Form of calf diet and the rumen. II: Impact on volatile fatty acid absorption.

Diet is known to affect rumen growth and development. Calves fed an all-liquid diet have smaller and less developed rumens and a decreased ability to absorb volatile fatty acids (VFA) compared to calves fed both liquid and dry feed. However, it is unknown how rumens respond when challenged with a defined concentration of VFA. The objective of this study was to assess the effects of 2 different feeding programs on VFA absorption in preweaned calves. Neonatal Holstein bull calves were individually housed and randomly assigned to 1 of 2 diets. The diets were milk replacer only (MRO; n = 5) or milk replacer with starter (MRS; n = 6). Diets were isoenergetic (3.87 ± 0.06 Mcal of metabolizable energy per day) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Milk replacer was 22% crude protein, 21.5% fat (dry matter basis). The textured calf starter was 21.5% crude protein (dry matter basis). Feed and ad libitum water intakes were recorded daily. Calves were exposed to a defined concentration of VFA buffer (acetate 143 mM, propionate 100 mM, butyrate 40.5 mM) 6 h before euthanasia on d 43 ± 1. Rumen fluid samples were obtained every 15 to 30 min for 6 h to measure the rate of VFA absorption. Rumen tissues were obtained from the ventral sac region and processed for morphological and immunohistochemical analyses of the VFA transporters monocarboxylate transporter 1 (MCT1) and 4 (MCT4). Body growth did not differ between diets, but empty reticulorumens were heavier in MRS than MRO calves (0.67 vs. 0.39 ± 0.04 kg) and MRS calves had larger papillae areas (0.76 vs. 15 ± 0.08 mm2). We observed no differences between diets in terms of the abundance of MCT1 and MCT4 per unit area. These results indicate that the extrapolated increase in total abundance of MCT1 or MCT4 in MRS calves was not due to increased transporter density per unit area. Modeled VFA absorption metrics (flux, mmol/h, or 6 h absorbed VFA in mmol) were not different across diets. These results demonstrate that the form of calfhood diet, whether solely MR or MR and starter, does not alter VFA absorption capacity when the rumen is exposed to a defined concentration of VFA at 6 wk of age.

Form of calf diet and the rumen. I: Impact on growth and development.

Preweaning diet is known to affect rumen tissue appearance at the gross level. The objectives of this experiment were to investigate effects of different preweaning diets on the growth and development of the rumen epithelium and on putative rumen epithelial stem and progenitor cell measurements at the gene and cell levels. Neonatal Holstein bull calves (n = 11) were individually housed and randomly assigned to 1 of 2 diets. The diets were milk replacer only (MRO; n = 5) or milk replacer with starter (MRS; n = 6). Diets were isoenergetic (3.87 ± 0.06 Mcal of metabolizable energy per day) and isonitrogenous (0.17 ± 0.003 kg/d of apparent digestible protein). Milk replacer was 22% crude protein, 21.5% fat (dry matter basis). The textured calf starter was 21.5% crude protein (dry matter basis). Water was available ad libitum and feed and water intake were recorded daily. Putative stem and progenitor cells were labeled by administering a thymidine analog (5-bromo-2'-deoxyuridine, BrdU; 5 mg/kg of body weight in sterile saline) for 5 consecutive days and allowed a 25-d washout period. Calves were killed at 43 ± 1 d after a 6 h exposure to a defined concentration of volatile fatty acids. We obtained rumen tissue from the ventral sac and used it for immunohistochemical analyses of BrdU (putative stem and progenitor cells) and Ki67 (cell proliferation), gene expression analysis, and morphological measurements via hematoxylin and eosin staining. Epithelial stem and progenitor cell gene markers of interest, analyzed by real-time quantitative PCR, were ß1-integrin, keratin-14, notch-1, tumor protein p63, and leucine-rich repeat-containing G protein-coupled receptor 5. Body growth did not differ by diet, but empty reticulorumens were heavier in MRS calves (MRS: 0.67 ± 0.04 kg; MRO: 0.39 ± 0.04 kg). The percentage of label-retaining BrdU basale cells was higher in MRO calves than in MRS calves (2.0 ± 0.3% vs. 0.3 ± 0.2%, respectively). We observed a higher percentage of basale cells undergoing proliferation in MRS calves than in MRO calves (18.4 ± 2.6% vs. 10.8 ± 2.8%, respectively). Rumen epithelial gene expression was not affected by diet, but the submucosa was thicker in MRO calves and the epithelium and corneum/keratin layers were thicker in MRS calves. Presumptive stem and progenitor cells in the rumen epithelium were identifiable by their ability to retain labeled DNA in the long term, changed proliferative status in response to diet, and likely contributed to observed treatment differences in rumen tissue thickness.

Effects of Staphylococcus aureus intramammary infection on the expression of estrogen receptor α and progesterone receptor in mammary glands of nonlactating cows administered estradiol and progesterone to stimulate mammary growth.

Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to alter mammary structure and negatively affect the amount of mammary epithelium in the gland. Mechanisms responsible for the observed changes in mammary growth during an IMI are poorly understood, yet the importance of the key mammogenic hormones driving mammary growth is well recognized. This study's objective was to characterize the expression of estrogen receptor α (ESR1) and progesterone receptor (PGR) in mammary glands stimulated to grow and develop in the presence or absence of an IMI as well as preliminarily characterize myoepithelial cell response to IMI. Mammary growth was stimulated in 18 nonpregnant, nonlactating dairy cows using subcutaneous estradiol and progesterone injections, and 2 culture-negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) postchallenge and examined using immunofluorescence microscopy to quantify positive nuclei and characterize staining features. There tended to be a greater number of ESR1-positive nuclei observed across 8 random mammary parenchyma fields of view in saline quarters than in Staph. aureus quarters (201 vs. 163 ± 44 nuclei). Saline quarters also contained a greater number of PGR-positive nuclei (520 vs. 440 ± 45 nuclei) and myoepithelial cells (971 vs. 863 ± 48 nuclei) than Staph. aureus-challenged quarters. However, when ESR1, PGR, and myoepithelial nuclei counts were adjusted for Staph. aureus quarters containing less epithelium, differences between quarter treatments abated. The examined ESR1 and PGR staining characteristics were similar between saline and Staph. aureus quarters but were differentially affected by day of tissue collection. Additionally, nuclear staining area of myoepithelial cells was greater in Staph. aureus quarters than in saline quarters. These results indicate that IMI had little effect on the number or staining characteristics of ESR1- or PGR-positive nuclei relative to epithelial area, but myoepithelial cells appear to be affected by IMI and the associated inflammation in nonlactating mammary glands that were stimulated to grow rapidly using mammogenic hormones. Accordingly, reductions in mammary epithelium in affected glands are not suspected to be resultant of alterations in the number or staining characteristics of ESR1- or PGR-positive mammary epithelial cells.

Apoptosis and proliferation in Staphylococcus aureus-challenged, nonlactating mammary glands stimulated to grow rapidly and develop with estradiol and progesterone.

Bovine mastitis is a common and costly disease in the dairy industry and is known to negatively affect the amount of epithelium in nonlactating mammary glands. Despite this recognition, an understanding of the mechanisms contributing to reductions in epithelium is lacking. The objective of this study was to evaluate cellular apoptosis and proliferation in uninfected and Staphylococcus aureus-infected mammary glands that were stimulated to rapidly grow and develop. Estradiol and progesterone injections were administered to 18 nonlactating dairy cows to induce mammary growth, and 2 quarters from each animal were infused with saline or Staph. aureus. Mammary tissues were collected at 5 (n = 9) and 10 d (n = 9) postinfusion and examined using quantitative bright field and florescent immunohistochemistry. Staphylococcus aureus mammary glands tended to have a greater number of mammary epithelial cells undergoing apoptosis than saline quarters. In the stromal compartment, challenged quarters contained a lower proportion of cells undergoing apoptosis than saline quarters overall; however, cell types undergoing apoptosis were differentially affected. Staphylococcus aureus quarters contained a lesser percentage of apoptotic fibroblasts while also containing more nonapoptotic immune cells than saline quarters in the intralobular stroma compartment. A similar number of proliferating epithelial cells were present in Staph. aureus and saline mammary tissues, but more proliferating cells were present in the intralobular stroma compartment of Staph. aureus-infused quarters than those infused with saline. When these cellular responses are considered together, it indicates that changes in cellular apoptosis and proliferation contribute to changes in the gland structure by potentiating the expansion of the intralobular stromal compartment, via cellular accumulation, and limiting the amount of epithelium due to increases in cellular apoptosis in affected glands. Reductions in mammary epithelium are expected to reduce future milk yields and productive herd life.

Excess deposition of collagen in mammary glands of tamoxifen-treated Holstein heifers is associated with impaired mammary growth.

