In order to collect the blood from the sow for the endocrine research, the antithrombogenic catheter was inserted from the auricular vein to the external jugular vein or anterior vena cava under restraint of the snout. The proximal end of catheter was led to the root of the ear under the auricular skin and fixed at the back of the intrascapular space. This method does not require general anesthesia and can be achieved with less surgical damage than the cervical insertion method. Using this catheterization, the blood is able to be collected for more than 20 days without restraint. It is concluded that this method is usuful for the blood collection in the sow to investigate the hormonal changes consecutively.

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Estrus was induced in ten ewes and they were artificially inseminated during the non-breeding season. Their ovarian activities were examined by laparoscopy every five days from day 5 to day 25 of the induced estrus. Concentrations of plasma progesterone (P₄) were measured by the radioim-munoassay at the day of laparoscopy. Return of estrus was also examined.
Ovulation was not detected in one ewe and three ewes conceived by single hormonal treatment during the non-breeding season.
The ewes were classified into 3 groups based on their ovarian activities:
Group I (n=4): Corpora lutea (CL) regressed by day 20.
Group II (n=3): CL were maintained by day 25. Group
III (n=2): New CL were detected at day 20.
Color changes in CL with the secretoy activities were in agreement with those observed during the breeding season. The results of present experiments indicate that the color of CL is a better index of CL function than the size and shape of CL.

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Mouse morulae collected from superovulated ICR mice were stored at 0°C in PBS containing var-ious sugars. After 24-120 h, viability of the embryos was assessed by their ability to develop into ex-panded blastocysts during 48-60 h in culture.
When embryos were stored with glucose, the viability of the embryos was considerably extended especially with 0.5-0.75 M of concentration; this effect was similar to that reported on sucrose. 0.4 M-raffinose seems also effective but recrystalization occurred during storage. Ficoll 70 was not protec-tive at all. Galactose, fructose and lactose were as effective as glucose, but mannose and maltose were less protective.
Considering that the embryos stored with effective sugars were recovered shrunken, the beneficial effect of the sugars seems to be from proper dehydration ofthe cells caused by osmotic pressure af-forded by a non-toxic, non-permeable andnon-electrolytic agent.

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Diploid gynogenetic mouse eggs were produced by the following micromanipulation procedures. Fertilized eggs were obtained 15 hrs after hCG injection from superovulated C57BL/6 (GPI type: bb) and Fl (C57BL/6 × CBA, GPI type: bb) females mated with males of CD-1 strain (GPI type: aa). The eggs were incubated with the culture medium (M16) including cytochalasin B (5μg/m1) for 4 hrs to suppress the second polar body extrusion. The eggs which had 3 pronuclei were used in the present study. Male pronucleus was microsurgically removed from the eggs following the method of BORSUK³⁾ (Experiment 1) or of SURANI and BARTON⁴⁾ (Experiment 2). The treated eggs were cultured for 1 to 5 days in vitro, then transferred to recipients. In some eggs, one of 2 female pronuclei was removed by the same method to restore the normal diploid state. The results obtained are as follows.
Experiment 1. Thirty-one of 44 eggs (70%) from C57BL/6 and 39 of 51 eggs (76%) from F₁ fe-males were morphologically normal after micromanipulation, respectively. Sixteen of 39 eggs (41%) from the F₁ females developed to blastocysts in vitro. Five of 15 blastocysts (33%) implanted after transfer, and one of them developed to a live fetus with 25 somites, GPI type of which was not deter-mined.
Experiment 2. The eggs from the F1 females were used through out. Out of 69 eggs manipulated 57 (83%) had morphologically normal appearance. Twenty-eight of them (49%) developed to morula or blastocyst stage. Out of 28 eggs transferred, 16 (57%) implanted and one of them developed to alive fetus with 27 somites, GPI type of which was ab, indicating the mistake in the enucleation pro-cedure. After removal of one of two female pronuclei, 39 outof 43 eggs (91%) were morphologically normal. The proportion of morulae or blastocysts developed in vitro from these eggs was 67% (26/ 39), being significantly P (<0. 05) higher than those for the gynogenetic eggs in Experiments 1 and 2.After transfer to recipients, 7 of 26 eggs developed to live fetuses on Day 18 of pregnancy, GPI type of which was all ab as expected. These results support the assertion by McGrath and Soter (1984) that both female and male genomes are necessary for normal development.

