Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0-5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30-40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between middanuary and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.

A pathogenic Entamoeba histolytica-like variant (JSK2004 strain) with genetic variations and a novel isoenzyme pattern isolated from a De Brazza's guenon in a Tokyo zoo in Japan has previously been documented. In this study, a multiplex polymerase chain reaction (PCR) assay that could distinguish the JSK2004-type E. histolytica-like variant (JSK04-Eh-V) from E. histolytica and Entamoeba dispar using three newly designed primer sets for amplifying each specific DNA fragment from their small-subunit ribosomal RNA genes was developed and established. Forty-seven primates (11 species) from the zoo were surveyed by multiplex PCR to assess the prevalence of JSK04-Eh-V infection, which was recognized in six individuals of four species, including an Abyssinian colobus monkey, a De Brazza's guenon (including the individual from whom JSK2004 was isolated), a white-faced saki, and a Geoffroy's spider monkey. In addition, the autopsied individuals of an Abyssinian colobus and Geoffroy's spider monkey that died of amoebic liver abscess were also evaluated. DNA samples were also analyzed for specific genotypes based on the nucleotide sequencing of two protein-coding (chitinase and serine-rich E. histolytica protein) genes and the protein-noncoding locus 1-2 that was used for fingerprinting of the E. histolytica strain. These studies indicated that the E. histolytica-like variant infection in this zoo was caused by the same type (i.e., JSK04-Eh-V). An axenic culture medium (yeast extract-iron-maltose-dihydroxyacetone-serum) was developed based on the yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium, which is designed for axenic culture of E. dispar. This new medium could be used for axenically culturing E. histolytica, JSK04-Eh-V, and E. dispar in a single medium.

The levels of progesterone, testosterone and estradiol-17p in serum samples from two female Asian elephants were measured for the period of 32 months from February 1987 to September 1989. Serum samples were collected weekly from unanesthetized elephants. Each elephant showed eight ovarian cycles in 32 months. Ovarian cycles, characterized by changes in concentrations of serum progesterone, averaged 16.8 2 0.6 (mean * SEM, n = 14) weeks in length. The changes in concentrations of testosterone in the serum showed a similar pattern to those of progesterone with a striking increase noted during the luteal phase. The highest levels of serum estradiol-17p were noted when progesterone levels showed low basal values. These results suggest that estradiol-17P may be an index of follicular maturation during the estrous cycle in Asian elephants, and that the ovaries of Asian elephants may produce testosterone in the luteal phase.

We collected semen from a male Amur leopard cat using the transrectal electroejaculation method and investigated the semen qualities for about four years. In addition, the influence of the season on the spermatogenic function of the Amur leopard cat was investigated with regard to the semen qualities, testicular volume and serum testosterone level. As a result, we could collect semen with good sperm qualities that would be useable for artificial insemination. Some seasonality was noted in the testicular volume and serum testosterone level. We clarified that the semen qualities were favorable before and during the female breeding season compared with those after the breeding season.