The objective of this study was to investigate the totipotency of bovine embryonic disc (ED) cells by nuclear transfer. Therefore, the development in vitro and survival rate after transfer to recipient cows of bovine embryos reconstructed from two cell types (ED cells and blastomere cells of morula) were examined. The developmental rates to the compacted morula and blastocyst stages were determined at day 6 after nuclear transfer. Thereafter, a portion of the compacted morula and blastocyst stage embryo was transferred to synchronized Holstein heifers. The incidence of embryos reconstructed from ED cells developing to the morula/blastocyst stages was significantly lower than the incidence of embryos reconstructed from blastomeres of the morula. Among the six ED cellderived pregnancies, four cows gave birth to four calves. These results demonstrate that bovine ED cells are totipotent by nuclear transfer.
In the present study, weight of calves and gestation periods derived from embryos developed either in vivo or in vitro were investigated. There were significant differences in the birth weight of calves derived from different strains of Japanese Black Cow (Hyogo and Shimane) when in vivo developed embryos were collected and transferred. Moreover, it was found that the average birth weight of Hyogo male calves derived from embryos cultured in in-vitro was significantly heavier than that of calves derived from embryos developed in vivo (in vivo: male; 29.2 kg vs in vitro: male; 37.0 kg ). However, there was no significantly difference between average birth weight of Hyogo female calves developed in vitro and that of calves derived from embryos developed in vivo. Moreover, there was no difference in the gestation periods between calves derived from embryos developed in vivo and those in vitro. In conclusion, it is suggested that (1) the average weight of calves derived from embryos developed in vitro is heavier than that from embryos developed in vivo, (2) heavy calf weight is more frequently observed in male, (3) there is no difference in the body weight of calves derived from embryos developed in TCM-lgg medium or in m-SOF medium, and (4) there is no difference in the average gestation period of embryos developed in vivo or in vitro.
Heterologous radioimmunoassays for canine FSH and LH were developed and used to quantitate these hormones during proestrus, estrus and diestrus of the bitch. The assay for FSH consists of a rabbit anti-human FSH serum, radioiodinated rat FSH and dog FSH-LER-l685-3A as standard. Inhibition curves produced by the standard, crude extracts of canine pituitary glands and various canine plasma were parallel. Specificity of FSH assay was confirmed by testing for binding inhibition with canine LH (LER-l685-l). Binding was hardly affected by canine LH. The intra- and interassay coefficients of variation was 3.1% and 10.8%. Sensitivity of the FSH assay was 0.20 ng/tube which was adequate to quantify the concentration of FSH in 100 μl or less of canine plasma. The LH assay consists of a rabbit anti-ovine LH serum, radioiodinated rat LH and dog LH-LER-l685-l as standard. Crude extracts of canine pituitary glands and various canine plasma p roduced inhibition curves which were parallel to the standard curve. Binding inhibition was no affected by canine FSH(LER-l685-3A). The intra- and interassay coefficients of variation was 5.8% and 14.3%. Sensitivity of the LH assay was 10.7 pg/tube which allowed estimation of LH concentrations in 100 μl or less of canine plasma. During the reproductive cycle, levels of FSH were lowest during proestrus (51.1 ± 7.4 ng/ml) and increased to 305.5 ± 39.6 ng/ml on a day after LH peak. Levels of FSH declined by Day 4 (63.5 ± 10.4 ng/ml) then increased to 117.4 ± 11.9 ng/ml on Day 18 after LH peak. Levels of LH increased only during preovulatory LH surge, during proestrus and diestrus, were less than 0.5 ng/ml.
Further progress in the production of clones and transgenic animals required an efficient system to produce porcine embryos from follicular oocytes in vitro. The failure in male pronuclear formation is now considered to be due to oxidative stress during relatively later stage of oocyte maturation. Polyspermy is still serious problem in production of porcine embryos in vitro, whereas the incidence has been partially solved by reducing the number of spermatozoa binding to the oocytes. The developmental competence of embryos matured and fertilized in vitro has been improved dramatically through modification of culture conditions for oocyte maturation. Oocyte competence for early development seems to be improved by synchronized meiotic progress and active communication between the oocyte and cumulus cells in the presence of secretions from granulosa cells. Further understanding about what are happening in vivo is required to gain an improved culture condition for in vitro production of porcine embryos.
