A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.

Various canine breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of them are under genetic control. Although dopaminergic neurotransmission system is considered to affect animal behavior, little is known about related genes in canine. Relations between specific alleles in polymorphic regions of the dopamine receptor D4 gene (DRD4) and personality or psychiatric disorders have been reported in humans, and we first found polymorphism in exon III region of the gene in 4 canine breeds. In this study we surveyed allele frequency distribution in 23 breeds including a total of 1,535 unrelated individuals. In exon III, 8 alleles including a novel allele were identified. A group of breeds in which the alleles 447b, 498 and 549 were frequent tended toward high scores in aggression-related behavioral traits than that with frequent alleles 435 and 447a. Moreover, a polymorphism based on 24 bp insertion/deletion was found in exon I region for the first time in dogs. This information may be of use for candidate gene studies of behavioral variation in dogs.

Sperm preservation is a useful technique for the maintenance of biological resources in experimental and domestic animals, and in wild animals. A new preservation method has been developed that enables sperm to be stored for a long time in a refrigerator at 4°C. Sperm are freeze-dried in a solution containing 10 mM Tris and 1 mM EDTA. Using this method, liquid nitrogen is not required for the storage and transportation of sperm. We demonstrate that chimpanzee, giraffe, jaguar, weasel and the long-haired rat sperm remain viable after freeze-drying. In all species, pronuclei were formed after the injection of freeze-dried sperm into the mouse oocytes. Although preliminary, these results may be useful for the future establishment of “freeze-drying zoo” to conserve wild animals.

In wild animal conservation, knowing the age of an individual animal is extremely beneficial. However, estimating the age is difficult for many species. Recently, epigenetics-based methods of estimating age have been reported. These studies were predominantly on humans with few reports on other animals, especially wild animals. In the present study, a chimpanzee (Pan troglodytes) age prediction model was developed based on the ELOVL2, CCDC102B, and ZNF423 genes that may also have application in human age prediction. Pyrosequencing was used to measure methylation in 20 chimpanzee blood samples and correlation between age and methylation status was calculated. Age and methylation of sites in ELOVL2 and CCDC102B were significantly correlated and an age prediction model was created using these genes. In the regression equation using only ELOVL2, the highest correlation coefficient was 0.741, with a mean absolute deviation (MAD) of 5.41, compared with the combination of ELOVL2 and CCDC102B, where the highest correlation coefficient was 0.742 and the MAD was 5.41. Although larger MADs were observed in chimpanzees than in humans based on these genes, the results indicate the feasibility of estimating chimpanzee age using DNA methylation, and can have implications in understanding the ecology of chimpanzees and chimpanzee conservation.

In Japan, overgrazing by sika deer (Cervus nippon) has been suggested to cause a decline in forest understory vegetation. DNA barcoding has become an accepted method for analyzing the diets of animals and may be useful for evaluating the impact of sika deer on vegetation. However, the applicability of DNA barcoding in the dietary analysis of sika deer, particularly whether all of the food plants can be detected with sufficient taxonomic resolution and whether the results can be evaluated quantitatively, has not been investigated. We conducted a feeding trial by feeding five plant species to a captive sika deer and sequenced the chloroplast trnL P6 loop region from the sika deer’s fecal DNA using the Ion PGM sequencer. We detected the sequences of all of the food plants at the species level using the local (selfproduced) database and at the genus or family level with the global database. Although the sequences of some major food plants were detected with high frequency, the proportion of consumed food plants did not match the proportion of sequences obtained from fecal DNA. With further technical advances and the further completeness of the sequence database for vegetation, DNA barcoding will be a useful tool for the dietary study of sika deer.