Two in vitro culture methods, an oil covered culture method and an open culture method, were used to determine the effects of fetal calf serum (FCS) and epidermal growth factor (EGF) on the development of bovine oocytes fertilized in vitro. Immature bovine oocytes were cultured to maturity as follows, fertilized in vitro and cultured by standard procedures. The first experiment compared the open culture method with the oil covered method. The open culture method involved using 500 μl of medium per well. The oil covered method involved using 100 μl droplets of medium covered by paraffin oil. In this experiment, 25 mM HEPES buffeted TCM-199 medium (TCM-199) supplemented with FSH (2 μg/ml), E₂ (1 μg/ml) and FCS (10%) was used as the maturation medium. The differences between the two methods were not significant in both the cleavage rate (65.4 vs. 64.1%) and the development rate into blastocysts (22.8 vs. 17.6%). In the second experiment, development of immature oocytes was compared using three media, A) TCM-199 + FSH (2 μg/ml)+ E₂ (1 μg/ml), B) TCM-199 + FSH + E₂ + FCS (10%) and C) TCM-199 + FSH + E₂ + EGF (10 ng/ml), and cultured using the open culture method. Neither the cleavage rate (68.9, 67.4 and 73.1%) nor the development rate (23.0, 22.7 and 25.9%) was significantly different among the three media. The oocytes matured in the serum-free medium can develop as well as oocytes matured in the medium supplemented with the serum. The development rate of oocytes matured in a serum-free medium in combination with the open culture method was not inferior to oocytes matured on the conventional system using a medium supplemented with serum and covered with oil. It appears from the present results that the serum-free medium under the open culture system is stable and can be used for mass production of bovine embryos.

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This paper describes a new method for producing identical twin calves developed from pairs of sex-predicted triplet demi-embryos, one of which used for embryo sexing. In addition, we compare this method with the conventionally used twin-bisection method. Bovine embryos (48) were collected 7 d after artificial insemination and then bisected in triplet by micromanipulation, after which one of monozygotic triplet from each embryo was subjected to a sex analysis. Those sexes were determined using the polymerase chain reaction (PCR) method (8/9, 89%) or cytogenic analysis (15/39, 38%, P<0.05). The all remaining 48 pairs of triplet embryos were evaluated as transferable after 20 h of culture in vitro. Of the resultant 23 sex-predicted pairs and 25 sex-incompletelypredicted pairs of demi-embryos, 31 pairs including both groups were nonsurgically transferred one or two demi-embryos into every 41 Holstein recipient. Twenty-two (54%) of the recipients became pregnant and produced 22 live offspring, of which five pairs were identical twins. The pregnancy rate and number of calves were not significantly different from those of conventional twin-bisection method, because 57% (51/89) of pregnancy resulted from twin-bisected embryos with 20 offspring and 14 pairs of identical twins. The sex of the resultant offspring produced using our method coincide with that predicted. These results indicate that the method used here is feasible for producing sex-predicted identical twin.

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An etiological survey on stillbirth and neonatal death in Japanese Black cattle was carried out through 1987 to 1993. Incidences of stillbirth and neonatal death in 6,475 carvings were 2.4% and 2.1%, respectively. Twenty-one percent of the stillbirth was associated with dystocia, 5.1% with death of dams, 3.9% with congenital defects and other 69.7% with unknown reasons. The neonatal death was caused by neonatal weakness (59.4%), diarrhea (17.3%), injuries by dams (4.5%), pneumonia (3.8%), omphalitis (3.8%) and congenital defects (3.0%). Average birth weight of the fetus on stillborns was 21.0 ± 8.2 kg and one of the newborns was 21.7 ± 7.0 kg. The stillborns with unknown reasons had markedly low weight (19.2 ± 7.5 kg), as well as the newborns with weakness (19.7 ± 6.8 kg). There was a significant positive correlation between the birth weight and gestation period in stillborns (P<0.01), though no significant correlation was found in the newborns with weakness in which average gestation period was 283.7 ± 11.3 days. These findings suggest that the low birth weight termed, "small for dates", is one of the major causes of neonatal weakness and subsequent death in Japanese Black cattle.

