Effect of follicle size on in vitro maturation in alpaca oocytes (Vicugna pacos) and the first ICSI in alpaca species

Abstract

The development of reproductive biotechnologies is essential for the productive improvement of South American camelids. The present study was conducted to examinate the potential maturation of alpaca’s oocytes derived from small and large follicles, as well as perform intracytoplasmic sperm injection (ICSI) after in vitro maturation (IVM). Ovaries were collected from a slaughterhouse and transported at 10 °C for 22 h. Cumulus-oocyte complexes (COCs) were obtained by aspiration from large follicles (2–8 mm), while COCs from small follicles (<2 mm) were obtained by performing superficial sections on the ovary cortex. COCs of I and II categories were selected and subjected to IVM for 32–36 h at 38.5 °C. ICSI was performed according to standard protocols. IVF was performed as control for ICSI. After ICSI, oocytes were chemically activated by 5 min with 5 µM Ionomycin and 3 h with 6-DMAP. The results showed that yield of morphologically good-quality graded COCs was significantly higher in large follicles (2.1 ± 0.8) than from the small ones (1.2 ± 0.7). However, after IVM no significant differences were observed in the rate of metaphase II (18.1% vs 22.1%) and degenerated oocytes (48% vs 57%). In the following experiment, ICSI (n = 27) was performed and compared against IVF (n = 46). The 44% of chemically activated oocytes cleaved after ICSI vs 37% after IVF. DNA staining of the presumptive zygotes after 168 h of embryo culture showed that 11% of IVF and 19% of ICSI embryos were at pronuclear stage; then 7% of IVF and 12.5% of ICSI zygotes reached the 2-cell stage. Subsequently, 15% of IVF and none ICSI reached the 4–8 cell stage. Finally, 4% of IVF and 12.5% of ICSI were at morula stage after 7 days of culture. Thus, our findings indicate that larger follicles yield a greater amount of morphologically high-quality oocytes, but also oocytes from smaller follicles might reach similar IVM competence. In addition, this study represents preliminary development of IVF and ICSI embryos in alpaca; however, more research is needed to optimize IVP conditions in this species.