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The purpose of the present study was to exploit a simple radioimmunoassay (RIA) of progesterone (P), testosterone (T) and estradio1-17β (E₂) using₁₂₅ I-labelled radioligands prepared by Amersham (En-gland) and to examine their use in comparison with corresponding 3H-labelled materials. The specific steps in the procedure were the following: ¹²⁵I labelled steroids were diluted with 0.01 M phosphate buffered O. 14 M NaCl, pH 7. 0 (PBS) containing 1% BSA. Antisera against P, T and E² were diluted with PBS containing 0.05 M ethylenediaminetetraacetic acid, disodium salt (EDTA) and 1% normal rabbit serum (NRS) or 1% normal sheep serum (NSS) to give the final working solution. Goat anti-rabbit gamma globulin serum (Anti-RGG) and rabbit anti-sheep gamma globulin serum (Anti-SGG) were diluted with 0.01 M PBS to give the final working solution. Plasma and standard were diluted to 500 μl with distilled water in13 x 100 mm culture tubes and then extracted once with 2-3 ml ether. The sampleswere allowed to settle for 5 min to ensure separation of the ether and aqueous phases. The tubes were then placed in a dry ice-ethanol bath that froze the aqueous phase. The ether phase was then decanted into 12 x 75 mm culture tube and dried under reduced pressure with gentle mixing at 50°C. Plasma extracts were assayed without chromatography. One hundred μl of appropriate antisera and 100 μl iodinated steroid (4, 000-5, 000 cpm/tube) were added to each tube and the contents were mixed and incubated at 4°C for 24 h. Then, 200 μl of the anti-RGG or the anti-SGG were added to each tube at a dilution which optimally precipitated the RGG or SGG. After further incubation of 24 h, the antibody bound hormone was separated from the free hormone by centrifuga-tion at 1, 700 g for 30 min. The supernatant was carefully decanted and the precipitate was counted in an automaticgamma-spectrometer.
The major advantages using 125I labelled steroids in RIA in comparison with tritiated ones were:
1) Antisera can be used in considerably higher dilutions with the 125I-histamine tracer than with the ³H-tracer.
2) Standard curves obtained using iodinated radioligands were more sensitive than those obtained using tritiated steroids.
3) Although 1251 has a short half life as compared with ³H, these tracer can be used for at least 3 to 4 months without renurifucation.
4) The use of the ¹²⁵I-labelled materials was more convenient, particularly when a large number of analyses were required, since the double antibody method can be used.
5) Steroid RIAs employing ¹²⁵I-labelled radioligands were much more economical with regard to counting time and counting expenses than RIAs using tritiated steroids because scintillation fluids and counting vials were not required.
6) In P RIA system, usable standard curves were obtained with both homologous and heterologous systems using P-3-oxime-¹²⁵I-radioligand (IM 139). However, there was an instance in which a homologous radioligand (P-11a-hemisuccinate-¹²⁵I, IM 138) bound so tightly to the antibody (P-11β-hemisuccinate-BSA serum) that the binding could not be inhibited by the radioinert P. It was found that all antisera used except an antiserumderived from P-11β-hemisuccinate-linked immu-nogen were incapable of binding appreciable quantities of the P-11α-glucuronide-125I radioligand (IM 140).

