Animal Reproduction Science 111 (2009) 8092

Embryo production from superovulated

Holstein-Friesian dairy heifers and cows after
insemination with frozen-thawed sex-sorted X
spermatozoa or unsorted semen
J. Peippo a,, K. Vartia b, K. Kananen-Anttila c,1, M. Raty a, K. Korhonen a,
T. Hurme d , H. Myllymaki e , A. Sairanen f , A. Maki-Tanila a
a
MTT Agrifood Research Finland, Biotechnology and Food Research, Building H,
FI-31600 Jokioinen, Finland
b ProAgria Cooperative Breeding Service, FI-76101 Pieksamaki, Finland
c University of Kuopio, Department of Biosciences, FI-70211 Kuopio, Finland
d MTT Agrifood Research Finland, Services Unit, FI-31600 Jokioinen, Finland
e Embryocenter Ltd., FI-01301 Vantaa, Finland
f MTT Agrifood Research Finland, Animal Production Research, FI-71750 Maaninka, Finland
Received 26 June 2007; received in revised form 1 February 2008; accepted 6 February 2008
Available online 13 February 2008

Abstract
The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers
and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose).
Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All
transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial
dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and
unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were
induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 812
of the estrus cycle. Inseminations began 12 h after the onset of estrus and were performed two to four times
at 915 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen
was placed into the uterine body.
In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of
embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively.

Corresponding author. Tel.: +358 3 4188 3617; fax: +358 3 4188 3618.
E-mail address: jaana.peippo@mtt.fi (J. Peippo).
1 Present address: Ovumia Oy, FI-33520 Tampere, Finland.

0378-4320/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.02.002
 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092 81

The proportion of transferable female embryos produced approximately doubled when insemination was
with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%;
cows 81.1% versus 39.8%).
In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5%
of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respec-
tively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows
in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermato-
zoa produced significantly smaller proportions of transferable embryos (p < 0.005) and significantly larger
proportions of unfertilized oocytes (p < 0.001) than those inseminated with unsorted semen. Proportions
of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within
treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated
oocytes/embryos.
It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for
the insemination of superovulated embryo donors can improve the proportion of transferable female embryos
produced but this potential may not be achieved in commercial practice, particularly in cows, because of
reduced fertilization rates when using low doses of X-sorted spermatozoa.
2008 Elsevier B.V. All rights reserved.

Keywords: Cattle; Fertilization; In vivo embryo production; Sex pre-selection

1. Introduction

On dairy farms, the ability to select the sex of offspring of heifers and cows is of great practical
and economic importance. With sex diagnosis, more female calves can be produced for replace-
ments from the best cows of the herd while the average cows and heifers can act as embryo
recipients within the herd or as dams for male beef calves.
Embryo sexing from a biopsy is a routine technique in dairy cattle breeding in Finland. The
procedure is simple and highly accurate even in farm conditions (Bredbacka et al., 1995, 1996).
However, the method is not economically optimal because on average, 50% of the recovered
embryos are of undesired sex and wasted. Also, the survival rate of biopsied embryos is com-
promised compared to that of intact embryos although some embryo transfer groups report good
pregnancy rates after transfer of frozen-thawed biopsied embryos (Hasler et al., 2002).
If female embryos are produced with X-sorted spermatozoa, practically all embryos are
of desired sex and disadvantages of embryo biopsy can be avoided. Due to success in cry-
opreservation of flow-cytometrically sorted spermatozoa, X-sorted spermatozoa are currently
commercially available from selected AI bulls (Seidel, 2003). Published research on sex-sorted
spermatozoa has focused on the use of X spermatozoa for the insemination of non-stimulated
(or non-superovulated) heifers and cows (Seidel et al., 1999; Bodmer et al., 2005; Andersson
et al., 2006; Kurykin et al., 2007; Cerchiaro et al., 2007). A few studies have also reported
in vivo embryo production results in superovulated heifers and cows (Panarace et al., 2003;
Sartori et al., 2004; Schenk et al., 2006). To date, only one study has reported embryo pro-
duction results from commercial embryo transfer collections in cows (Darrow, 2006), although
the top breeding cows of dairy farms are in practice the most wanted donor animals giving
the best profit of using sex-sorted spermatozoa to produce embryos for embryo transfer and
trade.
The aims of this study were to compare embryo production rates and embryo quality in heifers
and cows inseminated with either low doses of X-sorted spermatozoa or high doses of unsorted
82 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092

semen using the techniques and protocols commonly practised with each type of semen. Specific
interests were to evaluate the true sex ratio of transferable embryos at the research farm and the
efficiency of embryo production on commercial dairy farms.

