Superovulation (SO) with FSH was induced in 23 dairy cows and 12 dairy heifers which were treated on the day of embryo collection (Day 7) with a single luteolytic injection of either 500 mg or 1000 mg of Cloprostenol (Clp). The animals were clinically examined and their plasma progesterone (P₄) levels were monitored for 30 days after Clp injection. Estrus signs were checked twice daily. The ovaries were scanned by ultrasonography to follow corpora luteal (CL) regression, follicular, development and ovulation. The CLs regressed in all 34 animals responding to SO. Estrus signs occurred within 16 days after Clp injection in 91.2% of the population (19 cows and 12 heifers); ovulation occurred in 55.9% of the 34 animals responding to SO (12 cows and 7 heifers ). Of the animals showing estrus, the 7 cows and 5 heifers (38.7%) that did not ovulate either became cystic (4 cows and 3 heifers) or had follicular growth waves which never reached ovulation(3 cows and 2 heifers). Furthermore, 3 cows (8.8%) did not show estrus signs during this period. In the second cycle following Clp administration, the interestrus interval was within normal ranges (17 to 24 days) and estrus was followed by ovulation even in most of those animals with previously non-ovulated follicular waves. The cystic cases (20.6%) and 1 anestrus case, however, remained. Heifers had a significantly shorter interval from embryo collection to first estrus than cows. No significant differences in the effect of the 2 Clp dosages assayed on the interval from embryo collection to the first ovulatory estrus were found in those animals having a normal ovulatory estrus. The P₄ plasma profiles confirmed both luteolysis after Clp injection and the occurrence of ovulation. In heifers, the concentration of P₄ on the day of embryo collection and 24 h thereafter was positively correlated with the interval from collection to first estrus. Besides the satisfactory luteolytic effect of Clp, the resumption of cyclicity seemed to be more influenced by the disturbed ovarian dynamics at the time when Clp was injected than by its dosage.

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Two trials were conducted to investigate the effects of the number of follicle stimulating hormone (FSH) injections combined with pregnant mare serum gonadotropin (PMSG) on ovulation and recovery rates, normality and freezability of recovered embryos. Eleven Corridale ewes (Trial 1: October 1994, Trial 2: December 1994 [1 ewe excluded]) were treated with a vaginal sponge containing 60 mg 6-methyl-17-acetoxyprogesterone (MAP) for 12 days. In 2 trials, 3 days before sponge removal, superovulation was induced using 20 mg of FSH, administered in either a single injection or twice daily over a 3-day period in a decreasing dosage (multiple), in combination with PMSG (500 I.U.). In Trial 1, estradiol-17β (E₂) and luteinizing hormone (LH) were measured to evaluate the endocrine response to FSH injections in the 2 superovulation methods. In both trials, there were no significant differences in ovulation and recovery rates (Trial 1: single=17.5 ± 4.1, 69.4% vs multiple=23.0 ± 1.9, 57.4%; Trial 2: single=19.2 ± 5.5, 58.3% vs multiple=19.6 ± 5.7, 57.1%) between the treatments. There were also no significant differences on the numbers of normal and freezable embryos. There were no significant differences in the levels of E₂ and LH except the levels of LH at 6 h and 8 h after sponge removal for multiple FSH injection were higher than they were for the single injection (P<0.05). This suggested that the number of FSH injections did not affected the endocrine response in the superovulated ewes during the breeding season. In conclusion, FSH administered in a single injection together with PMSG was as effective as the same total dosage of FSH administered in 6 injections over a 3-day period.

