Browsing by Author "Corl, Benjamin A."
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- Abundance and Localization of (Yes-associated protein) YAP in Prepubertal Bovine Mammary TissueGranger, Paulnisha Davida (Virginia Tech, 2018-07-09)Most mammary development is postnatal. Mammary growth that occurs before puberty is diminutive in amount but consequential for future milk production, especially in dairy heifers. With advanced knowledge on fundamental aspects that govern prepubertal mammary development, scientists and farmers alike can ensure that heifers perform their best once they become cows. The Hippo pathway has been identified as an evolutionarily conserved pathway that regulates organ size in many animal species; it might contribute to mammary growth in dairy heifers. This pathway is mediated by yes-associated protein (YAP) and through downstream gene transcription activation, results in cell proliferation. Because YAP has never been identified in bovine mammary tissue, questions examined in this body of work mainly focused on the abundance and localization of YAP in mammary tissue of prepubertal heifers. The first trial investigated effects of in vivo estradiol administration on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. While YAP was present in nuclei and cytoplasm of both cell types, it was also discovered that estrogen did not influence YAP abundance or location. The second research trial focused on determining the effects of in vivo estradiol blockade on YAP abundance and localization in prepubertal bovine mammary epithelial and myoepithelial cells. Similar to the first experiment, results indicate that YAP abundance and localization was not influenced by estrogen blockade. Despite not being responsive to in vivo estradiol administration (experiment 1) or estradiol blockade (experiment 2) under the conditions of our experiments, YAP was present in nearly all mammary epithelial cells and myoepithelial cells of the 21 total prepubertal heifers examined. Its presence hints at an underlying biological function but that function was not ascertained here. It will be up to the next researcher to deduce what YAP contributes to mammary growth in prepubertal dairy heifers.
- Acute and chronic heat stress alters the metabolic profile of skeletal muscle in growing swineWon, Samantha Gwai Lan (Virginia Tech, 2012-08-02)Heat stress (HS) causes significant losses to the U.S. swine industry in several production and health areas including efficient lean tissue accretion. Perturbations in skeletal muscle metabolism may participate in this defect. The study objectives were to examine the cellular bioenergetic profile in skeletal muscle of piglets subjected to thermal stress in utero and/or during postnatal life. To accomplish this, 96 offspring from 14 sows were prenatally exposed to 1 of 4 environmental treatments involving thermal neutral (TN, 25°C) or HS conditions (cyclical 28-34°C). Sows exposed to TN or HS throughout gestation are denoted TNTN and HSHS, respectively whereas sows heat-stressed for the first or second half of gestation are denoted HSTN and TNHS, respectively. At 14 weeks of age, offspring were exposed to one of two postnatal thermal environments, constant TN (21°C) or HS (35°C) for 24 hrs (acute study) or 5 weeks (chronic study). Pigs were sacrificed after treatment and longissimus dorsi skeletal muscle samples collected for molecular analyses. Differences (p<0.05) were observed in protein abundance of p-4eBP1 and total Rs6 and gene expression of Cox5B, CytB, EEF2, HK2, MURF, ND1, PGC-1α, SDHA, and TFAM during the acute heat stress study. Differences (p<0.05) were observed in protein abundance of 4eBP1, total Akt, and p-Rs6 and gene expression of CytB, MURF, and PGC-1α during the chronic heat stress study. These data indicate that acute postnatal HS alters skeletal muscle metabolism, which may favor a reduction in mitochondrial respiration and protein synthesis potentially via the mTOR pathway.
- Challenges in enriching milk fat with polyunsaturated fatty acidsLanier, Jennifer S.; Corl, Benjamin A. (Biomed Central, 2015-06-12)Milk fatty acid composition is determined by several factors including diet. The milk fatty acid profile of dairy cows is low in polyunsaturated fatty acids, especially those of the n-3 series. Efforts to change and influence fatty acid profile with longer chain polyunsaturated fatty acids have proven challenging. Several barriers prevent easy transfer of dietary polyunsaturated fatty acids to milk fat including rumen biohydrogenation and fatty acid esterification. The potential for cellular uptake and differences in fatty acid incorporation into milk fat might also have an effect, though this has received less research effort. Given physiological impediments to enriching milk fat with polyunsaturated fatty acids, manipulating the genome of the cow might provide a greater increase than diet alone, but this too may be challenged by the physiology of the cow.
- Cyclical heat stress during lactation influences the microstructure of the bovine mammary glandPerez-Hernandez, G.; Ellett, Mark D.; Banda, L. J.; Dougherty, D.; Parsons, Catherine L. M.; Lengi, A. J.; Daniels, Kristy M.; Corl, Benjamin A. (Elsevier, 2024-05-31)This study aimed to evaluate the effect of heat stress on mammary epithelial cell (MEC) losses into milk, secretory mammary tissue structure, and mammary epithelial cell activity. Sixteen multiparous Holstein cows (632 ± 12 kg BW) approximately 100 DIM housed in climate-controlled rooms were paired by BW and randomly allocated to one of 2 treatments, heat stress (HS) or pair-feeding thermoneutral (PFTN) using 2 cohorts. Each cohort was subjected to 2 periods of 4 d each. In period 1, both treatments had ad libitum access to a common TMR and were exposed to a controlled daily temperature-humidity index (THI) of 64. In period 2, HS cows were exposed to controlled cyclical heat stress (THI: 74–80), while PFTN cows remained at 64 THI and daily DMI was matched to that of the HS cows. Cows were milked twice daily, and milk yield was recorded at each milking. Individual milk samples on the last day of each period were used to quantify MEC losses by flow cytometry using butyrophilin as a cell surface marker. On the final day of period 2, individual bovine mammary tissue samples were obtained for histomorphology analysis, assessment of protein abundance, and evaluation of gene expression of targets associated with cellular capacity for milk and milk component synthesis, heat response, cellular proliferation, and autophagy. Statistical analysis was performed using the GLIMMIX procedure of SAS. Milk yield was reduced by 4.3 kg by HS (n = 7) compared with PFTN (n = 8). Independent of treatment, MEC in milk averaged 174 cells/mL (2.9% of total cells). There was no difference between HS and PFTN cows for MEC shed or concentration in milk. Alveolar area was reduced 25% by HS, and HS had 4.1 more alveoli than PFTN. The total number of nucleated MEC per area was greater in HS cows (389 ± 1.05; mean ± SE) compared with PFTN (321 ± 1.05); however, cell number per alveolus was similar between groups (25 ± 1.5 vs. 26 ± 1.4). There were no differences in relative fold expression for GLUT1, GLUT8, CSN2, CSN3, LALBA, FASN, HSPA5, and HSPA8 in HS cows compared with PFTN cows. Immunoblotting analyses showed a decrease in abundance for phosphorylated STAT5 and S6K1, and an increase in LC3 II in HS cows compared with PFTN cows. These results suggest that even if milk yield differences and histological changes occur in the bovine mammary gland after 4 d of heat exposure, MEC loss into milk, nucleated MEC number per alveolus, and gene expression of nutrient transport, milk component synthesis, and heat-stress-related targets are unaffected. In contrast, the abundance of proteins related to protein synthesis and cell survival decreased significantly, whereas proteins associated with autophagy were upregulated in HS cows compared with PFTN cows.
