Assessing pregnancy outcomes in cow-calf operations after administration of modified-live or killed virus vaccinations at the initiation of synchronization for fixed-time AI.

The objective of this study was to compare the reproductive outcomes (artificial insemination [AI] pregnancy rates, season pregnancy rates, AI pregnancy losses) and calf traits (birth and weaning weights) after vaccination of suckled beef cows against bovine herpesvirus 1 and bovine viral diarrhea virus using commercially-available modified-live virus (MLV) or killed virus (KV) vaccine at the initiation of a fixed-time AI program. Previously-vaccinated cows (n = 2138) on 14 farms throughout Virginia were enrolled in the study during the Fall 2017 and Spring 2018 breeding seasons. Animals received a single vaccination injection at 10 d pre-breeding, corresponding with time of CIDR insertion at initiation of the 7-d CO-Synch + CIDR synchronization protocol. Cows were inseminated at a fixed time (60-66 h after removal of the CIDR insert) and subsequently turned out with bulls approximately 1 wk after insemination for a natural service. Cows treated with the MLV vaccine had greater AI pregnancy rates than cows treated with the KV vaccine during the fall (P = 0.008; 54% vs. 46%, respectively), but not during the spring breeding season (P = 0.62; 48 vs. 49%). Season pregnancy rates were greater (P = 0.01) in the fall (95-96%) than in the spring breeding season (89-90%), but were not affected by vaccine treatment (P = 0.49) or treatment by season (P = 0.30) interactions. Percentage of AI pregnancy losses was not affected by season (P = 0.85), vaccine treatment (P = 0.83), or treatment by season interactions (P = 0.68). The number of cycles it took for cows to become pregnant by natural service differed by season (P = 0.006) but not treatment (P = 0.87) or treatment by season interaction (P = 0.997). Cows treated with the MLV vaccine gave birth earlier in the calving season (8.36 ± 0.6 d) than those treated with the KV vaccine (10.31 ± 0.6 d; P = 0.02). There was a main effect of season on birth weights (P = 0.008), weaning weights (P < 0.001), and ADG at weaning (P < 0.001), but no effects of treatment (P ≥ 0.26) or treatment by season interaction (P ≥ 0.10) on any of these parameters. Overall, this study demonstrated that the administration of an MLV vaccine at 10 d before fixed-time AI did not have any adverse effects on pregnancy or calf outcomes compared with KV vaccine administration.

Management of Reproductive Diseases in Male Small Ruminants.

Reproductive tract diseases can be a significant cause of subfertility or infertility in male small ruminants. Affected males can usually be identified early by performing routine serologic screening and yearly breeding soundness examinations. Early diagnosis will either maximize treatment success or expedite culling decisions. Once clinical signs of diseases develop, prognosis for breeding ability declines. Veterinary practitioners should used ancillary diagnostic techniques, such as ultrasound examination and laboratory testing to acquire a diagnosis and determine prognosis of reproductive lesions. Some diseases (eg, brucellosis) should be ruled out immediately to cull affected animals and minimize transmission within the herd.

Effects of Nerve Growth Factor-ß From Bull Seminal Plasma on Steroidogenesis and Angiogenic Markers of the Bovine Pre-ovulatory Follicle Wall Cell Culture.

