My dissertation hypothesis is that bovine trophoblast cells express cell-surface and secreted non-classical major histocompatibility complex class I (MHC-Ib) proteins which inhibit NK cells and other leukocytes by binding to inhibitory receptors (e.g., LILRB1, LILRB2, KIR2DL4, and/or CD94/NKG2A). Extremely polymorphic and ubiquitously expressed classical MHC class I (MHC-Ia) proteins, which present foreign antigenic peptides to CD8+ T lymphocytes, are involved in acceptance or rejection of tissue grafts. Non-classical MHC class I (MHC-Ib) glycoproteins, such as Human Leukocyte Antigen-G (HLA-G) and murine Qa-2, are important modulators of the maternal immune system during pregnancy. MHC-Ib proteins are: (a) oligomorphic or monomorphic, (b) expressed in specific tissues under specific condtions, and (c) produced as surface and/or soluble isoforms due to alternative splicing. Third trimester-bovine trophoblast cells express both MHC-Ia and MHC-Ib proteins. The MHC-Ib proteins expressed by trophoblast cells during the third trimester of pregnancy are encoded by four bovine leukocyte antigen (BoLA) loci: BoLA-NC1, BoLA-NC2, BoLA-NC3, and BoLA-NC4. Two MHC-Ia (N*01701 and N*01802) and three MHC-Ib (NC1*00501, NC3*00101 and NC4*00201) proteins showed cell-surface expression in transfection studies performed in murine P815 and human K562 cells. Two additional isoforms, NC1*00401 and NC2*00102, were not detected on the surface of these cells. Nevertheless, both class Ia proteins, N*01701 and N*01802, and five class Ib proteins, NC1*00401, NC1*00501, NC2*00102, NC3*00101, and NC4*00201, were detected in crude cell lysates on Western blots. Precipitation of proteins from culture supernatants showed that cell-surface MHC-Ia (N*01701 and N*01802) and MHC-Ib proteins (NC1*00501, NC3*00101, and NC4*00201) are shed from the surface of these cells into the media. The mechanism of shedding of these proteins is, however, not known. Monoclonal antibodies W6/32, IL-A88, H1A, H6A, H11A, H58A, and PT-85A recognized surface MHC-I isoforms with varying affinity. We were able to develop a sandwich enzyme-linked immunosorbent assay (ELISA) using either H1A or IL-A88 antibody as the capture antibody and the W6/32 antibody for detection. We produced monoclonal antibodies against cattle NC1*00501 and NC3*00101 proteins. One monoclonal antibody generated against BoLA-NC3*00101 was highly specific. Unfortunately, due to failure to clone the NC3*00101- hybridoma, we no longer have an infinite source of this monoclonal antibody for NC3*00101. We eluted peptides from NC3*00101-transfected MHC-null K562 cells and identified peptides using liquid chromatography-mass spectrum (LC-MS) analysis. Analysis of peptide binding data using the SAS Proc mixed statistical program, suggested that the peptide EVTNQLVVL is a potential peptide ligand, which can be used to make tetramers for enumeration of antigen-specific leukocytes.

Toxaphene is reported to cause defects in the collagen of fish. Chronic exposure to toxhaphene weakens the backbone of fish by decreasing the amount of collagen and usually increasing the amount of calcium in the bone which results in a more brittle and fragile bone. We investigated the possible direct action of toxaphene on collagen synthesis by exposing vertebral and swim bladder organ cultures obtained from unexposed rainbow trout (Salmo gairdneri) fingerlings to the same lot of toxaphene found to cause this defect in vivio. Collagen produced by these organ cultures was measured by: (1) total 3H-proline incorporated into the matrix; (2) 3H-proline released during collagenase digestion of acid-precipitated protein; (3) 3H-hydroxyproline extracted from the acid hydrolysate, and (4) tritiated water produced during the hydroxylation of 4-3H-proline. At a relatively high concentration of toxaphene (2.4 mM) these indices of collagen production were reduced, but this was probably caused by a decrease in tissue viability rather than by a direct affect on collagen synthesis. At 240 μM cellular protein synthesis was reduced. Generally no effects were found at toxaphene concentrations below 240 μM. From these studies it was concluded that toxaphene does not have a direct inhibitory effect upon collagen production at the tissue level. For comparison to the in vivo were measured. All three of these parameters were significantly reduced in comparison to controls (α = 0.05) in those fish exposed to the highest concentration of toxaphene (200 ng/1). fish exposed to 150 ng/l toxaphene also had reduced prolyl hydroxylase activity. These results indicated that vertebral prolyl hydroxylase activity may be a sensitive indicator of toxaphene exposure in fish, and inhibition of that enzyme may be involved in the mechanism of toxaphene-induced collagen effects.

