Theriogenology 80 (2013) 950954

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Theriogenology
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Embryo production with sex-sorted semen in superovulated dairy heifers

and cows
I. Kaimio a, *,1, M. Mikkola a, e,1, H. Lindeberg b, J. Heikkinen c, J.F. Hasler d, J. Taponen e
a
Faba Co., Hollola, Finland
b
Department of Medicine, University of Eastern Finland, School of Biomedicine, Kuopio, Finland
c
Department of Biology, University of Eastern Finland, Kuopio, Finland
d
Bioniche Animal Health, Inc., Pullman, Washington, USA
e
Department of Production Animal Medicine, University of Helsinki, Saarentaus, Finland

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to examine the effect of sex-sorted semen on the number and
Received 17 April 2013 quality of embryos recovered from superovulated heifers and cows on commercial dairy
Received in revised form 16 July 2013 farm conditions in Finland. The data consist of 1487 commercial embryo collections per-
Accepted 16 July 2013
formed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively.
Superovulation was induced by eight intramuscular injections of follicle-stimulating hor-
Keywords:
mone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days
Cattle
after the onset of standing estrus. The donors were inseminated at 9 to 15hour intervals
Sex-sorted semen
Superovulation beginning 12 hours after the onset of estrus with 2 2 (1) doses of sex-sorted frozen-
Deep uterine articial insemination thawed semen (N 218) into the uterine horns or with 1 1 (1) doses of conven-
Embryo recovery tional frozen-thawed semen (N 1269) into the uterine corpus. Most conventional semen
(222 bulls) straws contained 15 million sperm (total number 3045 million per donor).
Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 814 million
per donor). Mean number of transferable embryos in recoveries from cows bred with sex-
sorted semen was 4.9, which is signicantly lower than 9.1 transferable embryos recovered
when using conventional semen (P  0.001). In heifers, no signicant difference was
detected between mean number of transferable embryos in recoveries using sex-sorted
semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized
ova was higher when using sex-sorted semen than when using conventional semen in
heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows
(P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed
to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted
semen remains to be found.
2013 Elsevier Inc. All rights reserved.

1. Introduction Generally, conception rates with sex-sorted semen are

Since the introduction of commercially available sex-
sorted bovine semen early in the 2000s, there have been
numerous reports published on conception rates [17].
* Corresponding author. Tel.: 358 40 311 5312.
E-mail address: iris.kaimio@fabapalvelu. (I. Kaimio).
1
These authors contributed equally to this article.
lower than with conventional semen, because of the
adverse effects of the ow cytometry sorting procedure on
sperm viability and the low number of sperm per dose of
sex-sorted semen [8]. The decline in conception rates
varies among these reports, depending on factors such as
sperm dose, site of sperm deposition, parity, estrus syn-
chronization, and management and generally ranged be-
tween 70% and 80% of that achieved with conventional
semen.