Nerve growth factor-ß (NGF) is critical for ovulation in the mammalian ovary and is luteotrophic when administered systemically to camelids and cattle. This study aimed to assess the direct effects of purified bovine NGF on steroidogenesis and angiogenic markers in the bovine pre-ovulatory follicle. Holstein heifers (n = 2) were synchronized with a standard protocol, and heifers with a preovulatory follicle (≥ 12 mm) had the ovary containing the dominant follicle removed via colpotomy. Pre-ovulatory follicles were dissected into 24 pieces containing theca and granulosa cells that were randomly allocated into culture media supplemented with either purified bovine NGF (100 ng/mL) or untreated (control) for 72 h. The supernatant media was harvested for quantification of progesterone, testosterone, and estradiol concentrations, whereas explants were subjected to mRNA analyses to assess expression of steroidogenic and angiogenic markers. Treatment of follicle wall pieces with NGF upregulated gene expression of steroidogenic enzyme HDS17B (P = 0.04) and increased testosterone production (P < 0.01). However, NGF treatment did not alter production of progesterone (P = 0.81) or estradiol (P = 0.14). Consistently, gene expression of steroidogenic enzymes responsible for producing these hormones (STAR, CYP11A1, HSD3B, CYP17A1, CYP19A1) were unaffected by NGF treatment (P ≥ 0.31). Treatment with NGF downregulated gene expression of the angiogenic enzyme FGF2 (P = 0.02) but did not alter PGES (P = 0.63), VEGFA (P = 0.44), and ESR1 (P = 0.77). Collectively, these results demonstrate that NGF from seminal plasma may interact directly on the theca and granulosa cells of the bovine pre-ovulatory follicle to stimulate testosterone production, which may be secondary to theca cell proliferation. Additionally, decreased FGF2 expression in NGF-treated follicle wall cells suggests hastened onset of follicle wall cellular remodeling that occurs during early luteal development.

Effects of mid-gestational l-citrulline supplementation to twin-bearing ewes on umbilical blood flow, placental development, and lamb production traits.

The objective of the study was to examine how l-citrulline supplementation to ewes during mid-gestation influences placental activity, placental blood flow, lamb body weight, and carcass characteristics. Two studies were completed. A pharmacokinetic study to compare circulating plasma amino acid concentrations after a single intravenous injection of 155 µmol/kg BW l-citrulline or after an isonitrogenous amount of l-alanine (control; 465 µmol/kg BW). Increases (P < 0.05) in circulating citrulline concentrations were detected for 8 h after l-citrulline injection versus the control. Similarly, increases (P < 0.05) in circulating arginine concentrations were detected for 24 h after l-citrulline treatment. The second study used 12 ewes with twin pregnancies. Daily intravenous injections of either l-citrulline or l-alanine were administered for 39 d from d 42-45 to 81-84 of gestation. Ewes were limit-fed at 85% daily energy requirements during the injection period. A decrease (P < 0.0001) in body weight was observed in both treatment groups during this period. No treatment differences were observed in circulating pregnancy-specific protein B concentrations or placental blood flow during the treatment and post-treatment gestational period. No treatment differences were observed in lamb survival nor in lamb birth, weaning and slaughter weights. Treatment did not influence lamb carcass composition or organ weights. However, there was a tendency (P = 0.10) for an increase in antral follicle numbers in ovaries from ewe lambs derived from ewes treated with l-citrulline. In summary, a daily l-citrulline injection increased both circulating citrulline and arginine concentrations in ewes, but daily l-citrulline injections during mid-gestation did not produce any detectable changes in placental activity and blood flow, neonatal and postnatal lamb development, and lamb carcass composition at slaughter. In conclusion, no benefits in placental function and lamb development were observed after providing l-citrulline during mid-gestation in ewes exposed to a mild energy restriction, but there was an indication that follicle numbers in ewe lambs were positively influenced by l-citrulline treatment during fetal development.

Comparison of reproductive performance of AI- and natural service-sired beef females under commercial management.

The objective of this study was to assess differences in reproductive performance of natural service and artificial insemination (AI) sired beef females based on pregnancy outcomes, age at first calving, and calving interval. Data were sourced from 8,938 cows sired by AI bulls and 3,320 cows sired by natural service bulls between 2010 and 2017. All cows were in a commercial Angus herd with 17 management units located throughout Virginia and represented spring and fall calving seasons. All calves were born to dams managed with estrus synchronization. Pregnancy was analyzed with generalized linear mixed models and other reproductive measures with linear mixed models in R. Six models were evaluated with the dependent variables of pregnancy status at the first diagnosis, pregnancy status at the second diagnosis, pregnancy type (AI or natural service) at the first diagnosis, pregnancy type at the second diagnosis, calving interval, and age at first calving. Independent variables differed by model but included sire type of the female (AI or natural service), prebreeding measures of age, weight, and body condition score, postpartum interval, sex of the calf nursing the cow, and management group. No differences were observed between AI- and natural service-sired females based on pregnancy status at first and second pregnancy diagnosis (P > 0.05). Sire type was only found to be significant for age at first calving (P < 0.05) with AI-sired females being 26.6 ± 1.6 d older at their first calving, which was expected because AI-sired females were born early in the calving season making them older at breeding. Surprisingly, age and body condition score were not significant predictors of pregnancy (P > 0.05). Body weight at breeding was not significant for pregnancy (P > 0.05) but was significant for age at first calving (P < 0.05). These data suggested that lighter heifers calved earlier which contradicts our original hypothesis. Overall, commercial Angus females sired by AI or natural service bulls had similar reproductive performance. Factors that were commonly associated with reproductive success were not significant in this commercial Angus herd managed with estrus synchronization. Given the size of these data, the importance of body condition, age, and weight should be reassessed in modern genetics and management practices.

