13 0.18 0.25

Adaptado de Martin-Rosset et al., 2006 43

Protena Cruda Digestible De la misma forma que para ED las elevaciones de las necesidades de PCD para gestacin son importantes solo a partir del octavo mes de gestacin. El aumento en los requerimientos llega a ser hasta de 20%,24,25 ya que la concentracin de protena cruda en el feto incrementa de 100 a 171 gramos por kilogramo de peso.50 Tabla 5. Comparacin de los requerimientos totales de PCD para gestacin y su valor proporcional de los requerimientos de mantenimiento de yeguas de 450 kg de PV de acuerdo con cinco sistemas de alimentacin. NRC Mes de gestacin g PCD P M 8 9 10 11 330.5 330.5 370.5 375.5 446.0 INRA g PCD 295 1.00 340 1.12 340 1.14 460 1.35 485 P Alemn g PCD 450 1.15 450 1.15 505 1.56 505 1.64 580 P Holands g PCD 317 1.00 369 1.12 446 1.12 446 1.29 541 P Escandinavo g PCD 317 1.16 377 1.41 442 1.41 482 1.71 567 1.19 1.39 1.52 1.79 P

M, mantenimiento P, incremento por arriba de mantenimiento (PCD Mantenimiento * P = PCD Gestacin)

Con base en lo presentado por el NRC y el INRA, se recomienda que para ajustar los requerimientos de PCD se adicionen al requerimiento calculado de mantenimiento 1.23, 1.45 y 1.68 gramos por kilogramo de peso metablico de la yegua para el 9, 10 y 11 mes de gestacin, respectivamente.51 Esto en una yegua de 500 kg correspondera a 130, 153 y 177 gramos de PCD extras a su requerimiento de mantenimiento, respectivamente. El NRC recomienda 44 g de PC por MCal de ED que corresponde a un 10 % extra al requerimiento de mantenimiento. El ajuste con base en el factor 19.19 g de PCD por MCal de ED, resulta en 21.10 g de PCD por MCal de ED. As, los requerimientos de PCD para cada uno de los tres ltimos meses de gestacin se calculan con el modelo siguiente: PCD = (19.19 g * MCalEDM) + (21.10*MCalEDG)

Al igual que para ED, los sistemas francs, alemn, escandinavo y holands trabajan con ecuaciones que consideran la ganancia de peso del producto y su composicin bioqumica a diferentes edades de gestacin. En el rubro de PCD las diferencias de un sistema a otro son notables; sobre todo porque los europeos, a excepcin de INRA, estiman requerimientos mayores al NRC (Tabla 5). Como proporcin de los requerimientos de mantenimiento, las elevaciones calculadas por los sistemas: francs, holands y escandinavo, son sustanciales.

LACTACIN
Los requerimientos de lactacin estn relacionados al gasto fisiolgico debido a la secrecin lctea. El requerimiento de energa depende de la composicin y cantidad de leche producida, ya que una buena proporcin de nutrientes son convertidos en leche. 7, 10, 52, 53, 54 Puesto que el potro debe haber alcanzado el 45 % de su peso adulto al momento de ser destetado, la lactancia es una etapa especialmente importante en la produccin equina tanto para la yegua reproductora, como para su cra. Adems, la nutricin de la yegua en esta etapa es crucial porque tambin de ello depender que presente un celo frtil durante las primeras semanas postparto y que el embrin encuentre las condiciones ideales para implantarse y desarrollar una nueva gestacin, asegurando as la paricin anual de potros. La sntesis de leche en la glndula mamaria tiene un doble origen: nutrientes de la dieta y reservas corporales, y/o sntesis de novo. 54, 55, 56 La lactosa proviene de la glucosa absorbida en el intestino delgado. Los cidos grasos son producidos por sntesis de novo y captacin directa por la glndula mamaria. Los precursores para la sntesis de novo de cidos grasos C4 a C18 son el acetato y B-hidroxibutirato producidos durante la fermentacin de los carbohidratos en el intestino grueso. 54, 55, 56 Ni el propionato en el intestino grueso, ni la glucosa en el intestino delgado son precursores de cidos grasos de la leche. El tejido adiposo puede proveer de cidos grasos para la leche cuando la alimentacin de la yegua es limitada en energa. Los cidos grasos de la leche provienen de la captacin de cidos grasos circulantes en la sangre y de triglicridos transportados desde el hgado.52,
53, 54, 55, 56

Energa Digestible Para lactacin, los requerimientos se estiman con base en la produccin lctea, dependiendo de la produccin promedio diaria, contenido de nutrientes de la leche y la tasa de conversin de estos nutrientes.52 La energa neta requerida para produccin de leche es el producto de la produccin y la energa bruta contenida en cada kilogramo de leche. Se ha demostrado que esto est afectado por la etapa de lactacin. Las yeguas con ms de 300 kg de peso secretan alrededor del 3 % de su peso en leche en la primera mitad de la lactacin, mientras que en la segunda mitad secretan el 2 %. Las hembras menores a 300 Kg de peso producen ms leche, aproximadamente 4 % PV durante la lactancia temprana y 3 % PV al final. 24, 25, 51 El requerimiento de energa para lactacin se eleva inmediatamente despus del parto pero decrece conforme avanza. El NRC calcula el requerimiento extra de ED apoyado en el hecho de que la leche de yegua contiene 1.987 MJ de EB kg-1 y que la ED es convertida en energa de leche con una eficiencia de 60 %, resultando en un factor 0.792 que tiene que ser multiplicado por el potencial de produccin lctea (PPL) en kilogramos. De tal modo: EDL = PPL * 0.792 El requerimiento se estima como MCal de ED y se tiene que sumar al de mantenimiento para el animal en cuestin; tomando en cuenta que para estimar los requerimientos de una yegua de ms de 600 kg se debe utilizar la ecuacin propuesta para tales propsitos.

Los sistemas europeos hacen una divisin ms fina de la lactacin. Ellos consideran que el pico de produccin en una yegua se alcanza al segundo mes, por lo que los requerimientos deben calcularse para el primero, segundo-tercero y cuarto-sexto mes de lactacin. El Sistema Alemn calcula los requerimientos diarios para produccin lctea de yeguas entre 100 y 800 kg considerando que la produccin de leche en la yegua para cada uno de los mencionados perodos va de 0.14, 0.17 y 0.12 kilos de leche por Kg de PM; que el contenido de energa bruta por Kg de leche es de 0.58, 0.55 y 0.50 MCal, respectivamente; 57 y que la tasa de eficiencia de utilizacin de energa para produccin de leche es del 66 por ciento. El modelo que proponen para el clculo de energa digestible para lactacin es el siguiente: 52 EDL = (0.12 a 0.17 * PV0.75) * (0.5 a 0.58 MCal/kg leche) 0.66

El sistema INRA propone ecuaciones (Tabla 6) con base en las suposiciones siguientes: Produccin de leche de 3.0, 2.5 y 2.0 % PV para meses 1, 2-3 y 4-6 de lactacin. Que, en el mismo orden, la energa bruta por Kg de leche es 0.57, 0.50 y 0.47 MCal. Que la tasa de eficiencia de utilizacin de energa para mantenimiento es de 0.785 Que la tasa de eficiencia de utilizacin de energa metabolizable es de 0.65. Tabla 6. Ecuaciones para estimar el requerimiento extra de ED en yeguas en lactacin de acuerdo con el sistema INRA. Etapa de Ecuacin lactacin 0.792 1 mes ED = 0.03PV * 0.57 * 0.65 0.792 2 mes ED = 0.025PV * 0.50 * 0.65 0.792 3 mes ED = 0.025PV * 0.50 * 0.65 0.792 4 a 6 mes ED = 0.02PV * 0.47 * 0.65 El resultado de cada ecuacin se divide entre 0.70 para llevar de energa neta (EN) a energa metabolizable (EM) y ese segundo resultado entre 0.87 para llevar de EM a ED. La Tabla 7 compara los requerimientos de ED para lactacin calculados con tres sistemas diferentes. En la mayora de los casos los requerimientos son de ms del cincuenta por ciento por arriba de mantenimiento; de hecho, los clculos del Sistema Francs estiman el doble de requerimientos de mantenimiento para el primer mes de lactacin.

Tabla 7. Comparacin de los requerimientos totales de energa digestible para lactacin y su valor proporcional de los requerimientos de mantenimiento de acuerdo con tres sistemas de alimentacin en yeguas de 450 kg de peso vivo. Sistema Mes de lactacin Mantenimiento 1 mes 2 mes 3 mes 4- 6 mes 15.7 26.4 26.4 26.4 22.9 1.68 1.68 1.68 1.46 NRC MCal ED P Alemn MCal ED 15.7 28.7 30.7 30.7 25.3 P INRA MCal ED 14.4 29.7 25.6 25.6 22.8 P

1.83 1.96 1.96 1.61

2.06 1.78 1.78 1.58

P, incremento por arriba de mantenimiento (ED Mantenimiento*P = ED Lactacin)

Protena Cruda Digestible El mayor contenido de protena en leche de yeguas se ve inmediatamente despus del parto y disminuye gradualmente conforme avanza la lactacin. Las casenas representan de 50 a 65 % de la protena verdadera. Es poco lo que se sabe del metabolismo del nitrgeno en la glndula mamaria de la yegua. Los resultados en cuanto al efecto del nivel y fuente de protena de la dieta sobre la concentracin y calidad de la protena de la leche en yeguas son inconsistentes,52 por lo que de manera general se maneja que la composicin de la leche se afecta por la dieta, pero que el nivel de produccin depende del consumo. 51, 52, 53, 54, 55, 56 Considerando los potenciales de produccin de leche de acuerdo al PV, que el requerimiento de PCD es de 0.6 g por kilogramo de PV, que el contenido de protena cruda en leche es de 2.1 % en la primera y 1.8 % en la segunda mitad, que la eficiencia en la utilizacin de PCD a protena de la leche es de 0.65 y que la digestibilidad de la PC de las dietas para lactacin es de 55 por ciento, el NRC 58, 59 presenta las ecuaciones que producen los siguientes factores: 33 g de PCD por Kg de leche para la primera mitad de la lactacin 28 g de PCD por Kg de leche para la segunda mitad de la lactacin

Con el mismo dato de que la eficiencia en la conversin de PCD a leche es del 55 %, Martin-Rosset et al.,58 deducen que para el sistema INRA los g de PCD requeridos por kilogramo de leche producida son: 44 g de PCD por Kg de leche para el mes 1 38 g de PCD por Kg de leche para los meses 2 y 3 36 g de PCD por Kg de leche para los meses 4 a 6

Con estos factores es sencillo calcular el requerimiento de PCD para leche de acuerdo al potencial de produccin de la yegua y sumarlo al requerimiento de mantenimiento calculado con la ecuacin presentada para tal efecto. Recurdese que el INRA considera potenciales de produccin de 3.0, 2.5 y 2.0 por ciento para los meses 1, 2-3 y 4-6, respectivamente.52

El Sistema Alemn considera que el contenido de PC de la leche es de 27, 22 y 18 g por Kg para los meses 1, 2 y 5, respectivamente, y que la eficiencia de utilizacin de protena para la secrecin lctea es de 0.5. Con base en ello proponen una ecuacin a partir de la cual se generan los siguientes factores para ser multiplicados por la produccin de leche y estimar as el requerimiento de PCD para lactacin a distintas etapas: 1 2 3 4- 6 mes mes mes mes 54 44 44 36 * * * * 0.14 PV0.75 0.17 PV0.75 0.17 PV0.75 0.12 PV0.75

La comparacin de los clculos del requerimiento total de PCD para lactacin por cada uno de los sistemas de alimentacin se presenta en la Tabla 8. Tabla 8. Comparacin de los requerimientos totales de PCD para lactacin y su valor proporcional de los requerimientos de mantenimiento de acuerdo con tres sistemas de alimentacin en yeguas de 450 kg de peso vivo. Sistema Mes de lactacin gPCD Mantenimiento 1 mes 2 mes 3 mes 4- 6 mes 300 746 746 746 552 2.5 2.5 2.5 1.9 NRC P gPCD 294 1032 1024 1024 716 3.5 3.5 3.5 2.4 Alemn P gPCD 254 594 682 682 578 2.3 2.7 2.7 2.3 INRA P

P, incremento por arriba de mantenimiento (PCD Mantenimiento*P = PCD Lactacin)

EL GARAN
Es poca la informacin disponible especfica para nutricin del semental equino. Puesto que la madurez sexual y la habilidad de producir espermatozoides, que son los parmetros utilizados para medir la fertilidad del garan, se encuentran bajo estricto control endcrino, las interacciones entre nutricin y funcin endcrina son las responsables de efectos en la fertilidad del garan, particularmente con respecto a la calidad del semen.59 Por tal motivo es necesario asegurar un manejo nutricional adecuado para cubrir todos los requerimientos con relacin a su raza, lnea, edad, peso y nivel de actividad. El nivel de actividad es importante, pues un semental que se ejercita regularmente estar ms sano y guardar un mejor equilibrio entre la captacin y utilizacin de nutrientes con sus niveles hormonales. Energa Digestible En requerimientos de energa digestible para reproduccin conviene tomar en cuenta que las necesidades del garan son mayores. El clculo inicia con aproximaciones similares a las propuestas para el mantenimiento de yeguas. Sin embargo, puesto que los requerimientos de mantenimiento se han encontrado de un 10 a 20 % 37 mayores en machos enteros y hasta de un 40 % cuando estn en actividad sexual, debern considerarse los incrementos mostrados en las Tablas 9 y 10.