2. Materials and methods

2.1. Source of sperm and inseminations

Flow-cytometrically sorted X spermatozoa from three Holstein bulls were used: Picston Shaker
FFF 93568 B, Cogent Courier ET FFF 93588 C and Overside Dreamer ET FFF 93796 B. Each
frozen X-sorted dose contained 2 million spermatozoa. All sorted semen was purchased from
Cogent Ltd. in Chester, UK.
In control groups, unsorted semen was used. In Experiment 1, the unsorted semen of Picston
Shaker was used. In Experiment 2, unsorted semen of 32 different Holstein AI bulls was used.
Each frozen unsorted dose contained 15 million spermatozoa.
Inseminations were started 12 h after estrus onset was detected (standing heat) and the donors
were inseminated two to four times at 915 h intervals. With X-sorted spermatozoa, both of
the uterine horns were inseminated with single straws of semen at the first insemination. In the
remaining one to two inseminations a single straw was split between the two uterine horns resulting
total dose of 68 million X-sorted spermatozoa per embryo donor. Deep-uterine inseminations
with X-sorted spermatozoa were carried out by AI technicians trained for embryo transfer. In the
control groups, donors were inseminated to the uterine body with unsorted semen two to three
times at 12 h intervals.

2.2. Superovulation of embryo donors

In the both treatment groups, Holstein-Friesian embryo donors were superovulated with eight
declining doses of FSH (Folltrophin, Oriola, Espoo, Finland). Superovulation induction was
started on days 812 of the estrus cycle. In total, 12 and 19 ml of FSH was administrated to
heifers and cows, respectively.

2.3. Experimental designs

In Experiment 1, embryo production was investigated at the MTT research farm using X-
sorted spermatozoa and unsorted semen of a single bull (Picston Shaker). Superovulated embryo
donors were randomly allocated into two groups. Four heifers and four cows were allocated to the
X-sorted treatment group, and four heifers and two cows were allocated to the unsorted semen
(control) group. All transferable embryos were diagnosed for sex by PCR.
In Experiment 2, embryo production from X-sorted spermatozoa was evaluated under farm
conditions with data collected from commercial dairy farms. The data included embryo recoveries
of Holstein-Friesian donors flushed between June 2003 and January 2005 in Finland. Results of
59 embryo flushings with X-sorted spermatozoa using three bulls (Picston Shaker, Cogent Courier
ET and Overside Dreamer ET) at 28 farms were compared to results obtained from 179 embryo
flushings with unsorted Holstein AI bull semen from 32 bulls at 79 farms during the same time
period. The farms were located throughout Finland and were experienced in embryo production
in vivo.
 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092 83

2.4. Embryo recovery, classication and sex diagnosis

Embryos were recovered nonsurgically on day 7 after the first insemination and classified
according to IETS recommendations (Robertson and Nelson, 1998). Statistical analyses were
focused on quality 1 embryos as they are the best candidates for freezing and trade (Hasler et al.,
2002). All transferable embryos from the research farm were diagnosed for sex by PCR using
Y-specific BRY.1 (Peura et al., 1991) and autosomal -casein (Velmala et al., 1993) primers.

2.5. Statistical analyses

In Experiment 1, results for the percentages of transferable embryos, unfertilized oocytes and
degenerated embryos were clear without statistical testing and therefore statistical analyses were
not performed for these variables. Data are presented here as means and standard deviations
(S.D.). Variables for the percentages of quality 1, 2 and 3 embryos were analyzed with a
two-sample t-test performed with the GLM procedure of SAS 9 (SAS Institute, Cary, NC, USA).
In Experiment 2, a mixed model with covariates (Littell et al., 2006) was used to analyze the
data of the percentages of transferable embryos, unfertilized oocytes and degenerated embryos.
Analyses were conducted on percentages, in order to give all the flushes/donors an equal weight.
This is because the number of oocytes ovulated depends on the superovulation success of the
donor. Each farm consisted of 122 inseminated animals that produced embryos. The animals
were divided into donor types. Each donor was flushed one to six times during the study and for
each insemination the treatment (X-sorted spermatozoa or unsorted semen) and the bull (within
treatment) could change. The treatment effect was examined separately for each donor type. The
statistical model was

