A study was conducted to determine the feasibility of concentrating the wastewater of a butter/powder factory for use as an animal feed. Two concentrated products were evaluated: recovered milk product (RMP), which is the direct factory waste stream; and formulated recovered milk product (FRMP), which is a product made by combining RMP and separator de-sludge in a 3:1 ratio. Three pilot scale dryer systems were used to concentrate the product: a spray dryer, a roller dryer, and a pulse combustion dryer. Dried samples were analyzed for fat, moisture, protein, ash, nitrates, chloride, pH, calcium, phosphorus, sodium and amino acids. Values for total digestible nutrients, nitrogen free extract and lactose were calculated. Functional properties were measured by free-fat, insolubility index, wettability, and particle size distribution. Additionally, the microstructure was examined using environmental scanning electron microscopy (ESEM). The objectives to this study were to: 1) further concentrate and dry RMP into a powder to reduce hauling cost, improve consistency, and increase the storage life of the product; 2) add other by-products to the RMP to improve the nutrient profile; and 3) compare three drying systems as to the effect to the nutrient profile, functional properties, and microstructure of the dried product. The protein content in the FRMP was significantly (P<0.05) higher than in the RMP. The nitrate (NO3) concentration in the RMP was significantly (P<0.05) higher than the NO3 concentration in the FRMP. The Mojonnier fat determination test resulted in higher fat content for the products dried on the spray and pulse dryer compared to the Soxhlet fat determination method. The roller drier produced a powder that was significantly (P<0.05) higher in free-fat compared to the other two drier systems. The pulse combustion dryer produced the most soluble product as determined by the insolubility index. The available lysine concentration in the product dried on the roller dryer was significantly (P<0.05) lower than the available lysine concentrations in the product dried on the spray and pulse combustion dryers. The microstructure of the powders was different for each of the dryers when examined using ESEM.

Two experiments were conducted to determine the effects of growth hormone (GH) or melatonin on the reproductive axis of the stallion. In Experiment 1, nine stallions were treated with GH (20 µg/kg BW) or saline for 21 d starting in January. During the last week of treatment, stallions were subjected to low and high dose injections of luteinizing hormone (LH), as well as low and high dose combined injections of gonadotropin releasing hormone (GnRH) and thyrotropin releasing hormone (TRH). Two months after the onset of GH treatment, semen was collected from all stallions every other day for 2 weeks. Treatment with recombinant equine GH increased (P < 0.001) daily IGF-I concentrations, but had no effect (P > 0.1) on concentrations of LH, follicle stimulating hormone (FSH), or testosterone. The testosterone responses to injections of LH were similar (P > 0.1) between treatments. Likewise, the LH, FSH, prolactin, and testosterone responses to the injections of GnRH/TRH were similar (P > 0.1) between groups. Stallions treated with GH exhibited greater volumes of gel-free semen (P < 0.01) and gel (P < 0.05) and had decreased time until ejaculation (P < 0.05). In Experiment 2, nine stallions were given corn syrup containing either melatonin (0.06 mg/kg BW) or nothing for 90 d starting in July. Between d 68 and 75 of treatment, stallions were given injections of LH and combined injections of GnRH and TRH, similar to Experiment I. Semen was collected from all stallions for three days during the last week of treatment. Treated stallions exhibited decreased daily concentrations of prolactin (P < 0.01) and FSH (P < 0.05), and tended to have lower (P = 0.07) LH concentrations for the first 30 d. Testosterone concentrations were similar between groups. In treated stallions, the low dose administration of GnRH/TRH was not as effective (P < 0.01) at increasing plasma concentrations of FSH and testosterone, and the response in plasma prolactin concentrations to a high dose administration of GnRH/TRH was decreased (P < 0.01). Melatonin treatment did not alter seminal characteristics or libido. In conclusion, GH may alter the long-term accessory gland contribution to seminal volume, but does not appear to interact with other constituents of the reproductive axis in the stallion. Long-term melatonin administration decreases plasma concentrations of gonadotropins and prolactin, but the role of melatonin in perturbation of hypothalamic interaction with the pituitary deserves further study.

