In vitro maintenance, cooling and cryopreservation of red wolf (Canis rufus) spermatozoa.

A current priority for the preservation of the endangered red wolf (Canis rufus) is the development of a sperm-based genome resource bank. The aims of this study were to examine the effects of (i) holding temperature on the motility of spermatozoa over time, and (ii) cooling methods on the characteristics of spermatozoa after cooling and cryopreservation. Electroejaculates (n = 11; fresh) were evaluated for the percentage of motile spermatozoa, cell and acrosome morphology (Spermac (Meditech 1st Canada Inc, Montreal, Ontario) and fluorescein isothiocyanate-labelled Pisum sativum agglutinin lectin (PSA/FITC; Sigma Diagnostics, Oakville, Ontario) staining), and zona penetration. Semen samples were then divided into two equal samples and centrifuged to remove seminal plasma. One half of the ejaculate sample was re-suspended in sperm-Tyrode's albumin lactate pyruvate (TALP), divided into three aliquots and maintained either at room temperature (approximately 21-23 degrees C), 0 degree C or 37 degrees C. Sperm motility was examined at 0.5 and 1.0 h, and subsequently every hour for 10 h. Motility of spermatozoa decreased after 2 h, but was consistently greater at room temperature than at 37 degrees C or 0 degree C. The other half of the ejaculate sample was re-suspended in an egg yolk-based extender and divided into two aliquots. One aliquot was cooled in a refrigerator (5 degrees C) for 30 min, whereas the second aliquot was put into a beaker containing water at 37 degrees C, which was then placed into an ice bath until the sample reached 0 degree C (approximately 120 min). Spermatozoa were evaluated after cooling and after freezing and thawing treatments. No differences were observed between cooling treatments either after cooling or freezing and thawing. However, marked decreases in intact acrosomes, post-thaw motility and normal morphology of spermatozoa after treatment demonstrate that further investigations are necessary to improve cryopreservation methods in this species.

Characteristics of fresh and frozen-thawed red wolf (Canis rufus) spermatozoa.

Ejaculates of the red wolf (Canis rufus) were evaluated immediately after collection and freeze-thawing to initiate a reproductive database for this endangered species. Electroejaculates from 13 adult red wolves collected during the breeding season (February-March; n=25; 1-3 collections/male) had a mean volume of 4.7+/-0.7 ml, 146.5+/-25.7 x 10(6) spermatozoa/ml and 71.2% motile spermatozoa. The mean proportion of cells with normal morphology was 73.6+/-3.2% (range, 20.3-93.7%), with 64% of ejaculates (16/25) containing 70-90% normal spermatozoa. The four most predominant abnormalities were a coiled flagellum (8.1%), a bent flagellum (4.7%), a bent midpiece with no cytoplasmic droplet (3.3%;), and a detached head defect (6.4%). After cooling in glycerolated extender, semen was frozen using a pelleting method on dry ice before plunging into liquid nitrogen. Pellets were thawed in phosphate buffered saline and examined for % sperm motility, normal morphology, viability and intact acrosomes. There was a decline (P < 0.05) in sperm motility (approximately 40%) and percentage of normal sperm (11.9%) after freezing, but no change in the proportion of viable cells. After freezing, there was a marked decline (P < 0.05) in the proportion of intact acrosomes from 74.5% to 55.5% which was accompanied by an increased proportion (P < 0.05) of partial acrosomes from 11.9% to 35.8%. These data demonstrate that, although red wolf spermatozoa can survive freeze-thawing using a technique common for domestic dog sperm, the finding of significant acrosome damage reveals (1) likely species specificity in the Canis genus and (2) the need for refining sperm cryopreservation technology for the red wolf.

Semen parameters and electron microscope observations of spermatozoa of the red wolf, Canis rufus.

