The isolation and purification of early pregnancy factor (EPF) in the serum of pregnant bovine is described. The serum of 10.4 1 was obtained from a pregnant bovine of 8 days after artificial insemination. The rosette inhibition titer (RIT) of the serum was 6. EPF was isolated using the diafiltration, ion-exchange chromatography (CM-Sepharose, DEAE-Sepharose) and FPLC-gel permeation chromatography. EPF active fraction was recognized in the elute (RIT=7) of 50 mM NaCl on the CM-Sepharose and the unadsorbed fraction (RIT ?? 8) on the DEAE-Sepharose. The unadsorbed fraction of DEAE-Sepharose had 4 bands by SDS-PAGE analysis. The molecular weight of 4 bands were 23, 22, 21.5, 21 KD respectively. Further, the fraction No. 32 of the FPLC had a high EPF activity (RIT ?? 8) and the molecular weight of this fraction was estimated as 2122 KD. The iso-electoric point of EPF was near 5.0 by 2D-SDS-PAGE analysis.

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The effects of cooling rates and plunging temperature into liquid nitrogen (LN₂) on the survival of rapidly thawed bovine blastocysts derived from oocytes matured, fertilized in vitro and cultured in vitro or in rabbit oviduct were examined. The samples were cooled (0.3C/min for blastocysts developed in rabbit oviduct, 0.3 to 0.7C/min for blastocysts developed in vitro ) in 1.4M Glycerol from -5.2C to temperatures between -21 to -39C before transfer to LN₂. The highest levels of survival in vitro of blastocysts cooled at 0.3C/min were obtained after transfer to LN₂ from -24 to -39C in the blastocysts developed in rabbit oviduct. By contrast, the blastocysts developed in vitro survived as high as those developed in rabbit oviduct after plunging into LN₂ from -30C. The highest levels of survival and development in vitro of blastocysts developed in vitro were obtained after transfer to LN₂ from -30C (0.3C/min: 86% survival and 47% development) and from -33 to -36C (0.5C/min: 84 to 87% and 41%). The results indicate that blastocysts produced by in vitro system were more sensitive to cryoinjury than those developed in rabbit oviduct. The range of cooling rate and plunging temperature into LN₂ was limited when applied to blastocysts developedin vitro.

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The effect of α-tocopherol on freezing-thawing process of boar spermatozoa was studied. The ejaculated semen was added α-tocopherol then the semen was taken for freezing. Addition of α-tocopherol decreased the malondialdehyde content and prevented the loss of motility by the freezing-thawing process. After thawing, addition of α-tocopherol inhibited to increase of lactate content, but the lactate content of control increased with time. In the presence of antimycin A, respiratory inhibitor, lactate content of addition of α-tocopherol was same as control. Addition of ethanol reduces cytosol and increases the ratio of lactate to pyruvate. Addition of α-tocopherol declined the ratio of lactate to pyruvate in the presence of ethanol and the effect was inhibited by antimycin A. These data suggest that α-tocopherol prevents peroxidation during freezing-thawing process, as a result, aerobic respiration is maintained and the loss of motility is prevented.

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In vitro exposure of bovine immature oocytes to Streptococcus agalactiae, Escherichia coli, Actinomyces pyogenes and Staphylococcus aureus were conducted to determine if these bacteria were removed from bovine immature oocytes by the standard procedures for washing, or the bacteria were recovered from the embryos during in vitro culture. All the exposed bacteria were recovered from the immature oocytes after in vitro exposure and subsequent washing. E.coli were recovered from matured oocytes and S.aureus were recovered from matured oocytes and the embryos during in vitro culture. The rates of cleavage and development to blastocyst of embryos were not different between bacteria exposed groups and controls (51.4-62.6% vs 52.5-58.4%, 4.6-14.4% vs 7.5-15.6%, respectively). It was concluded that neither of the standard procedures currently used for cleaning immature oocytes should be relied upon for insuring freedom exposed bacteria, thus indicating that effective antibiotics should be added to in vitro culture system for removing or killing bacteria adhered to oocytes or embryos.