- Dairy Pipeline, September 2018Hanling, Haylee; Corl, Benjamin A.; Martel, Cynthia (Virginia Cooperative Extension, 2018-08-24)This issue has two articles. The first article discusses hairy heel warts caused by bacteria, symptoms including lameness, and treatment. The second article focuses on agriculture education in the public schools.
- Effect of drought stress on in vitro neutral detergent fiber digestibility of corn for silageFerreira, G.; Martin, L. L.; Teets, C. L.; Corl, Benjamin A.; Hines, S. L.; Shewmaker, G. E.; De Haro-Marti, M. E.; Chahine, M. (2021-03)The objective of this study was to determine the effect of drought stress on neutral detergent fiber (NDF) and lignin (LIG) concentrations and on in vitro dry matter digestibility (IVDMD) and in vitro neutral detergent fiber digestibility (IVNDFD) of leaf blades and stem internodes of corn for silage. Eight plots were blocked (i.e., 4 blocks) and randomly subjected to a watered (W) or non watered (NW) treatment. Within each block, plots were split into 7 sub-plots, to which 1 of 7 corn hybrids were randomly assigned. Before planting, all plots were irrigated with 150 mm of water to ensure a consistent emergence of corn seedlings. After this pre-planting irrigation, NW plots were not irrigated ever again. After planting, W plots were irrigated with 225 and 360 mm of water pre-tasseling and post-tasseling, respectively. Stem internodes and leaf blades from the second phytomer below (LOWER) and the second phytomer above (UPPER) the ear insertion were collected to determine tissue composition and digestibility. Drought stress increased the concentration of NDF in both leaf blades (628 vs. 613 mg NDF/g DM) and stem internodes (625 vs. 572 mg NDF/g DM). Drought stress decreased IVDMD in stem internodes (0.575 vs. 0.525 IVDMD) but had no effect on IVDMD of leaf blades (0.561 IVDMD). Similarly, drought stress decreased IVNDFD in stem internodes (0.422 vs. 0.391 IVNDFD) but had no effect on IVNDFD of leaf blades (0.536 IVNDFD). Drought stress increased the concentration of lignin in the cell wall of leaf blades (161 vs. 141 mg/g CW) but had no effect on stem internodes (266 mg/g CW). Under the conditions of this study, water supply had a minimal effect on lignin concentration in the cell wall and did not increase the in vitro digestibility of fiber in corn for silage. The latter observation is contrary to the general industry belief that water stress increases fiber digestibility in forages.
- Effect of probiotics or high incubation temperature on gene expression and cell organization of the small intestine and yolk sac of chicksJia, Meiting (Virginia Tech, 2021-11-30)The small intestine and yolk sac (YS) are important organs for nutrient absorption and innate immunity in chickens during the post-hatch or prehatch periods. These organs share a similar structure of epithelial cell-lined villi with tight junctions between adjacent cells. Probiotics have been reported to improve chicken growth performance and gut health including promotion of intestinal morphology. However, there are few studies that show the effect of probiotics on ontogeny of intestinal epithelial cells and antimicrobial peptides, or intestinal integrity in young healthy chicks. Heat stress during incubation was shown to increase mortality and decrease hatchability of chicks, while no studies have investigated the effect of heat stress on the integrity of the YS, which might be related to hatching performance. There were four studies conducted in this research: 1) a comparison of the effect of two probiotics on the ontogeny of small intestinal epithelial cells in young chicks; 2) the effect of two probiotics on mRNA abundance of tight junction proteins in the small intestine of young chicks; 3) the effect of high incubation temperature on mRNA abundance of tight junction proteins in the YS of broiler embryos; and 4) comparison of avian defense peptide mRNA abundance in the YS of broilers and layers. In study 1, Probiotics transiently decreased body weight gain (BWG) from day 2 to day 4, but did not affect body weight (BW) from day 2 to day 8, and small intestinal weight and intestinal morphology from day 2 to day 6. Probiotics did not affect marker gene expression of intestinal stem cells (Olfm4) and goblet cells (Muc2) in all small intestinal segments, but did increase expression of a marker gene of proliferating cells (Ki67), and decreased an antimicrobial peptide (liver-enriched antimicrobial peptide 2, LEAP2) in the jejunum at day 4. Probiotic 1 decreased PepT1, a marker of enterocytes in the duodenum at day 4. These results suggest that probiotics did not improve growth performance and intestinal morphology in young healthy chicks, but temporarily promoted intestinal epithelial cell proliferation and decreased LEAP2 antimicrobial peptide expression in the jejunum. In situ hybridization (ISH) showed that Ki67+ proliferating cells were mainly located in the crypt region and the blood vessels of villi. In study 2, Probiotic supplementation to newly hatched chicks for less than one week did not affect mRNA abundance of the tight junction proteins in the small intestine. Occludin (OCLN) mRNA, which was detected by ISH to be expressed in intestinal epithelial cells in both the villus and crypt regions, was greater in the duodenum of female chicks than males. In study 3, high incubation temperature starting from embryonic day 12 (E12) affected mRNA abundance of the tight junction proteins in the YS, including increased zonula occluden 1 (ZO1) at E13, increased junctional adhesion molecule A (JAMA) and heat shock protein 90 (HSP90) at E17, but decreased tight junction protein JAMA at E19 and OCLN at day of hatch (DOH). These results showed that the YS tight junction proteins were increased by short term heat exposure but decreased by long term heat exposure. In study 4, the expression of avian β defensin 10 (AvBD10), CATHs and toll-like receptors in the YS was examined. Toll-like receptors were highly expressed in the YS at early incubation stages (E7), while CATHs showed a peak expression from E9 to E13, which was similar to the expression pattern of AvBD10. CATHs and AvBD10 mRNA temporal expression patterns were similar in broilers and layers, while their expression levels were different. Layers, especially brown layers, had greater mRNA abundance for antimicrobial peptides such as AvBD10, CATH1, and CATH2 in the YS. These results demonstrate that the antimicrobial peptide temporal expression patterns in the YS are not affected by breed, but their expression levels are affected by breed. In summary, the small intestine and the YS are essential for nutrient uptake, innate immunity, and maintenance of integrity. The ontogeny of intestinal epithelial cells, such as proliferating cells can be modulated by probiotic supplementation. Similar to the small intestine, the YS can also express tight junction proteins, which can be affected by high incubation temperature. Antimicrobial peptide expression in the intestine of healthy young chicks is also transiently decreased by probiotic supplements. Avian defensin and cathelicidin expression patterns in the YS were not affected by breed.
- Effects of Feeding Hulless Barley (Hordeum vulgare L.) and Supplementing a Fibrolytic Enzyme on Production Performance, Nutrient Digestibility, and Milk Fatty Acid Composition of Lactating Dairy CowsYang, Yang (Virginia Tech, 2018-11-07)The overall objective of this study was to evaluate the effects of feeding hulless barley and supplementing a xylanase enzyme on production performance and nutrient utilization of lactating dairy cows. In study 1, we evaluated production performance, milk fatty acid composition, and nutrient digestibility in high-producing dairy cows consuming diets containing corn and hulless barley in different proportions as the grain source. We hypothesized that a plausible reduction in production performance would be explained by an altered rumen function, which would be reflected in a reduction of the proportion of de novo fatty acids in milk fat. The inclusion of hulless barley grain as the energy source in diets for lactating dairy cows resulted in similar production performance and nutrient utilization as corn grain. We concluded that hulless barley is as good as corn grain as an energy source and increasing NDF concentration in hulless barley-based diet is not necessary. In study 2, we evaluated production performance, nutrient digestibility, and milk fatty acid composition of high-producing dairy cows consuming diets containing hulled or hulless barley as the grain source. We hypothesized that rumen function is altered when cows are fed low-forage diets containing barley grains, and this altered rumen function would be reflected in lower production performance and a reduction of fatty acids synthesis in the mammary gland. Contrary to our expectations, feeding hulled barley or hulless barely based diets with different forage to concentrate ratios to lactating dairy cows resulted in similar production performance and nutrient utilization. We concluded that both hulled or hulless barley grains are good energy sources for sustaining high milk production and there is no need to increase NDF concentration in diet when using barley grain as the grain source. In study 3, we evaluated the effects of supplementing a xylanase enzyme on production performance and nutrient digestibility of lactating dairy cows fed diets containing corn or sorghum silage as the forage source. We hypothesized that supplementing a xylanase enzyme product in diets containing corn or sorghum silage increases NDF digestibility, and production performance of lactating dairy cows would also be improved due to enhanced fiber digestion. Supplementation of xylanase for 19 d did not affect cow performance and nutrient utilization. Supplementation of xylanase may require a longer period of time to show any response in production performance and nutrient digestibility. We concluded that supplementing xylanase to cows fed corn or sorghum silage-based diets did not improve fiber digestion. But for feeding hulled or hulless barley grains to lactating dairy cows, increased NDF concentration in diets is not necessary and hulless barley is good as corn grain for feeding lactating dairy cows as the grain source.
