The Effects of Frequent Electroejaculation on the Semen Characteristics of a Captive Siberian Tiger (Panthera tigris altaica)

Artificial insemination (AI) can help to avoid inbreeding and genetic degeneration for sustaining genetically healthy populations of endangered species in captivity. Collection of a sufficient quantity of viable sperm is an essential first step in the AI process. In the present study, we examined the effects of frequent electroejaculation on semen characteristics in a Siberian tiger. We collected semen in all 17 trials during 6 breeding seasons (6 years). The mean number of sperm and the percentage of motile sperm were 294.3 ± 250.2×10⁶/ejaculate and 82.4 ± 11.4%, respectively. The number of motile sperm tended to increase during frequent electroejaculation in the same breeding season. Semen collection by electroejaculation can be performed effectively up to the fourth sequential ejaculate, which contained the most sperm in the study. In conclusion, frequent collection of sperm by electroejaculation from tigers may be effective for collection of a large number of motile sperm.     

Insulin-like Growth Factor 1 mRNA Expression in the Uterus of Streptozotocin-treated Diabetic Mice

Reproductive functions decline with the onset of diabetes in female mice. Diabetic mice have smaller uteri with an underdeveloped endometrium, suggesting diminished estrogen-induced growth. We aimed to clarify the changes in the estrous cycle and in insulin-like growth factor 1 (IGF1) expression in the uteri of streptozotocin (STZ)-treated diabetic mice, because IGF1 is one of the main growth factors involved in estrogen-induced uterine growth. ICR female mice were intraperitoneally administered STZ (10 mg/100 g BW), and blood glucose levels were determined. Mice with blood glucose levels > 200 mg/dl were classified as diabetic mice. The onset of diabetes was associated with acyclic estrous cycles. Diabetes was also induced with STZ in ovariectomized mice. Uterine Igf1 mRNA levels were reduced in ovariectomized STZ-treated diabetic mice. Estrogen is known to stimulate Igf1 mRNA expression in the uterus, but estrogen action was abolished in the uteri of STZ-treated diabetic mice. mRNA expressions of estrogen receptor α (ERα) and steroid hormone receptor coactivators (SRC-1/Ncoa1, SRC-2/Ncoa2, SRC-3/Ncoa3 and CBP/p300/Crebbp) were reduced in the uteri of ovariectomized STZ-treated diabetic mice. The present study demonstrates that diabetes induces a decline in female reproductive functions in mice. Igf1 expression in ovariectomized diabetic female mice was decreased, and decreased responsiveness to estrogen in the uteri of diabetic mice is probably associated with a reduction in ERα and steroid receptor coactivator mRNA expression.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

Comparison of cholesterol levels among lipoprotein fractions separated by anion-exchange high-performance liquid chromatography in periparturient Holstein–Friesian dairy cows

Changes in lipoprotein profiles occur in dairy cows during the periparturient period and in cows with transition cow disease. Here, the lipoprotein profiles of Holstein–Friesian dairy cows during the periparturient period were obtained by anion-exchange, high-performance liquid chromatography to evaluate the usefulness of lipoprotein profile evaluation during the periparturient period and in cows with fatty liver and milk fever. Lipoprotein levels (including total and high- (HDL-C) and low-density lipoprotein (LDL-C) cholesterol) in 10 healthy cows were low 4 weeks prepartum, with the lowest values at calving or within 1 week of calving; the values increased at 8 weeks postpartum. The lipoprotein levels were measured in 16 cows diagnosed with fatty liver (n=10) or milk fever (n=6) and compared to 10 healthy dairy cows. A significant difference was observed in HDL-C between healthy cows (at calving and 1 week postpartum), and the fatty liver and milk fever cows. Cows with fatty liver and milk fever had a lower mean HDL-C than the 10 healthy dairy cows at calving and 1 week postpartum. HDL-C might be a good indicator of energy balance for differentiating healthy cows from those with transition cow disease.     

Deficits of learning and memory in Hemojuvelin knockout mice

Iron is involved in various physiological processes of the human body to maintain normal functions. Abnormal iron accumulation in brain has been reported as a pathogenesis of several neurodegenerative disorders and cognitive impairments. Hemojuvelin (HVJ) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis. Although iron accumulation in brain has been related to neurodegenerative diseases, it remains unknown the effect of mutation of HVJ gene on cognitive performance. In our studies, HJV(−/−) mice showed deficits in novel object recognition and Morris water maze tests. Furthermore, the expression ration of apoptotic marker Bax and anti-apoptotic marker Bcl-2 in the hippocampus and prefrontal cortex showed higher levels in HJV(−/−) mice. Our results suggested that deletion of HJV gene could increase apoptosis in brain which might contribute to learning and memory deficits in mutant mice. These results indicated that HJV(−/−) mice would be a useful model to study cognitive impairment induced by iron overload in brain.     

Affected homologous chromosome pairing and phosphorylation of testis specific histone, H2AX, in male meiosis under FKBP6 deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.     

Endocrine status and development of porcine testicular tissues in host mice

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.     

Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.