A series of experiments were conducted to produce transgenic goats by somatic cell nuclear transfer (NT). In all experiments, donor cells were electrically fused to enucleated metaphase II oocytes, then chemically activated. In a preliminary study to evaluate embryonic development following NT with proliferating or quiescent fibroblast or cumulus cells, significantly more embryos reconstructed with quiescent cumulus cells fused (77%) compared with proliferating cumulus cells (41%), proliferating fibroblasts (36%) or quiescent fibroblasts (37%). Improved development to the eight- to sixteen-cell stage was observed when fibroblast cells were serum starved (serum starved 39% vs. serum fed 15%). However, there was no benefit of serum starvation for cumulus cells nor was there a difference among the treatments for development to the blastocyst stage. Next, the in vivo developmental potential of NT embryos produced from the fusion of quiescent transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries was compared. There was no difference in the number of transferable embryos produced, nor was there a difference in the number of pregnancies established per recipient between either treatment. All pregnancies from both groups culminated in the births of five healthy female kids. In the third and fourth experiments, proliferating and quiescent donor cells from two different transfected fibroblast cell lines were used to generate cloned goats capable of producing human recombinant antibodies in milk. There was no difference in the number of transferable embryos produced from proliferating donor cells compared with quiescent cells, nor was there a difference in the number of pregnancies established per recipient between either treatment. A twin pregnancy from the quiescent treatment resulted in the birth of two healthy transgenic kids. In the final study, oocytes were harvested either from FSH-stimulated ovaries or from nonstimulated abattoir-derived ovaries to generate transgenic goats by NT using fetal fibroblast cells transfected with the MSP-142 gene. Following transfer of the reconstructed embryos to recipient females, one healthy transgenic kid was produced. There was no effect of oocyte source on the number of pregnancies established or on the number of offspring produced. In total, eight transgenic goats were produced.

An experiment was conducted to determine the effects of dihydrotestosterone (DHT) and estradiol benzoate (EB) given simultaneously on concentrations of FSH and LH and the associated follicular activity, and to monitor the subsequent recovery of the gonadotropes and follicular growth following treatment with either DHT or EB. Twelve mares were assigned to daily treatments of DHT (150 Fg/kg BW) plus EB (22 Fg/kg BW) from d 0 through 30 (Phase I). Beginning on d 31, mares were randomly assigned to one of three treatment groups: 1) oil, 2) DHT (150 Fg/kg BW), or 3) EB (22 Fg/kg BW). On d 30, 44, and 58, GnRH was administered to determine the response of FSH and LH. In phase I, DHT plus EB decreased (P = 0.0001) concentrations of LH and FSH. In phase II (d 31 to 66), DHT decreased (P = 0.0001) LH and FSH concentrations. Estradiol benzoate decreased (P= 0.0001) concentrations of FSH but increased (P = 0.0001) concentrations of LH. There was a significant treatment x day x minute interaction of FSH (P = 0.0001) and LH (P = 0.0002) concentrations in response to administration of GnRH on d 30, 44, and 58. The number of medium follicles (P = 0.0001), large follicles (P = 0.0012) and the size of the largest follicle per day (P = 0.0001) decreased during phase I. During phase II, there was a treatment x day interaction (P = 0.0001) for medium follicles and large follicles. Control mares had more large follicles on d 57 through d 63 whereas EB treated mares had more large follicles on d 45, 49, and 51. Control mares had increased size of the largest follicles at d 57 to 63. Mares in the DHT and EB groups had prolonged (P = 0.01) ovulation intervals as compared with controls. It is concluded that the combination of DHT plus EB suppresses gonadotropin concentrations and the associated follicular activity in mares. Also, concentrations of both gonadotropins must be restored to normal for growth of an ovulatory size follicle and the subsequent ovulation of that follicle.

The first objective of these two studies was to evaluate genetic trends for bulls that have comprised the LSU AgCenter Dean Lee performance bull test for the past 55 years. Data included birth weight (BW), initial weight, 112-day weight, average daily gain (ADG), adjusted yearling weight, and scrotal circumference (SC), on 7,488 yearling bulls of 34 breeds on 112d of test for the last 55 years. The top 4 represented bull breeds with greater than 500 animals (Angus, Charolais, Hereford, and Simmental) were included in this analysis. Analyses revealed that growth traits for all bulls regardless of breed demonstrated a linear increase across the years with BW and SC being the lone exceptions. Birth weight demonstrated a decrease over the years. Hereford and Simmental breeds displayed the greatest decrease in birth weight. For start weight and 112-day weight, Simmental and Angus exhibited the greatest increase in weight over the years. Adjusted yearling weight had the greatest increase in the Simmental breed. Simmental breed exhibited the greatest decrease for SC over the years. The second objective was to test the association of single nucleotide polymorphisms (SNPs) on three candidate genes calpastatin (CAST), somatotropin (GH1), and insulin-like growth factor 1 (IGF-1) with growth and performance traits in bulls participating in a forage based performance bull test. Of the 49 SNP genotyped, 20 were chosen for CAST, 9 for GH1, and 20 for IGF-1. These SNP were genotyped on 47 purebred Angus, Braford, and Brahman bulls against traits including average daily gain, birth weight, weaning weight, initial weight, final weight, hip height, backfat (BF), intramuscular fat %, ribeye area (REA), and scrotal circumference (SC). The mixed model procedure of SAS was utilized to evaluate associations of the 49 SNPs and measured traits. Insulin-like growth factor 1 markers (rs133980322, rs137651874, rs132665612, rs132951819, rs110959643, rs109022910, rs110266103, rs109199979 and rs109327701) were determined to be associated with growth and performance traits, including weaning weight, initial weight, final weight, average daily gain, backfat, intramuscular fat %, hip height and scrotal circumference. GH1 marker rs10927590 was significantly associated with weaning weight, initial weight, and final weight.

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.