- Effects of low linolenic soy oil on pre-malignant human mammary epithelial cell progressionMcCall, Elaine Teresa (Virginia Tech, 2008-12-10)Beginning January 1, 2006 the U.S. Food and Drug Administration mandated that the amount of trans fats per serving be listed on the Nutrition Facts panel. Consequently new soybean breeds that would no longer be subject to the hydrogenation process, thus reducing trans fats, were developed. By traditional plant breeding techniques, plant breeders have developed a low linolenic soybean with 83.36% less alpha-linolenic acid (ALA; omega-3) than conventional soybean. A number of studies have demonstrated that the influence of dietary fats on cancer depends on the quantity as well as the type of lipids and diets with a disproportionately high omega-6 (n-6)/omega-3 (n-3) ratio are thought to contribute to cardiovascular disease, inflammation and cancer. Conventional soybean oil (SO) has an n-6/n-3 ratio of 8/1 while the new low linolenic soy bean oil (LLSO) has an n-6/n-3 ratio of 56/1. In this study, we evaluated the effects of dietary LLSO, SO and lard on the progression of breast cancer (BC). Thirty-five, 6-wk old, ovariectomized, athymic mice received human pre-malignant breast cells (MCF-10AT1 1 x 105 cells/40μl/ Matrigel/spot, 4 spots/mouse). Mice were divided into three groups and then fed isocaloric and isonitrogenous diets with disparate fat sources: LLSO (20% of total energy intake), SO (20%) and lard (20%). The dietary treatment lasted 24 weeks upon which the study was terminated and tumors, tissues and blood samples were analyzed. Average tumor surface area at termination for the LLSO group was 45.11 ± 4.46 mm2, 40.08 ± 4.2 mm2 for lard and 56.63 ± 5.42 mm2 for SO. Messenger RNA (mRNA) expression of HER2/neu, epidermal growth factor receptor (EGFR), H-ras, Bcl-2, cyclooxygenase-2 (COX-2), vascular epidermal growth factor (VEGF), and fatty acid synthase (FAS) in tumors were analyzed using quantitative real time-polymerase chain reaction (qRT-PCR). We found that dietary LLSO supplementation significantly (p < 0.05, Tukey's test) increased tumor expression of oncogenes HER2/neu, EGFR, FAS, and H-ras, but not in the SO or lard supplemented groups. Relative mRNA expression was also significantly increased in both LLSO and SO groups, however, there was no marked difference in mRNA expression for Bcl-2 and COX-2. Removed tumors were evaluated microscopically for histologic lesion progression corresponding to human breast cancer progression. Tumors from the LLSO group showed more advanced lesions (grade 2) (p < 0.05, Chi Square test) with areas of four or more layers of epithelial cells and irregularly shaped lumens. These data suggest that dietary intake of LLSO may accelerate mammary tumor progression at a faster rate than conventional SO or lard.
- Effects of Maturity at Harvest of Triticale and Dietary Forage Inclusion on Production Performance, Nutrient Utilization, and Milk Fatty Acid Profile of Lactating Dairy CowsSchultz, Milton Emanuel (Virginia Tech, 2024-01-12)The objective of this study was to evaluate the impact of maturity at harvest and dietary inclusion rate of triticale silage on the production performance and nutrient utilization of lactating dairy cows. The hypothesis was that the production performance of lactating dairy cows would increase when consuming triticale harvested and ensiled at the boot stage of maturity (BS), as compared to consuming triticale harvested and ensiled at the soft-dough stage of maturity (SDS), and that the difference would be greater in high-forage diets. A single field of triticale was planted, and harvested at the BS or SDS of maturity, and the harvested forage was ensiled in separate bunker silos. Eight primiparous and 16 multiparous Holstein cows were assigned to 1 of 4 diets in a replicated 4 × 4 Latin square design with 21-d periods. Cows were fed once daily (10:00 a.m.) using a Calan gate system (American Calan Inc., Northwood, NH). Diets included BS or SDS triticale silage with dietary forage inclusion of 54% (high-forage; HF) or 36% (low-forage; LF). Cows consuming diets containing BS silage produced more milk per day than cows consuming diets containing SDS silage. This response was observed when feeding both LF and HF diets. The maturity of the forage (BS vs. SDS) did not affect the apparent total tract digestibility of neutral detergent fiber (NDF) or the yield of energy-corrected milk. Cows consuming diets containing BS silage yielded more milk protein and lactose per day than cows consuming diets containing SDS silage, and we also observed this response when feeding both LF and HF diets. When fed the LF diets, cows had a higher apparent total-tract digestibility on CP and NDF, whereas apparent total-tract starch digestibility was higher in cows fed HF diets. Cows consuming LF diets exhibited higher levels of de novo fatty acid synthesis compared to those on HF diets. Feeding LF diets yielded more C14:1, C18:1 trans-9, and unknown fatty acids, while C16:0, C18:1 trans-10, CLA cis-9, trans-11, and CLA trans-10, cis-12 fatty acids yielded more in cows fed with SDS diets. Notably, CLA cis-9, trans-11, and CLA trans-10, cis-12 fatty acids were highest in SDS diets. Additionally, stearic fatty acid (C18:0) concentrations increased in HF diets and SDS silage. In conclusion, the study found that triticale maturity at harvest minimally affects dairy cow performance, with consistent milk production across harvest stages. While nutrient variations were observed, the conclusion emphasizes the importance of considering not only forage quality but also agronomic management and planting schedules for subsequent crops when deciding on harvest timing.
- The Enhanced Milk Yield Effect of Early Lactation Increased Milking Frequency and Bovine Somatotropin Is Additive and Not SynergisticHanling, Haylee H.; McGilliard, Michael L.; Corl, Benjamin A. (MDPI, 2023-07-05)Dairy farm profitability depends on milk yield, so the dairy industry manages cows to improve their productivity. Both bovine somatotropin (bST) and early lactation increased milking frequency (IMF) and milk yield (MY) in dairy cows. The objective of this study was to evaluate the effects of mid-lactation bST administration on milk production in established lactation when combined with the milk yield carry-over effect from early lactation IMF. Thirteen multiparous Holstein cows were milked unilaterally for 20 days in early lactation. The left udder halves were milked twice daily (2X) and the right udder halves were milked four times daily (4X). Udder halves milked 4X produced 8.60 ± 1.40 kg more than 2X on the final day of IMF treatment. Cows were then returned to 2X milking for the remainder of lactation and sampled on alternate days from 74–94 days in milk (DIM). Bovine somatotropin was administered to all cows at 80 DIM. The 4X halves continued to make 2.66 ± 0.12 kg/d more milk than 2X through 94 DIM. Fat, protein, and lactose yields were significantly greater in the 4X halves compared to the 2X from 74–94 DIM. Overall milk yield increased by 2.71 kg/d with bST administration. However, there was no significant interaction between MF and bST administration. We can infer from these data that the mechanisms by which bST and IMF in early lactation increase milk yield are complementary due to their non-synergistic nature of enhancing MY.
