Akihisa Shinjo

Protection of Porcine Oocytes Against Cell Damage Caused by Oxidative Stress During In Vitro Maturation: Role of Superoxide Dismutase Activity in Porcine Follicular Fluid

Abstract To elucidate the beneficial effects of porcine follicular fluid (pFF) added to maturation medium on the sustenance of cytoplasmic maturation responsible for the subsequent developmental competence after in vitro fertilization (IVF) of porcine oocytes, we focused on the antioxidative role of pFF in its function of protecting oocytes from reactive oxygen species (ROS)-induced cell damage. Porcine follicular fluid collected from small (2–6 mm) follicles had about 7.2-fold higher levels of superoxide dismutase (SOD) activity than that of fetal bovine serum (FBS), and this activity was markedly blocked by the CuZn-SOD inhibitor, diethyldithiocarbamate (DETC). The interruption of meiotic progression and the increasing intracellular glutathione (GSH) content throughout the maturation period, as well as an outbreak of DNA damage in oocytes and cumulus cells were difficult to detect in oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system, whereas cell damage encompassed by ROS was prominent in oocytes cultured with 10% FBS and 10% pFF plus 100 μM DETC. Similarly, significant enhancement to the degree of transformation of the sperm nucleus into the male pronucleus (MPN) after in vitro fertilization was shown by the addition of pFF to the maturation medium. The presence of DETC during in vitro maturation reduced the ability of oocytes to promote MPN formation to the same extent as oocytes matured with FBS. The proportion developing to the blastocyst stage was increased in oocytes that matured with pFF, but this developmental competence was significantly lowered by treatment with DETC (P < 0.05). These findings suggest that pFF plays a critical role in protecting oocytes from oxidative stress through a higher level of radical scavenging activity elicited from SOD isoenzymes, resulting in the enhancement of cytoplasmic maturation responsible for developmental competence postfertilization.

Genetic diversity of the Yonaguni horse based on polymorphisms in microsatellites and mitochondrial DNA

Thirty-two microsatellites and a mitochondrial DNA haplotypes of endangered Yonaguni horses were analyzed to establish a pedigree registration system and to understand their genetic diversity for planning effective conservation. Blood samples were collected from 78 of the 130 horses in existence, and DNA was extracted and genotyped. There were two major findings. One is that it is possible to use microsatellites for Yonaguni horse pedigree registration in the future because the power of exclusion of parentage testing is reliable at 0.999998. The second is the clarification of the current genetic diversity of Yonaguni horses. The average number of alleles, observed heterozygosity, expected heterozygosity and fixation index were 4.4, 0.591, 0.601 and 0.016, respectively, for the analyzed horses. The probability of a genetic bottleneck, under the assumptions of the stepwise mutation model, was 0.432, suggesting that the genetic structure of the horses was not influenced by a recent bottleneck. Genetic distance between individuals was visualized by a phylogenetic tree based on the proportion of shared alleles. Structure analysis based on Bayesian clustering revealed the possibility that Yonaguni horses comprise four or five subpopulations. Consequently, although only two haplotypes were identified in the mitochondrial analysis, genetic diversity of Yonaguni horses was not particularly low in comparison with that of other breeds that are at risk of extinction.

Effects of Magnetic Pole on Orientation of Bull Sperm Treated by Dithiothreitol or Heparin

Effect of the north (N) or south pole (S) of a magnet and dithiothreitol (DTT) or heparin sodium (H) on the orientation of bull sperm were studied. Sperm were collected from four Japanese Black bulls of Okinawa Prefectural Livestock Experimental Station were treated with various concentrations of DTT or H during the first and sixth days and were then exposed them to N or S with a magnetic field of 3,000 Gauss for 24 hours. Experimental results showed that both N and S significantly increased the perpendicular oriented percentage of bull sperm in most treatments not treated or treated with DTT or H. Anisotropy of magnetic field of bull sperm was the main cause which induced them to orient perpendicular to the magnetic direction. The results of the experiment also showed that, in most cases, the two polarities had the same effect on the orientation of bull sperm. However, in some cases, N exerted a stronger effect on the perpendicular percentage of bull sperm than S, such as with the 20 mM DTT treatment at the first and sixth days and the 100 units H treatment at the sixth day. These cases showed that the perpendicular oriented percentage of bull sperm at N was significantly higher than that of S. It was only the 100 units heparin treatment at S both at the first and sixth days that upward perpendicular orientation was higher than that at N. This might be due to the fact that protamin-DNA structure of a small number of bull sperm nuclei slightly changed because of heparin action. Keywords : Magnetic Pole, Bull Sperm, Orientation, Dithiothreitol, Heparin

The Trend of Sires' Meat Productivity in the Island Population of Japanese Black Cattle in Okinawa

沖縄県内で供用された種雄牛の産肉能力について,その年次的傾向と島嶼間比較を行なう目的で分析を行なった.使用したデータは,1978年から1986年まで沖縄県で屠畜された去勢牛1182頭の枝肉格付成績である.分析対象形質は枝肉重量,DCG(1日枝肉生産量),ロース芯面積,脂肪交雑評点および格付等級である.分析対象期間は,1期:1978-80年,2期:1981-83年および3期:1984-85年の3期とした.伊江島では,供用可能な種雄牛の平均能力(種雄牛の平均能力)は特定の形質に偏ることがなく,比較的バランスのとれたものであった.また,実際に供用された種雄牛の能力(供用種雄牛の能力)は,種雄牛の平均能力以下の水準に留まり,供用方法の非効率性が明らかとなった.国頭地域では,種雄牛の平均能力および供用種雄牛の能力が,共に増体性に関する形質において高く,増体性の改良を重視した種雄牛の選択および供用が行なわれていた.宮古島では,種雄牛の選択と供用に食い違いがみられるなど,非効率的な供用が示唆された,石塩島の種雄牛の平均能力は4地域の中で最も高くかつバランスがとれていたが,供用種雄牛の能力をみると,種雄牛の供用は増体性重視の方向に大きく偏っていた.肉質に関する形質の改良は,すべての地域において低迷していたが,これは肉質に関する能力の把握が充分ではないことを示し,精度の高い遺伝的評価システムの必要性が示唆された.