A direct enzyme immunoassay of progesterone in skim milk was established.
1. 11α-hydroxyprogesterone-hemisuccinate was conjugated with β-D-galactosidase from E. coli by a mixed anhidride method. After purification, the conjugate was used as tracer of immunoassay.
2. Anti-11α-hydroxyprogesterone-hemisuccinate-bovine serum albumin rabbit serum and anti-rabbit γ-globulin goat serum were used as the first and the second antibodies, respectively.
3. Milk sampled from one of any udder quarter regardless of time of milking was defatted by centrifugation (3, 000 rpm, 10 minutes) and subjected to progesterone measurement.
4. Skim milk (0.05 ml) was diluted 4 times with 0.1M phosphate buffer pH 7.0, added with 0.1 ml of the first antibody and incubated at 4C for 1h. To this mixture 0.1 ml of diluted normal rab-bit serum and 0.1ml of the diluted second antibody solution was added. After incubation at 4 C overnight, it was centrifuged at 3, 000 rpm for 20 minutes to separate the bound from free form. The precipitate was washed once and resuspended to the buffer. Then the enzyme activity was deter-mined. Skim milk from cows in estrus treated with dextran and charcoal, which were diluted 4 times with the buffer containing standard progesterone (0-1, 000 pg), were also assayed.
5. The sensitivity of this enzyme immunoassay was 4 pg/tube. Inter- and intra-assay coefficients of variation were 10.7% and 8.6% each. Milk progesterone values obtained by the present direct enzyme immunoassay and the previous technique with extraction of progesterone from milk were highly correlated (r=0.95, P<O.01).
It is concluded that the direct enzyme immunoassay of skim milk progesterone is highly sensitive, reliable, and practical, and therefore applicable for the assessment of luteal activity of cows.

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Histochemical observations were made on the lipid droplets and glycogen granules in delayed im-planting mouse blastocysts. Immediately after the removal of ovaries from mice on Day 3 of preg-nancy, 1 mg of progesterone (Nakarai Chemicals) dissolved in 0.05ml of sesame oil was daily injected subcutaneously. Mice thus treated were killed on Day 10, 17, 24 and 30 respectively. As controls, normally pregnant mice were killed on Day 4. For the demonstration of lipid droplets, the blasto-cysts collected by flushing the uteri were stained with sudan IV. For the demonstration of glycogen granules, the blastocysts in the serial paraffin sections of the uteri were treated by the periodic acid-Schiff method. The results obtained were as follows.
1. Lipid droplets. Most (94.3%) of the inner cell masses of blastocysts from normally pregnant mice on Day 4 contained lipid droplets, whereas most (76.7-94.6%) of those from delayed implanting blastocysts on Day 10 to 30 contained no lipid droplets. Trophoblasts of blastocysts both from nor-mally pregnant mice and from delayed implanting mice contained lipid droplets. The ratio of blasto-cysts containing a large amount of lipid droplets in the trophoblasts was much higher in the delayed implanting blastocysts (70.3-90.6%) than in the control blastocysts (31.4%).
2. Glycogen granules. Most (86.1%) of the inner cell masses of blastocysts from normally preg-nant mice on Day 4 contained glycogen granules, whereas most (74.2-94.1%) of those from delayed implanting blastocysts on Day 10 through 30 contained no glycogen granules. About one third (34.7%) of the trophoblasts of blastocysts from normally pregnant mice contained glycogen granules. As for the delayed implanting blastocysts, however, a few trophoblasts (0-11.8%) contained glycogen granules.
There were no differences observed among the groups of different delaying period (Day 10 through 30) concerning the amount of lipid droplets and glycogen granules neither in inner cell masses nor in trophoblasts.

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In 10 of 19 cows in which follicular cysts (FC) were rectally palpated as single or multiple fol-licular structures 2.5 cm or larger in diameter, and persisted more than 10 days unilaterally or bilat-erally, a Graafian follicle (GF), 1.0-2.0 cm in diameter coexisted with FC. Both the FC only group and the FC plus GF group were examined for changes in ovaries, estrous behavior and progesterone (P₄) levels in skim milk on 0, 1, 5, 10, 15 and 20 days after the first observation without any treat-ment.
Milk samples were centrifuged and P₄ concentration in skim milk was measured by enzyme im-munoassay.
All of the 10 cows in the FC plus GF group showed a normal estrus and then returned to estrus 20-24 days later, while the 9 cows in the FC only group exhibited continuous estrus (6 cows) or re-mained anestrus (3 cows).
The coexisting GF in the former group spontaneously ruptured on the day after the first observa-tion (9 of 10 cows), luteinized 5-15 days later (all cows), and then the corpus luteum regressed and a new GF developed on 20-24 days (all cows).
In the FC only group, however, none of FC coexisting with GF ruptured, luteinized or regressed. In all cows of this group no morphological changes in FC were observed during the same period.
Average concentrations of P4 in skim milk on 0, 5, 10, 15 and 20 days after the first observation were 0.6±0.2, 2.0±1.4, 4.5±2.1, 1.9±0.7, and 0.6±0.2ng/ml (Mean±SD) in the FC p¹as GF group, and 0.5±0.3, 0.6±0.2, 0.6±0.3, 0.7±0.4 and 0.8±0.3ng/ml in the FC only group.
These results indicate that cows having both follicular cysts and a normal Graafian follicle with normal estrous sign may be possible to conceive with no hormonal treatment for the cystic ovary, if the animals would be properly inseminated during the estrus.