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Effect of prifinium bromide in Beagle bitches in reducing side effects of a long-acting prostaglandin analog, fenprostalene administered subcutaneously to induce parturition was investigated. Seventeen pregnant Beagle bitches, 1 to 6 years old and pregnant for 56 to 58 days after the last mating, were divided at random into three groups. Bitches of groups I (n=6) and II (n=5) were first injected intravenously with 7.5 mg prifinium bromide and 5 minutes later, 20 μg /kg (group I ) or 5 μg/kg (group II) fenprostalene was injected subcutaneously. Other 6 bitches (group III; controls) were injected with 20 μg/kg fenprostalene alone without pretreatment with prifinium bromide. In controls (group III), 83% and 100% of bitches showed severe diarrhea and salivation, respectively, following fenprostalene administration, while none in groups I and II showed such side effects after the fenprostalene treatment. Other side effects caused by fenprostalene such as vomiting and colic in terms of the occurrence and its duration were also reduced in the groups pretreated with prifinium bromide. The reduction of the side effects by the pretreament was more remarkable in bitches treated with a relatively smaller dose of fenprostalene. An increase of plasma cortisol associated with p hysiological stresses caused by the side effects of fenprostalene was less in bitches pretreated with prifinium bromide than those not pretreated. There were no significant differences in the effects of fenprostalene in inducing parturition and the mortality rates of new-born puppies within one week after birth among the three treated groups. It may be concluded that an administration of 5 μg/kg of fenprostalene with a pretreatment with 7.5 mg prifinium bromide is a reliable and safe way of inducing parturition in the bitches.

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Thirty Beagle and one mixed breed bitches were treated with exogenous gonadotropin to induce super ovulation. Twenty bitches were injected intramuscularly with 44 IU/kg of pregnant mare serum gonadotropin (PMSG) daily for 5 or 9 days from the beginning of proestrus. Thirteen of the 20 bitches were administered intramuscularly with 500 IU human chorionic gonadotropin (hCG) 2 to 3 days after the last PMSG injection. Eleven anestous bitches were also treated with PMSG for 9 days followed by hCG. All the animals were subjected to midventral laparotomy under general anesthesia, and ovarian response to the treatment was investigated. The bitches treated with gonadotropin during pro estrus as well as anestrus showed a significant increase in the size of the ovaries (P<0.01), the number of follicles (P<0.05 - 0.001), and plasma concentrations of progesterone, compared with non-treated bitches in estrus (controls). The oocytes were collected by incision of the ovarian bursa and aspiration of the follicular fluid with a needle and syringe. Average number of follicles developed after the gonadotropin treatment and number of oocytes collected from each group of the animals ranged between 24 and 34, and 5 and 12 respectively. Morphological features of the oocytes examined by light microscopy as well as scanning electromicroscopy were similar to the ones observed in bitches during normal estrus. One and three days after collection of the oocytes, all the bitches were subjected to natural breeding. Ten of 18 bitches that were treated during proestrus and mated became pregnant, giving birth to five puppies on the average. Nine bitches were mated during the next estrus, and eight bitches conceived. Thus, the gonadotropin treatment during proestrus was shown to be effective in inducing super ovulation and the ovulated oocytes were considered to be fertile. On the other hand, seven of the 11 anestrous bitches exhibited estrus following the gonadotropin treatment and they were bred naturally. Six of the 7 bitches did not conceive; only one bitch conceived, but fetuses were absorbed at the middle of pregnancy. It may be concluded that super ovulation can be successfully induced with PMSG treatment for 5 days from the beginning of proestrus, fertile oocytes can be picked up by laparotomy, and donors can conceive following mating after the oocyte collection. Nevertheless, induction of estrus as well as super ovulation in anestrous bitches with PMSG could not be recommended because of pregnancy failure.

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Sex of early bovine embryos was determined by polymerase chain reaction (PCR) to amplified DNA using double primer (Y-specific DNA primer BOV97M, 157 bp; Bα-Lactalbumin primer in male and female, 109 bp). The sex determination accuracy of biopsied half embryo (20-30 blastomere) and 1/3 biopsied embryo (10-15 blastomere) by PCR method with BOV97M primer were 100% and 96.4% respectively, in accordance with chromosome analysis. In addition, double primer of BOV97M and Bα-Lactalbumin were used for DNA amplification for PCR. Detection rate of embryonic DNA was 95.2-100% with 8-16 cells, 72.0-83.3% with 2-4 cells, respectively. The result showed that the detection rate of embryonic DNA of 2-4 cells was significantly lower than that of 8-16 cells (P<0.05). It suggested that using of Bα-Lactalbumin primer is necessary and the effective number of blastomere was conjectured about 10-15 cells for sex determination with double primer by PCR method.

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Pulsatile LHRH/LH secretion plays a key role in the maintainance of reproductive functions in mammals. The present study suggests that LHRH pulse generator is located in the anterior part of the mediobasal hypothalamus and consists of non-LHRH neurons. Environmental stimuli, such as suckling stimulus and fasting stress profoundly suppresses pulsatile LH secretion. The neural pathway involved in suckling- or fasting-induced suppression of LH pulses was discussed.