It is established that the ovary and estrogen are essential to bovine mammary development with the onset of puberty. Recent studies have shown that ovariectomy in the very early prepubertal period, well before onset of puberty, also dramatically impairs mammary growth. Similarly, prepubertal heifers treated with the antiestrogen tamoxifen (TAM) also exhibit markedly impaired mammary growth in correspondence with reduced estrogen receptor α (ESR1) expression. Our objective was to evaluate the effect of TAM on the mammary stroma and specifically to determine if the reported decrease in mammary development was related to changes in TAM-induced alterations in the stroma surrounding the mammary parenchyma. Briefly, 16 Holstein heifers calves were randomly assigned to one of 2 treatment groups: TAM-injected or control. Calves were administered TAM (0.3 mg kg1 d1) or placebo from 28 to 120 d of age. At day 120, calves were euthanized and udders removed. Mammary tissue from near the boundary between the parenchyma and surrounding mammary fat pad was collected for histology and morphometric analysis, expression of selected extracellular matrix-related genes, and quantitation of stromal collagen deposition by study of Sirius Red-stained tissue sections imaged with polarized light. Compared with tissue from control heifers, TAM heifers frequently exhibited areas with abundant fibroblasts and mesenchymal cells especially within the intralobular stroma, as well as less complex ductal structures. Among the array of extracellular matrix-related genes tested, only a small difference (P < 0.05) in expression of laminin was found between treatments. The relative tissue area occupied by stromal tissue was not impacted by treatment. However, the deposition of collagen within the stromal tissue was more than doubled (P < 0.0001) in TAM-treated heifers. These data suggest that blocking ESR1 expression with TAM allows for excessive collagen deposition in the stroma surrounding the developing epithelial structures and that this interferes with both the degree of overall mammary parenchymal development, as well as the pattern of normal ductal morphogenesis.

Technical note: p40 antibody as a replacement for p63 antibody in bovine mammary immunohistochemistry.

Tumor protein 63 (p63) is a nuclear antigen found in basal epithelial cells. To date, 10 isoforms of p63 have been identified, falling into 2 major groups identified by presence or absence of an N-terminal transactivation domain (TAp63 and ΔNp63, respectively). Literature suggests that ΔNp63 is the predominant form expressed in basal epithelial cells and myoepithelial cells (MYEC). The mouse anti-p63 antibody, clone 4B1E12, has been used as a specific nuclear marker for bovine MYEC. Unfortunately, this antibody is no longer commercially available. A new mouse monoclonal antibody, clone BC28, specific to ΔNp63 (designated p40) has been developed. We hypothesized that the p40 antibody would be an appropriate substitution as a MYEC and epithelial basal cell marker. An array of archived formalin-fixed, paraffin-embedded bovine tissues were subjected to immunohistochemical staining for either p40 or p63, with a subset being dual stained for direct comparison. Positive staining for p40 and p63 was observed in serial sections of mammary, skin, rumen, salivary gland, ureter, and bladder. As predicted, negative staining for p40 and p63 was observed in testis and intestine. Dual staining for p40 and p63 in calf mammary (n = 4), lactating mammary (n = 4), rumen (n = 4), and skin (n = 4) showed nearly 100% agreement. Thus, we established that the mouse monoclonal antibody, clone BC28, is a suitable replacement for anti-p63, clone 4B1E12, as a marker of MYEC and basal epithelial cells in bovine tissues.

Short communication: Does early-life administration of a Megasphaera elsdenii probiotic affect long-term establishment of the organism in the rumen and alter rumen metabolism in the dairy calf?

Megasphaera elsdenii is a bacterial species of the rumen that can utilize lactate to produce butyrate, a key volatile fatty acid often implicated in driving calf rumen development. Because lactate is abundant in the rumen of young calves, administration of M. elsdenii to increase butyrate production and thus promote calf rumen development is an appealing possibility. The main objective of this study was to determine whether M. elsdenii administration to calves via oral drench at 14 d of age affected its long-term establishment at 70 d postadministration. Ruminal volatile fatty acid and lactate profiles and blood glucose and ß-hydroxybutyrate concentrations were also examined to determine potential influence on rumen metabolism. Six neonatal Holstein heifer calves were blocked on d 1 by body weight (41.3 ± 1.8 kg) and total serum protein (5.23 ± 0.16 g/dL) and assigned to either the M. elsdenii (n = 3) or control (n = 3) treatment groups. On d 14, calves in the M. elsdenii group orally received 25 mL of a commercially available M. elsdenii suspension, whereas calves in the control group received 25 mL of the same product that had been autoclaved. Rumen contents and blood samples were collected weekly from each animal until 84 d of age. The oral administration of M. elsdenii at 14 d did not increase the abundance of M. elsdenii 70 d postdosing, alter rumen fermentation, or change blood metabolites associated with butyrate. These results suggest that a single administration of the M. elsdenii probiotic may not affect the rumen establishment of the organism.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells.

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Short communication: Initial evidence supporting existence of potential rumen epidermal stem and progenitor cells.

