Semen collection by urethral catheterization and electro-ejaculation with different voltages, and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata).

In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.

Evolution of the sperm methylome of primates is associated with retrotransposon insertions and genome instability.

Changes in gene expression resulting from epigenetic and/or genetic changes play an important role in the evolutionary divergence of phenotypes. To explore how epigenetic and genetic changes are linked during primate evolution, we have compared the genome-wide DNA methylation profiles (methylomes) of humans and chimpanzees, which have a 1.2% DNA sequence divergence, of sperm, the frontal cortices, B cells, and neutrophils. We revealed that species-specific differentially methylated regions (S-DMRs), ranging from several hundred base pairs (bp) to several kilo base pairs (kb), were frequently associated with sequence changes in transcription factor-binding sites and insertions of Alu and SVA retrotransposons. We then generated a reference macaque sperm methylome map and revealed, in sperm, that both human and chimpanzee S-DMRs arose more frequently owing to methylation loss rather than gain. Moreover, we observed that the sperm methylomes contained many more hypomethylated domains (HMDs), ranging from 20 to 500 kb, than did the somatic methylomes. Interestingly, the sperm HMDs changed rapidly during primate evolution; hundreds of sperm HMDs were specific to humans, whereas most somatic HMDs were highly conserved between humans and chimpanzees. Notably, these human-specific sperm HMDs frequently occurred in regions exhibiting copy number variations. Our findings indicate that primate evolution, particularly in the germline, is significantly impacted by reciprocal changes in the genome and epigenome.

First detection of Echinococcus multilocularis infection in two species of nonhuman primates raised in a zoo: a fatal case in Cercopithecus diana and a strongly suspected case of spontaneous recovery in Macaca nigra.

The causative parasite of alveolar echinococcosis, Echinococcus multilocularis, maintains its life cycle between red foxes (Vulpes vulples, the definitive hosts) and voles (the intermediate hosts) in Hokkaido, Japan. Primates, including humans, and some other mammal species can be infected by the accidental ingestion of eggs in the feces of red foxes. In August 2011, a 6-year-old zoo-raised female Diana monkey (Cercopithecus diana) died from alveolar echinococcosis. E. multilocularis infection was confirmed by histopathological examination and detection of the E. multilocularis DNA by polymerase chain reaction (PCR). A field survey in the zoo showed that fox intrusion was common, and serodiagnosis of various nonhuman primates using western blotting detected a case of a 14-year-old female Celebes crested macaque (Macaca nigra) that was weakly positive for E. multilocularis. Computed tomography revealed only one small calcified lesion (approximately 8mm) in the macaque's liver, and both western blotting and enzyme-linked immunosorbent assay (ELISA) showed a gradual decline of antibody titer. These findings strongly suggest that the animal had recovered spontaneously. Until this study, spontaneous recovery from E. multilocularis infection in a nonhuman primate had never been reported.