66 Effect of sex in bovine twin production by blastomere separation and zona-free embryo culture

Monozygotic (MZ) twin production is used to obtain two embryos from a single zygote of a high genetic value or from a limited number of oocytes. Interestingly, when cows naturally have MZ twins, they tend to have more female than male twins. This difference could be related to sex differences in early embryo development. There are sex dimorphisms in early embryo kinetics, gene expression, or apoptosis, and while the cause is not clear, one plausible explanation is a temporal double dosage of the X chromosome in females. This could have implications in the blastomere separation technique used to obtain MZ twins or zona pellucida (ZP)-free embryo culture. The objective of this study was to evaluate the effect of ZP-free embryo culture and blastomere separation in the sex ratio of bovine embryos cultured in vitro. Ovaries were collected from a slaughterhouse and transferred to the laboratory. Cumulus–oocyte complexes (COCs) were aspirated from follicles of 2–8 mm using 18 G needles. COCs were searched in Tyrode’s albumin lactate pyruvate HEPES, with 1% penicillin-streptomycin fungizone (Gibco), then maturated in vitro in tissue culture medium 199 (Gibco) supplemented with FSH 10 µg mL⁻¹, 100-µL drops under mineral oil (MO) 22 h, in humidified air at 38.5°C (Gibco). Thawed bull sperm were washed in Brackett-Oliphant medium and co-incubated with matured COCs, 16 × 10⁶ sperm mL⁻¹, 100-µL drops under MO 5 h, in humidified air at 38.5°C. Presumptive zygotes were vortexed in hyaluronidase (Sigma) for 1 min to remove cumulus cells, and in vitro cultured in 50 µL drops of synthetic oviductal fluid, with 2.5% FBS, 38.5°C, 5% O₂ under MO. Thirty hours post-IVF cleavage rates were evaluated, and cleaved embryos were separated into three groups. G1 (control), in which embryos were cultured to Day 7 with no further treatment; G2 (ZP-free), in which embryos were treated 45–90 s with 1.5 mg mL⁻¹ pronase (P8811) to remove ZP and cultured in a handmade microwell system; and G3 (hemi-embryos), in which embryos were treated as in G2 to remove ZP, after which ZP-free 2- and 4-cell embryos were transferred to 50-μL drops of Dulbecco’s phosphate-buffered saline medium (Gibco) -Ca⁺ -Mg²⁺ and 20% FBS to produce two hemi-embryos. Blastomeres were separated by gently pippeting (4-cell embryos divided into two pairs). Hemi-embryos were cultured in handmade microwells. At Day 7, blastocyst rates were evaluated, and blastocyst sex was evaluated by nested PCR assay with SRY for sex diagnosis and amelogenin or GADPH as control. The blastocyst production results by group were the following: G1, 63/210 (30.00%); G2, 27/105 (25.61%); and G3, 55/107 (51.40%). Sex proportion for blastocysts by group was the following: G1, 42M:21F (2:1); G2, 16M:9F (1.77:1; two were not sexed); and G3, 36M:15F (2.33:1; four were not sexed). Blastocyst rate was evaluated by Fisher test (P < 0.05), and sex rate was evaluated by Chi-square test (P < 0.05). We reported twice as many male embryos as female. This male bias is probably related to Day 7 embryo sex evaluation and later development of female embryo. Because no differences were observed between groups, this bias is not affected by ZP removal or hemi-embryo production, contrary to what is observed in natural and twin offspring.