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Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1) identify the optimal inactivation and reactivation solutions for toad spermatozoa colleted in urine; 2) establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3) evaluate the consequence of inactivation on specific sperm characteristics; and 4) characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10–50 mM) produced inferior results. SAR-inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (P<0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure. J. Exp. Zool. 295A:172–182, 2003. © 2003 Wiley-Liss, Inc.

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P< 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ∼24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.

The purpose of this study was to evaluate cryopreserved fringe-eared (FE) oryx (Oryx gazella callotis) sperm function using a heterologous in vitro fertilization (IVF) system previously developed to study scimitar-horned (SH) oryx (Oryx dammah) spermatozoa. Semen was collected by electroejaculation from FE oryx (n = 2) and SH oryx (n = 2), evaluated immediately postcollection, and cryopreserved. Thawed spermatozoa were evaluated for motility, forward progression, and acrosomal status immediately post-thaw, after Percoll-separation, and 1, 2, 3, and 8 h after culture in IVF medium. In vitro-matured cow oocytes (n = 924) were inseminated with either domestic bull, FE, or SH oryx spermatozoa and after an 8-h coincubation period, half the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were placed into in vitro culture and evaluated for cleavage after 48 h. Overall, there were no between-species differences in sperm motility and acrosome integrity. However, an effect of time (P< 0.05) and a species-by-time interaction (P< 0.05) were detected for both parameters. Penetration, male pronuclear formation, and embryo cleavage were high (>90%, >85%, and >70%, respectively) for oocytes inseminated with domestic bull and SH oryx spermatozoa and did not differ (P> 0.05) between species. In contrast, very few oocytes (2.8%, 4 of 141) inseminated with FE oryx sperm were penetrated. Cleavage was rare (8.0%, 16 of 200) in oocytes inseminated with FE oryx spermatozoa and did not differ (P> 0.05) from that in parthenogenetic controls (4.2%, 3 of 72). Furthermore, FE oryx spermatozoa were incapable of penetrating zona-free cow oocytes. These results indicate that species-specific differences in gamete interaction may exist even between very closely related nondomestic bovids.

As many as 20% of all assessed amphibian species are threatened with extinction, and captive breeding programs are becoming important components of conservation strategies for this taxon. For some species, exogenous hormone administration has been integrated into breeding protocols to improve propagation. However, most treatments are administered by an intraperitoneal injection that can be associated with some risks. The general goal of this study was to identify a non‐invasive method of applying luteinizing hormone‐releasing hormone (LHRH), which reliably induces sperm release in toads. Specific objectives were to 1) test the spermiation response after topical application of different LHRH doses to the abdominal seat region, 2) evaluate the effects of adding the absorption enhancers dimethyl sulfoxide (DMSO), acetone, and glyceryl monocaprylate (GMC) to the LHRH, 3) assess the spermiation response after oral delivery of LHRH in a mealworm vehicle, and 4) compare sperm characteristics and spermiation responses to treatments in two different toad species. Male American (n= 9) and Gulf Coast (n= 7) toads were rotated systematically through a series of treatments. Urine was collected and evaluated for the presence of sperm at 0, 3, 7, 12, and 24 hours post‐treatment. There were no statistical differences in spermiation induction or sperm characteristics between American and Gulf Coast toads after the treatments. Oral administration of 100 μg LHRH was occasionally successful in inducing spermiation, but results appeared largely unreliable. Ventral dermal application of 100 or 10 μg LHRH in 40% DMSO were more effective (P< 0.05) at inducing spermiation compared with the other treatments tested, eliciting sperm release in more than 70% of toads tested. In breeding programs for rare and/or fragile anurans, these non‐invasive methods of exogenous hormone administration might be preferred over intraperitoneal injections. Zoo Biol 20:63–74, 2001. © 2001 Wiley‐Liss, Inc.