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The inhibitory effect of prifinium bromide on the rectal peristalic movement, after intrarectal administration (200 mg) or intravenous administration (75 mg) was studied in two heifers and two cows or two cows, respectively in examination 1. After intrarectal or intravenous administration, the inhibition of rectal peristalic movement was rapidly initiated within 20 sec to 44 sec or 5 sec to 10 sec and continued for 10 to 50 minutes or 43 to 50 minutes, respectively.
The contraction of rectum was recorded using devised monitoring apparatus. The contraction of rectum occurred 2 to 8 times (18-40 mmHg/contraction), intermittently, before recovery to normal peristalic movement.
In examination 2, 25 and 17 donors or 77 and 26 recipients were treated intrarectally with 200 mg and intravenously with 75 mg of prifinium bromide respectively. Among 19 of 25 (76%) and 15 of 17 (88%) donors, 68 of 77 (88%) and 25 of 26 (96%) recipients the treatment showed good efficacy for inhibition of the rectal peristalic movement for intrarectal and intravenous administration respectively. However there were some individual variations.

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The effects of freezing and thawing process on the capacity of malate-aspartate shuttle were studied in the boar spermatozoa. Shuttle capacity was indexed by measuring the ratio of lactate to pyruvate during ethanol oxidation. In the presence of glucose and ethanol, the ratio of lactate to pyruvate of washed spermatozoa was 15.1, while the ratio of frozen-thawed spermatozoa rose to 32.9. Addition of aminooxyacetate, inhibitor of malate-aspartate shuttle, caused an increase of the ratio of lactate to pyruvate in the washed spermatozoa (37.0) and the frozen-thawed spermatozoa (36.1). These results suggested that the capacity of malate-aspartate shuttle, transferring the reducing equivalents of cytosolic NADH produced during ethanol oxidation to mitochondoria, is higher in the washed spermatozoa, and is lower in the frozen-thawed spermatozoa.

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The 23 donor cows were administered with 3000 IU of PMSG and a total of 2440 AU FSH followed by PGF_2αanalog at about 80 days intervals by turns. Thereafter, the efficacy of the 2 treatments were compared using the same cows on the number of corpora lutea, embryos recovered and returned estrus. The cows treated with PMSG resulted in more corpora lutea than with FSH (11.3 vs 7.5; P<0.05). However, there were no differences between 22 PMSG-treated cows and 17 FSH-treated cows with more than 2 corpora lutea, on the average number of corpora lutea (11.8 vs 10.1), recovered eggs (6.7 vs 6.8), fertilized eggs (5.9 vs 5.4) and normal embryos (4.9 vs 4.6), respectively. Through the experiment, two out of 23 cows showed a poor response (with less than 5 corpora lutea) while 12 cows showed a good response (with more than 6 corpora lutea) in either treatment. On the other hand, the remainders had an opposite response in each treatment. There was no difference between 2 treatments on the days from superovulation to estrus. Consequently, it seems that the efficacy of the next treatment can be predicted from the results of the first treatment.

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A radioimmunoassay (RIA) of corticosterone was developed and characterized using ¹²⁵I labeled radioligand (ICN Biomedicals, U.S.A.) and a single ether extraction procedure. The intra- and interassay coefficients of variation was 7.2% and 14.6% and assay sensitivity was 16.4±3.5 pg per tube. Utilizing this assay, the antiserum for corticosterone (GDN377) made small samples (0.1-1.0μl) use possible. The ease of this technique allows handling of large numbers of samples as compared with RIA using ³H-corticosterone.
The specific steps in the procedure of assay were following: ¹²⁵I-corticosterone was diluted with 0.05M phosphate buffered saline (PBS) containing 1 % bovine serum albumin (BSA). Antiserum (GDN377) was diluted with 0.05M PBS containing 0.05mM ethylenediamine tetraacetic acid, 2Na salt, dihydrate (EDTA) and 0.4% normal rabbit serum (NRS) to 1 : 15000-1 : 20000 dilution. Goat anti-rabbit gamma globulin serum (ARGG) was diluted with 0.05M PBS containing 3.5% polyethylene glycol 6000 (PEG) to 1:150 dilution. Plasma or serum (0.1-1μl) and standard were diluted to 400μl with distilled water in 13 × 100mm culture tubes and extracted once with 2ml of diethyl ether. The samples were allowed to settle for 5 min to ensure separation of ether and aqueous phases. The tubes were then placed in dry ice-ethanol bath to freeze the aqueous phase. The ether phase was then decanted into 12 × 75mm culture tube and dried under reduced pressure with gentle mixing at 50C. One hundred μl of PBS containing 1% BSA, antiserum and labelled ligand (50-100Bq/100μ) were added to each tube and the extract was dissolved by agitation and incubated at 4C for 24 h. Then, 100μl of ARGG was added to each tube. After further incubation for 24 h, the antibody bound hormone was separated from the free hormone by centrifugation at 4C 1700g for 30 min. The supernatant was carefully decanted and the precipitate was counted in automatic gammacounter. Plasma or serum concentrations of corticosterone were calculated using a standard curve.

