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.     

Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Changes in sexual behavior and fecal steroid hormone concentrations during the breeding season in female Hokkaido brown bears (Ursus arctos yesoensis) under captive condition

The longitudinal changes in fecal steroid hormone concentrations and sexual behavior in 2 mated/pregnant and 3 non-mated female Hokkaido brown bears were investigated during the breeding season. Behavioral estrus (standing) lasted for 14 and 32 days in the mated females and for 25 to 36 days in the non-mated females. In non-mated females, sexual behavior, such as female-female mounting and masturbation, was observed for several days before and after the estrous period. In mated females, mean fecal estradiol-17beta concentrations were higher in the estrous period than in the post-estrous period, while fecal progesterone concentrations were higher in the post-estrous period than in the estrous period. The similar trends of steroid hormone changes were observed in the non-mated females.     

A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions.

Cross-protection between Haemophilus parasuis serovars 2 and 5 was examined in pigs using a bacterin based vaccine, and subsequently the safety and efficacy of a bivalent vaccine were evaluated. Upon intratracheal challenge of a serovar 2 or 5 strain, pigs immunized with a monovalent vaccine were protected against challenge with a homologous serovar strain, but not with a heterologous serovar strain. Immunization with a bivalent vaccine containing both serovars 2 and 5 bacterins conferred protection in pigs against lethal challenge with each of the serovar strains. A total of 86 pigs from two SPF herds were injected with the bivalent vaccine intramuscularly twice at a four-week interval. No adverse reactions following the vaccination were observed. On day 7 after the second vaccination, vaccinated and non-vaccinated control pigs from herd A were transferred to herd B, where Glasser's disease had broken out. Pigs in the control group developed clinical signs of the disease, and 6 of 8 (75%) pigs died until slaughter, in contrast with only 4 of 46 (9%) pigs in the vaccinated group. In herd C, where there was no outbreak of Glasser's disease, complement fixation antibody titer was raised only in the vaccinated group. A challenge experiment on days 20 and 79 after the second vaccination showed that only the vaccinated pigs were protected. From these findings, the safety and efficacy of the bivalent vaccine were confirmed under laboratory and field conditions.