Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Cross-species efficacy of a chemically-defined, soy lecithin-based cryomedium for semen banking in imperiled wild felids.

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium. However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. In the present study, our aim was to compare a chemically-defined, soy-based medium (SOY) to a commercial egg yolk-based medium (TEY) for the cryopreservation of sperm in four imperiled small cat species. Semen was collected from adult male cats (n = 6 black-footed cats; n = 6 sand cats; n = 4 fishing cats; and n = 7 Pallas' cats) via electroejaculation, split into two aliquots, and cryopreserved in SOY or TEY. Frozen-thawed samples were evaluated for sperm motility and rate of progressive motility (up to 24 h post-thaw) and acrosome status (0 and 6 h). No difference in post-thaw traits were observed between treatments in all four species. Heterologous IVF using oocytes collected laparoscopically from domestic cats demonstrated no difference among freezing treatments in percentage of mature oocytes that cleaved or the mean number of blastomeres at 48 h post-insemination. More spermatozoa frozen with SOY were bound to the zona pellucida in the sand cat (P = 0.018), but no treatment effect was observed in the other three species. These findings collectively demonstrate that SOY may be a preferable alternative to TEY for sperm cryopreservation in these four wild felid species.

Characterization of the behavior and reproductive endocrinology of giant river otters (Pteronura brasiliensis) in managed care.

Propagation of giant river otters (GRO) in zoos is inconsistent: some pairs never reproduce while others are prolific in producing young but can be hindered by low cub survival. Developing effective breeding programs requires understanding normal reproductive parameters and behavior. Fecal samples were collected for 6-16 months from five breeding pairs, two individual females, and one female pair at seven zoos, and analyzed for fecal progesterone, estrogen, testosterone, and glucocorticoid (FGM) metabolites via enzyme immunoassay. Enclosure characteristics and management routines were recorded at six facilities where behavior was assessed over 1 week. Median fecal progestogens during pregnancy and pseudopregnancy were ∼2.5-3.8× greater than basal concentrations. Gestation lasted 66.5 ± 3.5 days (62-70 days); pseudopregnancies lasted 58 ± 11.6 days (41-69 days). Elevated progestogens indicate ovulation but cannot distinguish pregnancy from pseudopregnancy. Periodically sustained, elevated progestogens observed in two females housed without a male indicated spontaneous ovulation. Elevations in fecal estrogens were not associated with estrus, and seasonality in male testosterone was not observed. Wavering scream and contact call vocalizations among reproductively successful males and females, respectively, suggested the importance of social communication. Most facilities housing successful pairs had larger enclosures with more water than land area, vegetation, and limited public exposure. Baseline FGM were negatively correlated with enclosure size and percentage of water area (p < 0.05), and lower baseline FGM were associated with reproductive success (p < 0.05). These results suggest that housing GRO in spacious enclosures with open water and some insulation from disturbance might promote appropriate behavior, lower FGM, and reproduction.

Interaction of extender composition and freezing method for effective semen cryopreservation in the North American river otter (Lontra canadensis).

Semen cryopreservation and storage in genome resource banks (GRBs), in combination with artificial insemination (AI), could be invaluable for genetic management and conservation of endangered otter species. For any applied conservation benefit, effective methods for otter sperm processing and cryopreservation first must be established. In this study, our objective was to develop an effective semen cryopreservation method for the North American river otter, evaluating the effect of extender composition (i.e., glycerol concentration, Equex STM paste supplementation) and freezing protocol (timing of glycerol addition, pre-freeze cooling rate, freezing/packaging method) on post-thaw sperm motility, longevity and acrosome status. Semen was collected from 14 otters housed at 9 zoos, and following cryopreservation in an egg-yolk based extender, thawed to assess sperm motility and acrosome status immediately post-thaw and during 6 h of in vitro culture. Results indicated that extender containing 4% glycerol was preferable (p < 0.05) to 8% glycerol but the temperature/timing of extender addition containing 4% glycerol did not affect (p > 0.05) post-thaw sperm parameters. Treatments with extender containing Equex and frozen by pelleting on dry ice showed greater (p < 0.05) motility and percentage of intact acrosomes compared to treatments frozen in extender without Equex, regardless of pre-freeze cooling rate. In the absence of Equex, pelleting provided superior post-thaw sperm motility (p < 0.01) and higher (p < 0.001) percentage of sperm with intact acrosomes compared to samples frozen in straws over liquid nitrogen vapor. Results of this study indicate that cryopreservation of otter sperm using an egg-yolk -TEST based extender containing 4% glycerol and 1% Equex, with the pellet freezing method, provided superior post-thaw sperm motility, longevity and acrosomal integrity compared to other combinations. Neither alterations in timing of glycerolated extender addition nor pre-freeze cooling rate had a discernable effect on post-thaw otter sperm parameters. These findings represent the first assessment of semen cryopreservation in any otter species and may be of value as a model for development of semen cryopreservation strategies in other endangered otter species.

