The rat embryos at 8-12 cell stages, obtained on Day 4 of pregnancy (the day of detection of vaginal plug is designated as pregnancy Day 1), were cut into two halves and their developmental potential to blastocysts and fetus were studied.
Each embryo was bisectomized by micromanipulation using a fine glass needle, and the "half" embryos were cultured in modified B. W. W. medium (MAYER & FRITZ, 1974) for 16 to 24 h in a 5% moistured air incubator. After incubation developed blastocysts were transferred into the uterus of pseudopregnant Day 5 rats.
One hundred and seventy three embryos were bisectomized and 163 "half" embryos developed into blastocysts in vitro during culture for 16 to 24 h. Seventy nine out of 122 transferred blastocysts were implanted and 5 of them were born normally.

View full abstract

Ninety-nine Holstein-Friesian heifers, 11 to 15 months in age, had superovulated with PMSG and PGF_2α. Factors affecting superovulation, nonsurgical egg recovery and egg quality were examined.
Ninety (90.9%) of the heifers were in estrus within 2 days after the PGF_2α treatment. Ninety-five heifers came into estrus, were subjected to AI, and were classified into four groups according to the CL (number of corpus luteum) estimated; 1-5 CL, 6-10 CL, 11-15 CL and 15<CL. Ten (10.5%) of the heifers had 1-5 CL, 29 (30.5%) presented 6-10 CL, 30 (31.6%) produced 11-15 CL and 26 (27.4%) were estimated as having 15<CL. There were no differences in the rates of superovulation or number of CL between heifers treated during the summer pasturing season and the winter housing season, nor were there differences in heifers 11-13 months and 14-15 months in age.
In 90 heifers nonsurgical recovery was performed between days 6 to 9 after estrus. It was not possible to pass a 16 FG Foley catheter or Sugie's 2-way apparatus through the cervix in 5 (5.3%) of 95 heifers. Eggs were collected in 82/90 (91.1%), averaging 7.3 per successful heifer and 6.6 per flushed. In 4 (4.9%) of 82 heifers only uncleavaged eggs were obtained. The number of eggs recovered tended to increase with the rate of superovulation.
The percentage of normal embryos, which showed no signs of degeneration or which were at the expected developmental stage according to estrus, decreased from days 6 to 7 (67.2% as against 53.5%; significant difference: P<0.05). Effects of the season at treatment, the age of the heifer and the rate of superovulation on the eggs recovered were not found.

View full abstract

To clarify the effect of superovulation and nonsurgical embryo collection on the fertility of the donor animals, the records of 134 donors were analyzed.
Holstein-Friesian heifers aged 11 to 15 months, had been superovulated with PMSG or FSH followed by synchronization with PGF_2α, treatments. In 122 of the treated heifers, nonsurgical recovery was performed between 6 to 9 days after estrus. Three of 122 flushed heifers, and 3 of 6 nonflushed heifers whose services could not be penetrated by the embryo collector became pregnant by the AI performing at superovulatory estrus. Abortion occurred in 3 of 6, and all of the other 3 produced a single calf.
One hundred and one (95.3%) of 105 treated heifers were subjected to AI with various intervals from the time of superovulation. They were diagnosed as pregnant after an average of 1.8 times of AI services per conception. Two hundred and seventy-seven (97.9%) of 283 contemporary control heifers became pregnant after an average of 1.6 services per conception. The difference between the two groups was not significant. Five of another 22 heifers those received embryo transfer became pregnant by embryo transfer and 15 of 17 heifers which faild in implanting the embryo became pregnant by the subsequent AI.
The lengths of estrous cycle that immediately preceded the superovulation-treatment, that of the estrous cycle of the embryo recovery and that of the subsequent estrous were 19.7, 27.9 and 18.5 days on the average. The average length of the superovulatory estrous cycle was significantly longer than the others (P<0.01) probably due to excess in the number of corpora lutea.

View full abstract

Lochiometra is a puerperium disease characterized by the distention of uterus with retained lochia. It occurs frequently in dairy cows with signs of the rise in body temperature and loss of appetite. Treatment must be aimed at an acceleration of involution of the uterus and the discharge of lochia. In this study, 24 Holstein-Freasian cows suffering from lochiometra in a period of 3 to 25 days after parturition were treated by two different administration routes of Prostagrandin F₂ alpha as the tromethamine salt solution (PGF_2α).
Twelve cows were treated by an intramuscular injection of 25mg PGF_2α (group I) and another 12 cows were treated with an intravulvosubmucosal injection of 10mg PGF_2α (group II).
The results obtained were as follows.
1. The lochia was discharged from the uterus 1-2 days after PGF_2α treatment in all cows of group I and 4 out of the 12 cows in group II.
2. General symptoms disappeared in all cows of group I and 11 out of the 12 cows in group II.
3. Ten out of the 12 cows were artificially inseminated with an average of 87.1 days after parturition in group I and 9 out of the 12 were inseminated with at average of 75.6 days in group II.
4. Eight out of the 10 cows conceived with an average of 131.9 days after parturition in group I and 8 out of the 9 cows conceived with an average of 95.8 days in group II.
From these results, it could be considered that the PGF_2α treatment with either an intramuscular or intravulvosubmucosal injection was effective for the cows with lochiometra.