The morphological and pathological status of the reproductive organs in cattle and horses were observed using a liner-array ultrasound scanner with a 5.0 MHz transducer, and then their image were confirmed by examination in a water- bath and/or post-slaughter. Several pathological conditions and the ultrasonic properties of the reproductive organs were characterized. From 22 days after artificial insemination, the fetal sac of cattle could be image d in uterine cavity ipsilateral to the ovary with a echogenic corpus luteum. The pregnancy of mare was also detected as early as 11 days after final breeding. Ovarian follicular cysts, cystic corpus luteum and luteal cyst in cattle were visualized, clearly, and luteinized cysts were also observed after treatment. The change in shape and size of granulosa-cell tumor, ovaritis, ovarian bleeding, and other ovarian diseases in cattle and horses were accurately observed by ultrasound. Cysts contained nonechogenic fluid in endometrium in mares were usually compartmentalized. Ultrasonography may be a good prediction of myometritis and papiloma in cows. In cases in which CO2 escaped into abdominal cavity through the oviduct in cattle, it was verified by ultrasound. The hypoechoic image was useful in monitoring one sided comptete blockage of the oviduct. Penile hematoma, oscheitis, testitis, testicular tumor, cryptorchism, and seminal vesiculitis in cattle and horses could be easily monitored using ultrasound. The results indicate that ultrasound technology provides noninvasive tools of visual access to the reproductive organ to evaluate normal morphological changes and to detect certain pathological processes.
Being one of the most important base areas for animal husbandry, Inner Mongolia has possessed total 71.12 million heads of farm animals by end of 1997. Government of Inner Mongolia has focused on the import of fine breeds from other countries through modern breeding technologies in recent years. From 1991 to 1997, about 10,000 heads of farm animal have born from embryo transfer Among which, the pregnancies of cattle in both pasture and sheds were 41.1%(241/323) and 51.6%(157/304); that of sheep and goats were 74.6%(241/323) and 79.9%(314/393), respectively. The other project is production of bovine IVF embryos and ET. From 1995 to 1996, we have produced 10,000 IVF embryos of fine breed cattle in Australia and transfered part of them mainly in Inner Mongolia. The average pregnancy and birth in from 1996 to 1997 were 19.9%(33/166) and 16.9%(28/166).In another project, the 1.5 or 3.0 month o1d kids of goats were used as oocytes donor treated with FSH-LH, and the number of follic1es, recovery of oocytes and enab1es for IVF were 42~56, 28~31, 22, respectively. The optimum duration for goat oocytes maturation is 16~20 hrs. The 2-to 4-ce11s embryos from 48 h cu1ture after IVF were transferred. By this way the F3 kids were born, and the development potential of the oocytes from kids was confirmed.
Follicles starting to grow reach a certain stage of development and then degenerate or mature and ovulate. The whole process must be under both exogenous as well as endogenous control. Under exogenous control, signals controlling the development of follicles must be translocated in the tissue, especially via vascular networks and/or tissue fluid. When observed histologically, there is a striking difference in the distribution of blood vessels in the mammalian ovary. Smaller follicles possess principally circumferential vessels with few branches, while the dominant follicles are supplied with numerous polyhedral networks of vessels that project downward to the basement membrane surrounding the granulosa cells. These morphological features suggest that microvascular networks and angiogenic factors affect the follicular growth, particularly the selective growth of follicles. In this article, the role of microvascular networks and angiogenic factors on the selective growth of follicles is discussed.
The brain mechanism governing pulsatile GnRH release, which induces pulsatile LH secretion from the pituitary, plays a key role to maintain the ovarian cyclicity. The frequency of GnRH/LH pulses is much higher at follicular phase than that at luteal phase. The increase in the pulse frequency is accepted to contribute to ovarian follicular development. Low level of estrogen secreted from immature follicles has a negative feedback effect on GnRH/LH release, while high level estrogen from matured follicles has a positive feedback effect on the release, resulting in the LH surge. One of the positive feedback action sites of estrogen has been revealed to be located in the medial preoptic area, where abundant estrogen receptors (ER) have been found. The arcuate nucleus, where many ERs is also distributed, could be another action site. It is suggested that GnRH pulse and surge generator are separately located and function in the brain, since multiple unit activity reflecting the GnRH pulse generator activity is not associated with the LH surge. Environmental factors, such as stress, malnutrition and suckling stimulus, suppress LH pulses, but not affect LH surge induced by exogenous estrogen treatment. This fact may be well explained by the above-mentioned concept that the mechanisms regulating LH pulse and surge modes of LH secretion are independent. The inhibition of LH surge and ovulation by the inhibitory environmental factors is due to the suppression the activity of GnRH pulse generator but not that of surge generator.