Laparoscopic oviductal artificial insemination improves pregnancy success in exogenous gonadotropin-treated domestic cats as a model for endangered felids.

Artificial insemination (AI) in cats traditionally uses equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) to induce follicular development and ovulation, with subsequent bilateral laparoscopic intrauterine insemination. However, long-acting hCG generates undesirable secondary ovulations in cats. Uterine AI also requires relatively high numbers of spermatozoa for fertilization (~8 × 10(6) sperm), and unfortunately, sperm recovery from felids is frequently poor. Using short-acting porcine luteinizing hormone (pLH) instead of hCG, and using the oviduct as the site of sperm deposition, could improve fertilization success while requiring fewer spermatozoa. Our objectives were to compare pregnancy and fertilization success between 1) uterine and oviductal inseminations and 2) eCG/hCG and eCG/pLH regimens in domestic cats. Sixteen females received either eCG (100 IU)/hCG (75 IU) or eCG (100 IU)/pLH (1000 IU). All females ovulated and were inseminated in one uterine horn and the contralateral oviduct using fresh semen (1 × 10(6) motile sperm/site) from a different male for each site. Pregnant females (11/16; 69%) were spayed approximately 20 days post-AI, and fetal paternity was genetically determined. The number of corpora lutea (CL) at AI was similar between hormone regimens, but hCG increased the number of CL at 20 days post-AI. Numbers of pregnancies and normal fetuses were similar between regimens. Implantation abnormalities were observed in the hCG group only. Finally, oviductal AI produced more fetuses than uterine AI. In summary, laparoscopic oviductal AI with low sperm numbers in eCG/hCG- or eCG/pLH-treated females resulted in high pregnancy and fertilization percentages in domestic cats. Our subsequent successes with oviductal AI in eCG/pLH-treated nondomestic felids to produce healthy offspring supports cross-species applicability.

Attempted in vitro maturation and fertilization of postmortem Sumatran rhinoceros (Dicerorhinus sumatrensis) oocytes.

A study was conducted opportunistically to evaluate the potential of rescuing immature oocytes from the ovaries of the Sumatran rhinoceros postmortem. Recovered oocytes (n = 30) were placed in maturation culture for 36 hr and inseminated with frozen-thawed homologous spermatozoa. After culture, evaluation of nuclear maturation status revealed that a large number of oocytes were degenerated (n = 21), but nine oocytes were assessed at the germinal vesicle (n = 3), metaphase I (n = 3), and metaphase II (n = 3) stages. Frozen-thawed Sumatran rhinoceros spermatozoa were capable of binding to the zona pellucida of in vitro matured oocytes, but no fertilization or cleavage resulted. In conclusion, relatively large numbers of oocytes can be obtained by ovarian follicular aspiration postmortem in the Sumatran rhinoceros, and some of these oocytes are capable of achieving nuclear maturation in vitro. However, additional studies are required to improve maturation success and achieve fertilization in culture.

Characterization of basal seminal traits and reproductive endocrine profiles in North American river otters and Asian small-clawed otters.

