The conception rate by artificial insemination (AI) was compared between two groups of cows bearing follicular cysts (FC): Cows of group 1 were cyclic and accompanying a Graafian follicle (GF) and those of group 2 were not accompanying GF and apparently non-cyclic. Behavioral estrous cycles and skim milk progesterone levels were monitored throughout the experimental period.
In all of 21 cows of the first group (FC-GF), estrus ceased within a couple of days after AI, and in most cases the GF ruptured spontaneously to form the corpus luteum. Fourteen Cows (66.7%) of this group were conceived by the first AI.
In contrast, in 9 cows of the second group (FC only) abnormal sexual behaviour continued after AI, and no one was conceived. HCG or GnRH analog treatment was required for successful insemi-nation in this group.
With respect to progesterone levels, the first group exhibited patterns similar to those in normal pregnant or cyclic cows, whereas the pattern of the second group was characterized by the continuous low levels (less than 1 ng/ml) throughout the observation period.
These results indicate that any treatment is not required before AI in cows of FC-GF type, while hormonal treatment is prerequisite to expect a good conception rate in cystic cows without GF.

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The purpose of this study was to examine the effect of β-adrenoceptor agonist, clenbuterol, on parturition time and uterine activity during parturition in rats.
The rats were intramuscularly injected with 0.15 μg clenbuterol twice a day at the specified times from day 20 of pregnancy to the delivery, and the parturition time was investigated. Twenty two of 26 intact and saline-treated control rats (84.6%) delivered before 12:00 on day 22 of pregnancy. On the contrary, 10 of 14 rats (71.4%), treated at 08:30 and 11:30, and 9 of 14 rats (64.3%), treated at 18:00 and 21:00 delivered after 12:00 on day 22 of pregnancy. In another series of experiments, the rats were intramuscularly injected with clenbuterol (0.45-2.25 pg) during parturition and the change of uterine contraction was investigated. In 4 of 5 rats, the frequency and the amplitude of uterine con-traction decreased and the deliveries interrupted for 194-384 min. After the interruption, the uterine contraction recoveried and the deliveries resumed. The noenates were physiologically and morpho-logically normal.

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Diagnosis of ovarian quiescence were made in five Holstein heifers by repeated rectal palpations on the basis of the absence of corpus luteum (CL) and abnormal follicle in the ovaries. The heifers were treated with 750-3, 000 IU of human chorionic gonadotrophin (hCG). When the treatment failed to induce the ovarian cyclic activity, second and third treatments were conducted with 2-4 times dosages of the hormone 25-26 days and 59 days, respectively, after the preceding treatment. A total of 11 cases of treatment were obtained from the five heifers. They were divided into four groups, I-IV, according to the dosage. Group I, II and III consisted of 3 cases each and received a single intra-muscular (IM) injection of 750, 1, 500 and 3, 000 IU of hCG, respectively. Group IV consisted of 2 cases and received a single IM injection of 6, 000 IU of hCG. Mean age was 18.2±2.7 (SD) months and mean weight 337.1±21.8 kg at the time of treatment in the 11 cases. Ovarian changes were traced by rectal palpations at intervals of 6 hr to 2 days during the experimental period extending from 6-7 days before to 25-46 days after the treatment. Plasma progesterone (P) and estradiol-17β (E₂) concentra-tions were determined by radioimmunoassay on samples collected at intervals of 12-48 hr during the experimental period.
The following results were obtained.
1. Ovulations were induced 37.8±2.7 hr after hCG treatment in 10 cases (90.9%). Second ovula-tions occurred spontaneously 9, 10 and 15 days after the induced ovulation (ind. OV) in one case each of Groups II, IV and III, respectively. The normal ovarian function began in these 3 cases thereafter. In the other 7 cases no second ovulation occurred and ovarian quiescence resulted again. In the remaining case of Group I, the ovaries continued to be quiescent, no ovulation being induced.
2. Corpora lutea developed after ind. OV (i.e. induced CL) in all the 10 cases. They were classi-fied into 2 types. One type was subnormal in size (<18 mm in diameter), accompanied with a small P rise (3.2±1.4 ng/ml), and degenerated as early as 5-8 days after ind. OV. The other type was generally normal in size ( ?? 18 mm in diameter), went with an obvious high P peak (8.5±3.3 ng/ml), and began to degenerate relatively early, or 10-12 days after ind. OV. A well-formed induced CL appeared frequently as the dosage was increased from 750 to 3, 000 IU. Average P peaks accompany-ing the formation of induced CL tended to be enhanced according to an increase in dosage, being 2.3±0.7, 4.1±2.9, 6.9±4.1, and 7.9±4.2 ng/ml in Groups I, II, III and IV, respectively.
3. Follicles developed in each of the 10 cases during the degeneration of the induced CL. They ovulated in 3 cases in which sharp E₂ peaks were observed with the advance of follicular development and maturation. In the other 7 cases, no E₂ rise was observed with the advance of follicular develop-ment and then follicles became atrophic.
4. Second spontaneous ovulations occurred and then the ovaries began to show their normal func-tion in 3 cases. In them P increased in blood level in coincidence with the formation of induced CL and E2 increased sharply in blood level with the follicular development and maturation accompanying the degeneration of induced CL.
These results indicate that (1) the ovarian response was more remarkable to the dosage of hCG of 1, 500 IU or over than to that of 750 IU. (2) It was found necessary for ovarian activation following hCG treatment that the induced CL had to be functional enough to exhibit a P peak in plasma and that a follicle had to develop and mature, secreting E2 actively with the degeneration of the induced CL. (3) It was also recognized that ovarian activation following hCG treatment was not always achieved only by the good development and sufficient P-secreting function of induced CL.