Mastitis is one of the most common and expensive dairy cattle diseases. Mastitis prevention and management are key factors in herd health and improved milk quality. One objective of this research was to evaluate management solutions to maintain a low somatic cell count, based on survey responses from Kentucky dairy producers. Because hyperkeratosis may increase mastitis incidence, another objective of this research was to examine changes in teat end hyperkeratosis in a herd transitioning from a standard pulsation milking system to an individual quarter pulsation milking system. The last objective of this research was to evaluate technologies that monitored rumination time, neck activity, reticulorumen temperature, and milk yield as potential mastitis detection devices.

Poor estrus detection can limit the reproductive performance of a dairy herd. One objective of this research was to evaluate an alternative method to traditional estrus detection in the form of automated monitoring technologies. To accomplish this, the first study considered the ability of automatically monitored parameters (activity, number of steps, lying bouts, lying time, feeding time, rumination time, and temperature) to detect estrus. A second study compared automated activity monitoring to timed artificial insemination as reproductive management strategies on commercial herds. The other objective of this research was to evaluate the economic potential of automated estrus detection technologies. This was accomplished by creating and evaluating a farm specific decision support tool to determine the net present value of adopting an automated estrus detection technology.

Precision dairy farming technologies provide a variety of functions to dairy farmers. Little is known about dairy producer perception of these technologies. A study was performed to understand dairy producer perception of parameters monitored by precision dairy farming technologies. Calving has potential to be predicted using these same parameters and technologies. A second study was performed using two commercially marketed technologies in calving prediction. In order for these technologies to generate accurate and useful information for dairy farm use, they must accurately quantify these parameters. The final study evaluated the accuracy of five commercially marketed technologies in monitoring feeding, rumination, and lying behaviors.

Bovine somatotropin (bST) is a protein synthesized at the base of the brain and released by the pituitary gland into the circulatory system. BST is transported by the circulatory system and absorbed only by cells of target organs that possess cell surface receptors for the protein (11, 41). Its effect is initiated via a protein receptor initiation and cyclic AMP cascade . This effect on the cell continues well past the degradation of the bST molecule. BST is likely transported into the cell, where it is degraded . Its constituent blocks, amino acids, are used to synthesize new proteins or converted to other metabolites such as sugars (1).

Enterovirus D68 (EV-D68) virus has become more prevalent over the last 15 to 20 years. EV-D68 attacks the respiratory system and can cause severe disease in individuals who have underlying respiratory problems. There have also been reports of individuals with EV-D68 showing signs of neurological system problems and acute flaccid paralysis. Because of the increase in patients with EV-D68 and also the potential for neurological disease, an animal model is needed to study the disease and to evaluate experimental therapies for EV-D68 infection. To develop the animal model, 4-week old AG129 mice that lack alpha and beta interferon receptors, making them immunosuppressed, were used. The mice were infected with EV-D68 by the intranasal route to allow infection of the lungs. On day-3 post-infection the mice were euthanized and lungs were removed and homogenized for collection of virus. The newly collected virus was then used to infect another set of mice. This procedure was repeated 30 times. As passage number increased so did the amount of virus that was collected from the lungs of mice. The virus titer increased 320-fold between mouse passage 0 to 30. At the end of the thirtieth passage, multiple tissues (lungs, liver, kidney, spleen, blood, brain, spinal cord and leg muscle) were collected from infected mice over several days and titered to demonstrate how quickly the virus spread to various tissues within the mouse. The virus replicated most rapidly in the lungs and remained in the lungs longer than the other tissues evaluated. However, large quantities of virus were found in all tissues evaluated. Finally, several experimental antiviral compounds were evaluated: rupintrivir, pleconaril, ribavirin, enviroxime and guanidine, all of which showed therapeutic potential in cell culture. In the animal model rupintrivir, pleconaril, ribavirin and enviroxime did not show any therapeutic effect. Only guanidine reduced the amount of virus that was found in the lungs as well as in whole blood.