Insights into nerve growth factor-ß role in bovine reproduction - Review.

Nerve growth factor-ß (NGF), initially recognized as a neurotrophin involved in regulating neuronal survival and differentiation, was also later revealed as a ubiquitous seminal plasma protein in mammals. In South American camelids, NGF was initially named ovulation-inducing factor and a dose-dependent luteotropic effect was also reported in llamas. Although NGF was present in the seminal plasma of bulls, the first studies only indicated a potential role on regulation of sperm physiology. The breakthrough discovery of NGF ability to induce ovulation in camelids led to a series of studies investigating the potential functions of NGF within the female reproductive system. In the bovine, a potential luteotropic effect of NGF was perceived as potential tool to overcome the current issues with early embryonic losses attributed at least in part to luteal insufficiency and failed maternal recognition of pregnancy. The aims of this review are to discuss recent advancements in the understanding of the biological roles of NGF in the bovine species. The insights of recent studies with NGF administered in cattle include enhancement of steroidogenesis, luteal formation, and function through increased release of LH, and downstream effect of increased expression of interferon-stimulated genes. In addition, a positive association with sire conception rates; the determination that is produced in the ampulla and vesicular glands of bulls and that is secreted into the sperm-rich fraction of the ejaculate; and the absence of improved post-thaw sperm motility, viability, acrosome integrity, or chromatin stability in ejaculated or epididymal derived sperm supplemented with purified NGF is also discussed.

Administration of nerve growth factor-ß to heifers with a pre-ovulatory follicle enhanced luteal formation and function and promoted LH release.

The objective of this study was to determine the effects of bovine nerve growth factor-ß (NGF) on pre-ovulatory follicle vascular area, LH release, ovulation, and luteal function when administered systemically to heifers. Post-pubertal Holstein heifers (n = 12) received an intravaginal progesterone-releasing device (CIDR) and GnRH agonist (100 µg IM). The CIDR was removed 5 d later, and heifers were given dinoprost (25 mg IM) at CIDR removal and 24 h later, followed by a second dose of GnRH agonist 48 h later. Heifers were randomly assigned to treatments using a cross-over design. For example, heifers assigned to NGF (250 µg reconstituted in 12 mL PBS IM) in replicate 1 were assigned to control (12 mL PBS IM) in replicate 2. Transrectal ultrasonography was performed before treatment and repeated every 4 h up to 32 h to determine the pre-ovulatory follicle diameter, vascular area, and ovulation. Serum samples were obtained to assess LH concentrations during the periovulatory period and every 2 d post-ovulation for measuring progesterone concentrations. A subset of heifers had luteal biopsies performed on days 9 (n = 6 per treatment) and 14 (n = 6 per treatment) post-ovulation to count luteal cell numbers and measure relative mRNA abundance for steroidogenic and angiogenic enzymes and LH receptor. Treatment with NGF increased pre-ovulatory follicle diameter (P = 0.02) and serum LH concentrations (P = 0.03) but did not affect time to ovulation (P = 0.42). Heifers treated with NGF had increased serum progesterone concentrations in the subsequent luteal phase (P = 0.03), but no change in vascular area of the follicle (P = 0.16) or CL (P = 0.20). Heifers treated with NGF had a greater number of small luteal cells (P < 0.01) and a tendency for increased LH receptor (LHR) mRNA abundance in the CL (P = 0.10). There was also increased steroidogenic acute regulatory protein (STAR; P = 0.05) and a tendency for increased cytochrome P450 family 11 (CYP11A1; P = 0.10) mRNA abundance in the CL of NGF-treated heifers. There was decreased prostaglandin E2 synthase (PGES; P = 0.03) and its receptor (PGER; P = 0.05) mRNA abundance and a tendency for decreased cytochrome P450 family 17 subfamily A member 1 (CYP17A1; P = 0.08) and hydroxysteroid 17-beta dehydrogenase (HSD17B; P = 0.06) mRNA abundance in the CL of NGF-treated heifers. Administration of NGF improved CL function in heifers potentially as a result of increased LH release.