View full abstract

To assess the possible source of estrogens in the cow during perinatal period, the authors measured estrogen contents, secretion rate of estrogens and progesterone by the method of in vitro perifusion, and progesterone metabolites using [¹⁴C]-progesterone in the placental tissue.
Estrone, estradiol-17α and -17β in the bovine placentae were detected by gas liquid chromatography. Total estrogen quantities present were about 10μg/1kg of tissue at 5 months of pregnancy. They increased during pregnancy, and reached to about 55μg/1kg of tissue at 9 months of pregnancy.
Fetal placental cells were prepared using collagenase in a Eagle MEM medium. Estrogens and progesterone secreted by dispersed cells were determined by radioimmunoassay during 3 to 5 hours of perifusion. Fetal placental cells at 6.5 months of pregnancy and term produced higher estrogens in the effluent media, while those at 7.5-8 months of pregnancy and term produced higher progesterone.
Each 100mg of fetal placental tissue was incubated with [4-¹⁴C]-progesterone (0.2μCi/3.6nmol) and NADPH. Radioactive metabolites were then separated by thin layer chromatographies and identified with the method of derivative formation and recrystallization to a constant specific activity. Placenta after 7 months of gestation converted significant amounts (up to 33%) of progesterone to 20-hydroxy-progesterone. However, no estrogens were shown to be formed in all fetal placental tissues examined. Thus, it appears unlikely that estrogens secreted by fetal placental tissue during perinatal period are synthesized from progesterone, but it could be synthesized from other steroid substrate, such as pregnenolone.

View full abstract

We reported previously that boar and goat spermatozoa incubated in the isolated uterus from a maturing gilt were able to penetrate into zona-free hamster eggs and that only acrosome reacted boar spermatozoa fused with such eggs. The present experiment was to investigate ultrastructural changes that occurred in boar and goat spermatozoa during incubation.
Ejaculated spermatozoa from a Landrace boar and Saanen goat were washed twice, resuspended and incubated for 5h in a modified Krebs-Ringer bicarbonate (m-KRB) solution or in the isolated uterus from a maturing gilt. They were recovered from the uterus by flushing and the flushings were gently centrifuged at 50g for 5min to remove blood cells and epithelial cell debris. The super-natants containing the spermatozoa were centrifuged again at 500g for 10min. The sedimented spermatozoa were fixed in 2.5% cold glutaraldehyde for 1h, followed by fixation with 1% osmium tetroxide for 1h, and dehydrate in acetone. Ultrathin sections of the spermatozoa embedded in a mixture of Epon 812 and Epon 815 were stained with lead citrate and uranyl acetate, then examined with a JEM 100C electron microscope.
Boar spermatozoa incubated in the m-KRB medium retained intact acrosomes. In contrast, boar and goat spermatozoa incubated in the isolated uterus from a maturing gilt exhibited various morphologi-cal changes, e.g., vacuolation of the acrosomal contents, appearance of microtuble like structures in the acrosomal cap region, and membrane vesiculation of the acrosomal cap region similar to the acrosome reaction. Such acrosome reactions were observed in 42.5% of boar and 20% of goat spermatozoa. The equatorial segment of acrosome reacted boar spermatozoa remained intact, but that of goat was almost lost. These results suggest that the uterus isolated from a maturing gilt has a capacity of inducing an acrosome reaction in spermatozoa of homologous (boar) or heterogous (goat) species.

View full abstract

Plasma cortisol and corticosterone were measured by competitive protein binding assay in 8 normal dogs. Blood was collected from the dogs by venipuncture at intervals of 2 hours throughout 24 hours period.
Resting plasma cortisol concentrations averaged 14.4±1.0 (SE) ng/ml and ranged from 3.3 to 43.4ng/ml. Resting plasma corticosterone concentrations averaged 4.7±0.3(SE) ng/ml and ranged from 1.2 to 11.1ng/ml. Of 79 plasma samples, 95% contained less than 36ng of cortisol/ml amount and less than 10 ng of corticosterone/ml. Episodic blood cortisol and corticosterone patterns were obtained as reported in the human, monkey, and cow. Circadian rhythm of cortisol secretion was found with the acrophase around 8:00-9:00 AM and the trough around 5:00 AM, while daily variation of corticosterone was obscure.

View full abstract

Hexestrol dicaprylate (H₈), a long-acting estrogen, was used in an attempt to induce temporary sterilization of adult male dogs. Each dog received a single intramuscular injection of H₈ in oil. The doses were ranged from 0.1 to 2.0 mg/kg body weight. The morphological and histological changes in the testis, and androgen content of the testis were examined after H₈ injection.
In cases of 0.5 to 2.0 mg/kg dose levels, spermatozoa in each seminiferous tubule disappeared within 30 days, and this seemed to continue over 150 days. In a dog treated with 0.5 mg/kg, sperma-tozoa were not found in the testis on day 180, but were found on day 240. Testosterone and andros-tenedione were assayed in H₈-treated and non-treated dog's testicles by GLC. The content of both androgens decreased markedly within 48 hours after injection with a dose of 2.0 mg/kg of H₈, and further decreased to undetectable amounts in the 72 hours samples. Up to 60 days after H₈ injection, both androgens were in the undetectable level of our assay system.
This type of estrogen may probably be used for temporary sterilization of male dogs for about 6 month at the 0.5mg/kg dose level.

