Introduction
Cryopreservation is a method that maintains cellular viability under low temperatures, resulting in diminished intracellular enzymatic activity and reduced cellular metabolism that ultimately allows preserving genetic material for indefinite periods of time (Seidel, 1996). This technique has applicability both on scientific and commercial settings (Dattena et al., 2004). Despite the differences in cryopreservation methodologies, all include temperature lowering, dehydration by cryoprotectants, freezing and thawing (Mara et al., 2013). Vitrification was developed as an alternative to increase cryotolerance for early cleavage embryos. This process consists of embryo dehydration by rapid exposure to a cryoprotectant solution of high molecular weight and direct immersion in liquid nitrogen (Vajta et al., 2000). Embryos submitted to cryopreservation suffer from considerable morphological and functional damage, resulting in reduced survival and developmental rates. Embryonic viability after cryopreservation depends upon embryo source (in vivo vs in vitro), quality, and developmental stage (Leibo et al., 1993, 1996). In vitro produced (IVP) embryos submitted to cryopreservation yield lower pregnancy rates than their in vivo counter-parts, possibly due to increased lipid content and smaller inner cell mass (Massip, 2001). Bovine embryo production system (in vitro vs in vivo), and breed (Bos indicus or Bos taurus breeds) are two major factors that affect embryo cryotolerance (Visitin et al., 2002). Embryo vitrification has received great attention in the last decade as an attractive alternative to conventional freezing due to reduced costs, labour and overall simplicity (Kuwayama et al., 2005; Vajta et al., 2006; Sanches et al., 2013; Kocyigit and Cevik, 2015). Moreover, it is considered an efficient technique by avoiding the formation of intra and extracellular ice crystals, diminishing damage to membranes and cellular organelles. However, despite significant progress in recent years, embryo cryopreservation in different domestic species does not hold standard practices and similar results (Prentice and Anzar, 2010), since IVP embryos remain less tolerant to cryopreservation. Due to the widespread of IVP system worldwide, and increasing demand for vitrification of surplus embryos from cattle producers at increasing distances from IVP laboratories, the development of protocols under less rigorous conditions (i.e. field conditions) remains as a research topic worth exploring (Pereira et al., 2016). Therefore, the objective of this study was to evaluate both pregnancy and delivery rates of in vitro produced (IVP) Nellore (Bos indicus) embryos after vitrification under field conditions.
Materials and Methods
Oocyte recovery
Ovum pick-up (OPU) was performed as described by Pereira et al. (2016). Ovarian follicles >2 mm diameter were aspirated by ultrasound (DP 2200 VET, Mindray, Nanshan, Shenzhen, China) using a 7.5 Mhz micro transducer convex, vacuum pump (WTA VET), and a 0.9 x 50 mm hypodermic needle 20G x 2” (Terumo Europe, Leuven, Belgium). The needle was connected to a 50 mL cone through a silicon tube (0.8 m long; 2 mm internal diameter). The medium used on OPU was TCM-199 (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 25 mM HEPES (Sigma, St Louis, MO, USA), 5% fetal bovine serum (FBS), 50µg/mL gentamycin sulfate (Schering Plough, São Paulo, SP, Brazil), and 10 IU/mL sodium heparin (Sigma, St Louis, MO, USA).
In vitro maturation (IVM)
Only oocytes with normal morphology, with at least 3 compact layers of cumulus cells and homogenous cytoplasm, were used (Pereira et al., 2016). Immediately after, the oocytes were washed 3 times in HEPES-containing TCM-199 medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS, 0.20 mM sodium pyruvate and 83.4 µg/mL amycacyn (Sigma, St Louis, MO, USA). For IVM, TCM-199 (Sigma, St Louis, MO, USA) supplemented with 10% FBS, 1µg/mL FSH (Sigma, St Louis, MO, USA), 50 µg/ mL hCG, 1 µg/mL 17-β estradiol (Sigma, St Louis, MO, USA), 0.20 mM sodium pyruvate (Sigma, St Louis, MO, USA) and 83.4 µg/mL amycacyn (Sigma, St Louis, MO, USA) were used. Drops (100 μL) of embryo culture medium containing 25 to 30 oocytes were placed under mineral oil at 39 oC in a humid atmosphere of 5% CO2 during 22 to 26 hours.