Nerve growth factor-ß effects on post-thaw bull semen quality: Effects of nerve growth factor-ß added to extenders for cryopreservation of electro-ejaculated and epididymal bull semen.

Nerve growth factor-ß (NGF) is a seminal plasma protein associated with improved sperm membrane integrity and motility in mammalian species. The objective of this study was to compare post-thaw semen quality from both ejaculated and pididymal-collected bull sperm incubated with purified NGF prior to cryopreservation. Semen was obtained from Angus × Simmental crossbred bulls (n = 10) collected by electroejaculation, followed by castration and epididymal sperm collections 3 days later. Semen samples were incubated with extender having 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) of purified NGF prior to cryopreservation. Sperm motility was assessed in each sample prior to treatment and cryopreservation and at post-thaw. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Values for post-thaw sperm motility and velocity variables were decreased, while linearity was increased in samples of the HIGH compared with CONT group (P < 0.01), but there were no differences in epididymal samples (P> 0.05). Samples from the HIGH group also had a lesser amplitude of lateral head displacement at 2.5 and 3 h post-thaw (P < 0.01). Post-thaw sperm viability, acrosome integrity, and DNA fragmentation index were not affected by NGF treatment in either ejaculated or epididymal sperm (P> 0.05). In conclusion, supplementation of freezing extender with NGF had minimal effects on post-thaw sperm quality in bulls. Results indicate NGF may have a function in preventing premature sperm hyperactivation in ejaculated, but not epididymal-collected spermatozoa. Fertility studies, both in vitro and in vivo, are warranted to ascertain the relevancy of these findings.

Nerve Growth Factor-ß production in the bull: Gene expression, immunolocalization, seminal plasma constitution, and association with sire conception rates.

Nerve Growth Factor-ß (NGF) is a seminal plasma protein that regulates sperm physiology in bulls, yet its production and association with fertility remain unclear. The objective of this study was to describe NGF distribution in bull accessory sex glands, quantify its seminal plasma concentrations, and determine its association with sire conception rates. Accessory sex glands were collected post-mortem from mature bulls (n = 3). Quantitative PCR and immunohistochemistry were performed in tissue samples. Pre-ejaculate and sperm-rich fraction of seminal plasma from Angus (n = 42) and sperm-rich fraction from Holstein (n = 10) bulls were collected by electroejaculation. Holstein (n = 76) and Jersey (n = 14) bulls were collected by artificial vagina, and those with available sire conception rate scores were allocated to: (1) negative/0 (n = 15); or (2) positive (n = 45). Seminal plasma NGF concentrations were measured using ELISA. Relative abundance of NGF mRNA was greatest in the vesicular, intermediate in ampulla, and lowest in prostate and bulbourethral glands (P ≤ 0.03). The most intense NGF staining was detected in the ampulla and vesicular glands (P < 0.05). Concentrations of NGF were greater in the sperm-rich fraction than in the pre-ejaculate and in samples collected by artificial vagina (P < 0.01). Seminal plasma NGF concentrations were greater in bulls with positive sire conception rate than those with negative/0 (P = 0.05). This study determined that NGF is produced in the ampulla and vesicular glands of bulls and is secreted into the sperm-rich fraction of the ejaculate. The positive association of NGF with bull fertility warrants further investigation.

