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Reprod Dom Anim 50, 807–811 (2015); doi: 10.1111/rda.

12591
ISSN 0936–6768

Pregnancy Rates to Fixed Embryo Transfer of Vitrified IVP Bos indicus, Bos taurus
or Bos indicus 3 Bos taurus Embryos
LSR Marinho1, BV Sanches1,2, CO Rosa1, JH Tannura2, AG Rigo2, AC Basso2, JHF Pontes2 and MM Seneda1
1
orio de Reproducßa~o Animal, State University of Londrina (UEL), Londrina, PR, Brazil; 2In Vitro Brasil Ltda, Mogi Mirim, SP, Brazil
Laborat

Contents The high sensitivity of these embryos to cooling appears


The pregnancy rates obtained after the transfer of to be mainly due to the greater lipid accumulation
cryopreserved in vitro-produced (IVP) embryos are usually caused by in vitro culture (Abe et al. 2002).
low and/or inconsistent. The objective of this study was to To reduce the amount of lipids in IVP porcine
evaluate the pregnancy rates of Holstein, Gyr and embryos, Men et al. (2006) added a lipolytic agent,
Holstein 9 Gyr cattle after the transfer of vitrified IVP namely forskolin, to the culture media. This agent
embryos produced with X-sorted sperm. Seventy-two Gyr
and 703 Holstein females were subjected to ovum pickup
increased cryotolerance and pregnancy rates after
(OPU) sessions, followed by in vitro embryo production using transfer of IVP Bos indicus (Nelore) embryos (Sanches
semen from sires of the same breeds. Embryos (1636 Holstein, et al. 2013). However, there are no reports of the
241 Gyr and 1515 Holstein 9 Gyr) were exposed to forskolin pregnancy rates obtained with the use of IVP dairy
for 48 h prior to vitrification. The pregnancy rate achieved embryos after exposure to forskolin.
with Gyr dam and sire was 46.1%, which was similar Therefore, the purpose of this study was to evaluate
(p = 0.11) to that of Holstein dam and Gyr sire (40.3%). the pregnancy rates following transfer of vitrified,
Crossing Gyr dams with Holstein sires resulted in a pregnancy rewarmed and forskolin-exposed IVP embryos from
rate of 38.9% and did not differ (p = 0.58) from the pregnancy Holstein (Bos taurus), Gyr (Bos indicus) and
rate obtained with the cross between Holstein dams and Gyr Holstein 9 Gyr cattle.
sires. The rate obtained with Holstein dam and sire was
32.5%. The average pregnancy rate was 36.6%, and no
difference was found in the proportion of female foetuses
(88.8%, in average) among breeds (p > 0.05). In conclusion, Materials and Methods
transfer of cryopreserved X-sorted embryos represents an All the chemicals used in this study were purchased from
interesting choice for dairy cattle. Despite the small differences Sigma-Aldrich (St. Louis, MO, USA) unless stated
between pregnancy rates, we highlight the efficiency of this otherwise.
strategy for all of the racial groups studied.

Cattle
Introduction Oocyte donors were healthy cyclic cows selected on the
The reproductive performance of milk herds has basis of genetic merit. Seventy-two Gyr (Bos indicus)
severely declined over the last 50 years. In periods and 703 Holstein (Bos taurus) cows with a mean ( SD)
and/or regions with a high average temperature, the body condition score of 3.0  0.5 (scale, 1–5) and mean
pregnancy rates resulting from artificial insemination age of 5  2.3 years (range, 3–7 years) were used. No
(AI) are usually even lower (Ferreira 2013). hormonal treatment was given to cows prior to OPU
To overcome this problem and maintain acceptable sessions, which were performed at least between 15 days
pregnancy rates throughout the year, embryo transfer apart. Crossbred heifers (15–19 months old) and cows
(ET) has been effectively employed (Stewart et al. 2011). (24–48 months old) with an average ( SD) body score
Embryos are known to be more resistant than gametes of 2.7  0.6 (scale, 1–5), with a healthy status and
to increased body temperatures, justifying the replace- regular oestrous cycles, were used as embryo recipients.
ment of AI by ET during periods of heat stress (Chebel The recipients were kept on pasture, with mineral
et al. 2008). supplementation available ad libitum, and were handled
In tropical areas, there is a growing interest in Bos in the same way. These females were located in farms in
indicus dairy breeds due to their ability to adapt and southern and south-eastern Brazil (states of Paran
a, S~
ao
produce milk under hot weather conditions. In this Paulo and Mato Grosso).
context, in vitro embryo production (IVEP) represents
an interesting choice. Compared with in vivo produc-
tion, IVEP requires fewer viable sperm cells and thus Follicle aspiration
allows the efficient use of sex-sorted semen (Pontes et al. Faeces were removed from the rectum, and the perineal
2010). area was washed with tap water and prepared with 70%
Despite the advantages offered by IVEP, cryopreser- ethanol. Each cow received epidural anaesthesia (7 ml
vation of these embryos represents a great challenge. of 2% lidocaine; Anestesico L, Pearson, S~ao Paulo, SP,