Some rodent species such as rats and mice recur incomplete estrous cycles, which are characterized by the lack of the luteal phase. Among animals that recur complete estrous cycles, ruminants have been shown to exhibit one or two events of follicular growth not resulting in ovulation (latent follicular waves) during the luteal phase. We investigated follicular dynamics ultrasonically in Shiba goats and found that they also showed predominantly two latent follicular waves with the third being the ovulatory wave. When goats were treated with a luteolytic dose of prostaglandin (PG) F2α 3 and 4 days after spontaneous ovulation, dominant follicles of the first wave ovulated 2 or 3 days later. Further, ovulation could be repeated at least four times at the interval of around 6 days by successive PGF2α treatments. Thus, the female goat appear to be furnished with every necessary mechanisms for recurring incomplete estrous cycles like rodents if progesterone levels are depleted shortly after the formation of corpora lutea. In rodents, 20α-hydroxysteroid dehydrogenase (20α-HSD), an enzyme which catabolizes progesterone to a bio logically inactive steroid 20α-dihydroprogesterone, plays a critical role in the maintenance of incomplete estrous cycles. Comparative analysis of genomic DNA for 20α-HSD, especially its promoter regions, will provide molecular basis for the differences in phenotypes of the estrous cycle and the evolution of estrous cyclicity in mammals.
Ovarian disjunction is a serious cause of reproductive failure in dairy cows. The present investigation was aimed at studying the clinical findings associated with their disorders. Ovarian subfunction in 5 cows were diagnosed one month following the last carving. In 3 out of 5 cows, ultrasonography over a 3 week period revealed a wave-like pattern follicular growth in the ovaries. Plasma progesterone (P4) concentration was determined by enzyme-immunoassay. Four cows had P4 of less than 1.0 ng/ml during the experimental period. However, one cow in this group had a corpus luteum during clinical examination and P4 level also increased later. The ovulatory failure in 5 cows were diagnosed by rectal palpation following 2 or 3 services among estrous days. Ultrasonography also showed the presence of follicles >1.5 cm in diameter in the ovaries. Two cows in this group had a hyper-echoic corpus luteum in addition to follicles 1.0-2.0 cm in diameter. Their P4 level were 1.5, 3.5 ng/ml, respectively. However, ultrasonography failed to show a Graafian follicle and an altertic follicle. Three cows with ovarian follicular cysts which showed nymphomaniac syndrome were diagnosed by ultrasound. Cystic follicles in all 3 cows had a wave-like pattern of growth and regression for 20 days similar to ovarian follicular wave. However, the granulosa layer and/or altered cystic follicular structure could not be imaged in all cases. Twenty days after first observation, the cows were treated with hCG 10,000 IU, intramuscularly. In 2 cows, P4 level increased to more than 1.0 ng/ml after treatment 3 days. The results indicates that cows with ovarian subfunction and follicular cysts have follicular wave in ovaries similar to a normal cycle. However, the degenerative changes and subsequent shedding of granulosa cells from the follicular wall which led to the ovulatory failure in the cows could not be imaged by ultrasound.
Infertility in cattle is often caused by poor estrus detection which results in insemination timing error. Estrus detection failure is also a major cause of missing heat and delayed start of re-breeding after carving. Synchronization of estrus by using gestagen or prostaglandin (PG) F2α has been extensively used for improving estrus detection efficiency. However, variation in intervals from treatment to estrus was too large for the existing estrus synchronization methods to be of practical value, for a timed artificial insemination after treatment with gestagen or PGF2α results in disappointing conception rates. Recently, a program for synchronization of estrus using GnRH and PGF2α has been developed. GnRH is administered at first irrespective of estrus cycle to induce ovulation of dominant follicle and a follicular wave, and then PGF2α is given 7 days later for luteal regression, followed by second GnRH injection 30 to 48 hours after the PGF2α. Cattle can be inseminated artificially at a fixed time, 16 to 24 hours after the second GnRH, since ovulation is usually synchronized 24 to 32 hours after the second GnRH. Satisfactory pregnancy rates after synchronization of ovulation and timed AI have been reported in north America as well as in Japan. The greatest advantage of the ovulation synchronization and timed AI program is that virtually 100% of the animals can be inseminated within a certain period with a satisfactory pregnancy rate. Moreover, the program does not require estrus detection. Further studies on the synchronization programs and on factors influencing conception rate after timed AI would significantly improve reproductive management of cattle.