Investigation of captive red wolf ejaculate characteristics in relation to age and inbreeding.

An evaluation of a large database of red wolf fresh ejaculate characteristics (n = 427 ejaculates from 64 wolves) was undertaken to increase knowledge of seminal characteristics in the red wolf and evaluate possible relationships between inbreeding, age, and seminal quality. Phase microscopy analysis of electroejaculates collected over 14 natural breeding seasons was compared with animal ages and inbreeding coefficients. Ejaculate volume increased and sperm concentration and total count decreased as wolves aged (P < 0.01, 0.001, and 0.05, respectively), and the proportion of sperm cell morphological abnormalities was greater in animals with higher coefficients of inbreeding (P < 0.001), particularly for older animals (P < 0.001). Moreover, the mean coefficient of inbreeding of animals that had failed to reproduce given at least one opportunity during their lifetimes was significantly greater than that of wolves with proven fertility, and wolves of proven fertility exhibited higher sperm concentrations and total counts than nonproven wolves. Thus, as the captive red wolf population becomes more inbred, the maximum age of reproduction is likely to decrease; an important finding to consider when projecting population dynamics and determining pairing recommendations.

The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis).

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America's most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or "wrestling" was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.

Retrospective investigation of captive red wolf reproductive success in relation to age and inbreeding.

The critically endangered red wolf (Canis rufus) has been subject to a strictly managed captive breeding program for three decades. A retrospective demographic analysis of the captive population was performed based on data from the red wolf studbook. Data analyses revealed a decrease in the effective population size relative to the total population size, and changes in age structure and inbreeding coefficients over time. To varying degrees, the probability of successful breeding and litter sizes declined in association with increasing dam age and sire inbreeding coefficients. Neonate survival also declined with increasing dam age. Recent changes in strategies regarding breed-pair recommendations have resulted in moderate increases in reproductive success.

Comparison of different osmolalities and egg-yolk composition in processing media for the cryopreservation of red wolf (Canis rufus) sperm.

Successful cryopreservation of sperm and the maintenance of a sperm-based genome resource bank have been identified as priorities for the recovery of the endangered red wolf (Canis rufus). The objectives were to improve sperm processing and to determine the relative timing of damage to red wolf sperm during freezing and thawing. Fresh ejaculates (n=37) from adult red wolves (n=15, aged 2-13 y) were collected via electroejaculation and subjected to cooling, freezing and thawing in four TRIS-egg-yolk extender treatments varying in osmolality ( approximately 305 mOsm versus approximately 350 mOsm) and egg-yolk composition (0.8 microm-filtered versus unfiltered). Ejaculates were evaluated for sperm percentage motility, forward progressive motion, and morphological characteristics immediately upon collection and following extension, cooling (prior to freezing) and thawing. Although no single treatment consistently produced superior results, sperm suspended in approximately 305 mOsm extenders exhibited slight losses in motility post-thawing (13 and 7%). Also, sperm suspended in approximately 350 mOsm extenders tended to have slower rates of decline in motility in vitro post-thawing than those stored in approximately 305 mOsm extenders (P=0.55). Finally, extenders incorporating unfiltered egg yolk exhibited a slightly larger ratio of absent to partial acrosomes than did sperm frozen in extenders prepared with clarified egg yolk. For approximately 350 mOsm extenders, most motility loss occurred during the cooling rather than freezing and thawing. In conclusion, these data contribute to knowledge regarding cryopreservation of red wolf sperm.

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.

Piecing together the puzzle of carnivore reproduction.

Recent advances in feline and canine reproductive studies demonstrate how methodically piecing this information together is beginning to reap rewards for wildlife conservation programs. Non-invasive endocrinology can be used to monitor female reproductive function, time con-specific introductions or AI, and diagnose pregnancy. Sperm morphology characteristics and cell membrane function may be genetically inherited and differ between genetically diverse and inbred species/populations in felids. It is not clear if the same is true for the endangered red wolf. While standards exist for freezing feline and canine sperm, new information using fluorescent staining and zona penetration assays (ZPA) indicates that significant damage can occur during pre-freeze cooling, and may also be related to a species' genetic diversity. Posthumous gamete salvage from genetically valuable animals not only provides a means to study sperm and oocyte physiology but also to assist with genetic management of populations. Using the knowledge gained, IVM/IVF and ICSI have been successful in the domestic cat and AI has resulted in offspring in numerous non-domestic felids. However, understanding the processes of IVM/IVF is still not well understood in canids. New information reveals that sperm and the cumulus cells may be integral to oocyte maturation and that canine epididymal sperm are not capable of undergoing fertilization. The acquisition of knowledge and application of biotechnologies lags behind for non-domestic canid conservation programs.