0093-691X/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2013.07.025
 I. Kaimio et al. / Theriogenology 80 (2013) 950954
In the dairy industry, there is a strong interest toward
applications that increase the production of female calves
from genetically superior cows. The use of sex-sorted X-
sperm in multiple ovulation and embryo transfer programs
can be instrumental for the production of female progeny.
Before sex-sorted semen was available, polymerase chain
reaction-based embryo sex determination was the only
approach to preselect the sex of calves and reduce the
number of recipients needed. However, sex determination
of an embryo demands skills and equipment for biopsy and
polymerase chain reaction analysis, increases costs, and
results in a decline in embryo vitality when freezing bio-
psied embryos [9,10]. The use of sex-sorted semen bypasses
these disadvantages of embryo sexing.
Despite the advantages of sex-sorted semen, it has not
been used widely in inseminations of superovulated do-
nors. The compromised fertility because of low doses of
sex-sorted semen, combined with the higher incidence of
fertilization failure on superovulated versus single-
ovulating cattle [1113] counteracts the use of sex-sorted
semen on embryo donors. Inevitably, all authors have re-
ported a decline in the fertilization rate and in the pro-
portion of transferable embryos using sex-sorted semen
compared with conventional semen on donor cows
[1417]. In heifers, this decline is usually smaller
[14,15,18,19]. However, comparison of the results is chal-
lenging, because of different insemination protocols, sperm
dosages, site and timing of insemination, parity of donors,
and other variables.
The dose of sex-sorted semen used for superovulated
donors has varied from 2 to 20  106 sperm [1419]. Most
previous experiments were performed using conventional
insemination techniques, with sex-sorted semen deposited
into the uterine body, but some also involved deep uterine
horn insemination [16,18,19]. The timing of inseminations
using sex-sorted semen can have a more important role in
superovulated than in single-ovulating animals because of
the time interval between the rst and last ovulations
[2,19]. It has been suggested that sex-sorted sperm have a
shorter functional life than conventional sperm [2]. In
addition to the variation in these factors, most previous
experiments were performed using a relative low number
of animals (559 donors per treatment group). This, along
with the high level of individual variation present in
superovulatory responses and embryo yield, exacerbates
the interpretation of the results. The optimal sperm dose
and the site and timing of inseminations to optimize
commercially satisfactory embryo production with sex-
sorted semen remain to be determined.
The objective of this study was to examine the effect of
sex-sorted semen on the number and quality of embryos
recovered from a large number of superovulated heifers
and cows on commercial dairy farms in Finland. In this
study, a deep uterine insemination technique was used.
2. Materials and methods
2.1. Donors and experimental protocol
The data consist of 1487 commercial embryo collections
performed in Finnish dairy herds between January 2008
951
and December 2011. The collections were performed in 633
and 854 animals of Holstein and Finnish Ayrshire breeds,
respectively. The donors were inseminated with sex-sorted
frozen-thawed semen (N 218) or with conventional
frozen-thawed semen (N 1269, controls). Donors
inseminated with sex-sorted semen consisted of 130
heifers and 88 cows (22 rst parity and 66 older animals),
whereas in the control group, there were 945 heifers and
324 cows.
2.2. Superovulation protocol and articial inseminations
Superovulation and embryo recovery were performed
by standard multiple ovulation and embryo transfer pro-
tocols. Superovulation was induced by eight intramuscular
injections of follicle-stimulating hormone (FSH), Folltropin
(Bioniche Teo, Inverin, Co., Galway, Ireland) or Pluset
(Laboratorios Calier, S.A., Barcelona, Spain), at 12-hour
intervals over 4 days, involving declining doses, 630 IU
(Folltropin) or 800 to 900 IU (Pluset) FSH in total for cows,
with heifers receiving 60% to 75% of cow doses beginning
on 9 to 12 days after the onset of standing estrus. Our un-
published data showed no difference in the superovulatory
response between these two FSH preparations. The donors
were treated with a generally recommended dose of
prostaglandin F2a or a synthetic agonist along with the
sixth (cows) or seventh (heifers) FSH treatment. In-
seminations were started 12 hours after the onset of
standing estrus. The donors were inseminated two or three
times 9 to 15 hours apart. When using conventional semen,
an articial insemination (AI) technician or a farmer
inseminated the animal with one dose of semen at a time
into the uterine corpus. When using sex-sorted semen, an
embryo transfer (ET) technician performed the in-
seminations. Instructions for the use of sex-sorted semen
were to inseminate three times with 2 2 1 straws.
When two straws were used at a time, each uterine horn
received one straw, and when one straw was used, the
content of the straw was divided between the two uterine
horns. In 61 cases, the estrus was so short that the donor
was inseminated only twice. In seven cases, the estrus was
prolonged and the donor was inseminated four times. In
the remaining 150 cases, the donor was inseminated three
times. Embryo ushing was performed 7 days after AI.
Recovered ova/embryos were evaluated according to the
International Embryo Transfer Society classication system
[20]. Flushing was performed by 10 experienced veteri-
narians and one ET technician.
2.3. Semen
Semen doses used for AI were commercially produced
at several AI centers. Conventional semen was collected
from 222 different bulls worldwide. Most conventional
semen straws contained 15 million spermatozoa. The total
number of conventional sperm used per donor was 30 to 45
million. Sex-sorted semen from 61 different bulls was used.
Sex-sorted semen was produced using the XY sperm sort-
ing protocol [21] at 10 AI centers in Canada, Denmark, Great
Britain, Italy, Switzerland, and the United States. The straws
contained 2 million spermatozoa. The total number of
952 I. Kaimio et al. / Theriogenology 80 (2013) 950954

Table 1
Mean numbers of transferable embryos, degenerate embryos, and UFO in recoveries from heifers and cows bred with sex-sorted versus conventional semen
(% per total embryos/UFO).