Yijklmn = + Farmi + Donortypej + Farm Donortypeij + Donork(ij) + Treatmentl

+ Donortype Treatmentjl + Bullm(l) + ijklmn ,

where was constant. Donortypej , Treatmentl , Donortype Treatmentjl and Bullm(l)

were fixed effects. Farmi , Farm Donortypeij , Donork(ij) (identifies an inseminated ani-
mal within ith farm and donor type j) and ijklmn (error term) were random effects.
Random effects were assumed to be mutually independent and normally distributed with
zero means and variances var(Farmi ) = F2 , var(Farm Donortypei ) = FDt2 , var(Donor
k(ij) ) =
D , and var(ijklmn ) = . Correlations between measurements from the same donor animal
2 2

were taken into account using compound symmetry covariance structure. Arcsine transformation
was applied to normalize the data for statistical analyses. The estimated means and confidence
intervals were back-transformed to the original scale of measurement (percents) for the presen-
tation of the results.
Because several animals did not produce transferable embryos, the data for quality variables
(quality 1 and 2) were scarce. For the statistical analyses the data were therefore simplified by
calculating a mean of multiple observations for each animal. This mean was then used as an
experimental unit and the statistical model was modified accordingly. The proportions of quality
3 embryos were not analyzed, because most of the values were zero.
Analyses of Experiment 2 were conducted with the MIXED procedure of SAS 9 (SAS Institute,
Cary, NC, USA) using REML and degrees of freedom were calculated according to Kenward and
Roger (1997). Model assumptions were checked graphically.
84 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092

3. Results

3.1. Embryo production rates, qualities and sex ratios after inseminations with
ow-cytometrically X-sorted spermatozoa and unsorted semen (Experiment 1)

Altogether, eight and six donors were successfully superovulated, inseminated and flushed on
day 7 in X-sorted spermatozoa and unsorted semen groups, respectively. Two other heifers, one in
the X-sorted spermatozoa treatment group and one in the unsorted control group, were originally
included in the experiment but neither oocytes nor embryos were recovered from these animals.
Data from these two heifers were excluded from the numbers of animals and calculation of the
data in this experiment (Table 1).
The four heifers inseminated with X-sorted spermatozoa produced a total of 38 embryos, with
on average (S.D.) 9.5 (5.8) embryos per animal of which 70.3 (17.7)% were transferable
quality. There were total of 25 transferable embryos, including 10 quality 1 embryos. The mean
percentage of quality 1 embryos of transferable embryos was 42.0 (21.3). The other four heifers
inseminated with unsorted semen produced a total of 27 embryos. One of these heifers pro-
duced only one embryo, which was degenerated. The other three heifers produced 26 transferable
embryos of which 18 were quality 1. The mean percentage of quality one embryos of transferable
embryos for these three animals was 59.5 (30.0).
The four cows inseminated with X-sorted spermatozoa produced in total 51 embryos, with
an average (S.D.) of 12.8 (3.0) embryos per animal. Two of these cows failed to produce
any transferable embryos and two produced mostly transferable embryos (100 and 94% of the
embryos were transferable). These two cows produced a total of 24 transferable embryos of
which 13 were of quality 1. For these cows on average 52.2 (11.0)% of transferable embryos
were quality 1. The two cows inseminated with unsorted semen produced a total of 15 (11
and 4) embryos, which were all transferable. In total 10 (8 and 2) of the embryos were of
quality 1.
Because of the above described data distribution and the small number of cows in the control
group, statistical analysis in Experiment 1 was limited to data on the quality of transferable
embryos produced by heifers. The use of X-sorted spermatozoa in heifers did not affect the
proportion of recovered quality 1 embryos (p = 0.40).
On average, 100 and 92.3% of transferable embryos were successfully diagnosed for sex in
heifers, and 79.2 and 100% in cows from X-sorted spermatozoa and unsorted semen groups,
respectively. The proportion of transferable female embryos obtained with X-sorted spermatozoa
was 96.4% for heifers and 81.1% for cows (Table 1). For insemination with unsorted semen the
proportion of transferable female embryos was 41.1% for heifers and 39.8% for cows (Table 1).
Consequently, when only considering those recoveries that produced transferable embryos, X-
sorted spermatozoa produced approximately twice as large proportion of transferable female
embryos compared to unsorted semen.

