Superstimulation in donor cows increases the number of cumulus-oocyte complexes (COC), but when compared to in vivo maturation, in vitro maturation results in only half as many blastocysts after prolonged in vitro culture. The objective of this study was to establish a superstimulation protocol that would produce a maximal number of competent COC for standard in vitro embryo production. During experiment 1, eight cyclic Holstein heifers were superstimulated with four doses of FSH. Half the heifers received an injection of LH 6 h before ovum pick-up (OPU). The COC were collected following OPU either 33 or 48 h following the last FSH injection (coasting period). During experiment 2, six cyclic Holstein heifers were superstimulated with six doses of FSH, and in half the heifers, LH was administered 6 h before OPU. The COC were collected following ultrasound-guided transvaginal aspiration of both ovaries 48 h after the last FSH injection (coasting period). The COC originating from follicles with a diameter of 5 mm or more (n = 180 for experiment 1 and 57 for experiment 2) were subjected to standard in vitro maturation, fertilization, and development. When animals were administered four doses of FSH, 48 h of coasting resulted in significantly more 5- to 10-mm follicles (P < 0.01) than 33 h of coasting. If a 33-h coasting period was used, administration of LH 6 h before OPU resulted in a significant increase in both percentage of blastocysts and embryo production rate at Days 7 and 8 (P < or = 0.05) of in vitro culture. If a 48-h coasting period was used, LH injection did not affect the rates of blastocyst production. When donors were administered six doses of FSH with a 48-h coasting period, the highest results, although not significant (P < 0.08), were obtained when animals received LH 6 h before OPU, with 80% +/- 9% (mean +/- SEM) blastocysts and 0.8 +/- 0.09 embryo produced per COC retrieved per heifer at Day 8 of culture. Never has in vitro technology been so close to producing 100% developmentally competent COC.

A repeatable procedure for fertilization of bovine ova in vitro is described. Oocytes were recovered from ovarian follicles or from oviducts near the time of ovulation following treatment of donors with pregnant mare's serum gonadotropin (PMSG) and prostaglandin F2 alpha (PGF2 alpha). For in vitro capacitation semen was incubated, then high ionic strength treated and subsequently incubated in defined medium prior to insemination of oocytes. In one experiment frozen bull semen was successfully used. In experiments with 4 bulls (B, C, D, F), 34 (43.6%) of 78 ova and 13 (19.7%) of 66 follicular oocytes were fertilized in vitro. In the last series (spermatozoa from Bull F) the fertilization of 22 (62.9%) of 35 tubal ova was achieved. In vitro development proceeded to the 8-cell stage. No fertilization in vitro followed use of one male (Bull E), even though his spermatozoa could penetrate zona-free hamster ova in vitro, and higher than usual bacterial contamination of his semen was implicated as the probable cause. Findings suggested vigorous progressive sperm motility and acrosome integrity to be important features of good sperm samples. In one experiment a 4-cell stage embryo was transferred with the result that the recipient gave birth to a normal bull calf on June 9, 1981. The first calf resulting from in vitro fertilization has been found to be completely normal.

The main objective of this study was to identify mRNA expressed in the granulosa cells characterizing differentiated follicles bearing developmentally competent bovine oocytes. Analytical comparisons were made on mRNA pools of granulosa cells using differential display reverse transcription polymerase chain reaction (DDRT) analysis and suppressive subtractive hybridization (SSH). With DDRT, mRNA patterns of granulosa cells from small (< 4 mm) and large (> 8 mm) follicles cultured in the presence or absence of LH were compared to identify mRNA associated with follicular size or with the LH response. Nine clones were sequenced, and two were identified. One of the clones, DRAK 1, was associated with the presence of LH in the medium. Other comparisons directed toward the identification of mRNA associated with the presence of a competent oocyte were done on granulosa cells collected in vivo from superstimulated heifers. With the DDRT analysis, four clones associated with the oocyte developmental competence status were identified. With the SSH analysis, four clones specific to the presence of an incompetent oocyte were sequenced and none were identified, whereas 49 clones specific to the presence of a competent oocyte were sequenced and 18 were identified. Among these clones, early growth response 1, sprouty 2, cytochrome C oxidase, matrix metalloproteinase inducer, matrix metalloproteinase, epiregulin, prostaglandin receptor, and progesterone receptor were the most relevant to the ovarian physiology being examined.

Bovine seminal plasma (BSP) contains a family of novel phospholipid-binding proteins (BSP-A1/-A2, BSP-A3, and BSP-30-kDa; collectively called BSP proteins) that potentiate sperm capacitation induced by heparin or by serum high-density lipoprotein (HDL). BSP proteins stimulate lipid efflux from sperm that may occur during the early events of capacitation. Here, we investigated the role of BSP proteins, bovine follicular fluid (FF), and bovine follicular fluid HDL (FF-HDL) in sperm capacitation. FF and FF-HDL alone stimulated epididymal sperm capacitation (19.5% +/- 0.8% and 18.2% +/- 2.8%, respectively, control, 9.0% +/- 1.9%) that was increased by preincubation with BSP-A1/-A2 proteins (30.2% +/- 0.4% and 30.9% +/- 1.5%, respectively). In contrast, lipoprotein-depleted follicular fluid (LD-FF) alone was ineffective, and a preincubation with BSP-A1/-A2 proteins was necessary before sperm capacitation was stimulated (up to 22.8% +/- 1.4%). The interaction of BSP proteins with FF components was analyzed using ultracentrifugation, Lipo-Gel electrophoresis, SDS-PAGE, and gel filtration. We established that the BSP proteins interact with factors present in FF including FF-HDL. Additionally, we obtained evidence that BSP proteins, found associated with FF-HDL, were released from the sperm membrane during capacitation. These results confirm that the BSP proteins and the FF-HDL play a role in sperm capacitation.

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