No abstract

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

A major obstacle of large-scale commercial application of bovine in vitro fertilization is the lack of a suitable cryopreservation method for supernumerary embryos produced. The traditional slow-freezing method has proven to be effective for embryos of a wide range of mammalian species; however, the formation of intracellular ice is still a challenge and the efficiency needs to be improved. Over the past decade, several advances have taken place in vitrification technologies, such that it can provide high efficiency with better pregnancy outcome due to its high cooling rates and the lack of crystals formed inside the cells. Most vitrification methods have been evaluated in Bos taurus cattle but more still remains to be investigated in Bos indicus races predominant in the tropics. There are several vitrification protocols and holders, including CryoLoop, open pulled straw (OPS), MS Grids, and Cryotop, among others. The CryoLoop method uses a nylon loop attached to a metal Cryovial lid were blastocysts are placed on an equilibration solution film. CryoLoop cooling rates are approximately 20.000°C min–1 and have shown very good results in humans. The OPS is a well-known support for bovine blastocysts; the embryos are taken by capillarity into the OPS and use a 1- to 2-μL drop of final equilibration solution. Cooling rates using this method are approximately 2.000°C min–1. The aim of this work was to prove CryoLoop and OPS vitrification methods in Bos indicus blastocyst and compare re-expansion and hatching rates 24 h after warming. Ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes (COC) were treated for the standard IVF method. A total of 60 blastocysts were vitrified in CryoLoops and 68 blastocysts in OPS (within 4 repeats). For CryoLoops, groups of 2 blastocysts were placed in a solution of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3 min, and then were placed in a solution of 15% EG, 15% DMSO, 10 mg mL–1 of Ficoll 70, and 0.65 M sucrose for 20 s, and rapidly were put into the nylon loop and taken to the LN. For OPS, groups of 2 to 3 blastocysts were placed in a solution of 10% EG and 10% DMSO for 1 min, and then were placed in a solution of 20% EG and 20% DMSO for 20 s, and rapidly were taken by capillarity into the OPS and taken to the LN. Thawing was the same for both treatments; vitrified blastocysts were taken out from the LN and rapidly put into a solution of 0.3 M sucrose for 2 min and then put into a solution of 0.2 M sucrose for 3 min, were washed twice in TCM199 supplemented with 10% FCS, and cultured for 24 h in CR1aa media. Data were analysed using the R language. Media comparison for proportions was done using a chi-squared test. No significant difference was observed in re-expansion or hatching rates between CryoLoop and OPS supports (P = 0.01 for both); however, the CryoLoop method showed more efficiency than OPS in re-expansion rate (65 v. 44.4%, respectively) and hatching rate (30.8 v. 20%, respectively). In all cases, the CryoLoop method showed much better outcomes. The results indicate that vitrification in CryoLoops is a suitable method for cryopreservation of Bos indicus blastocysts.

The ability of bovine embryos to develop to the blastocyst stage, to implant, and to generate healthy offspring, depends greatly on the oocyte contribution. Oocyte competence is attributed to its close communication with the follicular environment and to its capacity to synthesise and store great amounts of mRNA. Higher developmental competence of bovine oocytes has been associated with the expression of certain genes and with the steroid concentration in the follicular fluid. Hence, the aim of this study was to establish the influence of OCT-4 and MATER mRNA abundance in the oocyte and the influence of progesterone and oestradiol follicular fluid concentration on the competence of bovine oocytes retrieved 30 min or 4 h after slaughter. Cumulus–oocyte complexes (COC) were left in postmortem ovaries for 30 min (Group I) or 4 h (Group II) at 30°C before aspiration. Progesterone and oestradiol concentrations were measured in the follicular fluid in both groups by immunoassay using an Immulite 2000 analyzer. Immature oocytes were evaluated for MATER and OCT-4 mRNA abundance by real-time PCR (total RNA isolated from pools of 100 oocytes per repeat) or were subjected to in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC). For in vitro embryo production, 455 (Group I) and 470 (Group II) COC were used in three repeats. Progesterone concentration was lower (P ≤ 0.05) in Group II than in Group I. Conversely, oestradiol concentration did not vary between groups. Similarly, Group II oocytes exhibited the highest (P < 0.05) MATER and OCT-4 abundance. For embryo development, there were no significant differences between cleavage rates (72 h post-insemination) between both groups. However, blastocyst (168 h post-insemination) and hatching (216 h post-insemination) rates in Group II were greater (P < 0.05) with 21.3 compared with 30.7% and 54.2 compared with 75.3%, respectively. These results indicate that progesterone concentration in the follicle and the abundance of MATER and OCT-4 transcripts could be good predictors of embryo developmental competence and that retrieving COC 4 h after slaughter could increase blastocyst and hatching rates. This work was supported by COLCIENCIAS COD 122852128473 Colombia.