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The present study was carried out to clarify the mechanisms involved in the regulationof FSH secretion and folliculogenesis in cattle. The major findings are summarized as follows. 1) Changes in plasma concentrations of inhibin reflected the population of the antral follicles in the ovaries, and not solely the dominant follicle(s), while plasma estradiol reflected the growth and population of the dominant follicle(s). 2) There was a clear inverse relationship between the concentrations of FSH and inhibin in the intact cyclic and eCG primed cows. 3) Passive immunoneutralization of endogenous inhibin resulted in a marked increase plasma concentrations of FSH with a coincident increase in the number of antral follicles during both the follicular and mid-luteal phases. 4) Passive immunization against estradiol alone did not affect concentrations of plasma FSH during the follicular phase, however immunization against both estradiol and inhibin produced a prolonged increase in plasma FSH as compared with the increase after inhibin immunization alone. These findings indicate that ovarian inhibin has a primary role in the inhibitory regulation of FSH secretion during the bovine estrous cycle, and also suggest that estradiol, on the other hand, has a synergistic effect with inhibin on FSH secretion.

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Ethological studies of vertebrates in Japan has been started by scientists of physiological background, and their interest was mainly focussed on proximal causes rather than ultimate ones of animal behavior. Thus bias is being improved towards more balanced form now, but problems are still remaining. Here, the history of ethology in Japan was briefly reviewed with expectations of the progress in future.

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Brood parasitism is a breeding habit of birds, in which eggs are laid into the nests of other birds. Cuculus cuckoos, representative of such brood parasites, visit the laying nests of hosts, and remove one egg from the nest before laying their own egg. A particular female cuckoo lays one egg in one nest. It takes only a few seconds for the cuckoo to lay an egg. Cuckoo eggs are larger in size and similar in color to host eggs. Cuckoo young eject host eggs or young from the nest by placing them onto the scrape of their back. All of these characteristics are adaptive and contribute to higher breeding success. Many of them evolved through interactions with host species. One female cuckoo may lay up to 25 eggs in one breeding season. This large number of eggs to be laid is probably due to the greater contribution of energy released from nesting and feeding into egg formation.

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The goldfish Carassius auratus is a gonochoristic teleosts and exhibits sex-typical behavior during spawning. Female sex behavior (oviposition) is elicited by prostaglandin produced in the ovary, and the ovarian steroids are not essential to this female sex behavior to occur. In contrast, male sex behavior (chasing and ejaculation) is triggered by sex pheromones from ovulatory females, and androgen is considered to be a requirement for the behavior. Sexual behavior of opposite sex do not normally occur in the goldfish but could be induced in adult fish by hormone treatment. Prostaglandin injection induced female sex behavior in males and androgen implantation induced male sex behavior in females. Interestingly, these hormone treatment did not affect sexual behavior of the original sex. These results indicate that adult goldfish retain a bipotential brain sex. If this bipotentiality is common among gonochoristic teleosts, then bipotentialities of hermaphroditic teleosts could be viewed not as innovations distinct from those of other teleosts, but as variations on a basic teleost pattern.

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In sheep, rapid onset of maternal behaviour occurs at parturition and an exclusive bond between a mother and her lambs is established within a few hours after birth. Parturient ewes discriminate between their own lambs and alien lambs: they allow the former to access and suckle the udder, while they reject suckling attempts from the latter, often violently. Olfactory signals from the lambs are the most important cues in this context. Microdialysis measurement of neurochemical substances in the sheep olfactory bulb (OB) reveals increased release of intrinsic (glutamate (GLU), Y-aminobutyric acid (GABA) and dopamine) and centrifugal (noradrenaline (NA) and acetylcholine) neurotransmitters at parturition. Increased release of these transmitters also occurs after artificial vaginocervical stimulation (VCS), which induces ewes to accept alien lambs. Therefore VCS which takes place at parturition plays a key role in triggering the release of these transmitters in the OB and establishing a selective maternal bond. Rapid changes in effectiveness of VCS on release of neurotransmitters occur after the first experience of parturition in primiparous ewes, suggesting that maternal experience with lambs enhances the establishment of a lamb recognition memory at the time of subsequent parturitions. The formation of maternal olfactory memory depends in part on release of nitric oxide (NO) and NA, because inhibition of NO synthase activity or blockade of β-adrenergic receptors or NA release during labour prevents selective bonding and allows ewes t o accept alien lambs. Following selective bonding, the release of GLU and GABA in the OB increases in response to own lamb odours, but not to alien ones, indicating that the importance of these transmitters for mediating the recall of the olfactory memory. Moreover, blockade of GABA_A receptors in the OB overcomes a selective bond with own lambs and induces acceptance of alien lambs.

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[in Japanese]

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