- Estrogen signaling interacts with Sirt1 in adipocyte autophagyTao, Zhipeng (Virginia Tech, 2019-06-18)Obesity is a rapidly growing epidemic. It is associated with preventable chronic disease and vast healthcare cost in the United States (about 200 billion per year). Therefore, dissecting pathogenic mechanisms of obesity would provide effective strategies to prevent its development and reduce related cost. Obesity is characterized by excessive expansion of white adipose tissue (WAT). Autophagy, a cellular self-digestive process, is associated with WAT expansion and may be a promising target for combating obesity. Both hormone signaling (e.g., ERα) and energy sensing factors (e.g., Sirt1) control metabolism and prevent adiposity, and in which they have been shown to play collaborate roles. However, how autophagy is involved in ERα and Sirt1's inhibitory roles on adiposity is unknown. These questions have been addressed in my dissertation studies. To address this fundamental questions, I have established a method to monitor autophagy flux during adipocyte differentiation, which better reflected the dynamic process of autophagy. Compared with preadipocytes, autophagy flux activity was increased in mature adipocytes after differentiation. And then, my thesis project has addressed three main questions. Firstly, the gender difference in visceral fat distribution (Males have higher deposit of visceral fat than females) is controlled by an estradiol (E2)-autophagy axis. In C57BL/6J and wild type control mice, a higher visceral fat mass was detected in the males than in the females, which was associated with lower expression of estrogen receptor (ER) and more active autophagy in males vs. females. ER knockout normalized this difference. Mechanistically, E2-ER- mTOR-ULK1-autophagy signaling contributed to the gender difference in visceral fat distribution. Secondly, in vitro and in vivo studies demonstrated that Sirt1 suppressed autophagy and reduced adipogenesis and adiposity via inducing mTOR-ULK1 signaling. Specific activation and overexpression of Sirt1 induced mTOR-ULK1 signaling to suppress autophagy and adipogenesis. And knockdown of Sirt1 exhibited opposite effects. The first and second studies revealed that ER and Sirt1 acted on mTOR-ULK1 signaling pathway, underlying the importance of their interaction in inhibiting autophagy and adipogenesis. As such, the third study was conducted and it unraveled that ER acted as upstream of Sirt1, possibly through its direct binding to Sirt1 promoter. Specifically, E2 signaling suppressed autophagy and adipogenesis. But when Sirt1 was knockdown, the effects of E2 on autophagy and adipogenesis were abolished. Taken together, my dissertation project underscores the importance for future research to consider gender difference and how E2-ER-autophagy axis contributes to this difference in other metabolic diseases. Also, the unraveled interaction between ERα and Sirt1 might lead to new therapeutic approach to adiposity and metabolic dysfunction in post-menopausal women or individuals with abnormal estrogen secretion. For example, dietary intervention or exercise challenge to activate Sirt1 may partially compensate estrogen deficiency.
- Evaluating the duration of increased milking frequency during early lactation for increased yield through lactationTate, Kaley Renee (Virginia Tech, 2018-07-11)Increasing the milking frequency of early lactation dairy cows increases their milk yield, both during increased milking and after the cows are returned to a normal twice-daily milking schedule. When milked four times a day (4X) for only the first twenty-one days of lactation, the right udder half produces 3 kg/d more milk than the left half, which is milked only two times a day (2X) over the course of lactation (Hale et al., 2003). Alterations to this increased milking frequency (IMF) approach have been investigated in order to maximize production of the animals and determine the most efficient practice for producers. The aim of this study was to determine the appropriate duration of early lactation IMF treatment by increasing milking frequency of early lactation cows for various lengths of time, and subsequently increasing the use of this management practice on Virginia dairy farms. The right udder half of twenty-three primiparous and multiparous Holstein cows were milked 4X for 10, 20, or 40 days at the beginning of lactation, and the left udder half 2X for the entire lactation. Udder-half milk yields were measured at various time points throughout lactation and used to calculate the difference between right (4X) and left (2X) udder halves. Overall, treatment did not have a significant effect on milk yield difference throughout the entire lactation; the udder half differences for each group were -0.45 kg, 1.92 kg and 4.62 kg for the 10 d, 20 d and 40 d treatments (P > 0.05). In addition to the IMF portion of the experiment, two different methodologies were used to investigate the possible mechanism of local regulation of milk yield in response to IMF treatment. Milk fatty acid analysis was performed on milk samples obtained from the above experiment. Three different groups of fatty acids were analyzed to detect potential changes in the right udder half (4X) when compared to the left (2X); the three groups were denovo, C16, and preformed fatty acids. There was no significant effect of treatment on fatty acid composition of right and left udder halves for any of the three groups (P > 0.05). The second methodology used to explore a possible mechanism behind increased milk yield following IMF treatment was immunohistochemistry of mammary gland tissue samples obtained after IMF treatment in a previous experiment. The key target investigated was a component of the Hippo signaling pathway, Yes-associated protein (YAP). Intensity of YAP staining in the cytoplasmic area of mammary epithelial cells (MEC) and number of YAP-positive stained nuclei located in the MEC were quantified for each of the images obtained. There was no effect of treatment or day on intensity of staining (P > 0.05) with no difference in the intensity of staining between 4X and 2X samples or d 21 and d 60 samples. However, the interaction for treatment x day tended to be significant (P < 0.06), with the d 60 samples tending to have higher intensity of staining than d 21 samples. For YAP-positive nuclei, there was a significant effect of day (P < 0.05), with d 60 samples having significantly more YAP positive nuclei. There was not a significant effect of treatment or treatment x day interaction (P > 0.05) with 2X and 4X samples having the same number of YAP positive nuclei. Results from the first experiment reveal that 40 d of IMF during early lactation is sufficient to produce an increase in milk and component yields throughout lactation. This practice could be implemented on Virginia dairy farms as a way to increase efficiency and milk yield per cow. Results from the second half of this research indicates that further research is needed to investigate the fatty acid content of milk from cows subject to IMF treatment during early lactation. In addition, YAP potentially plays a role in the changes occurring in the mammary gland, with increased intensity of YAP staining and increased number of YAP positive nuclei observed at 60 DIM. Understanding of this protein and its involvement in the mammary gland could lead to identifying a mechanism for which this increase in milk yield and components following IMF is occurring. Further research needs to be done to provide results supporting the current experiment.