Type of embryo/ova Heifers Cows

Sex-sorted (N 130) Conventional (N 945) P Sex-sorted (N 88) Conventional (N 324) P

Transferable 6.1 (59) 7.2 (63) ns 4.9 (45) 9.1 (70) <0.001
Degenerated 1.6 (15) 1.9 (17) ns 2.4 (22) 1.5 (11) <0.05
UFO 2.8 (26) 2.3 (21) <0.01 3.6 (33) 2.5 (19) <0.01
All 10.6 (100) 11.4 (100) 10.9 (100) 13.0 (100)

Abbreviations: ns, nonsignicant; UFO, unfertilized ova.

sex-sorted sperm used for each embryo recovery attempt
was 8 million sperm in 64 cases, 10 million sperm in 76
cases, 12 million sperm in 71 cases, and 14 million sperm in
seven cases.
2.4. Statistical analysis
The nonparametric Mann-Whitney U test was used to
analyze possible differences in numbers of transferable
embryos, degenerated embryos, and unfertilized ova (UFO)
according to the type of semen (sex-sorted/conventional)
among heifers and cows. To control the possible bias
caused by the variation in the number of all embryos/ova
recovered in each of the four groups, the same analysis was
performed using the relative proportions of the classes
instead of absolute numbers. However, this revealed no
signicant differences between the analyses and thus, re-
sults expressed are based on the former approach. Fisher
exact test was used to analyze whether the relative pro-
portion of failed ushings (no transferable embryos) from
all ushings was independent from parity (heifer/cow) and
type of semen (sex-sorted/conventional). Kruskal-Wallis
test was used to detect a further possible effect of parity
(heifer/rst parity/older cows), AI centers (AI centers that
used 10 or more bulls were selected for analysis), and bulls
(bulls that had inseminated nine or more animals were
selected for analysis) to numbers of transferable embryos,
degenerated embryos, and UFO. All statistical analyses
were performed with IBM SPSS Statistics v.19.
UFO are presented in Table 1. In heifers, the mean number
of transferable embryos was numerically 1.1 embryos less
when sex-sorted semen was used compared with conven-
tional semen. However, this was not statistically different
(P > 0.05). In cows, there was a clear decline (P  0.001) of
4.2 transferable embryos when using sex-sorted semen
compared with conventional semen. The number of UFO
was greater when using sex-sorted semen than when using
conventional semen in heifers (P < 0.01) and in cows (P <
0.05). In addition, the number of degenerate embryos was
greater in cows (P < 0.01) when using sex-sorted semen. As
a consequence, in cows the percentage of transferable
embryos out of all embryos/ova recovered was only 45%
using sex-sorted semen, whereas it was 70% using con-
ventional semen. When the embryo recovery results were
compared between the rst parity and older cows, no sig-
nicant differences were found. Bull or AI center had no
signicant effect (P > 0.05) on embryo recovery results
when using sex-sorted semen.
The number and proportion of failed ushings, i.e.,
ushings that contained no transferable embryos, using
sex-sorted and conventional semen are shown in Table 2. In
cows inseminated with sex-sorted semen, there was a
larger risk of recovering no transferable embryos than in
heifers (P < 0.05). The use of sex-sorted semen did not
result in more failed ushings than the use of conventional
semen.
4. Discussion

3. Results This research provides the largest published dataset to

The average numbers of transferable embryos, degen-
erate embryos, and UFO in ushings after the use of
sex-sorted and conventional semen in heifers and cows
separately and their percentages of all recovered embryos/
date of commercial embryo recoveries from superovulated
cattle bred with sex-sorted semen (N 218) compared
with control animals bred with conventional semen (N
1269). The data include results from 61 sex-sorted bulls
and 222 bulls with conventional semen. The semen from

Table 2
The number and proportion of recoveries with no transferable embryos (failed recoveries) compared to recoveries with at least one transferable embryo
(successful recoveries) in heifers and cows.

