No description available.

Establishment and maintenance of pregnancy is a central concern to the cattle industry, as it strongly impacts efficiency and profitability of beef cow-calf operations. The objective of this study was to determine if nutrient restriction impacts ovulatory follicle size and corpus luteum (CL) development and function of Bos taurus cows enrolled in estrous synchronization. A total of 26 Angus cows were housed in a facility equipped with a Calan gate system for individual animal intake. Cows were stratified by body weight (BW), and randomly assigned one of two nutritional treatments: 1) 100% of nutrient requirements (MTN; n=13) or 2) 70% of nutrient requirements (REST; n=13). Individual daily intakes were measured and adjusted weekly based on BW. Cattle underwent an acclimation period of 14 days and were exposed to nutritional treatments for 30 days prior to estrous synchronization. Body weight was measured daily using an automated scale and a conventional livestock scale at the beginning and end of the experiment. Cows were synchronized using a 7-day CO-synch + CIDR protocol beginning on day -10. Ultrasonography of the ovaries was performed at each event of the estrous synchronization protocol on days -10, -3, 0, 5, and 7. Blood samples were taken on days -10, -3, and daily from day 0 through 7 to observe changes in progesterone (P4). Data were analyzed as repeated measures using the MIXED procedure of SAS. Initial BW tended to differ between treatments (P = 0.07; MTN 597 ± 32 kg, REST 604 ± 32 kg), but MTN had greater final BW (P < 0.001; 687 ± 24 and 556 ± 27 kg, respectively) and greater average daily gain (1.35 ± 0.18 and -0.72 ± 0.21 kg/d, respectively, P < 0.001) than REST. Diameter of the largest follicle was similar (P = 0.851) between treatments at CIDR insertion (12.6 ± 0.6 mm) and CIDR removal (12.9 ± 0.4 mm) but was greater (P < 0.05) for MTN than REST cows at 60 hrs after CIDR removal (14.01 ± 0.6 and 12.37 ± 0.5 mm, respectively). Volume of CL was similar (P > 0.1) at 5 (3211 ± 113 mm3) and 7 (5280.3 ± 212 mm3) days after ovulation. Concentration of P4 did not differ on days -10, -3, or 0-5. However, on days 6 and 7, P4 was greater (P < 0.05) for MTN than REST (2.07 ± 0.15 and 1.65 ± 0.15, and 2.27 ± 0.15 and 1.83 ± 0.15 ng/mL, respectively). In conclusion, nutrient restriction to 70% of maintenance during estrous synchronization negatively affects diameter of the ovulatory follicle and circulating P4, but it did not affect CL volume in multiparous Bos taurus beef cows. / Master of Science / Ensuring successful pregnancy in beef cow-calf operations is crucial for the efficiency and profitability of the cattle industry. This study investigates the effects of nutrient restriction on ovulatory follicle size and corpus luteum (CL) volume in Angus cows undergoing estrous synchronization. A total of 26 cows were subjected to either a maintenance diet meeting 100% of nutrient requirements (MTN) or a diet providing 70% of nutrient requirements (REST). Intakes were updated weekly using computer software. The cows underwent a 30-day nutritional treatment before synchronization of ovulation. Results revealed that cows on the maintenance diet exhibited greater final body weight and average daily gain compared to those on the restricted diet. While estrus expression showed a numerical increase in MTN cows, the impact was not statistically significant. Analysis of ovulatory follicle size demonstrated that MTN cows had larger follicles 60 hours after synchronization compared to REST cows. Surprisingly, corpus luteum volume did not differ between the two groups at 5 and 7 days after ovulation. Additionally, circulating progesterone (P4) levels were affected by nutrient restriction, with notable differences observed on days 6 and 7. In summary, nutrient restriction during ovulation synchronization negatively influenced ovulatory follicle size and P4 levels, but did not affect corpus luteum volume in mature Angus cows. These findings contribute valuable insights for the cattle industry, emphasizing the importance of proper nutrition for optimal reproductive health in beef cows.

No description available.

No description available.

The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD. / Ph. D.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes. / Master of Science

Pre-pubertal bovine mammary development involves ductal and stromal tissue changes. In mice, this process is impacted by presence of innate immune cells. Whether or not such immune cells are present or involved in bovine mammary development is unknown. We studied the presence, location and changes in numbers of eosinophils, mast cells and macrophages in pre-pubertal bovine mammary tissue. Chemical stains and immunofluorescence were used to identify the cells in formalin fixed, paraffin embedded mammary tissue. The first set (ONT) included samples (n=4/week) from birth to 6 weeks of age. Another set (OVX) determined the influence of ovaries, 19 animals were intact or ovariectomized 30 days before sampling. They were 90, 120 or 150 days old at examination. The third set (EST) allowed examination of the potential influence of exogenous estrogen on innate immune cells in the mammary gland. Samples were from calves given estrogen implants (n=6) or placebo (n=4) at 56 days old, and sampled at 70 days old. We examined 20 images each of NEAR and FAR stroma from every animal. More eosinophils were observed in NEAR versus FAR in the ONT and OVX , more mast cells observed in NEAR versus FAR in ONT. More macrophages were observed in NEAR versus FAR in ONT and EST. We show, for the first time, that innate immune cells are present in prepubertal bovine mammary tissue and that abundance is related to the epithelial structure. We suggest a possible role for these cells in control of bovine mammary development. / Master of Science

Three studies were conducted to assess if the ability of Enterococcus spp. surviving the ensiling process and bovine gastrointestinal tract could impact risk of bovine mastitis. The first study determined ability of enterococci to survive 3 wk ensiling. Grass and corn crops were divided into 3 treatments: 2 commercial silage inoculants, 1 negative control. After wk 1, 2, and 3 of ensiling, dry matter and bacterial enumeration were performed. Addition of silage inoculant led to greater levels of enterococci in grass silage compared with negative control levels, but showed less difference in inoculated corn silage. The second study quantified enterococci shedding rates in lactating dairy cows. Using a 4 x 4 Latin Square design, lactating, ruminally fistulated Holsteins were inoculated with enterococcal isolates from silage inoculants, ensiled forages, or clinical mastitis cases. Over the 4-period study, each period consisted of rumen and fecal sampling (2 wk) followed by a wash period (10 d). There were no significant differences in rumen or fecal enterococci levels between the 4 treatments. Both rumen and fecal enterococci levels showed significant differences between baseline and treatment periods (period 3, 4). The third study analyzed similarity in enterococcal isolates of silage and bovine origin using pulsed-field gel electrophoresis patterns from SmaI restrictions. Dendogram analysis showed none of the isolates met or were greater than a 75% genetic similarity and produced a genetically diverse population. Results suggest Enterococcus spp. from silage inoculants are not likely to contribute to an increased risk of enterococcal bovine mastitis. / Master of Science

The objective of this study was to examine the effects of presynchronization and recombinant bovine somatotropin (rbST) on conception rates following a timed insemination (TAI) protocol in lactating dairy cows. A further objective included the evaluation of the efficacy of the Early Conception Factor (ECF) test kit. Recombinant bST may offer some benefit when used in conjunction with estrus synchronization and TAI. Presynchronization treatment consisted of two injections of PGF2α given 14 d apart, with the second dose administered 14 d prior to the initiation of a TAI protocol. A total of 216 lactating Holstein cows were presynchronized with PGF2α and then received GnRH (100μg) at 67 ± 7 d post partum (PP), administration of PGF2α (25 mg) 7 d later, another GnRH (100μg) administration 2 d after PGF2α, and were inseminated 8-18h later (OvSynch). First service conception rate (CR) was determined by rectal palpation at 42 ± 7 d after artificial insemination (AI). Treated cows (n=113) received rbST 67 ± 7 d PP whereas control cows (n = 113) were presynchronized without rbST. The cycling status of all cows was determined by paired milk P4 levels at 53 and 67 ± 7 d PP. No differences (P > 0.10) in conception rate were observed between control and rbST treated cows (44.7 and 40.7% respectively), nor was there any interaction of cyclicity and rbST. Milk samples were collected 7 d following AI for use in ECF test kit evaluation. Samples were stored at -20ºC (n=216) and at 5ºC (n=113) until assayed. Test results for frozen and refrigerated samples were compared to conception rates determined by rectal palpation at 42 ± 7 d after AI. The rate of false positive and negative results for frozen milk samples were 36.1 and 14.8% respectively, and 40.7 and 7.1% for refrigerated milk samples. Treatment with rbST at the time of the first GnRH injection of an OvSynch protocol did not significantly alter first service conception rates. Additionally, an acceptable 92.9% accuracy of the ECF test for the detection of open cows 7 d after AI using milk samples stored at 5ºC was obtained. / Master of Science