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It has been known that the life span of the corpus luteum is prolonged after hysterectomy in various species of animals. In the goat, however, the effects of hysterectomy have been studied only on the corpus luteum of pregnancy.
In the present study, the effect of hysterectomy on the function of luteum of estrous cycle was investigated by measuring progesterone (P) levels in the peripheral venous blood in shiba-goats.
Three animals were used, one was on Day 2 and next one was on Day 4 of the estrous cycle, and the last one was in anestrus (Table 1). They were hysterectomized under xylazine and fluothane anesthesia (Figs. 1-3).
After hysterectomy, P levels in the peripheral venous blood were measured by radioimmunoassay (RIA) every day for 25 consecutive days after operation and at 2-3 days interval thereafter.
In two cyclic goats in which corpus luteum or corpus hemorrhagicum was found at operation, P levels were maintained in a range of 0.7-3.9 ng/ml for about 50 days after hysterectomy (Figs. 4, 5) and during which time the estrus did not return. In an anestrous goat in which no corpus luteum was present, low P levels (about 0.5ng/ml) were maintained.
From these results, it was suggested that the luteolytic factors should have its origin in the uterus in case of shiba-goats in the cycle.

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In order to elucidate vascular connections between the uterus and ovary, colored latex was infused into abdominal aorta and vena cava to make casting specimens.
It was shown that the vascular system of the uterus and ovary in the shiba-goat closely resembled that of the ewe. In both specise, the ovarian artery branched off from abdominal aorta (Figs. 1 &3), then ran tortuously over the surface of the ovarian vein. Of three branches of the ovarian artery (ovarian, uterine and tubal), the ovarian branch is the major one and the other two are judged as minor branches (Figs. 2 & 4).
It is suggested that in the shiba-goat most of the venous blood coming from the uterus flows into the uterine branch of the ovarian vein through the venous plexus which are formed adjacent to the uterine horn. The remaining venous blood of the uterus seemed to flow into the uterine vein.
From these results, putative route by which luteolytic factor produced by the uterus is transmitted down to the ovary was discussed.
The results of the present study suggested that the transfer of luteolytic factor derived from the uterus to the ovary in the shiba-goat may depend upon "Counter Current Mechanism" as presumed previously in the ewe by BARRETT et al. (1971).

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Ejaculated bull spermatozoa from two sires were washed, preincubated in a synthetic culture medium (BRACKETT & OLIPHANT, 1975; BO medium) for 4 to 5h in a CO₂ incubator at 37C, and thereafter treated with calcium ionophore A 23187 (I-A) to investigate the effect of I-A for the induction of sperm capacitation and acrosome reaction in vitro. The effect was evaluated by sperm penetration into zona-free hamster eggs in vitro. When spermatozoa were treated with high ionic strength BO medium (376 mOsmol/kg) for 15min before their preincubation, none of the eggs were penetrated after subsequent incubation for 3 to 3.5h. The penetrated eggs were observed only by spermatozoa treated with I-A, and the effect of I-A differed according to the medium used and to I-A concentration and its treating time. In BSA-free medium the effect of I-A was more obvious with lower I-A concentra-tion and with shorter treating time than in the medium containing BSA. Sperm motility was im-paired by the increasing I-A concentration and by prolongating treating time. Whiplash-like move-ment of spermatozoa was observed in every sperm suspension after I-A treatment. Caffeine (2mM) appeared to enhance the effect of I-A by shortening the time required for I-A treatment. Clear dif-ference in the rates of penetrated eggs was observed between spermatozoa from two sires, and the difference did not appear to be related directly with sperm motility and with the condition of I-A treatment. Highest rate of penetrated eggs was obtained by spermatozoa from one bull after treat-ment with 0.5μM I-A for 2.5min in BSA-free medium, and penetration of spermatozoa into the eggs was achieved within 3 h after I-A treatment. Preincubation of sperm before I-A treatment was not essential for sperm penetration.
These results suggest that the capacitation and acrosome reaction of ejaculated bull spermatozoa can be induced in vitro within 3 h by treating spermatozoa with I-A.

