224 Superstimulation effect on in vitro embryo production and relationship with anti-Müllerian hormone in buffaloes (Bubalus bubalis) aspirated 7 or 14 days after follicular ablation

Abstract

The success of buffalo in vitro embryo production (IVEP) depends, in part, on the number and competence of oocytes. Superstimulation with FSH has been linked to oocyte quality and embryo yield, and anti-Müllerian hormone (AMH) has been proposed as an indicator of ovarian reserve. The objective was to evaluate FSH stimulation in one ovum pick-up (OPU) cycle conducted 7 or 14 days after follicular ablation; we additionally analysed residual FSH treatment effect carried over to a subsequent aspiration cycle. Finally, we evaluated the relationship between plasma AMH and FSH response. Nine multiparous, cycling Murrah donors (603 ± 7.0 kg, body condition score = 3.5) were subjected to complete follicular ablation followed by OPU 1 week later (T0 Control), and plasma AMH was determined at this point. They were then randomly allocated to FSH stimulation (3 days b.i.d. in descending doses, total 300 mg of pFSH, Vetpharm, with the last injection 48 h before OPU) with aspiration conducted 7 (T7 FSH, n = 5) or 14 days (T14 FSH, n = 4) post-follicular ablation. The residual effect (T Residual, n = 5) was evaluated in one subsequent, nonstimulated OPU cycle conducted 1 week later (9 days after last FSH administration). For plasma AMH, concentration was measured using ELISA kit (cat. no. AL114, AnshLabs; sensitivity 1.0 pg mL⁻¹, interassay variation <5%, additional validation conducted with serial dilutions of buffalo plasma, r = 0.98 against standard curve). Continuous data were analysed with ANOVA and Tukey's post hoc comparisons; categorical data were analysed using Chi-squared test. Pearson coefficient was used to evaluate correlation between AMH, follicular reserve, and in vitro performance. Superovulatory treatment resulted in a higher number of follicles and good quality oocytes (grades 1 and 2) per donor, irrespective of OPU interval (T7 FSH: 3.0 ± 2.55 and T14 FSH: 1.5 ± 0.5 vs. T0 Control: 0.45 ± 0.38 and T Residual: 0.4 ± 0.4; P < 0.05). There were more cleaved embryos (T7 FSH: 26%, T14 FSH; 30% vs. T0 Control: 7% and T Residual: 20%) and blastocysts (T7 FSH: 21%, T14 FSH; 30% vs. T0 Control: 5% and T Residual: 0; P < 0.05) from FSH-treated donors. There was a strong correlation (r = 0.9439) between follicle count and plasma AMH, and weaker relationships for oocytes recovered (r = 0.3975), cleaved embryos (r = 0.2317), or blastocysts (r = 0.0106). Results indicate that FSH treatment with a 48-h coasting period results in significant improvements in follicular population, oocyte quality, and embryonic development; these effects are observed independently of the interval between initial follicular ablation and OPU. No residual effects of FSH treatment were observed, indicating that the beneficial effects of superstimulation are not carried over to the following OPU cycle. In conclusion, a strong correlation coefficient was established between initial plasma AMH level and follicular response to FSH treatment, indicating that it may be used as a selection criterion for animals likely to exhibit a good response to superstimulatory treatment.