Type of recovery Heifers Cows

Sex-sorted (N 130) Conventional (N 945) Sex-sorted (N 88) Conventional (N 324)

Failed recoveries (N) 10 115 16 42
Successful recoveries (N) 120 830 72 282
Failed recoveries (%) 7.7a,c 12.2d 18.2b,e 13.0f
a,b
P < 0.05.
c,d
Nonsignicant.
e,f
Nonsignicant.
 I. Kaimio et al. / Theriogenology 80 (2013) 950954
different bulls was used randomly according to the donor
owners selection. Earlier publications of embryo recoveries
using sex-sorted semen have primarily concentrated on
small-scale experiments in which numbers of embryo re-
coveries have varied from 5 to 59 [1419]. In these exper-
iments, excluding the study by An et al. [19] in which the
number of bulls was not mentioned, sex-sorted semen was
used from fewer than ve different bulls. Only Peippo et al.
[16] published results of a eld trial based on commercial
embryo recoveries with sex-sorted and conventional
semen.
Comparisons of embryo recovery results among different
experiments are difcult. For example, semen sorting pro-
tocols differ and not all articles mention the sorting pressure,
which was reported to be important for semen viability [2].
All articles do mention other inuencing factors such as
semen dosage, insemination protocols, and insemination
sites for sex-sorted and conventional semen, but the pro-
tocols differ considerably. In several superovulation protocols,
in contrast to the present study, ovulation was stimulated by
using GnRH or agonistic analogues, which further compli-
cates the interpretation of results.
It has been reported that the timing of inseminations of
superovulated donors is of utmost importance and that the
amount of semen deposited 24 hours after the detection of
standing estrus is the key factor for successful embryo re-
covery in superovulated donors [18]. This is supported by
the fact that ovulation occurs approximately 30 hours after
the onset of standing estrus. In the earlier publications,
semen deposited 24 hours after the detection of standing
estrus varied from a total of 2 million sperm split between
the uterine horns [16], 2 million [14,15], more than 2.1
million sperm into the uterine body [17], a total of 4 million
sperm split between the uterine horns [19], 5 million sperm
into the uterine body [15] to 10 million sperm into the
uterine horns [18], or 10 million sperm into the uterine
body [14,15].
With a very small number of sex-sorted sperm
(approximately 2 million) 24 hours after detection of
standing estrus of cows, the mean number of transferable
embryos per recovery attempt remained relatively low in
previous studies: 3.8 [17], 3.1 [14], 2.5 [15], and 2.1 [16].
Larson et al. [17] and Schenk et al. [14] inseminated into the
uterine body and their donors were Angus cows, whereas
Hayakawa et al. [15] and Peippo et al. [16] inseminated into
the uterine horns of dairy cows. Therefore, the insemina-
tion site did not seem to be of great importance when low
numbers of sperm were used. These studies seem to sup-
port the importance of the breed of cattle (beef vs. dairy) as
being more important than the insemination site. However,
these embryo recovery results are not necessarily satis-
factory from a commercial point of view.
An et al. [19] inseminated 4 million sex-sorted sperm 24
hours after detected standing estrus of heifers and recov-
ered a mean of 4.8 transferable embryos. Hayakawa et al.
[15] inseminated 5 million sex-sorted sperm 24 hours after
detected standing estrus and recovered means of 5.55
and 5.16 transferable embryos from heifers and cows,
respectively. These numbers are very similar to the results
in the present study, in which 4 million sex-sorted sperm
inseminated 24 hours after detected standing estrus
953
resulted in a mean of 6.1 transferable embryos from heifers
and 4.9 from cows. However, the study by Hayakawa et al.
[15] used only sex-sorted semen with no control recoveries
involving conventional semen. Hayakawa et al. [15] ins-
eminated into the uterine body, whereas in the present
study and in the study by An et al. [19], insemination was
into the uterine horns. Therefore, the number of sperm
seemed to be more important than the insemination site.
Although 4 to 5 million sex-sorted sperm 24 hours after
detected standing estrus resulted in quite satisfactory
numbers of embryos recovered in heifers, the question
remains whether using higher sperm numbers will
improve the results in cows. Schenk et al. [14] inseminated
10 million sex-sorted sperm 24 hours after detected
standing estrus into the uterine body, resulting in means of
3.7 transferable embryos from heifers and 4.6 from cows.
Hayakawa et al. [15] inseminated 10 million sex-sorted