3.2. Embryo production with ow-cytometrically X-sorted spermatozoa and unsorted semen
on commercial dairy farms (Experiment 2)

Within X-sorted spermatozoa groups neither oocytes nor embryos were recovered from four
heifers and one cow. Within unsorted semen groups neither oocytes nor embryos were recovered
from 14 heifers and nine cows. All these animals were excluded from the numbers of animals and
calculation of the data in Experiment 2 (Tables 2 and 3).
 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092
Table 1
Embryo production from dairy cattle following superovulation and insemination with low-dose X-sorted spermatozoa or normal-dose unsorted semen in Experiment 1 on the
MTT research farm
Inseminations Number of flushes Total number Transferable Unfertilized Degenerated Female embryos
(% 0 flushesa ) recovered/flushb embryos (%)b (of oocytes (%)b embryos (%)b (%)c
quality 1%b )

Heifers
X-sorted spermatozoad 4 (0) 9.5 5.8 70.3 17.7 (42.0 21.3) 15.7 25.2 14.0 14.0 96.4
Unsorted semene 4 (25.0) 6.8 5.6 75.0 50.0 (44.6 38.5) 00 25.0 50.0 41.1
Cows
X-sorted spermatozoad 4 (50.0) 12.8 3.0 48.4 56.0 (26.1 30.8) 28.6 29.8 22.9 26.7 81.1
Unsorted semene 2 (0) 7.5 4.9 100.0 0 (61.4 16.1) 00 00 39.8
a Flushes producing zero transferable embryos.
b Means standard deviations.
c Transferable female embryos calculated from embryo flushings that produced transferable embryos.
d Picston Shaker FFF 93568 B (2 million/dose).
e Picston Shaker FFF 93568 B (15 million/dose).

85
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Table 2
Values for F-test, degrees of freedom and levels of statistical significance for effects of donor type (heifer, cow), treatment
(inseminated with low-dose X-sorted spermatozoa or normal-dose unsorted semen), donor type treatment interaction
and bull (semen donor) in superovulated dairy cattle in Experiment 2 on commercial dairy farms
Effect Transferable embryos (%) Unfertilized oocytes (%) Degenerated embryos (%)

Donor type F1,78.9 = 3.55a , p = 0.06 F1,93.2 = 6.06, p = 0.02 F1,45.7 = 0.39, p = 0.53
Treatment F1,186 = 7.62, p < 0.01 F1,189 = 10.21, p < 0.005 F1,186 = 0.10, p = 0.75
Donor type treatment F1,194 = 3.77, p = 0.05 F1,195 = 3.93, p = 0.05 F1,190 = 0.00, p = 1.00
Bull (treatment) F33,177 = 1.10, p = 0.34 F33,176 = 1.05, p = 0.41 F33,161 = 0.87, p = 0.67
a Notation: in F
A,B = C, A, B and C represent numerator degrees of freedom, denominator degrees of freedom and F-test
statistic, respectively.

In Experiment 2, heifers inseminated with X-sorted spermatozoa produced on average (S.D.)

