The effects of doses of porcine follicle-stimulating hormone (pFSH) and equine chorionic gonadotropin (eCG) for a simple superovulation method in ewes were investigated on ovulation rate, proportions of fertilized oocytes and normal embryos at recovery. The ewes were treated with intravaginal sponges impregnated with fluorogesterone acetate (FGA) for 12 days. For superovulation, an intramuscular injection of pFSH was given at 48 h before sponge removal, and eCG was given at 24 h after the pFSH injection. In Trial 1, the ewes (n=28) were divided into two groups. Group A (n=14) received 20 mg pFSH and 250 IU eCG, and Group B (n=14) was treated with 15 mg pFSH and 500 IU eCG. The effect on different breeds (Suffolk: Sf, Merino × polled Dorset: MD, South-Down: SD) was also investigated. In Trial 2, the ewes (n=20) were divided into 4 groups with different doses (0: control, 100, 200, 300 IU) of eCG combined with a 20 mg pFSH injection. All ewes were inseminated into the uterus with fresh-diluted or frozen-thawed semen by laparoscope, and the numbers of newly formed and regressed corpora lutea were recorded. Embryos were recovered on Day 6 or 7, and morula and blastocyst stage embryos were observed as being normal. There were no significant differences in the mean ovulation rates between Groups A (23.7 ± 21.5) and B (16.3 ± 9.8), and among the three breeds of ewes. The rates of fertilized oocytes and normal embryos were significantly higher in Group A than Group B (Trial 1). The ewes treated with 200 or 300 IU eCG had significantly shortened time-intervals from sponge removal to the onset of estrus and had higher ovulation rates than controls (Trial 2). The present study indicates that an appropriate eCG dose combined with a single injection of 20 mg pFSH would be 200-300 IU for a simple superovlation method in ewes pre-treated with progestogen-impregnated vaginal sponges.

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Buffalo ovaries were collected from a slaughter house and cumulus oocyte complexes were aspirated from follicles 2-6 mm in diameter. A portable CO₂ incubator and a CR1aa medium were used for in vitro production of embryos. In the first experiment, numbers of follicles and oocytes per ovary were counted, and classified according to the appearance of the cumulus cells. In the second experiment, in vitro fertilization and cultivation of buffalo oocytes matured in vitro were carried out using semen from three different bulls, which had different sperm motilities (Bull 1: 20%, Bull 2: 60% and Bull 3: 15%, respectively). In the third experiment, the cleavage and transferable embryos of fertilized oocytes from denuded oocytes, and oocytes with intact cumulus cell layers were compared. The percentages of good, fair and poor quality oocytes were 60.9%, 17.8% and 21.4%, respectively. The rates of cleavage and transferable embryos were significantly higher after incubation with semen from Bull 2 (81% and 40.2%) than those after incubation with semen from Bulls 1 (52% and 13.5%, p<0.05 and p<0.01) and 3 (15.8% and 0%, p<0.01). The rates of cleavage and transferable embryos were significantly higher (p<0.01) for oocytes with an intact cumulus than for oocytes without cumulus cells at fertilization.

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20α-Hydroxysteroid dehydrogenase (20α-HSD) converts progesterone to a biologically inactive steroid, 20α-dihydroprogesterone. To examine a potential role of 20α-HSD in tissues other than corpus luteum (CL) during pregnancy, mouse 20α-HSD cDNA was cloned and was used for the examination of its mRNA localization in the conceptus and uterus at different stages of gestation. Using probes based on the rat sequence, mouse 20α-HSD cDNA clone was isolated from a cDNA library prepared from ovaries of day 12 pseudopregnant mice. The mouse 20α-HSD cDNA was 1193 bp in length and encoded 323 amino acids with an estimated molecular weight of 37 kD. The mouse 20α-HSD was homologous to the rat 20α-HSD in nucleotide and amino acid sequences (both in 93% homology), and to other members of aldo-keto reductase superfamily at the cofactor (NADP(H)) binding site. By in situ hybridization, 20α-HSD mRNA was localized in endometrial epithelial cells on days 10, 15 and 18 of pregnancy, maternal placental endothelial cells on day 10, and fetal epidermal cells on day 15. During pseudopregnancy as well as early pregnancy, however, 20α-HSD mRNA was not detected in endometrial cells. Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation.

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Transitional multiparous dairy cows were used to estimate the relationships between the interval from parturition to the first ovulation (1st-OV), and the interval from parturition to the first day when the plasma metabolite concentration reached the steady range (within two standard deviations from the mean value for the post-ovulational 2 weeks). Blood samples were collected from 13 Holstein dairy cows 4 times every week from 14 days prepartum to about 60 days postpartum in order to analyze plasma concentrations of progesterone and the following eight metabolites: glucose, ketone bodies, free fatty acids (FFA), urea nitrogen (UN), free cholesterol, cholesterol ester, glutamic-oxaloacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT). The interval to 1st-OV averaged 25.6 ± 8.7 days, and correlated respectively with the intervals to the days when plasma concentrations of almost all the metabolites, except GPT and UN, reached the steady range. We concluded that the interval to the 1st-OV of dairy cows is closely related to the interval to the steady range of metabolites concerned with the energy status, but not protein intake. Additionally, the interval to the 1st-OV also correlated significantly with the mean concentration of free cholesterol before the 1st-OV.