Correlation of periovulatory serum and fecal progestins in the domestic dog.

This study was initiated to determine the relationship between canine ovarian steroids detected in serum and feces during the periovulatory interval in domestic dogs, and to examine the feasibility of a non-invasive method to estimate the time of ovulation in canid species. When bitches (n = 14) were observed to enter proestrus (based on vulvar enlargement or serosanguineous vaginal discharge), paired daily serum and fecal samples were collected for a 15- to 20-day period and stored at -20 degrees C. After extraction, progestin concentrations in both substrates were measured using an established enzyme immunoassay procedure. All samples were aligned to Day 0, the first day in which fecal progestins reached a sustained rise above 100 ng/g feces. Mean fecal progestin concentrations increased in parallel with mean serum progesterone values (r = 0.78), rising from 44.6+/-2.6 ng/g feces to 409.6+/-90.9 ng/g feces, and 5.4+/-0.9 nmol/L to 81.2+/-18.5 nmol/L, on Day -5 and Day 5, respectively. Individual fecal progestin concentrations varied markedly, but plotted against serum progesterone concentrations demonstrated correlation coefficients ranging from 0.41 to 0.97 (P<0.05). These results demonstrate that sequential changes in domestic dog serum ovarian steroid concentrations are paralleled in the feces, and that it is feasible to non-invasively monitor individual progestin changes in the periovulatory interval using fecal hormone analysis.

Evaluation of epidural morphine for postoperative analgesia in ferrets (Mustela putorius furo).

We evaluated the analgesic efficacy of epidural morphine for relieving postoperative pain in domestic ferrets by evaluating behavior and fecal cortisol concentrations. The 12 laboratory-reared, intact, female, domestic ferrets were anesthetized then underwent ovariohysterectomy and bilateral anal sacculectomy. Using a double-blind procedure, we provided epidural morphine (0.1 mg/kg) to six ferrets and epidural saline (0.1 mL/ferret) to the remaining animals prior to surgery. Compared to the animals that received saline, the morphine-treated ferrets were more likely to have attenuated pain responses, and they returned more rapidly to preoperative behavior. Although fecal cortisol concentrations during the first 24 h after surgery increased in all animals, the increase was statistically significant only in the ferrets that received saline epidurals. These data suggest that morphine epidurals administered to ferrets prior to surgery may attenuate both the physiologic and behavioral manifestations of surgically induced pain.

Hormonal control of estrous cyclicity and attempted superovulation in wood bison (Bison bison athabascae).

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).

Faecal progesterone metabolite analysis for non-invasive monitoring of reproductive function in the white rhinoceros (Ceratotherium simum).

The two subspecies of white rhinoceros, southern (Ceratotherium simum simum) and northern (Ceratotherium simum cottoni), breed poorly in captivity, and estimates of oestrous cycle length vary considerably (range, 25-90 days). To characterise reproductive patterns, faecal samples were collected 2-3 times/week for up to 56 months from non-pregnant animals (n=21) of both subspecies. Immununoreactive pregnanes containing a 20-oxo-group (20-oxo-P) were analysed in a group-specific enzyme immunoassay using an antibody against 5alpha-pregnane-3beta-ol-20-one 3HS:BSA. Reproductive patterns were highly variable among and within individual animals. However, rhinoceroses could be classified into four major categories on the basis of oestrous cycle length and luteal phase 20-oxo-P concentrations: (1) regular oestrous cycles of 10 weeks duration and > 800 ng/g (n=2 animals); (2) oestrous cycles between 4-10 weeks and 250-750 ng/g (n=6); (3) no apparent cycle regularity, but luteal activity indicated by 20-oxo-P concentrations of 100-200 ng/g (n=6); (4) no apparent luteal activity as indicated by 20-oxo-P of < 100 ng/g (n=7). In two attempts to induce ovarian activity, chlormadinone acetate was fed daily to one animal for 35 and 45 days, respectively. Each treatment was followed by a subsequent hCG injection which resulted in luteal phases of 17 and 18 days, respectively, beginning about 10 days after hCG. Concentration of faecal 20-oxo-P in one pregnant animal during the 4th and 5th month of gestation were markedly higher than those observed during the luteal phase of the cycle. In conclusion, two thirds of white rhinoceroses in this study had erratic or missing luteal activity, whereas variable cycles of 4-10 weeks in length were evident in six females, and regular oestrous cycles of 10 weeks in length were found in two animals.

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.

Changes in the number of follicles and of oocytes in ovaries of prepubertal, peripubertal and mature bitches.

