Urethral catheterization and sperm vitrification for simplified semen banking in felids.

Semen banking of domestic cats and wild felids represents a vital resource for their long-term conservation, but current methods require access to advanced training and specialized equipment. A newer method of semen collection, urethral catheterization of medetomidine-treated cats, allows recovery of high sperm numbers, but it is unclear if this approach permits maximal sperm recovery or is feasible using less expensive alpha-2 agonists. Similarly, a newer sperm preservation approach, vitrification, offers advantages of simplicity and minimal equipment needs, but its efficacy in combination with urethral catheterization has not been investigated. Our specific objectives were to (i) evaluate sequential semen recovery with urethral catheterization and electroejaculation in domestic cats, (ii) assess the effectiveness of a weak (xylazine) versus strong (dexmedetomidine) alpha-2 agonist for inducing sperm release, and (iii) compare post-thaw sperm motility, acrosome status and fertilizing capacity of catheter-recovered samples after vitrification or straw freezing. Results indicated that electroejaculation following repeated catheterization allowed recovery of additional spermatozoa (range, 11-32 × 106  sperm/male) and that xylazine was ineffective for inducing meaningful sperm release (range, 0-0.4 × 106 sperm/male). Post-thaw motility and acrosome status of vitrified catheter samples did not differ (p > .05) from that of straw frozen samples. Preliminary results indicated that in vitro fertilization success (9/30, 30%) of vitrified catheter sperm did not differ (p > .05) from that observed with straw frozen samples (17/30, 57%). In conclusion, urethral catheterization of dexmedetomidine-treated cats allows recovery of substantial sperm numbers but electroejaculation still may be warranted for maximal sperm recovery. Xylazine is not suitable as an inexpensive alternative to dexmedetomidine for catheterization. Vitrification of catheter samples results in comparable post-thaw parameters to straw freezing and may be adequate for use with oviductal insemination procedures.

Temporal changes in serum luteinizing hormone following ovariohysterectomy and gonadotropin-releasing hormone vaccination in domestic cats.

Measurement of circulating luteinizing hormone (LH) concentrations in cats and temporal changes following ovariohysterectomy (OHE) or possibly GnRH vaccination may be informative for assessing their fertility, contraception or sterilization status. In this study, serum LH concentrations were measured in domestic cats (n = 6) immediately prior to and up to 120 days post-OHE. Basal LH concentrations of females previously subjected to OHE (n = 4; ~1.5 years post-OHE) were compared pre- and post-vaccination with a GnRH immunocontraceptive, and to LH concentrations in intact females. Basal serum LH concentrations (2.67 ± 0.43 ng/ml; mean ± SEM) in intact females increased (p < .01) by 30 days post-OHE (5.65 ± 0.87 ng/ml) but then declined (p < .05) to pre-OHE levels (mean range, 3.26-3.62 ng/ml) at days 60-120 post-OHE. Serum LH (3.84 ± 0.51 ng/ml) in four females ~1.5 years after OHE tended to be higher (p = .10) than those of intact females prior to OHE. Three months following first or second GnRH immunocontraceptive vaccine treatment, serum LH values in females previously subjected to OHE decreased (p < .05) to concentrations similar to those observed in intact females. Our preliminary results suggest that OHE of domestic cats causes a marked increase in basal LH levels within the first few weeks after ovariohysterectomy followed by a return to pre-OHE basal values over the next several months. Reduced LH concentrations after GnRH vaccine may indicate the effectiveness of the immunocontraceptive in reducing the circulating levels of GnRH, thereby reducing secretion of LH.

Safety and effectiveness of a single and repeat intramuscular injection of a GnRH vaccine (GonaCon™) in adult female domestic cats.

