Abstract

The black-and-white, or Yunnan snub-nosed monkey (Rhinopithecus bieti) is among the world’s top 25 most endangered primates. It occurs only in forests above 2,700 meters in a tiny range in Southeast Tibet and Northwest Yunnan, between upper Yangtze and Mekong. Only 14 groups remain with the whole population consisting of about 1,500 individuals. The Yunnan snub-nosed monkey was mistaken as a subspecies of Rhinopithecus roxella, when a French missionary, Biet, brought the first skull and body specimen to Paris in 1890. In 1962, eight pelts bought by professor Hongshou in a Tibetan village again confirmed the existence of the Yunnan snub-nosed monkey. In 1979, the Chinese scientists proved the existence of this very shy primate species living in the rough Tibetan forest. Since the rediscovery of this magnificent monkey, an intensive research project was initiated by the Chinese Academy of Sciences. Shortly thereafter, captive breeding centers were established at the Zoological Institute of Kunming (KIZ), at the Kunming Zoo and at the Beijing Zoological Park. Currently there are 3.5 adult snub-nosed monkeys with 4 offspring at the Breeding Center of Endangered Primates of KIZ and the Kunming Zoo. As part of a Sino-German research program on reproductive biology in Yunnan snub-nosed monkeys, first time ultrasonographic examinations were performed in males and a new electroejaculation technique was developed. One of the major goals of this program is the establishment of a sperm bank for this highly endangered species.

The investigations were performed in December 2002 and 2003 which is the peak of the breeding season. Breeding season lasts from the end of November through the beginning of March. A total of nine examinations and semen collections were performed in three adult males over the 2-yr period. The dominant sire was examined twice in 2002 and three times in 2003 while the two subdominant adults were each examined twice in 2002 and 2003. Former difficulties with the semen collection were overcome by applying a new electrostimulation technique with a customized transrectal probe, which was developed based on the sonomorphologic findings of the ultrasound investigations. The entire procedure was performed under general anesthesia. Animals were anesthetized by darting with ketamine hydrochloride (10 mg/kg) (Ketamine 10%, Essex GmbH, Germany). Body weights ranged from 17.0–19.5 kg. Total time of anesthesia ranged from 30–40 min. The preparations for the transcutaneous ultrasound exam (heart, liver, kidney, spleen, testis) and transrectal ultrasonography (accessory sex glands) included shaving specific scanning windows and the genital area, as well as a rectal enema with lukewarm water. To prepare the animal for semen collection, the urinary bladder was emptied by catherization and refilled with cell culture medium M199 (Sigma GmbH, Germany).

Due to the extensive digestive tract, typical of leaf-eating monkeys, the transcutaneous ultrasonography was limited. Heart and liver were imaged but the kidneys and spleen were easier to visualize by transrectal ultrasound. One of the subdominant males showed a pseudomembrane formation in the urinary bladder, which split the bladder nearly completely into two parts, a larger cranial part (2/3), and a smaller caudal part (1/3). The cranial compartment seemed static without inflow from the ureters or draining through the urethra. Even under ultrasound-guided catherization it was impossible to drain this compartment. Medical records from this male were not indicative of a former urinary bladder infection. A teratogenic malformation of the bladder could not be excluded but seemed unlikely. Even with this dramatic reduction of bladder capacity, there was no obviously different urination behavior observed. However, the extensive pathologic alteration detected led to the recommendation to exclude this male from the breeding program.

Ultrasound findings on the genital tract reflected the social rank of the three males. The dominant male showed the largest testicles with a moderate echogenic parenchyma indicating an active spermatogenesis. This interpretation was supported by color-Doppler investigations showing several intraparenchymal blood vessels. The testes of the two subdominant males were smaller but showed equal tissue activity. In contrast to equal-sized macaque species or baboons, the dimensions of the internal accessory sex glands (bulbo-urethral gland, prostate, seminal vesicle) were relatively small, which resulted in a smaller amount of the typical coagulum in the ejaculate of this species. The liquid phase of the ejaculate contained a total sperm cell number comparable to other primate species.^1,2 However, the spermatogenic recovery time seemed to be extremely long in snub-nosed monkeys. Second and third electroejaculations in the same male with a time interval of 3–5 days resulted in a nearly aspermatic ejaculates with almost similar ejaculatory volumes, even in the proven breeder. This suggested that the time of the spermatogenic cycle is extended in this primate species. The very rigid social structure of small family groups with one male and 2–5 females may play a role in this phenomenon. Further investigations of spermatogenesis are planned including future ultrasound-guided testicular biopsies and flow cytometric analyses.

Acknowledgments

The authors thank the animal keepers from the Kunming Breeding Center of Endangered Primates.

Literature Cited

1.  Schaffer N., M. Cranfield, T. Meehan and S. Kempske. 1989. Semen collection and analysis in the conservation of endangered nonhuman primates. Zoo Biol. Suppl. 1:47–60.

2.  Si W, Zheng P, Tang X, He X, Wang H, Bavister BD, Ji W. 2000. Cryopreservation of rhesus monkey (Macaca mulatta) spermatozoa and functional assessment by in vitro fertilization. Cryobiology. 41:232–240.