Serum concentrations of free testosterone in beagle dogs were determined. Dextran-coated char-coal was used for separating testosterone-binding globulin (TBG)-bound steroid from the unbound fraction. Having a measure of TBG levels together with a knowledge of the total endogenous testo-sterone permitted the estimation of free testosterone based on the binding affinities of serum proteins. The concentrations of free testosterone (pg/ml, mean±Em) in mature males (391±37) were signi-ficantly higher (P<0.001) than those in mature females (47±16). No annual variations of free testo-sterone concentrations were observed in mature male dogs. Serum concentrations of free testosterone were low (<100 pg/ml) at 3 months of age, increased with days of age and appeared constantly from 1 year of age through 10 years in male dogs.

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Ovarian quiescence was diagnosed in 5 Holstein heifers by repeated rectal palpations. The heifers were injected intramuscularly with 100-400 μg of LH-RH analogue (LH-RH-A) i.e. [Des-Gly-NH2 10, Pro-ethylamid9]-LH-RH. When the treatment failed to induce the ovarian cyclic activity, a second or third treatment was conducted with the same or larger dosage of the hormone 21 or 41 days after the preceding treatment. A total of 10 cases were treated using 5 heifers. They were divided into divisions I to IV. Division I consisted of 3 cases each received a single injection of 100 μg of LH-RH-A. Division II consisted of 3 cases each received double injections of 100 μg at 1 h interval. Division III consisted of 3 cases each received a single 200 μg injection. Division IV was 1 case which received a single 400 μg injection. At the time of treatments heifers were 18.9±1.7 (Mean±SD) months in age and 328.2±14.0 kg in weight. Ovarian changes were traced by rectal palpation at inter-vals of 6 h to 2 days. Plasma progesterone (P) and estradiol-17 β (E2) concentrations were determined by radioimmunoassay using samples collected at 12-48 h intervals during the experimental period.
Results: 1. Ovulations were induced 37.5±5.3 (in a range of 30-48) h after LH-RH-A treatment in 8 cases (80.0%) in all, or 2, 2, 3 and 1 cases of divisions I, II, III and IV, respectively. Second ovulation occurred spontaneously 8-9 days after the induced ovulation in 3 out of 8 cases (or 1 case each of divisions II, III and IV). The normal ovarian function began in these 3 cases after the 2nd ovulation. Ovarian quiescence occurred again in the other 5 cases. In the remaining 2 cases of divi-sions I and II, no ovulation was induced and the ovaries remained quiescent. 2. Development of corpus luteum (CL) was observed in 7 of the 8 cases after the induced ovulation, although this induced CL was always subnormal in size and began to degenerate as early as 5-7 days after the ovulation. Plasma P concentration increased in 4 cases of these 7 showing small peaks (1.2-2.8 ng/ml) after the induced ovulation. This increase coincided with the development of the induced CL. In the other 3 cases, no P increase was seen after the ovulation. Neither development of CL nor increase of P was observed in the remaining case after the induced ovulation. 3. Follicles developed in each of the 7 cases before and during the period when the induced CL degenerated, and the follicles ovulated 8-9 days after the induced ovulation in 3 cases. In the other 4 cases they became atrophic and degenerated. Plasma E₂ concentration increased in 4 cases of these 7 to reach obvious peaks (8.7-19.5 pg/ml). This increase coincided with development and maturation of the follicles. In the other 3 cases no increase of E2 was noted during the period of follicular development. 4. In all 3 cases in which following events were observed after the induced ovulation, second ovulation occurred and normal ovarian cyclic activity started subsequently; plasma P concentration reached small peak with development of the induced CL, plasma E₂ concentration reached obvious peak with development and maturation of the follicle during the period of degeneration of the induced CL..
These results indicate that good ovarian responses to LH-RH-A treatment required at least 200 μg. It is also indicated that induced CL must be functional enough to form a small P peak in plasma, and follicle must reach maturity and secrete E₂ activity in coincidence with the degeneration of the induced CL for LH-RH-A treatment to induce ovarian cyclic activity in ovarian quiescent heifer.

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The ultrasonic linear electronic scanning technique was applied for the detection of early preg-nancy and for the estimation of litter size in bitches. Pregnancy was detected as early as 17 to 22 days after the last mating in all of five bitches used, and four of these delivered normal puppies on full term, while the remaining one aborted. The percentages of the litter sizes at parturition to those estimated by ultrasonic echography at 19 to 24 days after the last mating were 83-100% (average 92%).
Developmental investigation on the early stage of pregnancy in the dog was also carried out in this experiment. Very clear echographs of the embryo with accessory organs or fetal fluids were obtained. This technique may be applicable as a practical tool for early pregnancy diagnosis and as auseful method of developmental studies in the bitch.

