The objective of this study was to investigate the effect of calcium ionophore A23187 (IA) on the isolation of inner cell mass (ICM) and trophoblastic tissue of bovine blastocyst derived from in vitro fertilization. Oocytes were aspirated from follicles of 2-5 mm in diameter of the ovaries and then were matured for 21h and fertilized in vitro. In vitro fertilized ova were co-cultured with cumulus cells for 7 or 10 days. In first experiment, Day 7 blastocysts were treated with IA. Treatments were 1) 10-30 min, 20μM; 2)10-30min, 100μM; 3) 40-60 min, 20μM; 4) 40-60 min, 100μM. In next experiment, Day 10 blastocysts were treated with IA. Treatments were 1) 10-30 min, 20 μM; 2) 10-30 min, 100μM; 3) 10-30 min, 200μM; 4) 40-60 min, 10μM; 5) 40-60 min, 100μ M; 6) 40-60 min, 200μM. When Day 7 blastocysts were treated for 10-30 min, 20μM, the isolation rate (81.8%) was significantly higher (P<0.05) than the other treatments. On the other hand, using Day 10 blastocysts significantly higher isolation rate (80.0-91.2%) were obtained with 40-60min, 100μM or 200μM. In conclusion, our experiment demonstrated that bovine ICM can be isolated with IA treatment and the treatment conditions were different with the age of embryos. embryos.
The present study was carried out to investigate the effect of glucose on the development of bovine embryos fertilized in vitro up to the blastocyst stage using CR1aa medium. In vitro fertilized ova were randomly assigned to 4 groups and then these were co-cultured with cumulus cells for 8 days. The groups were 1) CRlaa + 2% calf serum (CS); 2) CR1aa + 2% CS + 0.1 mM glucose (day 0 to 3), then CRlaa + 2% CS (day 3 to 8); 3) CR1aa + 2% CS + 0.1 mM glucose (day 0 to 8); 4) CRlaa + 2% CS (day 0 to 3), then CR1aa + 2% CS + 0.1 mM glucose (day 3 to 8). Developmental rate up to the blastocyst stage of the group cultured in CR1aa + 2% CS + 0.1 mM glucose from day 3 to day 8 was higher (P<0.05) than the other culture groups. In the second experiment in vitro fertilized ova were co-cultured with cumulus cells in CRlaa + 2% CS (day 0 to 3), and further cultured for 7 days in CR1aa + 2% CS + 5 different glucose levels (0 mM, 0.1 mM, 0.5 mM, 1.0 mM, 10 mM or 20 mM). In the three levels of glucose (0.1 mM, 0.5 mM and 1.0 mM) development rates up to the hatching blastocyst stage (11.5, 17.4 and 17.2%, respectively) at day 10 were higher (P<0.05) than in the 0 mM level (5.6%). Two high levels of glucose (10mM and 20 mM) reduced significantly the rate of blastocyst development (2.2 and 0%, at day 7). This study demonstrates that 0.1 mM1.0 mM levels of glucose in CRlaa + 2% CS from day 3 to day 10 in co-culture with cumulus cells are beneficial to bovine embryo development to the hatching blastocyst stage.
Testes of Japanese black bears (Selenarctos thibetanus japonicus) were collected from 27 bears shot by hunters and biopsied from 13 bears captured by traps. The size and weight of the testis, and the diameter of seminiferous tubules increased in 2 or 3 year-old bears. The percentage of sexually matured bears in each age group was 0% (1 year-old), 50% (2 year-old) and 100% (over 3 year-old), respectively. Therefore, puberty was assumed to be at 2 or 3 year of age. Only Sertoli and large round cells were the cell population in the seminiferous epithelium of prepuberal testis. The morphological characteristics of the large round cells were identified with that of gonocyte as reported in other species and were judged to be the stem cells in the testis of the prepuberal bear. The large cells resembling gonocyte were present in the seminiferous tubules of the puberal testis. In the present study, these cells were referred to as gonocyte-like cell. These cells increased in number during the non-breeding season. The gonocyte-like cells were morphologically similar to those of undifferentiated type A-spermatogonia. Therefore, these cells may be equivalent to undifferentiated type A-spermatogonia, that is, the stem cells in the testis of the puberal bear for resumption of spermatogenesis.