Ovaries were collected from prepubertal (< 6 months of age, n = 4 ovaries), peripubertal (6 to 10 months of age, n = 12 ovaries) and mature (> 10 months, n = 12 ovaries) bitches after routine ovariohysterectomies and fixed in formalin. Ovaries were bisected, embedded in paraffin wax and 20 serial sections were made at intervals of 10 microns. Sections were stained with haematoxylin and eosin to examine follicles and oocytes in a cross-section of cortex of known size. Counts were made on all sections, resulting in examination of the entire cortical area present in the sections. Oocytes were counted and classified as nucleate or anucleate. Follicles were counted and classified as large (> 100 microns in diameter) or small (< 100 microns in diameter), containing one oocyte (monovular), more than one oocyte (polyovular) or no oocytes (anovular). It was concluded that at first oestrus there was an increase (P < 0.05) per section in number of total oocytes and small monovular follicles and a significant increase (P < 0.05) in the number of nucleated oocytes in monovular follicles, suggesting that oogenesis or folliculogenesis is still occurring at this age. At pre- and peripubertal ages polyovular follicles were found which persist into maturity. There was no difference in numbers of anovular follicles and total number of polyovular follicles among different age groups.

Effects of cooling, freezing and glycerol on penetration of oocytes by spermatozoa in dogs.

A homologous zona penetration assay was used to evaluate dog spermatozoa after selected treatments and associated gamete interaction with sperm characteristics. Canine semen, diluted in egg-yolk extender, was treated to compare samples quickly cooled to 0 degree C (after 0.5 and 3 h), slowly cooled to 0 degree C after 3 h or held for 3 h at room temperature. In a second study, slowly cooled spermatozoa with or without the addition of 4% glycerol were studied. After each treatment, spermatozoa were incubated with canine oocytes for 18 h, stained with Hoechst and examined for bound sperm heads. After 3 h, cells that had been cooled quickly demonstrated lower numbers of spermatozoa per ovum than fresh or slowly cooled treatments (2.4 +/- 0.6 versus 5.2 +/- 1.1 and 3.6 +/- 0.9 sperm per oocyte, respectively) and coincided with a reduction in acrosomal integrity and motility (58.6% fast cool 3 h versus 83.1% fresh). Addition of glycerol effected no change in acrosomal status or motility, but resulted in a decline in spermatozoa bound per oocyte to 1.5 +/- 0.3. While freezing caused a further decline in motility, acrosomal status and oocyte penetration, there was no difference between freezing with and without seeding (0.3 +/- 0.08 versus 0.2 +/- 0.04 spermatozoa per ovum). Evaluation of sperm penetration of homologous oocytes provides new insights into the effects of cooling, seeding and glycerol on canine spermatozoa.

Reproductive activity in captive female cheetahs (Acinonyx jubatus) assessed by faecal steroids.

Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (Acinonyx jubatus) (n = 26) for 1-24 months. Increased faecal oestradiol excretion was associated with mating or equine chorionic gonadotrophin (eCG) administration for artificial insemination, whereas increased progestogen metabolites were observed during natural and human chorionic gonadotrophin (hCG)-induced pregnant and nonpregnant luteal phases. On the basis of oestradiol excretory patterns, duration of the oestrous cycle (mean +/- SEM) was 13.6 +/- 1.2 days with high oestradiol concentrations lasting for 4.1 +/- 0.8 days. In non-gonadotrophin-treated cheetahs, 75% showed evidence of oestrous cyclicity; however, none evaluated for 1 year or longer were continuously cyclic. Rather, cyclicity was interrupted by periods of anoestrus, often exceeding several months in duration. These inactive ovarian periods were unrelated to season and were not synchronous among females. Mean duration of gestation (breeding to parturition) was 94.2 +/- 0.5 days, whereas duration of faecal progestogen metabolite excretion during the nonpregnant luteal phase was 51.2 +/- 3.5 days. On the basis of progestogen metabolite evaluations, spontaneous ovulation (non-mating induced) occurred only once in two females (2 of 184 oestrous cycles; 1.1%). Peak eCG-stimulated, preovulatory oestradiol concentrations were similar to those associated with natural oestrus, whereas progestogen metabolite profiles after hCG resembled those during pregnant and nonpregnant luteal phases after natural mating. In summary, results confirm that the cheetah is polyoestrus and ovulation is almost always induced. However, new evidence suggests that many females inexplicably experience periods of anoestrus unrelated to season, while 25% of the cheetahs examined expressed no ovarian activity during the study period.

Characteristics and zona binding ability of FRESH and cooled domestic cat epididymal spermatozoa.

