Attempts were made to collect amniotic fluid for the purpose of prenatal diagnosis of sex or chromosomal anomalies in cattle.
1) Two amniotic fluid samples were collected by inserting an aspirating needle (Fig. 1-1, 1-4) through the incised upper right flank.
2) Four samples were collected by using a fiberscope in which a teflon cannula containing a piano wire was inserted (Fig. 1-2, 1-3).
3) Eight samples were collected by inserting a teflon cannula and a piano wire, which was intro-duced into the abdominal cavity via the trocared vaginal wall or the upper right flank.
Two cases of abortion and one case of premature birth occurred. In spite of the risks involved, 2) and 3) technique may be considered to be a practical method for the collection of amniotic fluid in cattle for diagnostic purposes.

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For the purpose of prenatal diagnosis of sex in cattle, amniotic fluid was asepticaly collected, and the amniotic cells were cultured in vitro in 70 cases; 64 cases were obtained from the pregnant uterus at the time of slaughter, and 6 cases were collected from a pregnant cow.
As the number of amniotic cells per 1ml of amniotic fluid was 5×10³-10⁵ (2-6 months of preg-nancy), 10-20ml of amniotic fluid was centrifuged at 1, 000rpm for 5 minutes, and the cell button was dispersed and suspended in a medium of 3ml. The cell cultures were prepared in an incubator at 37C from 8 to 37 days.
Successful cultivation of amniotic cells was obtained by a medium supplemented with bovine fetal serum and N-2-Hydroxyethylpiperadine-N'-2-ethanesulfornic Acid rather than by the other pre-pared mediums.
Amniotic cells from the 3 to 5 months of pregnancy attained the fullest growth in vitro as com-pared with those of the other months.
Twelve cases of successful cultures from the pregnant uterus could be examined chromosomally, and the chromosomal sex and the gonadal sex of the fetus were in good agreement. In 5 out of 6 cases from the pregnant cows, the prenatal chromosomal sex and the gonadal sex of the calf after birth was in agreement, however, in 1 case, a discordance was observed between the prenatal chromo-somal sex and the neonatal gonadal sex.
In addition, a rapid, simple dividing method between amniotic fluid and the allantoic fluid was designed to prevent the false aspiration of allantoic fluid in place of amniotic fluid.

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A total of twenty-two cows having at least one follicular cyst (FC) which was sized more than 2.5 cm in diameter and remained in the ovary for more than 10 days, were used in the present experiment. Of these cows, 12 had apparently normal Graafian follicle (GF) 1.0 to 2.0 cm in diameter in the same or contralateral ovary in which FCs were found at the start of the treatment.
All the cows were injected intramuscularly with 100 or 200μg of LH-RH analogue (LH-RH-A). If the follicular cysts were luteinized and the cows did not return to estrus within 23 days following LH-RH-A treatment PGF_2α-tham salt at a dose of 10 or 20 mg were injected intramuscularly.
Changes in serum progesterone levels before and after the treatments were examined by an enzyme immunoassay to monitor the luteinization of FC and/or GF.
The results obtained were as follows:
1) Four of the 12 cows having FC and GF together and 4 of 10 cows with FC only exhibited normal esturus.
2) Nine (75%) of the 12 cows with FC and GF had GF in the ovary contralateral to the ovary with FC.
3) Spontaneous rupture of GF was detected by palpation per rectum in 9 of the cows with FC and GF one day after the treatment, while no FC rupture was found either in the 12 cows with FC and GF or in 10 cows with FC only.
4) Of the 12 cows having FC and GF together, 11 showed an increase in serum progesterone levels 10 days after the treatment and 8 showed a decline of the progesterone levels 20 days after the treatment. Similarly, 8 of 10 cows having FC only showed an increase in serum progesterone after the treatment and in four of them the level decreased thereafter.
5) Eleven of the 12 cows having both FC and GF exhibited normal estrous signs 21+3 days (mean±SD) after LH-RH-A treatment, and 6 of 8 cows inseminated on the day of estrus were result-ed in conception.
6) In the other 10 cows with FC, only 2 came into estrus within 23 days after LH-RH-A treat-ment. The interval from the treatment to estrus was 29±11 days.
7) The conception rate at the first insemination in the cows bearing FC and GF together was 75% (6/8) and overall conception rate was 92% (11/12), while the corresponding figures in the cows with FC only were 57% (4/7) and 50% (5/10).
These findings indicate that if the cows those having FC and GF together exhibit normal estrous sign, they may have a possibility to conceive when inseminated, since FC themselves do not seem to impair basic function of ovary.

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The temporal relationship of the change in peripheral plasma level of gonadotropins to the onset of estrus and the occurrence of ovulation was studied in 3 cyclic Japanese Black heifers. Blood sam-ples were obtained from the juglar vein at intervals of 1 or 2 h for consective 4 to 5 days coveringthe period of estrus and ovulation. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were determined by the respective double antibody radioimmunoassay.
Plasma FSH concentrations remained low before and after the estrus period. The FSH surge with the peak level of 318.4±29.6ng/ml (mean±SD) was observed concurrent with the LH surge 5.3±2.3h after the onset of estrus and the duration of the surge was 9.3±0.6h. Ovulation was detected 25.7±0.6h after the FSH peak or 31.0±2.6h after the onset of estrus.
Besides the prominent FSH surge during the estrus period small but noticeable peaks were fre-quently observed in the plasma FSH concentration. The fluctuations of FSH before the onset of estrus tended to synchronize with those of LH. After the estrus period, however, LH stayed at a constantly low level irrespective of the changes in FSH.
Thus, there is a possibility that FSH might be involved in the ovulatory process in the heifer and that the responsiveness of pituitary cells for FSH and LH to gonadotropin releasing hormone (GnRH) might be differently modified by ovarian steroids.

