The present study was conducted to examine the effect of ascorbic acid (AsA) on the cytoplasmic maturation of cumulus-denuded porcine oocytes during in vitro maturation (IVM) culture. After mechanical removal of cumulus cells, cumulus-denuded oocytes (DOs) were cultured for 44 h in BSA-free NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and 0 to 750 μM ascorbic acid 2-O-α-glucoside (AA-2G), a stable ascorbate derivative, and then assessed for their developmental changes, including the progression of meiosis, in vitro fertilization (IVF) parameters and subsequent embryo development. Treatment with 250 μM AA-2G during IVM culture in DOs showed no influence on the rates of maturation (60-61%), sperm penetration (86%), and polyspermy (69-71%) following IVF, but enhanced the ability (43%) to form male pronucleus (MPN) compared to that of DOs treated without AA-2G (20%) (P<0.05). Although this rate of MPN formation of DOs was still lower than that of COCs (66%) (P<0.05), the addition of 250 μM AA-2G to maturation medium in DOs apparently enabled IVM-IVF-derived zygotes to develop to the blastocyst stage (3%). The levels of intracellular AsA and glutathione in DOs matured with AA-2G were significantly higher than those in DOs matured without AA-2G (P<0.05). Moreover, the length of DNA migration, analyzed using the comet assay, was increased by oxidative damage in DOs treated without AA-2G during IVM culture, but treatment with AA-2G abated DNA damage, indicating that intracellular AsA supplied by AA-2G can potentiate the cellular protection of oocytes against oxidative stress through its scavenging activity during IVM culture. Thus, the present findings suggest that the preventing oxidative stress in oocytes during meiotic maturation supports the cytoplasmic maturation responsible for the subsequent developmental competence post-fertilization.

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This study was carried out to investigate changes in gonadotrophin secretion in response to estradiol, and to clarify the change of the positive feedback action of estradiol in prepubertal heifers. Four prepubertal Holstein-Friesian heifers were treated with 2 μg/kg estradiol benzoate intramuscularly at 1, 3, 5, 7 and 9 months of age, and plasma samples were collected every 3 h for 36 h after estradiol treatment. Plasma concentrations of LH, FSH and estradiol were measured by RIA. The changes in peripheral estradiol concentration after estradiol treatment were similar in pattern with peaks at 3 h in all ages. LH and FSH rise induced by estradiol were observed in heifers after 3 months of age, and the time from the estradiol administration to the appearance of the peak of gonadotrophin rise gradually shortened with the age. The peak concentration of LH rise increased, while the concentration of FSH rise did not change with age. These results indicate that the hypothalamus and pituitary gland already have reactivity to estradiol in heifers by 3 months of age, and that estradiol positive feedback action on LH secretion develops with age in prepubertal heifers. We concluded that the development of estradiol positive feedback action on LH secretion before puberty may be one of the essential factors for deciding the time of the onset of puberty in heifers.

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We investigated translation kinetics after enhanced green fluorescent protein (EGFP) mRNA injection into maturing porcine oocytes and then established a method for selecting the oocytes that were translating the injected mRNA without fixing and staining. The EGFP signal was observed within 3 h of the mRNA injection, and the EGFP expression rate reached a maximum (about 30%) at 6 h after injection. The high EGFP expression rate increased gradually until 18 h after injection. In the EGFP expressing group, degenerated oocytes were absent and the maturation rate was comparable to that of non-injected oocytes, while the maturation rate was low and some oocytes were degenerated in the non-translating group. There were a significant number of morphologically normal oocytes without EGFP expression, therefore, we tried to select the oocytes translating the injected mRNA by co-injecting objective mRNA with EGFP mRNA as a marker. We found that the oocytes expressing EGFP also highly translated the co-injected mRNAs. These results suggest that translation of injected mRNA starts within 3 h and continues for 18 h after injection in maturing porcine oocytes, and that co-injection of objective mRNA with EGFP mRNA is a useful method for finding the oocytes that are translating objective mRNAs.

