Liquid Storage of Goat Semen in Chemically Defined Extenders

The suitability of certain commercial and self‐made chemically defined extenders for liquid storage of goat semen was tested and the effects of storage temperatures, dilution rates and sperm washing and pH of extenders on the goat sperm during liquid storage were observed. Semen was collected from nine goat bucks of the Lubei White and Boer breeds using an artificial vagina. Each ejaculate after initial evaluation was diluted with a specific extender, cooled and stored at a desired temperature. Stored semen was evaluated for sperm motility and other parameters every 24 or 48 h of storage. The ranking order of the existing milk‐ and yolk‐free extenders in sustaining goat sperm motility was Androhep > Zorlesco > Beltsville thawing solution > the Tris–glucose medium. The new extender (mZA) which was formulated based on Zorlesco and Androhep was more suitable for goat sperm than Androhep. The mZAP extender with Bovine Serum Albumin (BSA) replaced with polyvinyl alcohol (PVA) worked as efficiently as the mZA in maintaining sperm motility, membrane integrity, acrosome intactness and capacitation status. Goat sperm motility was best maintained at 5°C during liquid preservation, but decreased significantly as the temperature increased. When semen was sixfold diluted, sperm motility was maintained longer (p < 0.05) after centrifugation, but sperm motility did not differ between the centrifuged and non‐centrifuged groups when semen was 11‐fold diluted. When the extender pH was adjusted from 6.6 to 6.04, the efficiency increased significantly in both Androhep and mZAP. A forward sperm motility of 34% was maintained for 9 days when buck semen was 11‐fold diluted and stored at 5°C in mZAP, with pH adjusted to 6.04. It is concluded that for liquid storage of buck semen, the mZA extender was more suitable than other extenders; BSA can be replaced with PVA in mZA; centrifugation to remove seminal plasma can be omitted by adequate dilution; and the storage temperature and pH of extenders affected sperm motility significantly.     

Alpha1-adrenoceptor-mediated negative inotropic effect caused by intracellular ionic activities in guinea-pig papillary muscle

In guinea-pig papillary muscle, phenylephrine (PE), an agonist of alpha1-adrenoceptor (alpha1-AR), led to a transient negative inotropic effect (NIE) and a subsequent sustained positive inotropic effect (PIE). To clarify the ionic mechanisms underlying the NIE, we measured the [Na+]i or [pH]i by ion-selective microelectrodes. PE produced a decrease in the intracellular Na+ concentration ([Na+]i) and an increase in intracellular pH ([pH]i). During the phase of NIE, PE produced only a (-) change of [Na+]i (Delta[Na+]i). With a decrease in extracellular Na+ or an increase in extracellular Ca2+, the PE-induced NIE was attenuated and PE produced (+)Delta[Na+]i. The PE-induced NIE and (-)Delta[Na+]i were definitely strengthened by lowering the bath temperature or increasing the stimulation frequency. 2-(2,6-di-methoxyphenoxyethyl)amino-methyl-1,4-benzidioxane HCl, an antagonist of alpha1A-AR, completely abolished the PE-induced NIE and (-)Delta[Na+]i. Phorbol 12,13-dibutyrate, an activator of protein kinase C (PKC), decreased the baseline [Na+]i and twitch force and increased the baseline [pH]i in mimicry of PE. Pretreatment with 1-5(isoquinolinesulphonyl)-2-methylpiperazine, an inhibitor of PKC, abolished the PE-induced NIE and (-)Delta[Na+]i. During pretreatment with benzamil, an inhibitor of Na+/Ca2+ exchange, we found that the PE-induced NIE and (-)Delta[Na+]i were reversibly abolished. Our results indicate the PE-induced NIE may be elicited upon the activation of Na+/Ca2+ exchange which can be attributed to the (-)Delta[Na+]i. (-)Delta[Na+]i is mediated through a PKC-dependent pathway via an activation of alpha1A-AR subtype and its effect could be strengthened remarkably at high [Na+]i and [Ca2+]i values.     

Blocking polar body with cytochalasin B in the fertilized eggs of the small abalone, Haliotis diversicolor supertexta (Lischke), and the development and ploidy of the resultant embryos

The effects of blocking polar body I (PB1) or polar body II (PB2) with four different dosages of cytochalasin B (CB) on the development and ploidy of resultant embryos were studied in the small abalone, Halitis diversicolor supertexta (Lischke). To block the release of PBI, the fertilized eggs were treated with 0.25, 0.5, 1.0 or 2.0 mgL^?1 of CB for 10min beginning at 3 min post-fertilization at 24°C. To block the release of PB2, the fertilized eggs were treated under the same conditions as PB1, except that the treatment was begun 10min post-fertilization. In the control group, only 41.8% of the cells had a diploid number of 32 chromosomes, although spontaneous haploids (9.0%). tripolids (7.5%) and aneuploids (41.7%) were also observed. In CB treatment of PB1 and PB2 groups. 5.0-28.6% of the cells remained as diploid. triploids (10.0-18.9%) and aneuploids (41-1-61.0%). With regard to the development of the resultant embryos, the proportion of normal embryos in the control group was 87%, while in the treatment groups, the proportions of normal embryos in the FBI and PB2 groups were 57-58% and 53-56% in the 0.25 mg L^?1 and 0.5mg L^?1 CB treatments, respectively. From this data on induced triploids and the resultant development of normal embryos, the proportions suggest that 0.25-0.5 mg L^?1 of CB for 10min was sufficient for blocking the release of FB1 or PB2 to produce triploids in the small abalone.     