Tabla 9. Factores de ajuste de requerimientos de mantenimiento dependiendo del tipo y nivel de actividad del equino. Nivel de actividad Pesado Garan inactivo Garan activo
Adaptado de Vermorel et al., 1984 34

Tipo de equino Silla 1.15 1.25-1.35 Pura sangre 1.20 1.30-1.40

1.10 1.20-1.30

Protena Cruda Digestible El clculo de requerimientos de protena cruda puede hacerse directamente con el factor 19.19 g de PCD por MCal de ED total para reproduccin, o bien, calcular el requerimiento de mantenimiento y agregarle el incremento porcentual propuesto en la Tabla 10. Tabla 10. Incremento porcentual en los requerimientos de garaones de acuerdo con dos sistemas de alimentacin (NRC e INRA). Rubro NRC Energa digestible Protena Cruda Digestible
Adaptado de Ellis et al., 2006 59

Sistema INRA 25.9 37.5

25 25

Podra considerarse que los requerimientos del garan son de moderados. Sin embargo debe tenerse cuidado en evitar prdidas de peso o disminucin en condicin corporal debido a seleccin de alimentos de mala calidad. Solo en casos de desnutricin severa o prolongada debe esperarse ineficiencia reproductiva en el macho desde el punto de vista nutricional, sobre todo debido a supresin de la produccin hormonal. Aunque la mayora de los casos de infertilidad en el garan son atribuidles a factores distintos a la nutricin.59

CONCLUSIN
El plano nutricional del equino es prioritario para lograr eficiencia reproductiva, sobre todo en aquellos casos en que los animales son explotados en esquemas o ciclos distintos a las condiciones en que evolucionaron. Los requerimientos deben ser cubiertos en cantidad y calidad de acuerdo con la etapa reproductiva. Los mayores incrementos se observan en las necesidades de energa digestible y protena cruda digestible sobre todo en hembras en la primera mitad de la lactancia. La hembra gestante tiene incrementos discretos, sobre todo hacia el final de la gestacin. En el caso del semental equino, basta con asegurar el aporte de energa y protena con base en su tasa metablica. Es importante que durante la actividad reproductiva los requerimientos nutricionales se cubran con alimentos de buena calidad para asegurar la provisin de nutrientes especficos para cada etapa de la fisiologa reproductiva del quido.

Adems del plano energtico y proteico, la nutricin mineral y vitamnica de los equinos debe tambin ser considerada al momento de disear estrategias de alimentacin. Sobre todo en lo que respecta a desequilibrios que resultan en compromiso de la fertilidad o el desarrollo del producto durante la gestacin.

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SEASONAL OVULATORY INACTIVITY IN MARES: RESPECTIVE PART OF PHOTOPERIOD AND BODY CONDITION. PART 1) PHOTOPERIOD AND MELATONIN EFFECTS. PART 2) ENDOGENOUS RHYTHM AND BODY CONDITION EFFECTS.
Daniel GUILLAUME1 and Juan SALAZAR-ORTIZ2 . UMR 6175 INRA/CNRS-Universit F. Rabelais of Tours-Haras Nationaux, Centre of research of Tours 37380 Nouzilly, France. 2 Colegio de Postgraduados, Campus Crdoba. Carretera Federal Crdoba-Veracruz Km. 348, Congregacin Manuel Len 94946 Amatln de los Reyes, Veracruz , Mxico. Apdo. Postal 143 Col. Centro 94500 Crdoba, Veracruz, Mxico.
1

ABSTRACT The aim of the present review is to outline the respective effects of photoperiod and nutrition level on mare's seasonal anoestrus. Mares present a winter ovarian inactivity, which takes place from fall to spring. This winter inactivity is synchronised by the photoperiod, which is mediated by melatonin to the GnRH neuron through an unknown pathway. 50 percent of fat mares which have not nursed a foal during the previous summer continue to cycle all along the year whereas the other have a very short inactivity and miss only 1 or 2 cycles. Conversely restricted mares systematically exhibit long winter inactivity and only have few sexual cycles during summer. Increasing the feeding level in the early autumn cannot reactivate the cyclicity. Mares have an annual reproductive rhythm synchronised by photoperiod, with a phase of reproductive inactivity centred on midwinter and a peak of reproductive activity centred on midsummer. Nutrition-body condition are the main causes of the occurrence and length of winter ovarian inactivity. The photoperiod is only responsible for the timing of that inactivity. MY LABORATORY STRUCTURE. The main part of the results presented in this paper are obtained at the French National Institute of Agronomical Research, (http://www.inra.fr/). This institute employ directly around 8500 workers (1800 scientists, 2400 engineers and 4250 technical assistants and administrators. Further more around 500 employees for other government institutes or private organizations work in different INRA centres. This institute is located in 150 different places and divided into 218 research unites. In its different experimental unites, INRA used 10500 Ha and breed around 94000 animals. Our research centre is the middle of France, in a deep countrysite, near a town named Tours (http://www.tours.inra.fr). This centre uses 580 hectares to breed 200 horses, 220 cattle, 2400 sheep, 350 goats 1150 pigs 67000 poultry, 3800 rabbits 12000 mice and rats. The permanent staff includes 239 researchers and engineers and 313 technical assistants. Each year 250 students make their end of studies internships or PhD theses. The research at the Tours research centre focuses on reproductive and behavioural physiology and animal health. Our "joint research unit for reproductive and behavioural physiology" (UMR-PRC) (http://www.tours.inra.fr/physiologie_reproduction_comportements) has a permanent staff of 150 workers. This unit is associated with the university and between 100 to 200 students

work in its laboratory. On the last years some Mexican students have done their PhD theses in this unit. In equine physiology research, our lab was one of the pioneers for ultrasonography, light treatment or in vitro fecundation. Our spermatozoa middle for artificial insemination is commercialized every where in the word. PART 1) PHOTOPERIOD AND MELATONIN EFFECTS. I) Seasonal variation of the birth date in temperate zone In the French breeding system an important peak of births occurs in April and May (Figure 1). This period of birth are approximately the same than in feral horses: 14 April (n=118) which live in the south of France (Grange et al 2009). This result is probably generalised at all the temperate zones in the word. This peak of birth occurs at the optimum moment when food bio-availability is maximal.

Month of birth in the main horses breeds in France.


Figure 1

70000 60000 50000 40000 30000 20000 10000 0

Cumulated total of births between 1992 and 2002

PONEY Warmblood Horses French Saddle Horses and Anglo-Arabian French Troter Draught Horses

M ay

ry

ry

Langlois and Blouin personal communication 2005

This strong annual birth rhythm is the consequence of a annual reproduction rhythm with a breeding season which occurred 11 months before. In horses, the natural breeding season is centred approximately, on the longest day of the year, 20th June in the northern hemisphere. The breeding season starts in spring when the hours of daylight, ambient temperature and availability of food increase to maxima at midsummer before decreasing. In wild species matting had an important energetic cost. In horse it occurs when energy allowance is maximal. So horses hold the 2 seasonal advantages for matting and for birth. It is not generally the case. For example, ovine, goats had their breeding season in fall and winter and the birth season in spring and summer. Another specie which can take advantage of these 2 seasonal

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ua

Ju l

events is the European roe deer (Capreolus capreolus). In this species; matting, fertilization and birth occur in spring and the blastocyst shows a variable diapauses between August and December (Lambert et al 2001). Furthermore, in equine specie, the length of pregnancy is influenced by the time of fertilization. This length is longer for mares bred in winter 338 days and spring 342 days, than for mares bred in summer 331 days or fall 329 days (Ginther 1993). In equine species all the reproduction physiology is organized to order the birth in April or May. For wild mares living in temperate or cold zones, this winter ovarian inactivity constitutes a tremendous adaptation. This winter ovarian inactivity should be overcome by problems the breeders who want to get the mare pregnant as soon as possible in the year to get an age advantage over the foals born later in the year (Langlois and Blouin 1996, 1997, 1998). Further more the numbers of usable cycles and so the odds to be pregnant at the end of the year increase when the mares can be matted soon in the year (Langlois and Blouin 2004). This breeding challenge requests to develop methods for induction of an early onset of the breeding season in mares. II) Effect of light in the regulation of the annual reproduction rhythm. Most of mares show an anovulatory period approximately from October to May. The length of this winter inactivity depends on the age of the mare and its physiological state. It is long and systematic in young 2 or 3 years old mares. Adult mares which have nursed a foal on the previous summer have also a long and systematic ovulatory inactivity on the next winter. In other mature mares, this inactivity is observed in only half of them (Palmer and Driancourt 1983). It is easy to show this inactivity by a weekly progesterone assay on blood sample (Figure 2). In ovariectomized mares, this winter inactivity is characterized by an important decreased in LH plasma concentrations (Figure 2).
Figure 2

Natural Photoperiod

4 Oct.

188 D. 8 Avr.
luteales phases, in draught mares which had suckled a foal during summer.

GUILLAUME et al 1995. Biol. Reprod Monograph Series Ip: 435-442.

eLH in ovariectomised poney mares


Mean S.E.M . n=6

PALMER E., GUILLAUME D., 1998 Reprod in Domestic Animals 33, 205-208 d j f m a m j j a s o n d j f m a m j j a s o n d j f

months

The first investigations on the effects of light on this annual rhythm of reproduction were made by Burkhard (1947) followed by Nishikava (1959). The discovery of melatonin, the main hormone involved in this effect, occured one year later by Albert (1958) and its chemical formulation by Lerner et al (1959). The important role of this pineal hormone in the photoperiodic effects on reproduction began to be investigated in the eighties in equine species. The photoperiod is independent of the weather and it is the more stable parameter from one year to the other. Under temperate zones a lot of animals and plants have chosen this parameter to adapt their physiology to the season. It is well accepted that in the horses, photoperiod is the most important external factor that influences the circannual reproductive rhythm (Ginther, 1992). Additional light exposure during winter and early spring stimulates ovarian activity in anoestrous mares and is commonly used to advance the onset of the breeding season (Burkhardt, 1947). A lot of experiments have implemented different light treatments, beginning around the winter solstice, on mares previously chosen to be in ovarian inactivity. These experiments have clearly indicated that the length of the light phase efficient to advance the first ovulation of the year is 14.5 h under our temperate latitude. Using this light treatment, the first ovulation of the year occurs around 2 months before the control groups subjected to natural photoperiod. The same effect is obtained with 1 or 2 h of a light flash during a long night, 9.5 to 10 hours after the abrupt beginning of darkness (Palmer and Driancourt, 1981; Palmer et al 1982; Malinowski et al 1985). Some conditions have been recently specified to implement this treatment (Guillaume et al 2000). A light intensity of 10 lux is sufficient to advance the first ovulation, and the light-treatment began around the winter solstice can be stopped 35 days after, when the natural photoperiod is short, without adverse effect on the treatment (figure 3). The effects of this light treatment are not dramatically disrupted by some disruption of the light dark cycle induced by some breakdowns of the clock which monitors the artificial light. (Legros et al 2003, Guillaume et al 2006). The light treatment performed during the anoestrus of mares in good body condition is very secure.

Figure 3

Photostimulation and Winter anoestrus 6 March 30 April

Duration of light treatement


Individual progesterone curve

Light treatement 14.5L 9.5D

Nat. Phot.

-30

30

60

90

120

150

180

Numbers of days after January the 1st


Guillaume et al 2000. J. R. F. supplement 56, 205-216.

III) Effect of melatonin in the regulation of the annual reproductive rhythm. The role of melatonin on the seasonal variations of reproduction has been strongly investigated in a lot of other species (Malpaux et al 1999). Mares, like other photoperiodic species, transduce photoperiodic information into a neuroendocrine signal in the pineal gland trough its product, melatonin. The pineal gland is involved in the control of seasonal reproduction and conveys the photoperiodic signals registered by the eyes to endocrine signals. In mares, elevated plasma melatonin concentrations are strongly associated with the dark phase. Melatonin secretion increases at the beginning of the dark phase and decreases rapidly at the end of the night. The secretion of melatonin is proportional to the duration of the night (Guillaume et al 1995). A short exposure to light during the dark phase results in an immediate decrease in melatonin concentrations; the return to dark-phase is followed by a new increase in melatonin concentrations (Guillaume et al 2000). The role of melatonin in the regulation of mares' cyclicity was demonstrated by Guillaume and Palmer (1991), who reported that exogenous melatonin, administered 4 hours before the beginning of short nights (14.5L:9.5D), prevents the stimulatory effect of long days. Similarly, mares in seasonal anoestrus subjected to artificial photoperiod (14.5L:9.5D), do not respond to the stimulatory photoperiod when melatonin is administered every 2 hours, during a 12-hours period, that includes the 9.5-hour dark period (Guillaume and Palmer 1992). In these mares, the high levels of exogenous melatonin mask the onset of darkness, marked by an increase in melatonin secretion, and the onset of daylight, marked by a decrease in melatonin secretion. Seasonal reproduction in horses is affected as well as by a daily melatonin administration as a permanent melatonin application. Melatonin implants, inserted near the shortest day of the year, suppress the stimulating effect of long days, but do not prevent the occurrence of cyclic ovarian activity (Guillaume et al 1995).