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The objective of this experiment was to examine the effect of progesterone and gonadotropins on the ovulation and fertilization in feeble estrus and anestrus cows. Group 1 consisted of six cows with feeble estrus, and anestrus cows, 4 Holstein and 2 Japanese Black breed, were allotted to Group 2. Long-term sterile Japanese Black heifers were assigned to Group 3.
Cows in Group 1 were administered subcutaneously 20 mg of progesterone in oil daily for 6 days between day 9 to 14 of the estrus cycle. In Group 2, progesterone treatment, 50 mg/day for 6 days, started on a given day and 1, 000 IU of PMSG were administered intramuscularly 48 hours after the final injection of progesterone. Human chorionic gonadotropin (hCG), 1, 000 MU, were injected in-travenously within 12 hours after the onset of estrus. In Group 3, 20 mg/day for 6 days of proge-sterone treatment were performed two times at an interval of 10 days, then followed by 2, 000 IU of PMSG 24 hours after the final injection of progesterone. One thousand MU of hCG were administered in the same manner as in Group 2. Ova were recovered 6 days after the artificial insemination by non-surgical uterine flushing. All animals in these three groups were inseminated with the frozen Holstein and Japanese Black bull semen respectively after the onset of estrus.
The administration of progesterone timed to the luteal phase in estrous cycle caused an intense or normal estrus and ovulation in Group 1. Five of six cows in this group conceived in the first in-semination with the frozen semen. Induction of estrus and ovulation succeeded in Group 2 by the administration of progesterone and gonadotropins, but only one of six cows become pregnant. Fer-tilized ova were obtained in two of three animals by uterine flushing in Group 3.
These results imply that a short-term progesterone treatment in feeble estrus cows may improve the fertility, and that induction of estrus and ovulation in acyclic anestrus cows with a combined treatment of progesterone and gonadotropins is possible.

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Sexual behavior of the Hokkaido brown bears in captivity was observed during the breeding sea-sons in 1982 and 1983. Pairing between the males and females was found to be random and occurred most frequently at around 7 AM and again 7 PM. Chance of pairing peaked at the beginning of June during the breeding season. Copulation took place in the sequence of mounting, pelvic thrusting, quivering and dismounting, and average copulation time was 23 minutes. Ejaculation was suspected to have occurred during quivering. Females in heat were observed to be receptive to the males for a long period during the breeding season. Estimated durations of estrus varied widely between animals. A decrease in mounting frequency with age was observed in the males but the receptivity of the females was not influenced by age.

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Present study was undertaken to examine the synergistic effect of progesterone and estrogen on the physical properties of the chronically distended rat uterus. Ovariectomy and insertion of a par-affin pellet into a uterine horn were performed within 24 hours after delivery (Day 1). The rats were injected with vehicle alone (Group I), 4 mg progesterone (Group II), 0. 2 μg estradiol-17β (Group III) or both progesterone and estradiol-17β (Group IV) from Day 3 through 7. Stress relaxation curves and passive length-tension curves of the circular uterine strips obtained from rats given no hormonal in-jection on Day 3 (Group V) and from animals injected with vehicle or the hormone on Day 8 were determined.
The stress decline was slower in Group I than in Group V. The stress relaxation in any of the hormone-treated groups was similar to that in Group I. The length of the collagenous framework (L_z) and distensibility (D), which were obtained from the linear part of the passive length-tension curve, were significantly greater, while tnesion at in vivo length (T_v) in passive length-tension curve was smaller, in Group V than in Group I. L_z, D and T_v in Groups II and III were similar to those in Group I. In Group IV, however, L_z was greater, and T_v was smaller, compared with those in Groups I, II and III. L_s and T_v in Group IV were similar to those in Group V. D in Group IV was smaller than that in Group V and significantly greater compared with the value in either Group I or III.
These results suggest that progesterone and estrogen act synergistically in maintaining collagenous framework and distensibility of the distended rat uterus and keep the uterine tension lower.