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An improved culture method for the hamster ovary was developed to study the mechanism of ovulation. Hamsters used in this study spontaneously ovulated a mean number of 6.2 ova per ovary between 00:00 h and 02:00 h of the day of estrus. The ovaries were excised between 17:00 h of the day of proestrus and 01:00 h of the day of estrus, placed on a stainless steel wire table and cultivated at the surface of the culture medium of modified M-199 until 10:30 h of the following morning. Ova shed into the medium were counted. Among ten ovaries explanted at 17:00 h only one ovary ovulat-ed. The ovulation rate increased when the ovary was explanted at 18:00 h (47%) and 19:00 h (78%)and reached 100% at 21:00 h or later. When the ovaries were explanted at 21:00 h and 23:00 h of proestrus, the average number of shed ova was 4.7 and 4.6, respectively. These values are 75% of the value in in vivo ovulations. The ovaries explanted at 21:00 h of proestrus ovulated between 02:00 h and 06:00 h while the ovaries explanted at 00:00 h completed ovulation by 02:00 h.

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This experiment was conducted in order to clarify the effects of underfeeding on estrous be-havior and ovarian function in postpubertal beef heifers. Five Japanese Black heifers, 13.5-months-old and having repeated at least two normal estrous cycles after puberty, were underfed with the maintenance level (2.30kg TDN) ration for three estrous cycles. Observations for estrous behavior were made at 1:00, 5:00, 9:00 A, M., and 13:00, 17:00 and 21:00P.M, for an hour each on a herd including one sterile bull and four cows nursing their calves besides the experimental heifers. Ovula-tion was examined by rectal palpation every four hours. Progresterone concentration was measured by radioimmunoassay in blood collected twice a week during an estrous cycle before the underfeeding period and in the three estrous cycles during the underfeeding periods. Data on estrous behavior during the underfeeding period were compared with the data obtained at the estrus before the under-feeding.
The body weights of heifers remained unchanged through the underfeeding period. The mean of 15 estrous cycle length for 5 heifers during the underfeeding period was 20.1 days and it was same as the mean of 18 estrous cycles before the underfeeding (20.0 days). Duration of estrus during the underfeeding period, however, was shorter by 4 to 8 hours than that before the underfeeding (20.2 hours), and intensity of sexual activity tended to decline during the underfeeding period. Fur-therfore, the number of standing to be mounted by other cows decreased from 17.6 times in the estrus before underfeeding to 7.4, 4.4 (P<.05) and 5.8 times (P<.05) per estrus in the first, second and third estrus during the underfeeding period, respectively. Estimated mean interval from the end of estrus to ovulation before the underfeeding was 14.1 hours. On the other hand, these intervals were extend. ed by 3.5 hours at the first estrus and reduced by 2.0 and 3.9 hours at the second and third estrus, respectively, during the underfeeding period. Although serum progesterone concentrations in the first and second estrous cycles during the underfeeding were lower than that before the underfeeding, values in the third estrous cycle increased to the values before the underfeeding. There were, how-ever, no significant differences in time of ovulation and progesterone concentration between the periods during and before the underfeeding. Four heifers out of five conceived at the third estrus in the underfeeding period. It seemed that there was no deleterious effect on conception. The results of this experiment suggest that reduced duration of estrus and weak estrous behavior caused by underfeeding may decrease the efficiency of estrus detection in breeding practice.

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Two experiments were conducted to examine the effect of carbon dioxide (CO₂ gas) on the survival of ram spermatozoa frozen by the pellet method. Frozen-thawed semen was flushed with CO₂ gas for various periods and the motility of spermatozoa after thawing was investigated. The longer periods for more than 135 seconds of CO₂ gas treatment significantly (P<0.01) depressed the motility of spermatozoa after thawing. However, flushing with CO₂ gas for less than 90 seconds did not affect the motility. It appears that flushing with CO₂ gas for a limited time, 90 seconds at the time of insemi-nation could be effectively utilized for the rapid method of non-surgical intrauterine in-semination with frozen-thawed semen in sheep.

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During the breeding sepson in 1980 a previously unrecognized form of equine metritis was observ-ed among Thoroughbred mares in a farm in Iburi district of Hokkaido, Japan. This farm had about 240 mares and 8 stallions. Infected mares developed grayish white vaginal discharge and associated inflammatory changes of the cervix and vagina 2-4 days after mating. Early in May, 1980, Haemo-philus equigenitalis (H.e.), causative of contagious equine metritis (CEM), was first isolated from cervical swabs of an infected mare and the disease was diagnosed as CEM.
H.e. was isolated from 20 mares by bacteriological examinations of cervical or clitorial swabs during a period from May, 1980 to February, 1981. Fourteen of the infected mares showed the same clinical signs, while others did not. Five of the 6 mares without clinical signs had been pregnant. In four of these pregnant mares abortion or early embryonic death took place, but H.e. was isolated neither from placentae, aborted fetuses nor cervices after abortion.
One stallion was mated with 29 mares from 20 April to 10 May. After mating sixteen of the mares showed clinical signs of which 12 were H.e. positive and only three became pregnant suggest-ing that he was a carrier of CEM, but all stallions including him were H.e. negative by the examina-tions of urethral fossa swabs or semen.
The infected mares were treated with intrauterine infusion of antibiotics (ampicillin, mycillin or teramycin). The pregnant infected mares were treated by washing of the vagina, clitoris and clitorial fossa with antibiotics. Stallions received intraurethral infusion of mycillin and systemic treatment with ampicillin or penicillin.
In addition, the external genitals of both the sexes were washed with 0.5% chlorohexidine before and after mating. This seemed effective in preventing spread of the disease.
By these treatments and care, CEM was cleared from this farm before the commencement of the next breeding season.

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