The objective of this study was to evaluate the time of blastulation monitored by time-lapse technology to predict in vitro viability of bovine blastocysts. This technology can be a powerful tool for bovine embryos selection with higher implantation capacity and competence. Also, in humans an early blastulation is associated with higher quality and pregnancy rate. Cumulus oocyte complexes (COCs) were matured for 20 to 22 h and then fertilized by co-incubation of COCs and spermatozoa (10,000 sperm per oocyte) for 18 h. Presumptive zygotes were placed individually in microwells, in droplets of commercial culture medium. The Primo Vision TL system (EVO+; Vitrolife) captured digital images of developing embryos every 15 minutes. The time frame from IVF to the start of blastulation (tSB) and to blastocyst development (tB) was recorded. After day 7.5, the blastocysts were in vitro culture for 48 h until day 9.5 after IVF to evaluate post hatching development. In vitro viability was evaluated at day 9.5: those with a diameter greater than 200 μm and a total cell count greater than 180 were classified as viable (value 1), while the rest were classified as non in vitro viable (value 0). The area under the ROC curve (AUC) was estimated to determine the predictive power of in vitro viability through blastulation time. In addition, binary logistic regression analysis was used to generate a mathematical model with morphokinetic variables that allow the best prediction of in vitro viability. In 13 sessions, the blastocyst production rate was 46.2% (96/208). The cut-off time to discriminate early or late blastulation was 149.8 h. The post-hatching development of the embryos with early blastulation was 63.3% (31/49), being statistically superior (p = 0.001) than the late blastulation group 14.9% (7/47). Likewise, the time of blastulation showed an accuracy of 90.8% (p < 0.001) in predicting in vitro viability of bovine blastocysts. In conclusion, the selection of blastocysts based on blastulation time (< 155 h) and blastocyst diameter measured on day 7.5 after IVF (> 180 μm) maximizes the in vitro viability.

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P&lt;0.05). The values of sperm kinetic parameters obtained with swim-up (VCL 132.5µm/s; VSL 73.5µm/s) and microfluidic technique (VCL 129.5µm/s; VSL 64.4µm/s) were significantly higher (P&lt;0.05) than those obtained by density gradient (VCL 98.3µm/s; VSL 45.01µm/s). However, the total and progressive motility by density gradient method was slightly higher (89% and 57%) compared with that assessed by swim-up (64% and 43%) or microfluidic technique (74% and 54%) respectively. Microfluidic sorting (11.3%) showed lower (P&lt;0.05) DNA fragmentation levels compared with density gradient method (16.6%), whereas the swim-up technique (12.5%) was similar between both groups. No significant difference was detected between the 3 groups for sperm morphometric and vitality parameters. Moreover, cleavage rates were similar (P&gt;0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P&lt;0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.

Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

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