- Evaluating the inclusion of alfalfa hay in diets fed to pregnant and non-lactating Holstein cows during the prepartum periodThompson, Ahmerah Unique (Virginia Tech, 2023-02-07)The study objectives were to determine the dry matter intake, urine pH, Ca concentration in blood, Ca output in urine, and incidence of hypocalcemia from pregnant, non-lactating dairy cows during the prepartum period consuming diets containing either grass hay (GH) or alfalfa hay (AH) with the inclusion of either calcium chloride (CL) or polyhalite mineral (PO). Eighty Holstein cows in their 2nd parity or greater were fed an experimental diet according to a 2 2 factorial arrangement of treatments during the prepartum period (21 d before calving). All diets had a dietary cation-anion difference (DCAD) below -190 mEq/kg/DM. Grass hay contained 7.5% CP, 74.9% NDF, 0.36% Ca, 0.02% Na, 1.88% K, 0.38% Cl, and 0.15% S. Alfalfa hay contained 19.6% CP, 45.6% NDF, 1.52% Ca, 0.16% Na, 2.5% K, 0.77% Cl, and 0.32% S. Cows consuming grass hay tended to consume more dry matter than cows consuming alfalfa hay (11.6 vs 10.8 kg/d), but dry matter intake (DMI) was not affected by the acidogenic products. Urine pH decreased below 6.5 for all diets and was greatest for cows consuming the GHPO diet. The concentration of calcium in plasma decreased significantly (P < 0.01) around calving but neither the hay type (P=0.86) nor the acidogenic product (P =0.81) affected it. Urinary calcium output was less for cows consuming the GHPO diet. Cows consuming diets containing alfalfa hay had a greater incidence of normocalcemia (37 and 40% for AHCL and AHPO, respectively) than cows consuming diets containing grass hay (20 and 25% for GHCL and GHPO, respectively). In conclusion, alfalfa hay can be included in prepartum diets without necessarily increasing the incidence of hypocalcemia, and the cation-anion difference of alfalfa hay is a determinant of whether it can be included in the prepartum diet.
- Extracellular Proteoglycan Decorin in Bovine Mammary PhysiologyTucker, Hannah L. (Virginia Tech, 2017-09-27)The majority of bovine mammary gland research focuses on the main cell types - mammary epithelial cells and fibroblasts. However, the extracellular matrix (ECM) within the mammary gland is also of importance for its ability to regulate cell shape, proliferation, polarity, differentiation, gene transcription, protein synthesis, and secretion. Decorin is an ECM proteoglycan known to impact mammary cell proliferation in humans and rodents. Prior to this work, very little was known about decorin in bovine mammary biology. A series of bovine mammary cell culture experiments was conducted. The first experiment demonstrated existence of decorin pathway molecules in immortalized bovine mammary cells, but stopped short of demonstrating mature decorin proteoglycan deposition into the extracellular space. During the investigation it was noted that when cultured under basal conditions, intracellular decorin core protein (DCP) localization patterns appeared to be coordinated with specific phases of the cell cycle. Therefore, the objective of the second set of experiments was to characterize DCP localization patterns in bovine mammary epithelial cells (BME) at known phases of the cell cycle. The work was carried out in two sequential experiments. The hypothesis of the first experiment was that DCP accumulates in BME during S-phase of the cell cycle; the research rejected this hypothesis. The hypothesis of the second experiment, formulated after completion of the first experiment for this objective, was that DCP accumulates in BME during metaphase of the cell cycle. However, the experiment was unable to confirm of reject this hypothesis. Major findings were that both BME and mammary fibroblasts produce DCP and known decorin pathway molecules. BME produce intracellular DCP, but it is not accumulated during the S-phase of the cell cycle. However, it is still unknown if DCP is accumulated in BME during metaphase. Future research should focus on further characterization of decorin and its associated pathway molecules to learn if decorin induces proliferation or apoptosis of bovine mammary epithelial cells. This is important because number and activity of mammary epithelial cells ultimately determine milk yield in dairy cows. Fundamental knowledge gained in this research area may one day be applied at the animal-level and lead to gains in milk production efficiency by altering the cellular composition of mammary glands.
- Factors affecting the nutritional composition and digestibility of corn for silage: Cover crops and cell wall compositionBrown, Alston Neal (Virginia Tech, 2017-09-15)Corn silage is one of the major components in dairy cattle rations in the United States. Many factors affect the nutritional composition of corn for silage, such as cropping system, including cover crops, and the composition of the corn plant cell wall. The objectives of the first study were to determine the nutritional quality of different winter crops for silage and to determine the impact of the various winter crops on the succeeding productivity of corn and sorghum. Experimental plots were planted with 15 different winter crop treatments: 5 winter annual grasses in monoculture or with one of two winter annual legumes (crimson clover [CC] and hairy vetch [HV]). After harvesting the winter crops, each plot was planted with either corn or forage sorghum. Crimson clover increased DM yield compared to monocultures but HV did not. Adding legumes increased the crude protein concentration, but reduced the fiber and sugar concentrations of the forages. Even though in vitro neutral detergent fiber digestibility was reduced with the addition of legumes, the concentration of highly digestible non-fibrous components is greater in the mixtures than the monocultures, increasing the nutritive value of the silage. The objective of the second study was to determine the cell wall (CW) composition along the corn stalk. Three phytomers of corn plants were examined: center (C) of ear insertion, upper (U) and lower (L) phytomers. Each phytomer was cut into 4 sections: top (T), middle (M), bottom (B), and node (N). The CW, uronic acid (UA), glucose (GLU), and lignin concentrations did not change among phytomers. The concentrations of arabinose (ARA) and xylose (XYL) were greater in the U than in the L phytomers. Concentrations of CW, ARA, and XYL increased from B to T within the phytomer, but UA and GLU concentrations decreased from B to T. Lignin did not change within the phytomer. In mature corn for silage, changes within the corn internode may be more useful in determining how the environment changes the CW.