The role of ammonia in reproductive inefficiency in early lactation dairy cows was studied in a series of experiments designed to determine the concentrations of ammonia in normal bovine follicular fluid (bFF), to determine the effects of ammonia on the bovine embryo during specific stages of development, and to test the hypothesis that elevated plasma urea nitrogen concentration is associated with elevated ammonia and urea nitrogen concentrations in the reproductive fluids. In the first study, ammonia concentration in different size follicles and the effect of ammonia during in vitro maturation on embryo development were determined. Ammonia concentration in the bFF was significantly different (P<0.001) between each follicle size (< 1 mm, 2-4 mm, 5-8 mm, and >10 mm) and ammonia concentration decreased as follicle size increased. There was no difference (P>0.05) in cleavage rates, morula development, and blastocyst development when oocytes were exposed to various concentrations of ammonia during in vitro maturation. In the second study, the effects of addition of ammonia in the media during in vitro fertilization (IVF), culture (IVC), and throughout maturation (IVM), IVF, and IVC were evaluated. Addition of moderate concentrations of ammonia to IVF media resulted in a significant (P<0.05) increase in embryos that developed to blastocysts and to expanding and hatching blastocysts. Ammonia in the IVC media significantly (P<0.05) decreased the proportion of ova that developed to blastocysts. When exposed to ammonia throughout IVM, IVF, and IVC, ova developing to morulae was significantly (P<0.05) higher in media containing moderate concentrations of ammonia compared to control. In the third study, plasma urea nitrogen (PUN) concentrations were related to follicular and uterine fluid ammonia and urea concentrations in early lactation dairy cows. Mean PUN concentrations were used to distribute the cows into two groups: 1) cows with PUN ≥ 20 mg/dl (HPUN), and 2) cows with PUN<20mg/dl (LPUN). Follicular fluid ammonia and follicular fluid urea were significantly (P<0.01) higher in HPUN cows compared to LPUN cows. Uterine fluid ammonia concentration was significantly (P=0.05) higher in HPUN cows than in LPUN cows during the luteal phase, but not during the estrus phase of the estrous cycle, but not on Day 0. Uterine fluid urea nitrogen concentrations were significantly (p<0.001) higher in HPUN cows than in LPUN cows during both the luteal and estrus phases.

The objective of this thesis was to determine how pregnant, lactating Holstein cows on pasture interact with shade and sprinklers when offered a choice or no-choice between methods. Authors hypothesized that cows would use shade more then sprinklers, but sprinklers would be more effective at cooling cows. Additionally, authors predicted cows using shade more would have decreased lying bouts and steps and an increase in time lying, compared to cows choosing sprinkler. Results indicate cows will use both methods when offered alone or together. However, cows will choose to be exposed or in shade at lower temperature humidity index values, but as the heat load increases, cows will shift to using sprinkler just as much as shade, and decrease overall time exposed. Overall, sprinklers were more effective at reducing vaginal temperature in heat stressed cows on pasture. Cows in shade had decreased lying bouts and steps, and increased time lying.

Dairy producers play a central role in evaluating and seeking treatment or care for animal pain. The primary aim of this study is to examine dairy producers’ emotional states and professional quality of life and analyze the relationship between these variables and their perception of pain in dairy cattle. Dairy farm owners and managers of 65 southeastern U.S. herds participated in a survey that included demographic information, the Depression, Anxiety, and Stress Scale, a modified Professional Quality of Life Scale, and 23 items requiring participants to evaluate various painful conditions in cattle.