The ovaries of 10 Japanese black cows were examined by ultrasound twice weekly from Day 6 to Week 10 postpartum. The mean (± S.D.) number of days from calving to detection of the first dominant follicle was 10.7 ± 3.9, and the first postpartum dominant follicle ovulated in half of the cows (5/10). The interval from calving to first ovulation (25.8 ± 21.0 days) was characterized by the growth and regression of various dominant follicles (2.2 ±1.5; range 1-6). Estrous behavior was not detected at the first ovulation but was detected in 7/10 cows at the second ovulation. Following the first ovulation, the subsequent cycle length shortened (15.5 days) in 2/10 cows, remained normal (19.7 days) in 3/10 cows, and lengthened (46.2 days) in 5/10 cows. The growth rate and maximum diameter of ovulatory dominant follicles during follicular development were greater than in non-ovulatory atretic follicles prior to the first and third ovulations (P<0.01). These data show that in beef suckler cows, follicular development and dominant follicle formation occur soon after calving, but the incidence of ovulation of the first dominant follicle and occurrence of estrus at the first ovulation are low.
The role of the seminal vesicle in the fertility of mice and the effect of supernatant fluid of seminal vesicles and coagulating glands (SSC) on both in vitro fertilization and artificial insemi-nation were investigated. Removal of seminal vesicles along with coagulating glands caused a severe reduction of fertility in mice. When oocytes were flushed from the oviducts of females mated with treated males 2 or 10 h after mating, none of 100 and 2 of 60 eggs were fertilized, respectively. Although each vaginal smear showed the presence of spermatozoa, few or no sper-matozoa were detected in uterine flushings. No detrimental or beneficial effects of adding SSC to the preincubation and/or fertilization media were detected in in vitro fertilization. In the case of artificial insemination, however, the addition of SSC to the medium significantly increased the fertilization rate. When females were inseminated with a sperm suspension supplemented with 10% SSC at less than 2 × 105, 2-30 × 105 or greater than 3 × 106 spermatozoa, 11, 48 and 85% of eggs were fertilized 24 h after insemination, respectively. In controls, the fertilization rates were 2, 26 and 63% at less than 2 × 105, 2-30 × 105 or greater than 3 × 106 sperm 24 h after insemination, respectively. It is clear that SSC does not affect in vitro fertilization, but it does promote in vivo fertility. Seminal vesicle fluid is an essential factor for the uterine transport of sperm in mice.
Changes in the GnRH pulse generator activity were examined during the programmed treatment with ovarian steroids, which mimicked endocrine events in the luteal and follicular phases, by recording the hypothalamic multiple unit activity specifically associated with the pulsa-tile LH secretion (MUA volleys) in ovariectomized goats. The steroidal milieu of the luteal phase was simulated by subcutaneously implanting silastic capsules containing progesterone and estra-diol, and that of follicular phase by intravenously infusing estradiol at a gradually increasing infusion-rate after removal of the progesterone capsules. The administration of progesterone alone for 3 days had little influence on MUA volleys, but there was a significant reduction (p<0.05) in the MUA volley frequency following the additional administration of estradiol, which resulted in the low pulse frequency observed in the normal luteal phase. In the mimicked follicular phase the MUA volley frequency was increased from 10.0± 2.9 to 13.7±1.9 volleys/12 h after the proges-terone removal, but then decreased gradually in parallel with an increase in the plasma estradiol concentration. There was no increase in MUA volley frequency during the transition from the basal to the surge mode of LH secretion. These results suggest that the hypothalamic GnRH pulse generator activity is suppressed during the luteal phase by a synergetic action of progesterone and estradiol, and that the acceleration of the GnRH pulse generator activity does not take place during the preovulatory LH surge in the female goat.
Enzymatic dispersed small naked mouse oocytes from ovaries at birth were co-cultured with ovarian somatic cells for eight days in vitro, and the growth of oocytes and the formation of zona pellucida were examined. After enzymatic treatment of the ovaries at birth (Day 0 of culture), oocytes and ovarian cells were dispersed completely. The oocytes were 11-22 μm in diameter (average: 16.9±2.2 μm) and had no zona pellucida around them. From Day 1 of culture, oocytes and ovarian somatic cells aggregated, formed several clumps and oocytes grew inside or became attached to them. On Days 4, 6 and 8 of culture, 3.0, 6.0 and 35.3% of oocytes grew to 30 μm or more in diameter, respectively. Even on Day 8 of culture, no follicle formation was observed. At the periphery of the grown oocytes without surrounding somatic cells on the clumps, PAS-positive, indented zona pellucida-like materials were deposited. The materials reacted strongly with anti-mouse zona pellucida antibody in immunogold staining. Immuno-reactive materials were also observed in the oocyte cytoplasm, whereas no reaction was found in ovarian somatic cells. These results suggest that the zona pellucida was formed by the oocyte and deposited its periphery without follicle formation in mice.