- A flow cytometric method for measuring and isolating mammary epithelial cells from bovine milkLengi, Andrea; Makris, Melissa; Corl, Benjamin A. (2021-11-01)Sampling frequent timepoints of mammary signaling pathways is not possible with tissue biopsies. We have validated a flow cytometry and cell sorting procedure for isolating live bovine mammary epithelial cells from the somatic cell populations in milk using butyrophilin 1A1 as a marker for mammary epithelial cells and CD45 as a marker for hematopoietic cells. Hoechst 33342 staining and propidium iodide exclusion were used to select for nucleated live cells. Positive selection of butyrophilin (BTN) expressing cells was performed by Fluorescence Activated Cell Sorting. Quantitative Real-Time PCR performed on mRNA isolated from these cells showed a 226-fold increase in kappa casein mRNA expression in BTN single positive cells compared to unsorted cells, while CD45 single positive cells showed a significant decrease. A negative selection strategy for cells not expressing the hematopoietic cell marker CD45 also resulted in a cell population with a 196-fold increase in kappa casein mRNA expression compared to unsorted cells. We found no enrichment of kappa casein mRNA expression after sorting cells using cytokeratin antibodies. The non-invasive assays described here can allow for daily or more frequent sampling timepoints for measurement of mammary epithelial cells during the course of lactation.
- FoxO1 in the regulation of adipocyte autophagy and biologyLiu, Longhua (Virginia Tech, 2016-12-08)Obesity is a rapidly growing epidemic in the USA and worldwide. While the molecular and cellular mechanism of obesity is incompletely understood, studies have shown that excess adiposity may arise from increased adipogenesis (hyperplasia) and adipocyte size (hypertrophy) . Emerging evidence underscores autophagy as an important mediator of adipogenesis and adiposity. We are interested in the upstream regulator of adipocyte autophagy and how it impacts adipocyte biology. Given that metabolic stress activates transcription factor FoxO1 in obesity, my dissertation project is designed to depict the role of FoxO1 in adipocyte autophagy and biology. We found that FoxO1 upregulation was concomitant with elevation of autophagy activity during adipogenesis. Inhibition of FoxO1 suppressed autophagy flux and almost completely prevented adipocyte differentiation. For the first time, we found that the kinetics of FoxO1 activation followed a series of sigmoid curves that showed multiple activation-inactivation transitions during adipogenesis. Our study provides critical evidence casting light on the controversy in the literature that either persistent inhibition or activation of FoxO1 suppresses adipogenesis. In addition, we identified two central pathways that FoxO1-mediated autophagy regulated adipocyte biology: (1) to control lipid droplet growth via fat specific protein 27 (FSP27) in adipocytes; and (2) to differentially regulate mitochondrial uncoupling proteins (UCP) that have been implicated in browning of white adipose tissue and redox homeostasis. Mechanistically, FoxO1 appears to induce autophagy through the transcription factor EB (Tfeb), which was previously shown to regulate both autophagosome and lysosome. Chromatin immunoprecipitation assay demonstrated that FoxO1 directly bound to the promoter of Tfeb, and inhibition of FoxO1 attenuated the binding, which resulted in reduced Tfeb expression. To investigate the role of FoxO1 in vivo, we have developed mouse models to modulate FoxO1 in adipose tissue using an inducible Cre-loxP system. Tamoxifen is widely used to activate the inducible Cre recombinase that spatiotemporally control target gene expression in animal models, but it was unclear whether tamoxifen itself may affect adiposity and confounds phenotyping. Part of my dissertation work was to address this important question. We found that tamoxifen led to reduced fat mass independent of Cre, which lasted for 4-5 weeks. Mechanistically, Tamoxifen induced reactive oxygen species (ROS) and augmented apoptosis. Our data reveals a critical period of recovery following tamoxifen treatment in the study of inducible knockout mice. Together, my dissertation work demonstrates FoxO1 as a critical regulator of adipocyte autophagy via Tfeb during adipogenesis. FoxO1-mediated autophagy controls FSP27, lipid droplet growth, and mitochondrial uncoupling proteins. Further study of FoxO1-autophagy axis in obese subjects is of physiological significance, and the investigation is under way.
- Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryosCarey, Kayla; Ryu, Junghyun; Uh, Kyungjun; Lengi, Andrea J.; Clark-Deener, Sherrie; Corl, Benjamin A.; Lee, Kiho (2019-05-06)Background The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.