6.4 (4.2) embryos per recovery of which estimated 53.9% were transferable and 21.1% were
unfertilized oocytes. Heifers inseminated with unsorted semen produced on average 8.6 (6.5)
embryos per recovery of which estimated 65.5% were transferable and 10.6% were unfertilized.
Cows inseminated with X-sorted spermatozoa produced an average 10.4 (9.6) embryo per recov-
ery with estimated 21.1% of these embryos being of transferable quality and 56.0% unfertilized.
Cows inseminated with unsorted semen produced on average 9.4 (6.7) total embryos of which
estimated 64.5% were transferable and 14.4% were unfertilized.
Significant donor type insemination type interactions were observed for the proportion of
transferable embryos and the proportion of unfertilized oocytes (p = 0.05, Table 2). The proportion
of transferable embryos was significantly smaller (p < 0.005) and the proportion of unfertilized
oocytes significantly larger (p < 0.001) for cows inseminated with X-sorted spermatozoa than
for those inseminated with unsorted semen (Table 3). In heifers, the insemination type did not
affect the proportions of transferable embryos or unfertilized oocytes recovered (p = 0.32 and
p = 0.18, respectively, Table 3). No significant differences were observed between the insemination
types (p = 0.75) or between the donor types (p = 0.53) in the proportion of degenerated embryos
recovered (Table 3). Within insemination treatments, the bulls used did not have a significant
effect on proportions of unfertilized oocytes (p = 0.41), transferable (p = 0.34) or degenerated
(p = 0.67) embryos (Tables 2 and 4). Differences were observed between the bulls in estimated
means for unfertilized oocytes, transferable and degenerated embryos, but due to small number
of observations per each bull and high degree of variation, the observed differences were not
statistically significant.
Heifers inseminated with X-sorted spermatozoa produced on average (S.D.) 3.1 (3.2) trans-
ferable embryos of which an estimated 89.1% were quality 1 and 4.8% quality 2 (Table 5). Heifers
inseminated with unsorted semen produced on average 5.7 (5.4) transferable embryos per recov-
ery of which an estimated 87.5% were quality 1 and 7.7% quality 2. Inseminating cows with
X-sorted spermatozoa produced on average 2.1 (2.7) transferable embryos with an estimated
76.7% of these were quality 1 and 7.6% of quality 2. Cows inseminated with unsorted semen
produced on average 6.3 (5.7) transferable embryos, estimated 80.9% of these were of quality
1 and 13.8% of quality 2. Picston Shaker produced on average 3.0 (3.8) transferable embryos
of which an estimated 91.1% were quality 1 and 4.6% were quality 2. Cogent Courier produced
on average 2.9 (2.9) transferable embryos per recovery, of these an estimated 76.2% were qual-
ity 1 and 17.2% were quality 2. Overside Dreamer produced on average 2.0 (1.2) transferable
embryos, of these, an estimated 81.2% were quality 1 and 1.1% were quality 2.
Table 3
Embryo production from dairy cattle following superovulation and insemination with low-dose X-sorted spermatozoa or normal-dose unsorted semen in Experiment 2 on

J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092

commercial dairy farms
Inseminations Number of flushes Total number Transferable Unfertilized oocytes Degenerated
(% 0 flushesa ) recovered/flushb embryos (%)c,f (%)c,g embryos (%)c,h

Heifers
X-sorted spermatozoad 42 (19.0) 6.4 4.2 53.9 [33.8, 73.4] 21.1 [8.3, 37.8] 12.3 [4.4, 23.6]
Unsorted semene 108 (12.0) 8.6 6.5 65.6 [50.2, 79.4] 10.6 [3.6, 20.8] 10.8 [4.9, 18.8]
Cows
X-sorted spermatozoad 17 (41.2) 10.4 9.6 21.1 [4.7, 45.1] 56.0 [33.0, 77.7] 9.6 [1.6, 23.2]
Unsorted semene 71 (9.9) 9.4 6.7 64.5 [49.8, 78.0] 14.4 [6.3, 25.2] 8.3 [3.4, 15.1]
a Proportion of flushes producing zero transferable embryos.
b Means standard deviations.
c Estimated means and their 95% confidence intervals [lower CI, upper CI].
d Low-dose (2 million) inseminations from Picston Shaker FFF 93568 B, Cogent Courier ET FFF 93588 C and Overside Dreamer ET FFF 93796 B at 28 farms.
e Normal-dose (15 million) inseminations from 32 Holstein AI bulls at 79 farms.
f Donor type by treatment interaction was statistically significant (Table 2). Therefore, the difference between treatments was tested separately for cows and heifers. The

difference between treatments was statistically significant for cows (p < 0.005).
g In unfertilized oocytes the donor type by treatment interaction was statistically significant (Table 2). Therefore, the difference between treatments was tested separately for

cows and heifers. The difference between treatments was statistically significant for cows (p < 0.001).
h In degenerated embryos neither the interaction nor the main effects of donor type or treatment were statistically significant (Table 2). Therefore, no further pairwise

comparisons were made.

87
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J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092
Table 4
Embryo production from dairy cattle following superovulation and insemination with low-dose sex-sorted spermatozoa from different bulls in Experiment 2 on commercial
dairy farms
Bulls Number of flushes Number of embryos Transferable Unfertilized oocytes Degenerated
(% 0 flushesa ) recovered/flushb embryos (%)c,d (%)c,d embryos (%)c,d

