Kefir is a fermented milk traditionally made from a unique starter culture, which consists of numerous bacteria and yeast species bound together in an exopolysaccharide matrix produced by certain lactic acid bacteria. Many health benefits are associated with traditionally produced kefir; however, bulging and leaking packaging, caused by secondary yeast fermentation during storage, has limited large scale manufacture traditionally produced kefir. Commercial kefir products have been designed to reduce these effects by using a pure starter culture consisting of a mixture of bacteria and yeast species that give a flavor similar to traditional kefir, but some health benefits may be lost in commercial production due to reduced microbial diversity and lack of beneficial exopolysaccharides. In this study, traditional and commercial kefir was frozen to study the effects of frozen storage on the viability of probiotic bacteria over time. The traditional kefir was prepared by inoculating 1 L of pasteurized whole goats milk with approximately 30 g of kefir grains. Commercial kefir was prepared by inoculating 1 L of full fat, pasteurized goat milk with a commercial kefir starter. The milk was allowed to ferment at room temperature (24-28°C) until pH 4.6 was reached. Samples were frozen (-8 to -14°C.) immediately following the completion of fermentation and were thawed and plated for lactobacilli, lactococci and yeasts on day 0, day 7, day 14 and day 30 of frozen storage. Statistical analysis was preformed by statistical analysis software (SAS®) using the variance analysis (ANOVA) f-test, with a confidence interval of 95% (P<0.05). Means were compared by the least significant difference (LSD) test. Lactobacilli, lactococci and yeasts were significantly (P<0.05) reduced in number during frozen storage; however, the traditionally produced kefir was shown to have significantly (P<0.05) higher counts of bacteria and yeast at each sampling. It was concluded that frozen storage and the development of frozen kefir products could eliminate most packaging concerns associated with the large scale manufacture of traditionally produced kefir, resulting in increased production and marketability of this healthful product.

Bovine respiratory coronavirus (BRCoV) is a group 2a coronavirus expressing both hemagglutinin-esterase and spike (S) envelope glycoproteins. The S glycoprotein is a primary coronavirus virulence factor responsible for both receptor specificity and membrane fusion-mediated entry into host cells. In addition, the S glycoprotein serves as a major antigen targeted by both the cellular and humoral immune responses and, as such, is an important target for antibody-facilitated virus neutralization. The objective of this research was the design of a safe and effective vaccine against BRCoV using a prime-boost vaccination approach. This method utilized an initial DNA vaccine encoding either the soluble portion of the spike glycoprotein, or the soluble portion of the spike glycoprotein fused in-frame to bovine CD154, administered intramuscularly. The initial priming was followed 14 days later by vaccination with purified immunogenic extracellular portion of S glycoprotein alone or this portion fused in-frame to the soluble portion of the bovine CD40 ligand (CD40L; CD154). The bovine CD40L was included to enhance the immunogenicity of the S glycoprotein and elicit protective immune response against BRCoV infection. Both of the recombinant proteins were expressed in insect Sf9 cells via recombinant baculovirus expression and purified using affinity chromatography. The efficacy of these vaccine approaches in eliciting neutralizing antibody responses, preventing virus replication and spread and the onset of respiratory disease in cattle was then investigated in animal experimental infections. An ELISA was developed and utilized to screen 129 cattle for animals that did not have appreciable antibody titers to BRCoV. In addition, BRCoV-specific serum was obtained from one cow immunized with commercially available vaccine and high-titer anti BRCoV S-specific serum was obtained by immunization of rabbits with the S-CD154-fusion protein. As expected, animals responded to vaccination with the soluble portion of spike. Furthermore, fusion of CD154 to the soluble portion of the spike glycoprotein resulted in a pronounced increase in circulating and neutralizing serum antibody specific for the BRCoV spike glycoprotein.