© 2015 Blackwell Verlag GmbH


808 LSR Marinho, BV Sanches, CO Rosa, JH Tannura, AG Rigo, AC Basso, JHF Pontes and MM Seneda

Brazil) to decrease peristalsis and discomfort. The previous utilization for IVF) were used. The straws were
follicular aspiration was performed as previously thawed for 20 s in a water bath at 35°C. The sperm were
described (Seneda et al. 2001). Briefly, each visible washed by centrifugation at 200 9 g for 30 min through
follicle was aspirated using a real-time B-mode a Percoll gradient of 45 and 90%. The sperm were
ultrasound scanner (Scanner 200 Vet, Pie Medical, capacitated using heparin (30 lg/ml), and their motility
Maastricht, the Netherlands) with a 7.5-MHz convex was stimulated by the addition of 40 ll/ml penicil-
array transducer fitted into an intravaginal adaptor (Pie lamine, hypotaurine and epinephrine (PHE). Visual
Medical) containing a stainless steel needle guide. The assessment of sperm motility was carried out. Then,
follicular puncture was performed using a disposable sperm concentration was adjusted to 25 9 106 motile
19-gauge ½” hypodermic needle (Becton Dickinson, sperm per ml, and each fertilization drop received 4 ll
Curitiba, PR, Brazil) connected to a 50-ml conical tube of sperm (final concentration of 1 9 105 sperms per
(Corning, Acton, MA, USA) via a silicon tube (0.8 m in drop; Seneda et al. 2001).
length; 2 mm id). The aspiration was performed using a
vacuum pump (Cook Veterinary Products, Queensland,
Australia) with a negative pressure of 10–12 ml of In Vitro culture
water/min. The collection medium was TCM 199 Cumulus cells were removed from presumptive zygotes,
(Gibco Life Technologies, Grand Island, NY, USA) which were transferred to 100-ll drops of culture
supplemented with 25 mM HEPES (Sigma H-0763), 5% medium consisting of a modified synthetic oviduct fluid
foetal calf serum (FCS), 50 ll/ml gentamicin sulphate (SOFaa BSA containing 8 mg/ml fatty acid-free BSA
(Schering-Plough, S~ ao Paulo, SP, Brazil) and and 1 mM glutamine) under the same temperature and
10 000 IU/l sodium heparin (Sigma H-3149). gaseous atmospheric conditions that were used in the
IVF procedure. After three (D3) and five (D5) days of
culture, 50% of the culture medium was replaced with
In Vitro maturation fresh medium (feeding) from a stock of the same
Immediately after recovery, oocytes were classified into medium that was used at the beginning of the culture.
one of the following morphological categories: good, On D5, the embryos were also exposed to 10 lM
with more than three layers of cumulus cells; regular, forskolin (7b-acetoxy-8,13-epoxy-la,6b,9a-trihydroxy-
with at least one layer; denuded, partly covered with labd-14-en-11-one, C22H34O7) for 48 h prior to