Picston Shaker FFF 93568 B 23 (30.4) 9.7 7.9 35.8 [17.5, 56.6] 45.8 [27.6, 64.7] 6.6 [1.2, 16.0]
Cogent Courier ET FFF 93588 C 29 (27.6) 6.6 5.3 24.8 [8.7, 45.9] 47.0 [28.0, 66.4] 11.2 [3.2, 23.0]
Overside Dreamer ET FFF 93796 B 7 (0) 4.1 1.8 50.4 [14.9, 85.7] 21.7 [1.6, 55.7] 16.0 [1.5, 41.5]
a Proportion of flushes producing zero transferable embryos.
b Means standard deviations.
c Estimated means and their 95% confidence intervals [lower CI, upper CI].
d Bull within treatment effects were not statistically significant for proportions of transferable embryos, unfertilized oocytes or degenerated embryos.
 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092 89

Table 5
Quality of embryos recovered from dairy cattle following superovulation and insemination with low-dose X-sorted
spermatozoa or normal-dose unsorted semen in Experiment 2 on commercial dairy farms
Inseminations Number of transferable % quality 1 % quality 2
embryos recovered/flusha embryosb embryosb

X-sorted spermatozoa
Heifers 3.1 3.2 89.1 [76.6, 97.2] 4.8 [0.4, 13.9]
Cows 2.1 2.7 76.7 [52.1, 94.2] 7.6 [0.2, 24.5]
Unsorted semen
Heifers 5.7 5.4 87.5 [77.4, 94.9] 7.7 [2.4, 15.7]
Cows 6.3 5.7 80.9 [70.1, 89.7] 13.8 [6.9, 22.7]
Bulls (X spermatozoa)
Picston Shaker FFF 93568 B 3.0 3.8 91.1 [76.3, 99.1] 4.6 [0.1, 15.6]
Cogent Courier ET FFF 93588 C 2.9 2.9 76.2 [54.4, 92.5] 17.2 [3.6, 38.0]
Overside Dreamer ET FFF 93796 B 2.0 1.2 81.2 [51.0, 98.5] 1.1 [0, 15.5]
a Means standard deviations.
b Estimated means and their 95% confidence intervals [lower CI, upper CI].

For the proportion of quality 1 embryos, donor type insemination type interaction (p = 0.66),
donor type (p = 0.14), and insemination type (p = 0.89) effects were not statistically significant.
Although the effect of bull within treatment was close to significance (p = 0.06), there were no sig-
nificant differences between the bulls within the X-sorted spermatozoa group (results not shown).
For proportion of quality 2 embryos, donor type insemination type interaction (p = 0.80), donor
type (p = 0.31), insemination type (p = 0.36) and bull within treatment effects (p = 0.12) were not
significant.

4. Discussion

Farmers interest in the use of sex-sorted spermatozoa for embryo production is determined by
the breeding value of bulls from which the spermatozoa are available from, and by the quantity and
the quality of the produced embryos. Moreover, an increased risk of not getting any transferable
embryos from a flushing may also guide embryo production strategies on commercial dairy farms.
In Experiment 1, approximately twice the proportion of transferable female embryos were
recovered from heifers and cows after inseminations with X-sorted spermatozoa compared to
inseminations with unsorted semen. Embryo diagnosis was not possible on commercial dairy
farms (Experiment 2), and therefore we do not know, if similar increase in the proportion of
transferable female embryos observed in a small scale study at the research farm also occurred
in a larger scale. However, due to severely compromised X-sorted spermatozoa fertilization rates
in cows, the use of X-sorted spermatozoa likely did not increase the proportion of transferable
female embryos produced in cows on commercial dairy farms to the extent to which it occurred
at the research farm.
Shorter life span and subsequently reduced fertility of sex-sorted spermatozoa has been sug-
gested by the thermo-resistance test, where the motility of sex-sorted bull spermatozoa has been
observed to decline faster compared to unsorted controls (Rath et al., 2003). In addition, indi-
vidual bulls semen have been reported to have different susceptibility to the sorting procedure
(Gardner and Suh, 2002; Suh et al., 2005). In our study, deep-uterine inseminations of X-sorted
spermatozoa did not compensate for the reduced number of spermatozoa per insemination dose
90 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092