Semen parameters were evaluated on ejaculates of a captive population of red wolves (Canis rufus) sampled over two consecutive mating seasons. A total of 31 samples from 15 animals yielded mean sperm motility of 69.6 +/- 19.4%, mean sperm density of 131 +/- 124 x 10(6) ml-1, mean total number of spermatozoa of 470 +/- 465 x 10(6) and mean percentage morphologically abnormal spermatozoa of 35 +/- 11.8%. Restricting the data to animals sampled three times or more or limiting the samples to proven breeders resulted in statistically non-significant differences in these numbers (P < 0.05). When compared with data from other canines the seminal parameters of red wolves are at the lower extremes of the range. In particular the proportion of morphologically abnormal spermatozoa (35%) is approximately twice that seen in other canine species. Light microscopic analysis of abnormal forms revealed that almost half (45%) were bent defects, another 40% were secondary defects (coiled, detached and immature) and 15% were primary defects. Electron microscopy confirmed the presence of substantial numbers of morphologically abnormal forms including double-headed and double-flagellar cells, bent or kinked forms especially in the neck region, acrosomal abnormalities and bizarre spermatids. Approximately one-third of the samples also showed the presence of white blood cells, in some cases demonstrating sperm phagocytosis (spermophagy). These results are consistent with the concept of declining sperm parameters associated with restricted gene pools in numerically limited populations. However, alternative explanations are also explored.

Conception by use of postmortem epididymal semen extraction in a dog.

A 9-year-old sexually intact male Boxer was euthanatized because of progressive, generalized seizures that were refractory to medical treatment. To try to preserve the breeding line, postmortem epididymal semen extraction was attempted. The testes were excised after the dog was euthanatized, and semen was extracted, frozen, and stored in liquid nitrogen. Approximately 3.5 months later, a suitable recipient was identified, and surgical intrauterine insemination was performed. Fifty-seven days after insemination, a viable pup was born.

Spermophagy in semen in the red wolf, Canis rufus.

The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electro-ejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality.

Electroejaculation and semen analysis and freezing in the giant panda (Ailuropoda melanoleuca).

Semen was collected by a standardized electroejaculation procedure from a giant panda on 4 occasions. Ejaculate volume, sperm count and % sperm motility were 2.3-3.6 ml, 62-562 x 10(6) spermatozoa/ml and 45-85%, respectively. The results, although limited to a single male, suggested a seasonal influence on ejaculate and gonadal parameters with improved ejaculate volume, sperm motility and increased testicular size in the season proximate to the female's oestrous period. Frozen-thawed spermatozoa were motile with no apparent abnormalities induced by the freezing procedure.

Oestrous and ovarian activity in a female jaguar (Panthera onca).

The mean +/- s.e.m. duration of oestrus was 12.0 +/- 1.0 days (n = 7) and the oestrous cycle length was 47.2 +/- 5.4 days (n = 6) for a captive jaguar. Ovulation did not occur spontaneously, but was induced by treatment with hCG or LH-RH.

Semen collection by electroejaculation in the domestic cat.

A procedure for electroejaculation of domestic cats was developed. The method involved anesthetization of adult male cats with ketamine HC1 (33 mg/kg) or CI 744 (5.5 mg/kg). Optimal results were obtained with a 120-V ac electric stimulator capable of a continuous range of 0 to 60 V and 0 to 1 A. Using a 1- by 12-cm rectal probe constructed of teflon and stainless steel electrodes, ejaculation was obtained with an electrical stimulus over a range of 2 to 8 V and 5 to 220 mA. Voltage and amperage were increased at specific intervals in order to compensate for refractory ejaculatory response due to continued nerve and muscle stimulation. The procedure provided a means of obtaining ejaculates from domestic cats for purposes of semen evaluation and artificial insemination.

Pregnancy in the domestic cat after artificial insemination with previously frozen spermatozoa.

Methods for collection, freeze preservation and artificial insemination of domestic cat semen were developed. Total sperm count and pre-freeze and post-thaw percent motility were significantly greater (P is less than 0.05) for samples collected by an artificial vagina method than for those collected by electroejaculation. Although conception rate was low (10.6%), 6 pregnancies were achieved, 2 after hormonal induction of oestrus.

Successful pregnancies with concentrated frozen canine semen.

A method for concentrating canine semen prior to freezing was developed. Concentration was by centrifugation at 1470 g at room temperature, after which the majority of seminal fluid was removed. Motility, speed of progression, and morphology of sperm were not affected by concentration. Subsequent artificial insemination using concentrated semen resulted in a conception rate of 92%. The offspring were normal and healthy. The mean litter size (6.7) and birth weight (256.3 g) were comparable to those reported by a commercial supplier (6.2 and 255.7 g) of the same breed (beagle).