This study was conducted to determine if cryopreservation and thawing reduces the quality of scimitar-horned oryx spermatozoa and thus might be responsible for sub-optimal artificial insemination (AI) efficiency. Functional capacity of frozen–thawed oryx spermatozoa was compared in a heterologous bovine in vitro fertilization (IVF) system after being prepared by four methods. Fertilizing longevity was also assessed after thawing and pre-incubating spermatozoa for 12 or 24 h before IVF. Sperm characteristics (viability, morphology, acrosomal and capacitation status) were superior for samples prepared by Percoll centrifugation and standard swim-up compared with microdrop swim-up and wash methods. Regardless of variation in sperm characteristics over time, fertilization success and embryo development were high and did not differ among treatments. Fertilization and cleavage success for spermatozoa pre-incubated for 12 h before IVF were comparable with that achieved with non-incubated spermatozoa. Even 24 h after thawing, spermatozoa were capable of fertilizing oocytes, but percentage fertilization and embryo cleavage were significantly lower than for spermatozoa pre-incubated for 12 h. Overall, functional capacity of oryx spermatozoa after thawing appears comparable with that of domestic bull spermatozoa. When used for AI, frozen&mdash;thawed oryx spermatozoa should be capable of fertilizing oocytes in females ovulating 12 or even 24 h after insemination, providing sperm transport mechanisms are adequate. The functional capacity and fertilizing longevity of oryx sperm after thawing is high, and therefore unlikely to be responsible for decreased AI efficiency in the scimitar-horned oryx. Extra keywords: antelope, artificial insemination, capacitation.

Spermiation and LH release in response to several methods of LHRH administration were assessed in the American toad (Bufo americanus), and the most successful method was tested in the endangered Wyoming toad (Bufo baxteri). Specific objectives were to: (1) compare spermiation responses and plasma LH concentration after invasive and non-invasive LHRH treatments; (2) evaluate sperm production in response to different LHRH dosages; (3) characterize the timing of sperm release post LHRH treatment; and (4) assess sperm quality (motility, viability, morphology and acrosomal status). Male American toads were administered 4 g LHRH by one of four routes: (1) intraperitoneal injection (IP); (2) subcutaneous injection (SQ); (3) dorsal dermis absorption (DDA); and (4) ventral dermis absorption (VDA). Aspermic urine only was collected from saline-treated controls and DDA animals. Several VDA animals released spermic urine; however, all LHRH-injected toads released spermatozoa. IP animals produced higher sperm and LH concentrations than SQ animals. The spermiation response in animals treated IP with 1 g LHRH was similar to that in animals treated with 4 g, but lower LHRH dosages tested produced inferior responses. Sperm production in responsive animals increased over time during the 12-h sampling interval. Regardless of treatment, most American toad spermatozoa were motile, viable, and acrosome-intact. Endangered Wyoming toads were treated IP with 4 g LHRH, and spermic urine was collected. Although most spermatozoa were viable and acrosome-intact, a considerable percentage possessed structurally abnormal heads. A single IP injection of LHRH appears to be a reliable and safe method for controlling spermiation in toads and may be useful for assisting endangered amphibian propagation. Extra keywords: amphibian, gonadotropin-releasing hormone, reproduction, testes.

In vivo embryogenesis, embryo migration, and survival were studied in the domestic cat. Queens were naturally mated (three times daily) on the second and third days of behavioral estrus and, if ovulation occurred, ovariohysterectomized at 64 (n = 8), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), and 480 h (n = 8) after first copulation. Of 52 cats mated, 48 (92.3%) ovulated (as evidenced by the presence of ovarian CL), and of these, 38 (79.2%) either produced good-quality embryos or had implantation sites. From the remaining cats, only unfertilized oocytes (n = 5), degenerating embryos (n = 4), or no oocytes/embryos (n = 1) were recovered. Embryos at 64, 76, 100, and 124 h after the first copulation typically were 1 to 4 cells (17 of 20; 85.0%), 5 to 8 cells (18 of 28; 64.3%), 9 to 16 cells (14 of 24; 58.3%), and morulae (15 of 21; 71.4%), respectively; all were within the oviducts. At 148 h, embryos primarily were compact morulae or early blastocysts (15 of 18; 83.3%), and all were within the uterus. For the preimplantation groups, the overall recovery of embryos plus oocytes per CL was 80.6%, and the mean (± SEM) numbers of CL and embryos were 64 h, 4.8 ± 0.3, 3.1 ± 0.8; 76 h, 4.7 ± 0.3, 3.9 ± 0.6; 100 h, 5.8 ± 0.5, 3.3 ± 0.8; 124 h, 4.4 ± 0.5, 4.0 ± 0.6; and 148 h, 6.5 ± 1.1, 3.7 ± 0.7, respectively. Cats in the 480-h group produced a mean of 5.6 ± 0.5 CL and 3.9 ± 0.5 implantation sites. In six of eight cats in this group, there was a disparity between CL number on a given ovary and number of implantation sites in the ipsilateral horn, supporting the concept of transuterine embryo migration. In summary, results indicated that 1) more than 90% of cats ovulated following this multiple mating regimen, but ~ 21% of these failed to produce any fertilized or viable embryos; 2) embryo developmental rate in vivo was biphasic, with a rapid cleavage rate to the 5- to 8-cell stage followed by a slower cleavage rate to the morula stage; 3) cat embryos entered the uterus as compact morulae or early blastocysts ~ 5.5 days after the first copulation; and 4) on the basis of implantation/CL ratio, ~ 30% of all ovulated cat oocytes underwent either fertilization failure or preimplantation embryonic mortality.