The effect of various O2 and CO2 mixtures on development of bovine embryos produced by in vitro maturation and in vitro fertilization (IVM/IVF) was examined in a newly formulated simple KSOM medium versus a complex Menezo B2 serum-free medium and in co-culture. Numbers of cells comprising blastocysts were also counted using Hoechst 33342 stain. Following routine IVM/IVF procedures oocytes and zygotes were cultured for 40 to 44 h in various media. Then 2-to 8-cell embryos had cumulus cells removed and were alotted randomly for continued culture to the same experimental culture media, but with various gas conditions. In Experiment 1, the percentage of embryos developing to and beyond morula stages in 5, 10 and 20% O2 with 5% CO2 in humidified air was 22, 15 and 12%, respectively, with the 22 versus 12% being different (P<0.05). Blastocyst cell number in the 5 and 10% O2 treatments was higher than in 20% O2 (P<0.05). Also, the simple KSOM and complex Menezo B2 media were compared and more embryos developed into blastocysts (10%) in KSOM medium than in B2 medium (5%; P<0.05), but cell number did not differ (P>0.05). In Experiment 2, the proportions of embryos developing into blastocysts in 5 and 10% CO2 were 15 and 6% (P<0.05), respectively, and in 5 and 10% O2 were 15 and 7%, respectively (P<0.05). Mean blastocyst cell number (82) was highest with 5% CO2 and 10% O2 even though more blastocysts were obtained with 5% CO2 and 5% O2 (P<0.05). In Experiment 3, embryos were co-cultured on monolayers of bovine oviduct epithelial cells (BOEC) or buffalo rat liver cells with KSOM medium in 5 and 10% O2 and 5% CO2. Treatment effects did not differ except 10% O2 was superior to 5% O2 when considering both morulae and blastocysts (41 vs 33%; P<0.05). These experiments indicate that the O2-CO2 concentrations can affect the development of bovine embryos produced by IVM/IVF when cultured in a simple serum-free medium with and without co-culture and that the simple KSOM medium can serve as a defined medium to study required supplementation for culturing IVM/IVF bovine oocytes.
After acute testicular irradiation in adult male cynomolgus monkey semen was periodically collected, and sperm concentration was examined as an index of radiation damage to the seminiferous epithelium. Sperm concentrations decreased after irradiation with 1, 2, or 3 Gy. Concentrations fell below the minimum normal level for unirradiated monkeys, (107 sperm /ml of semen ) at 16.0, 8.5, and 8.0 weeks after irradiation, respectively. The lowest sperm concentra-tion was observed 17.0, 12.5, and 14.0 weeks after irradiation, and the percentages of the lowest sperm concentration relative to the pre-irradiation concentration were 1.20, 1.37, and 0.20%, respectively. The sperm concentration recovered to pre-irradiation levels after 35.5, 36.5 and 41.0 weeks at doses of 1, 2 and 3 Gy, respectively. These results indicate that spermatogenesis decreases markedly after irradiation with 1-3 Gy but resumes after a definite period that depends on the radiation dose. Sperm concentration can be utilized as an effective index of radiation damage to the seminiferous epithelium and as an index of recovery.
The membrane fraction of anterior pituitary in laying hens was found to possess a vasoactive intestinal polypeptide (VIP)-binding component having the properties of a receptor. Scatchard analysis of saturation studies revealed that the binding component had a single class of binding site with an equilibrium dissociation constant (Kd) of 0.092 ± 0.008 (mean ± SEM; n=5) nM and the maximum binding capacity (Bmax) of 631 ± 41 fmol/mg protein. The Kd value determined from kinetic analysis (0.091 ± 0.002 nM; n=3) was very similar to that obtained from Scatchard analysis. The Kd value obtained from Scatchard analysis was not significantly different between the laying and nonlaying hens. The Bmax in laying hens was greater than that in nonlaying hens. The administration of chicken VIP to nonlaying hens caused a marked decrease in the VIP receptor binding in the anterior pituitary with a concomitant increase in the blood serum concentrations of prolactin. The results may provide an evidence for a direct action of VIP on the anterior pituitary of female chickens.
