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.

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.

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.

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.

Pituitary and gonadal response to exogenous LH-releasing hormone in the male domestic cat.

To determine the influence of exogenous LH-releasing hormone (LHRH) on serum LH and testosterone, ten adult male domestic cats received three treatments on a rotating schedule at 10-day intervals as follows: (I) 0.1 ml saline i.m. (control); (II) 10 micrograms LHRH i.m., single injection; (III) 10 micrograms LHRH i.m., two injections given at a 2-h interval. Serial blood samples collected over a 360-min interval were analysed by radio-immunoassay for LH and testosterone. Although baseline serum LH values in saline-treated animals (treatment I) varied markedly among individual cats (2.2-29.2 micrograms/l), there was no evidence of pulsatile LH release or alterations in testosterone over time within individual males. In treatment II, the single injection of LHRH induced a rapid rise in mean serum LH within 30 min in all cats (mean peak, 88.2 +/- 9.8 micrograms/l), which returned to baseline by 120 min after LHRH. Mean testosterone increased within 30 min in this group (from 6.03 +/- 2.18 to 18.55 +/- 3.36 nmol/l), peaked at the 60-min collection (19.76 +/- 2.77 nmol/l) and returned to baseline by the 150-min sample. After treatment III, serum LH peaked at 131.6 +/- 13.6 micrograms/l within 30 min of the initial LHRH injection. A second injection of LHRH produced another LH surge within 30 min, but in all cats this second response was of a lower magnitude (mean peak, 69.0 +/- 14.5 micrograms/l) and shorter duration (P less than 0.05). The second LHRH injection sustained peripheral testosterone levels for approximately 1 additional h.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Ovarian response to human chorionic gonadotropin or gonadotropin releasing hormone in cats in natural or induced estrus.

Human chorionic gonadotropin (hOG) or gonadotropin releasing hormone (GnRH) was given alone or with repeated coital stimuli to study ovarian activity and ovulation in the domestic cat. Adult cats in natural estrus (NE) or treated with follicle stimulating hormone (FSH-P) to induce estrus (2.0 mg/d for 5 d; IE) were assigned to one of five treatments: I, mating (M) only (three times daily for the first 3 d of estrus); II, M + hOG (250 IU, i.m. on Days 2 and 3 of estrus); III M + GnRH (25 mug, i.m. on Days 2 and 3 of estrus); IV, hOG only (250 IU, i.m. on Days 2 and 3 of estrus); or V, GnRH only (25 mug on Day 2 and 3 of estrus). Overall, IE females produced a greater (P < 0.05) number of corpora lutea (7.6 +/- 0.9) and unovulated follicles (18.9 +/- 2.1) than NE cats (4.9 +/- 0.6 and 3.6 +/- 0.9, respectively). For both NE and IE females, the M + hOG treatment (II) produced a greater number (P < 0.05) of ovulations (9.1 and 13.9, respectively) than any other ovulatory regimen (I, 4.1, 6.6; III, 4.1, 7.8; IV, 4.0, 6.2; V, 4.1, 5.6, respectively). These results indicate that 1) the excessive follicle number resulting from FSH-P treatment cannot be reduced with any of the hOG or GnRH treatments tested and 2) the use of hOG with copulatory stimuli synergistically enhances the ovulatory response of cats experiencing a natural estrus or those treated with FSH-P.

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.

Human menopausal gonadotropin induces ovulation in sheep, but embryo recovery after prostaglandin F2alpha synchronization is compromised by premature luteal regression.

Using pregnant mares' serum gonadotropin (PMSG) and follicle stimulating hormone (FSH-P) as conventional gonadotropins, human menopausal gonadotropin (hMG) was tested for its comparative ability to induce multiple ovulations in sheep. Estrous cycles were synchronized using either prostaglandin F2alpha (PGF2alpha) or progestogen (MAP)-impregnated pessaries. During the mid-luteal phase, control ewes received serial saline injections, whereas test females (which also served as embryo donors) received either a single PMSG injection (1200 IU) or serial injections of FSH-P (total, 21 mg) or hMG (total, 1350 IU) over 3.5 d. These sheep were naturally mated and artificially inseminated (AI) in utero. Number of CL and transferable-quality embryos 5 d after AI was greater (P<0.05) in FSH-P-and hMG-treated donors than in PMSG-treated ewes. The lower number of transferable-quality embryos produced by PMSG-treated donors was attributed to a reduced (P<0.05) fertilization rate compared with that of the other treatment groups. There were no differences (P>0.05) in daily circulating estradiol-17beta and progesterone concentrations among the gonadotropin treatment groups. Gonadotropin-treated ewes demonstrated estrus approximately 24 h earlier than control ewes and, therefore, exhibited an accelerated estradiol-17beta surge and rise in circulating progesterone. Progesterone production in gonadotropin-treated ewes was also more variable than in the controls; this was due, in part, to premature luteal regression which occurred in 4 of 10 PMSG-, 3 of 10 FSH-P- and 6 of 10 hMG-treated ewes also given PGF2alpha. Ewes with prematurely regressing CL experienced transient luteal tissue development within 4 d of ovulation and produced no embryos. Overall results 1) demonstrate that serial administration of hMG induces multiple ovulations in sheep comparable to FSH-P, and 2) suggest that PGF2alpha treatment during ovulation induction adversely affects newly formed luteal tissue compromising subsequent embryo recovery.