Development of in vitro-fertilized (IVF) cat embryos was compared to that of naturally produced cat embryos in vivo and in vitro. To obtain in vivo-fertilized embryos, queens were mated three times daily on the second and third days of natural estrus and ovariohysterectomized at 64, 76, 100, 124, or 148 h after the first copulation. Embryos were flushed from the reproductive tract, evaluated for developmental stage, and cultured. For IVF, oocytes from gonadotropin-stimulated queens were inseminated with electroejaculated cat sperm in Ham’s F-10 and evaluated for fertilization (cleavage to ≥ 2 cells) at 30 h. In vitro development of embryos fertilized in vivo (n = 109) and in vitro (n = 46) was evaluated every 24 h for up to 10 days. High-quality embryos recovered at 64, 76, 100, 124, and 148 h after the first copulation were typically 1 to 2 cells (13 of 20), 5 to 8 cells (18 of 28), 9 to 16 cells (14 of 24), morulae (15 of 21), and compact morulae (11 of 18), respectively, suggesting blastomere cleavage once per day in vivo after the first three rapid cell divisions. A similar developmental rate to the morula stage (p≥ 0.05) was achieved in vitro by embryos derived from both in vitro and in vivo fertilization. Additionally, the proportion (p≥ 0.05) of in vivo-generated embryos (2 to 16 cells) that developed to morulae (64 of 83; 77.1%) was similar to that of IVF embryos (28 of 46; 60.9%). However, none of the IVF embryos (0/46), but 70.6% (77 of 109) of the in vivo-produced embryos, achieved blastocyst formation in culture (p≤ 0.05). Furthermore, 66.2% (51 of 77) of these blastocysts exhibited zona hatching. Incidence of morula and blastocyst formation in the in vivo group was influenced by stage of the embryo at collection. Embryos that were at the 9- to 16-cell stage at recovery were more likely (p≤ 0.05) to achieve morula or blastocyst status and emerge from the zona pellucida than younger-stage counterparts. In summary, the in vivo and in vitro growth rate of cat embryos produced after natural mating was comparable to that of embryos fertilized and cultured in vitro. However, developmental ability to the blastocyst stage was superior for embryos produced in vivo after natural mating. In vitro conditions during fertilization or early embryogenesis appeared to compromise developmental competence from the morula to the blastocyst stage of IVF or early-stage in vivo-produced cat embryos.

Pregnancy success and embryo survival are low with the use of assisted reproduction in felids treated with exogenous gonadotropins. In this study, the pharmacokinetics and ovarian-stimulatory effects of eCG and hCG were evaluated in the domestic cat. Catheterized anestrual queens (n = 4 per treatment [Trt] group) were given 100 IU eCG i.v. (Trt 1), 100 IU eCG i.m. (Trt 2), 75 IU hCG i.v. (Trt 3), 75 IU hCG i.m. (Trt 4), or 100 IU eCG i.m. followed 80 h later by 75 IU hCG i.m. (Trt 5). Blood samples were collected at 0, 5, 30, and 60 min and 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h postinjection, and serum samples were analyzed for estradiol-17β, progesterone, eCG, and hCG. Pharmacokinetic traits (volume of distribution, Vd; elimination half-life, t1/2β; clearance rate, Clr) were calculated for eCG and hCG. When i.v. and i.m. administration were compared, no differences (p> 0.05) were observed in follicle or corpus luteum (CL) number or hormone concentrations for queens receiving eCG or hCG alone. Number of mature ovarian follicles (≥ 2 mm diameter) observed at 168 h postinjection did not differ (p> 0.05) for eCG (mean ± SEM, 10.5 ± 2.0) vs. hCG (11.1 ± 3.0), indicating that these were equally effective in inducing follicular growth. In most queens (> 90%) given single gonadotropins (i.m. or i.v.), eCG and hCG persisted in circulation for at least 120 h and 96 h after injection, respectively, reflecting similar (p> 0.05) pharmacokinetic (i.v.) values for Vd (eCG, 91.4 ± 24.8 ml/kg; hCG, 59.1 ± 7.9 ml/kg), t1/2β(eCG, 23.0 ± 2.4 h; hCG, 22.9 ± 4.1 h), and Clr (eCG, 2.7 ± 0.5 ml/h per kg; hCG, 1.8 ± 0.1 ml/h per kg). Sequential treatment with eCG+hCG did not affect (p> 0.05) the t1/2βof individual gonadotropins. In summary, eCG and hCG have comparable pharmacokinetics and ovarian-stimulatory activity when administered alone to the domestic cat. These findings suggest that hCG promotes the ancillary follicle formation that is frequently observed after ovulation in cats treated with eCG+hCG regimens, possibly disrupting the maternal environment and decreasing fecundity following assisted reproductive procedures.