The objective of this study was to test the association of single nucleotide polymorphisms (SNPs) with fertility in two populations consisting of Holstein cows and multi-generational Angus cows. The candidate gene approach was utilized and previously described SNPs were tested for possible associations with fertility. Single nucleotide polymorphisms on three genes were evaluated including leptin receptor LEPR, calpastatin CAST, and DGAT1. Fertility traits were evaluated in conjunction with production traits for Holstein females and growth traits for Angus females. One SNP was significantly associated with birth weight (P < 0.05) in Angus females while a trend (P < 0.10) was observed for two markers influencing birth weight performance and three markers influencing weaning weight performance. An association of two SNP for birth weight and back fat thickness in Angus females was identified.!A trend (P < 0.10) was observed for one marker within LEPR influencing average services to conception, two markers within CAST influencing average days open, two markers within CAST and one marker within DGAT1 influencing average protein production, and one marker within CAST and one marker within DGAT1 influencing average milk production. One SNP within LEPR was significantly associated with average milk production (P < 0.05) in Holstein females. An association of one SNP within CAST and one SNP within DGAT1 for average protein production and average milk production in Holstein females was identified. An association of one SNP within CAST for average days open and average protein production in Holstein females was also identified. The association of these markers indicates that the evaluated quantitative trait loci (QTL) region may harbor causative mutations responsible for the variation observed in fertility and production traits. Further evaluation of SNP in these regions is necessary in order to identify mutations accounting for the largest degree of variation for fertility and production traits.

Salvaging of epididymal sperm from injured or deceased animals allows for the propagation of favorable traits from endangered or genetically superior males. Techniques developed in domestic species can also serve as the foundation for the collection, cryopreservation and utilization of epididymal sperm in exotic breeds. The need to preserve and utilize epididymal sperm, in the most efficient manner, is of the utmost importance. In a series of experiments, ejaculated and epididymal sperm from the same four mature, fertile, Holstein bulls were collected, cryopreserved, cultured and used for in vitro fertilization. In Experiment I, epididymal sperm was found to have higher post-thaw motility compared to ejaculated sperm. During cryopreservation, the membrane permeability of ejaculated and epididymal sperm was found to be similar. In addition, the membrane permeability of both ejaculated and epididymal sperm was decreased by the inclusion of glycerol during freezing. The optimal cooling rate for ejaculated and epididymal sperm was determined to be between 50 and 60⁰C/min. In Experiment II, we demonstrated that following castration, circulating concentration of plasma cholesterol increased. In Experiment III, the percentage of post-thaw auto-acrosome reacted ejaculated sperm was found to be higher than epididymal sperm. During in vitro culture, the percentage of auto-acrosome reacted ejaculated sperm remained relatively stable compared with epididymal sperm that significantly increased over time. The percentage of capacitation significantly increased over time for ejaculated sperm but not epididymal sperm, which only slightly increased following 6-hours of culture. In Experiment IV, cryopreserved ejaculated Holstein bull sperm was unaffected by the inclusion of PIF to the culture medium. PIF was also unable to improve or inhibit the in vitro fertility of cryopreserved ejaculated bull sperm. In Experiment V, ejaculated sperm with heparin and epididymal sperm with and without heparin were similar in their ability to fertilize oocytes in vitro and in their cleavage rates compared with ejaculated sperm without heparin, which was significantly lower. The number of embryos developing to the 8-cell stage was higher for the epididymal sperm plus heparin group compared with epididymal sperm without heparin. The number of blastocyst was similar for both ejaculated and epididymal sperm when heparin was added to the fertilization medium, compared with when heparin was not included in the fertilization medium. In summary, epididymal sperm was better able to endure cryopreservation. Ejaculated and epididymal sperm also displayed different in vitro dynamics. However, in vitro fertility of ejaculated and epididymal sperm was found to be similar and the inclusion of heparin increases blastocyst development in vitro. This may be useful when using epididymal sperm for in vitro reproductive techniques.