cumulus cells or without cumulus cells; and atretic, with vitrification. Cleavage and blastocyst production rates
dark cumulus oophorus and signs of cytoplasmic were recorded on D3 and D7 of culture, respectively.
degeneration (Seneda et al. 2001). Both good and
regular oocytes were used in this study.
Prior to in vitro maturation (IVM), cumulus oocyte Vitrification and warming
complexes (COCs) were washed three times in TCM-199 Expanded blastocysts of excellent quality were subjected
HEPES supplemented with 10% FCS, 0.2 mM sodium to Cryotop vitrification, as previously described
pyruvate and 83.4 lg/ml amikacin. The COCs were (Kuwayama et al. 2005). The embryos were placed in
matured for 24 h in 100-ll drops of TCM 199 (Gibco a solution of 10% ethylene glycol (EG; WakoPure
Life Technologies) supplemented with 10% foetal calf Chemical Industries Co., Osaka, Japan) and 10%
serum (FCS; Gibco Life Technologies), 1 lg/ml folli- dimethyl sulphoxide (DMSO; Wako) in TCM-HEPES
cle-stimulating hormone (FSH; Folltropin, Bioniche base medium (TCM-199, 25 mM HEPES supplemented
Animal Health, Belleville, ON, Canada), 50 lg/ml with 20% FCS) for 1 min at room temperature.
human chorionic gonadotropin (hCG; Profasi, Serono, Embryos were then transferred to a vitrification solution
S~ao Paulo, Brazil), 1 lg/ml estradiol (estradiol-17b), consisting of 20% EG, 20% DMSO and 0.5 M sucrose
0.2 mM sodium pyruvate and 83.4 lg/ml amikacin in the base medium and maintained for 20 s at room
(Instituto Bioquımico, Rio de Janeiro, Brazil). The temperature. During this period, the blastocysts were
medium drops were covered in mineral oil (D’Altomare, loaded in groups of 3–5 embryos onto the top of a
Santo Amaro, SP, Brazil) at 39°C and 5% atmospheric polypropylene strip of a Cryotop (Kitazato BioPharma
CO2 (25–30 oocytes per microdrop). Co., Shizuoka, Japan) with a minimal amount of
vitrification solution. Then, the blastocysts were quickly
immersed in liquid nitrogen (N2).
In Vitro fertilization For rewarming, the blastocysts were exposed to air
After maturation, COCs were washed three times in for 4 s, then immersed into the base medium
TCM 199 pre-fertilization medium supplemented with (TCM-HEPES and sucrose) at approximately 35°C
25 mM HEPES (Gibco Life Technologies) and 0.3% and maintained in this temperature for 1 min. The
BSA (Sigma A-9647) and then washed once in TALP blastocysts were transferred to the base medium at
fertilization medium supplemented with 10 lg/ml room temperature in a stepwise manner (0.3 and 0.15 M
heparin and 40 ll/ml PHE solution. Frozen–thawed sucrose for 5 min each) (Vieira et al. 2001; Mezzalira
sexed sperm (X chromosome bearing; 2 9 106/dose) et al. 2004). After warming, the embryos were
from Gyr and Holstein sires of known fertility (based on transferred to the recipients.