The effects of gonadotropin treatment and laparoscopic artificial insemination (AI) on embryo quality, serum progesterone and estradiol concentrations, and luteal progesterone content were examined in the domestic cat. These data were compared to similar historical data reported for naturally estrual, mated queens. All queens in this study (n = 32) were treated with eCG followed by 1) natural breeding (eCG-NB), 2) NB and hCG (eCG-NB-hCG), 3) NB and a sham AI procedure (eCG-NB-sham AI), or 4) hCG and actual AI (eCG-hCG-AI). Queens ovulating in response to treatment were ovariohysterectomized, and oviducts and uteri were flushed to collect embryos. Ovarian structures were recorded, corpora lutea (CL) were excised and evaluated for progesterone content, and serum was analyzed for estradiol-17β and progesterone. Follicle and CL numbers ranged from 0 to 28 and 2 to 42 per cat, respectively, and treatment means did not differ (p ≥ 0.05) among groups. Embryos were recovered from oviducts and uterine horns in all treatment groups, and recovery ranged from 60-96%. Mean embryo number per queen ranged from 8.2 ± 2.6 to 23.2 ± 3.8 and did not differ (p ≥ 0.05) among groups. However, the proportions of unfertilized oocytes were greater (p < 0.05) for groups treated with hCG and/or artificially inseminated, and the proportion of blastocysts produced (31 of 107, 29.0%) was lower (p < 0.05) in the eCG-hCG-AI group than for any other treatment (range, 59 of 116 [50.9%] to 67 of 116 [57.8%]). Not all queens in each group produced good-quality embryos (eCG-NB, 5 of 5; eCG-NB-hCG, 5 of 8; eCG-NB-sham AI, 2 of 5; and eCG-hCG-AI, 3 of 6). Serum progesterone and estradiol-17β, and total luteal progesterone per ovary did not differ (p ≥ 0.05) among treatments. Compared to historical controls (naturally estrual, mated queens), eCG-NB queens produced > 4 times as many good-quality embryos and blastocysts. Similarly, eCG-hCG-AI-treated queens produced > 4 times the number of oocytes and embryos, although a high proportion of these were poor quality and did not develop to blastocysts. Together, these results indicate that queens treated with eCG are capable of consistently producing many good-quality embryos, at least half of which develop to blastocysts in culture. These data support the use of eCG in felids and suggest that other factors are responsible for reduced pregnancy success and small litter sizes following assisted reproduction.

Serum hormones, corpus luteum (CL) progesterone, and CL LH receptors were characterized and interrelated to ovarian follicle and CL number and preimplantation embryo quality/survival in the cat. Blood samples were collected from queens ovariohysterectomized at 64 (n = 7), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), or 480 (n = 8) h after first copulation (3-times-daily matings on Days 2 and 3 of estrus). Ovarian CL were enucleated, weighed, and bisected; one hemi-CL was assayed for progesterone and the other for LH receptors. Serum was assessed for estradiol-17β and progesterone. Serum estradiol-17β concentrations did not return (p> 0.05) to baseline ∼ 20 pg/ml) until 124 h after first copulation, whereas serum progesterone began to increase (> 1 ng/ml) by 76 h, was elevated (p< 0.05) by 124 h, and continued to rise thereafter. Serum progesterone was highly correlated with CL mass and LH receptor and progesterone concentration (range, r= 0.69–0.82; p< 0.01). CL progesterone and LH receptor concentrations were similar (p> 0.05) at 64 and 76 h, and both increased (p< 0.05) at subsequent time intervals and were correlated closely (r= 0.65; p< 0.01). Although ovarian CL were distinct and well organized by 64 h, pronounced elevations in serum progesterone and CL LH receptors and progesterone did not occur until at least 36 h later. The rapid increase in serum progesterone concentrations between 100 and 148 h was related to accelerated LH receptor synthesis and CL progesterone production and not entirely to enhanced CL growth.