Five experiments were conducted to assess potential improvements in a protocol for inducing ovulation in seasonally anovulatory mares based upon estradiol pretreatment followed by dopamine antagonist injection. The first experiment compared various doses of estradiol cypionate (ECP) and domperidone (in biodegradable microparticles), as well as additions and deletions to the protocol. It was concluded that as little as 75 mg ECP and as little as 1.5 g of domperidone could be used with success, but that both components were required. In the second experiment, timing of the injection (1, 6, or 11 days apart) and ECP dose were assessed. It was concluded that administration of domperidone 1 day after ECP injection provided the best results, and that 50 and 100 mg ECP provided similar results. The third experiment, using geldings as a model for prolactin secretion, compared the prolactin responses to an alternate dopamine antagonist, sulpiride, to those with domperidone, both in biodegradable particles. The magnitude of the prolactin responses were similar for both antagonists, however the sulpiride effect was quicker to occur (1 day) and was shorter-lived (<10 days) than that of domperidone (about 18 days). In the fourth experiment, two doses of ECP (25 and 50 mg) were compared followed immediately (same day) by injection of domperidone or sulpiride in biodegradable microparticles. Both the luteinizing hormone (LH) and prolactin responses to treatment were poor or absent in all but a few mares, and only two mares had early ovulation (one control mare and one mare receiving 25 mg ECP and domperidone). The reason for the poor responses was unknown, but the experiment confirmed the need for responses in LH and prolactin for positive ovarian responses. The last experiment evaluated domperidone versus a new non-particle formulation of sulpiride given at two doses (0.75 versus 1.5 g) on days 1, 6, and 11 relative to ECP (100 mg). Also factored across those treatments was the administration of 50 mg thyroxin in microparticles administered 6 days before ECP injection. High success rates (prolactin and ovulation) were obtained with the higher sulpiride dose. Thyroxin treatment had no effect.

Bovine adipose-derived stem cells (ADS), a form of adult stem cells, are somatic cells that have similar characteristics of embryonic stem (ES) cells. Bovine ADS cells possess multipotent capabilities and have been found to express pluripotency genes associated with ES cells. The unique properties of ADS cells make them a desirable source for reprogramming experiments. The goal of reprogramming experiments is to transform somatic cells from a differentiated state to a pluripotent state. When somatic cells reprogram, there are certain epigenetic changes or modifications that must occur in order to successfully reprogram the nucleus. Epigenetic modifications will change the chromatin configuration without changing the DNA sequence. Somatic cells can be exposed to small molecules that may be able to reduce the chances of having incomplete chromatin modification. Two epigenetic modifying factors are a DNA methyltranferase inhibitor, zebularine (Zeb), and a histone deacetylase inhibitor, valproic acid (VPA). By inducing gene expression with the epigenetic modifiers, the cells may be stimulated to reprogram more efficiently than cells with lower gene expression. In the first experiment, three bovine ADS cell lines were treated with VPA or Zeb to observe the changes in expression levels of Oct4, Sox2, and Nanog (pluripotency-associated genes). The cells were treated for a period of 5, 7,10, or 14 days. VPA led to the highest increase of the pluripotency genes; however, both treatments may have produced a partial reprogramming. This partial reprogramming may result in the bovine ADS cells reaching complete pluripotency when combined with a reprogramming technique. In the second experiment, three bovine ADS cell lines were treated with VPA or Zeb for five days then followed with transduction using lentivirus. Oct4, Sox2, and Nanog were increased the highest when using epigenetic modifiers. Statistical differences for expression of the pluripotency-associated genes were found for cells treated with zebularine. While it was thought that viral transduction in combination with epigenetic modifiers would produce higher expression levels of the pluripotency-associated genes, this was not found to be true in this experiment.