Sterilization is a key strategy to reduce the number of domestic cats entering and killed in shelters each year. However, surgical sterilization is expensive and labour-intensive and cannot fully address the 70 million free-roaming cats estimated to exist in the United States. GonaCon™ is a gonadotropin-releasing hormone vaccine originally developed for use as a wildlife immunocontraceptive. An earlier formulation was tested in domestic cats and found to be safe and effective for long-term contraception. However, the current Environmental Protection Agency (EPA)-registered formulation consists of a different antigen-carrier protein and increased antigen concentration and has never been tested in cats. A pilot study was undertaken to evaluate the short-term safety of a single GonaCon immunization, assess the consequences of vaccinated cats receiving an accidental second GonaCon injection and determine the humoral immune response to immunization. During Phase 1, cats in Group A (n = 3) received a single intramuscular injection of GonaCon and Group B (n = 3) received a single intramuscular injection of saline. During Phase 2, Group A received a second GonaCon injection and Group B received their initial GonaCon injection. All cats developed GnRH antibodies within 30 days of vaccine administration. The endpoint titre (1:1,024,000) was similar among all cats, and levels remained high throughout the duration of the study. Four cats developed a sterile, painless, self-limiting mass at the site of injection. The mean number of days to mass development was 110.3 (range, 18-249 days). In conclusion, this preliminary study suggests that the EPA-registered GonaCon formulation is safe for continued testing in domestic cats, an accidental revaccination should not increase the risk of a vaccine reaction and the EPA-registered formulation effectively elicits a strong humoral immune response.

Sperm Morphology Assessment in Captive Neotropical Primates.

The main objective of this study was to evaluate sperm morphology in four neotropical primate species to compare the sperm morphological traits and the sperm morphometric parameters as a basis for establishing normative sperm standards for each species. Data from 80 ejaculates collected from four primate species, Callithrix jacchus, Callimico goeldii, Alouatta caraya and Ateles geoffroyi, were analysed for detection of sperm morphological alterations using subjective World Health Organization (WHO-2010) standards and Sperm Deformity Index (SDI) criteria, objective computer-assisted sperm morphometry analysis (CASMA) and subpopulation sperm determination (SSD) methods. There were multiple differences (p < 0.01) observed among primate species in values obtained from WHO-2010, SDI, CASMA and SSD sperm analysis methods. In addition, multiple significant positive and negative correlations were observed between the sperm morphological traits (SDI, Sperm Deformity Index Head Defects, Sperm Deformity Index Midpiece Defects, Sperm Deformity Index Tail Defects, Normal Sperm, Head Defects, Midpiece Defects and Tail Defects) and the sperm morphometric parameters (SSD, Area (A), Perimeter (P), Length (L), Width (W), Ellipticity, Elongation and Rugosity) (p ≤ 0.046). In conclusion, our findings using different evaluation methods indicate that pronounced sperm morphological variation exists among these four neotropical primate species. Because of the strong relationship observed among morphological and morphometric parameters, these results suggest that application of objective analysis methods could substantially improve the reliability of comparative studies and help to establish valid normative sperm values for neotropical primates.

Laparoscopic oviductal embryo transfer and artificial insemination in felids--challenges, strategies and successes.

Embryo transfer (ET) and artificial insemination (AI) are potentially invaluable techniques for the propagation and management of genetically valuable domestic cat and endangered nondomestic cat populations. Many of the challenges that impair the effective application of ET and AI in felids may be overcome by using laparoscopic oviductal (LO) approaches. LO-ET and LO-AI are minimally-invasive procedures, requiring only two small skin incisions for insertion of a laparoscope and grasping forceps into the abdominal cavity to permit visualization and catheterization of the oviduct for embryo or semen deposition. With concurrent improvements in embryo culture systems and ovarian synchronization protocols, LO-ET has proven effective over the past decade for propagation of laboratory cats, cat models of hereditary disease and nondomestic cats. To date, viable offspring have been produced following LO-ET of non-frozen and frozen-thawed IVF-derived embryos in eight cat hereditary disease models and two nondomestic cat species, the ocelot and sand cat. LO-AI with low sperm numbers (c. 2-8 million motile) has shown similar efficacy to LO-ET, resulting in high pregnancy percentages (50-70%) following insemination of gonadotropin-treated domestic cats. Multiple kittens also have been produced in two hereditary disease models following LO-AI with frozen semen, and both ocelot and Pallas' cat kittens have been born after LO-AI with freshly-collected semen. The application of LO-ET and LO-AI to felids has resulted in substantial improvement in the efficiency of assisted reproduction for genetic management of these invaluable domestic cat and wild cat populations.