© 2015 Blackwell Verlag GmbH


Pregnancy Rates of Cryopreserved IVP Dairy Embryos 809

Embryo transfer However, we could obtain a general overview of the


A synchronization treatment protocol for fixed-time farmers, reporting no large offspring syndrome or
embryo transfer was used to ensure recipient oestrous congenital malformations.
synchrony with stage of embryo development. On D0,
each recipient received an intravaginal progesterone
Discussion
device (CIDR, Pfizer, Hamilton, New Zealand) and
2 mg of estradiol benzoate (Estrogin, Farmavet, S~ao In this study, we presented data from 3392 vitrified/
Paulo, Brazil). On D8, the progesterone devices were warmed IVP embryos from dairy breeds that were
removed, and 300 IU of eCG (Novormon, Syntex, produced with sex-sorted sperm and cultured with
Buenos Aires, Argentina), 150 lg of d-cloprostenol forskolin prior to cryopreservation. The results show
(Preloban, Intervet, S~ao Paulo, Brazil) and 1 mg of that acceptable pregnancy rates can be obtained after
estradiol cypionate (ECP; Pfizer, Guarulhos, S~ao Paulo, the transfer of vitrified/warmed Holstein (Bos taurus),
Brazil) were administered. D10 was considered to be the Gyr (Bos indicus) and Holstein 9 Gyr embryos.
day of oestrus. The embryos were transferred to The pregnancy rate obtained with the transfer of
recipients on D17. Prior to embryo transfer, each vitrified Holstein embryos (32.5%) was similar to those
recipient was subjected to an ovary examination by described by Block et al. (2010) and Stewart et al.
ultrasound scanning (Aloka SSD 500Ò, 5 MHz linear (2011) with vitrified IVP Holstein (27.7%) and Holstein
transducer, Tokyo, Japan) to confirm the presence and and Jersey (29.3%) embryos, respectively, showing that
size of the corpus luteum (CL). Only recipients with a our conditions were suitable to assess the cryopreserva-
CL ≥ 13 mm received an embryo. The pregnancy status tion method with the established technique. However,
of each recipient was determined by ultrasonography on Xu et al. (2006) reported a greater pregnancy rate of
D60. 40.9% after the transfer of vitrified/warmed IVP
Holstein embryos. The transfer of vitrified IVP embryos
with X-sorted sperm was also shown to result in
Statistical analysis acceptable pregnancy rates, with reports of 24%
For statistical analysis of the pregnancy rates, the (Rasmussen et al. 2013) and 29.3% (Stewart et al.
variable responses were presented as percentage and 2011). The pregnancy rates obtained in this trial were
subjected to a logistic regression test using the Car achieved through the cryopreservation of only excellent
statistical package of ‘R’ software (R Development Core quality embryos.
Team, 2008; Fox and Weisberg 2011). The level of Holstein 9 Gyr cross cattle appear to be beneficial
significance was set at 5%. for dairy farms in regions with high temperatures and
high humidity. The higher resistance of the Gyr breed to
adverse conditions results in reduced drug-related
Results expenses and provides greater ease of handling.
A total of 3392 IVP embryos were transferred, and the Furthermore, the high oocyte yield of Bos indicus
pregnancy rates varied from 32.5% to 46.1% (Table 1). females ensures positive outcomes in the reproductive
Due to the commercial conditions, the foetal sex of performance of dairy herds (Pontes et al. 2010). There-
only 47% (581) of the pregnancies was assessed by fore, the approximately 40% pregnancy rate obtained in
ultrasonography. The average percentage of female this study with Holstein 9 Gyr embryos appears to be
calves was 88.8%, varying from 84.7 (Gyr dam and highly promising. The overall sex ratio obtained in this
Holstein sire) to 94.7 (Gyr dam and sire; Table 1). As study was 88.8%, and this value ranged from 84.7% to
most of the pregnant donors in this study were dispersed 94.7%, with no difference between the different breeds
widely after the pregnancy diagnosis, we could not (p > 0.05). This ratio is comparable to that obtained in
properly evaluate embryonic and foetal problems. a previous study in which the sex ratio observed after
the transfer of IVP embryos produced with X-sorted
Table 1. Pregnancy rates and percentage of female foetuses of IVP
sperm was 84.3% (Stewart et al. 2011). In another
Bos indicus, Bos taurus or Bos indicus 9 Bos taurus embryos treated experiment, Xu et al. (2006) reported a sex ratio of
with forskolin prior to vitrification and then transferred to Bos 96.5% females born after the transfer of vitrified IVP
indicus 9 Bos taurus recipients Holstein embryos. Interestingly, Rasmussen et al.
(2013) observed different sex ratios for fresh (90.0%)
Transferred Pregnancy Female
Dam Sire embryos, n rate % (n) foetuses % (n)
and vitrified (67%) embryos of dairy cows.
In addition to increasing the rusticity of the cattle and,
Gyr Gyr 241 46.1a (111) 94.7a (36/38) therefore, easing their handling, a great advantage of
Gyr Holstein 733 38.9b (285) 84.7a (83/98) breeding Bos indicus breeds with Bos taurus cattle is a
Holstein Gyr 782 40.3ab (315) 89.3a (192/215) resulting high fertility. In this study, a pregnancy rate of
Holstein Holstein 1636 32.5c (531) 89.1a (205/230)
Total 3392 36.6 (1242) 88.8 (516/581)
46.1% was achieved with Gyr cattle and a rate of 32.5%
was achieved with Holstein cattle. Pontes et al. (2010)
Within a column, rates without a common superscript differ (p < 0.05).
a,b,c also reported quantitatively higher pregnancy rates with