Research has found conflicting results on animal performance and carcass traits associated with the use of forage as the primary feed source for finishing cattle. Consumer interest in forage-fed products has grown and little research has been done comparing performance of forage-fed animals and beef finished on different forages. Spring weaned calves (n=54; 257 ± 2.5 kg; 3/8 Gelbvieh, 3/8 Red Angus, and 1/4 Brahman) were used in the evaluation of three forage systems (S1, S2, and S3) on a 100% forage diet in two consecutive years (June 2009 and 2010). Steers were divided into 9 groups based on initial body weight (d0) and randomly assigned to replicates within system (3 replicates per system). Pastures were rotationally stocked at 1.01 ha/steer. Steers in S1 grazed bermudagrass (45% of area) during summer, and ryegrass (35% of area) and ryegrass sod-seeded into bermudagrass paddocks (20% of area) in winter. Steers in S2 grazed bermudagrass (45% of area) during summer, dallisgrass/clover mix (20% of area) during fall and spring, and ryegrass/cereal rye/clover mix (35% of area) during winter. Those in S3 had access to bermudagrass (20% of area) and sorghum-sudan hybrid/forage soybean during summer (7.5% of area each), dallisgrass/clover mix (20% of area) during fall and spring, and ryegrass/cereal rye/clover mix (45% of area) during winter. Excess forage was cut for hay and fed within system when necessary. Average daily gain (ADG) for summer, winter and for the whole study were not different (P > 0.05) between systems. Animals in Y2 (0.5 kg/d) gained more (P > 0.05) than those in Y1 during summer (0.21 kg/d), but ADG in Y1 (1.5 kg/d) was greater (P > 0.05) than in Y2 (1.3 kg/d) during winter. No significant differences (P > 0.05, Table 3.12) were found in final weights, LM area, KPH, YG, lean color and marbling. Dressing percentage and hot carcass weight were greater (P < 0.05) for S3 than those for S1 and S2 was intermediate. Backfat and PYG was affected by YxS interaction (P < 0.05). Cooking loss and Warner-Bratzler shear force were greater (P < 0.01) for Y1 than for Y2.

There has been thorough documentation to support the role of dopamine in the control of prolactin production and secretion in various mammalian species, including the horse. However, there is evidence that other factors are involved in prolactin secretion. Seven experiments were conducted to assess factors that potentially might affect prolactin secretion in the horse. The first two experiments were conducted (separately) to test whether arginine vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) affected prolactin secretion. In each experiment, AVP or VIP was administered intravenously and blood samples were collected to determine the effect on prolactin secretion. Neither peptide produced any alteration in plasma prolactin concentrations compared to simultaneous saline-injected controls (P > 0.1). Five subsequent experiments were conducted to assess the effects of various drugs on prolactin secretion in response to acute exercise. Pre-exercise treatments included dexamethasone (a glucocorticoid analog, administered 15 h before exercise), naloxone (an opiod antagonist, administered 2 min before exercise), cabergoline (a dopaminergic agonist, administered 15 h before exercise), flunixin meglumine (a prostaglandin inhibitor, administered 15 min before exercise), and sulpiride (a dopamine antagonist that causes the release of prolactin, administered 1.5 h before exercise). In all experiments, exercise induced an immediate increase (P < 0.05) in plasma prolactin concentrations in control horses. Pretreatment with dexamethasone, naloxone, or flunixin meglumine did not alter (P > 0.1) plasma prolactin concentrations relative to saline-treated controls. Pretreatment with cabergoline completely obliterated (P < 0.01) the exercise induced rise in prolactin concentrations. Pretreatment with sulpiride caused an immediate increase (P < 0.001) in prolactin concentrations relative to controls, but resulted in no change in prolactin response to exercise 90 min later relative to controls. It is concluded that the only drug that had a significant effect on prolactin secretion was the dopaminergic agonist cabergoline. Direct administration of AVP or VIP, or perturbations of the adrenal cortical axis, the opioid system, or the prostaglandin system, had no effect on prolactin secretion as has been reported previously for other species.