Comparative fertility of freshly collected vs frozen-thawed semen with laparoscopic oviductal artificial insemination in domestic cats.

Artificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO-AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO-AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO-AI in domestic and nondomestic cats.

In vitro fertilization and sperm cryopreservation in the black-footed cat (Felis nigripes) and sand cat (Felis margarita).

Studies of in vitro fertilization (IVF) and sperm cryopreservation have been conducted in several small cat species, but virtually no data exist for black-footed cats (Felis nigripes) (BFCs) or sand cats (Felis margarita) (SCs). The objectives of this study were 1) to compare in vitro motility and acrosome status of fresh and cryopreserved (frozen in pellets on dry ice or in straws in liquid nitrogen vapor) BFC and SC spermatozoa cultured in feline-optimized culture medium (FOCM) or Ham F-10, 2) to assess ovarian responsiveness in BFCs and SCs following exogenous gonadotropin treatment and laparoscopic oocyte recovery, and 3) to evaluate the fertility of fresh and frozen-thawed spermatozoa from both species using homologous and heterologous (domestic cat oocytes) IVF in the two culture media. Motility and acrosomal integrity of fresh and frozen-thawed spermatozoa from BFCs and SCs were similar (P > 0.05) in both media during 6 h of culture. Although effects were more pronounced in SCs, cryopreservation in straws was superior (P < 0.05) to cryopreservation in pellets for both species. Gonadotropin stimulation produced approximately 16 ovarian follicles per female, and >80% of recovered oocytes were of optimal (grade 1) quality. The BFC and SC spermatozoa fertilized 60.0%-79.4% of homologous and 37.7%-42.7% of heterologous oocytes in both culture media, with increased (P < 0.05) cleavage of homologous (SC) and heterologous (BFC and SC) oocytes in FOCM. These results provide the first information to date on the gamete biology of two imperiled cat species and further our capacity to apply reproductive technologies for their conservation.

Fecal endocrine profiles and ejaculate traits in black-footed cats (Felis nigripes) and sand cats (Felis margarita).

Information regarding the reproductive biology of black-footed cats (BFC) and sand cats (SC) is extremely limited. Our objectives were to: (1) validate fecal hormone analysis (estrogens, E; progestagens, P; androgens, T) for noninvasive monitoring of gonadal activity; (2) characterize estrous cyclicity, ovulatory mechanisms, gestation, and seasonality; and (3) evaluate male reproductive activity via fecal androgen metabolites and ejaculate traits. In both species, the estrous cycle averaged 11-12 days. In BFC (n=8), estrus lasted 2.2+/-0.2 days with peak concentrations of E (2962.8+/-166.3 ng/g feces) increasing 2.7-fold above basal concentrations. In SC (n=6), peak concentrations of E (1669.9+/-83.5 ng/g feces) during estrus (2.9+/-0.2 days) were 4.0-fold higher than basal concentrations. Nonpregnant luteal phases occurred in 26.5% (26 of 98) of BFC estrous cycles, but were not observed in SC (0 of 109 cycles). In both species, P concentrations during pregnancy were elevated (32.3+/-3.0 microg/g feces BFC; 8.5+/-0.7 microg/g feces SC) approximately 10-fold above basal concentrations. Fecal T concentrations in males averaged 3.1+/-0.1 microg/g feces in BFC and 2.3+/-0.0 microg/g feces in SC. Following electroejaculation, 200 to 250 microl of semen was collected containing 29.9 (BFC) to 36.5 (SC)x10(6) spermatozoa with 40.4 (SC) to 46.8 (BFC)% normal morphology. All females exhibited estrous cycles during the study and spermatozoa were recovered from all males on every collection attempt, suggesting poor reproductive success in these species may not be due to physiological infertility.

Assisted reproduction mediated resurrection of a feline model for Chediak-Higashi syndrome caused by a large duplication in LYST.

Chediak-Higashi Syndrome (CHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). The feline model for CHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile,  CHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. This study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat.

Effect of housing and environmental enrichment on adrenocortical activity, behavior and reproductive cyclicity in the female tigrina (Leopardus tigrinus) and margay (Leopardus wiedii).