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We examined the motility and sperm viability of frozen/thawed epididymal spermatozoa in the Japanese serow (Capricornis crispus) in order to develop a method for cryopreservation of genetic resources in this species. Thirty-four males captured in February and March of 1998 and 1999 were used. Epididymides were transported to the laboratory at 15 C and dissected in the medium I which contained egg-yolk (23.0%) and lactose (8.0%) and was buffered with 10% Tris solution (pH 7.4). Semen with sperm motility of more than 50% was used for the experiments. The semen was cooled to 5 C and diluted with freezing medium II which was freezing medium I supplemented with Equex Stem (1.4%) and glycerol (7.0 or 14.0%). The semen mixed with medium II was loaded into 0.25-ml plastic straws, and exposed to liquid nitrogen vapor for 10 min. The straws were then plunged into liquid nitrogen. Straws were thawed in a water bath kept at 37 C. Thawed spermatozoa were treated with a combination of fluorescent dyes, SYBR-14 and propidium iodide, to assess the sperm viability. The post-thaw motility of spermatozoa frozen with 3.5% and 7.0% glycerol were 10.0% and 11.2%, respectively; the proportions of living spermatozoa frozen with 3.5% and 7.0% glycerol were 6.9% and 6.1%. These results demonstrated that epididymal spermatozoa of the Japanese serow can be frozen in freezing medium containing glycerol, Equex Stem and egg-yolk.

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In vitro matured bovine and porcine oocytes were enucleated by aspiration of the first polar body and adjacent cytoplasm after removing the cumulus cells by vortexing and the factors affecting enucleation rate were investigated. The results indicate that vortexing cumulus-oocyte complexes for long periods in hyperosmotic media moves the first polar body and reduces the enucleation rate. Removal of 20% cytoplasmic volume increased enucleation rate compared with that of 10% and showed no deleterious effect on subsequent developmental capacity of reconstructed bovine embryos. Aging of oocytes could also reduce enucleation efficiency by inducing the migration of the chromatin material to the center of the oocyte. Therefore, we recommend to enucleate newly matured bovine and porcine oocytes by removing the first polar body and adjacent 20% of ooplasm after the removal of cumulus cells by vortexing for a short period using a hypo-osmotic media to achieve a high enucleation rate .

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Published research indicates that few of the sperm deposited in the female tract are recovered after insemination. Experiments were performed in rabbits to determine if sperm recovery could be improved by intensive flushing, and to examine leucocytic response as a factor affecting recovery. Freshly ejaculated and washed sperm were compared with sperm aged for 10 to 12 h in vitro prior to insemination. More aged sperm (54%) were recovered from the uterus than fresh sperm (36%) within 10 to 15 min of uterine deposition. Use of whole semen instead of washed sperm reduced recovery of both types of sperm (33 to 37% recovery). Increasing concentrations of Triton X-100 and multiple flushes increased recovery of fresh sperm, but not of aged sperm. Only 6.6% of the aged sperm and 11.5% of the fresh sperm were engulfed by neutrophils after 12 h in ligated uteri, and many sperm were unaccounted for. A breakthrough in recovery was achieved by developing a method to solubilize neutrophils, leaving sperm heads intact. This increased recovery of sperm held for 12 h in the female from 48% before to 93 to 95% after neutrophil dissolution. This technique will enhance studies involving sperm interactions in the female.

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We previously showed that luteal cytosolic phospholipase A₂ and prostaglandin F₂α level which rises during functional luteolysis persisted during the following structural luteolysis in rats. To investigate the possible role of the sustained activation of luteal prostaglandin synthesis in the accomplishment of luteolysis, we studied the influence of systematic injection with a phospholipase A₂ inhibitor, dexamethasone, and a cyclooxygenase inhibitor, indomethacin, on structural luteolysis. Repeated administration of the inhibitors to mothers for seven days postpartum partially reversed suckling stimuli-induced decline in pregnant corpus luteum weight. The inhibitors also inhibited exogenous prolactin-induced structural regression in immature pseudopregnant rats, and the inhibition of luteal cytosolic phospholipase A₂ activity and prostaglandin F₂α level was confirmed in this experimental model. Immunohistochemical study revealed that dexamethasone-treated corpus luteum had a significantly lower number of infiltrating macrophages. Moreover, fibroblastic proliferation in that corpus luteum seemed to be attenuated by both dexamethasone and indomethacin. Considered together with our previous findings, the data suggest that the persistent activation of luteal phospholipase A₂ activity and prostaglandin generation may function to facilitate the luteolytic process and that this mechanism mediates prolactin-induced structural luteolysis in rats.

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To investigate a means for evaluating bull fertility, analysis of frozen-thawed bull sperm migration in the medium with or without bovine cervical mucus was carried out. Sperm were prepared by thawing frozen-semen from 4 bulls, which showed different conception rates of 71, 43, 43 and 1% by non-return rate post AI. Media with 50% cervical mucus and with 1% BSA were prepared to make a test column. A horizontal column of 21 cm length had specific shape and composition on a 60-mm Petri dish, and was used to analyze the migration characteristics of the sperm. After 2 h incubation, viability and migration of the sperm were examined. There was correlation between bull fertility and sperm migration in the media containing cervical mucus: the migration in the medium with cervical mucus of sperm from the bull with low fertility was lower than those of the other bulls with high fertility. After a single wash, the migration in medium without cervical mucus of sperm with high fertility was significantly higher than that of sperm with low fertility. The migration of sperm increased with the number of washes. This study confirmed that sperm viability after migration in a medium varied with different media used and was positively correlation with bull fertility in the medium containing cervical mucus. We concluded that the Petri dish-based horizontal column may be a useful tool for analyzing sperm migration as a predictor of bull fertility.