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Inhibin activity and concentrations of sex steroid hormones were determined in normal follicular fluid and ovarian cystic fluid of individual dairy cows. Inhibin activity was measured using the primary monolayer culture system of rat anterior pituitary cells and expressed as potencies relative to the working standard solution of our laboratory by the parallel line assay. A pooled bovine folli-cular fluid after removal of steroids by charcoal treatment (working standard) was divided into small aliquots and kept frozen until use. One unit was defined as the potency of FSH-inhibiting activity of 1 ml working solution. Estradiol-17β (E2), progesterone (P) and testosterone (T) were measured by radioimmunoassay. The results were given in Table 1.
1) Inhibin activity in almost all the luteal cysts and cystic corpora lutea was below the sensi-tivity of the assay and the concentration of E2 was low, whereas huge amounts of P were found. Histological examinations of the luteal cyst showed no granulosa cell around the wall of antrum and extensive formation of fibroid tissue with the luteinized theca interna.
2) Inhibin activity was still noted in the follicular fluid of a silent heat cow with low concen-trations of E2 and T, suggesting that granulosa cells may wane the activity of estrogen secretion earlier than that of inhibin secretion.
3) Follicular cysts can be divided into three groups. One is the cystic follicle with hyper-func-tion of granulosa cells. Second is the follicles with rather degenerating granulosa cells with higher amounts of P. Third is the one with little endocrine function.
The presence of inhibin activity in some of the cystic follicles may be responsible for the low levels of FSH in the plasma of cows with follicular cysts.

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This study was conducted to investigate the effect of times of PMSG injection and ram introduc-tion on estrus incidence and lambing rate in ewes treated during the non-breeding season. During April to May, 85 Suffolk ewes were treated with an intravaginal sponge containing 60 mg medroxyprogesterone acetate for 9 days. Therefore, a factorial study (2×2) was designed for times of PMSG injection and ram introduction (The day of sponge removal: Day 0 and 2 days before sponge removal: Day-2); group I: 600 IU PMSG injection (Day 0) + ram introduction (Day 0), group II: 600 IU PMSG ineection (Day 0)+ ram introduction (Day-2), group III: 600 IU PMSG injection (Day-2) + ram introduction (Day 0) and group IV: 600 IU PMSG injection (Day-2)+ ram introduction (Day-2). Estrus was examined at 6 hr intervals after joining for 4 days. Ewes were injected a 100 μg LH-RH analogue at estrus detection, and after 9 hr mated by a hand service with one of four fertile rams. Semen characteristics of each ram at 57 hr after sponge removal examined. The results were as follows.
1) The rate of estrus incidence within 4 days after treatment were higher in groups where PMSG was injected at Day 0 than Day-2, regardless of the times of teaser rams introduction (100%, 95.2%, 90.0% and 85.0% for groups I, II, III and IV, respectively). One ewe in group IV exhibited silent estrus 24 hr before sponge removal.
2) The lambing rates were 75.0%, 60.0%, 66.7% and 87.5% for groups I, II, III and IV, respec-tively. Group IV showed a significantly higher lambing ratethan group II (P<0.05).
3) Semen characteristics and increased times of services did not affect lambing rates and prolifi-cacy in the 4 rams used.
These results suggested that the decrease of estrus incidence when PMSG was injected at 2 days before sponge removal involved an undeterminate or silent estrus. The hand service method could provide satisfactory lambing rates as effective as natural-service method in ewes for out-of-season breeding.

