EFFECT OF SEASONAL CHANGES ON TESTICULAR MORPHOLOGY AND THE EXPRESSION OF CIRCADIAN CLOCK GENES IN JAPANESE WOOD MICE (APODEMUS SPECIOSUS).

This study aimed to determine the seasonality of reproduction throughout the year in Japanese wood mice (Apodemus speciosus). The effect of seasonal changes on testicular morphology and the periodic expression of circadian clock genes in the hypothalamus and testes of male individuals was evaluated. We also examined the morphology of the testes and caudae epididymides of male mice. In addition, RT-PCR analysis was carried out with mRNA extracted from the hypothalamus and testes to evaluate the expression of the circadian clock genes Clock, Bmal1, Per1, and Cry1. The complete induction of testicular activity was detected from February to April and from August to October, with testes weight increasing with the completion of spermatogenesis (reproductive season). From May to early June and from November to early January, testicular weight declined, the seminiferous tubules reduced in size, spermatogenesis was arrested, and sperm were not produced (non-reproductive season). From mid- June to July and mid-January, the re-induction of testicular activity for spermatogenesis was observed in the seminiferous tubules (transitional season). Out of the four examined genes, Cry1 had the highest expression level in both the hypothalamus and testes throughout the year, followed by Bmal1, Per1, and Clock. The expression of Bmal1 was significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive and non-reproductive seasons. Cry1 transcript levels were also significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive season. In conclusion, the results indicating changes in testicular morphology revealed annual reproductive, non-reproductive, and transmission periods in Japanese wood mice. When an increase in testicular activity was observed indicating the onset of the reproductive season, the mean day length was approximately 11–13 h. The expression of the circadian clock genes Bmal1 and Cry1 in the hypothalamus and testes during the reproductive season was significantly higher than that of the same genes during the transitional season. Consequently, completion of spermatogenesis occurred in the seminiferous tubules of Japanese wood mice testes during the reproductive period.

Semen collection and polymerase chain reaction-based sex determination of black-headed and straw-necked ibis.

This study aimed to develop a polymerase chain reaction (PCR)-based sexing and effective semen collection methods for black-headed and straw-necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black-headed and 4 straw-necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W-chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro-ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw-necked ibis using the massage method. Limited motility, viability and concentration of straw-necked ibis sperm were observed. The sperm length was 180 µm and that of the nucleus was 30 µm with acrosome located at the tip of the nucleus. Thus, the PCR-based sexing proved to be an accurate molecular sexing method for black-headed and straw-necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw-necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.