In this study, fecal samples were collected from 24 North American river (NARO) and 17 Asian small-clawed otters (ASCO) for 6-36 months and semen collected seasonally from NARO males (n=4/season) via electroejaculation. Our main objectives were to: (1) characterize endocrine parameters by longitudinal monitoring of fecal hormone metabolites and (2) investigate semen collection and basal seminal traits in NARO. NARO demonstrated a distinct seasonality in the spring, with females having a monoestrual estrogen elevation lasting 15.33+/-1.98 (mean+/-SEM) days and males peaking in testosterone production for 25.50+/-7.51 days. Pregnancy was characterized by 7-9 months of basal fecal progesterone, presumably corresponding to embryonic diapause, followed by a rapid increase over the final 68-73 days to term. Pseudopregnancy exhibited a similar late winter progesterone peak of 68-72 days, which could not be differentiated from pregnancy. Geographic latitude possibly influenced the timing of increased testosterone in males and increased progesterone in pregnant/pseudopregnant females. In ASCO, monitoring of fecal estrogens did not allow consistent detection of peak values associated with behavioral estrus. Both pregnancy and pseudopregnancy were characterized by a moderate rise in fecal progesterone for 14-16 days postovulation followed by a marked increase. Total gestation length was 67-77 days compared with 62-84 days for pseudopregnancy. In NARO, optimal sperm recovery and quality occurred only in the spring, corresponding with seasonal increases in testicular volume and fecal testosterone. These findings represent the first comprehensive information on normative endocrine and seminal traits in freshwater otter species.

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis).

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.

Toward a feline-optimized culture medium: impact of ions, carbohydrates, essential amino acids, vitamins, and serum on development and metabolism of in vitro fertilization-derived feline embryos relative to embryos grown in vivo.

The objective of this study was to define the physiologic needs of domestic cat embryos to facilitate development of a feline-specific culture medium. In a series of factorial experiments, in vivo-matured oocytes (n = 2040) from gonadotropin-treated domestic cats were inseminated in vitro to generate embryos (n = 1464) for culture. In the initial study, concentrations of NaCl (100.0 vs. 120.0 mM), KCl (4.0 vs. 8.0 mM), KH(2)PO(4) (0.25 vs. 1.0 mM), and the ratio of CaCl(2) to MgSO(4)-7H(2)O (1.0:2.0 mM vs. 2.0:1.0 mM) in the medium were evaluated during Days 1-6 (Day 0: oocyte recovery and in vitro fertilization [IVF]) of culture. Subsequent experiments assessed the effects of varying concentrations of carbohydrate (glucose, 1.5, 3.0, or 6.0 mM; l-lactate, 3.0, 6.0, or 12.0 mM; and pyruvate, 0.1 or 1.0 mM) and essential amino acids (EAAs; 0, 0.5, or 1.0x) in the medium during Days 1-3 and Days 3-6 of culture. Inclusion of vitamins (0 vs. 1.0x) and fetal calf serum (FCS; 0 vs. 5% [v/v]) in the medium also was evaluated during Days 3-6. Development and metabolism of IVF embryos on Day 3 or Day 6 were compared to age-matched in vivo embryos recovered from naturally mated queens. A feline-optimized culture medium (FOCM) was formulated based on these results (100.0 mM NaCl, 8.0 mM KCl, 1.0 mM KH(2)PO(4), 2.0 mM CaCl(2), 1.0 mM MgSO(4), 1.5 mM glucose, 6.0 mM L-lactate, 0.1 mM pyruvate, and 0x EAAs with 25.0 mM NaHCO(3), 1.0 mM alanyl-glutamine, 0.1 mM taurine, and 1.0x nonessential amino acids) with 0.4% (w/v) BSA from Days 0-3 and 5% FCS from Days 3-6. Using this medium, ~70% of cleaved embryos developed into blastocysts with profiles of carbohydrate metabolism similar to in vivo embryos. Our results suggest that feline embryos have stage-specific responses to carbohydrates and are sensitive to EAAs but are still reliant on one or more unidentified components of FCS for optimal blastocyst development.

Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus.

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.