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.

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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.

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.

Relaxin, a 6-kDa polypeptide hormone, is excreted in the urine during pregnancy in several mammalian species. A recent study showed that detection of urinary relaxin using a bench-top serum assay (Witness relaxin kit, Synbiotics Corp., San Diego, California 92127, USA) can be diagnostic for pregnancy in domestic cats (Felis silvestris catus), but it is unknown whether the bench-top kit is applicable with urine across felid species. Our objectives were to 1) examine modifications in urine processing to improve kit reliability in pregnant cats, 2) evaluate the impact of concentrating urine via filtration on relaxin detection, 3) assess the effect of sample freezing on relaxin concentrations, and 4) begin quantifying urinary relaxin levels in nondomestic felids. Urine and serum were collected from domestic cats and nondomestic cat species (Pallas' cat, Otocolobus manul; sand cat, Felis margarita; cheetah, Acinonyx jubatus; and lion, Panthera leo) at several times after breeding. Urine samples, subjected to various processing methods, were tested using the bench-top kit, and relaxin levels were later quantified via radioimmunoassay. For domestic cat urine samples, filtration and addition of protein/phosphate buffer improved the consistency of the relaxin kit for early pregnancy diagnosis. Urine freezing caused a slight (approximately 13%) but significant decrease in relaxin concentrations, but frozen-thawed samples still tested positive with the bench-top kit. In nondomestic felids, urinary relaxin immunoreactivity during pregnancy was similar to or higher than that of pregnant domestic cats, suggesting that relaxin is a reliable cross-species marker of pregnancy. Urinary relaxin was detectable using the bench-top kit in pregnant Pallas' cats, but urine samples from other species tested negative, regardless of processing methods. Findings suggest that measurement of urinary relaxin is a promising approach for noninvasive pregnancy diagnosis in exotic felids, but further assessment of urinary relaxin profiles among cat species and modification of the bench-top relaxin kit are warranted to improve cross-species utility.

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.

The superstimulation protocol of Blondin et al. (2002; Biol. Reprod. 66, 38-43) produces cumulus-oocyte complexes (COCs) of high developmental competence for IVP. Using a similar protocol we assessed the affects of alterations in oocyte donor carbohydrate and lipid metabolism during ovarian stimulation on the production and viability of blastocysts in vitro. A 2 × 2 factorial experiment offered two diets: Fiber (F) and Starch (S) alone (0) or with 6% w/w (6) protected lipid (calcium soaps of fatty acids). Thirty-two heifers ranked by body condition score (scale: 1 = thin, 5 = obese) were allocated within score to one of the 4 treatments: F0, F6, S0 and S6. COCs were collected 5 days after estrus by OPU for lipid analysis. Ovarian stimulation (4 doses of FSH (9 mg NIADDK oFSH) given 12 h apart) commenced 2 days later. COCs were collected 40 h after the last FSH injection. GnRH (0.012 mg Buserelin) was administered i.v. 6 h prior to OPU. A second period of ovarian stimulation and OPU then followed. Following IVM/IVF, zygotes were cultured in SOF with 0.3% w/v fatty acid-free BSA under oil (38.8 • C, 5% CO 2 , 5% O 2 , 90% N) until Day 8 of development, when blastocysts were subjected to total cell counts and TUNEL analysis. Data were analyzed by ANOVA. Neither follicles aspirated (25.9 ± 1.87) nor oocytes recovered (12.1 ± 0.92) differed between treatments. Total fatty acids in plasma were greater (P < 0.001) for the F than for the S diets and increased with the inclusion of protected lipid (0.75, 1.82, 0.50 and 1.39 µg mL −1 for F0, F6, S0 and S6, respectively; SED = 0.076). The dietary lipid-induced increase in plasma fatty acids was reflected in an increase (P < 0.05) in total fatty acids within the oocyte (70.4, 74.7, 69.9 and 78.4 ng/oocyte; SED = 3.41). Retrospective analysis by body condition indicated that S diets reduced (P = 0.006) blastocyst yields in thin heifers and reduced (P = 0.02) cleavage rates in fat heifers (Table 1). Blastocyst yields were lower (P = 0.1) for fat heifers on the F0 diet. Total cell numbers