and possible shorter life span and/or damage of sex-sorted spermatozoa, as the proportions of
unfertilized oocytes were larger in all of the X-sorted spermatozoa groups when compared to
the unsorted semen groups. At the research farm, unfertilized oocytes were only recovered from
inseminations with X-sorted spermatozoa in both heifers and cows. On commercial dairy farms,
higher proportion of unfertilized oocytes were also found in both heifers (11%-units more) and
cows (42%-units more) in X-sorted spermatozoa groups compared to the unsorted groups, but the
difference was only statistically significant for cows. Bulls susceptibility was not a factor in this
study, because no significant differences were observed between the bulls for the proportions of
unfertilized oocytes recovered on commercial dairy farms. In general, it cannot be concluded from
our data whether the sorting procedure itself, the low insemination doses used, or the combination
of these two factors reduced the fertilization rates of X-sorted spermatozoa.
By using deep-uterine inseminations with 20 times more X-sorted spermatozoa per insemi-
nation than in our study, Sartori et al. (2004) also reported a significant increase in the number
of unfertilized oocytes recovered in superovulated heifers compared to the control inseminations
using equal amounts of unsorted semen. Schenk et al. (2006) also reported that depositing low
doses (2 million) of sex-sorted spermatozoa to the uterine body, significantly increased the pro-
portion of unfertilized oocytes obtained from a group of heifers and cows compared to unsorted
inseminations to the uterine body using 20 times higher concentrations of spermatozoa. The
increase in the proportion of unfertilized oocytes obtained was, however, smaller between heifers
and cows than in our study. In addition, Panarace et al. (2003) showed a 22%-units increase in the
proportion of unfertilized oocytes obtained from a group of heifers and cows after inseminating
five times more X-sorted spermatozoa to the uterine body per inseminate than Schenk et al. (2006).
It can be concluded from these studies that neither the number of spermatozoa per insemination
dose nor the site of insemination are the factors determining the proportion of unfertilized oocytes
obtained when X-sorted spermatozoa are used in embryo production.
The differences in fertilization rates between sex-sorted spermatozoa and unsorted semen
inseminations have been reported to be higher in cows than in heifers (Panarace et al., 2003; Schenk
et al., 2006). This may result from the interactions of the low insemination doses, large postpartum
uterus (Darrow, 2006) and weak heat symptoms (Yoshida and Nakao, 2005) of cows. The optimal
insemination dose and the number of inseminations required in cows may subsequently be higher
than those in heifers. The increase both in the number of inseminations and in the number of
spermatozoa per dose substantially increase costs of sex-sorted spermatozoa inseminations. On
the other hand, reduced fertilization rates also elevate the costs of embryo production.
In this study, smaller proportions of transferable embryos were recovered from cows after
inseminations with X-sorted spermatozoa compared to inseminations with unsorted semen. Since
the proportions of degenerated embryos did not differ between the insemination treatments,
smaller number of transferable embryos obtained was due to reduced fertilization rates rather
than due to reduced zygote developmental potential. Between the bulls, no significant differences
were observed in the proportions of transferable, quality 1, or degenerated embryos recovered on
commercial dairy farms. In contrast to our findings, Sartori et al. (2004) reported a significant
increase in the proportion of degenerated embryos after using X-sorted spermatozoa compared to
the unsorted semen in superovulated heifers. Bacterial contamination of sex-sorted spermatozoa
has been reported (Andersson et al., 2006), which has been shown to affect embryo quality in
vitro (Kim et al., 1998).
One of the advantages of using sex-sorted spermatozoa for production of sex diagnosed
embryos is that biopsy is not required and, subsequently, intact embryos have better post-thaw sur-
vival rates compared to embryos that have been biopsied for sex diagnosis (Hasler et al., 2002).
 J. Peippo et al. / Animal Reproduction Science 111 (2009) 8092 91

After cryopreservation, pregnancy rates are best for quality 1 embryos (Hasler et al., 2002).
According to this study, embryo quality after successful fertilization with X-sorted spermatozoa
is similar to the embryo quality with unsorted semen. The average proportion of quality 1 embryos
among transferable embryos was not significantly different between X-sorted spermatozoa and
unsorted semen groups either in heifers or in cows. In addition, within the X-sorted spermatozoa
group, the bull did not have significant effect on the proportion of quality 1 embryos.

5. Conclusions

Compared to normal-dose unsorted semen the use of low-dose X-sorted spermatozoa for the
insemination of superovulated embryo donors can improve the proportion of transferable female
embryos produced, but this potential may not be achieved in commercial practice, particularly in
cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.

Acknowledgments

Authors thank Usko Kokkonen for AIs at the MTT research farm. Commercial dairy farms,
embryo collection teams and Faba Breeding are thanked for providing data for this study. Magnus
Andersson, Peter Bredbacka and Juhani Taponen are acknowledged for useful comments on the
manuscript.

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