Effect of Follicle Stimulation Hormone and Luteinizing Hormone on Cumulus Cell Expansion and In Vitro Nuclear Maturation of Canine Oocytes

In general, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in the regulation of cumulus cell expansion and oocyte maturation. We investigated the effects of supplementation of FSH or LH in in vitro maturation (IVM) medium on the incidence of cumulus cell expansion and nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with 10% foetal bovine serum (FBS), 1 mg/ml cysteine, 0.2 mm pyruvic acid and different concentrations of FSH or LH (control, 0.5, 5 or 50 microg/ml) at 38.5 degrees C, 5% CO(2) in air for 72 h. The cumulus cell expansion was measured by microscopic visualization, and nuclear maturation of denuded oocytes was determined by staining with 10 microg/ml Hoechst33342 for 30 min. The cumulus cell expansion in the 5 microg/ml FSH group (397.2 +/- 64.3 microm) was significantly higher than those in the control, 0.5, and 50 microg/ml FSH groups (168.3 +/- 19.1, 286.0 +/- 69.7 and 300.0 +/- 84.3 microm, respectively; p < 0.05). However, there was no difference in cumulus cell expansion among the control, 0.5, 5 and 50 microg/ml LH groups (165.6 +/- 20.2, 160 +/- 26.5, 172 +/- 20.5 and 168 +/- 23.1 microm, respectively; p > 0.05). After 72 h of IVM, the proportion of nuclear development to the MI-MII stage in the 0.5 microg/ml FSH group (15.1%) was higher than those in the control, 0.5 and 50 microg/ml FSH groups (0.9%, 6.5% and 8.0%, respectively; p < 0.05). However, there was no significant difference in nuclear maturation to the MI-MII stage among control, 0.5, 5 and 50 microg/ml LH groups (4.6%, 2.3%, 5.4% and 8.6%, respectively; p > 0.05). This study indicated that a FSH supplement in IVM medium can increase cumulus cell expansion and nuclear maturation, while the nuclear maturation rate remained low. Further studies are required to improve the nuclear development to the MI-MII stages in canine oocytes.     

Age-related change of somatostatin-immunoreactive neurones in the main olfactory bulb of the rat

Somatostatin is found in the olfactory system, including the main olfactory bulb (MOB), and is thought to be one of the neuroactive substances for olfaction. However, somatostatin immunoreactivity in the olfactory system has not been determined during ageing. Hence, we examined the age-related changes of somatostatin-immunoreactive (IR) neurones in the rat MOB over a period of 2 years, at the following various ageing stages: post-natal month 1 (PM 1), PM 3, PM 6, PM 12 and PM 24. In PM 1 group, a few somatostatin-IR neurones were detected in the granule cell layer (GCL), and had slender or oval somata and short processes. At PM 3, somatostatin-IR neurones were observed in the glomerular, external plexiform and GCL. The size of somatostatin-IR somata was larger than that at PM 1. In PM 6 group, the number and size of somatostatin-IR neurones increased, and their processes became longer while running in various directions. At PM 12, somatostatin-IR neurones increased in number, and their processes became markedly longer than those at PM 6. At this stage, somatostatin-IR neurones had multipolar somata, and were the largest in size. In PM 24 group, somatostatin-IR neurones were most numerous. However, the processes of somatostatin-IR neurones were shorter than those at PM 12. This study suggests that the increased number of somatostatin-IR neurones in the MOB of aged rats may play a role to compensate for any decrease of olfactory function.     

Diagnostic survey of bovine abortion in Korea: with special emphasis on Neospora caninum

Causes of bovine abortion were surveyed in Korea within a designated period from the cases submitted to the Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University. One hundred and eighty aborted fetuses and maternal sera were evaluated by necropsy, histopathology, bacteriology, virology, PCR, and serologic tests. The causes of abortion were identified in 108 (60%) cases, of which 38 (21.1%) were due to the infection with Neospora caninum. None of the 38 cases showed any co-infection with either virus or bacteria. Viral and bacterial causes were diagnosed in 28 (15.5%) and 13 (7.2%) aborted fetuses, respectively. Non-infectious causes such as multiple pregnancy, maternal weakness or torsion of umbilical cord were observed in 22 (12.3%) cases. Results of the present study suggest that N. caninum is believed to be the leading cause of bovine abortion in Korea. Thus, more attention should be paid to this emerging disease in Korea. However, the causes of many aborted fetuses remain undiagnosed in this study. Therefore, this enigma should be clarified through further studies such as chromosomal analysis.