Follicular fluid obtained after the LH surge-stimulates meiotic maturation of bovine oocytes

FOLLICULAR FLUID OBTAINED AFTER TItELPISURGE STIMULATES NEIOTIC MATURATION OF BOVINE OOCTTFS A. Romero. U.K. Thomas, S.E. Olson and G.E. Seidel, Jr. Animal Reproduction Laboratory, Colorado State University, Fort Collins, CO 80523 USA. This study was designed to determine whether normal meiotic maturation of bovine oocytes is due in part to a stimulatory factor in follicular fluid, rather than simply removal of an inhibitor. To obtain bovine follicular fluid (BFF), cows were given prostaglandin Faa on day 15 or 16 of the estrous cycle and checked hourly for estrus, starting 42 h post-injection. At the first sign of standing estrus, animals received 100 pg of GnRH i.m. Ovaries were obtained per vagina, and follicular fluid was collected from the dominant follicle at 0 h (no GnRH), 8 h, or 20 h post-LH surge. Follicular fluid from animals with a premature LH surge or that did not Cumulus-enclosed respond to GnRH was excluded from the experiment. oocytes were collected from l-6 mm follicles from slaughterhouse ovaries and cultured in 5% CO, in air at 39C. Follicular fluid from each time point (0, 8, and 20 h) was mixed (40%) with Hepes-buffered TCM-199 (Sigma cat. M2520) plus 0.8% BSA, and used as culture medium. A negative control designed to inhibit meiotic maturation (TCM-199 + 2.0 mM hypoxanthine + 0.2 mM adenosine) and a control with no additives were included. After 18 h of incubation, oocytes were stripped of cumulus cells and fixed in ethanol/acetic acid (3:l) for 4 days. Oocytes were stained with 1% orcein in 45% acetic acid, and the stage of meiosis was determined with bright field optics at 1562X magnifications. The experiment was replicated four times; 416 oocytes were used. The percentages of oocytes at different stages of meiosis follows: Stage of Meiosis

n

mtment

199 (no additive) HA (neg control) 0 h BFF 8 h BFF 20hBFF a.b.c

88 105 65 83 75

Degan

18 24 14 8 11

GV

17' 4gb 18' 20' 9'

Diak

Met-I

8 5 6 0 1

6 3 11 8 8

Ana-

6 9 9 4 3

Tel-I

7 0 0 5 4

Met-II

39b 11" 41b 54b" 64'

Means within columns without common superscripts differ (P
A similar percentage of oocytes remained in the Germinal Vesicle (GV) stage for all treatments except the negative control (HA). Also, fewer oocytes reached metaphase II with the negative control than with any other treatment (PcO.05). 0 h BFF and the no additive control had a similar proportion of oocytes at Metaphase II. More oocytes reached Metaphase II in the 20 h BFF treatment than in any other treatment (P
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JANUARY 1990 VOL. 33 NO. t