The present study was conducted to evaluate effects of oxygen concentration and free-radical scavengers on in vitro development of bovine oocytes matured and fertilized in vitro. Oocytes were matured in TCM-199+10% FCS supplemented with hormones and granulosa cells, and were fertilized in vitro with frozen-thawed, swim-up separated, and heparin-treated spermatozoa. After insemination, oocytes were cultured for 8 days in SOFM supplemented with 10% HS and with 5 different SOD (super-oxide dismutase) levels (0 to 750 μg/ml) [Experiment 1] and 5 different catalase levels (0 to 100 μg/ml) [Experiment 2]. In Experiment 1, high levels of SOD (500 and 750 μg/ml) significantly (P<0.01) reduced the rates of blastocyst development. In Experiment 2, there was no significant difference on the develop-ment to the blastocysts stage among the catalase levels. In Experiments 3 and 4, effects of the addition of SOD+ catalase under two gas atmospheres (5% CO2, 5% O2, 90% N2 vs 5% CO2 in Air) were examined. The addition of the free-radical scavengers in the medium under 5% CO2, 5% O2, 90% N2 resulted in sig-nificantly (P<0.05) higher rate of blastocyst development than embryos cultured under 5% CO2 in Air. The free-radical scavengers did not improve the blastocyst development under 5% CO2 in Air. It appeared that the cause of the stress of high oxygen tension (20% O2) was not only due to the free radicals but also other undetermined inhibiting factors.
We discussed possibility of conception and blood progesterone concentration of recipients for embryo transfer in cows with the coexistences of corpus luteum and follicles, and cystic corpus luteum. As for blood progesterone concentration, both the coexistences of corpus luteum and follicles and cystic corpus luteum indicated the similar value as corpus luteum without follicles. Moreover, investigated changes of blood progesterone concentration from the previous day of transfer to the very day of transfer, in each case, the coexistences of corpus luteum and follicles, cystic corpus luteum and cor-pus luteum without follicles, high rise of blood progesterone concentration was recognized in pregnancy cows. The corpus luteum and follicles, if they are A rank corpus luteum, were higher conception rate whether there were follicle rupture and observation and diagnosis of external genital organs or not. By the treatment of exclusion of luminal fluid in cystic corpus luteum, the normalization of morphological investigation of corpus luteum and rise of blood progesterone concentraion were recognized. It was thought that the corpus luteum and follicles, if morphological investigation of their corpus luteum are normal, could be used as recipients. It was also thought that cystic corpus luteum could be used by removing luminal fluid.
An enzyme immunoassay (EIA) kit for bovine milk progesterone was used for measuring progesterone levels in saliva from sow. Accuracy of early pregnacy diagnosis by the saliva progesterone EIA was also examined. The saliva standard of 0.5 to 30.0 ng/ml progesterone was prepared in the authors' laboratory. The measurable range of saliva progesterone was between 1.25 and 30.0 ng/ml. Intra-assay coefficients of variation (CVs) (n = 6) of saliva progesterone levels from 2 pooled saliva samples were 6.7% (mean = 2.0 ng/ml) and 4.1% (14.9 ng/ml). The inter-assay CVs of progesterone levels from same pooled samples were 17.5% (2.0 ng/ml) and 10.5% (13.6 ng/ml). The time required for the progesterone measurement was less than 2 h. A significant correlation (r = 0.937, P<0.01, n = 20) was observed between saliva proges-terone levels determined by this kit and the values obtained by a double antibody EIA. The early pregnancy diagnosis of sows based on progesterone levels 17 to 24 days after last mating showed an accuracy rate of 96.9% (31/32) for negative results and 96.2% (227/236) for positive cases. The saliva progesterone EIA may be successfully applied to the sow as an aid for early pregnancy diagnosis.
The effect of estrogen administration on eosinophil infiltration into the uterus was examined. To investigate the role of mast cells in eosinophil infiltration, genetically mast cell-deficient (W/Wv) mice were used. The ovariectomized W/Wv mice were injected subcutaneously with 7 doses (0.001-0.25μg/g BW) of estradiol-17β (E2) once a day for 2 days. The treatment resulted in significant increases in the uter-ine weight and the number of uterine eosinophils compared to the vehicle-injected control W/Wv mice 24 hrs after the second injection. Although the increases were dependent on the dose of E2 administered, the magnitude of the responses was maximal in the uterus treated with the doses over 0.05μg/g BW. Fur-thermore the uterine weight in the treated mice was not increased between 24 and 48 hrs after the second injection of E2 (0.15 μg/g BW). On the other hand, the number of eosinophils was increased until 48 hrs after the injection. The E2 treatment at a dose of 0.15 μg/g BW resulted in a significant increase in histamine content of the uterus than that of the vehicle-injected control. However, there was no difference in the con-centration of the uterine histamine between the treated and control groups. These data suggest that the E2 induced uterine eosinophilia is not mediated by mast cells in the W/Wv mice.