Although, the effect of photoperiod is well documented, the site of action of melatonin has not been studied in horses. We know from studies in other species, that melatonin does not influence GnRH-secretion directly, but acts through a complex network of interneurons involving a number of different neurotransmitters. Its target sites appear to be located within the hypothalamus (Malpaux et al 1993). In horses, specific melatonin binding was found in the pars tuberalis, in the median eminence and in the suprachiasmatic nucleus (Stankov et al 1991). The nocturnal plasmatic melatonin concentration appears to be unrelated to the reproductive status, and some mares, showing no cyclicity in winter do not exhibit a rise in plasma melatonin during darkness. Plasma melatonin concentrations are sometimes difficult to distinguish at the beginning of the dark phase and can even be absent during the entire dark period (Fitzgerald Schmidt, 1995; Guillaume et al 2006). This very low or absence of melatonin concentrations in peripheral blood cannot be interpreted as a lack of physiological effect of this hormone in equine. In sheep, melatonin concentrations are many-fold higher in the third ventricle than in peripheral blood (Skinner and Malpaux, 1999) and in this species, the route of melatonin to this specific site of action in the mediobasal hypothalamus (Malpaux et al 1993) can be the blood or the cerebrospinal fluid through the third ventricle (Tricoire et al 2003). These observations suggest that, in equine species, the main route of melatonin to the reproduction axis is not the general blood circulation but probably the cerebrospinal fluid, through the third ventricle and circulating melatonin concentrations are a poor reflection of efficient melatonin secretion. PART 2) ENDOGENOUS RHYTHM AND BODY CONDITION EFFECTS. IV) Evidence of a strong endogenous component of the annual reproductive rhythm. In ewes, Karsch et al (1989) provided clear evidence for the existence of an endogenous circannual reproductive cycle. Ovariectomized, estrogens-implanted ewes maintained in constant light conditions for 5 years, maintain circannual changes in LH and prolactin secretion. However, the period of elevated LH levels, representative of the breeding season, differs among individuals, reflecting the lack of synchronising environmental factors. The seasonal reproductive pattern is the result of a circannual endogenous rhythm that is synchronised by external environmental factors, which the main one is photoperiod. In horses, available data indicate that the annual reproductive rhythm has a strong endogenous component. The first direct evidence for the existence of an endogenous circannual rhythm was given by the removal of the pineal gland or the cervical superior ganglion (Sharp et al 1979; Grubaugh et al 1982). In the 2 cases, the lesions did not result in the disappearance of seasonal reproductive activity. In pinealectomized mares, placed under extended photoperiod, the onset of reproductive activity was not advanced by artificial photoperiod and these mares resumed cyclic ovarian activity significantly later than controls, during the second season after surgery. Another evidence for the existence of an annual endogenous rhythm is the variation of ovarian activity in mares kept under constant photoperiod or constant melatonin level during several seasons. Mares, maintained under constant long day photoperiod (16h light), beginning at the summer solstice, enter into anoestrus and mares maintained under short days photoperiod (8.5h light) beginning at the winter solstice resume cyclic ovarian activity (Kooistra Ginther, 1975; Palmer and Driancourt, 1981; Palmer et al 1982; Scraba and Ginther, 1985). This is interpreted as an inability to continue to respond to the current type of photoperiod and is described as a refractory state. For this reason, mares kept under 16h

light starting in winter (Palmer et al 1982) or summer (Kooistra and Ginther, 1975; Scraba and Ginther, 1985), still return to winter anoestrous. Melatonin implants, inserted near the summer solstice, on adults draught mares a few days before the birth of their foal (very fat at this moment), do not induce or advance the beginning of winter inactivity but advance the ovulatory season of the following year (Guillaume et al 1995). The likely explanation for this observation is a sequence of an early short-day perception, caused by the continuous high melatonin concentrations, followed by an early onset of the refractory state caused by continuous elevated levels of melatonin. In the same experiment, the same melatonin treatment applied to adults mares, which had not nursed a foal during summer, did not induce winter inactivity and these treated mares had permanent reproduction. These previous results were verified by Fitzgerald and McManus (2000) and Peltier et al (1998). Under this refractory state, the annual reproductive cycle is phase advanced which is characterized by an earlier occurrence of the next ovulatory season. On another hand, the same melatonin treatment, applied on young haflinger mares (1.5 years old), induced an advanced of 2 months of the beginning of winter inactivity but its end was only advanced of one month and appears to be not significant (Guillaume et al 1995). At this age, at the moment of their melatonin implantation, these young haflinger mares had not finished their growth. These differences between young and adult mares can be interpreted consecutively to the recent results on body score and winter inactivity (see below). These observations support the concept that photoperiod and rhythm of melatonin secretion drive the endogenous circannual rhythm but do not influence directly the reproductive activity. This settlement of a photo-refractoriness states against the constant photoperiod or constant melatonin level highlights the existence of an endogenous rhythm (endogenous calendar). It seems that this endogenous circannual rhythm can be monitored by photoperiod transmitted by melatonin just a few months in advance. The effect of photoperiod or exogenous melatonin on the time of the last ovulation of the breeding season is controversial. In adult mares, those which have nursed a foal in summer, kept under natural photoperiod and given subcutaneous melatonin implants around the summer solstice or in autumn, showed no change in the date of their last ovulation (Guillaume et al 1995, McManus and Fitzgerald 2003). Alternatively, in young Haflinger pony mares, that were still growing (1.5 years old), the same melatonin implants administrated in summer advanced the date of the last ovulation by two months in comparison with untreated mares (Guillaume et al 1995). The same non-significant trend was obtained with melatonin administered in June; via an osmotic pump but, the age and the body condition score of the experimental mares were not given (Peltier et al 1998). These data suggest that the annual endogenous rhythm, which is regulated by photoperiod or its mediator melatonin, do not directly induce winter ovarian inactivity but permissively determine the season of anestrus. When one mare presents permanent reproduction, without winter inactivity, it does not mean that this mare has no circannual rhythm but only that this rhythm is not extrapolated on its cyclicity. Some other environmental factors are necessary to highlight this annual rhythm. V) Modulation of the annual rhythm of reproduction by body condition Effects on the end of winter inactivity. Several authors suspected the effects of nutrition and body condition on the resumption of winter inactivity. Van Niekerk and Van Heerden (1972) showed that mares, which are supplemented diet with concentrates, ovulate earlier after winter anoestrus than control mares without supplementation. Ginther (1974) also noted that the anovulatory period is shorter in mares, which gain weight during early spring. McDaniel et al (1979) reported an additive effect of nutritional supplementation and artificially extended photoperiod on the onset of reproductive activity. Henneke et al (1984) observed that the average date of the first

ovulation was significantly latter in mares with body condition score less than 5.0 (scale from 1, poor to 9, extremely fat) compared to mares with body condition score above 5.0. The interaction between energy intake and body condition in the reproductive performance of nonpregnant mares was evaluated by Kubiak et al (1987). A high-energy intake shortens the interval to the first ovulation in transitional mares with a low level of body fat but does not have any effect in mares in moderate or fat body condition. Mares with a body fat content greater than 15% had a shorter interval to the first ovulation compared to those with a body fat content lower than 15%. The authors suggest that non-lactating mares should be brought into the breeding season with a body fat content above 15% e.g. a body condition score above 5.0 and then maintained in a positive energy balance to obtain an earlier onset of ovulation. The quality of dietary proteins influence the onset of the breeding season. Mares fed with a high-quality protein diet exhibit increased FSH secretion and ovulate approximately 2-3 weeks earlier than mares fed low-quality protein diet (Van Niekerk Van Niekerk, 1997). The stimulatory effect of pasture grazing on the time of the first ovulation has also been reported. First ovulation occurs over a large period of time in Thoroughbred mares that are housed inside at night and are allowed to eat grass on pasture for 4-6 hours per day, whereas pony mares that are kept in concrete yards during winter ovulate in synchrony after they are turned out to graze lush grass (Allen, 1987). Carnevale and Ginther (1997) demonstrated the beneficial effect of pasturing on the onset of cyclic ovarian activity. Anoestrous mares pastured on green grass from early May ovulate sooner than mares housed on dry lot and fed hay. It is possible that the earlier onset of anoestrus in young mares (Ginther, 1992) and the high incidence of anoestrus in lactating mares (Palmer and Driancourt, 1983) are related to nutritional factors. In a 10-year survey of breeding records in a thoroughbred farm in Australia, Guerin and Wang (1994) reported significant differences between years for the time of the first ovulation. The authors concluded that the onset of reproductive activity was closely related to minimum and maximum environmental temperatures. Field data for thoroughbred mares in the United Kingdom suggest that the spring transition is slowed by cold weather (Allen, 1987). Thus, it appears that under similar conditions of photoperiod, nutrition, management system and temperature play a role in the timing of the circannual reproductive rhythm. The mechanism of the effect of temperature is completely unknown. But, the lack of energy supplementation or/and body reserves to meet the extra energy cost due to thermoregulation would be responsible. EFFECT ON THE BEGINNING AND DURATION OF WINTER INACTIVITY. In our laboratory (Guillaume et al 2002), some recent data indicate the important role of nutrition and body condition on the onset of anoestrus in mares. In a more recent experiment, the influence of feed allowances on the annual rhythm of reproduction was studied in adult ponies and the changes in the plasma melatonin, insulin, GH and glucose daily patterns were determined to identify a possible signal between nutritional status and ovulatory activity. The mares were randomly assigned in 3 groups: well feed group (WF), variable group (V) and restricted group (R) referring to INRA 1990 recommendations. A special attention was paid to the R mares, to keep them healthy but very thin all along the experiment, which lasted 3 years. The group V was fed to mimic the seasonal variations of the available forage resources in natural foraging system under temperate latitudes, but with an important out of phase with these natural variations with the aim to advance the ovulatory activity. In the R group, the winter ovarian inactivity was systematically advanced, longer and ended later than in the WF group (figure 4). During the 3 years of the study, in the WF group, 5 mares never presented any ovulatory inactivity, 2 mares systematically had a short inactivity and the 2 last mares had a short inactivity for 2 successive winters. The high level of feed allowance doubled the number of cycles in comparison with the low level. The occurrence or the lack and the duration of ovulatory inactivity were repeatable in adult mares fed with a constant and

high feeding level. These results support those of Fitzgerald et al' (2000), which observed a tendency of adult mares with a higher percent of body fat and a higher concentration of plasma leptin to exhibit a continuous cyclicity. These results are comparable with those obtained in cows (Bossis et al 1999). The V group presented an intermediate body weight between the R and WF groups. In the V group, despite an important increase of body condition score during the late fall and winter no difference was observed in the end of winter inactivity. In this group, 2 mares never presented winter inactivity. The lack in the response to the change of feeding level is contradictory with studies previously mentioned (Van Niekerk and Van Herden, 1972, Ginther, 1974, Mac Daniel et al 1979, Allen, 1987, Carnevale and Ginther, 1997). But these studies are likely confusing the global effect of breeding system and the temporary effect of feeding level. In the 3 groups, the observation of Nagy et al (1998): "later is the last ovulation of the year, earlier is the first ovulation of the following year" is clearly supported by our data.
Figure 4

Food effect on winter anoestrus

% of cyclic mares (n =10/group)


100 90 80 70 60 50 40 30 20 10 0 a s o n d j f m a m j j a s o n d j f m a m j j a s o n d j f m a m j a s o

Well fed group restricted group

1st year

2nd year

3rd year Salazar-Ortiz et al 2005

In our experiment, GH concentrations were higher in R and V groups than in WF and were correlated with the body condition score. The IGF-I level was reduced by about 50% in the R group compared with the WF group. In horses, the plasma rates of leptin are correlated with the body condition score (r = 0,64 p<0,0001, Buff et al 2002). So, in our experiment, we can assume than leptin level should be higher in the WF group than in the 2 other ones. Important postprandial peaks of glucose and insulin were observed in the WF group and the V group fed as the WF group. But these postprandial peaks completely disappear in the R group and in the V group when there were fed as the R groups. CONCLUSION ON INTERACTION BETWEEN FEEDING LEVEL AND SEASON OF REPRODUCTION. The ovulatory activity of the mare fits a sinusodal curve (Figure 5). During spring and summer the ovulatory activity represents the visible part of the annual endogenous rhythm. The spring-summer ovulatory activity is regulated by the photoperiod, mediated by melatonin. The highest feeding level is important, the highest the phase of the ovulatory activity is long. The period when the feeding level should be adjusted to reduce the length of winter inactivity

is still to be defined; but it is likely during the previous season to winter. The peripheral hormones indicating the optimal nutritional status to be met for optimising reproduction is no yet defined but leptin should be the more interesting candidate.
Figure 5

Annual rhythm of reproduction

Food ood energy available

Restricted Restricted mares

Winter ovarian inactivity

Winter ovarian inactivity

Well fed mares Fat mares

Summer ovarian activity

Summer ovarian activity

d j f m a m j j a s o n d j f m a m j j a s o n d j f

RFRENCES:
Albert C. 1958. Melatonin: the first hormone isolated from the pineal body. Farmaco [Prat]. Aug; 13(8): 604-605. Allen WR. 1987. Endogenous hormonal control of the mare's oestrus cycle. Proceedings of the BainFallon Memorial Lectures. 2-13. Bossis I., Wettemann RP., Welty SD., Vizcarra JA., Spicer LJ. Diskin MG. 1999. Nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation. J. Anim. Sci. 77: 15361546. Buff PR., Dodds AC., Morrison CD., Whitley NC., McFadin EL., Daniel JA., Djiane J. Keisler DH. 2002. Leptin in horses: tissue localisation and relationship between peripheral concentrations of leptin and body condition. J.Anim. Sci 80: 2943-2948. Burkhardt 1947. Transition from anestrus in the mare and the effects of artificial lighting Journal of Agricultural Science 37: 64-68. Carnevale EM. Ginther OJ. 1997. Age and pasture effects on vernal transition in mares Theriogenology. 47: 1009-1018. Fitzgerald BP. McManus CJ. 2000. Photoperiodic versus metabolic signals as determinants of seasonal anestrus in the mare. Biol. Reprod. 63: 335-40. Fitzgerald BP. Schmidt MJ. 1995. Absence of an association between melatonin and reproductive activity in mares during the nonbreeding season. Biol. Reprod. Mono. 1: 425-434. Ginther OJ. 1974. Occurrence of anestrous, estrus, diestrus and ovulation over a twelve-month period in mares American Journal of Veterinary Research. 35: 1173-1179.