Maintenance of genetic diversity within endangered species is important for ensuring healthy populations. Because unexpected deaths can occur, it would be advantageous to salvage gametes to effect posthumous participation in species reproduction. Using the domestic cat as a model for nondomestic felids, this investigation was undertaken to determine epididymal sperm cell characteristics, capacitation timing and the effects of storage temperature on fertilizing ability. In Study 1, the timing of capacitation was evaluated by examining zona attachment of spermatozoa to in vitro matured oocytes at 30-min intervals for 5 h. In Study 2, the ability of freshly collected (FRESH) and overnight cooled (COOL) epididymal spermatozoa to undergo capacitation and nuclear decondensation was evaluated using the zona attachment and zona-free hamster ova penetration assays. From Study 1, mean characteristics (n=29) for epididymal sperm cell motility and progressive status were 51.9% and 3.1+/-0.1, respectively, with a concentration of 80.3 x 10(6) spermatozoa/ml and 51% morphologically normal cells. Zona attachment (n >/= 25 ova/time interval) by sperm cells occurred at each time interval, but both the mean number of attached sperm cells/zona and the percentage of zonae with attached spermatozoa reached maximum values at 240 min (12.0+/-2.1 and 89.7%, respectively; P<0.05). In Study 2, overnight cooling did not affect progressive status of motility (3.3+/-0.1) or the percentage of morphologically normal spermatozoa (53.2+/-4.4) compared with that of FRESH (2.9+/-0.1, 50.7+/-3.2%) samples; however, motility was 14% lower (P<0.05) in the COOL vs FRESH group. Hamster ova penetration and the mean number of sperm cells attached/zona were greater in the COOL (28%, 18.6+/-5.7) than in the FRESH (5%, 7.4+/-2.0) group (P<0.05). However, it is speculated that the increased sperm-zonae interaction may have been the result of acrosomal damage. Nevertheless, these data demonstrate that domestic cat epididymal sperm cells have the ability to capacitate and undergo the first stages of fertilization.

Influence of day of oestrus on egg viability and comparative efficiency of in vitro fertilization in domestic cats in natural or gonadotrophin-induced oestrus.

Thirty-six domestic cats received 100 iu hCG (i.m.) on day 1, 2 or 3 of a natural, behavioural oestrus. Twenty-two anoestrous cats were injected with 150 iu pregnant mares' serum gonadotrophin (PMSG; i.m.) followed 84 h later by 100 iu hCG. Twenty-four to 26 h after hCG, all cats were examined laparoscopically to determine the number of ovarian follicles and to recover follicular eggs. Mature eggs were cultured with conspecific spermatozoa and examined 30 h later for cleavage. Within the natural oestrus group, cats on day 1 produced fewer (P < 0.05) follicles and total eggs than females on day 2 or 3, and 88.9% of the day 1 eggs were degenerate or immature and unsuitable for in vitro fertilization (IVF). Although only 54.5% of the cats in the PMSG/hCG group exhibited overt oestrus, mean (+/- SEM) numbers of follicles (9.7 +/- 0.8) and oocytes recovered (8.7 +/- 0.8) were at least twofold greater (P < 0.001) than those measured in the natural oestrus group (3.7 +/- 0.6; 3.4 +/- 0.6, respectively) or subgroups on day 2 (3.7 +/- 0.4; 3.3 +/- 0.4) and day 3 (5.7 +/- 0.8; 5.3 +/- 0.8). Overall, the proportion of eggs cleaving in vitro was similar (P > 0.05) between the natural oestrus group (48.3%) and the PMSG/hCG group (50.9%), but the latter group produced more than twice the number of embryos per donor. Embryo quality was unaffected (P > 0.05) by day of hormone treatment, and more than 80% of all two-cell embryos were rated good-to-excellent quality.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurements of faecal oestradiol and progesterone in non-pregnant and pregnant domestic and exotic cats.

Faecal samples collected for variable periods from 12 animals and five species of cats were assayed for progesterone and oestradiol content by application of standard radioimmunoassays to aliquots (50 microliters) of methanol extracts (4 ml) of a mixture of 0.5 g sample, 0.5 ml water and 1 g aluminium oxide, following partitioning of the total extract with petroleum ether (3 ml), further dilution of assay aliquots and drying. Recoveries averaged 100 and 72% for oestradiol and progesterone, respectively. Results included increases in progesterone during luteal phases or pregnancies to 7688 ng g-1 (tiger, Panthera tigris), 2594 ng g-1 (lion, P. leo), 3000 ng g-1 (cheetah, Acinonyx jubatus) and 4915 ng g-1 (caracal, Felis caracal). Faecal oestradiol peaks near oestrus included 246 ng g-1 (tiger), 175 ng g-1 (lion) 190 ng g-1 (cheetah), 23 ng g-1 (caracal) and 190 ng g-1 (domestic cat, F. catus).