A radioimmunoassay of plasma ACTH, useful for various mammalian species was devel-oped. The RIA for ACTH involves use of commercially available antiserum and 125I labelled hormone, and permits measurement of immunoreactive ACTH in unextracted plasma of various mammals. Physiologi-cal validation of the method was obtained from the following studies. Intact male rats had elevations of immunoreactive plasma concentrations of ACTH after the onset of immobilization stress. A dose related increase in plasma concentrations of ACTH was obtained in adult male rats by the injection of ACTH releasing hormone (CRH). The specific steps in the procedure of assay were following. All steps were performed under cool condi-tion (4C). Basal buffer was 0.063 M Na2HPO4 containing 0.013M ethylenediaminetetraacetic acid 2Na salt (EDTA) and 0.02% NaN3 adjusted to pH 7.4. RIA buffer (basal buffer containing 0.1% Triton X-100 and 250 KIU/ml aprotinin) was made into BSA•RIA buffer (for dilution of standards or samples: containing 1% protease free bovine serum albumin), NRS•RIA buffer (for dilution of antiserum to ACTH: contain-ing 0.4% normal rabbit serum) and PEG•RIA buffer (for dilution of antiserum to rabbit gamma globulin: containing 4% polyethylene glycol). BSA•EDTA buffer (separation buffer) was also prepared by adding 1% BSA to basal buffer. Plasma or serum (1-40 μl) were diluted to 100μl with BSA•RIA buffer. Standards or samples (100μl) were put in the polystylene tubes and 100 μl of antiserum were added to each tubes. After incubation at 4C for 24 h, 100μl of labelled ligand were added and incubated for another 24 h. Then, antiserum to rabbit gamma globulin was added to each tube. After further incubation for 24 h, 1 ml of separation buffer was added to each tube and antibody bound hormone was separated from the free hormone by centrifugation at 4C, 1700 g for 30 min. The supernatant was carefully decanted and the precipitate was counted in auto-matic gamma counter. Plasma or serum concentrations of ACTH were calculated using a standard curve.
This study outlines environmental and internal factors affecting the seasonal breeding of Japanese monkeys (Macaca fuscata). 1) Females housed indoors exhibited the prolonged breeding season as well as the reduced inhibition of ovarian functions during the nonbreeding season in comparison with those housed out-doors, as judged from reproductive endocrine profiles. 2) Artificial manipulation of photoperiod alone had no effect on the manifestation of annual reproductive cyclicity, whereas simultaneous manipulation of photoperiod and ambient temperature was at least to some extent effective in modifying the ovarian functions. 3) Estradiol-treated ovariectomized monkeys exhibited marked seasonal changes in response to the negative feedback action of estradiol on LH secretion. 4) Thyroidectomy resulted in significantly earlier termination of the breeding sea-son. 5) Analysis of hormonal profiles in socially housed male and female monkeys revealed that onset of the breeding season was preceded by the increase in plasma testosterone and inhibin in male monkeys, which sug-gested that social factor is also involved in the manifestation of the breeding seasonality. From these studies, it is concluded that seasonal breeding of Japanese monkeys is governed by biannual changes in the response of the hypothalamo-hypophysial axis to the negative feedback action of estradiol, and that multiple annually cyclic environmental factors appears to influence the seasonal breeding of this animal.
The present study was carried out to establish the reproductive performance of Japanese Black Cows in their lifetimes. The major results were summarized as follows. 1)Fetal development: Growth of fetal body weight and crown-rump length was characterized for conceptuses ranging 39 to 268 days of gestation. 2)Growth of female genital tract from birth to puberty: Heifers whose body weight reached 257-269 kg, had matured sexually. Uterine and cervical weights at puberty, distributed 78. 0-139.0g and 22. 0-85.0g, respectively. The body weight of heifers showed continual increase after puberty, while there were a little increase in the uterine and cervical weight, and no growth of the oviduct either in weight or length. 3) Uterine involution postpartum: Primiparous Japanese Black Cows were slaughtered at 18, 23, 29, 46 and 54 days postpartum to examine the histological patterns of involution of uterus. At 18 and 23 days postpartum, the uterine endometrium contained many phagocytes and nodular aggregations of lymphocytes. Capillaries located under the surface epithelium were not yet completely contracted. At 46 and 54 days postpartum, however, the endometrium contained only a little amount of lymphocytes and phagocytes, uterine glands evenly distributed with a tall glandular epithelium. It may be concluded that uterine involution was completed histologically at approximately 40 days or later postpartum. Using the ultrasonic linear scanner, it was confirmed that uterine involution was completed at approximately 40 days postpartum. 4) Lifelong reproductive performance of Japanese Black Cows: The mean duration for the first postpartum estrus was 67.7 days. Mean time interval from parturition to conception, that was days open, was 125.5 days, so mean calving interval was 417.5 days. First and final calving ages of the cows were 2.53 and 8.14 years old on average, respectively, and they produced 5. 4 calves on average in their lifetimes. Cows in which primiparous age ranged from 2.01 to 3.00 years old, produced more calves (over 6) in their lifetimes than those in which primiparous ages was under 2.00 or over 3.01 years old. Rate of culled cows at every calving number reached to 51 and 83 % of total cows after fifth and eight calving number, respectively. Seventy eight % of the culled cows were due to some reasons of reproduc-tive disorders. A reasonable age for beginning of calf production in Japanese Black Cows was estimated to be in the range from 15 to 20 months old, and economical limit of age for reproductive use was estimat-ed to be 9 to 10 years old when they will have more than 8 calves.