Ginther OJ. 1993. Reproductive biology of the mare; basic and applied aspects. Ed. Library of Congress Catalog Cross plains Equiservices Publishing, 2nd ed. Grange S, Duncan P Gaillard JM 2009 Poor horse traders: large mammals trade survival for reproduction during the process of feralization Proceeding of the Royal Society Biological Sciences 10.1098 Grubaugh WR, Sharp DC, Berglund LA, McDowell KJ, Kilmer DM, Peck LS Seamans KW 1982. Effects of pinealectomy in pony mares. Journal of Reproduction and Fertility Suppl. 32: 293-295. Guerin MV Wang XJ. 1994. Environmental temperature has an influence on timing of the first ovulation of seasonal estrus in the mare. Theriogenology. 42: 1053-1060. Guillaume D Palmer E. 1991. Effect of oral melatonin on the date of the first ovulation after ovarian inactivity in mares under artificial photoperiod Journal of Reproduction and Fertility Suppl. 44: 249257. Guillaume D Palmer E., 1992. Lumire, mlatonine et reproduction chez la jument. Annales de Zootechnie 41; 263-269. Guillaume D., Arnaud G., Camillo F., Duchamp G Palmer E. 1995. Effects of melatonin implants on reproductive status of mares. Biology of Reproduction Monograph 1: 435-442. Guillaume D., Duchamp G., Nagy P. Palmer E. 2000. Approach of the minimum lighting conditions for photostimulating mares in winter inactivity. Journal of Reproduction and Fertility suppl. 56: 205-216. Guillaume D., Duchamp G., Salazar-Ortiz J. Nagy P., 2002. Nutrition influences the winter ovarian inactivity in mares. 8th International Symposium on Equine Reproduction, Colorado USA. Theriogenology. 6642: 593-597. Guillaume D., Nagy P., Duchamp G. Palmer E. 2000. Approach of the minimum lighting conditions to photostimulate mares in winter inactivity. Journal of reproduction and fertility supplement 56: 205-216. Guillaume D., Regnier F., Arnaud F., Larry JL., Chesnau D. Malpaux B. 2006. Effet d'un clairement permanent sur la reprise de l'activit sexuelle chez la jument. 32me journe d'tude sur les quids, 1er mars 2006, Paris. ed. Les Haras Nationaux. 33-38 Guillaume D., Rio N. Toutain P.L. 1995. Kinetic studies and production rate of melatonin in pony mares. American Journal of Physiology 268; R1236-1241. Guillaume D., Zarazaga L., Malpaux B. P. Chemineau. 2006. Variability of plasma melatonin level in pony mares (Equus caballus), comparison with the hybrid: mules and with jennies (Equus asinus). Accepted for publication in Reprod. Nutr. Dev. Henneke DR., Potter GD. Kreider JL. 1984. Body condition during pregnancy and lactation and reproductive efficiency of mares. Theriogenology. 21 897-909. Karsch FJ., Robinson JE., Woodfill CJI. Brown MB. 1989. Circannual cycles of luteinizing hormone and prolactin secretion in ewes during prolonged exposure to a fixed photoperiod: Evidence for an endogenous reproductive rhythm. Biology of Reproduction. 41: 1034-1046. Kooistra LH Ginther OJ. 1975. Effect of photoperiod on reproductive activity and hair in mares. American Journal of Veterinary Research. 36: 1413-1419. Kubiak JR. Crawford BH. Squires EL. Wrigley RH. Ward GM. 1987. The influence of energy intake and percentage body fat on the reproductive performance of nonpregnant mare Theriogenology. 28: 587-598.

Lambert RT, Ashworth CJ, Beattie L, Gebbie FE, Hutchinson JS, Kyle DJ, Racey PA. 2001 Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus). Reproduction 121: 863-871.
Langlois B. Blouin C. 2004. Statistical analysis of some factors affecting the number of horse birth in France. Reproduction Nutrition Development 44: 583-596.

Langlois B, Blouin C 1997 Effect of a horse's month of birth on its future sport performance. I Effect on annual phenotypic indices AnnZootech 46:393-398
Langlois B, Blouin C 1998 Effect of a horse's month of birth on its future sport performance. II Effect on annual earning and annual earning per start AnnZootech 47:67-74 Langlois B, Blouin C. 1996. Effect of a horse's month of birth on its future performances Proceedings of the 47th Annual Meeting of the European Association for Animal Production. Legros C., Salazar-Ortis J. Guillaume D., 2003. Photostimulation de la jument : la technique supportet-elle des accidents durant l'clairement ? 29me Journe de la Recherche Equine. 26 fvrier, Paris dition: Les Haras Nationaux 16 rue Claude Bernard 75231 Paris Cedex; 1-10. Lerner AB., Case JD., Mori W. Wright MR.. 1959. Melatonin in peripheral nerve. Nature. 27: 18181821. Malinowski K., Johnson AL. Scanes CG. 1985. Effects of interrupted photoperiods on the induction of ovulation in anestrous mares. Journal of Animal Science 61 951. Malpaux B., Thiry JC. Chemineau P. 1999. Melatonin and the seasonal control of reproduction. Reproduction Nutrition Development. 39 355-366. Malpaux B., A. Daveau, F. Maurice, V. Gayard JC. Thiery, 1993. Short days effects of melatonin on LH secretion in the ewe : evidence for central sites of action in the mediobasal hypothalamus. Biol. Reprod. 48: 752-760. McDaniel J.B., Kreider J.L. Thrasher D.M. 1979. The influence of artificial light and a nutritional supplement on the onset of the breeding season in mares. Journal of Animal Science Supplement. 1: 141-145. McManus CJ. Fitzgerald BP, 2000. Effects of a single day of feed restriction on changes in serum leptin, gonadotropins, prolactin, and metabolites in aged and young mares. Domest Anim Endocrinol. 19: 1-13. Nagy P., Huszenicza Gy., Juhasz J., Solti L. Kulcsar M. 1998. Diagnostic problems associated with ovarian activity in barren and postpartum mares early in the breeding season Reproduction in Domestic Animals 33 187-192. Nishikawa Y., 1959. Studies on reproduction in horses. Jap. Racing Ass., Shiba Tamuracho Minatokou, Tokio. Palmer E, Driancourt MA Ortavant R. 1982. Photoperiodic stimulation of the mare during winter anoestrus Journal of Reproduction and Fertility 32: 275-282. Palmer E. Driancourt MA. 1981. Photoperiodic stimulation of the winter aneostrous mare: what is a long day? In Photoperiodism and reproduction in vertebrates (International Colloquium No 6) pp 6582 Eds R Ortavant, J Pelletier J-P Ravault. Intitut National de la Recherche Agronomique, Versailles (France). Palmer E. Driancourt MA. 1983. Some interactions of season of foaling, photoperiod and ovarian activity in the equine Livestock Production Sciences 10: 197-210. Palmer E. Guillaume D. 1992. Photoperiodism in the equine species -- what is a long night? Animal Reproduction Science 28: 21-30. Peltier MR., Robinson G. Sharp DC. 1998. Effects of melatonin implants in pony mares. 2. Long-term effects. Theriogenology. 49: 1125-42. Scraba ST. Ginther OJ. 1985. Effects of lighting programs on onset of the ovulatory season in mares. Theriogenology. 24: 667-679. Sharp DC., Vernon MW. Zavy MT. 1979. Alterations of seasonal reproductive patterns in mares following superior cervical ganglionectomy. Journal of Reproduction and Fertility Supplement 27: 87-93.

Skinner DC. Malpaux B. 1999. High melatonin concentrations in the third ventricular cerebrospinal fluid are not due to galen vein blood recirculating through the choroid plexus. Endocrinology. 140: 43994405. Stankov B., Cozzi B., Lucini V., Fumagalli P., Scaglione F. Fraschini F. 1991. Characterization and mapping of melatonin receptors in the brain of three mammalian species: rabbit, horse and sheep. Neuroendocrinology. 53: 214-221. Tricoire H., Moller M., Chemineau P. Malpaux B., 2003. Origin of cerebrospinal fluid melatonin and possible function in the integration of photoperiod. Reprod. Suppl. 61: 311-321. Van Niekerk CH. Van Heerden JS. 1972. Nutrition and ovarian activity of mares early in the breeding season Journal of the South African Veterinary Medical Association. 43: 351-360. Van Niekerk FE. Van Niekerk CH. 1997. The effect of dietary protein on reproduction in the mare. III. Ovarian and uterine changes during the anovulatory season, transitional and ovulatory periods in the non-pregnant mare. Journal of the South African Veterinary Medical Association. 68: 86-92.

THE PERIPARTURIENT MARE: PROBLEMS AND MANAGEMENT


Patrick McCue, DVM, PhD, Diplomate American College of Theriogenologists Equine Reproduction Laboratory Colorado State University Fort Collins, Colorado 80525, USA

UTERINE TORSION
A uterine torsion refers to the twisting of the uterus in a clock-wise or counterclock-wise direction as viewed from behind the horse. The rotation occurs in either direction with approximately equal frequency. The torsion can be from 180o to 540o. A majority of uterine torsions occur between 8 and 11 months, although torsions have been described from 4 months to term. There is no age or breed predilection in the horse. In general, uterine torsions are infrequent in the mare. Uterine torsions have been reported to account for approximately 3 % of cases of colic in pregnant mares and 5 to 10 % of serious equine dystocias. The cause(s) of uterine torsion in the mare are not known. It has been suggested that a combination of vigorous fetal movements and rolling by the mare may lead to torsion. The most common clinical sign of uterine torsion in a mare is mild to moderate, chronic or intermittent abdominal pain. The severity of pain expressed by the mare is related to the degree of uterine torsion or the concurrent involvement of the gastrointestinal tract. Diagnosis of uterine torsion is made by palpation of the reproductive tract per rectum. The broad ligaments in the normal mare are located symmetrically in the abdomen. With a uterine torsion, the broad ligaments spiral in the direction of the torsion. One broad ligament passes across the dorsal aspect of the uterine body and the opposite broad ligament passes ventrally as the uterus rotates. The degree of torsion is indicated by the amount of tension on the broad ligaments. A visual or digital examination of the vaginal cavity may not be helpful as only about 4 % of equine cases involve the cervix or cranial vagina. Once a diagnosis of uterine torsion is made, immediate intervention and correction is necessary to optimize the chances of fetal and maternal survival. Treatment options include manual repositioning and surgical repositioning. The choice of techniques is dictated by stage of pregnancy, severity and duration of clinical signs, economics and the expertise and facilities available. If the mare is several weeks from her projected foaling date, a standing flank surgery or a rolling technique may be optimal. The flank surgery is usually done on the side toward which the uterus is rotated. For example, if the torsion is in a clock-wise direction, the incision is typically made on the right flank. The torsion is corrected by gently grasping the uterus ventrally and with repeated rocking movements eventually flipping the uterus over into a normal position. The technique of rolling a mare to correct a uterine torsion can be successful if performed correctly. However, it should not be used if the mare is near term because of the increased risk of uterine rupture. The mare is anesthetized and laid on the ground with the side toward which the torsion is directed down. For example, if the torsion is in a clock-wise direction, the right side of the mare would be placed down. Ropes are attached to the distal limbs and the mare is rapidly turned over in the direction of the torsion. The principle of the rolling technique is that inertia of the pregnant uterus maintains the fetal position constant while the maternal position changes. A modification of the rolling technique utilizes a wooden plank to stabilize the uterus while the mare is rolled. The mare is positioned as previously described. A large plank (approximately 2 in. by 10 in. by 10 ft.) is laid across the abdomen of the mare and a person sits on the middle of the board. The mare is slowly rolled over while

the plank is maintained in position on the abdomen. The rolling is continued in the same direction until the mare is back in her original position. The position of the uterus should be evaluated by palpation after each 360o rotation. The rolling techniques may have to be repeated several times to successfully correct the torsion. When performed correctly, rolling is a practical and less expensive alternative to surgery. If the mare is at term and the cervix is dilated, the torsion may be corrected manually using a vaginal approach or by a ventral midline surgery. The vaginal approach for manual correction of a uterine torsion in a term mare can only be done if the cervix is dilated and not involved in the torsion. An arm is inserted into the uterus via the cervix and the fetus is grasped. Rocking movements are again used to detorse the uterus. If successful, the mare should start the second stage of labor spontaneously or labor should be induced. An alternative procedure for the term mare is a ventral midline surgery. The mare is anesthetized and placed in dorsal recumbancy. This approach allows for good access to the uterus for detorsion and a cesarean section, if necessary. A cesarean is indicated when the torsion cannot be corrected by rolling or when the fetus is dead and cannot be delivered by vaginal extraction. Complications include rupture of the uterus, premature separation of the placenta, abortion, retained placenta, peritonitis and endotoxic shock. The prognosis for survival and future fertility of the mare is good if the torsion is diagnosed and corrected in a timely manner. The prognosis is poor if the torsion is extensive and treatment is delayed due to occlusion of the blood vessels supplying the uterus. Uterine rupture may lead to visceral herniation, peritonitis, hemorrhage, shock and death.