© 2015 Blackwell Verlag GmbH


810 LSR Marinho, BV Sanches, CO Rosa, JH Tannura, AG Rigo, AC Basso, JHF Pontes and MM Seneda

Gyr cattle than with Holstein cattle (40 vs 36%, disadvantage: the lack of an efficient method to cryop-
respectively). reserve these embryos due to their higher sensitivity
Due to the advantages arising from the use of (Havlicek et al. 2009; Yu et al. 2010). The benefits of
embryos, some dairy farms might consider to being able to store these structures for prolonged
completely replace AI with ET. In these situations, the periods are numerous. The possibility of having embryo
cows with the best genetic potential would be used as banks that can be transferred while maintaining satis-
oocyte and embryo donors, and the remaining would be factory pregnancy rates throughout the year is definitely
used as recipients. Compared with AI, embryo transfer the most interesting aspect of the embryo cryopreserva-
not only allows higher pregnancy rates throughout the tion of dairy cattle due to the well-documented negative
year (Stewart et al. 2011) but also enhances the genetic effects caused by heat stress (Roth 2008). Furthermore,
gain between generations (Marinho et al. 2013). These this technique favours large-scale embryo production by
benefits are possible because this technique increases the simplifying the transportation of the embryos over long
number of descendants from sires and cows with high distances. Finally, the cryopreservation of IVP embryos
genetic merit. In contrast, AI only propagates the is a great tool for the preservation of endangered species
desirable characteristics of males. or breeds.
Nevertheless, the in vitro technique provides some In summary, the results show that acceptable
interesting advantages over the in vivo production of pregnancy rates can be obtained after the transfer of
embryos. The advantages of OPU/IVEP include the vitrified/warmed Holstein (Bos taurus), Gyr (Bos
following: (i) embryos can be obtained without indicus) and Holstein 9 Gyr cattle. The use of sex-
exogenous hormone stimulation, (ii) females in early sorted semen efficiently provided a percentage of
pregnancy can also be used as donors, (iii) smaller female foetuses close to 90%, even though only 47%
intervals between sessions are required, and (iv) the of the pregnancies had the foetal sex confirmed.
semen dose can be optimized. However, one of the Additionally, the transfer of cryopreserved IVP dairy
major advantages of IVEP may be its efficient use of Bos indicus, Bos taurus and Bos indicus 9 Bos taurus
sexed semen. This feature is particularly useful in dairy embryos can result in pregnancy rates similar to the
cattle because male calves are usually not economically ones obtained after the transfer of fresh embryos. We
interesting. Although the sorting process often com- highlight that pregnancy rates consistent with a large-
promises the fertilization efficiency of a portion of the scale in vitro production of embryos of a determined
spermatozoa, the sperm are subjected to less demand- sex are of great assistance to dairy farmers. Until
ing conditions during the in vitro process (Carvalho recently, most strategies used to achieve good preg-
et al. 2010). As a result, IVEP provides better results nancy rates after the transfer of cryopreserved IVP
than AI and MOET (Carvalho et al. 2010; Norman embryos consisted of adjusting protocols, embryo
et al. 2010). containers and cooling rates (Rios et al. 2010; Ha
In addition to providing good blastocyst rates, the use et al. 2014; Lai et al. 2015). However, the most
of sex-sorted sperm is interesting for other reasons. promising approach seems to be the improvement of
First, it enables a more rational use of embryo recipients culture media so that embryo survival becomes similar
because twice as many recipients are required to achieve to that of in vivo-produced embryos.
the desired number of female products when unsorted
sperm is used. In addition, almost all the embryos
obtained using this technique may be of the desired sex Acknowledgements
and of the same quality as the embryos produced with The authors acknowledge Coordination for the
conventional semen (Xu et al. 2006) unlike those Improvement of Higher Education Personnel (CAPES),
obtained through biopsy followed by DNA analysis Post-Graduation Program in Animal Science at the
(Hasler et al. 2002). The rates currently obtained State University of Londrina and National Council for
through IVEP with sorted sperm are very encouraging Scientific and Technological Development (CNPq) for
(Pontes et al. 2010). However, the bull- and ejaculate- providing financial support.
related effects that are observed require rigorous
evaluation and selection of the best batches to obtain
better results (Pontes et al. 2010). Author contributions
It has been previously reported by our team that the Marinho analysed the data and drafted the manuscript. Sanches was
treatment with forskolin before vitrification improved involved in embryo production and designed the study. Rosa drafted
the manuscript. Tannura was involved in embryo and media produc-
cryotolerance of Bos indicus IVP embryos, resulting in tion. Rigo was involved in embryo transfer. Basso was involved in
good post-transfer pregnancy rates (Sanches et al. media production and designed the study. Pontes was a field and
2013). The pregnancy rates obtained in the present laboratory supervisor. Seneda was an experimental supervisor.
study after the use of forskolin prior to the vitrification
of dairy cattle embryos represent a great achievement
for the milk industry. Until recently, all the advantages Conflict of interest
of the IVEP technique offset the following major None of the authors have any conflict of interest to declare.

© 2015 Blackwell Verlag GmbH


Pregnancy Rates of Cryopreserved IVP Dairy Embryos 811

Lai D, Ding J, Smith GW, Smith GD, vival of in vitro-produced bovine


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© 2015 Blackwell Verlag GmbH

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