- Genetic and Maternal Factors Underlying Early Milk Production and Their Influence on Calf HealthNin-Velez, Alexandra Irma (Virginia Tech, 2020-09-11)The quality of early milk produced by dams is affected by various factors (i.e. breed, age, parity, environment, nutrition, management). The impact of these factors on the quality of milk then have subsequent effects on calf health and development. Producers are responsible for following guidelines in order to ensure that they feed calves optimal quality milk in order to produce a healthy animal. They can also regulate factors such as environment and nutrition of the dam in order to produce better quality early milk. However, even after maximizing these factors there is still high mortality rate among pre-weaned calves, therefore, other factors such as mode of birth and genetics need to be studied to determine impacts on early milk quality and make further improvements to calf health and decrease mortality. Two experiments were conducted in order to study the effects of maternal and genetic factors on early milk production and to determine relationships that exist with calf health. The objective of the first study was to determine the effects that the mode of delivery had on early milk composition, and on the rumen microbiome of calves. We hypothesized that mode of birth would impact early milk composition, and, in turn, influence the microbial phyla in the calf gut. The second study had three objectives: 1) establish phenotypic relationships between colostrum composition traits, milk production traits, and calf health, 2) determine impact of breed and season on colostrum production and 3) ) elucidate the genetic parameters (i.e. heritability, genotypic, and phenotypic correlations) among colostrum production and milk production We hypothesized that colostrum composition and production differ among breeds and by season and that individual components influence calf health. Additionally, we hypothesized that colostrum quality traits (i.e. Brix score and volume) are heritable. For the first study Charolaise (CHAR; n = 23) and Angus (ANG; n = 15) dams were divided into two experimental groups; dams underwent vaginal (VD; n= 25) or cesarean (CD; n= 13) deliveries. Early milk samples were collected and quantified for protein, lactose, somatic cell count, and fatty acid concentrations. After parturition calves were separated based on dams experimental group. Rumen fluid was collected from calves on d 1, 3, and 28 post-partum. Extracted DNA from fluid were used for metagenomic sequencing (ANG calves, n=11; CHAR calves, n=13). Samples were run on the HiSeq 2500 platform as paired end reads according to Ilumina's standard sequencing protocol. A regression analysis was done in SAS using PROC GLM and regressing mode of birth on milk components for d 1,3, and 28. After, milk components found to be significantly impacted by mode of birth were regressed against microbial counts. Results showed that VD dams were more likely to have increased (P 0.05) protein, solids non-fat, and lactose on d 1 and 3, but decreased (P < 0.05) urea concentrations. Similarly, short, medium, and long-chain fatty acids were increased (P 0.05) in VD d 3 milk. Changes in true protein elicited a decrease (P 0.05) in rumen fluid Actinobacteria and Proteobacteria; whereas, both solids non-fat and lactose were associated with an increased (P 0.05) response in d 1 transition milk. No significant results for d 28 of sampling were observed. Based on our results we suggest that mode of birth influences protein concentrations in early milk. However, only a slight impact on the overall dynamics of the calf rumen was observed with the microbiome remaining relatively stable on the phyla level in response to changes in protein concentration. The second study looked into relationships between colostrum composition traits, management practices, and calf health, as well as determined heritability and genetic correlations for colostrum quality traits. Values for test-day milk, protein, fat, and somatic cell count (SCS) for Holstein (HO, n= 250) and Jersey (JE, n=289) cows were obtained from the Animal Genomic and Improvement laboratory server at the USDA. Brix score, colostrum weight, dam age, parity, and 3-month season of calving were also recorded. After, colostrum samples from JE cows were sent to DHIA where compositional measurements were obtained (i.e. true protein, fat, lactose, SCS, solid non-fats). Lactoferrin concentration for JE colostrum samples was also determined via ELISA. Calf blood samples were collected within 72 h post-partum and total serum protein (TSP) quantified to determine success of passive immunity transfer. Additionally, farm staff were instructed to record colostrum source for 1st feeding (i.e. dam, mix, other), freshness for 1st feeding (frozen vs fresh), Brix score of colostrum fed, volume of colostrum fed, and birth weight. A PROC Mixed with LSMEANS was performed in SAS to determine relationships between colostrum components, test day components, and quality traits for season, breed, and the interaction between season and breed. Also, PROC Mixed with LSMEANS was used to determine relationships of calf health with environment, management, and colostrum components. Additionally, a Pearson correlation was used to determine relationships between colostrum components and quality traits. Results for Holstein and Jersey showed that both colostrum Brix and volume (P < 0.001) differed by breed. Colostrum volume (P < 0.001), lactose (P < 0.001), and lactoferrin (P = 0.002) varied significantly by season. Additionally, test day milk (P = 0.046), fat (P = 0.012), and protein (P = 0.003) varied significantly by season. Moreover, a significant season and breed interaction (P = 0.028) was observed solely for colostrum volume. Calf health models indicated that TSP, colostrum total protein and solid non-fats impacted incidence of respiratory illness, but no factor significantly impacted incidence of scours. Results for Pearson correlation indicated strong correlations between true protein and solid non-fats and Brix (r = 0.99; 0.86). Lactoferrin also had moderate negative correlations with volume and lactose (r = -0.35; -0.33). Heritability and repeatability's were calculated using BLUPF90 family of programs. A single-trait repeatability animal model was used and included a 1-vector phenotype (Brix or Colostrum weight), fixed effects (i.e. calving year, parity, 3-month season of calving, and age at calving), additive genetic variance, random permanent environment effects, and random residual effects. A series of bivariate models were used to calculate genetic correlations of Brix score and colostrum weight with test-day compositional traits. Heritability estimates results for Holstein cow Brix and colostrum weight, were 0.25 and 0.15. Jersey cow heritability estimates were 0.36 and 0.47 respectively. We also observed some significant genetic correlations with Holstein Brix score and test-day milk (-0.23), fat (0.54), and SCS (0.29) having moderate correlations. Holstein colostrum weight had a strong correlation with test-day milk (0.96). Jerseys had strong genetic correlation of Brix score with colostrum weight (-0.98). Low to moderately heritability was observed for Brix score and colostrum weight in both breeds making them receptive to genetic selection in order to improve breeding programs. In conclusion, mode of birth significantly impacted colostrum composition which had subsequent effects on abundance of rumen microbiota. Colostrum Brix and volume were impacted by breed, season, and interaction, and calf incidence of disease was impacted by colostrum composition and environment. Additionally, two factors influencing colostrum quality (Brix score and colostrum weight) were found to be low to moderately heritable and have moderate to strong genetic correlations to compositional traits. Strong significant relationships were also found between colostrum compositional traits and colostrum quality traits. Therefore, incorporating quality traits into breeding programs has the potential to influence compositional traits which, in turn, can impact calf health and development by the interactions that exist between composition and microbial abundance in the rumen.
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