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The aim of this study was to develop the transcervical method of pig embryo transfer using a rubber spiral catheter. A total of 587 ova and embryos were surgically collected on Days 4 to 6 (Day 0=onset of oestrus) from 36 donors, of which 456 transferable embryos were transcervically transferred to 25 synchronous (0 or ± 1 day) recipients, without any premedical treatment, using a sterile rubber spiral catheter covered with a vinyl sheath. Embryos (the mean number ± s.d.=17.8 ± 7.9) were transferred to each recipient through the catheter with either 30 ml or 50 ml of phosphate buffered saline, which was supplemented with 0.4 mg/ml amikacin sulfate and 10% fetal calf serum, by the 2- or 3-stepwise method. Out of 25 recipients, 16 recipients became pregnant (64%) and farrowed (64%). The mean number of piglets farrowed (± s.d.) was 3.1 ± 1.6, and the mean embryo survival rate (± s.d.) was 16.7 ± 7.6%. No backflow was detected during and immediately after embryo transfer in any cases. The pregnancy and farrowing rates were 44% (4/9), 60% (6/10) and 100% (6/6) as they related to the combination of resistances to rotation of the catheter and to infusion of PBS with a syringe, (+, +), (+, -) and (-, -) in order, respectively. The insertion depth of the catheter in the case of (-, -) was significantly larger (P<0.01) than that in the cases of (+, +) and (+, -). No differences in the number of piglets farrowed and in the embryo survival rate were observed between the three combinations with respect to the resistance. The results indicated that a high pregnancy rate can be achieved by the transcervical transfer of pig embryos using a rubber spiral catheter when no resistances both in the rotation of catheter and infusion of fluid containing embryos are detected during embryo transfer.

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The occurrence of early pregnancy-associated thrombocytopenia (EPAT) in adult female rabbits was examined in the present study. The rabbits were divided into 5 groups: (i) 5 rabbits were superovulated with follicle stimulating hormone (FSH) and human chorionic gonadotropin (hCG) and mated, but the embryos were not recovered; (ii) 6 rabbits were superovulated and mated, and the embryos were recovered; (iii) 8 rabbits were splenectomized and mated, and the embryos were recovered; (iv) 6 rabbits were splenectomized and injected with hCG; and (v) 3 control rabbits underwent sham operations and they were injected with hCG. In each case, blood (2 ml) was obtained by venipuncture from the marginal ear vein from the day of hCG injection to the day of embryo collection. A significant change of peripheral platelet concentration (PLT) was characterized either by a decrease or by an increase of >20% compared with the value of the initial sampling time. The proportions of rabbits which displayed a significant change in PLT were 80% (4/5) in Group i, 83% (5/6) in Group ii, 100% (8/8) in Group iii, 83% (5/6) in Group iv and 67% (2/3) in Group v(P>0.05). In Groups i and ii, 91% (10/11) of rabbits showed an increase of PLT. In Group iii, 75% (6/8) of rabbits showed a decrease of PLT (EPAT). In Groups iv and v, although each rabbit showed a decrease of PLT, the PLT patterns were variable. Effects of group (interaction of treatment/condition × pregnant status) and pregnant status on PLT were significant (P<0.0004 and P<0.0003 respectively). No effect of time after hCG injection on PLT was detected. Significant differences of PLT were observed only between Group i or ii and other groups, when least square mean was compared with a variance of time after hCG injection. From the results of the present study, splenectomy for the pregnant rabbits was requisite for an occurrence of EPAT. Also, the results suggest that EPAT does not occur in normal rabbits. Observations in splenectomized rabbits suggest that platelet activation may be occurring in early pregnancy but not to a sufficient degree to cause measurable thrombocytopenia in peripheral blood.

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Aromatase (EC 1.14.14.1.) is a key enzyme of feminizing hormone biosynthesis and catalyzes the conversion of androgens to estrogens. In birds, sex-steroid hormones, especially estrogens, play a critical role in the development of the gonadal glands and the aromatase is one of the most important factors in sex determination. Herein, we cloned the 5' upstream regions of chicken and quail aromatase genes and determined these sequences, which showed high homology between chicken and quail. Moreover, the transcription initiation site of the chicken aromatase gene in early development was determined by the 5'-RACE method. The findings showed that the transcription of the chicken aromatase gene starts from a more upstream site than previously reported.

