Академический Документы
Профессиональный Документы
Культура Документы
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
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,
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.
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.