The diagnosis of early stage of pregnancy in cows is very important for veterinarians. We diagnosed a total of 204 cows raised at the dairy farms of Kanagawa, Chiba and Ibaraki Prefectures by using Doppler ultrasound pregnancy detector (Rectal doctor, Medata, England).
Experimental cows were divided into 5 groups according to their pregnant term (number of days after artificial insemination). The following results were obtained; the accuracy of diagnosis is 90.6% for 1-39 days, 92.5% for 40-59 days, 91.9% for 60-79 days, 94.5% for 80-149 days and 100% for over 150 days, respectively. The diagnosis of pregnant and non-pregnant cow is very easy with the use of this detector.
Therefore, we can conclude that Doppler type ultrasound pregnancy detector is a very useful instrument for diagnosis of early pregnancy in cows. This detector is very compact and easy to carry, moreover compared with the rectal palpation technique this method can shorten the time for diagnosis of pregnancy.

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The effects of varying doses of gonadotropin (GTH) administration or thetransplantation of the pituitary gland on fertilization, embryonic development and serum steroid hormone levels of immature rats were studied to explore the cause of a low incidence of implantation of rats given a high dose of GTH.
Both the incidence of rats with ova and the number of ova recovered from the uterus decreased with increasing doses of GTH and the lapse of time after mating. In the pituitary transplanted rats, 24 embryos or more per rat were recovered from the uterus 84 to 108 h after coitus. The fertility rates and embryonic development were normal in all groups with increase of serum progesterone levels. Estradio1-17β concentrations were significantly higher in 20 and 40 IU GTH-treated rats 12 to 60 h after mating than those in 5 IU PMSG•hCG-treated rats, whereas the levels in the pituitarytransplanted rats were similar to those in 5 IU GTH-treated rats.
These results suggest that the incidence of implantation is closely related to serum estradio1-17β levels, and the low incidence of implantation in immature rats treated with high doses of GTH may be due to the embryonic loss from the uterus by accelerated transport. Furthermore the hormonal control through the pituitary gland should be important for a success of inducing superfecundation in immature rats.

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Methods for collecting cells in metaphase and spreading chromosomes (hypotonic treatment) were investigated in 2-cell bovine embryos derived from in vitro fer-tilization and the sex ratio was studied. The embryos were exposed to 0.08 or 0.16 μg/ml of vinblastin-podophyllotoxin (V-P) for 12 hr at 39°C before being treated with 0.75-1.0% of sodium citrate solution for 15 to 60 min. The exposure to 0.08 μg/ml V-P yielded analyzable metaphases in 69.1% of the embryos prepared, which was contrasted with 42.9% in the exposure to 0.04 μg/ml colcemid for 4 hr. A hypotonic treatment with 1.0% sodium citrate for 60 min was recommended to obtain a high quality of metaphase spread. Chromosomal sexing was made for 61 embryos with a sex ratio of 63.9% males.

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Diagnosis of pregnancy and ovarian function in 15 cows was made by using milk progesterone test kits (Ovucheck Cowside R, England). On the 20th day after servicing, a corpus luteum was rectally palpated in all the 15 cows. However, high serum progesterone levels (over 1 ng/ml) were determined in only 11 cows out of them with radioimmunoassay. The results of milk test was comparable to those of radio-immunoassay, showing high concentrations of milk progesterone in those 11 cows. On the 40th day, a well-developed corpus luteum was found in 14 of 15 cows by rectal palpation, and they were diagnosed as pregnant with milk test kit. But only 11 of 14 were confirmed as pregnant with rectal palpation on day 60th. This milk test kit is very easy in handling, and useful for measuring serum and/or milk progesterone levels. It was considered to be an useful means in the diagnosis of pregnancy and determination of ovarian function in the field work, because there is no need for any equipment or facility to use this test kit for scanning of cow's ovarian function.

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Effect of active immunization against androstenedione-human serum albumin conjugate (Fecundin), in combination with or without methyl-acetoxy-progesterone (MAP) and pregnant mare's serum gonadotropin (PMSG), on both ovulation rate and prolificacy was investigated in Suffolk ewes during the breeding (Experiment 1) and the non-breeding seasons (Experiment 2). Nineteen to 20 ewes were treated as follows: control (Group I), two injections of 2 ml Fecundin (Group II), administration of combined 60 mg MAP and 500 IU PMSG (Group III), and treatment with combined Fecundin, MAP and 250 IU PMSG (Group IV). In Experiment 1, the mean ovulation rates in all the treated groups were respectively higher than the rate in Group I. Prolificacy was significantly higher in Groups II and III than in Group I. In Experiment 2, ovulation occurred in Groups I and II without estrous behavior. The mean ovulation rates in Groups III and IV were respectively higher than the rate in Group I or II. Compared with Group III, prolificacy remained higher in Group IV. In Experiment 3, a single booster injection of Fecundin in the following breeding season elevated the prolificacy. These results indicate that the Fecundin treatment during the breeding season is effective in increasing both ovulation rate and prolificacy in the Suffolk ewe.

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Plasma concentrations of LH, testosterone, and estradio1-17β in male beagle dogs were measured.
The following results were obtained.
1. During the first 14 days of life, the LH and testosterone levels of both sexes were significantly lower(P<0.001) than those for mature male dogs. The testosterone level for male neonates was significantly higher (P<0.05) than that for female neonates.
2. The level of LH increased with days of age and appeared constantly from 8 years through 13 years of age in male dogs. The level of testosterone increased with days of age and appeared constantly from 1 year through 10 years of age in male dogs. Thereafter, the level of testosterone declined gradually. In contrast, there was noeffect of age upon the level of estradio1-17β in mature male dogs.
3. No annual variation of LH nor testosterone concentration was observed in mature male dogs.