Variation in post-thaw sperm quality of white-tailed deer bucks (Odocoileus virginianus) during rut.

White-tailed deer farming is a growing industry in the United States, with breeding operations contributing significantly to the industry's economic impact. Artificial insemination with frozen semen allows for selection and dissemination of valuable genetics, yet surprisingly little is known regarding the best time throughout rut to perform semen cryopreservation. The objective of this study was to compare semen quality following cryopreservation of white-tailed deer bucks collected early in the breeding season (September, n = 6), at peak rut (December, n = 8), and late season (March, n = 7). We hypothesized that post-thaw semen quality would be enhanced at peak rut. Mature bucks were anesthetized with tiletamine-zolazepam and xylazine administered intramuscularly via projector. Semen was collected by electroejaculation and cryopreserved using Optixcell extender. Overall and progressive sperm motilities were assessed for each sample before and after cryopreservation using a computer-aided sperm analyzer. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and DNA stability (acridine orange). Analysis of variance was applied to normalized data using a general linear mixed model with buck ID as a random variable, and a Tukey HSD test was performed as needed for post-hoc analysis. Pre-freeze overall and progressive sperm motilities were lowest in March, intermediate in September, and highest in December (p ≤ 0.04). Post-thaw overall and progressive motilities were lowest in September (p ≤ 0.02), but did not differ between December and March (p ≥ 0.12). The DNA Fragmentation Index was lowest in December, intermediate in September, and highest in March (p ≤ 0.05). The percentage of spermatozoa with intact plasma membrane was higher in December than September (p < 0.01), but the percentage of intact acrosomes per sperm with intact plasma membrane was highest in September (p = 0.03). This study confirms that post-thaw semen quality appears to be superior during peak rut (December) in bucks. Though semen collected early or late in rut may present acceptable motility, DNA stability is impaired, which could adversely affect fertility rates. This data suggests that semen cryopreservation during transitional periods should be avoided, though field studies evaluating the translation of these results into satisfactory pregnancy rates are warranted.

Physiological variations in reproductive and metabolic features of white-tailed deer (Odocoileus virginianus) bucks throughout the rutting season.

White-tailed deer farming is a growing industry in the United States, yet there is still a need to improve breeding practices in captive cervids. The objective of this study was to compare reproductive and somatic characteristics of white-tailed bucks early in the breeding season (September), at peak rut (December), and late breeding season (March). We hypothesized that reproductive parameters would improve at the peak of the breeding season. Young, mature bucks (n = 7-11, ages 2-3 yr) were anesthetized via projector with tiletamine-zolazepam and xylazine. Semen was collected by electroejaculation and evaluated for sperm output, motility, and morphology. The bulbourethral, vesicular, and prostate glands were measured by transrectal ultrasound. Body condition score, thoracic circumference, rump fat thickness, scrotal circumference, and testicular measurements were also recorded. Serum testosterone, estradiol, and IGF-I concentrations were measured. Body condition score and rump fat thickness were highest in September, intermediate in December, and lowest in March (p < 0.01), whereas thoracic circumference was lowest in September (p = 0.02). In September, serum IGF-I was highest (p < 0.01) and estradiol was lowest (p ≤ 0.02). Serum testosterone levels were lowest in March and highest in December (p < 0.01). Testicular volume and scrotal circumference were highest in September, intermediate in December, and lowest in March (p < 0.01). Bulbourethral gland length was greatest in March (p ≤ 0.05), whereas prostate length was greatest in September (p ≤ 0.02). Vesicular gland length and width were highest in December, intermediate in September, and lowest in March (p ≤ 0.02). Both overall and progressive sperm motilities were lowest in March (p < 0.01), but did not differ between September and December (p ≥ 0.12). In December, bucks had the highest percentage of normal sperm and lowest percentage of primary sperm defects (p ≤ 0.01). Collectively, our results demonstrated variation in reproductive and metabolic characteristics of white-tailed deer bucks throughout the breeding season at 40°N. The physiological variations described here will be useful for veterinarians and researchers performing reproductive evaluations in white-tailed deer bucks.