In vitro fertilization (IVF)
The semen used was collected from bulls of proven in vitro fertility. Semen straws (25 x 106 spermatozoa per straw) were thawed for 30 seconds at 35 ºC. Semen samples were washed twice by centrifugation at 200 g for 5 minutes in 2 mL TALP medium supplemented with 10 mM HEPES (Sigma, St Louis, MO, USA), 0.2 mM sodium pyruvate (Sigma, St Louis, MO, USA) and 83.4 g/mL amycacyn (Sigma, St Louis, MO, USA). Spermatozoa were capacitated with 10µg/mL heparin and motility was stimulated with addition of 18 M penicillamine (Sigma, St Louis, MO, USA), 10 M hypotaurine (Sigma, St Louis, MO, USA), and 8 M epinephrine (Sigma, St Louis, MO, USA) (PHE). After visual appraisal of sperm motility, spermatic concentration was adjusted to 1 x 106 viable spermatozoa/mL, and placed in medium containing 90µL TALP-IVF supplemented with 10 µg/mL sodium heparin (Sigma, St Louis, MO, USA) and PHE with 1x105 spermatozoa per drop (Viana et al., 2010).
Oocytes were washed 3 times after IVM in pre- IVF medium composed of TCM-199 (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 25 mM HEPES (Sigma, St Louis, MO, USA), and 0.3% BSA (Sigma, St Louis, MO, USA). Additionally, oocytes were co-incubated with spermatozoa in FERT- TALP medium supplemented with 10 µg/mL sodium heparin and PHE under mineral oil at 39 oC under saturated humidity and 5% CO2 during 18 to 20 hours.
In vitro embryo culture (IVC) and blastocyst vitrification
The presumptive zygotes had their cumulus cells removed and were transferred to 100 µL drops of culture medium (SOFaa containing 0.5% BSA and 2.5% FBS) under mineral oil at 39 oC in a humid atmosphere of 5% CO2, 5% O2 and 90% N2. Embryo development was assessed on day 3 (D3), day 5 (D5), and day 7 (D7) of IVC. At D3 and D5, 50% of culture medium was replaced with fresh medium (feeding). Fresh controls were randomly selected (n = 140) at day 7 (D7) and transferred to synchronized recipients, as described below.
Blastocyst vitrification
Blastocysts (n= 130) were submitted to vitrification by the “Cryotop” method described by Kuwayama et al. (2005). Grade I initial blastocysts, blastocysts, and expanded blastocysts were placed in 10% ethylene glycol (EG) (Sigma, St Louis, MO, USA) and 10% DMSO (Sigma, St Louis, MO, USA) in HEPES- containing TCM-199 ((Sigma, St Louis, MO, USA) supplemented with 20% FBS (HTCM-FBS) during 1 minute at room temperature (RT). Embryos were transferred to a vitrification solution containing 20% EG, 20% DMSO and 0.5 M sucrose (Sigma, St Louis, MO, USA) for 20 seconds in RT. During this incubation, blastocysts were loaded on top of polypropylene Cryotop tips (3 to 5 embryos) with minimum quantity of vitrification solution and immersed in liquid nitrogen.
Blastocyst warming
Blastocysts were removed from liquid nitrogen, exposed for 4 seconds in RT and subsequently warmed by immersion of polypropylene Cryotop tip (In Vitro Brasil, Mogi Mirim, Brazil) in medium (TCM- HEPES + sucrose) at 35 °C for 1 minute. Blastocysts were gradually transferred to HTCM-FBS medium containing 0.3 and 0.15 M sucrose (Sigma, St Louis, MO, USA) during 5 minutes each in RT (Morató et al, 2008; Vajta et al, 2010; Pereira et al, 2016). Embryos were loaded in 0.25 mL straws with HTCM-FBS medium and transferred to recipients after warming.
Embryo transfer
Embryo transfer was performed in a fixed-time manner. Recipients received intravaginal progesterone implants (CIDR, Pfizer, Hamilton, New Zealand) and 2 mg estradiol benzoate (Estrogin, Farmavet, São Paulo, SP, Brazil) on day 0. On day 7, recipients received 400 IU equine chorionic gonadotropin (eCG; Sincro eCG®; Ourofino Agronegócio) in association with 150 µg synthetic prostaglandin (D-Cloprostenol; Ciosin®; MSD Saúde Animal). The progesterone implant was removed on day 8, associated with an application of 2 mg estradiol. Before transfer, ovaries were evaluated by transrectal ultrasonography for the detection of corpus luteum (CL). Only recipients with a CL received an embryo on day 17. Pregnancy diagnosis was performed by transrectal ultrasonography on days 35 and 60 after IVF. Furthermore, all pregnancies were monitored monthly until delivery.