A heterologous bovine in vitro fertilization (IVF) system was used to study the functional competence of scimitar-horned oryx spermatozoa after cryopreservation. Four sperm-freezing methods were compared after dilution of ejaculates from six oryx with an equine semen extender: 1) dry ice, 2) dry shipper one-step, 3) dry shipper two-step, and 4) liquid nitrogen vapor. Post-thaw sperm motility, longevity, and acrosomal status were assessed and zona pellucida penetration, fertilization, and embryo cleavage were evaluated after coincubation of thawed oryx spermatozoa with in vitro-matured domestic cow oocytes. Sperm motility index (SMI) decreased (p< 0.05) over a 6-h period, but a high percentage (≥ 65%) of spermatozoa contained intact acrosomes in all treatments. Despite differences in sperm motility among methods, oocyte penetration, fertilization, and embryo cleavage did not differ (p≥ 0.05). However, cleavage success was < 50% across all treatments. There were positive correlations (p< 0.05; r= 0.81–0.97) between sample SMI at 3 and 6 h and fertilization, penetration, and cleavage, but no correlations (p≥ 0.05) between SMI at 0 or 1 h and IVF success. This study demonstrates that compatible heterologous gamete interaction allows thorough assessment of post-thaw sperm function in an endangered antelope. Scimitar-horned oryx spermatozoa appear relatively tolerant of varied cryopreservation methods, and preserved samples exhibit adequate post-thaw function to warrant use for assisted reproduction.

Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 µM heparin, 3) TALP + 20 µM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p< 0.05) sperm incubated in TALP + 10 µM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p< 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to ≥ 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting that the two bovid species have similar fertilization mechanisms.

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short‐term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long‐term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40‐day study period. Thirty‐eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P< 0.05) and the number of spermatozoa with a bent head or midpiece increased (P< 0.05) for both temperature groups over the 3‐hr storage interval. DMSO and EG were equally effective in maintaining penguin sperm quality parameters during the cryopreservation and thawing process. Frozen‐thawed semen maintained 69 ± 5 and 78 ± 3% of its pre‐freeze SMI and viability, respectively. SMI and viability decreased slightly during the cooling and equilibration phases but remained relatively stable during the 3‐hr storage interval post‐thaw. Frozen‐thawed semen also exhibited an increase (P< 0.05) in spermatozoa with a bent head or midpiece over time. The pre‐freeze SMI was higher (P< 0.05) for ejaculates with low levels of urates (clean ejaculates) compared with ejaculates with high levels of urate contamination, but sperm viability and morphology were similar (P> 0.05). Both SMI and viability of frozen‐thawed spermatozoa were higher (P< 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short‐ and long‐term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley‐Liss, Inc.

Ovaries of the seasonally-breeding snow leopard (Uncia uncia) were examined to determine whether they were responsive to exogenous gonadotrophins throughout the year. The potential of laparoscopic artificial insemination (AI) also was assessed for producing offspring. During the non-breeding, pre-breeding, breeding and post-breeding seasons, females (n = 20) were treated with a standardized, dual-hormone regimen given intramuscularly (600 I.U. of equine chorionic gonadotrophin followed 80–84 h later with 300 I.U. of human chorionic gonadotrophin (hCG)). Laparoscopy was performed 45–50 h after administration of hCG, and all ovarian structures were described. Females with fresh corpora lutea (CL) were inseminated, and anovulatory females were subjected to follicular aspiration to examine oocyte quality. Snow leopards responded to exogenous gonadotrophins throughout the year. Mean number of total ovarian structures (distinct follicles mature in appearance plus CL) did not differ (P ≥ 0 · 05) with season, but the proportion of CL : total ovarian structures was greater (P &lt; 0 · 01) for the breeding season compared with all other seasons. The proportion of females ovulating was greater (P&lt;0 · 05) during the breeding and post-breeding seasons than during the pre-breeding and non-breeding seasons respectively. No Grade-1 quality oocytes were recovered from follicles of anovulatory females. Serum concentrations of oestradiol-17b appeared elevated in all females, and neither oestradiol-17β concentrations nor progesterone concentrations differed (P ≥ 0 · 05) among seasons. Of 15 females artificially inseminated, the only one that was inseminated in the non-breeding season became pregnant and delivered a single cub. This is the first successful pregnancy resulting from AI in this endangered species. Extra keywords: felid, seasonality, ovulation induction, laparoscopy, ovary.