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Plasma samples for the determination of LH and ovarian steroids were collected from the jugular vein at 10-min intervals for 4 hr on Days 3 and 10 (luteal phase) and Day -1(follicular phase) of the estrous cycle in 5 animals. Distinct LH pulses with a half-life of about 20 min were detected throughout the cycle. Frequency of LH pulses decreased toward the mid-luteal phase and then increased during the follicular phase (P<0.05) with no significant variations in amplitude. The mean plasma concentrations of progesterone during each 4-hr sampling period were negatively correlated with the frequency of LH pulses (r=-0.85, P<0.001). Duringthe follicular phase, each LH pulse was followed by an increase in estradiol secretion: the mean plasma concentrations of estradiol increased (P<0.05) 20-40 minafter the preceding LH pulse and returned to the previous levels by 60 min afterthe LH pulse. These results demonstrate the presence of a pulsatile secretionof LH and the causal relation-ship between the LH pulses and the ovarian estradiol secretion in cyclic Shiba goats.

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The pulsatile secretion of LH was monitored in Shiba goats during the gestation (3 animals) and the postpartum suckling period (4 animals), by measuring plasma LH levels at 10-min intervals for 4 hr on three occasions in each reproductive phase: early-, mid-and late-stages, respectively. Except for one case at the mid-gestation stage, the LH secretion remained inactive throughout the gestation period showing none or one LH pulse/4 hr with low amplitude. The pulsatile secretion of LH with an amplitude of greater than 1 ng/ml resumed within a few weeks after kidding in 3 out of 4 animals, followed by the resumption of estrus at variable postpartum intervals (15 to 66 days), whereas the pulsatile LH secretion did not resume in the other animal which remained acyclic at least until day 78 postpartum. These results demonstrate that the pulsatile secretion of LH decreases throughout the gestation period and the increase of the LH pulse frequency after kidding would be a prerequisite for the resumption of the postpartum ovarian cycle in suckling Shiba goats.

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Histochemical investigation was carried out on the testis of the adult Japa-nese weasel (Mustela itatsi). The testis weight in the breeding season was larger than in the non-breeding season. The enlargement of the testis was related to the develop-ment of seminiferous tubules and tunica albuginea. The thickness of the tunica albuginea was inversely, and the diameter of the seminiferous tubules was directly related to increasing testicular activity. The disappearence of spermatids and spermatozoa as well as the degenerating primary spermatocytes and the small-sized Leydig cells were observed in the testes obtained in the non-breeding season. The localization and relative activity of dehydrogenases (lactate, glucose-6-phosphate, succinate, malate, iso-citrate, α-glycerophosphate, 3α-hydroxysteroid, steroid 3β-ol, 11β-hydroxysteroid, 17α-hydroxysteroid, 17β-hydroxysteroid, 20α-hydroxysteroid and 20β-hydroxysteroid dehydro-genases) and hydrolases (adenosine triphosphatase, alkaline and acid phosphatases, non-specific esterases) were histochemically evaluated in the non-breeding and breeding seasons. In the testis during breeding season, the interstitium showed very strong enzyme activities, particularly of lactate, glucose-6-phosphate, malate and isocitrate dehydrogenases, adenosine triphosphatase, alkaline and acid phosphatases, and non-specific esterases. The epithelial cells showed very strong activities of malate dehydrogenase, and moderately strong activities of lactate and glucose-6-phosphate dehydrogenases, and adenosine tri-phosphatase. Spermatozoa contained some dehydrogenases such as lactate, glucose-6-phosphate, α-glycerophosphate dehydrogenases. The interstitium showed very strong activities of 3α-hydroxysteroid dehydrogenase in the non-breeding and breeding seasons, however the activity of steroid 3β-ol dehydrogenase was moderately strong only in the breeding season. 11β-, 17α-, 17β-, 20α-and 20β-hydroxysteroid dehydrogenase activities were not observed in the testes.

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Pig hatched blastocysts, especially those which had been developed in vitro from expanding blastocysts, had a higher tolerance to cooling compared with pre-hatching stage embryos. The blastocysts which underwent hatching in vivo (vivo-HB) and in vitro (vitro-HB) were subjected to cooling compared with control pre-hatching stage embryos: expanding blastocysts, zona-removed blastocysts, early blastocysts and morulae. The embryos suspended in Dulbecco's PBS supplemented with 16% fetal calf serum were cooled in 0.25 ml plastic straws to 6°C for 1 hr or 16 hr. Survival of the embryos were assessed by testing the ability to develop in culture for 24 hr. All of the 26 (100%) vitro-HB and 51 of 65 (78.5%) vivo-HB, which had been exposed to 6°C for 1 hr, were survived. When the length of exposure to 6°C was prolonged from 1 hr to 16 hr, a remarkable difference (p<0.05) of the survival rate was found between the vitro-HB (20/21, 95.2%) and vivo-HB (22/41, 53.7%). This suggests that the vitro-HB were more tolerant of the cooling than the vivo-HB. The survival rate of the expanding blastocysts was significantly lower than that of the vitro-HB (p<0.001) and vivo-HB (p<0.05). None of the other control embryos showed any signs of survival, except for one small vesicle derived from one of the 15 zona-removed blastocysts.
Transfer of 18 vitro-HB, which had been cooled to 11°C, resulted in a normal fetus development, but none of the 22 morulae and 18 early blastocysts could survive this short exposure.

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