Eighteen neonatal Holstein bull calves (38.85 ± 4.71 kg) were assigned to one of two treatments at birth to determine the effect of feeding time on growth, nutrient intake, metabolic hormone secretion, and energy metabolism. Regularly fed calves (n = 9) were fed MR daily at 0630 h, and irregularly fed calves (n = 9) at 1030, 0800, 0630, 0830, 0530, 0930, and 0730 Monday through Sunday. Body weights were measured weekly from birth to 9 weeks. Water intake, fecal scores, and starter intake were measured daily. Serial blood collections were conducted at 2, 4, 6, and 8 weeks for ghrelin and GH concentrations. Blood sample collection began one hour prior to regular feeding time (0530 h) and ended one hour post regular feeding time (0730 h), at time points 0, 15, 30, 45, 50, 55, 60, 65, 70, 75, 90, 105, and 120 minutes. An IVGTT was performed at weeks 3, 6, and 9 to assess glucose metabolism. Water intake increased (P < 0.05) in irregularly fed calves at weeks 1 and 3. A treatment by week interaction and a main effect of week were observed for ghrelin concentrations (P < 0.05), and regularly fed calves exhibited increased ghrelin concentrations at week 4 (P < 0.10). Plasma ghrelin concentrations increased with age until weaning at week 6 then decreased at week 8. An increase in GH concentrations were observed at time points t= 75 (P < 0.05), 90 (P < 0.10), and 120 (P < 0.10) min. A treatment by week interaction and a main effect of week were observed for GH concentrations (P < 0.0001). Regularly fed calves had higher GH concentrations at weeks 2 and 4 (P < 0.05). No differences were observed (P > 0.10) for glucose concentrations. Peak insulin concentrations (P < 0.05) and AUC for insulin (P < 0.10) increased as calves aged, indicating that calves become less sensitive to insulin as they develop. Results indicated that feeding time does not have an overall effect on growth, feed intake, and glucose metabolism, but does affect growth hormone concentrations.

Six insulin sensitive and six insensitive mares were used in a replicated 3 x 3 Latin square design to determine the pituitary hormonal responses (compared to vehicle) to sulpiride and thyrotropin-releasing hormone (TRH), two compounds commonly used to diagnose pituitary pars intermedia dysfunction (PPID) in horses. Mares were classified as insulin sensitive or insensitive by their previous glucose responses to direct injection of human recombinant insulin. Treatment days were February 25 and March 10 and 24, 2012. Treatments were sulpiride (racemic mixture, 0.01 mg/kg BW), TRH (0.002 mg/kg BW), and vehicle (saline, 0.01 mL/kg BW) administered intravenously. Blood samples were collected via jugular catheters at -10, 0, 5, 10, 20, 30, 45, 60, 90, 120 min relative to treatment injection. Plasma ACTH concentrations were variable and were not affected by treatment or insulin sensitivity category. Plasma melanocyte stimulating hormone (MSH) concentrations responded (P < 0.01) to both sulpiride and TRH injection, and were greater (P < 0.05) in insulin insensitive mares than in sensitive mares. Plasma prolactin concentrations responded (P < 0.01) to both sulpiride and TRH injection, and the response was greater (P < 0.05) for sulpiride; there was no effect of insulin sensitivity. Plasma thyroid stimulating hormone (TSH) concentrations responded (P < 0.01) to TRH injection only, and were higher (P < 0.05) in insulin sensitive mares in almost all time periods. Plasma LH and FSH concentrations varied with time (P < 0.05), particularly in the first week of the experiment, but were not affected by treatment or insulin sensitivity category. Plasma GH concentrations were affected (P < 0.05) only by day of treatment. The greater MSH responses to sulpiride and TRH in insulin insensitive mares were similar to, but not as exaggerated as, those observed by others for PPID horses. Also, the reduced TSH concentrations in insulin insensitive mares are consistent with the previous observation of elevated plasma triiodothyronine concentrations in hyperleptinemic horses (later shown to be insulin insensitive as well).