Conception rates and plasma hormonal patterns were investigated after treating ewes with CIDRR (controlled internal drug release device) for different lengths of time during the non-breeding season. In Experiment 1, 101 Suffolk ewes were treated with CIDRR for 6, 9, 12 days; pregnant mare's serum gonadotropin (PMSG) was injected the day before CIDRR removal. At 42 to 50 h after treatment, ewes were inseminated with frozen-thawed semen using a laparoscope. Lambing rates were not significantly different among the groups (66.7, 76.5 and 85.3% for 6, 9, and 12 days, respectively). Seven days after insemination, the re-insertion of CIDRR for 14 days (n=51) did not improve the pregnancy and lambing rates or prolificacy compared to the controls (n=50). In Experiment 2, the mean plasma progesterone concentration on the day of CIDRR removal in Booroola-Dorset ewes treated for 12 days was significantly (P<0.01) lower than those in ewes treated with CIDRR for 6 or 9 days. However, the onset of estrus after treatment was similar in ewes treated for different periods with CIDRR. These results indicate that a satisfactory lambing rate (67 to 85%) can be obtained with frozen semen in ewes treated with CIDRR, regardless of the insertion period (6 to 12 days) during the non-breeding season.
The expression of the bovine growth hormone receptor (GHR) was detected in various tissues by a reverse transcription-polymerase chain reaction system (RT-PCR). For the quantifica-tion of the bovine 438 bp GHR cDNA fragments a HPLC-UV detection system was used. bGHR amplificates were well detectable in liver from cows, heifers and in fetal liver. bGHR expression was also demonstrated in mammary gland, kidney and in adipose tissue. Lower but significant amounts of bGHR amplificates were obtained in skeletal muscle and in rumen villi, indicating a potential new mode of action of GH via affecting the gastrointestinal tract. bGHR mRNA was detected in various female and male reproductive tissues, i.e. in uterus, oviduct, corpus luteum, and in the vesicular gland. No bGHR expression was detectable in testes from adult bulls. A long-term treatment of heifers with a slow-release preparation of bovine growth hormone had no effect on either bGHR-mRNA expression in liver or on GH binding capacity on liver membrane prepara-tions. These results demonstrate that the RT-PCR system together with a HPLC-UV detection system provides a useful strategy to study GHR expression and regulation in different tissues under various conditions.
The regulatory role of epidermal growth factor (EGF) in the rabbit placental growth has been elucidated by investigating the proliferative response and sensitivity of placental cells to EGF during pregnancy. Trophoblastic cells were isolated from rabbit placental tissues from days 11, 17, 23 and 29 of pregnancy. The cells were incubated with a varying concentration of EGF and exposed to 100μM bromodeoxyuridine (BrdU). The BrdU incorporation, evaluated by an enzyme-linked immunosorbent assay (ELISA), was significantly stimulated by EGF on days 11 and 17 when more than 2.5 and 5.0 nM was added, respectively, but not on days 23 and 29 up to 160 nM. Scatchard analysis of the specific 125I-labelled EGF binding to the cells revealed two classes of binding sites. Their dissociation constants (Kd) in both high-and low-affinity binding sites were not significantly different among days of pregnancy. However, the number of binding sites per cell (Bmax) significantly decreased with progress of pregnancy: 4, 154 ± 1, 255 sites/cell (mean ± SEM) to 834 ± 79 in the high-affinity site and 13, 064 ± 5, 983 to 2, 924 ± 785 in the low-affinity site from day 11 to 29. These results suggest that the signal mediated by EGF receptor is a regulatory factor for the rabbit placental growth and that EGF may be involved in the regulation.
This study was designed to investigate the relationship between embryonic volume changes in response to addition of glycerol and the in vitro survival of frozen-thawed equine blastocysts. Unexpanded blastocysts (diameter <200 μm; n=9) shrunk rapidly when embryos were transferred in 5 and 10% glycerol solutions at 30 C in 10 min steps. At the end of equilibration in the 10% glycerol solution, the mean embryonic volumes were regained up to 59% of initial values. Expanding blastocysts (diameter 200-300 μm; n=9) shrunk with a rather slower response, and regained 41% of initial volumes. Hatched blastocysts (diameter >300 μm; n=9) shrunk gradually down to 35% of initial volumes. Following the 2-step glycerol equilibration, single embryos loaded into 0.25 ml French-straws were seeded at -6 C and cooled to -35 C at the rate of 0.3 C/min before plunging into liquid nitrogen. The straws were thawed rapidly in 37 C waterbath and glycerol was removed from embryos at 30 C in 10 min 6-steps (8.3, 6.7, 5.0, 3.3, 1.7 and 0%). The embryos were cultured for 48 h in TCM199 + 10% FBS at 37 C under 5% CO2 in air. Severe fractures of embryonic coats (zona pellucida and/or capsule) were found in 0/9 (0%; unexpanded), 2/9 (22%; expanding) and 6/9 (67%; hatched) frozen-thawed embryos. All unexpanded blasto-cysts (9/9, 100%) could develop normally in vitro. Only 5/9 (56%) expanding blastocysts and 2/9 (22%) hatched blastocysts could develop in vitro. The overall survival for embryos with intact or slightly fractured coats were 9/9 (100%), 4/9 (44%), and 2/9 (22%) in unexpanded, expanding, and hatched blastocysts, respectively. The mean values of relative embryonic volumes before freezing were related with post-thaw viability; however, the predictability of freezability of a particular embryo was low.