PERIPARTURIENT HEMORRHAGE
Rupture of one of the major arteries supply blood to the uterus or ovaries is the major cause of death in mares in the period immediately after foaling. Blood vessels most commonly involved include the middle uterine, utero-ovarian, and external iliac arteries. The incidence of arterial rupture is highest in older mares, and an association between age, vascular degeneration and declining serum copper levels has been reported. The cause of rupture has been suggested to be a combination of factors such as stretching of the blood vessels by the gravid uterus, pressure associated with abdominal straining and uterine contractions during foaling. A majority of uterine artery ruptures occur on the right side. Two clinical scenarios may develop based on location and extent of the hemorrhage. Rupture of an artery within the broad ligament will often result in hemorrhage that is contained within the broad ligament or between the myometrium and serosal surface of the uterus. Affected mares develop a large hematoma within the broad ligament and exhibit significant abdominal pain, but many survive. In contrast, arterial rupture not contained within the broad ligament leads to rapid exanguination into the peritoneal cavity, shock and death. Clinical signs exhibited depend on location of the hemorrhage and amount of blood lost. Affected mares may show severe colic and profuse sweating. Evidence of hemorrhagic shock such as pale mucous membranes, increased pulse and respiratory rate, low body temperature, circling/staggering, weakness and prostration may become evident. Diagnosis can often be made based on clinical signs alone. However, diagnostic tests such as abdominocentesis, transabdominal ultrasonography, and periodic assessment of packed cell volume (hematocrit) and total protein levels may be valuable in determining the location and extent of the hemorrhage so that a treatment strategy and prognosis can be given. Palpation of

the uterus and broad ligament per rectum may be contraindicated due to the possibility of disrupting a clot and causing further hemorrhage. Mares should be confined to a darkened stall to limit activity and prevent excitement. It is generally recommended that the foal remain with the mare to minimize anxiety in the mare. Analgesics, such as flunixin meglumine (Banamine) and butorphanol tartrate (Torbugesic) may be administered to control pain. Tranquilization of mares with arterial ruptures should be performed with caution since many drugs will decrease blood pressure and accentuate hypovolemic shock. Corticosteroids may be administered for shock. Transfusion of whole blood should be considered when clinical and laboratory signs of severe blood loss are present. Administration of plasma and hypertonic saline and/or isotonic fluids may also be indicated. Additional medications such as naloxone hydrochloride, aminocaproic acid and ergonovine maleate may be administered to improve circulation and control hemorrhage. Administration of oxytocin to promote uterine involution is controversial, as uterine contractions may disrupt blood clots and reinitiate bleeding. Surgical intervention is usually not attempted due to the medically compromised status of affected mares and the difficulty in locating the source of the hemorrhage. Hematomas that remain contained within the broad ligament usually regress over a few weeks to months. Mares that have experienced an artery rupture and survived are usually fertile once the hematoma regresses and can usually deliver a foal without recurrence of hemorrhage. However, owners should be advised that fatal artery ruptures during a subsequent foaling have been documented in mares that survived a previous artery rupture episode.

PLACENTITIS
Placentitis, or inflammation of the placenta, is most often due to bacterial or fungal infection. Fetoplacental infection is the most common cause of abortion, stillbirth or perinatal death in the horse, accounting for approximately 30 % of all cases. The most common bacterial organisms associated with placentitis are Streptoccus spp., Escherichia coli and Leptospira spp. The most common organism causing fungal placentitis is Aspergillus spp. Bacterial and fungal organisms generally spread to the placenta by ascending through the cervix. Ascending infections may be acute and spread rapidly throughout the chorioallantois or chronic and remain localized around the cervical star and adjacent area of the chorioallantois. Acute bacterial infections of the placenta usually cause rapid death of the fetus and a subsequent abortion. In contrast, chronic placental disease results in placental insufficiency and a compromised maternal-fetal exchange of nutrients, resulting in a small, emaciated fetus. Both acute and chronic infections may cause a localized thickening of the placenta in the area of the cervix. Bacterial placentis often results in a thickened, ulcerated placenta coated with a fibrinonecrotic exudate. Mycotic infections are characterized by a thickened chorioallantois covered with a brown, viscid exudate that contains fungal hyphae. Placentitis can also be caused by bacteria that gain access to the placenta via the blood vascular system of the mare or by infectious organisms already in the uterus at the time of conception. A common sequelae to all forms of placentitis is abortion. Clinical signs of placentitis in the pregnant mare may include a cervical or vaginal discharge and an increase in the combined thickness of the uterus and placenta (CTUP) on transrectal or transabdominal ultrasound examination. An increased thickness has been associated with placental pathology. A definitive diagnosis of placentis can be made by examination of the fetus, fetal membranes and mare after an abortion or parturition Prevention and management of placentitis should begin with strict hygiene at breeding and elimination of any persistent endometritis after breeding. If a pregnant mare is diagnosed

with placentitis, a prolonged course of systemic antibiotic therapy should be initiated, along with nonsteroidal anti-inflammatory drugs (NSAIDs) and supplemental progesterone. Broadspectrum antibiotics should used initially until culture results are obtained from the cervical or vaginal discharge. The antibiotic can be changed once and organism and its antimicrobial sensitivity have been detemined. The NSAIDs are administered to decrease uterine inflammation and reduce uterine prostaglandin release. Elevated prostaglandin levels associated with placentitis and endometritis may stimulate uterine motility and induce premature delivery of the fetus. The efficacy of supplemental progesterone administration to maintain pregnancies in high-risk mares is controversial. Progesterone may be beneficial by inhibiting uterine contractility, decreasing uterine prostaglandin production and promoting closure of the cervix. However, the use of supplemental progesterone may be deleterious in mares with active septic processes. Stall rest to reduce movement and stress may also be beneficial. Some mares do not tolerate separation from the herd and isolation in a stall or small paddock. In that case, a companion should be placed into the paddock with the high-risk mare to reduce the separation anxiety.

RETAINED FETAL MEMBRANES (RETAINED PLACENTA)


Retained placenta is the most common problem in the early postpartum period in the mare. The percentage of broodmares that retain their placenta ranges from 2 to 10.6 %. The equine placenta is considered to be abnormally or pathologically retained in the mare after 3 hours postpartum. Retention may include the entire chorioallantois or only a portion of the chorioallantois. The most common site of partial retention is the tip of the nonpregnant horn. Retention of the placenta is associated with delayed uterine involution. Autolysis of the placenta promotes rapid bacterial growth within the uterine lumen, which may lead to toxic metritis. Toxic metris is characterized by accumulation of bacteria, toxins and inflammatory fluid within the uterine lumen. The uterine wall may be thin and is often necrotic. Absorption of toxins through the disrupted endometrium may lead to endotoxemia and laminitis. The most common bacteria cultured from mares with a retained placenta is Streptococcus zooepidemicus although infection with gram-negative bacteria capable of endotoxin production may be more clinically important. The incidence of retained placenta increases following abortion, dystocia, obstetrical manipulations, cesarean surgery, induction of parturition, placentitis, fescue toxicity, hydrops, and other factors that alter pregnancy or foaling. Retained placentas and serious sequelae are more common in draft horses than in light horse breeds. Mares that have had a retained placenta previously have an increased chance of having another. The cause(s) of placental retention are unknown, but have been hypothesized to be inadequate release of oxytocin or inadequate response of the myometrium to oxytocin. As a consequence, areas of chorioallantois near the tip of the nonpregnant horn fail to separate from the endometrium. Diagnosis of retained placenta is obvious if fetal membranes are visible protruding from the vulva. However, diagnosis may not be obvious if only a portion of the fetal membranes are retained within the uterus. Consequently, if the fetal membranes are examined after foaling and an area of tissue appears to be missing, a thorough examination of the uterus is indicated and appropriate therapy initiated. A fetid, red-brown vaginal discharge may be present. A retained placental horn tip can often be visualized on ultrasound examination as a piece of echogenic tissue floating in fluid within the uterine lumen. Systemic signs of illness are unusual early in the time course of a retained placenta. However, if the placenta has been retained for more than 12-24 hours affected mares may exhibit depression, anorexia, and an elevated body temperature.

Strategies for the management of a retained placenta vary with the duration of retention and health of the mare. Initial treatment often involves administration of oxytocin, either as low-dose intramuscular or intravenous boluses (5-20 Units) or as a prolonged intravenous infusion. Manual removal of a retained placenta is not usually recommended. Manual removal has been associated with severe hemorrhage, placental tearing, delayed uterine involution, endometrial damage, intussusception of a uterine horn and uterine prolapse. If initial oxytocin therapy is unsuccessful and the allantochorion is intact, a large volume (9-12 liters) of fluid (sterile saline or water with a small amount of povidone-iodine solution added) may be infused into the allantoic space. Distension of the allantoic cavity induces oxytocin release and subsequent uterine contractions and also stretches the endometrium facilitating the release of placental microvilli. The placenta may be expelled within 5-30 minutes following the fluid treatment. A single small piece of retained chorioallantois may often be successfully removed by application of gentle manual traction. Oxytocin therapy and uterine lavage may also be required. If the entire chorioallantois or a portion of the chorioallantois remains retained > 12 hours after foaling, additional medical treatments and management practices should be instituted to prevent toxic metritis and laminitis. Treatments may include systemic administration of broad-spectrum antibiotics (i.e. penicillin and gentamicin) and intrauterine infusion of antibiotics or antiseptics to prevent bacterial growth, oxytocin and uterine lavage to promote uterine involution and remove bacteria, debris and inflammatory fluid from the uterine lumen, nonsteroidal anti-inflammatory drugs (i.e. Banamine) to prevent endotoxemia and laminitis, and acepromazine and/or topical nitroglycerine (2 %) to promote peripheral vasodilation and prevent laminitis (Table 8.2). Frog support and housing in a deeply bedded stall are also recommended to prevent laminitis. Additional therapy may include administration of anti-endotoxin hyperimmune plasma and polymixin B to prevent endotoxemia, tetanus vaccination and possibly a Caslicks vulvoplasty to limit aspiration of air into the uterus. Other therapies for retained placenta have included administration of propranolol, a beta blocker and injection of the umbilical artery with collagenase. Further research is needed before routine use of the latter therapies can be recommended. Treatment should be instituted early, since complications such as toxic metritis and laminitis can be severe and life threatening. Mares with a history of retained placenta should be treated with oxytocin within 1-2 hours after foaling. Prognosis for mares with retained placenta is generally favorable, but decreases if retention is prolonged or treatment is delayed.

Table 1. Treatment protocol for management of retained placenta in the mare.

TREATMENT Oxytocin 5-20 IU, IM or IV every 1-2 hours up to 8 hours 100 IU in 1 liter of saline, IV, over 30-60 minutes Infusion of 9 to 12 liters of sterile fluid into allantoic cavity Uterine lavage Intrauterine antibiotics Systemic treatment Systemic antibiotics (i.e. penicillin and gentamicin) Nonsteroidal anti-inflammatory drugs (i.e. flunixin Banamine) Acepromazine and/or topical nitroglycerine (2%) Frog support pads Deep bedding in stall Tetanus vaccination Other considerations Hyperimmune anti-endotoxin plasma Polymixin (3 million Units, IV) Caslicks vulvoplasty UTERINE PROLAPSE
Prolapse of the uterus refers to displacement of the uterus outside of the vulva. Uterine prolapse is relatively rare in the mare and is most likely to occur within hours after foaling. However, prolapse may occur several days later. Factors that predispose mares to uterine prolapse include dystocia, vaginal trauma with associated straining or tenesmus, and retained placenta. Management of a mare with a uterine prolapse should be directed initially at preventing rupture of large uterine blood vessels and secondarily with replacement of the uterus into the abdomen. The mare should be kept restrained and quiet until veterinary help arrives. Straining should be decreased by administration of sedation (i.e. xylazine and butorphenol), a caudal epidural or general anesthesia, as needed. The uterus should be elevated to the level of the vulva to prevent further damage, relieve tension on the broad ligament and uterine blood vessels, improve circulation and reduce edema. The tail should be wrapped and the perineum and the exposed surface of the uterus cleaned with soap and water. Any placental remnants that have remained attached should be removed. Endometrial laceration should be closed with absorbable sutures if required. Topical application of sugar to the endometrium has been reported to aid in fluid loss and decrease the size of the uterus and allow for easier reduction. Obstetrical lubricant or petrolatum jelly may be applied to the endometrium and the uterus gently kneaded back through the vagina. Care must be taken to make sure that a section of intestine or the urinary bladder is not entrapped within the prolapsed uterus. Once the uterus has been replaced, the lumen may be distended with sterile saline to ensure that the horn tips are fully replaced. The fluid in the uterus is then allowed to exit by gravity flow.

meglumine,

The position of the horn tips should subsequently be confirmed by palpation per rectum. Failure to completely evert the uterine horns into a normal position may result in discomfort, straining, and recurrence of the prolapse. If fluid therapy is unsuccessful, administration of acepromazine may cause sufficient uterine relaxation to allow for eversion of the horn tip during fluid infusion. Low dose oxytocin therapy should then be initiated to promote uterine involution. Mild, controlled exercise may be beneficial in repositioning the uterus. Additional therapy may include broad spectrum systemic antibiotics, intrauterine antibiotics, nonsteroidal anti-inflammatory drugs, tetanus prophylaxis, and a Caslicks vulvoplasty in mares with poor vulvar conformation. Complications include internal hemorrhage, incarceration and ischemic damage of intestines, endometritis/metritis, septicemia, endotoxemia, laminitis and death. However, if treated promptly, mares generally recover and can be rebred the same season.