The objective of this study was to evaluate the effects of different captive housing conditions on reproductive cyclicity and adrenocortical activity in adult females of two small-sized felid species, the tigrina (Leopardus tigrinus; n = 3) and margay (Leopardus wiedii; n = 2). Females were housed as singletons and subjected to three enclosure conditions over successive time periods: Phase I-large, enriched enclosures for 3 months; Phase II-small, empty enclosures for 5.5 months; Phase III-the same small enclosures enriched with branches and nest boxes for 6.5 months. Fecal samples were collected five times weekly throughout the study for analysis of progestagen, estrogen, and corticoid metabolites. On the basis of observed behaviors, stereotypic pacing was more frequent before feeding for all cats, regardless of enclosure conditions. Both species displayed a bimodal activity pattern, with peaks occurring at nightfall and dawn. All animals exhibited agitated behavior, characterized by a high frequency and duration of stereotypic pacing, primarily during the first 3 days after moving to the small empty enclosures. On the basis of hormonal analyses, ovarian follicular activity decreased and corticoid concentrations increased in tigrinas after transfer to the small barren cages compared to the patterns observed in the initial large, enriched enclosures. Corticoid concentrations in tigrinas then declined after small cage enrichment. Margay females exhibited increased corticoid excretion during Phases II and III, but in contrast to tigrinas, concentrations remained high even after cage enrichment. It was further showed that enriching the small enclosures was insufficient to reestablish normal ovarian activity within the time frame of the study for both species. In summary, margay and tigrina females exhibited distinct elevations in corticoid concentrations after transfer from large enriched enclosures to smaller barren cages that corresponded with agitated behavior, especially immediately after transfer. Fecal corticoid concentrations were reduced after cage enrichment in tigrinas, but not in margays. Although only a few individuals were evaluated, data suggest there may be species differences in response to captive environmental conditions. Overall results emphasize the importance of enclosure dimensions and enrichment when designing species appropriate environments for improving the health and reproductive fitness of threatened species. Zool Biol 26:441-460, 2007. (c) 2007 Wiley-Liss, Inc.

Snow leopard (Panthera uncia) spermatozoa are sensitive to alkaline pH, but motility in vitro is not influenced by protein or energy supplements.

To better understand the biology of snow leopard spermatozoa and to facilitate developing assisted reproduction, a series of studies was conducted to: 1) identify the component(s) of complex culture media responsible for the detrimental effect on sperm survival in vitro, 2) optimize medium for supporting sperm viability, and 3) evaluate sperm capacitation in vitro. Constituents of complex media were added systematically to phosphate-buffered saline (PBS) to isolate the factor(s) influencing snow leopard sperm motility in vitro. Sperm capacitation was also assessed following incubation in PBS with bovine serum albumin (BSA), fetal calf serum (FCS), or heparin. For maintaining sperm motility, there was no benefit (P > or = 0.05) to supplementing PBS with low (5%) or high (20%) concentrations of snow leopard serum (SLS) versus FCS or BSA. Likewise, adding supplemental energy substrates (pyruvate, glucose, lactate, or glutamine) did not enhance or hinder (P > or = 0.05) sperm motility. However, motility rapidly decreased (P < 0.05) with the addition of NaHCO3 to PBS or Ham's F10 nutrient mixture. Surprisingly, Ham's F10 with no buffering component or with both NaHCO3 and N-Z-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) maintained sperm motility at levels similar (P > or = 0.05) to PBS. Although sperm motility in all treatments decreased with time, there was a strong inverse relationship (P < 0.01; r = 0.90) between motility and sample pH at 6 hours. Spermatozoa incubated in PBS containing FCS, BSA, or heparin did not undergo the acrosome reaction when exposed to calcium ionophore. In summary, alkaline pH has a profound detrimental effect on snow leopard sperm motility, and capacitation does not occur under conditions that normally promote this event in other felid species. These results clearly demonstrate a high degree of interspecific variation among felids in fundamental sperm function, and they provide evidence for the necessity of basic research when developing assisted reproduction in little-studied nondomestic species.

International training programs in reproductive sciences for conservation of Latin American felids.

