Isolated subpopulations of mass-harvested pig oocytes and their characterization by size, incidence of atresia and competence to mature in culture

Abstract

Pig oocytes were hand collected after follicular puncture or were mass harvested with stainless-steel screens and purified on sucrose density gradients. Most oocytes obtained by mass harvesting with a 75 μm screen and selecting on a 0 and 30% discontinuous sucrose gradient were meiotically incompetent and atretic after culture. Two enriched populations of oocytes were isolated from the 0-15% and 15-30% interfaces with a 106 μm screen and a 0%, 15% and 30% 2-step discontinuous sucrose gradient. These oocyte populations differed significantly in size (P < 0.001, t test) and in meiotic competence. More than 50% of the larger oocytes matured compared with 14% of the smaller oocytes (P < 0.002). Of the larger oocytes, 21% progressed beyond meiotic metaphase I, whereas all of the maturing small oocytes were arrested at first metaphase after culture (P < 0.002). Yields were 45 times greater for oocytes mass harvested with a 106 μm screen and a 15% sucrose density gradient than for oocytes hand collected in the same period of time. Layering these mass-harvested oocytes over a second 15% sucrose gradient or hand sorting the mass-harvested oocytes after culture increased the proportion of maturing oocytes compared with oocytes which were mass harvested before culture and produced higher proportions of oocytes at telophase I or beyond (P < 0.002). The results demonstrate that mass harvesting of pig oocytes is more efficient than hand selection, and that oocytes collected and selected in this way are at least as capable of meiotic maturation as are oocytes hand selected before culture.