PARTIAL INVERSION OF A UTERINE HORN


A single uterine horn tip may become inverted as a result of straining secondary to retained placenta or due to excessive traction on a piece of retained placenta. Inversion of a uterine horn may progress to complete uterine prolapse. Clinical signs include abdominal discomfort (mild colic) and straining within a few hours after foaling that is unresponsive to analgesics. Diagnosis is based on palpation of the tips of the uterine horn per rectum and palpation of the uterine lumen per vagina. Palpation per rectum will reveal a uterine horn that is shorter than normal and extremely thickened. Palpation per vagina will reveal a domeshaped inverted tip of the horn projecting into the uterine lumen. Eversion of the effected horn can be accomplished by one of several techniques. Gentle manual pressure can be used from within the uterine lumen to slowly evert the horn. If the mare is not large and the person has a long enough arm they may be able to reach completely to the horn tip. Alternatively, if the uterine horn tip is out of reach a clean 1-liter plastic bottle can be gently guided up the horn (bottom of the bottle first) once the process has been started manually to accomplish full eversion. A third option is to infusion several liters of fluid into the uterine lumen to accomplish eversion. Oxytocin should be administered to promote involution and the fluid drained from the uterus as it contracts. Nonsteroidal antiinflammatory drugs may be administered to relieve discomfort. If untreated, the invaginated uterine horn tip may become necrotic. Surgical resection of a portion of the uterus may subsequently be indicated.

ABRASIONS, LACERATIONS AND HEMATOMAS OF THE VAGINA AND VULVA.


Vaginal and vulvar injuries may occur during an unassisted foaling or during obstetrical procedures. Small to moderate lesions will usually resolve within a few days without treatment. Deeper lacerations or bruising may result in formation of hematomas that may require drainage 7-10 days later. Severe lacerations of the vagina may result in extensive scarring and formation of permanent adhesions. In addition, severe cranial vaginal injuries may lead to herniation of intestine into the vagina.

SAVING FOALS: THE VETERINARIANS ROLE IN EDUCATION OF HORSE OWNERS AND FOALING ATTENDANTS
Patrick McCue, DVM, PhD, Diplomate American College of Theriogenologists Equine Reproduction Laboratory Colorado State University Fort Collins, Colorado 80525, USA

The inherent economic value of foals has changed management practices regarding foaling and routine care of the newborn foal on many breeding farms. It is common practice to house mares in stalls with sophisticated camera systems, test the blood of the late-term pregnant mare to screen for the presence of anti-red blood cell antibodies, monitor milk calcium levels to predict foaling, utilize a labor alert device to indicate the onset of active labor, and to employ an on-site foaling attendant. Colostrum quality is evaluated immediately after foaling and the placenta is examined for presence of lesions. Newborn foals are monitored for suckling from the mare and passage of meconium. Enemas may be routinely administered to all foals within 1 to 2 hours of birth or only if straining to pass meconium is observed. Veterinarians have pre-packaged acetylcysteine enemas available for treatment of refractory meconium impactions. A blood sample is collected from the foal at 12 and/or 24 hours of age to evaluate passive transfer of colostral antibodies. On many farms, foals are routinely administered hyperimmune plasma at birth and possibly again 21 to 30 days later to prevent occurrence of endemic diseases, such as Rhodococcus equi. Veterinarians should be aware of the latest diagnostic and therapeutic options for broodmares and foals in order to provide management options for horse owners. Prediction of foaling based on changes in calcium concentrations in mammary secretions has been used successfully for many years. Calcium concentration in milk increases sharply as the mare approaches the time of foaling. Calcium levels above 40 mg/dl or 200 ppm indicate that the mare has a high probability of foaling within the next 48 hours. Conversely, mares with a milk calcium level of less than 40 mg/dl are unlikely to foal in the next 24 hours. Labor alert devices are intended to signal an owner or foaling attendant that the mare is in the process of giving birth. Devices are available that can be sutured to the vulva of the mare, attached to a halter or worn around the girth area. None of the labor alert devices are free of inherent problems associated with false alarms or failure of the system to be activated during labor. Labor alert devices may be most effective in allowing farm personnel to go about their routine chores or business during the daytime when one does not necessarily expect a mare to foal. Veterinarians should educate horse owners and foaling attendants as to when to intervene during a problem foaling, when to call for professional assistance, and how to attend to the immediate needs of the newborn foal. A foaling kit, emergency contact list, telephone, and emergency intervention protocol should be readily available. Personnel involved with foaling should be instructed on the ABCs of foal resuscitation (Airway, Breathing, and Circulation) and how to use a manual resuscitation device (i.e. Ambu bag and mask or a Foal ResuscitatorTM). The umbilical stump should be disinfected immediately after the cord has ruptured with a diluted chlorhexadine solution (i.e. 1:1 dilution with distilled water). Quality of the mares colostrum can be estimated using a Brix (sugar) refractometer to measure refractive index or a colostrometer to measure specific gravity. Both refractive index

and specific gravity are correlated with antibody content of colostrum. Foals born to mares with poor quality colostrum can be supplemented with frozen-thawed colostrum from another mare or administered a colostrum substitute orally within the first few hours of life to prevent failure of passive transfer. Meconium is the first feces of a foal and is comprised of digested amnionic fluid, gastrointestinal secretions, and cellular debris swallowed by the fetus while in utero. Meconium is generally passed within the first 2 to 3 hours after birth. A common initial treatment for foals with a meconium impaction is administration of a commercial sodium phosphate enema. Administration of an acetylcysteine retention enema may be successful in medically relieving refractory meconium impactions. Early testing for antibody levels in a neonatal foal can identify potential cases of failure of passive transfer and allow for early intervention and medical management. It is recommended that a blood sample be collected from a newborn foal approximately 12 hours after birth to evaluate circulating IgG levels prior to closure of the gastrointestinal tract to antibody absorption. If IgG levels are < 400 mg/dl at 12 hours, oral supplementation with frozen-thawed colostrum or a commercial colostrum substitute should be performed. If IgG levels are 400 to 800 mg/dl, the need for intervention and therapy is dependent on potential pathogen exposure and/or the medical condition of the foal. Foals with IgG levels 400 to 800 mg/dl at risk of developing infections may benefit from IgG supplementation, whereas similar foals born into a clean environment with low pathogen exposure potential and good preventive management practices may not need supplemental IgG. Antibody levels of > 800 mg/dl at 12 hours of age indicate that adequate passive transfer of immunoglobulins occurred and no additional testing or intervention is necessary under most management conditions. Testing a foal at 24 hours of age or more will determine the final extent of passive antibody absorption. Foals greater than 24 hours of age identified with FPT require intravenous administration of plasma or a commercial equine IgG preparation to successfully increase blood antibody levels. Although plasma transfusions are commonly performed in foals for disease prevention and medical therapy, oral administration of colostrum early in the first day of life is easier and safer to perform.

ENFERMEDADES RESPIRATORIAS DEL POTRO RECIN NACIDO HASTA LOS 3 MESES DE EDAD
Stephanie Retteg

GENERALIDADES La neumona es la principal causa de mortalidad en los potros hasta los 6 meses de edad y constituye una gran prdida econmica en la industria equina debido a la alta morbilidad y mortalidad, a los altos costos por tratamiento y profilaxis, retardo en el crecimiento y prdida en el valor del potro (Wilson, 1997). Generalmente las causas infecciosas y no infecciosas deben tomarse en consideracin, pero los agentes infecciosos representan la causa principal de problemas respiratorios en potros. Los virus y bacterias son los patgenos principales, siendo las infecciones virales consideradas como la causa primaria y que en muchas ocasiones es seguida de una infeccin bacteriana (Wilson, 1997). Existen varios factores predisponentes que conllevan a neumonas infecciosas incluyendo una alta temperatura ambiental, condiciones ambientales secas, irritantes ambientales, sobrepoblacin, parasitismo y otros factores que impiden la efectividad del mecanismo de defensa del sistema respiratorio (Lakritz et al., 1993). La mayora de las infecciones que causan neumonas en potros son adquiridas por inhalacin de los patgenos ambientales. Los agentes infecciosos suspendidos en el medio ambiente tienden a depositarse en la mucosa del tracto respiratorio en la unin broncoalveolar (Wilson, 1997). NEUMONAS NO INFECCIOSAS Aspiracin de meconio Asfixia en tero o una compresin prolongada del cordn umbilical resulta en evacuacin de meconio al interior del lquido amnitico. Estrs fetal y asfixia es reconocido al ver el potro y placenta teidos con meconio. Schoon y Kikovic (1987) sugirieron que la aspiracin de meconio puede deberse tambin a una placentitis bacteriana, debido a hipoxia uterina resultando en una disminucin en la perfusin placentaria. Normalmente, en un potro con aspiracin de meconio se puede observar una secrecin nasal de un color caf-amarillento. El meconio aspirado obstruye las vas respiratorias resultando en aire atrapado y atelectasia, tambin puede causar una irritacin qumica, bronconeumona y muerte. Los sonidos pulmonares, en la auscultacin pueden escucharse normales o estar aumentados en cuanto a intensidad, as como la frecuencia respiratoria (Paradis, 1999). En la radiografa torcica se puede observar un infiltrado granular as como consolidacin pulmonar en la regin caudo-ventral (Beech, 1985). En el anlisis de gases arteriales es posible obtener valores normales, as como tambin pueden observarse valores que indiquen hipoxia o acidosis respiratoria (Paradis, 1999). En casos severos, se puede observar atelectasia pulmonar, indicando una falla en la distensin pulmonar causado por una obstruccin mecnica y el efecto qumico que causa el meconio al surfactante. Microscpicamente se observa meconio y queratina en bronquios, bronquolos y alveolos, acompaado de alveolitis caracterizada por infiltracin de neutrfilos, macrfagos y ocasionalmente clulas gigantes multinucleadas, as como membranas hialinas (Schoon y Kikovic, 1987; McGavin et al., 2007).

Aspiracin de leche La aspiracin de leche se debe normalmente a un defecto o a un desplazamiento del paladar blando. El defecto es una falla del paladar blando al separar la orofaringe y la nasofaringe. El paladar hendido es una malformacin del paladar blando y ocasionalmente del paladar duro, habiendo un defecto en la unin formando una hendidura. La fusin ocurre en direccin de rostral a caudal y es por eso que los defectos ocurren con ms frecuencia en el paladar blando que en el paladar duro o en una combinacin de ambos (Almiar y Morris, 1993). El desplazamiento puede deberse a una flacidez en el paladar blando y la inhabilidad del potro de colocar el paladar nuevamente en su posicin normal al momento de la deglucin (Almiar y Morris, 1993). Ambos problemas, resultan en una aspiracin de leche, observndose leche en ollares despus de mamar. En la auscultacin se puede escuchar ligeros estertores en pulmn. En la radiografa torcica se observa consolidacin pulmonar en el lbulo accesorio (Paradis, 1999). Sndrome de estrs respiratorio del neonato El sndrome de estrs respiratorio del neonato es una condicin caracterizada por una falla en el intercambio gaseoso a nivel alveolar en el neonato (Paradis, 1999). Esta enfermedad esta caracterizada por un aumento en la permeabilidad y edema pulmonar. Clnicamente es reconocido por un aumento en el esfuerzo respiratorio, taquipnea, cianosis y es refractario a terapia de oxgeno. En la fase exudativa, hay dao en la barrera epitelial de los alveolos que conlleva a un edema alveolar y se puede manifestar radiogrficamente con un patrn alveolar-intersticial (Paradis, 1999). Los potros tambin puede mostrar hiperfibrinogenemia 9 (fibringeno > 4.0 G/L; rango de referencia [rr]: 2-4 G/L), y leucocitosis (leucocitos > 13 x 10 9 por l de sangre; rr: 6-12 x 10 por l de sangre). En la necropsia se observan pulmones aumentados de tamao y no se colapsan al momento de abrir la cavidad torcica. Los hallazgos histolgicos que se pueden observar incluyen congestin, edema intersticial, formacin de membranas hialinas, fibrosis intersticial y proliferacin de neumocitos tipo II. Esto resulta en la produccin de exudados intraalveolares e intersticiales que consisten en lquido y debris celular (Lakritz et al., 1993). NEUMONAS INFECCIOSAS Infecciones bacterianas Existe evidencia que las neumonas en potros mayores (> 1 mes de edad) son causadas por bacterias residentes del tracto respiratorio superior, siendo gram positivas; y las neumonas en neonatos estn asociadas a septicemia causada por bacterias aerbicas gram negativas (Koterba et al., 1984). La ruta de infeccin incluye aspiracin de bacterias vaginales durante el parto, infeccin placentaria, umbilical, nasal, oral o secundario a una mala tcnica de inyeccin (Beech, 1999). Falla completa o parcial de transferencia de anticuerpos maternos puede llevar a una infeccin posparto (Koterba et al., 1984). Signos clnicos, hallazgos patolgicos y mtodos de diagnstico para neumonas por bacterias gram negativas En la mayora de los casos de infecciones en neonatos los signos clnicos pueden ser no especficos. Los signos clnicos de estrs respiratorio pueden o no estar presentes. Los signos clnicos estn ms asociados a septicemia que incluyen debilidad, diarrea, hipotermia, estrs respiratorio, frecuencia respiratoria elevada y ocasionalmente secrecin nasal (Wilkins, 1997). El cambio radiogrfico mas comnmente observado es una opacidad alveolar difusa en la regin crneo-ventral (Lakritz et al., 1993). Bacterias pueden ser aisladas de lavados bronco-alveolares o aspirados traqueales por medio de endoscopa. Otra herramienta de diagnstico es un hemograma en donde se observa hiperfibrinogenemia

y leucocitosis. La mayora de estas bacterias son identificadas post mortem en muestras de pulmn. En la necropsia se aprecian pulmones pesados, aumentados de tamao con fibrosis intersticial, ocasionalmente formacin de abscesos y no se colapsan al momento de abrir la cavidad torcica. Histolgicamente se observa una bronconeumona caracterizada por necrosis epitelial, membranas hialinas y proliferacin de neumocitos tipo II. Tambin se puede observar una infiltracin celular en alveolos e intersticio de macrfagos, linfocitos, neutrfilos y colgena (Lakritz et al., 1993). Bacterias

Gram -

Gram +

Septicemia

Residentes del tracto respiratorio superior

- Escherichia coli - Actinobacillus equuli - Pasteurella haemolytica - Bordetella bronchiseptica - Chlamydophila psittaci

- Streptococcus equi spp. zooepidemicus - Streptococcus equi spp. equi - Streptococcus pneumoniae - Rhodococcus equi

Fig 1. Esquema de bacterias gram positivas (+) y gram negativas (-) causantes de neumonas en potros.