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Using 30 multiparous Japanese Black Cattle, two experiments were conducted to clarify the effects of restricted suckling on postpartum reproductive performance of cows and growth and ingestive behavior of their calves.
In Experiment 1, 21 cow-calf pairs were assigned at 4 days after calving to one of three treatment groups: once-daily suckling, twice-daily suckling and normal suckling. Calves on the restricted suck-ling regimes were kept in calf pens except for their suckling time of 30 minutes and had hay available there until weaning at 90 days of age. All cows and the normal suckled calves were put on pasture. In Experiment 2, in addition to the effect of concentrate supplements ingestive behavior was inves-tigated in once-daily and twice-daily suckled calves.
In Experiment 1, cows on the restricted suckling regimes slightly gained, whereas the normal suckled cows lost an average of 17 kg during the 3 postpartum months. Restricted suckling tended to shorten the postpartum intervals to first ovulation, first estrus and conception when compared to normal suckling, and there was a significant difference in the interval to first estrus between once-daily and normal suckling (23.6 vs. 44.4 days, P<0.05). Furthermore, restricted suckling increased the proportion of cows exhibiting estrous behavior at the first ovulation. There were no significant dif-ferences in the interval from first to second ovulation, the number of inseminations per conception and the peak serum progesterone levels after first and second ovulation among the three groups. Average daily gain from birth to 3 months of age was significantly lower for once-daily suckled calves than for twice-daily and normal suckled calves (0.53 vs. O.81 and O.87 kg, P<0.01).
In Experiment 2, once-daily suckled calves showed a growth rate similar to twice-daily suckled calves (0.82 vs. 0.88 kg). Although daily hay intake by the calves was almost the same in both groups throughout the experimental period, once-daily suckled calves consumed about twice the concentrate twice-daily suckled calves did. Ingestive behavior was similar for all calves at 10 and 25 days of age. As the calves grew older, once-daily suckled calves tended to spend more time in in-gesting concentrates, whereas twice-daily suckled calves spent more time in ingesting hay.
These results suggest that restricted suckling would have desirable effects on cow performance, and that even once-daily suckling would not reduce the growth rate of calves if creep feed were avail-able.

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Various cryoprotectans and freeze-thawing procedures were examined for the preservation of rab-bit embryos. Rabbit morulae were recovered 60-65h after mating from females which had been in-duced to superovulate by injections of FSH and hCG. The morulae were suspended in 0.2ml PBS (supplemented with 20% rabbit serum) containing 1.5M-DMSO, -ethylene glycol, -glycerol or -ery-thritol in a glass test-tube at room temperature. The samples were cooled at 1C/min to -4C and were seeded. Tubes were then cooled either (a) slowly to -75C at 1C/min and then plunged into liquid nitrogen, or (b) rapidly by transfer into -20C ethanol, kept for 10min, suspended in nitrogen vapor (-100C) for 10min, and then plunged into liquid nitrogen. After storage for 10-150 days, these samples were thawed either slowly at room temperature or rapidly in warm water at 30-35C. The cryoprotectants were removed by transferring the embryos into PBS containing the cryoprote-ctant and 0.5M-sucrose, PBS with 0.5M-sucrose, and finally into fresh PBS. The embryos were cul-tured for 48h in a modified Tyrode solution containing 20% rabbit serum to determined their survival rate, which was assessed by the ability to develop to expanded blastocysts. The results obtained are as follows.
In the freeze-thawing procedures examined, the highest survival rates were obtained when embryos were frozen in the presence of DMSO, followed by ethylene glycol, and the third glycerol. Slow thawing of slowly frozen samples gave good survival rates of 70% in DMSO, 38% in ethylene glycol and 24% in glycerol. When slowly frozen samples were thawed rapidly, survival rates of embryos stored with DMSO and glycerol decreased. Survival rates of embryos frozen-thawed rapidly were lower than those of slowly frozen embryos.
When erythritol was used as a cryoprotectant, morphologically normal embryos were not obtained after freeze-thawing except for the case of rapid freezing, and no embryos developed in culture.

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The present study was designed to examine the efficacy of ethylene glycol (E.G.) as a cryoprotectant in the frozen storage of rabbit morulae. The highest viability of frozen-thawed embryos in vitro was attained in 1.2 M-E.G., but it was significantly lower (P<0.05) than that in 1.5 M-DMSO (dimethyl sulfoxide) (69% vs. 88%). On the other hand, the viability of young produced by transfer of frozen-thawed embryos in 1.2 M-E.G. was equal to that in 1.5 M-DMSO (20% vs. 14%). The production of normal live young in the present study, by transfer of rabbit embryos frozen with a cryoprotectant other than DMSO, may be the first success. However, the results demonstrate that E.G. is less effective than DMSO to protect against freezing damage in the rabbit morulae under the present experimental conditions.

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