METHODS FOR COLLECTING AND MATURING EQUINE OOCYTES IN-VITRO

The objective of our study was to develop an effective method for coll ecting and maturing equine oocytes. In Experiments 1 and 2, oocytes we re collected from excised ovaries obtained via colpotomy. In Experimen t 3, oocytes were collected from ovaries obtained after slaughter. Fol licles were aspirated and flushed with various treatments to recover t he oocytes, which were then cultured and stained to observe the stage of meiosis. In Experiment 1, the aspiration treatments consisted of 0. 5 ml of modified Dulbecco's PBS with 0, 100 or 500 IU\ml hyaluronidase . There was no increase (P>0.05) in oocyte recovery with the addition of hyaluronidase. The oocytes were cultured in either TCM-199 or Ham's F-10 medium containing 0.5 ug/ml FSH, 1 ug/ml LH, 1 ug/ml estradiol 1 7 beta, 250 uM Na-pyruvate and 10% estrual mare serum for 0, 24, 36 or 48 h. Maturation rates were higher (P<0.05) at 36 h for oocytes cultu red in TCM-199 (79%) than for those in Ham's F-10 (21%). There was no difference (P>0.05) in the percentage of maturation of oocytes between the 2 media at 48 h of culture. In Experiment 2, a single aspiration was performed with no flushing medium (dry aspiration) in 0.5 ml of PB S or in PBS with 1000 IU/ml hyaluronidase. The oocytes were then cultu red in TCM-199 for 24, 30 or 36 h. There was an increase (P<0.05) in o ocyte recovery when follicles were flushed with PBS, with or without h yaluronidase. There was also a difference (P<0.05) in the percentage o f maturation of oocytes between 30 and 24 h (86 vs 48%), but no furthe r increase was seen by 36 h (84%). In Experiment 3, follicles were asp irated with PBS 5 to 6, 6 to 7 or 7 to 8 h after slaughter. The oocyte s were cultured for 30 h in TCM-199 either with or without 100 IU/ml e CG. There was no effect of eCG or time from slaughter on oocyte matura tion or cumulus expansion (P>0.05).