The objective of this study was to determine dairy cattle preference amongst four species of cool-season forage grasses: eight orchardgrasses (Dactylis glomerata L.), five tall fescues [Schedonorus arundinaceus (Schreb.) Dumort.], five perennial ryegrasses (Lolium perenne L.), and six festuloliums [xFestulolium braunii (K. Richt.) A. Camus.]; 24 cultivars in total. Each grazing trial utilized four Holstein-Friesian heifers over six hours. Maturity differences were eliminated by having animals graze only vegetative material. After six grazing trials (three each in 2014 and 2015), consistent results in animal preference were not found; three of the six trials did show preference (P

In vitro produced bovine embryos are critical to the cattle industry. However, these embryos have altered morphology, epigenetics, and metabolism when compared to their in vivo counterparts. The aim of this thesis was to alter maturation and culture media to improve the developmental competence of in vitro bovine embryos. This thesis is comprised of three experiments and one proof of concept study. Each experiment followed the same general layout: oocyte aspiration from Jersey or Holstein ovaries, oocyte maturation for 24 hours, fertilization with bull semen for 24 hours, then embryo culture for 7-8 days in 38.5 °C in 5% O2, 5% CO2 and 90% N2. A proportion of stage 7 grade 1 blastocysts were fixed and stained with Nile Red to evaluate lipid content, Mitotracker Red CMX-Rosamine to measure mitochondrial activity, or Cell Rox Green to assess reactive oxygen species (ROS). Using a confocal microscope, images were taken of each stained embryo to detect and measure fluorescence. Any stage 7 embryos that were not imaged were slow frozen and evaluated for re-expansion when thawed. Experiment 1 was a one-way treatment designed to compare the conventional maturation media (control) containing fetal bovine serum (FBS), to an alternative media replacing FBS with a human platelet lysate serum substitute (SS). Both abattoir and ovum pick up (OPU) oocytes were used. The results suggested that maturing in vitro and OPU oocytes with serum substitute maintained developmental competence, including a similar yield of embryos and re-expansion rate. Resulting in vitro SS embryos had lower lipid content (p<0.05) and ROS levels compared (p<0.05) to the FBS control. Experiment 2 was a 2x2 factorial design testing how the addition of FGF2, LIF, and IGF1 cytokines to maturation and culture media affected in vitro embryo development. The first factor was maturation media (Mcon: industry standard and Mcyt: added cytokines) and the second factor was culture media (Ccon: industry standard and Ccyt: added cytokines). The two maturation media crossed with the two culture media equated to four treatments, including a control. The results suggested that cytokine addition had no effect on blastocyst rate or re-expansion rate. The combination of MCyt x CCyt media produced the lowest lipid levels (p<0.05) while the MCon x CCon treatment led to the highest mitochondrial activity (p<0.05). Experiment 3 was a 2x2 factorial design testing how the addition of melatonin to cytokine supplemented maturation media affected embryo developmental competence. The maturation factor had two levels: no supplementation (NoM) and melatonin with cytokine supplementation (MM). The culture factor had two levels: no supplementation (NoC) and cytokine supplementation (CC). We found no difference in blastocyst or re-expansion rate between any treatments. NoM showed higher mitochondrial activity than MM (p<0.05). NoC showed higher mitochondrial activity than CC (p<0.05). The NoM x NoC treatment showed the highest lipid levels of any treatment (p<0.05). The NoM x NoC treatment showed the highest mitochondrial activity of any treatment (p<0.05). The final part to this thesis focused on the preliminary use of phasor-fluorescence lifetime imaging microscopy (FLIM) and deep imaging via emission recovery (DIVER) technologies to autofluoresce endogenous compounds and predict the viability of an embryo without the use of invasive labels. In conjunction with the University of California Irvine, we tested the technologies on morula and blastocyst stage embryos to see if developmental competence was altered. Results suggested FLIM successfully captured NADH levels and DIVER successfully captured ROS and lipid content. Future studies are planned to fully investigate the effects of the microscopes on development and to accurately predict bovine embryo viability for transfer. Overall, human platelet lysate was a successful replacement for FBS, likely due to its similar content of protein and growth factors. Neither cytokine nor melatonin supplementation had conclusive results, further trials are needed to fully determine effectiveness.

Twenty four cattle, six each of healthy cows and calves, and cows and calves with brisket disease, were obtained, examined and slaughtered, The concentrations of calcium, chloride, cobalt, copper, iron, magnesium, molybdenum, phosphorus, potassium, sodium and zinc; and percent absolute dry matter and percent ash were determined in tissues selected from the following: cardiac, hepatic, renal, osseous, whole blood and blood serum. In addition, certain physical and biological parameters were recorded for each animal. The results were analyzed as a 2 x 2 factorial, segregating the effects of age and brisket disease, and the age-disease interaction. The following statistically significant (P<0.05) differences were attributed to the effect of brisket disease: reduction in the percent dry matter and percent ash in all soft tissues studied; increase in cardiac, hepatic and renal calcium and sodium; decrease in serum total calcium; marked decrease in hepatic copper and increase in hepatic iron; decreased blood iron, hematocrit and hemoglobin; decreased hepatic potassium, magnesium and phosphorus; and increased hepatic zinc. The effects of brisket disease are superimposed upon these marked differences in the cattle in the present study as compared to those in a previous study of well nourished cattle of similar breeding from a similar environment: reduced cardiac, hepatic, serum and osseous calcium; reduced hepatic, osseous and serum magnesium and increased renal magnesium; reduced hepatic phosphorus and increased renal phosphorus; reduced hepatic, serum and osseous potassium and increased cardiac potassium; and reduced cardiac, osseous and serum sodium and zinc. The effects of age must be evaluated in view of the fact that half of the animals were diseased; moreover, some age effects occurred almost exclusively in the diseased animals. Statistically significant (P<0.05 ) differences attributed to the effect of age were: decreased phosphorus concentrations in hepatic and renal tissue and serum; increased percent dry matter in hepatic and osseous tissue; increased osseous percent ash; decreased hepatic and osseous potassium; increased serum ionic calcium; and decreased hepatic calcium, magnesium and sodium, all in cows as compared to calves. The interaction of increased age and brisket disease produced the following statistically significant (P<0.05) results: hepatic percent dry matter and iron concentration were increased; hepatic magnesium, potassium and sodium were decreased; and cardiac zinc was increased. Hypotheses regarding possible reasons for these results are formulated and discussed.

The intensity of the pressure of population on an area may be increased on three occasions. Economic and social opportunities may contract; population may increase; or both these factors may combine to increase the intensity of pressure. This pressure, naturally can be diminished either by an increase of opportunity or an dimunition of population.

Two factors that are important in the production of the typical body and texture of cheddar cheese are acid development and milk fat content. When either of these is absent or present in less-than-normal amounts, the resulting cheese shows marked defects of curdiness and firmness.

Properly dried sweet cream buttermilk, because of its high fat and lecithin contents, should make superior ice cream. Its use as a source of serum solids in ice cream. Its use as a source of serum solids in ice cream mix would open an outlet for this butter by-product and would serve the needs of ice cream manufacturers during nonfat dry milk solids shortage and it may make a higher score ice cream. The demand for milk solids shortage and it may make a higher score ice cream. The demand for milk solids has been increasing so rapidly that today the by-products of the dairy industry are being utilized as sources of human food to a much greater extent than before. In past years the greater bulk of creamery buttermilk has been utilized as animal feeds. Attempts are being made to convert more of this by-product into channels of human consumption. As the manufacture of sweet cream butter is increasing at a rapid pace, a larger supply of buttermilk product of high quality which is fit for human consumption is available in the market. Many previous investigators have used buttermilk products as a source of serum solids in ice cream mix with favorable results. The advantage claimed has been that it tends to improve the whipping ability of ice cream mixes and to impart richer flavor to the product. These beneficial qualities have been attributed to the butterfat and the phospholipids, of which lecithin is predominant. The work of Chapman and Supplee shows that buttermilk and cream contins several times as much licithin as skimmilk. The amounts of total phospholipids reported by Holm et al and Wright et al are approximately 1.77 per cent in dry buttermilk and 1.06 per cent in dry skim milk.

Rumen protected Methionine (MET) and Lysine (LYS) are critical for milk protein synthesis in dairy cows. N-acetyl-L-methionine (NALM) is a MET derivative that consists of L-Met protected with an acetyl group that is attached to the α-amino group.N-acetyl-L-lysine (NALL) is a LYS derivative that is similarly protected. The objectives of these studies were to quantify the gastrointestinal availability of NALM and NALL. Three experiments were run as 3 × 3 Latin square using 3 second lactation Holstein cows that have been fitted with cannulas in the rumen and duodenum. The cows were fed diets containing the supplements for two weeks prior to each experiment so that the rumen microbes had time to adjust to the supplement. Each period consisted of 10 d of adaptation followed by 2 d of sampling. A dose of 0, 30, or 60 g of NALM was placed under the rumen mat at the time of feeding every day during experiment 1. The cows were similarly supplied with 0, 60, or 120 g of ƐNALL during experiment 2. The cows were supplemented with 0 g, 120 g ofƐNALL, or 120 g of diNALL during experiment 3. On day one of sampling, a liquid marker (Co-EDTA) was also administered at the time of the protected AA administration. Blood, ruminal, and duodenal samples were taken at hours 0, 1, 3, 6, 9, 12, and 24 post-feeding. There were no differences for milk production, milk protein, milk fat, or DMI for NALM or either NALL. There were no differences for ruminal escape (69.1% and 46.2% respectively) and duodenal appearance (2.16% and 3.40% respectively). The ruminal escape of ƐNALL was not different between the 120 g dose (32.7%) and the 60 g dose (27.2%). Duodenal appearance was higher (P < 0.01) for the 60 g dose (2.86%) than for the 120 g dose (1.19%) of ƐNALL. Acetate, propionate, butyrate, and valerate were higher (P < 0.01) for the supplemented cows during experiment 1 with NALL. There were no differences between ƐNALL and diNALL for rumen escape, duodenal appearance, VFA production, or blood LYS AUC. Results of the experiment verify significant protection of the N-acetyl MET and LYS from rumen degradation.

Bovine milk fat globules naturally vary from less than 0.2 µm to 15 µm in diameter. Milk has at least two distinct distributions of fat globules. While the majority (~90%) of globules in milk are of the smaller distribution (average diameter of 0.4 µm), virtually all the fat is carried in the larger globules (average diameter 3.5 µm). This distribution suggests some compositional and/or functional significance might exist between the two populations of fat globules, which may be related to origin of these globules in the lactating cell. Milk fat globules have a unique structure, composed of a core droplet of non polar lipids (triacylglycerol) surrounded by a lipid bilayer membrane known as milk fat globule membrane (MFGM). Other than MFGM, there is another source of membrane that has been identified in skim milk. It has been hypothesized that this skim milk membrane (SMM) is derived from MFGM, but little data are available to support this idea, and the membrane may also have alternate origins. In this study, different aggregates of lipids (small and large fat globules, SMM, skim milk) from milk were isolated and characterized for their lipid contents. Isolation of small and large fat globules fractions was verified by laser diffraction particle size analysis. The lipids were extracted from isolated different lipid aggregates and individual classes were separated using thin layer chromatography. Lipids were transesterified to fatty acid methyl esters and analyzed by gas chromatography-mass spectrometry. The results indicate that there are some compositional differences between native milk fat globule membranes of different sizes. For example, the total phospholipid fraction of small fat globules (SFG) contained significantly more unsaturated C18:1n9 and C18:2n6 than large fat globules (LFG). Conversely, sphingomyelin composition of SFG contained less C18:1n9 and C18:2n6cc, but more long chain fatty acids C22:0, C23:0, and C24:0. Phosphatidylethanolamine composition of SMM contained more C17:1 than SFG and LFG. The composition of C18:1n9 in triacylglycerol increased with fat globule size. Clear differences were also found in lipid profile of SMM and small and large fat globules from milk. Composition differences between SMM and native milk fat globules of different sizes suggest that origin of this membrane material in skim milk might have some different source than that of MFGM.

Bovine mastitis, or inflammation of the mammary gland, has substantial economic and animal welfare implications. A genetic basis for mastitis resistance traits is recognized and can be used to guide selective breeding programs. The discovery of regions of the genome associated with mastitis resistance, and knowledge of the underlying molecular mechanisms responsible, can facilitate development of efficient mastitis control and therapeutic strategies. The objectives of this dissertation research were to identify sites of genetic variation associated with mastitis resistance, and to define the contributions of the milk-secreting epithelial cells to mammary gland immune responses and mastitis resistance. Twenty seven regions of the bovine genome potentially involved in mastitis resistance were identified in Holstein dairy cattle. Additionally, this research demonstrates a role of bovine mammary epithelial cells in mastitis resistance, and provides guidance for the use of an in vitro model for mastitis studies. Primary bovine mammary epithelial cells from mastitis-resistant cows have differential expression of 42 inflammatory genes compared with cells from mastitis-susceptible cows, highlighting the importance of epithelial cells in mastitis resistance. Bovine mammary epithelial cells display both similarities and differences in pro-inflammatory gene expression compared to fibroblasts, and their expression of inflammatory genes is influenced by administration of the enzyme phospholipase A2. The growth potential of milk-derived bovine mammary epithelial cells in vitro can be extended, facilitating their use in mastitis studies, by transfection with a viral protein. Collectively, this research contributes to current knowledge on bovine mastitis resistance and in vitro models.

A recombinat Chinese Hamster Ovary (rCHO) cell line designated as CHO SEAP was utilized in this investigation to optimize protein production. Two bench top stirred-tank bioreactors, namely a pitched-blade and a packed-bed basket bioreactor, were utilized for a comparative study to determine which bioreactor would produce the best results in terms of protein production. The objective of this research project was to provide basic data that shows cells cultured in a packed-bed basket bioreactor in perfusion mode will generate more protein product than cells in batch mode suspension culture with a pitched-blade bioreactor. The packed-bed bioreactor creates a homeostatic environment similar to the environment found in vivo, where waste products are constantly removed and fresh nutrients are replenished. Closed batch cultures do not provide a homeostatic environment. In batch culture systems, nutrients are depleted and waste products accumulate. The results from this experiment could help investigators involved in protein and/or vaccine production facilities select the appropriate bioreactor and mode of operation to optimize cell productivity for generation of a specific protein product. CHO cells have been used for the production of vaccines, recombinant therapeutic proteins, and monoclonal antibodies, and these cells are now the cell line of choice in the biopharmaceutical industry. Traditional vaccine production methods in egg embryos are slow and outdated, whereas roller bottle-based cell culture techniques are time consuming and have limited scalability. These limitations justify the need for development of stirred tank bioreactors. Cells cultured in a packed-bed bioreactor are not exposed to hydrodynamic forces, as is the case with pitched-blade bioreactors, allowing for maximum growth and protein expression. This mode of operation involves the constant removal of media depleted of nutrients and the addition of fresh media with more nutrients to keep the cells growing. Long run times decrease the constant need for re-seeding cells and re-establishing seed cultures, thus, reducing setup time and labor dramatically. Secreted products are automatically separated from cells in perfusion, eliminating filtration and membrane fouling. A detailed description of both modes of operation are discussed in this thesis.