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The present study was designed to clarify mechanisms responsible for regulation of FSH and LH secretion and ovarian follicular maturation during lactation in rats. Throughout lactation in rats, plasma levels of follicle-stimulating hormone (FSH) remain within the range normally found during the diestrous phase of the estrous cycle, regardless of litter size, although levels of luteinizing hormone (LH) in plasma are consistently lower in dams nursing 8 pups than in dams nursing 2 pups. All antral follicles larger than 401μm in diameter degenerate during the first half of lactation and do not reappear until the second half of lactation in rats nursing 8 pups. Healthy follicles of this size, however, are always present in mothers nursing 2 pups. These changes in follicular degeneration corresponded with a decrease in concentrations of plasma LH in the first half of lactation, and follicular development was noted with an increase in basal levels of plasma LH in the second half of lactation. Corpora lutea formed after postpartum ovulation secrete a large amount of progesterone under the influence of prolactin released as a result of suckling. Despite continued suckling, plasma concentrations of prolactin and progesterone decline gradually and a slight increase in plasma concentrations of estradiol-17β occurs during the second half of lactation. Concentrations of bioactive and immunoreactive inhibin in plasma vary with the number of healthy antral follicles and no positive correlation has been observed with luteal function throughout lactation in the rat.
On the basis of the present results it is indicated that 1) tonic secretion of LH is an important factor in normal follicular maturation and maintenance in lactating rats, 2) the suckling stimulus, rather than ovarian factors, is mainly responsible for the suppression of FSH as well as LH secretion during the first half of lactation in rats nursing 8 pups. On the other hand, during the second half of lactation in rats nursing 8 pups, ovarian inhibin plays a primary role in the suppression of FSH secretion, whereas ovarian steroids act to suppress LH secretion. The present findings also indicate that 3) the suckling stimulus alone suppressed the secretion of both LH and FSH and this effect may be mediated by the inhibition of LH releasing hormone (LH-RH) secretion from the hypothalamus, 4) the suckling-induced inhibition of LH-RH may be primarily mediated by endogenous corticotropin-releasing hormone and opioid peptides and 5) the pituitary-adrenal system is capable of influencing the maintenance of a normal secretion of gonadotropins and prolactin as well as the maintenance of ovarian function during the lactation in the rat.

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Cytogenetic and morphological study was carried out to assess the quality of bovine embryos fertilized in vitro. The major results were summarized as follows. 1) An optimal condition for the chromosome preparation of bovine preimplantation embryos was established using 2-cell embryos fertilized in vitro. 2) Chromosomal anomalies were observed in 7.1-36.4% of 89 two- to 32-cell embryos and these anomalies were caused by abnormal fertilization, especially by polyspermy, and abnormal cleavage. 3) At blastocyst stage, the incidence (18.6%) of chromosomal anomalies in the inner cell mass (ICM) separated from trophectoderm cells by immunosurgery was significantly lower than that (44.2%) in whole blastocyst. 4) Anti-bovine spleen cells rabbit serum was prepared and the method to count the cell numbers of ICM and trophectoderm of bovine blastocysts separately was developed by a differential fluorochrome staining technique using this antiserum. 5) Using this technique, it was found that the cell-cell contacts of ICM cells in blastocysts derived from in vitro fertilization were tighter than those from in vitro fertilization followed by culture in vitro and in a rabbit oviduct. 6) The delineation of each blastomere of ICM cells in blastocysts derived from in vitro fertilization of in vitro matured oocytes was improved by the transfer of the embryos to a rabbit oviduct from the 4-cell stage. 7) The dead ICM cells were observed in ICM cells from survived blastocysts after freezing and thawing by three-step or one-step methods. These findings show that the low developmental ability of bovine embryos fertilized in vitro is caused by an abnormal fertilization and their low pregnancy rates may be caused by the reduced cell proliferation and the loose cell-cell contact of ICM cells.

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[in Japanese]

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