View full abstract

We reported previously that glucose plays an important role in the acrosome reaction of hamster sperm and in fertilization of hamster eggs in vitro by acting synergistically with some component(s) in the postovulatory oviduct contents. The present study was to investigate whether this synergistical action also plays an important role in inducing hamster sperm capacitation in vitro. The basic medium consisted of 123.6mM NaCl, 1.71 mM CaCl₂ and 25.07mM NaHCO₃ was used for incubation (5% CO₂ in air at 37 C) of epididymal spermatozoa at a concentration of 3.1-5.1×10⁶/ml. Spermatozoa in the medium with either 5.56 mM glucose (Exp. II) or the postovulatory oviduct contents (Exp. III) are difficult to induce hyperactivation and the acrosome reaction during incubation for 5 h. However, when the postovulatory oviduct contents or 5.56 mM glucose were supplemented into the sperm suspensions 5 h after incubation, advanced inductions of hyperactivation and acrosome reaction of spermatozoa were observed; the rates of hyperactivated and acrosome reacted spermatozoa at 3h later were 67.1-77.5% and 69.6-86.8%, respectively. The equivalent rates were obtained in sper-matozoa more than 6-7 h after incubation in the medium with both glucose and the postovulatory oviduct contents (Exp. I). Increased rate of sperm penetration was also observed when the postovula-tory oviduct contents or 5.56 mM glucose were supplemented 5 h after incubation of spermatozoa in the medium with latter or former component. These results indicate that partial capacitation may be induced in the simple medium with only the postovulatory oviduct contents or glucose, but that both components are essential for completion of sperm capacitation and induction of hyperactivated motility of spermatozoa.

View full abstract

Inhibitory mechanisms of hexestrol dicaprylate (H₈) on testosterone production in canine testes were studied. Testicular homogenate (100 mg) was incubated with ³H-H₈ (10 nmol) for 2 h at 34 C. Following incubation, radioactive metabolites were extracted, isolated by TCL, and identified with the method of recrystallization. Approximately 30%of ³H-H₈ was metabolized to ³H-hexestrol (H₀) in testicular homo-genate.
One hundred mg of the sliced testis was incubated with 1 IU hCG and 0.01-1μg of H₈ or H₀ for 12h at 34C in an atomosphere O₂:CO₂ (95:5%). Testosterone, pregnenolone and cyclic AMP content in the medium were determined by radioimmunoassay or competitive protein binding assay. Testosterone production in testicular tissue was stimulated by hCG. The addition of H₀ markedly depressed the testosterone response of testis to hCG, while the addition of H₈ weakly depressed it. Formations of cyclic AMP and pregnenolone were not affected by H₀.
Male dogs were injected with a single dose of H₈ (2 or 20mg/kg). Each testis removed on the 90th day after H₈ injection was homogenized, and incubated with ¹⁴C-pregnenolone. H₈ treatment caused significant decreases in the convertion of pregnenolone to 4-androstenedione and testosterone, with the accumulation of dehydroepiandrosterone in vitro. By a high dose of H₈, the accumulation of 17 α-hydroxypregnenolone also was observed.
These results suggest that H₈ after being hydrolyzed to H₀ in testes directly inhibits the testicular steroidogenesis, and they will also imply that the principle site of H₈ inhibition is located at the metabolic pathway of dehydroepiandrosterone to testosterone.

View full abstract

The role of the cervix in maintenance of pregnancy was investigated follow-ing unilateral resection of the cervix and utero-vaginal anastomosis in 23 rabbits. Fertility and morphological studies were made to compare the operated and contralateral intact uterine horns. In 5 animals autopsied 2 days post coitum, 52.6% of the eggs ovulated from the ovaries on the operated side were fertilized compared with 92.9% on the intact side. At laparotomies or autopsies performed 9 days post coitum (18 does), 93 of 104 (89.3%) ova released from the ovaries on the intact side and 55 of 106 (52.1%) ova on the operated side became implanted. There was no marked difference in the spacing and size of implants between the operated and intact uterine horns, but distribution of implants in the operated horns had shifted slightly toward the vagina compared to the controls. When allowed to go to term, pregnant does delivered young normally from the intact uteri; but in the operated uteri dystocia was noted frequently. Morphological observations revealed no abnormality in the endometrium of either uterine horn following operation, but there was occasional inflammatory reaction in the newly formed vaginal portion. The present findings indicate that the rabbit cervix does not always play a significant role in holding embryos or fetuses in the uterine cavity once fertilization has been accomplished.

View full abstract