The Study On The Developmental Competence Of Equine Oocytes After ICSI And The Corpus Luteum Ultrastructure During Post-pregnancy Period In Mare

Embryo transplantation and ICSI have been developed overseas in mare. As a traditional country of horse breeding,China has wide markets foreground on horse meat,mare's milk and horse race,however,China is short of horses of fine breed. Embryo transplantation and IVF are the efficient paths to increase propagation frequence and accelerate the improvement of variety in horse.This study used mare ovary which from abattoir,investigate the affect pregnant or non-pregnant,ovary status to the weight and size of ovary and follicles distribution in mare. Following that,oocytes were collected from slaughterhouse-derived ovaries,matured in vitro,and injected with frozen-thawed stallion sperm,and then evaluate the effect of initial cumulus morphology (expanded or compact),systems of in vitro maturation (TCM199 or NCSU23) and duration of in vitro maturation (34h or 38h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI) in non-breeding season. We also investigate the affect ovary status (red body,corpus luteum,follicle,stillness) to the ovarian morphology and follicles distribution and the corpus luteum ultrastructure during post-pregnancy period in mare.The study on the ovary status and follicles distribution indicated that:(1)There is no significant difference for ovary weight,ovary status and follicles distribution between pregnant and non-pregnant mares(P>0.05);(2)Ovary status did not affect the weight of ovary(P>0.05),there is no significant difference for the size of ovary between different status(P>0.05);(3)The number of middle follicles in ovary with one big follicle was significantly higher than no big follicle,the diameter of big follicle increased with their number,the number of middle follicles and small follicles decrease when big follicles increased;(4)The ovary of follicles bigger than 20mm was about 19.02%,there was 4.65 follices in each ovary. Big follicles,middle follicles and small follicles was 9.00%,24.18% and 66.82%,respectively.From the study of the ultrastructure of corpus luteum during post-pregnaney period,the following conclusions were got:(1)LLC was cords distribution with round structures,SLC was fusiformis or polygonal structures;(2)Collagenous fibre were full of intercellular substance,most structures of cellular organ can not been seen. Corpus luteum stopping secreting progestational hormone during post-pregnaney period;(3)LLC and SLC were 26.06% and 73.94% in five fields of view at 3000×,respectively;(4)The longer diameter of LLC was 6.45μm,when SLC was 3.67μm. The shorter diameter of LLC was 3.29μm,when SLC was 1.67μm. The longer diameter of LLC nucleolus was 5.06μm,when SLC was 3.51μm. The shorter diameter of LLC nucleolus was 2.64μm,when SLC was 1.59μm;(5)Most luteal cells show the obvious regression during post-pregnancy period.The study on the developmental competence of equine oocytes after ICSI indicated that:(1)The maturation rates of expanded (Ex) oocytes were significantly higher than those of compact (Cp) oocytes (P<0.05,60.62% vs 40.68%). Thirty-six hours after ICSI of mature oocytes,the rate of cleavage with normal morphology was no significant difference with expanded or compact oocytes (P>0.05,47.00% vs 44.21%);(3)Two different systems of in vitro maturation did not affect development of embryos from expanded or compact oocytes at 36h after ICSI (P>0.05). The maturation rates of expanded oocytes were significantly higher than compact oocytes when matured in the same system (P<0.05),but there is no significant difference for the rate of cleavage with normal morphology at 36h after ICSI(P>0.05). The maturation rates were significantly higher for oocytes matured for 38h than for those matured for 34h (P<0.05,44.43%～68.87% vs 34.52%～58.90%). The rate of cleavage with normal morphology at 36h after ICSI was significantly higher for expanded oocytes matured in TCM199 for 34h than for expanded or compact oocytes matured in NCSU23 for 38h (P<0.05);(4)Combined activation with ionomysin and 6-DMAP did not affect the rate of cleavage with normal morphology at 36h after ICSI (P>0.05).These data indicate that developmental competence differs between expanded and compact equine oocytes,and is differentially affected by the duration of maturation.