Daniel Bousquet

Manipulation of Follicular Development to Produce Developmentally Competent Bovine Oocytes

Abstract 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 ≤ 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.

In vitro embryo production in the cow: An effective alternative to the conventional embryo production approach

Development of new technology related to in vitro embryo production has allowed for the commercial use of this method of reproduction. In the present work, we evaluate the efficiency of this technology compared with conventional embryo production based on results obtained with a standard procedure, including the sexing of embryos. The donor animals were mature nonlactating dairy cows (n = 92) kept under a constant environment and feeding program in an ET center. Ultrasound guided transvaginal ovum pick-up following 48 h pre-treatment with FSH has been used for the IVF-IVC protocol. A total of 437 oocyte recovery sessions performed on 92 cows yielded 4145 oocytes, which were used in an IVF-IVC protocol. Using the conventional approach, 156 embryo collections on 49 cows yielded 1652 ova and embryos. All Quality 1 and 2 embryos were sexed by a PCR procedure, and embryos of the desired sex were transferred to synchronized recipients located at the center. The results obtained in the IVF protocol showed that 4 oocyte collections per cow performed within 60 d, yielded 38 oocytes, which resulted in 18.8 viable embryos, of which 7.05 were female. After transfer of the female embryos, an average of 3.8 recipients were pregnant at 60 d. One embryo collection under the conventional approach yielded an average of 1.2 female pregnancies, which was confirmed during the same 60-d time period. These results indicate that IVF procedures can effectively replace conventional embryo production methods when a predetermined number of pregnancies of known sex are needed within a short period of time.

Differential Display and Suppressive Subtractive Hybridization Used to Identify Granulosa Cell Messenger RNA Associated with Bovine Oocyte Developmental Competence

Abstract 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.

Effect of Seminal Phospholipid-Binding Proteins and Follicular Fluid on Bovine Sperm Capacitation

Abstract 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.

Penetration of zona-free hamster ova as a test to assess fertilizing ability of bull sperm after frozen storage.

Frozen stored sperm samples of two Holstein bulls (A and B) were compared for their abilities to interact with zona-free hamster ova. The percentage of hamster vitelli interacting with sperm from Bull A was significantly higher than that interacting with sperm from Bull B (94.5% vs. 68.2%, P<0.01), and these results were in good agreement with 60 day non-return data for the same months (68.2% for Bull A vs. 64.3% for Bull B). Sperm from Bull A also excelled in average numbers attached to vitelli, and in average numbers of sperm penetrated into the zona-free hamster ova. However, of the penetrated vitelli, sperm of Bull B resulted in more pronuclei. In these experiments the percentage of progressively motile sperm at insemination was highly correlated with the percentage of vitelli interacting with sperm. The percentages and numbers of sperm cells with intact acrosomes were significantly correlated with the average number of sperm attached per vitellus. These observations encourage further development of this test for assessing sperm fertilizing ability.

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle.

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.

Efforts to correlate laboratory with field observations on bull sperm fertility.

This work represents efforts towards development of the zona-free hamster ovum sperm penetration assay for predicting relative levels of fertility for semen from individual bulls. Results reported here followed insemination of hamster vitelli with bull sperm, after frozen storage, with observations of sperm acrosomes and parallel inseminations of more than 1000 cows with semen from each bull. The average 75-day non-return rate for the four bulls was 74.0% (range 71.6 to 75.6). Laboratory studies indicated the following: the percentage of sperm with intact acrosomes varied from 55 to 73 between bulls, the percentage of motile sperm varied from 41 to 64, the percentage of sperm with progressive motility ranged from 24 to 40, the number of sperm interacting per (zona-free hamster) ovum ranged from 1.6 to 3.8, the number of sperm attached per ovum ranged from 1.4 to 2.9, the number of sperm within each penetrated ovum ranged from 1.5 to 1.8, the percentage of ova interacting with sperm ranged from 76 to 92, the percentage of ova penetrated ranged from 62 to 85, and the percentage of ova with male pronuclei ranged from 33 to 49. Although predictive ranking in the laboratory of these bulls with less than 4% variation in fertility levels was not possible, the zone-free hamster ovum test could be useful in identifying potentially subfertile bulls before they enter a young sire-sampling program.