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).

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.

Disposition of the manchette in the normal equine spermatid.

Bielanski and Kaczmarski (1979) reported the presence of microtubules in the neck region of mature stallion spermatozoa. It was hypothesized that these microtubules are derived from the manchette (a microtubular organelle present during spermiogenesis). In order to test this hypothesis, testes from 15 mature stallions were collected, perfused with 2% phosphate-buffered glutaraldehyde, and prepared for transmission electron microscopy. Spermatozoa from the caudae epididymides of each stallion were prepared in a similar manner. Spermiogenesis was observed in general, and the presence of a microtubular manchette was established in this species, juxtapositioned posterior to the nuclear ring and extending distally into the cytoplasmic collar which surrounds the prospective midpiece. Interlocking arms between the microtubules of the manchette were observed in transverse sections at all levels within the cytoplasmic collar before, during, and after caudal migration of the nuclear ring. Consequent to caudal migration of the nuclear ring and the annulus, as well as adluminal movement of the spermatid, the cytoplasmic collar was transformed into the residual cytoplasm. Within the residual cytoplasm, the manchette remained as a microtubular organelle which undergoes degeneration. The mature spermatozoa from the caudae epididymides of these stallions lacked the microtubules reported by Bielanski and Kaczmarski. The occurrence of such microtubules in the neck region of stallion spermatozoa is probably an abnormality.

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)

Comparison of embryo recovery, embryo quality, oestradiol-17 beta and progesterone profiles in domestic cats (Felis catus) at natural or induced oestrus.

Domestic cats experiencing a natural or FSH-induced oestrus were studied. Mated cats produced fewer (P less than 0.01) unfertilized oocytes and more (P less than 0.01) morulato blastocyst-stage embryos of better quality after a natural oestrus than after FSH treatment. Serum oestradiol-17 beta concentrations were lower (P less than 0.05) and progesterone levels rose earlier (P less than 0.05) in the induced oestrus compared to the natural oestrus group. Morula/blastocyst-stage embryos from both groups transferred to 15FSH/hCG-treated recipients produced 3 pregnancies and 2 live-born litters (1 from a natural oestrus donor and 1 from an FSH-treated donor). These results indicate that fertilization rates and embryo quality in domestic cats appear to be compromised by the FSH treatment, probably because of altered oestradiol-17 beta and progesterone concentrations.

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)

In vitro fertilization of gonadotropin-stimulated leopard cat (Felis bengalensis) follicular oocytes.

Based on techniques developed for the domestic cat, in vitro fertilization (IVF) studies were conducted in the taxonomically related leopard cat (Felis bengalensis). Adult females received pregnant mares' serum gonadotropin (PMSG) followed 80 or 84 h later by human chorionic gonadotropin (hCG) on two to four occasions over a 40-day to 27-month interval. Oocytes were collected laparoscopically from ovarian follicles 25-27 h after hCG and co-cultured with processed, homologous spermatozoa (37 degrees C, 5% CO2 in air, humidified atmosphere) for 30-36 h. There was no apparent ovarian refractoriness to repeated treatments with exogenous gonadotropins. Overall, the mean number of mature follicles present and the total number of oocytes and proportion of immature oocytes collected did not differ (P greater than 0.05) between the 80 h (4.9 +/- 0.9; 4.7 +/- 1.2; 14.9%, respectively) and 84 h (5.6 +/- 1.4; 5.4 +/- 1.7; 22.2%, respectively) gonadotropin interval groups. However, the proportion of mature leopard cat oocytes fertilized in vitro, as determined by embryonic cleavage, was increased (P less than 0.005) by extending the interval between PMSG and hCG from 80 (17.5%) to 84 (52.4%) h. These data 1) demonstrate that, compared to the domestic cat, the ovaries of the leopard cat are less responsive to a given PMSG/hCG treatment; 2) indicate that leopard cat follicular oocytes can be recovered readily by laparoscopy and are capable of becoming fertilized in vitro; and 3) suggest that IVF may be a viable approach for producing embryos and perhaps enhancing captive propagation of rare Felidae.

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.