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Embryos were collected non-surgically from superovulated Holstein cows on day 6 to 7.5 post-estrus. Fifty-three embryos classified as good-to-excellent were bisected microsurgically. Viability of the half-embryos was assessed by blastulation in vitro or development into calves.
Twenty-nine day 6-6.5 embryos were bisected with a fine glass needle, and 52 half-embryos (25 pairs) were produced. Twenty-nine (13 pairs) of these half-embryos were cultured for 24 to 36 hours, and 28 (12 pairs) of which were developed, including 4 trophectodermal vesicles. Ten pairs of half-embryos were transferred non-surgically to 10 recipients, and two recipients were pregnant at day 60. One of them calved only one female, but another aborted on day 60 to 120.
Day 7.5 embryos were bisected by three different methods. In method a), eight embryos were bisected with a fine glass needle, and 12 half-embryos (6 pairs) were produced. These half-embryos were cultured for 2 to 12 hours, and three (no pairs) of which were developed. Transfer was not carried out. In method b), three embryos were bisected with a machined surgical blade, and four half-embryos (2 pairs) were produced. These were cultured for 2 to 12 hours, and all of which were developed. Transfer was not carried out. In method c), thirteen embryos were bisected with a razor blade and a fine glass needle, and 25 half-embryos (12 pairs) were produced. Nineteen (9 pairs) of these half-embryos were cultured for 2 to 12 hours, and four (1 pair) of which were devel-oped. Two pairs of half-embryos were transferred non-surgically to two recipients, and one recipient calved a set of normal male monozygotic twins. Two half-embryos of a monozygotic pair were trans-ferred surgically to two recipients, but no pregnancy was obtained.

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The distribution of blastocysts in the uterine horn was examined in mice in delayed implantation. Female ICR mice were injected with 5 IU PMSG and 5 IU hCG 48 hrs apart and mated immediately after hCG injection with males of the same colony. The day when vaginal plug were found was des-ignated Day 1 of pregnancy. Intra-uterine distribution of the blastocysts was examined in the follow-ing 5 groups of the females; a) the females ovariectomized on Day 3, having no treatment thereafter and examined on Days 4 and 8, b) those ovariectomized on Day 3, having daily injection of progesterone thereafter and examined on Days 4 and 8, and c) the PMSG and hCG treated females examined on Day 4.
1. When the numbers of blastocysts were compared among the oviductal side, middle region and vaginal side, there were no significant differences in the mice on the Day 4 groups; only the PMSG and hCG treated, ovariectomized, and progesterone-injected ones. In the delayed implanting mice on Day 8, the ovariectomized, and progesterone-injected ones, the blastocysts were predominantly distributed in the vaginal side.
2. When the numbers were compared among the antimesometrial side, middle region and meso-metrial side of uterine cavities, the blastocysts were mostly distributed in the antimesometrial side in the mice on Day 4 of only the PMSG and hCG treated, and the ovariectomized ones and those on Day 8 of progesterone-injected ones. The similar tendency was found in the mice on Day 4 of progesterone-injected ones, though the numbers showed no significant difference. As for the ovariectomized mice on Day 8, the partial distribution of blastocysts in the antimesometrial side was not recognized.

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In an effort to find preselection criteria that can be used to show good ovarian response to superovulation treatment, the relationship between serum total cholesterol and progesterone levels and the number of transferable embryos that can be obtained per donor was investigated. Superovulation was induced by either PMSG or FSH in 106 Holstein heifers and cows between days 9 and 13 of the estrous cycle. Serum total cholesterol and progesterone were analyzed. Percentage of animals in which over 3 transferable embryos were obtained was significantly less in animals with serum total cholesterol level of less than 130 mg/dl as compared to the animals with 130 mg/dl and more. It was also found that percentage of animals in which no transferable embryos were obtained was significantly higher in animals with total cholesterol of less than 130 mg/dl. Animals with progesterone level of less than 1.0 ng/ml did not produce over 3 transferable embryos. These results indicate that serum total cholesterol and progesterone levels may be used as a criteria to indicate good ovarian response to superovulation.

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The sex ratio of quail embryos at 16 hours of incubation was measured on the basis of karyological sexing. Chromosome preparations were made from Colcemid-primed blastodiscs by the air-or fire-drying method followed by conventional Giemsa staining. A method for preparing chromosome slides from the telolecital eggs of birds was described in some detail. Sexing was made by ascertaining the sex chromosome constitution, ZZ (male) or ZW (female), for a minimum of five metaphase plates per embryo. A total of 305 embryos were prepared for cytological sexing. Of these 274 embryos, about 90% could be sexed successfully. The embryos sexed as male were 138 and those sexed as female were 136, a sex ratio of 50.4 (% males) being very close to the fifty-fifty ratio.

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