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The present study was designed to examine the effects of several factors on the survival of mouse embryos frozen rapidly by liquid nitrogen vapour based upon the two-step procedure. The mouse morulae in 2 M glycerol were frozen by direct transfer into liquid nitrogen vapour at -170-180°C after seeding at -7°C and then being plunged into liquid nitrogen 1015 min later. Frozen samples were thawed rapidly by agitating the tubes in a 3840°C water bath. The frozen-thawed embryos were placed in PBS containing 2 M glycerol plus 0.5 M sucrose and then placed in PBS containing 0.5 M sucrose each for 3 min at room temperature, followed by two washes in fresh PBS. The morulae were then cultured for 4045 h to assess their ability to develop into blastocysts. 1. Glycerol (survival rate: 78%), propylene glycol (70%) and ethylene glycol (67%) were effective cryoprotectants and some pro-tection was also afforded by erythritol (53%). In contrast, the degree of protection given by DMSO was very low and no embryos survived freezing in the presence of methanol or sucrose. 2. A higher survival rate (81%) was found after exposure to glycerol at 0°C for 10 min prior to freezing than after exposure at 20 (41%) or 38°C (19%). 3. Exposure of embryos to 0.5 M sucrose in PBS for 5 min at room temperature prior to freezing that makes partial dehydration of blastomeres osmotically prior to freezing did not improve the survival of frozen embryos as compared to the survival of embryos un-pretreated with sucrose. 4. There was no difference in the survival rate between embryos where ice-seeding was induced at -7°C and embryos where ice-seeding was omitted. 5. The temperature (0, 20 and 38°C) at which glycerol was diluted with sucrose out of frozen-thawed embryos did not affected the survival rate. 6. Glycerol was diluted out of the frozen-thawed embryos by three methods at room temperature. The survival rate improved after glycerol-dilution with sucrose described previously (74%) as compared to the stepwise dilution (addition of 0.2, 0.2 and 0.4 ml PBS at 1-min intervals) (51%) or the direct dilution (direct transfer of embryos into PBS) (8%). 7. Although the survival rate (76%) of embryos frozen in glycerol rapidly by liquid nitrogen vapour appeared a little lower than that (89%) of embryos frozen slowly, the difference was not significant. In conclusion, a relatively high proportion of mouse embryos survived rapid freezing by direct transfor from 0 (without ice-seeding) or -7°C (with ice-seeding) to liquid nitrogen vapour at -170180°C before plunging into liquid nitro-gen. Further experiments are needed to determine the optimum conditions for freezing mouse embryos by using liquid nitrogen vapour.

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Induction of twinning was attempted in 54 Japanese black cows and 57 Holstein heifers by ipsi-lateral transfer of a pair of fertilized fresh eggs or of two demi-embryos raised from fresh of frozen eggs.
Nineteen Japanese beef cows (35%) and 35 Holstein heifers (61%) were diagnosed as pregnant by rectal palpation at 40 to 60 days after the transfer. Two Japanese beef cows and 13 Holstein heifers gave birth to twins and 14 cows and 17 heifers gave birth to single. Consequently a total of 18 and 43 live calves was born; 113% and 143% crop with regard to a pregnant mother were obtained respec-tively. The remaining 3 cows and 5 heifers resulted in abortion. Twinning rate in Holstein heifers (43%) was higher (P<. 05) than that in Japanese beef cows (13%). Twin calves were lighter in birth weight (P<. 05) and shorter in gestation period than single birth in the two breeds.

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Mouse ova were fertilized and developed in vitro in protein-free media. Superovulated ova from (JCL-ICR strain) female mice were fertilized in vitro by epidi-dymal sperm of the same strain and cultured for 114 h under 5% CO₂ in air and the sub sequent development of the fertilized ova was examined.
In the separate experiment, 2-cell embryos were flushed out of the oviducts of super-ovulated JCL-ICR mice 48 h after hCG injection and mating. The 2-cell embryos were cultured for 68 h and the subsequent development of the embryos was examined.
Fertilization rates judged from the second polar body extrusion at 6 h after insemina-tion were 28.3 (45/159), 26.8 (71/265) and 62.5% (60/96) in protein-free medium containing 0, 50 and 100 μm EDTA, respectively. The development rates of these fertilized ova to bla-stocyst stage were 0 (0/45), 53.5 (38/71) and 76.7% (46/60) after 114 h of culture in protein-free medium containing 0, 50 and 100 μm EDTA, respectively. The development rate of the late 2-cell embryos collected from the oviducts to blastocyst stage was 95.7% (45/47) after 68 h of culture in protein-free medium. From these findings, it is suggested that mouse ova can be fertilized and be developed in protein-free media.

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Because it is difficult to visualize the three-dimensional shape of uterine cavities only through observation of transverse tissue specimens, careful casting was done for mouse uterine cavities and the casts were observed under a stereoscopic microscope.
Eighty-nine ICR strain female mice were divided into three groups: cyclic mice (proestrus, estrus, metestrus I, metestrus II, diestrus), ovariectomized mice (5, 10, 15 days after ovariectomy) and mice administered daily with progesterone after ovariectomy (5, 10, 15 days after ovariectomy). When their uteri were taken, casting of the uterine cavities was done by the method of MURAKAMI et al. (1984) which does not require partial polymerization by heating nor ultraviolet light treatment prior to injection.
The casts were different in length, width and configuration among those under physiological con-ditions. The casts were like thick ribbons or rather worn-out tapes with widths and thicknesses different at places and some were wrinkling irregularly all along the lengths. The casts were found in the uteri with one edge always toward the mesometrium throughout its length. These casts observed in this study help us imagine how uterine cavities lie in mouse uteri.

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