M. Konishi

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.

CHARACTERIZATION AND CYTOGENETIC ANALYSIS OF JAPANESE BLACK CALVES PRODUCED BY NUCLEAR TRANSFER

To confirm the normality of the Japanese Black calves produced by nuclear transfer, we examined the properties of such calves at parturition and analyzed their karyotypes. Thirty Japanese Black calves were produced by nuclear transfer; 3 of these calves (10.0%) required assisted delivery while 1 calf (3.3%) died soon after birth. Average birth weight was 31.0 +/- 1.8 kg and gestation period was 286.4 +/- 1.0 d (mean +/- SEM). None of the nuclear transfer calves showed external malformations. Within sets of cloned nuclear transfer calves, which were genetically identical, birth weights varied by up to 20.5 kg. Among singleton Japanese Black calves, the mean birth weight of nuclear transfer calves was significantly greater (P < 0.05) than that of calves produced by in vivo-derived embryo transfer. Cytogenetic analysis of 23 Japanese Black nuclear transfer calves revealed the presence of 2N 4N mosaicism in 21 of the nuclear transfer calves. The frequency of occurrence of tetraploidy was unrelated to birth weight. Endoreduplication was observed in 1 Japanese Black nuclear transfer calf, and the frequency of occurrence of the endoreduplication in this calf was 0.5% (1/209). We conclude that there was no external malformation or chromosomal aneuploidy in Japanese Black nuclear transfer calves, but the mean birth weight of nuclear transfer calves was heavier than that of in vivo-derived embryo transfer calves in both sexes, and a variation of birth weight within sets of nuclear transfer calves cloned from the same embryo was recognized.

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.

Superovulation of holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.

Effect of active immunization of cattle against inhibin on ovarian follicular development and ultrasound-guided transvaginal follicular aspiration

Abstract The effects of active immunization of cattle against inhibin on ovarian follicular development and ultrasound-guided transvaginal follicular aspiration were determined. Six Multiparous Japanese Black were actively immunized with a synthetic peptide replica of the amino acid sequence from 1 to 26 (numbering from the N-terminal end) position of the α-subunit of porcine inhibin (pINH) conjugated with rabbit serum albumin (RSA) using Freunds complete adjuvant. At 6, 10 and 14 wk after the primary injection, booster injections of the peptide were given. As controls, 6 Japanese Black received rabbit serum albumin as described above. Blood samples were taken at 0, 2, 6 wk and once a week from 10 to 17 wk after the primary injection to measure antibody titer. The changes in the numbers of recruited follicles of the 3 size categories (small, 2 to 3 mm; medium, 4 to 9 mm and large, ≥ 10 mm) and aspirated oocytes were examined once a week from 10 to 17 wk using ultrasound scanning. Antibody titers increased after the first booster in all immunized cattle. In comparison with controls, inhibin-immunized cattle had more small, medium and large follicles during the ultrasound scanning (16.0 ± 1.8, 4.0 ± 0.6 and 2.2 ± 0.3 versus 9.1 ± 0.5, 1.7 ± 0.2 and 1.1 ± 0.1, respectively; P

Unaged bovine oocytes successfully develop to blastocysts after parthenogenic activation or nuclear transfer

UNAGED BOVINE OOCYTES SUCCESSFULLY DEVELOP TO BLASTOCYSTS AFTER PARTHENOGENIC ACTIVATION OR NUCLEAR TRANSFER Y. Aoyagi, E Konishi, T. Wada and T. Takedomi Embryo Transfer Laboratory Zen-Noh, Tsukuba, Ibaraki, 300-33, Japan We have previously reported that activated bovine oocytes can develop to the blastocyst stage(Aoyagi et al.,Theriogenology 37:188,1992). The present study was carried out to investigate the development to blastocysts of in vitro matured and a r t i f i c i a l l y activated oocytes or nuclear transfer embryos. In vitro matured oocytes(24h) with cumulus cells removed by 0.1% hyaluronidase were treated with Ca-ionophore A23187(24h; 5~M, 5min) ÷ electr ic pulse(25h;lOOV/mm, 90~sec) ÷ cycloheximide(24 to 30h;10 ~g/ml). The activated ova were treated with cytochalasin D(24 to 42h;2.5~g/ml) and then were cultured in CRlaa + 5%CS for 7d. In vitro matured and enucleated oocytes were activated by treating with Caionophore(24h) ÷ electr ic pulse(25h) + cycloheximide(24 to 3Oh) as above. Nuclear (D5 in vivo blastomere) transfer ova were fused in a solution of 0.3Mmannitol, O. 1~ MgS04 and O. 05mM CaCl2 by DC pulse(lOOV/ram, 60 ~sec) at 31h and then were cultured in CRlaa ÷ 5%CS for 7d. One or two embryos derived from nuclear transfer were non-surgically transferred to each of 15 recipient heifers at 6 to 8 d post estrus. The activation rate as assessed by two pronuclei formation was 94.6%(35/37). The development rates to blastocysts of activated and nuclear transfer(NT) embryos were as follows: