Objective markers to identify higher fertility individuals are needed to maximize livestock breeding success. Two heparin-binding proteins, which are reflective of fertility in bulls, have been biochemically identified as fertility-associated antigen (FAA) and tissue inhibitor of metalloproteinases-2 (TIMP-2). These four studies were designed to examine the importance of those proteins in relation to reproduction in bulls and other livestock species. In the first study, indirect immuno-fluorescent microscopy was performed to localize FAA and TIMP-2 to livestock sperm. FAA was localized on spermatozoal acrosomes of bulls and rams, but no cross-reactivity was observed for stallions. TIMP-2 labeling was observed on acrosomes and posterior heads, which was species dependent. Localization patterns for FAA and TIMP-2 were further investigated during heparin-induced capacitation and acrosome reactions of bovine sperm. In study two, an enzyme-linked immunosorbent assay (ELISA) was developed to determine concentrations of FAA in bovine seminal plasma (SP). A commercially available TIMP-2 ELISA was utilized to quantify TIMP-2. Respective mean concentrations of FAA and TIMP-2 in SP were 6.661.487 ug/ml and 1.180.045 mg/ml. Concentrations of FAA in SP did not correspond to bull fertility potential, however, older bulls with higher concentrations of TIMP-2 in SP sired more calves. The third study evaluated utility of an amplified fragment length polymorphism with bovine TIMP-2 gene specific primers to amplify a 700 bp genomic DNA (gDNA) product from sperm. From 53 bulls screened, 22.6% were negative for the 700 bp amplicon. There was a three-fold likelihood for 700 bp negative bulls to not sire a calf compared to 700 bp positive bulls. The product was cloned and sequenced, but no homology to TIMP-2 was detected. Therefore, the product represented novel bovine gDNA sequence. The fourth study identified an equine homologue to the bovine FAA gene. Immuno-based diagnostics had not detected FAA in stallion semen. The equine DNA homologue was 88.5% identical in nucleotide and 86% in amino acid sequences to bovine FAA. Subtle differences in the amino acid sequence are likely responsible for the inability to detect FAA in stallion semen with FAA antibodies to bovine FAA.

No description available.

No description available.

No description available.

No description available.

No description available.

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.

No description available.

Bovine colostrum collected within 12h of parturition was de-fatted, de-caseinated, and ultrafiltered (UF) using a 5 kDa cut-off membrane; the resulting UF permeate was freeze dried to create a powder with possible use as a functional food ingredient. Samples representative of five lots of this powdered “colostrum low molecular weight fraction” (CLMWF) were analyzed for chemical composition and nutrient profile. The average contents of fat, moisture, and ash were 0.6%, 1.7%, and 8.3% w/w, respectively. Carbohydrate analysis showed an average of 58.2% w/w lactose monohydrate with no monosaccharides, other disaccharides, trioses, or tetroses detected. The total nitrogen content averaged 1.13% w/w, with 74% of this in the non-protein nitrogen fraction, producing a true protein content of 1.9% w/w. A significant mass fraction of the material (~29% w/w) remains to be characterized. The CLMWF powders were found to contain significant quantities of the minerals calcium (average 870 mg/100g), magnesium, (311 mg/100g), phosphorus (1473 mg/100g), potassium (1705 mg/100g) and sodium (690 mg/100g), the nutrients taurine (average 26.5 mg/100g), L-carnitine (40.5 mg/100g), thiamine (648 mcg/100g) and riboflavin (6991 mcg/100g), and the nucleos(t)ides uridine (55.2 mg/100g) and 5’UMP (18.8 mg/100g), cytidine (3.33 mg/100g) and 5’CMP (4.83 mg/100g) and guanosine (3.45 mg/100g) and 5’GMP (3.57 mg/100g).

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Brad J. White and Robert L. Larson / Bovine respiratory disease (BRD) is the most costly disease of cattle in US feedyards and diagnosis based on clinical signs of illness is challenging. Over the course of five independent studies we evaluated the precision of multiple observers assigning clinical illness scores (CIS) to calves with induced Mycoplasma bovis pneumonia. We also evaluated the accuracy of CIS in relation to lung lesions at necropsy. Agreement among observers over all five studies was slight ({kappa]= 0.16; 95% confidence interval, 0.10 to 0.24) and ranged from 0.10 to 0.21 for individual trials. The accuracy of CIS varied based on the pulmonary consolidation score chosen to represent a truly ill animal. Inflammation associated with BRD can lead to significant pulmonary damage and reduced lung function. Treatment for BRD frequently involves antimicrobial administration and occasionally non-steroidal anti-inflammatory drugs. We evaluated how calves experimentally challenged with Mannheimia haemolytica respond to treatment with flunixin meglumine, alone or in combination with the antimicrobial florfenicol. Individual calf response to bacterial pneumonia was highly variable in this study. None of the changes in serum biomarkers, CBC or chemistry parameters provided reliable indicators of the pulmonary inflammation associated with the mild severity of bronchopneumonia in our study. Metaphylaxis is frequently administered to manage the risk of BRD within cohorts of cattle. We evaluated the impact of metaphylactic antimicrobial administration 10 days prior to experimental Mannheimia haemolytica inoculation to mitigate pulmonary lesions. We found that calves receiving tildipirosin had less lung damage and fewer clinical signs of illness compared to calves treated with tulathromycin or saline. Finally, the ability to predict those animals that would not finish the production cycle normally would provide benefits in effectively managing cattle. We evaluated the ability of classification algorithms to accurately predict an individual calf’s outcome based on data available at first identification of and treatment for BRD. We found accuracy of classifiers was dependent on the data recorded by the feedyard and there are sub-groups of calves within feedyard populations where classifiers were highly accurate. These data suggest the importance of pairing the proper classifier with the data available.