sperm at the same time point into the uterine body and
recovered means of 3.38 and 5.04 transferable embryos
from heifers and cows, respectively. Sartori et al. [18]
ushed only heifers, which were inseminated with 10
million sex-sorted sperm into the uterine horns at the same
time point. The recovered mean number of transferable
embryos was 2.3. In these studies, however, the number of
superovulated donors was relatively low (ranging from 7 to
21). Furthermore, the technique of semen sorting has been
modied and improved since the earlier studies conducted
at the beginning of the 2000s. This might have contributed
to the variable results of some of the earlier studies. In view
of these results, inseminating 10 million sperm, instead of 4
to 5 million, 24 hours after the onset of standing estrus did
not increase the number of transferable embryos in
superovulated cows. However, when 10 million conven-
tional sperm were used 24 hours after detected standing
estrus, mean embryo production from cows was greater
than in heifers, supporting previous studies comparing
cows and heifers [22,23] and contrary to the results of
lower semen doses.
In the present study, the proportions of transferable
embryos, degenerate embryos, and UFO were similar in
heifers when sex-sorted or conventional semen was used.
In cows, the proportions of degenerate and UFO were
greater when using sex-sorted semen (22% and 33%,
respectively) than with conventional semen (11% and 19%,
respectively). The same pattern is seen in other publica-
tions. The use of a very small number (2 million) of sex-
sorted sperm 24 hours after detected standing estrus
seemed to result in a greater proportion of UFO, especially
in cows: 39% [15], 53% [17], and 70% [14]. However, the
largest number (10 million) of sex-sorted semen used at 24
hours did not seem to result in a greater proportion of
transferable embryos, but increased the proportion of
degenerate embryos: 44.6% [15]. The best proportion (45%)
of transferable embryos in cows inseminated with sex-
sorted semen was achieved in the present study with 4
million sperm 24 hours after detected standing estrus. In
the study by Hayakawa et al. [15], almost the same pro-
portion (44.1%) was achieved with 5 million sperm at 24
hours. However, this is still far less than the 70% proportion
of transferable embryos recovered from cows inseminated
with conventional semen in the present study.
954 I. Kaimio et al. / Theriogenology 80 (2013) 950954
From an economic point of view, recoveries with no
transferable embryos from superovulated cattle are highly
undesirable. In this study, the risk of recovery failures was
not increased with the use of 4 million sex-sorted sperm at
the 24-hour time point. However, 2 million sex-sorted
sperm at the 24-hour time point in cows [16] resulted in
a greatly increased risk of failed recoveries compared with
conventional semen (41.2% vs. 9.9%, respectively). Other
publications do not mention the number of recoveries with
no embryos, which would be worth reporting because
farmers partly base their decisions on the risk of not
recovering any embryos when making plans to use ET and
choosing between sex-sorted and conventional semen.
4.1. Conclusions
The use of sex-sorted semen did not signicantly
decrease the mean number of transferable embryos in
superovulated heifers compared with use of conventional
semen. However, in cows, the mean number of transferable
embryos was signicantly lower when sex-sorted semen
was used compared with conventional semen. Four million
sex-sorted sperm inseminated 24 hours after detected
standing estrus seemed to be an adequate number for
heifers. Several inseminations can still be recommended
because of the uncertainty of accurate estrus detection and
possible variation in the time of ovulation. In superovulated
cows, an optimal protocol for using sex-sorted semen re-
mains to be identied. Compromised quality of cow ova and
differences in the environment and the size of uterus of cows
versus heifers might explain the poor quality of the cow
embryos observed in this study. Distribution of the embryos
in the different quality classes, pregnancy results, and the
number and sex of the calves born after the use of sex-sorted
semen are areas of interest for further publications.
Acknowledgments
The authors wish to thank the Faba embryo collection
teams with the veterinarians, Kirsi Vartia, Seija Vahtiala,
Henri Simonen and technician Juha-Pekka Seppnen for
embryo recoveries on farms, MTT Agrifood Research
Finland (Jaana Peippo and Paula Lidauer) for providing data
for this study, and Dr. George Seidel for valuable comments
on the manuscript.
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