Results
In vitro production of bovine blastocysts
A total of 750 oocytes were retrieved from 30 donor cows by OPU (mean of 25.0 oocytes/cow). After IVF, 638 presumptive zygotes were cultured (85.06%). The cleavage rate was 73.66% (470/638), and blastocyst development rate at day 7 was 41.37% (264/638).
Pregnancy and delivery rates after transfer of fresh or vitrified blastocysts
The overall pregnancy rates on days 35 and 60 were similar between embryos transferred either fresh or after vitrification (p>0.05; Table 1). Overall pregnancy loss at 60 d was similar between fresh (7.69%) and vitrified embryos (6.00%; Table 1).
Table 1
Pregnancy and delivery rates for Nellore (Bos indicus) in vitro-produced embryos transferred fresh or after vitrification.
Regarding fresh embryos, the stage of embryonic development affected pregnancy rate at day 35 (Table 2). Blastocysts transferred fresh displayed lower pregnancy rate when compared to expanded blastocysts (p<0.05; Table 2). The pregnancy rate after transfer of vitrified embryos was not affected by the stage of blastocyst development on both time points of development (Table 2). No difference was observed on pregnancy loss between blastocyst and expanded blastocyst stages, irrespectively if transferred fresh or after vitrification (Table 2). Moreover, delivery rates were similar between fresh and vitrified embryos (Table 1).
Discussion
Bovine breeds are grouped in two subspecies, namely Bos taurus and Bos indicus, which differ in various aspects of their reproductive physiology, including embryo production potential under in vitro and in vivo conditions (Viana et al., 2010). Differences between these subspecies can also be observed at the gene expression level in preimplantation embryos. Strikingly, these differences are somewhat attenuated by in vitro culture, suggesting a capacity for adaptive plasticity during early development (Wohlres-Viana et al., 2011). Moreover, embryo source (e.g. in vitro or in vivo) is a major factor to embryo cryotolerance (Mucci et al., 2006). It has been shown that IVP embryos have increased lipid content compared to their in vivo counterparts, which is associated with reduced survival after cryopreservation (Sudano et al., 2011).
Vitrification is being widely adopted as an alternative embryo cryopreservation method to conventional freezing, due to its simplicity, reduced costs, and less labor, while dispensing for sophisticated equipment (Vajta et al., 1996). Vitrification is characterized by high concentrations of cryoprotectants (ACP), resulting in high viscosity of the vitrification solution, which leads to physical properties similar to solid compounds, despite the absence of crystallization (Chian et al., 2004).
The data described here demonstrated that vitrification was as efficient as fresh embryos to establish pregnancy in recipient cows. During vitrification, the freezing rate, viscosity and volume of vitrification solution where embryos are placed, are important factors to consider (Arav et al., 2002; Yavin and Arav, 2007). A small volume of embryos improves heat transfer, increasing cryopreservation rate (Yavin et al., 2009). The high concentration of cryoprotectants used during vitrification could cause toxicity; however, due to the small volume and short period of exposure to cryoprotectants in the present study, no effect was expected on pregnancy rate.
Our results show that embryos transferred fresh during blastocyst and expanded blastocyst stages contributed differently to the overall pregnancy rate at 35 d. However, this difference was not observed on day 60, where vitrification was as efficient as fresh embryos. This discrepancy is probably due to the smaller cell number of the blastocysts transferred. Alternatively, the increased time of exposure to supplemented embryo culture medium after warming may have offered increased energy support and higher cellular metabolism that ultimately led to improved embryo quality and higher survival in vivo.
Scanavez et al. (2013) did not observe any effect of developmental stage of IVP embryos (morulae and initial blastocyst) compared to embryos at later stages (blastocyst, expanded blastocyst and hatched blastocyst). In the present study, pairwise comparisons (vitrification and fresh controls) between developmental stages resulted in different pregnancy rates for blastocysts and expanded blastocysts. It is important to note that all blastocysts were transferred at day 7 of development, irrespectively of their developmental stage. Putative differences in cellular metabolism and proliferation between embryonic developmental stages may have resulted in such differences in pregnancy rates of fresh controls.
Pregnancy loss was similar after transfer of blastocysts and expanded blastocysts at 60 d, which disagrees with Andreoti et al. (2009), who reported that embryonic loss of d7 embryos at earlier stages was higher than more developed embryos. This result is probably due to the variable correlation between embryo development kinetics and developmental potential. Another factor that probably contributed to high pregnancy rates was the improved synchrony between uterine environment and embryonic stage due to embryo transfer in a fixed-time manner (Randi et al., 2015
In conclusion, both pregnancy and delivery rates of Bos indicus IVP embryos after vitrification under field conditions is indistinguishable from fresh embryos.