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Sixty Holstein heifers, aged between 25 and 28 months, were used. Blood collection was conducted weekly from 8 weeks before calving to 10 weeks after calving. The serum chemical components studies include total cholesterol, GOT, total protein, albumin, globulin, A/G, glucose, Ca, inorganic P, Ca/P, γ-GTP, NEFA, phospholipid and triglyceride. Days open in cows which suffered diseases, especially those related to reproductive organs, was significantly longer than that in normal cows (P<0.01). Correlation between fertility status and serum chemical conponents was observed to be more significant within 3 weeks after calving in the periods of 8 weeks before calving to 10 weeks after calving. A negative correlation between total cholesterol level and the number of services per conception was observed within 3 weeks after calving. Similar correlation was observed between the level of albumin, Ca/P and days open. As for γ-GTP, a positive correlation was observed with days open within 3 weeks after calving. Cows which showed the initiation of ovarian activities within 4 weeks after calving as detected by changes in progesterone levels had significantly less disease occur-rence (P < 0.05).

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Serum progesterone levels in pregnant sows can be determined by radioimmunoassay (RIA) and the method has been used successfully to the early pregnancy diagnosis of this species. However, RIA has some disadvantages, mainly due to restrictions on the use of radioisotope. The present study is concerned with early pregnancy diagnosis in swine by serum progesterone levels using enzyme immunoassay (EIA), in which β-galactosidase from E. coli was used as a label instead of radioisotope.
A total of 442 blood samples (2 ml) was taken from the ear veins or Vena cava cranialis of 184 sows and gilts in a farm during 18-21 days after service (day 0=day of first service). The blood was placed for 2 h at room temperature and then centrifuged (3, 000 rpm, 10 min). Sera obtained were kept at -30C until EIA was carried out. Serum progesterone levels of 5.0 ng/ml or more were diagnosed to be pregnant and levels less than 5.0 ng/ml were determined not to be pregnant. Accuracy of diagnosis was based on parturition.
The results diagnosed as pregnant by serum progesterone levels on 18, 19, 20 and 21 days after service were 87.9 (87/99), 93.6 (88/94), 95.3 (143/150) and 96.3% (26/27), respectively. On the other hand, the results diagnosed as non-pregnant by serum progesterone levels were all 100% on the abovementioned days after service. Thus, a total accuracy of diagnosis was 90.1, 94.4, 96.2 and 96.4% on the respective days after service.
In the same herd, the accuracy in pregnancy diagnosis based on non-return rate (NR) by 21 days after service was 81.6% (191/234) in positive cases and 100% (24/24) in negative cases, respectively.
Statistically, a highly significant difference (P<0.01) was obtained between pregnancy diagnosis by progesterone assay on day 19 or 20 and those by the NR method. Significance (P<0.05) was also observed between diagnostic accuracy of progesterone assay on day 18 and that on day 20.
In conclusion, early pregnancy diagnosis about 20 days after service by serum progesterone levels by EIA can be used successfully in swine practice.

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Sex identification of ddY mouse morulae by immunological means was investigated.
Anti male spleen cell serum was produced by 3 intraperitoneal injections of C57BL/6 male spleen cells into females of the same strain at a dose of 3 x 107 spleen cells per injection. Five days after the last injection, serum was collected and absorbed with ddY female mouse spleen cells.
Day-3 morulae of ddY mice were cultured in BMOC-3 containing 10% antiserum, which was pooled from six immunized mice, and 10% guinea pig serum as a source complement.
These embryos were classified as normal or degenetated after 12 h of culture. Out of 207 embryos treated, 94 (45.4%) were found to be degenerated. Approximately 89.7% of the embryos which were not degenerated were found to be females by chromosomal analysis. A significantly higher percent-age of females was obtained from embryos that were cultured in antiserum, than untreated embryos (P < 0.01).
The results indicate that the sex of ddY mouse morulae can be identified clearly by using anti male spleen cell serum.

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One-celled eggs were recovered from mated, colchicine-primed dd female mice, and chromosome preparations were made by the air-drying and C-staining methods. Their mitotic stages were mostly at late prometaphase with two haploid groups of chromosomes and at early metaphase, a stage of syngamy, and partly at the chromatid stage of metaphase. The Y, a small chromosome with no C-band, was distinguished not only at the chromatid stage of metaphase but also at the other stages above-mentioned. Out of 2, 053 eggs examined 767 eggs could be sexed, sexing rate being 37.4%. Of these, 334 eggs were identified as males and 433 females, with a sex ratio of 43.5% males. The sex ratio was further classified according to the stages above-mentioned. Sex ratio before syngamy was significantly higher than that at syngamy and the chromatid stage of metaphase.

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A new silver-colored plumage mutation was found in a closed colony of the Japa-nese quail. This mutation was inherited by a simple, autosomal gene(s) recessive to the wild-type. The female homozygotes were able to ovulate, but they were unable to secrete albumen in spite of immunoelectrophoretic and histochemical evidence that their oviducal tissues contained conspicuous amounts of albumen. Reproductive performance of the homo-zygous males and heterozygotes of both sexes were judged as normal, and all the functional defects were localized upon the Miillerian duct system. Segregation of the plumage color dilution from the secretory defects in the oviduct has not been observed in the subsequent generations.

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Pregnancy diagnosis by the ultrasonogram method was carried out in a captive 13 years old female Hokkaido brown bear. Two fetuses were detected in the left abdomen. The fetus transverse diameter was 5.2 cm and the heart rate was 180 beats/min. Further improvement is necessary to diagnose the number of fetuses by our method, for she gave birth to 3 cubs 4 days later.

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