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Comparative studies of four different intravaginal devices containing progesterone or synthetic progestogens on estrous incidence, LH surge and fertility of synchronized ewes by an intrauterine insemination with frozen semen by laparoscopy were conducted during the breeding season. A total of 161 Suffolk or Suffolk-crossed ewes at two sheep farms (Farms A and B) were used. In Farm A, the mean times of estrous onset after the removal of four intravaginal devices were 27.3, 31.2, 21.8 and 22.8 h for MAP, FGA, CIDR and P sponge (self-made progesterone sponge), respectively. The progesterone-impregnated intravaginal devices (CIDR and P sponge) induced estrus significantly earlier (P<0.05) than the devices impregnated with the synthetic progestogens (MAP and FGA) (22.3 ± 2.0 and 29.4 ± 2.3 h, respectively, after the removal of the intravaginal device). CIDR induced estrus (P<0.01) and LH surge (P<0.05) significantly earlier than FGA and MAP sponges, respectively. Pregnancy determined by the plasma progesterone concentration (>2.0 ng/ml) on Day 21 (Farm B) and lambing rates (Farms A and B) of ewes inseminated once on a fixed-time basis between 44 and 52 h after removal of intravaginal devices were not significantly different: 45.0, 41.5, 57.9 and 39.5% in lambing rates for MAP, FGA, CIDR and P sponge, respectively. Also, no significant differences in prolificacy were observed among the four intravaginal devices. These results indicate that the fertility of ewes treated with the four different intravaginal devices including a self-made P sponge and inseminated with frozen semen is similar, although the onset time of estrus and the LH surge varies with the use of progesterone- or progestogen-impregnated intravaginal devices.

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The ability of frozen-thawed boar spermatozoa obtained from epididymides stored at 4 C for 1 day to produce piglets after Fallopian insemination or in vitro fertilization (IVF) of in vitro matured (IVM) oocytes was examined. To test their in vivo fertilization ability, frozen-thawed spermatozoa from 2 boars, which had already shown IVF ability, were introduced into the Fallopian tubes of estrus-synchronized gilts. Embryo collection on the 7th day post-insemination revealed that one of two batches of spermatozoa had an ability of fertilization in vivo and that fertilized oocytes could develop to blastocysts. Three of 6 gilts inseminated with one batch of spermatozoa became pregnant and one of them farrowed 2 normal piglets (1 male and 1 female). IVM oocytes fertilized in vitro by the same spermatozoa were transferred to 3 recipients (200 oocytes per recipient). One of them became pregnant and farrowed 5 normal piglets (3 males and 2 females). The results indicate that boar spermatozoa collected from epididymides stored for 1 day at 4 C and then frozen have the ability to produce piglets. The new cryopreservation protocol and reproductive technology described here can enhance conservation of boar genetic resources when the collection site of the epididymides is far from a laboratory.

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The aim of this study was to improve the cryosurvival of bovine blastocysts following biopsy. In vitro-derived bovine blastocysts were biopsied with a metal blade, then cultured for 0 to 20 h in m-SOF and then vitrified in the presence of ethylene glycol and dimethyl sulfoxide. The post-warm survival rates after 24 h of in vitro culturing of the biopsied embryos were significantly higher at 1.25, 2.5, 5 and 10 h than that of 0 and 20 h (69, 73, 78 and 64% vs 34 and 27%, p<0.05, respectively). The survival rate of embryos vitrified at 2.5 and 5 h after biopsy was not different from that of the non-vitrified control group (73 and 78% vs 96%, p=0.08 vs 0.07, respectively). These results indicate that 1.25 to 10 h culture between biopsy and vitrification improves the viability of bovine blastocysts produced in vitro.

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Following ovulation, the follicle undergoes a period of rapid cellular remodeling that results in major capillary invasion and formation of the corpus luteum (CL). The production of angiogenic factors is responsible for CL development. The biologically active form of angiotensin (Ang), Ang II, has been shown to induce neovascularization, and the objective of the present study was to observe Ang II production and its action on secretory functions in the process of luteinization of granulosa cells in culture. Bovine luteinized granulosa cells (LGC) in culture were treated with Ang I (10^-11, 10^-10, and 10^-9 M) three times (Days 1-3, 3-5, and 5-7). None of these treatments resulted in a conversion of Ang I to Ang II. Ang II did not have any effect on the production of progesterone, prostaglandin E₂ or oxytocin at any of the doses used (10^-9, 10^-8, and 10^-7 M) at any time during the culture period. In addition, the effect of Ang II on cell proliferation was not significantly different from that of the control. These data indicate that Ang II may not be involved in regulating the secretory function of LGC or in regulating the rapid proliferation of LGC during development of the CL.

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