Comparative analysis of four commercial on-farm culture methods to identify bacteria associated with clinical mastitis in dairy cattle.

Several multiple-media culture systems have become commercially available for on-farm identification of mastitis-associated pathogens. However, the accuracy of these systems has not been thoroughly and independently validated against microbiological evaluations performed by referral laboratories. Therefore, the purpose of the present study was to evaluate the performance of commercially available culture plates (Accumast, Minnesota Easy System, SSGN and SSGNC Quad plates) to identify pathogens associated with clinical mastitis in dairy cows. Milk samples from the affected quarter with clinical mastitis were aerobically cultured with the on-farm culture systems and by two additional reference laboratories. Agreeing results from both standard laboratories were denoted as the reference standard (RS). Accuracy (Ac), sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV, respectively) and Cohen's kappa coefficient (k) of on-farm plates were determined based on the RS culture of 211 milk samples. All four plate-systems correctly identified ≥ 84.9% of milk samples with no bacterial growth. Accumast had greater values for all overall predictive factors (Ac, Se, Sp, PPV and NPV) and a substantial agreement (k = 0.79) with RS. The inter-rater agreements of Minnesota, SSGN, and SSGNC with RS were moderate (0.45 ≤ k ≤ 0.55). The effectiveness to categorize bacterial colonies at the genus and species was numerically different amongst the commercial plates. Our findings suggest that Accumast was the most accurate on-farm culture system for identification of mastitis-associated pathogens of the four systems included in the analysis.

Nerve Growth Factor-Beta, purified from bull seminal plasma, enhances corpus luteum formation and conceptus development in Bos taurus cows.

The objective of the current study was to determine if Nerve Growth Factor-Beta (NGF), purified from bovine seminal plasma, would improve corpus luteum function and enhance conceptus development when administered to cows at artificial insemination. Angus cows (n = 60) were synchronized using a GnRH-prostaglandin and intravaginal progesterone protocol (7-day CO-Synch/CIDR) and randomly allocated to 1 of 2 treatment groups: (1) CONT- 12 mL PBS; or (2) NGF- 296 µg purified NGF in 12 mL PBS administered intramuscularly at insemination (day 0). Blood collections were performed to measure plasma concentrations of progesterone (days 0, 3, 7, 10, 14, 19) and pregnancy-specific protein B (day 24) using immunoassays. Expression of interferon-stimulated genes (ISG15, MX1, MX2, RTP4) were assessed in peripheral blood leukocytes on day 19. Transrectal ultrasound was performed for measuring corpus luteum size (days 0, 3, 7, 10, 14, 19) and pregnancy diagnosis (days 28, 45, 66). Statistical analysis was performed using analysis of variance with repeated measures (SAS 9.4, Cary NC). Corpus luteum volume and diameter increased over time (p < 0.001), but did not differ between treatment groups (p = 0.46). Cows treated with NGF had increased plasma progesterone over CONT cows from days 10-19 (p = 0.04). Pregnancy rates at day 28 were 75% in NGF cows versus 59% in CONT cows (p = 0.13). In pregnant cows, pregnancy-specific protein B concentrations at day 24 were greater in NGF than CONT cows (p < 0.05). Additionally, fold-change expression of ISG15 and MX2 at day 19 were greater in pregnant NGF cows than in pregnant CONT cows (p < 0.05), but no differences for MX1 and RTP4 were present. Here we demonstrate that NGF administration to cows at insemination improved corpus luteum function, which translated to improved early conceptus development as determined by upregulation of interferon-stimulated genes and increased pregnancy-specific protein B concentrations. These results suggest that seminal plasma NGF could play a role in conceptus development and may be important to improve fertility in cattle.

Hyponatremia as the Presenting Feature of a Pituitary Abscess in a Calf.