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The effect of NaCl in maturation media on histone H1 kinase activity of porcine oocytes was examined at the end of culture for maturation. Oocyte-cumulus complexes were cultured in modified Whitten's medium containing different concentrations of NaCl (44.50, 68.49, 92.40, 116.40 or 140.35 mM). The media were supplemented with 10% porcine follicular fluid and hormones for 20 h and then without hormonal supplements for an additional 20 h. At the end of culture, oocytes were sampled for morphological observation, for glutathione assay and for histone H1 kinase assay. At the end of culture, the incidence of oocytes at each meiotic stage was not different among groups. However, histone H1 kinase activity and glutathione content were higher (P<0.01) in oocytes cultured in media with lower NaCl concentrations. Histone H1 kinase activity in the oocytes at the end of maturation culture was correlated with intracellular glutathione content (r=0.899, P<0.01). These data indicate that histone H1 kinase activity and glutathione content of porcine oocytes at the end of culture for maturation are reduced when a high salt medium is used for maturation.

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A specific decrease in the number of ultrasonographically identified follicles (uiFOL) in Japanese Black cows at 92 to 98 days after parturition had previously been reported. In the present study, the number of uiFOL in cows was observed until 500 days postpartum by an ultrasonic wave technique. The observation results showed that there are two specific decrease periods at about 303 to 308 days and 495 to 500 days after parturition. Also when superovulation treatments were done at these periods, many transferrable embryos were recovered. Therefore, these periods well resembled the previously reported decrease of uiFOL and embryo production at 92 to 98 days. To elucidate whether or not the variation of uiFOL including the specific decrease period resulted from a phenomenon with multiple periodicity or random variation, the ovarian follicular dynamics for 14 to 500 days after parturition were analyzed using the spectral analysis method based on the maximum entropy method (MEM). The variation in the number of uiFOL was constituted of a distinct periodic structure in each cow. In addition, the frequency distribution of the values estimated by MEM spectral analysis was investigated. As a result, five peaks were identified and a peak of 0.045 corresponded to 21 days postpartum. The other three peaks except for 0.03 corresponded to multiples of 21 days. Accordingly, the frequency distribution demonstrates that ovarian follicular dynamics is based on a 21-day cycle, and that it starts from parturition day.

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We have previously cloned 4 bovine male-specific and 3 gender-neutral repetitive DNA fragments [1] and successfully utilized them to sex bovine embryos [2]. Further characterization and mapping of these repetitive fragments were made here. All 4 male-specific repeats were mapped to the proximal region of the short arm of the Y-chromosome. Although a Southern blot analysis showed no specific hybridization to sheep, goat, and pig DNAs, the fluorescence in situ hybridization (FISH) identified weak signals in the proximal region of the short arm of the ovine and caprine Y-chromosomes. The copy number of the male-specific repeats was roughly estimated to be around 100 in the bovine genome. One of gender-neutral repeats was a part of mitochondrial DNA (NADH dehydrogenase5 gene), which showed crucial deviations from the known sequence [3] and thought to be a pseudo-gene in the nucleus. Another one was a combination of an autosomal sequence and satellite DNA, which was mapped to centromeric region of most bovine chromosomes excepting sex-chromosomes. This hybrid DNA was bovine-specific as revealed by the Southern blot analysis against the other domestic animals.

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The present study was undertaken to investigate whether bovine granulosa cells obtained from small antral follicles could differentiate into luteal-like cells in vitro. Granulosa cells obtained from small antral follicles (3-5 mm in diameter) were cultured up to 9 days in the presence or absence of insulin (2 μg/ml), forskolin (10 μM) and insulin (2 μg/ml) plus forskolin (10 μM). On days 1, 3, 5, 7 and 9 of culture, spent media were harvested for hormone assays. Progesterone production in the granulosa cells incubated with insulin plus forskolin increased continuously during the culture period. On the other hand, estradiol-17β production in the granulosa cells sharply decreased on day 3, and then continued to decrease. The size of granulosa cells cultured with insulin plus forskolin for 9 days was significantly larger than that of granulosa cells at the beginning of culture (P<0.001), and the size was similar to that of large luteal cells. Collectively, these data suggest that bovine granulosa cells obtained from small antral follicles can differentiate into large luteal-like cells during culture in the presence of insulin and forskolin.

