Effects of follicle-stimulating hormone followed by gonadotropin-releasing hormone on embryo production by ovum pick-up and in vitro fertilization in the river buffalo (Bubalus bubalis)

Abstract

In this study, we examined the effects of superstimulation using follicle-stimulating hormone (FSH) followed by gonadotropin-releasing hormone (GnRH) on buffalo embryo production by ultrasound-guided ovum pick-up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU-IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice-daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23-24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi-layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU-IVF in river buffaloes.

Keywords: FSH; GnRH; OPU-IVF; Water buffalo.

Figure 1

Schematic of ovum pick‐up (OPU) for in vitro embryo production. Nine Murrah buffaloes were used in this study. Buffaloes were injected with prostaglandin F_2α (PGF _2α) with a 12‐day interval for synchronization. Five days after the second PGF _2α injection, buffaloes were subjected to OPU without superstimulation (control), and the oocytes collected were subjected to in vitro maturation (IVM, 22 hr), in vitro fertilization (IVF, 6 hr), and in vitro culture (IVC, 6 days). Two days after the first OPU, buffaloes were subjected to a follicle‐stimulating hormone (FSH) treatment that consisted of twice‐daily intramuscular injections for 3 days with a decreasing dose of FSH (6, 6, 5, 5, 4, and 4 AU per injection in the morning and afternoon for a total of 30 AU). At the last FSH injection, PGF _2α was also administered, and, after 2 days, gonadotropin‐releasing hormone (GnRH) was injected. The second OPU with superstimulation (superstimulated group) was conducted 23‐24 hr after the GnRH injection. Collected oocytes with expanded cumulus cells were defined as in vivo‐matured oocytes, and subjected to IVM (3 hr), IVF (6 hr), and IVC (6 days). Other collected oocytes were allocated as in vitro‐matured oocytes, and subjected to IVM, IVF, and IVC, similar to the non‐stimulated control