Abstract

Background and Aim: Vitrification is a promising cryopreservation technique for conserving the genetic resources of Aceh cattle; however, it may induce cryoinjury and follicular apoptosis. Pituitary extract (PE), containing follicle-stimulating hormone (FSH) and luteinizing hormone (LH), can potentially mitigate apoptosis and maintain follicular viability. This study investigated the effect of bovine PE supplementation in vitrification media on the expression of caspase-3, FSH receptor (FSHR), and LH receptor (LHR) in vitrified ovarian tissue of Aceh cattle.

Materials and Methods: A factorial, completely randomized design was employed using five vitrification media, PE at 0, 200, 400, and 600 μg/mL, and commercial bovine PE (Gibco, 300 μg/mL), combined with three vitrification durations (0, 7, and 14 days), each in triplicate. Post-vitrification ovarian tissue was evaluated by immunohistochemistry for caspase-3, FSHR, and LHR expression. The intensity scores (IS) were analyzed using the Kruskal–Wallis and Mann–Whitney U tests (p < 0.05).

Results: PE supplementation significantly (p < 0.05) reduced caspase-3 expression across all follicular stages, indicating decreased apoptosis, whereas vitrification duration alone showed limited effects. The lowest caspase-3 IS (negative) occurred in the 600 μg/mL PE group after 7 days. Both PE concentration and vitrification duration significantly influenced FSHR and LHR expression (p < 0.05), with strong positive interactions between the two factors. FSHR and LHR expression increased progressively from primordial to antral follicles, suggesting preserved gonadotropin responsiveness. Optimal follicular preservation and receptor integrity were achieved at 600 μg/mL PE following 7 days of vitrification.

Conclusion: Supplementation of vitrification media with 600 μg/mL bovine PE effectively suppresses follicular apoptosis while enhancing FSHR and LHR expression, maintaining ovarian functionality after vitrification. These results highlight PE as a low-cost, multi-hormonal alternative to single-gonadotropin supplements in cryopreservation media, supporting sustainable genetic conservation of Aceh cattle. Integrating locally derived PE into cryopreservation protocols could improve post-thaw follicular survival, reduce dependency on commercial reagents, and strengthen reproductive biotechnology for indigenous livestock conservation.

Keywords: Aceh cattle, caspase-3, cryopreservation, follicle-stimulating hormone receptor, luteinizing hormone receptor, pituitary extract, vitrification.