A trial of semen collection by transrectal electroejaculation method from Amur leopard cat (Prionailurus bengalensis euptilurus).

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.

The quality of cryopreserved sperm collected from feline caudal epididymides using seminal plasma.

It is thought that differences in conception rate between feline epididymal sperm and ejaculate sperm occur because, unlike ejaculated sperm, caudal epididymal sperm have not been sensitized with seminal plasma (SP). In this study, we investigated whether collection of feline epididymal sperm with SP influences sperm qualities after freezing-thawing. Sperm were sensitized with SP for 10 min at room temperature. As a result, the motility of caudal epididymal sperm sensitized with SP immediately after collection was significantly lower than that of ejaculate sperm, and no difference was noted in sperm qualities after freezing-thawing. This shows that the qualities of caudal epididymal sperm cannot be improved to a level higher than those of ejaculate sperm by sensitization with SP.

The quality of cryopreserved sperm collected from feline caudal epididymides stored at room temperature.

On the assumption that animals of wild feline species died in the field, caudal epididymal sperm were cryopreserved following storage of the feline epididymides at 20°C for 0-24 hr, and their qualities were observed. Compared to the qualities at 0 hr, no significant differences were noted following 12 hr of storage at 20°C. On comparison of the qualities between caudal sperm cryopreserved after 24 hr storage at 4°C and after 12 hr at 20°C followed by 12 hr storage at 4°C, no significant differences were noted. These findings suggest that the cryopreserved sperm collected from epididymides of dead animals might be useful for artificial insemination if cryopreservation was performed within 12 hr exposure to ambient temperature.

Artificial insemination with cryopreserved sperm from feline epididymides stored at 4 °C.

Recovering and storing sperm from the epididymides of males of rare felidae is useful for preserving the species. The objective of the present study was to determine pregnancy rates following artificial insemination (AI) of frozen-thawed epididymal sperm, which were cryopreserved following low-temperature storage of the epididymides. In this study, these sperm were used for unilateral intrauterine AI (UIUAI) or unilateral intratubal AI (UITAI) using 40 × 10(6) and 10 × 10(6) sperm, respectively. The caudal epididymides of 17 cats were stored at 4 °C for 24 h after castration. Artificial insemination of seven female cats was performed on Days 3 or 4 (start of estrus = Day 1) by UIUAI, 20 h after injection of 100 IU hCG to induce ovulation. Furthermore, UITAI at 24 h (UITAI-24) or 30 h (UITAI-30) after hCG were also done (five cats per group). It was noteworthy that AI by UIUAI and UITAI-24 was performed before ovulation, whereas AI by UITAI-30 was performed after ovulation. Pregnancy rates were 28.6% (2/7) by UIUAI, 80% (4/5) by UITAI-24, and 20% (1/5) by UITAI-30. Litter size was one or two by UIUAI, and one to four by UITAI. Spontaneous abortion occurred on Days 25-30 of pregnancy in one of the two female cats pregnant following UIUAI, and in two of five female cats pregnant following UITAI. Based on the high pregnancy rate obtained with 10 × 10(6) sperm in the UITAI-24 group (AI performed before ovulation), we concluded that this was the most appropriate method for AI with frozen-thawed epididymal sperm after initial low-temperature storage of epididymides.

Characterisation of the carboxylesterase enzyme cauxin in the seminal fluid of the cat.

The carboxylesterase cauxin is a major urinary protein in cats that is also found in seminal fluid (SF). This study investigated cauxin in feline SF including biochemical features, concentration, distribution and gene expression in epididymal tissue, and its reaction with acylglycerol substrates. Monomeric, dimeric, and/or multimeric forms of cauxin carrying N-glycosylations were detected on Western blots of feline SF but most were monomeric. Cauxin concentrations were markedly lower in SF (0.042±0.020 mg/mL) than in urine (∼0.5 mg/mL) and cauxin gene expression was 60-fold lower in the epididymis than in the kidney. Immunohistochemical examination localised cauxin within the stereocilia and cytoplasm of epithelial cells lining the caput and corpus epididymis. Cauxin-positive spermatozoa were detected in the lumen of the cauda epididymis but not in the cytoplasm of the epithelial cell lining. Using an in vitro assay, cauxin hydrolysed saturated 1-mono- but not di- and tri-acylglycerols. The results suggest that cauxin secreted from the caput and corpus epididymis acts as an esterase on lipid within feline SF.

Distribution of glycoproteins on feline testicular sperm, epididymal sperm and ejaculated sperm.

Glycoproteins (GPs) are known to be involved in the phenomenon of sperm maturation and capacitation. In the present study, we investigated the attachment of GPs on sperm cell membrane during the process of feline sperm maturation from testicular sperm to ejaculated sperm by using 8 FITC-labeled lectins. The results showed that 3 types of GPs were presented on testicular sperm and 7 on caput epididymal sperm. Corpus and cauda epididymal sperm and ejaculated sperm had GPs detected by 8 FITC-labeled lectins used in the present study. This study demonstrates the part of the characteristic of GPs that are present on the feline sperm cell membrane during the process of sperm maturation.

Changes in qualities and quantities of consecutively ejaculated feline semen.

Cats show repeated copulation, but changes in semen qualities and quantities with repetition of ejaculation have not previously been clarified. We collected semen 4 times consecutively from 5 cats using the artificial vagina method and observed the semen qualities and quantities. No significant changes were noted in the semen volume, frequency of abnormal sperm or incidence of immature sperm, but the number of sperm and sperm motility and viability decreased with repetition, and in particular, the number of sperm in the first semen accounted for 55.0% of the total number in the 4 consecutive ejaculations, showing a significant difference from those in the 2nd-4th semen (P<0.01).

Surgical intrauterine insemination with cat semen cryopreserved with Orvus ES paste or sodium lauryl sulfate.

The mean post-thaw sperm motilities of feline frozen semen prepared with 1% OEP or 3 g/ml SLS as a cryoprotective agent, in addition to 7% glycerin, were 35.0 ± 2.4 and 37.0 ± 2.5%, respectively, showing no significant difference. On unilateral intrauterine insemination (UIUI) using these semen samples at a sperm number of 40 × 10(6), the conception rate was 70.0% (7/10) in the OEP group and 30% (3/10) in the SLS group, showing that the rate was higher in the OEP group, but the difference was not significant. It was suggested that sperm in frozen semen showing the above qualities were transferred to the contralateral uterine horn on UIUI.

The qualities of cryopreserved epididymal sperm collected from feline epididymides stored at low temperature.

We observed the influences of low-temperature storage of the feline epididymis on the epididymal semen qualities before and after cryopreservation to identify the optimal duration for low-temperature storage of the epididymis. After excision, the feline epididymis was stored at 4 degrees C for 0-72 hr and then subjected to epididymal sperm collection. When sperm from the refrigerated cauda epididymis were frozen and thawed, there was no significant difference in sperm motility between the 0- and 24-hr low-temperature storage groups, but sperm motility was significantly decreased in the 48-hr storage group. The above findings suggested that low-temperature storage of the epididymis until 24 hr is useful for frozen sperm collected from the feline cauda epididymis.