Survival of the ten non-domestic felid species endemic to Latin America is imperiled by habitat loss, poaching and poor captive management. Over the past 10 years, conservation of these felids has been the primary focus of a reproductive research and training program conducted in Brazil, Mexico, and the USA. The objectives of this program were to: (1) provide intensive training in reproductive sciences to Latin American scientists, (2) conduct collaborative studies investigating basic and applied reproduction in endangered felids, and (3) establish a highly-trained scientific cohort to conduct independent conservation-based research. Four formal training courses, consisting of didactic lectures and hands-on instruction in research techniques, including semen collection, sperm cryopreservation and laparoscopic artificial insemination (AI), were taught in Brazil and Mexico between 1995 and 1998. Several of these scientists received further training in conducting fecal hormone analysis in the USA, and a number of research studies, many in collaboration with American scientists, were initiated in Latin American felids. Research findings have characterized basal reproductive traits in several felid species, including ocelots, margay, tigrinas and jaguars, and established that Latin American felids exhibit only minimal seasonal variation in most reproductive traits. Other studies have explored the impact of acute and chronic stressors on adrenocortical activity and demonstrated the importance of environmental enrichment in captivity, especially in small felids. Additional research has examined ovarian and immunological responsiveness of Latin American felids to exogenous gonadotropins and assessed the impact of nutrition on sperm production and oocyte quality. Applied reproductive studies have investigated sperm cryopreservation in both captive and wild felid populations and demonstrated the production of viable offspring in ocelots and tigrinas following laparoscopic AI. Ongoing studies are investigating the potential of in vitro fertilization (IVF), embryo cryopreservation and embryo transfer for genetic management of ocelots and tigrinas. To date, over 75 Brazilian ocelot and 50 tigrina IVF embryos have been cryopreserved and two pregnancies have been established in ocelots following transfer of frozen-thawed embryos. Findings from these studies are helping to improve husbandry, population management, and breeding of Latin American felids in captivity. Continued advances in assisted reproduction eventually may provide an alternative route for exchanging genetic material among Latin American felid populations. Most importantly, this collaborative program has been essential for building scientific capacity, within Brazil and Mexico, in establishing a core group of highly-trained reproductive biologists that will continue applying their new knowledge and skills to the conservation of Latin American felids.

Gonadotropin exposure, salt storage and storage duration affect penetration of domestic cat oocytes by homologous spermatozoa.

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2-3 weeks) immature oocytes, or salt-stored (2-3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2-3 weeks, 2-3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2-3 weeks of storage than after 2-3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2-3 weeks or 2-3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.

Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport.

Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 +/- 0.4 d with estrus lasting 6.3 +/- 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r = -0.91 and r = -0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.

Using hormone-treated pregnant cows as a potential source of oocytes for in vitro fertilization.

In vivo collection of oocytes during pregnancy may be alternative method of obtaining gametes for in vitro fertilization (IVF) from genetically superior gestating cattle. The objectives of this experiment were to induce follicular growth in mature beef cows during each trimester of pregnancy, and then to collect oocytes and verify oocyte competency by IVF and subsequent embryo culture in vitro. Cyclic beef cows in Treatment A and pregnant cows in Treatment B were administered a total dose of 40 mg of FSH in descending dose levels (6, 5, 4, 3 and 2 mg) twice daily for 5 consecutive days. Cows in Treatment A were administered 25 mg of PGF(2)alpha and in Treatment B an equal volume of 0.9% saline at the seventh FSH injection. Pregnant cows in Treatment C were administered neither FSH nor PGF(2)alpha and served as a control group. Following a gonadotropin treatment, the ovaries of each female were evaluated for follicular development by ultrasonography. Oocytes were collected by follicle aspiration from cows in the first trimester. Following IVF procedures, the embryos were co-cultured on caprine oviductal cells, or in the chicken embryo co-culture system, or were placed in goat oviducts in vivo. The mean number of follicles per ovary 12 hours after FSH treatment was not different for cows in Treatments A and B, (8.1 vs 7.7) and both numbers were greater (P<0.05) than the 1.1 follicles per ovary for the control cows in Treatment C. Oocytes collected in vivo and exposed to IVF, resulted in 20% cleaving, and of these embryos 50% developed to the morula stage in culture. In summary, stimulating supplemental follicular development with FSH treatment during pregnancy and collecting the oocytes for IVF may be an alternative method for obtaining supplemental gametes from valuable donor cattle.