The bovine rumen epidermis is a keratinized multilayered tissue that experiences persistent cell turnover. Because of this constant cell turnover, epidermal stem cells and their slightly more differentiated daughter cells, epidermal progenitor cells, must exist in the stratum basale of rumen epidermis. To date, these 2 epidermal cell populations and any unique cellular markers they may possess remain completely uncharacterized in the bovine rumen. An important first step in this new research area is the demonstration of the relative abundance and existence of markers for these cells in rumen tissue. A related second step is to document rumen epidermal proliferative responses to an extrinsic signal such as nutrient concentration within the rumen. The objectives of this experiment were to evaluate the extrinsic effect of diet on (1) gene expression of 6 potential rumen epidermal stem or progenitor cell markers and (2) rumen epidermal cell proliferation within the stratum basale. Twelve preweaned Holstein heifers were fed either a restricted diet (R) or an enhanced diet (EH). Animals on R received a milk replacer (MR) diet fed at 0.44kg of powder dry matter (DM)/d (20.9% crude protein, 29.8% fat, DM basis) and EH received MR at 1.08kg of powder dry matter/d (28.9% crude protein, 26.2% fat, DM basis). All calves had access to a 20% crude protein starter and were weaned during wk 7 of the experiment. Lifetime DM intake was 0.73kg of DM/calf per day for R (5.88 Mcal of net energy/calf per day) and 1.26kg of DM/calf per day for EH (10.68 Mcal of net energy/calf per day). Twenty-four hours before slaughter heifers received an intravenous dose of 5-bromo-2'-deoxyuridine to label proliferating cells. Heifers were slaughtered at 8 wk of age, and rumen samples from the ventral sac region were obtained and stored in RNA preservative and processed for routine histology. Quantitative real-time reverse transcriptase PCR was used to analyze relative abundance of genes. Candidate genes for markers of epidermal stem and progenitor cells were ß1-integrin (ITGB1), tumor protein p63 (TP63), keratin-14 (KRT14), Notch-1 (NOTCH1), Leu-rich repeat-containing G protein-coupled receptor 5-expressing (LGR5), and musashi-1 (MSI1). All genes were detected in the rumen tissue; ITGB1 was increased in EH compared with R. 5-Bromo-2'-deoxyuridine immunohistochemistry revealed that both R and EH rumen tissue had proliferating cells within the stratum basale of the rumen epidermis at the time of analysis. The EH diet resulted in an additive effect on cell proliferation. The percentage of cells in the stratum basale synthesizing DNA in preparation for mitosis nearly doubled (23.8±2.4% for EH vs. 14.7±2.0% for R) compared with calves fed R. This work represents the first attempt at characterizing rumen epidermal stem and progenitor cells. We demonstrated the relative abundance and existence of potential markers in rumen tissue and showed a rumen epidermal proliferative response to the extrinsic stimulus of nutrient concentration in the form of diet.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways.

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and ß (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

Tamoxifen impairs prepubertal mammary development and alters expression of estrogen receptor α (ESR1) and progesterone receptors (PGR).

Research has shown that prepubertal heifers experience allometric mammary growth that is influenced by the ovaries. Our purpose was to determine the role of estrogen in prepubertal mammary gland development. Sixteen Holstein calves were randomly assigned to 1 of 2 treatment groups: tamoxifen-injected (TAM) or control (CON). Calves were administered the antiestrogen tamoxifen (0.3 mg kg(1) d(1)) or placebo from 28 to 120 d of age. At 120 d, calves were euthanized and udders removed. Weight and DNA content of trimmed parenchymal tissue were halved (P ≤ 0.0001) in TAM compared with CON calves. Parenchymal samples from 3 zones of the left rear mammary gland (lower, middle, and outer regions) were processed for immunohistochemical staining for estrogen receptor α (ESR1) and progesterone receptor (PGR), Ki67-positive cells, and 5-bromo-2'-deoxyuridine label retaining cells (LRCs). Overall, neither the percentage nor location within the epithelial tissue layer of either ESR1- or PGR-positive cells was impacted by TAM treatment. However, image analysis indicated a 6.2-fold lower (P = 0.0001) level of ESR1 protein expression in TAM calves. Similarly, messenger RNA expression of ESR1 was also reduced (P = 0.0001) in TAM heifers. In contrast, expression of PGR protein was greater by 43% (P = 0.03) in TAM calves, but messenger RNA expression did not differ between treatments. Overall, TAM calves had a higher (P ≤ 0.03) percentage and density (cells per tissue area) of Ki67-positive cells. Irrespective of treatment, there were also more Ki67-labeled cells in the outer zones of the mammary gland (P ≤ 0.001). We were able to effectively use multispectral imaging to identify positive cells and quantify the expression of ESR1 and PGR protein. We also identified and counted the proportion of label retaining cells (LCR) (putative epithelial stem cells). We noted an overall 2.9-fold greater number of LRCs in TAM heifers and more LRCs in the outer sampling zones. This suggests that a cohort of LCR cells in TAM remained inactivated in comparison with CON heifers, which exhibited markedly increased growth of the mammary parenchymal tissue over the treatment period. These results suggest that the impacts of ovariectomy are partially explained by loss of ESR1 expression and/or estrogen receptor signaling in the prepubertal bovine mammary gland. The significance of mammary expression of PGR in control of prepubertal bovine mammary development remains unresolved.