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The major ganglioside compositions of placental tissues from several species (rat, sea lion, porpoise, cow and horse) were compared. The samples were extracted with chloroform/methanol/water and the glycosphingolipid fractions were isolated through sequential treatment by peracetylation with pyridine/acetic anhydride, Florisil column chromatography, and deacetylation with sodium hydroxide. Gangliosides were isolated using DEAE Sephadex A-25 column chromatography. Their structures were identified by silica-gel thin-layer chromatography (TLC) and TLC/immunostaining using eight monoclonal antibodies that specifically recognize GM3, GM2, GD3, GD1a and IV³NeuAc-nLc₄Cer (SPG). The major ganglioside in each placenta was GM3, comprising 50-80% of the total gangliosides. GM3 containing N-acetylneuraminic acid (NeuAc) was the major placental ganglioside in the sea lion and horse, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major ganglioside in the porpoise. In rat and cow placentas, the expression levels of both types of GM3 were equal. GD3 was detected in all five species. SPG was detected in them all except the rat. TLC/immunostaining detected GM2 (NeuAc) in the sea lion and cow, and GD1a (NeuAc, NeuAc) in the sea lion. We previously reported that the major gangliosides present in the rat placenta were GM3 and GD3. Therefore, GM3 and GD3 was commonly expressed in the placenta of six animal species examined.

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The effects of Medroxyprogesterone acetate (MPA) on suppressive of menstrual cycles and sexual behavior were studied in female macaque monkeys. In Experiment 1, 8 female cynomolgus monkeys (Macaca fascicularis) were used. Five monkeys received a single s.c. injection of 15 mg/kg of MPA, whereas the remaining 3 monkeys received vehicle (water) as controls. Blood samples were collected three times a week, and changes in plasma concentrations of estradiol, progesterone, and LH were monitored as an indicator of ovulatory cyclicity. Before treatments, all monkeys exhibited normal menstrual cycles with mid-cycle increase in plasma estradiol and LH, followed by luteal increase in plasma progesterone and onset of menses. After MPA-treatments, menstrual cycles completely disappeared and anovulation persisted for 161 ± 17 days, followed by the spontaneous recovery of normal menstrual cycles. Control monkeys did not show any change after treatment. In Experiment 2, 39 multiparae female Japanese monkeys (Macaca fuscata) of the provisioned free-ranging Arashiyama E troop at the Iwatayama Monkey Park were used for behavioral and contraceptive studies. Fifteen monkeys received a single s.c. injection of 7.5 mg/kg of MPA in early autumn, just before the onset of breeding season, whereas the remaining 24 monkeys served as intact controls. Both groups of monkeys exhibited copulatory behaviors during the ensuing breeding season, and delivered live babies during the subsequent birth season. In Experiment 3, 19.4 mg/kg of MPA was injected to five monkeys of the same troop in the following year. These monkeys did not show copulation or delivery. These results showed that a single s.c. injection of MPA suppressed ovulatory cycles in dose-dependent and reversible manners. Thus, MPA appears one of the most effective means for fertility control in macaque monkeys and possibly in other wild mammals.

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The aim of the present study was to assess whether or not a modified Whitten's medium lacking glucose and supplemented with 0.4% (w/v) bovine serum albumin (BSA) can sustain the viability of early-stage pig embryos during long-term culture followed by transfer to recipient gilts. Embryo collection was surgically carried out on Days 2, 3 or 4 (Day 0 = onset of estrus) by midventral incision. The 2-cell and 4-cell embryos recovered were placed in 100-μl droplets of Whitten's medium under mineral oil in an incubator gassed with 5% CO₂ in air at 38.5 C. During culture, the embryos were morphologically evaluated every 24 h. Two-cell embryos from Day 2 donors developed to blastocysts and expanded blastocysts beyond the 4-cell block (11/11, 100%). Four-cell embryos from Day 3 and Day 4 donors also developed to blastocysts at high rates (85% [41/48] and 100% [34/34] respectively). When the embryos from Day 3 donors were surgically transferred to 3 recipient gilts following in vitro culture for 108 or 120 h in modified Whitten's medium, 1 recipient, which received 10 expanded and 5 hatching blastocysts, became pregnant and farrowed three piglets (3/15=20%). Similarly, when the embryos from Day 4 donors were transferred to 2 recipients after culture for 72 h, 1 recipient which received 17 expanded and 1 hatching blastocysts became pregnant and farrowed 8 piglets (8/18 = 44%).

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