Influence of oral melatonin on natural and gonadotropin-induced ovarian function in the domestic cat.

Ovarian hyperstimulation after exogenous gonadotropin stimulation is believed to be a cause of poor success after artificial insemination (AI) in felids. The objectives of this study were to assess the effect of oral melatonin on endogenous ovarian activity in the domestic cat and subsequent eCG/hCG-induced ovarian activity. Serum melatonin concentrations peaked approximately 1h after a single oral dose of 30 mg melatonin and remained elevated above endogenous day-time concentrations for >8h. The calculated circulating half-life (mean +/- S.E.M) of oral melatonin was 45.4+/-3.5 min, and the elimination rate constant (k(10)) was 55.2+/-4.2 min(-1). Oral melatonin (30 mg per day) administered 3h before lights-off effectively and reversibly suppressed estrous elevations in fecal estrogens after 25 days of treatment. There was a progressive decrease in baseline estrogen concentrations from inter-estrous concentrations after 25 days of treatment to below inter-estrous concentrations after 35 days of treatment. Oral melatonin treatment (30 mg per day for 30 days) prior to eCG/hCG administration only marginally reduced ancillary follicle development and had no significant effect on the quantity or quality of embryos produced by AI. Thus, oral melatonin effectively inhibited endogenous ovarian activity and had no adverse impact on embryo quality after AI in the domestic cat; however, this treatment was only marginally effective in minimizing eCG/hCG-induced ovarian hyperstimulation.

Ancillary follicle and secondary corpora lutea formation following exogenous gonadotropin treatment in the domestic cat and effect of passive transfer of gonadotropin-neutralizing antisera.

A combination regimen of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) was used to stimulate ovarian follicular development in domestic cats. The rate of elimination of eCG from circulation was estimated, and, following follicular aspiration, the formation of ancillary follicles and secondary CL was characterized. The effect of gonadotropin-neutralizing antisera on the development of secondary ovarian structures, CL function and humoral immune responses also was evaluated. After intramuscular injection, initial serum eCG concentrations were variable, with the elimination half-life estimated at 39 to 55 h and eCG persisting in circulation for several days. Following follicular aspiration, queens formed CL equal to the number of aspirated follicles and exhibited a rapid increase in progesterone concentration but developed high numbers of ancillary follicles by 5 d post aspiration. By 15 d post aspiration, all ancillary follicles had luteinized to form secondary CL. Treatment with neutralizing antisera at the time of follicular aspiration slowed (P < 0.05) CL formation but did not decrease (P > 0.05) the number of ancillary follicles or secondary CL. Progesterone concentrations did not differ (P > 0.05) from control queens while secondary humoral immune responses to eCG were qualitatively similar between groups. In summary, eCG was eliminated slowly from cats following intramuscular injection and this persistence in circulation may have contributed to the development of ancillary follicles and secondary CL. However, the administration of neutralizing antisera at the time of follicular aspiration was ineffective in preventing the formation of these secondary ovarian structures.

Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus).

Captive adult male ocelots (Leopardus pardalis, n = 3), margays (L. wiedii, n = 3) and tigrinas (L. tigrinus, n = 4) in two locations in southern Brazil were studied for 14 consecutive months to evaluate the effect of season on testicular function. Reproductive evaluations, including testicular measurements, electroejaculation and blood collection were conducted monthly. Fecal samples were collected weekly for androgen metabolite analysis to assess testicular steroidogenic activity. Ocelots had the highest number of motile spermatozoa in the ejaculate (114.7+/-15.8 x 10(6); P < 0.05), the highest percentage of morphologically normal spermatozoa (82.4+/-1.2%; P < 0.05) and the highest concentration of fecal androgens (1.71 vs. 0.14 microg/g; P < 0.05). Margays and tigrinas had lower numbers of motile spermatozoa (23.4+/-2.8 x 10(6), 74.2+/-8.9 x 10(6), respectively), lower percentages of morphologically normal spermatozoa (57.4+/-2.8, 59.2+/-3.5%, respectively), and lower fecal androgen concentrations (0.15+/-0.01, 0.23+/-0.01 microg/g, respectively). Serum testosterone concentrations were similar among the three species. Fecal androgen concentrations were not affected by season, with the exception of the ocelot where concentrations were higher (P < 0.05) in the summer. Ejaculates were collected throughout the year; however, peaks in average sperm production were observed during the summer for all species. In summary, this study has identified several species differences in male testicular traits among ocelots, margays and tigrinas. Results of longitudinal reproductive assessments suggest males of each species are capable of breeding throughout the year.