The effects of season (autumn or spring), device type (MAP sponge or CIDR device) and the number of devices (one or two) were evaluated by the superovulatory responses of ewes in a 2 × 2 × 2 factorial design. Plasma progesterone (P4) and estradio1-17β (E2) were measured to evalu-ate the characteristics of the different progestogen treatments and seasons. The embryos (1 to 8 cell stage) recovered from superovulated ewes in both seasons were cultured to examine their developmental capacity. The mean ovulation rate of ewes treated during spring was significantly (P<0.01) lower than that of ewes treated during autumn (8.4 and 14.7, respectively), but there were no significant differences in the number of recovered ova and normal embryos between autumn and spring. Replacing the CIDR device on Day 9 of the 12-day insertion period maintained the P4 concentra-tions during gonadotropin treatment but did not improve ovulation rate, number of recovered ova and normal embryos of ewes treated in both autumn and spring. Developmental capacity of the early embryos (1 to 8 cell stage) to morulae or blastocysts did not significantly differ between autumn (45.7%) and spring (34.4%). These results indicate that the type (MAP sponge or CIDR device) and the number (one or two) of the progestogen devices did not affect ovulation rate, number of recovered ova or normal embryos, and a seasonal effect was found only in the ovula-tion rate but not in the number of recovered ova, normal embryos or the subsequent developmen-tal capacity of cultured embryos.
Three trials were conducted to investigate the superovulatory response of Control Inter-nal Drug Releasing device (CIDRR). Forty-six Holstein cows were divided into three treatment groups per trial; CIDRR without progesterone (P4) (Control), CIDRR containing 1.9 g P4 for 12 days (CIDRR-1) and the two CIDRR with P4 replacing with a new CIDRR on day 9 of the 12 day insertion period (CIDRR-2). Follicle stimulating hormone (FSH) were administered on day 9 up to the time of CIDRR removal and additional prostaglandin F2α injections were done on day 11 of the 12 day insertion period. Ovulation rate, recovered and transferable embryos, and P4 and estradiol-17β (E2) levels in blood plasma were evaluated and compared among the treatment groups. There were no statistical differences on the mean (± SEM) ovulation rate, recovered and transfer-able embryos. An encouraging results of transferable embryos were obtained in the 3 treatment groups (80.7%, 69.8%, and 61.4% for Control, CIDRR-1 and CIDRR-2 respectively). No existing evidence of correlation between plasma P4 and ovulation rate at the time of AI (day 13) was observed. Plasma P4 and E2 concentrations were characterized by the physiological variability of individual cow and heterogeneous follicular development following superovulatory treatments. The results suggest that the use of CIDRR presents a satisfactory superovulatory regimen which could prove applicable for embryo production under field conditions. However, further research is necessary to elucidate factors involved in the improvements of the regimen.
Viability of pig blastocysts following in vitro culture for 0-3 day(s) was assessed after transfer to recipients. Embryos collected on Day 4, 5 and 6 were cultured in a modified Brinster's medium +10% fetal calf serum for 3, 2 and 1 day(s) respectively. Embryos were transferred to Day 6 recipients. Day 7 noncultured blastocysts were transferred as a control. Nine (90%) of 10 recipients with noncultured embryos, 6 (75%) of 8 recipients with embryos cultured for 1 day, 5 (83%) of 6 recipients with embryos cultured for 2 days, and 2 (29%) of 7 recipients with embryos cultured for 3 days farrowed. Embryo survival was 50%, 34%, 35% and 12%, respectively. Sur-vival of noncultured embryos was higher (P<0.05) than that of embryos cultured for 1-3 day(s). Survival of embryos cultured for 3 days was lower (P<0.01) than that of those cultured for 1 or 2 day(s). These results indicate that the viability of cultured pig embryos following transfer de-creases with increasing length of culture in vitro.