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In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and development rates. The objectives of this study were to evaluate 1) the effect of timing of ethanol activation after ICSI on cleavage and subsequent development (experiment 1), and 2) the effect of aging of bovine oocytes fertilized by ICSI followed by ethanol activation treatment (experiment 2). In experiment 1, cleavage rates were significantly higher in the groups receiving activation treatment 2 or 4 h after ICSI (64% or 70%) than that in the non-activation group (41%). The blastocyst rate was also significantly higher in the group receiving activation treatment 4 h after ICSI (19%). In experiment 2, cleavage and blastocyst rates were significantly higher in 22 to 26 h matured sperm-injected oocytes than those in non-activation groups. On the other hand, cleavage rates in 28 and 30 h matured sperm-injected oocytes were similar regardless of activation treatment and were not different from those in 22 to 26 h matured bovine oocytes activated with ethanol. However, despite activation treatment blastocyst rates in the 30 h matured oocytes were significantly lower than those in 22 to 28 h matured oocytes activated with ethanol. These results indicate that the timing of activation treatment and the age of bovine oocytes affect the cleavage and subsequent development following ICSI.

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The objective of this study was to determine the parthenogenetic activation regimen of ovulated rat oocytes using strontium (Sr²⁺). Spontaneous activation of Wistar rat oocytes was triggered immediately after dissociation from the animals, but such activation seemed to be incomplete because no cleavage occurred even after 34 h culture in modified KRB medium. The survival rate of oocytes treated with 5 mM Sr²⁺ for 6 h (28%) was lower than that of those treated with 0, 0.31 and 1.25 mM Sr²⁺ (91-99%). Overall activation efficiency, as determined by the proportion of cleaved oocytes at 28 h of subsequent culture, was the highest in the oocytes treated with 1.25 mM Sr²⁺ (57%), followed by those treated with 0.31 mM Sr²⁺ (45%), 5 mM Sr²⁺ (20%) and 0 mM Sr²⁺ (8%). When oocytes from Sprague-Dawley (SD) or Wistar rats were incubated with 1.25 mM Sr²⁺ for 6 h, the oocytes from the SD strain tended to be more resistant to the Sr²⁺ treatment than the oocytes from the Wistar strain (survival rate 99 vs 87%), and the overall activation efficiency for SD oocytes was higher than that for Wistar oocytes (74 vs 45%). Therefore, nuclei of cumulus cells were injected into enucleated SD oocytes using piezo-driven micropipettes and the reconstructed oocytes were then activated with 1.25 mM Sr ²⁺ for 6 h. The cleavage rate of reconstructed oocytes 18 h post-activation was 22%. One implantation site but no live offspring was observed in the 9 surrogate mothers receiving a total of 224 presumptive cloned zygotes. This study contains fundamental data concerning oocyte activation and somatic cell nuclear transplantation in rats.

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Seasonal changes in testicular steroidogenesis and spermatogenesis were studied in a captive male northern fur seal, Callorhinus ursinus, of Izu-Mito Sea Paradise. Under anesthesia, monthly blood samplings and testicular biopsies at intervals of 3 months were performed to measure serum testosterone concentrations by radioimmunoassay and to observe spermatogenic activity histologically and the immunolocalization of steroidogenic enzymes. Histological observation of testes revealed full spermatogenesis, including spermatogonia to spermatozoa in the seminiferous epithelium in June and a limited generation of germ cells such as spermatogonia and primary spermatocytes in September and December. The seminiferous tubule diameter and serum testosterone concentrations showed maximum fluctuations corresponding to full spermatogenic activity in June. Three kinds of steroidogenic enzymes, P450scc, 3βHSD and P450c17, which are necessary for androgen synthesis, were immunolocalized in Leydig cells, and P450arom, the key enzyme for estrogen synthesis, was immunolocalized in spermatogonia in the testes in all 3 seasons. These results indicate a definite seasonality in high-level testicular steroidogenic and spermatogenic activity of this northern fur seal during the breeding season in spite of there being no changes in the immunostaining of intratesticular steroidogenic enzymes throughout the year.

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