Kinetics of the humoral immune response to multiple treatments with exogenous gonadotropins and relation to ovarian responsiveness in domestic cats.

OBJECTIVE: To investigate ovarian responses and kinetics of gonadotropin-binding immunoglobulin production in domestic cats repeatedly treated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) at short or long treatment intervals. DESIGN: Queens were treated 3 or 4 times with a standard eCG/hCG regimen at short (49 to 57 days) or long (130 to 135 days) intervals and subjected to laparoscopy after each treatment to evaluate ovarian follicular development. Serial serum samples were assessed by ELISA for the presence of eCG-binding immunoglobulins. ANIMALS: 11 clinically normal sexually mature female cats. RESULTS: Queens repeatedly stimulated with eCG/hCG at long intervals typically had no decrease (P > 0.05) in ovarian follicle production or in maturity of recovered oocytes, whereas queens treated at short intervals had reduced (P < 0.05) follicular development and compromised oocyte maturity by the third stimulation. For both interval groups, ELISA data indicated individual variability in seroconversion after eCG/hCG challenge exposure. In general, queens treated at short intervals had higher peak anti-eCG immunoglobulin titer than did queens treated at long intervals; high titer at the time of eCG/hCG injection, or rapid increases in titer immediately after injection were predictive (P < 0.05) of poor ovarian responses. CONCLUSIONS: Results suggest that individual variability in immune responses and intervals between repeated gonadotropin treatments determine whether queens develop immunologically mediated ovarian refractoriness to exogenous gonadotropins. Intervals of at least 4 months between successive eCG/hCG treatments are recommended for assisted reproductive procedures in domestic and nondomestic cats.

Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus.

Reproductive endocrine patterns were characterized in female ocelots (Leopardus pardalis; n = 3), tigrinas (Leopardus tigrinus; n = 2), and margays (Leopardus wiedii; n = 2) housed in captivity in southern Brazil. Females were maintained as singletons and exposed to natural fluctuations in photoperiod. Cyclic changes in ovarian steroids were monitored by analyzing estrogen and progestogen metabolites in fecal samples collected five times weekly for 14 to 18 months. Based on intervals between fecal estrogen peaks, mean (+/- SEM) duration of the estrous cycle was 18.4 +/- 1.6 days for the ocelots (range, 7-31 days; n = 75 cycles), 16.7 +/- 1.3 days for the tigrinas (range, 11-27 days; n = 23 cycles), and 17.6 +/- 1.5 days for the margays (range, 11-25 days; n = 32 cycles). Fecal progestogen analyses combined with two laparoscopic observations of the ovaries confirmed that ocelots and tigrinas did not ovulate spontaneously. In contrast, non-mating-induced luteal phases of 40.1 +/- 6.3 days in duration (range, 30-60 days) were observed frequently in both margays. There was no evidence of gonadal seasonality in margays in either follicular or luteal activity. In ocelots, cyclic changes in estrogen excretion were observed during each month of the year; however, only one female cycled continuously. In the other two ocelots, periods of acyclicity of several months' duration were observed. It was not possible to conclude whether tigrinas were aseasonal because estrous cyclicity was observed in only one of two individuals. In the female that cycled, a 3-month period of acyclicity was observed in the late fall/early winter. These data demonstrate similarities among three felid species of the genus Leopardus, including evidence they are polyestrous but experience unexplained periods of ovarian inactivity. Only the margays differed by exhibiting occasional spontaneous, non-mating-induced ovulations. Historically, these species have not bred well in captivity. However, it is hoped that understanding the biological similarities and differences among them could lead to improved management strategies that may one day result in increased reproductive success. Zoo Biol 20:103-116, 2001. Copyright 2001 Wiley-Liss, Inc.