The effects of heparin exposure on Equine oocytes matured in vitro

Abstract

Heparin is a glycosaminoglycan probably best known for its anti-coagulant activity, and its capacitation effects on spermatozoa, however its effects on the equine oocyte have not been thoroughly studied. Based on preliminary observations it was hypothesized-that heparin enhanced protease-induced post-culture cumulus cell loss for oocytes matured in vitro in equine follicular fluid, and that heparin exposure would have no effect on nuclear maturation. Over an eight-week period on nine separate occasions ovaries were obtained from an abattoir, 565 oocytes from 833 follicles were recovered from 177 ovaries. Oocytes were graded for gross cumulus morphology and randomly assigned to the control and treatment groups. All oocytes were cultured for 36 h in equine follicular fluid supplemented with gonadotropins and steroids, and in the case of the treatment group, two mg per ml heparin. Following culture, oocytes were treated with trypsin, scored for cumulus loss, and stained with aceto-orecein to assess nuclear maturation. Maturation rates for heparin-exposed and non-exposed oocytes were not significantly different: 26.83% and 27.01 % respectively (P- .968). Pre-culture cumulus morphology was slightly related to nuclear maturation (pooled data); cumulus grade one, two, and three oocytes had maturation rates of 31.58%, 18.45%, and 29.01 % respectively (P-.068); cumulus grade was related to maturation for control oocytes (P-.029), but not for oocytes exposed to heparin (P-.743). The nuclear status of the oocyte was slightly related to cumulus loss (P-.052). Heparin supplementation significantly increased (pooled data; P-.002) the rate of cumulus loss when compared to oocytes cultured similarly without heparin. Immature oocytes contributed more to this level of significance than did mature oocytes; more (P-.002) heparin-exposed immature oocytes (62%) were cumulus free than control immature oocytes (43.3 1 %). Heparin exposure did not seem to affect mature oocytes (P-.466). While prematuration cumulus grade is not related to post-maturation trypsin-induced cumulus loss for oocytes cultured in medium supplemented with heparin (P- .565), cumulus grade was related to cumulus loss for untreated oocytes (P-.003). Grade one oocytes had the highest (P-.00 1) rate of cumulus loss, and grade two oocytes had a higher (P-.042) rate of cumulus loss than grade three oocytes.