Caractersticas, signos clnicos, hallazgos patolgicos y mtodos de diagnstico para neumonas por bacterias gram positivas STREPTOCOCCUS EQUI SPP. ZOOEPIDEMICUS Streptococcus (S.) equi spp. zooepidemicus es una bacteria gram positiva, -hemoltica que pertenece al grupo Lancefield C. Es un comensal de las tonsilas y mucosa de la nasofaringe que puede llegar a actuar como oportunista e invadir el tracto respiratorio causando rinitis purulenta y bronquitis en potros, y neumonas en caballos de todas las edades (Beech, 1999). Como signos clnicos se observa fiebre, anorexia, depresin y secrecin nasal mucopurulenta. A la percusin de senos frontales, stos indican presencia de moco o lquido. En la auscultacin se puede llegar a escuchar silibancias y estertores resultantes de consolidacin y lquido pleural.

El diagnstico se basa en el aislamiento de la bacteria por medio de lavado bronco-alveolar o aspirado traqueal y posteriormente un cultivo. Histolgicamente se observa una bronconeumona supurativa que incluye tambin una neumona necrotizante con vasculitis y trombosis. STREPTOCOCCUS EQUI SPP. EQUI Es el agente causal de la Papera o Gurma equina altamente contagiosa e infecta el tracto respiratorio superior y ndulos linfticos asociados de los equinos. Como signos clnicos se observa fiebre, depresin, anorexia y secrecin nasal purulenta. Neumona no es comn, pero es una complicacin fatal (Timoney, 2004). Por ultrasonografa se puede llegar a observar una consolidacin nodular a nivel superficial caracterizada por ndulos de diversos tamaos de apariencia hipo o hiperecoica con material de diferentes ecogenicidades. En la radiografa torcica se observan mltiples ndulos opacos difusos por todo el pulmn. Para confirmacin del diagnstico se requiere un cultivo de secreciones nasales, aspirado traqueal o secrecin de un absceso. Tambin es muy til hacerlo por medio de PCR. A la necropsia se observa una consolidacin y los pulmones no se colapsan al momento de abrir cavidad torcica. Histolgicamente los abscesos contienen gran cantidad de neutrfilos y material necrtico. RHODOCOCCUS EQUI Rhodococcus (R.) equi es un coco bacilo pleomrfico, no mvil, facultativo intracelular que causa una neumona piogranulomatosa en potros de uno a seis meses de edad. Los potros se pueden infectar durante los primeros das de vida, pero los signos clnicos no se desarrollan hasta los 30 60 das de vida y no son aparentes por varios meses (Horowitz et al., 2001). Tiene una mortalidad del 10% y una morbilidad del 45% de las neumonas en potros. Como signos clnicos se puede observar anorexia, letargia, fiebre, taquipnea, deshidratacin, estrs respiratorio caracterizado por dilatacin de ollares y aumento de movimiento torcico y abdominal. En la auscultacin pulmonar se pueden llegar a escuchar sonidos aumentados as como estertores y silibancias. Tos y secrecin nasal mucopurulenta pueden ocurrir pero no siempre estn presentes (Prescott y Giguere, 2005). En la radiografa torcica se puede observar un patrn intersticial y ndulos. En la ultrasonografa pulmonar se observan mltiples ndulos hipoecognicos rodeados de una zona hiperecognica. En una muestra sangunea se puede encontrar hiperfibrinogenemia y leucocitosis. Cultivo o PCR del agente es necesario para confirmar el diagnstico por medio de lavado bronco-alveolar o aspirado traqueal. Otra herramienta de diagnstico es la inmunohistoqumica por medio de un anticuerpo especfico contra el antgeno 15 17 kDa (kilodalton) de R. equi (Mab 10G5) que se puede llevar a cabo con muestras de lavado bronco-alveolar, de una biopsia pulmonar, o de una muestra pulmonar post mortem (Takai et al., 1993). A la necropsia se observa una consolidacin difusa con mltiples abscesos que pueden tener un dimetro desde 1 cm hasta 6 o ms centmetros normalmente localizados en la porcin crneo-ventral del pulmn. Los abscesos consisten en reas que contienen material necrtico caseoso, y no cuentan con una cpsula fibrtica. El contenido de los abscesos puede variar desde un material espeso amarillento hasta material granuloso o caseoso. Lesiones similares son observadas en los ndulos linfticos bronquiales.

Histolgicamente se observa una bronconeumona piogranulomatosa. Las lesiones se caracterizan por una infiltracin difusa de clulas inflamatorias en alveolos con acumulacin de bacterias basfilas en el citoplasma de macrfagos y clulas gigantes multinucleadas. reas necrticas centrales se encuentran rodeadas por congestin, edema y clulas inflamatorias. Se pueden observar macrfagos, neutrfilos, material necrtico y moco en el interior de bronquios y bronquolos. Infecciones virales Virus ADN Adenovirus Las infecciones con adenovirus comnmente envuelven el tracto respiratorio y la conjuntiva ocular. Los potros adquieren la infeccin al momento de mamar calostro o leche de la madre infectada, por inhalacin de aerosoles infectados o va secrecin ocular o nasal (Powell, 1991). Como signos clnicos se observa secrecin nasal, disnea, conjuntivitis, pirexia y diarrea. Los potros pueden desarrollar una neumona intersticial que generalmente se resuelve por s sola, si no se complica con una infeccin bacteriana secundaria. Frotis de epitelio nasal o conjuntival revela cuerpos de inclusin intranucleares. Mtodos serolgicos para la confirmacin de la infeccin incluyen neutralizacin, inhibicin de la hemoaglutinacin y prueba de anticuerpos de precipitina (Powell, 1991). A la necropsia se observa el 25% del pulmn con consolidacin y de color prpura, atelectasia, enfisema alveolar predominantemente en la porcin crneo-ventral. Los pulmones no se colapsan al momento de abrir la cavidad torcica. Los bronquios contienen material espeso de color blanco-amarillento. Histolgicamente los bronquios y bronquolos pueden contener un exudado purulento que consiste de epitelio bronquiolar y gran cantidad de leucocitos que pueden contener cuerpos de inclusin intranucleares. Herpes virus equino (EHV) 1, 2, 4 y 5 El EHV1 y EHV4 son alfa-herpesvirus y EHV2 y EHV5 son gamma-herpesvirus. La infeccin del EHV lo puede adquirir el potro va secreciones nasales, fetos abortados o lquido amnitico. La infeccin por EHV5 se puede adquirir ms tarde en la vida. El EHV1 y EHV4 pueden causar una enfermedad respiratoria febril caracterizada por rinofaringitis y traqueo bronquitis. EHV2 causa una queratoconjuntivitis en caballos adultos y enfermedad respiratoria en potros, pero tambin se ha asociado a neumonas, faringitis, fiebre, inflamacin de ndulos linfticos, anorexia, malasia y pobre desempeo. Como signos clnicos en las etapas tempranas de la enfermedad se puede observar una secrecin nasal acuosa la cual puede progresar a una secrecin mucosa blanquecina. Otros signos clnicos incluyen secrecin ocular, letargia y anorexia (Allen 2002). Para la confirmacin del diagnstico es la demostracin del agente en secrecin nasal o leucocitos sanguneos. En cultivo celular EHV1 y EHV4 tienen un efecto citoptico caracterstico formando cuerpos de inclusin acidoflifos intranucleares, mientras que EHV2 tiene un efecto citoptico lento y no forma cuerpos de inclusin. Mtodos de laboratorio incluyen aislamiento del virus, PRC e inmunofluorescencia (Allen, 2002). A la necropsia en infecciones con EHV1 y EHV4 se observan pulmones que no se colapsan al momento de abrir cavidad torcica, con mltiples reas grises o rojo oscuro predominantemente localizadas en la porcin crneo-ventral. Histolgicamente las lesiones observadas van desde una ligera bronquitis supurativa linfo-histioctica con infiltracin

peribronquiolar intersticial de clulas inflamatorias hasta pulmones que contienen una severa acumulacin de histiocitos y neutrfilos con reas de fibrosis intersticial y una bronquiolitis necrotisante. VIRUS RNA Virus de Influenza El virus de influenza equina (EIV) es un Orthomyxovirus que es adquirido por inhalacin y es altamente contagioso. Tiene una morbilidad del 100%. Causa bronquitis y bronquiolitis que son seguidos por una neumona intersticial acompaada de congestin, edema e infiltracin de neutrfilos. El primer signo clnico que se llega observar es una dramtica elevacin de la temperatura corporal (hasta los 41C) seguido por una severa tos seca y descarga nasal serosa que puede pasar a ser mucopurulenta por una infeccin bacteriana secundaria. Otros signos clnicos observados incluyen anorexia, debilidad muscular, depresin, edema en miembros posteriores y aumento del tamao de los linfonodos submandibulares (Cullinane, 2005). Para la confirmacin por laboratorio se llevan a cabo pruebas como aislamientos del virus de una muestra de la nasofaringe por medio de un hisopo o por medio de serologa retrospectiva. En potros que llegan a morir por el virus de la influenza equina se observa necrosis del epitelio bronquial con exudado protenico hacia los espacios alveolares. VIRUS DE LA ARTERITIS VIRAL EQUINA El virus de la arteritis viral equina (EVA) es un togavirus del cual su reservorio principal es el semental (Timoney y McCollum, 1993). Este virus puede causar problemas respiratorios severos o muerte espontnea en potros y aborto en yeguas. La principal va de infeccin es por va nasal por inhalacin de aerosoles infectados. La infeccin tambin la puede adquirir el feto en tero resultando en aborto o al parto, el potro nace vivo pero muy dbil. Como signos clnicos se observa aborto, fiebre, edema, especialmente de miembros posteriores y alrededor de los ojos, secrecin nasal y ocular, anorexia, depresin, urticaria en la tabla del cuello y menos comn tos, diarrea y ataxia. (Powell, 1991). En el semental, es probable observar inflamacin del escroto y prepucio. Una infeccin por EVA es frecuentemente confirmada por medio de la demostracin del virus por seroconversion o por un severo aumento de anticuerpos (Timoney y McCollum, 1993). Tambin se utilizan diferentes pruebas de ELISA. A la necropsia, los pulmones se aprecian con un aspecto hmedo, pesados y con mltiples hemorragias. Microscpicamente se observa una ligera neumona intersticial acompaada de edema severo y hemorragias. Las clulas inflamatorias son predominantemente macrfagos y pocos neutrfilos. En una gran porcin del pulmn los bronquios, bronquolos y alveolos contienen grandes cantidades de membranas hialinas. INFECCIONES PARSITARIAS Parascaris equorum Parascaris (P.) equorum es un nemtodo del intestino delgado de los equinos. La larva migra por el pulmn del potro menor a los 6 meses de edad que es ms susceptible a la infeccin y es la principal fuente en produccin de huevos parasitarios (Clayton, 1985). El ciclo de vida