A 2-month-old Simmental heifer presented for acute onset of neurological behavior. Laboratory tests confirmed the presence of hyponatremia, hypochloremia, and hypokalemia that improved with intravenous fluid therapy. Despite an initial cessation of neurological signs, symptoms re-emerged, and the heifer was euthanized due to poor prognosis. A pituitary abscess (Trueperella pyogenes) was observed on gross necropsy, suggesting that the effects of panhypopituitarism (inappropriate anti-diuretic hormone (ADH), adrenocorticotropic hormone (ACTH), and/or thyroid-stimulating hormone (TSH) secretion) may have resulted in the clinical findings. Pituitary abscess syndrome carries a poor prognosis due to the inability to penetrate the area with systemic antibiotic therapy. These findings highlight the unusual clinical presentations that may occur following pituitary abscess syndrome in cattle that practitioners need to consider when determining prognosis.

Cryopreservation of white-tailed deer (Odocoileus virginianus) semen using soybean-, liposome-, and egg yolk-based extenders.

The objectives of the present study were to compare the use of soybean-based (Andromed), liposome-based (Optixcell), and egg yolk-based (Ovine Red, Triladyl, and Biladyl) extenders for cryopreservation of white-tailed deer semen. In experiment 1, ejaculates obtained from six bucks were aliquoted into the following extenders: Andromed, Ovine Red, Triladyl, and Biladyl (containing 4%, 6%, or 8% of glycerol). In experiment 2, ejaculates obtained from eight bucks were divided amongst Andromed, Ovine Red, and Optixcell extenders. Total and progressive sperm motility were assessed for each sample before and after cryopreservation using a computer-automated semen analyzer. In experiment 2, flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). In experiment 1, both Andromed and Ovine Red extenders exhibited higher post-thaw total motility than Biladyl containing 4% or 6% of glycerol (p<0.05). Andromed also produced higher progressive motility than all other extenders (p<0.01) before and after cryopreservation with no differences amongst the other extenders (p≥0.11). In experiment 2, there were no differences in total and progressive motility between Andromed, Ovine Red, or Optixcell extenders (p≥0.39). Additionally, there were no differences in sperm viability (p=0.18), acrosome integrity in viable sperm (p≥0.10), or DNA fragmentation index (p=0.15). These results demonstrated that soybean (Andromed) and liposome-based (Optixcell) extenders are equally as effective at cryopreserving white-tailed semen as egg yolk-based Ovine Red extender, but are superior to egg yolk-based Biladyl or Triladyl extenders.

Fecal endocrine monitoring of reproduction in female snow leopards (Uncia uncia).

Although the snow leopard (Uncia uncia) is a common endangered felid species in zoos, little is known about the complex endocrine interactions controlling ovarian function and conception in this species. The goal of this work was to characterize ovarian activity throughout the estrous cycle, nonpregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. This goal was accomplished using an enzyme immunoassay to measure fecal concentrations of estrogen metabolites (E) and progesterone metabolites (P). Fecal samples were collected from 12 female snow leopards (ages 18 months to 18 years) during one to three breeding seasons. In each breeding season, the majority of females (78%, 88%, and 100%, respectively) began to exhibit ovarian activity in December or January. The estrous cycle, defined by the first day of estrus (E ≥ 2 × basal concentration) to the first day of the subsequent estrus, was 12.7 ± 0.6 days (n = 145 cycles). Estrus lasted 4.3 ± 0.4 days with mean concentrations of fecal E during the follicular phase (1661 ± 139 ng/g feces) increasing 3.2-fold above basal concentrations (515 ± 32 ng/g feces). No spontaneous ovulations were observed in any of the cycling females. Nonpregnant luteal phases were observed in eight females that bred but did not become pregnant. The length of the nonpregnant luteal phase ranged from 11 to 72 days (45.7 ± 5.7 days; n = 10) with mean concentrations of fecal P during the luteal phase (12.46 ± 1.7 µg/g feces) increasing 6.2-fold above basal concentrations of P (2.01 ± 0.2 µg/g feces). Three of the females in the study became pregnant and gave birth after a gestation of 93 (n = 2) and 95 (n = 1) days. Fecal P concentrations during pregnancy increased to 11.64 ± 1.3 µg/g feces, or 5.8-fold above basal concentrations. The results of this study provide a comprehensive characterization of reproductive endocrinology in snow leopards, and confirm that fecal hormone monitoring is an effective way to monitor female snow leopards throughout the breeding season.