Comparative cryopreservation and capacitation of spermatozoa from epididymides and vasa deferentia of the domestic cat.

Methods of cryopreservation for spermatozoa from domestic cat epididymides and vasa deferentia were compared as models for posthumous gamete salvage from non-domestic felids. Spermatozoa were collected either immediately after castration (Fresh, n = 37) or after being cooled (5 degrees C) in tissue overnight (Cool, n = 37) and released into one of three extenders containing 20% egg yolk and 3% glycerol for cryopreservation: (1) TE: Tris buffer, citric acid and fructose; (2) TC: Tris buffer, citric acid and glucose, or (3) CP: lactose, and frozen over lipid nitrogen. Before and after freezing, each sperm cell sample was evaluated for motility and percentage morphologically normal cells. Samples were also evaluated for their ability to initiate fertilization using a zona attachment assay. Neither percentage morphologically normal spermatozoa nor percentage motility differed among the three diluents for prefreeze and post-thaw samples, regardless of the collection treatment. However, CP tended to provide lower post-thaw status than did the TE and TC cryoextenders. Before freezing, there was no difference in percentage motility between the Fresh and Cool groups (mean: 76 versus 72%, respectively); however, progressive status and normal morphology were lower (P < 0.05) in Cool (3.0 and 57%) than in Fresh (3.4 and 64%) samples. After thawing there was a greater decline (P < 0.05) in percentage motility in the Cool than in the Fresh group (34 versus 24%) and the number of intact acrosomes dropped from prefreeze values of 66.7 +/- 6.3 and 56.4 +/- 4.8% to 17.8 +/- 3.9 and 20.9 +/- 4.6% after thawing in the Fresh and Cool groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of sperm viability with gamete interaction and fertilization in vitro in the cheetah (Acinonyx jubatus).

Sperm-oocyte interaction in vitro was studied in the cheetah, a species known to produce poor quality ejaculates and to experience low rates of fertility. Twelve female cheetahs were injected (i.m.) with eCG followed by hCG 84 h later. Twenty-four to 26 h post hCG, each was subjected to laparoscopic oocyte aspiration. A sperm motility index (SMI) was calculated for each of 9 cheetah sperm donors that produced ejaculates averaging 41.3 +/- 22.9 x 10(6) motile sperm and 28.4 +/- 4.9% structurally normal sperm. Each ejaculate was used to inseminate cheetah oocytes from 1 or 2 females and salt-stored, domestic cat oocytes. The presence of ovarian follicles (greater than or equal to 1.5 mm in diameter) showed that all females responded to exogenous gonadotropins (range, 11-35 follicles/female). A total of 277 cheetah oocytes was collected from 292 follicles (94.9% recovery; 23.1 +/- 2.2 oocytes/female). Of these, 250 (90.3%) qualified as mature and 27 (9.7%) as degenerate. Of the 214 mature oocytes inseminated, 56 (26.2%) were fertilized, and 37 (17.3%) cleaved to the 2-cell stage in vitro; but the incidence of in vitro fertilization (IVF) varied from 0 to 73.3% (p less than 0.001) among individual males. When oocytes from individual cheetahs (n = 5) were separated into two groups and inseminated with sperm from a male with an SMI greater than 0 after 6 h coincubation versus an SMI = 0 at this time, the mean fertilization rates were 28/44 (63.6%) and 0/37 (0%), respectively (p less than 0.05). Of the 117 domestic cat oocytes coincubated with cheetah sperm, 96.6% contained 1 or more cheetah sperm in the outer half of the zona pellucida (ZP). Although the mean number of cheetah sperm penetrating the outer ZP of the cat oocyte was similar (p greater than 0.05) among all males, there was a positive correlation between the number of sperm reaching the inner half of the ZP and fertilization rate in vitro (r = 0.82; p less than 0.05). Compared to IVF efficiency in the domestic cat and tiger as reported in earlier studies, IVF efficiency in the cheetah is low. Because oocytes from 11 of 12 cheetahs were fertilized in vitro, there is no evidence that the female gamete is incompetent. Although sperm pleiomorphisms may contribute to poor reproductive performance, examination of the data on the basis of individual sperm donors reveals that effective gamete interaction in the cheetah is dictated, in part, by sperm motility.

Nuclear maturation of domestic cat ovarian oocytes in vitro.

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 micrograms/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degrees C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase II (MII); metaphase I (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p less than 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%).(ABSTRACT TRUNCATED AT 250 WORDS)