de P. equorum inicia cuando el huevo que contiene la larva 3 (L3) es deglutido por el potro, stos llegan a intestino y migran por las paredes del intestino delgado hacia la vena porta de ah migran hacia hgado, corazn y pulmones. Una vez en pulmones, al toser, la L3 es expulsada o deglutida con secreciones traqueales y regresa a intestino, la larva madura en duodeno y yeyuno proximal. Los primero huevos parasitarios se pueden observar en heces a partir de los 72 a 110 das post infeccin. En los potros infectados a una edad temprana se puede observar tos, secrecin nasal mucopurulenta y puede llegar a tener una frecuencia respiratoria elevada. A la necropsia se observan focos necrticos y petequias en hgado, pulmones y ndulos linfticos hepticos y traqueo bronquiales. Estos cambios se observan tambin histolgicamente, pero tambin se pueden observar eosinfilos como parte de una neumona granulomatosa asociada a la larva. Otro hallazgo es una alveolitis eosinoflica, perivasculitis, bronquitis y bronquiolitis (McGavin et al., 2007). Los ndulos crnicos consisten en agregados de tejido linfoide con fibrosis y mineralizacin. DICTYOCAULUS ARNFIELDI stos parsitos infectan los pulmones de los equinos. Los burros son considerados el reservorio principal y pueden tolerar grandes cantidades de parsitos sin mostrar signos clnicos (Gilbert, 1992). La larva infectante se desarrolla del huevo en los pastizales, una vez ingeridos, la larva pasa por va linftica del tracto gastrointestinal hacia los pulmones. En algunos casos severos puede observarse tos, elevacin de la frecuencia respiratoria y estertores en pulmones a la auscultacin. En un aspirado traqueal se puede llegar a observar eosinfilos, pero no es diagnstico de parasitismo. A la necropsia es posible encontrar parsitos adultos en la periferia de los bronquios. Se pueden observar reas plidas circunscritas en el parnquima pulmonar en la porcin caudal del pulmn (Colahan et al., 1999). En el centro de las lesiones pueden encontrarse larvas en los pequeos bronquios. Histolgicamente aparte de las larvas, se observan infiltrados linfocitarios. El parsitos adulto no causa mucha respuesta, pero la L3 estimula una reaccin muco purulenta o una bronquitis supurativa eosinoflica crnica. Infecciones por Hongos Aspergillus fumigatus Aspergillus (A.) fumigatus afecta a animales inmunosuprimidos, con tratamientos prolongados con antibiticos o corticosteroides o con un compromiso de la mucosa entrica. La aspergillosis pulmonar es poco comn en caballos, pero se ha asociado a enterocolitis debido a infecciones por Salmonella spp. Como signos clnicos se observan malasia, depresin, frecuencia respiratoria elevada con estertores y reas de consolidacin en la auscultacin. Adicionalmente se puede encontrar tambin fiebre, deshidratacin, taquipnea, hipoalbuminemia, hiperfibrinogenemia y neutropenia (Breshears et al., 2007). El diagnstico es comnmente hecho al momento de la necropsia, dnde los pulmones aparecen aumentados de tamao y firmes a la consistencia con una severa congestin y mltiples hemorragias y neumona piogranulomatosa o granulomatosa. Pequeos ndulos de color blanco de una dimetro de 0.5 a 1 cm estn rodeados de un halo de color rojo oscuro. Existe edema nter lobular y hemorragias, as como edema alveolar con

depsitos de fibrina. Las hifas micticas son aparentes mostrando paredes paralelas y ramificadas. PNEUMOCYSTIS CARINII Pneumocystis (P.) carinii es la infeccin ms comn en humanos con el virus de inmunodeficiencia, y en potros se ha asociado al sndrome de inmunodeficiencia combinado, a administracin de corticosteroides, CD+4 linfopenia e infecciones por R. equi (Rush y Mair, 2006). Los huspedes sanos son capaces de eliminar el agente de los plumones, pero pueden estar evacuando al agente por va nasal e infectar a huspedes inmunocompetentes como son los mamferos neonatos. Despus de la inhalacin del agente, las estructuras uninucleadas se establecen en los alveolos, los cuales despus de una divisin adquieren una apariencia multinucleada con 6 a 8 trofozoitos, los cuales se adhieren a los neumocitos tipo I. Como signos clnicos se observa tos no productiva, disnea, taquipnea y taquicardia. En la auscultacin se puede escuchar estertores y silibancias en todo el campo pulmonar. En el hemograma se puede encontrar leucocytosis, hyperfibrinogenemia y una ligera eosinofilia (Perron Lepage et al., 1999). En casos severos se puede observar mucosas orales cianticas. El anlisis de gases arteriales revela hipoxia e hipercapnia. El diagnstico ante mortem normalmente es realizado por medio de citologas del lquido del lavado bronqueo alveolar. Las laminillas de la citologa, de biopsias o de muestras pulmonares se pueden teir de con diferente tinciones que incluyen tincin de Grocott, tincin azul de toloudina, tincin de Gram, tincin de CEV o tincin de Giemsa. Las tinciones de Grocott, Gram, CEV, y azul de toloudina tien nicamente la pared de P. carinii dejando los trofozoitos y los esporozoitos internos sin teir, mientras que la tincin de Giemsa tie los trofozoitos y los esporozoitos internos dejando la pared sin teir. Con la inmunohistologa usando un anticuerpo especfico contra P. carinii tambin puede ser aplicado en muestras de pulmn, biopsias o citologas. A la necropsia se observan pulmones de consistencia firme y carnosa, aumentados de tamao que no se colapsan al momento de abrir cavidad torcica. En la superficie de corte la parte afectada se encuentra seca y a veces se encuentran focos rosas o amarilloscafs (Rush y Mair, 2006). Histolgicamente se observa una distensin alveolar, los bronquios terminales contienen macrfagos espumosos, material finamente vacuolizado protenico que da la apariencia de panal de abejas que contiene pequeas estructuras basoflicas representando el ncleo de los organismos (McGavin et al., 2007). Los espacios intersticiales son infiltrados por linfocitos y clulas plasmticas. Formacin de membranas hialinas es prominente, as como la proliferacin de neumocitos tipo II (Rush y Mair, 2006). MTODOS DE DIAGNSTICO Ultrasonido El ultrasonido de los pulmones en los potros se puede llevar a cabo con un transductor lineal o sectorial de 5.0 o 7. 5 MHz. Con el transductor de 7.5MHz se puede obtener una profundidad de 4 a 12 cm, la cual es ideal para la examinacin torcica de los potros (Slovis et al., 2005). El rea a evaluar debe incluir ambos lados del trax avanzando de caudal a craneal y de dorsal a ventral desde el decimosexto espacio intercostal hasta el tercer espacio intercostal. El rea a evaluar debe ser previamente rociado con alcohol al 99% o agua caliente para mejorar zona de contacto y se sugiere poner una lnea de gel para ultrasonido en la zona dorsal que se va jalando hacia ventral como se va avanzando para tener una mejor zona de contacto. (Reef. et al., 2004).

Las ondas de ultrasonido son completamente reflejadas en el pulmn sano y slo se observa la pleura visceral que se aprecia como una lnea hper ecoica en la porcin superior de la imagen (Fig. 1). nicamente cuando hay lquido o acumulacin de clulas debajo de la pleura visceral se crea una ventana acstica, observndose una patologa pulmonar. El rea de pulmn afectado aparece como una imagen hipoecoica (Slovis et al, 2005). Las colas de cometa son creadas por la presencia de lquido o infiltrados celulares en la periferia pulmonar. La onda de ultrasonido es transmitida por las reas de lquido o infiltrados celulares y luego se encuentra con el aire que es altamente reflectivo creando stas colas de cometa (Fig. 2a) (Reef, 2004). La consolidacin pulmonar se observa hipoecoica, y se debe a causa de un reemplazo del aire alveolar por clulas o lquido que producen una ventana acstica. Las zonas que tienen una apariencia hipo ecoicas hasta hper ecoicas son variables en tamao, pueden estar localizadas por todo el pulmn, siendo ms frecuentes craneales y ventrales, y son interpretados como abscesos pulmonares. Son identificados por una apariencia cavitatoria y por la ausencia de estructuras pulmonares normales (Fig. 2b). El centro de estas estructuras puede aparecer hipo ecoico o anecoico, dependiendo del tipo de lquido que contiene. La consolidacin nodular en el pulmn es rara vez encapsulado, siendo la excepcin los abscesos causados por R. equi. En stos potros se observa la cpsula ecoica rodeando lquido hipo ecoico a ecoico (Reef., 2004).

Piel

Msculo intercostal Artefactos

Pleura visceral Pulmn sano

Fig. 2: Imagen ultrasonogrfica mostrando las estructuras de un pulmn sano.

Absceso

Cola de cometa

3a

3b

Fig. 3: Imgenes mostrando a: colas de cometa formadas por lquido o infiltracin celular y b: absceso pulmonar de 1.7 cm de dimetro.

Anlisis de gases arteriales El anlisis de gases arteriales es til para definir la severidad del dao respiratorio, y para determinar el tratamiento a seguir. Es complicado administrar soporte respiratorio que sea efectivo y seguro sin llevar a cabo el anlisis de gases arteriales, monitoreando la presin parcial del oxigeno (PaO2) y la presin parcial del dixido de carbono (PaCO2). Arterias recomendadas para la muestra de sangre arterial incluyen la gran metatarsiana, la braquial en el punto donde cruza el aspecto medial del miembro anterior; la cartida y la facial transversa, caudal al cantus lateral del ojo. La muestra es obtenida mientras el potro es mantenido en decbito lateral o esternal. El sitio de puncin es desinfectado mediante el uso de antispticos. Es recomendado administrar anestesia local de forma subcutnea con lidocana al 2% en el sitio de puncin. La muestra es recolectada con un tubo Vacutainer con heparina como anticoagulante (Koterba et al., 1984). Edad pos parto Al nacimiento 30 minutos 1 hora 4 horas 12 horas 24 horas 48 horas 4 das pH 7.413 +- 0.019 7.354 +- 0.010 7.362 +- 0.013 7.355 +- 0.017 7,357 +- 0.024 7.393 +- 0.012 7.396 +- 0.008 7.396 +- 0.012 PaCO2 (mmHg) 45.7 +- 1.1 51.5 +- 1.5 47.3 +- 2.2 45.0 +- 1.9 44.3 +- 1.2 45.5 +- 1.5 46.1 +- 1.1 45.8 +- 1.1 PaO2 (mmHg) 39.7 +- 2.1 57.0 +- 1.8 60.9 +- 2.7 75.7 +- 4.9 73.5 +- 3.0 67.6 +- 4.4 74.9 +- 3.3 81.2 +- 3.1

Tabla 2: Valores normales del anlisis de gases arteriales del potro neonato.

LAVADO BRONCO-ALVEOLAR El potro deber de estar tranquilizado y preferentemente de pie. El broncoscopio se pasa por el pasaje nasal hacia la trquea con direccin hacia el lbulo bronquial izquierdo o derecho avanzando hacia los alvolos para as obtener del lavado una muestra para conteo celular. Administrando 200ml de solucin salina estril a una temperatura corporal. El aspirado debe

realizarse con una jeringa y lo obtenido debe almacenarse en hielo y debe ser llevado al laboratorio para su evaluacin. En un estudio se obtuvo que el valor medio de macrfagos en un potro de 2 das de edad fue de 3.9 x 103 +- 1.1, pero en un potro de 14 das de edad los valores se aproximan a los valores de una animal adulto (57.6 x 103). El valor promedio del tipo celular en el lquido obtenido de un potro de dos das de edad es como sigue: macrfagos 84.2%, neutrfilos 10.4% y linfocitos 5.6% (Koterba et al., 1984). TRATAMIENTO En cualquier tipo de enfermedad respiratoria lo ms importante es reposo en estabulacin y tratamiento de soporte. Los potros con neumona deben de mantenerse en un ambiente controlado, evitando temperaturas extremas y humedad. Es necesario una buena calidad de cama, humedecer si es necesario para evitar polvos irritantes. En casos severos dnde el potro no se puede poner de pie, se recomienda rotar al potro de posicin mnimo cada 2 horas. Si se presenta hipotermia es necesario calentarlo por medio de mantas o colocar botellas o bolsas con agua caliente a sus costados. Animales con una hipoxia severa (>65 mmHg), el objetivo principal es aumentar la cantidad de O2 inspirado y disminuir el CO2 en sangre. Ventilacin mecnica con oxgeno suplementario es la forma ms efectiva con 10L/min. con 100% de oxgeno. Tambin se puede suplementar oxgeno por medio de una sonda nasal. El tratamiento para las neumonas en potros es bsicamente basado en un antibitico de amplio espectro o la combinacin de 2 o ms, y tratamiento suplementario para mejorar la condicin general del potro. La combinacin ms utilizada es ampicilina (22mg/kg IV cada 6 hrs.) o penicilina (22000 UI/kg IV cada 6 hrs.) con gentamicina (6.6 mg/kg IV cada 24 hrs.). Tambin se utiliza mucho la combinacin de amicacina (21mg/kg IV cada 24 hrs) con ampicilina. Cefalosporinas de tercera generacin presentan ciertas ventajas antes los aminoglicosidos en el tratamiento de las neumonas en potros. Una combinacin de eritromicina (25mg/kg PO cada 6-8 hrs.) y rifampin (10 mg/kg PO cada 12-24 hrs.) fue un tratamiento muy utilizado en las neumonas por R. equi, en estos das azitromicina (10mg/kg PO cada 12hrs) o claritromicina (7.5 mg/kg PO cada 12 hrs.) son una alternativa para el tratamiento. La resolucin de signos clnicos, normalizacin de niveles de fibringeno y la evaluacin de las lesiones por radiografa o ultrasonografa son utilizados para guiar la duracin del tratamiento que puede ser desde 4 hasta 9 semanas. Es recomendable administrar tratamientos de soporte como broncodilatadores como tesofilina (4-7 mg/kg PO cada 8 hrs), aminofilina (2mg/kg PO), o un 2-adrenrgico como clenbuterol (0.8mg/kg PO); y mucolticos como acetilcistena al 10% (30ml en nebulizador por 30 min cada 12hrs) para mejorar las condiciones respiratorias del potro. BIBLIOGRAFA
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