15 The effect of different bovine oocyte recovery methods on oocyte ultrastructure pre- and post-in vitro maturation

Abstract

In current in vitro embryo production practices, method of oocyte recovery tends to be based primarily on the personal preference of the operators and availability of resources rather than on any practical understanding of the best practices for retrieving oocytes of the greatest competence for embryo production. There is little consensus on the effects of either postmortem follicular atresia or exogenous hormone-stimulated follicle development on the quality of oocytes, but it is expected that both interventions have effects. Cumulus-oocyte complexes were collected from beef cattle using 3 methods: unstimulated ovum pickup (OPU), OPU following superstimulation (FSH), and oocyte aspiration from postmortem-recovered ovaries (PM). Each collection method was repeated 3 times, with 25 individuals used in each. There were no interventions in place before oocyte recovery in either OPU or PM. In FSH, cows underwent dominant follicle removal and were treated with 200 mg of FSH in 6 doses before OPU. The majority of oocytes (66 OPU, 80 FSH, and 160 PM) were fertilized and cultured, with a portion being reserved for transmission electron microscopy (2 per treatment at each of germinal vesicle and metaphase II) and a portion being reserved for a complementary study. However, oocytes collected by OPU following FSH had notable ultrastructure differences from those collected by other methods, suggesting a potential effect of follicle stimulation on both the development and cytoplasmic maturation of oocytes. Although oocytes from all recovery methods and at both maturation levels exhibited mitochondria primarily in the hooded conformation, both OPU and FSH oocytes, at germinal vesicle and metaphase II, had several anomalous mitochondria exhibiting a lobulated, budding-type shape. This is potentially indicative of these oocytes having failed to complete the mitochondrial division phase before recovery. An average of 11.6, 10.6, and 11.9 oocytes per individual were recovered from PM, FSH, and OPU, respectively, resulting in oocyte recovery of 55, 61, and 57% of aspirated follicles, respectively (P > 0.05). Fewer oocytes from OPU developed to morulas compared with other recovery methods (chi-square; P < 0.01; 24% PM, 21% FSH, and 3% OPU). Cleaved embryos from OPU had decreased blastocyst production rates at 14% compared with 59% (PM) and 55% (FSH; chi-square; P < 0.01). Overall results suggest that although superstimulation may affect oocyte ultrastructure, this does not have a negative effect on oocyte competence, though further investigations are required. Oocyte recovery rates were not affected by recovery method, but oocytes recovered by unstimulated OPU had decreased developmental competence compared with those recovered either postmortem or following superstimulation.