Evaluation of feed restriction and abomasal infusion of resistant starch as models to induce intestinal barrier dysfunction in healthy lactating cows.

Intestinal hyperpermeability and subsequent immune activation alters nutrient partitioning and thus, decreases productivity. Developing experimental models of intestinal barrier dysfunction in heathy cows is a prerequisite in identifying nutritional strategies to mitigate it. Six cannulated Holstein cows (mean ± standard deviation, 37 ± 10 kg/d milk yield; 219 ± 97 d in milk; 691 ± 70 kg body weight) were used in a replicated 3 × 3 Latin square design experiment with 21-d periods (16-d wash-out and 5-d challenge) to evaluate either feed restriction or hindgut acidosis as potential models for inducing intestinal hyperpermeability. Cows were randomly assigned to treatment sequence within square and treatment sequences were balanced for carryover effects. Treatments during the challenge were (1) control (CTR; ad libitum feeding); (2) feed restriction (FR; total mixed ration fed at 50% of ad libitum feed intake); and (3) resistant starch (RS; 500 g of resistant starch infused in abomasum once a day as a pulse-dose 30 min before morning feeding). The RS (ActiStar RT 75330, Cargill Inc.) was tapioca starch that was expected to be resistant to enzymatic digestion in the small intestine and highly fermentable in the hindgut. Blood samples were collected 4 h after feeding on d 13 and 14 of the wash-out periods (baseline data used as covariate), and on d 1, 3, and 5 of the challenge periods. Fecal samples were collected 4 and 8 h after the morning feeding on d 14 of the wash-out periods and d 5 of the challenge periods. By design, FR decreased dry matter intake (48%) relative to CTR and RS, and this resulted in marked reductions in milk and 3.5% FCM yields over time, with the most pronounced decrease occurring on d 5 of the challenge (34 and 27%, respectively). Further, FR increased somatic cell count by 115% on d 5 of the challenge relative to CTR and RS. Overall, FR increased nonesterified fatty acids (159 vs. 79 mEq/L) and decreased BHB (8.5 vs. 11.2 mg/dL), but did not change circulating glucose relative to CTR. However, RS had no effect on production or metabolism metrics. Resistant starch decreased fecal pH 8 h after the morning feeding (6.26 vs. 6.81) relative to CTR and FR. Further, RS increased circulating lipopolysaccharide binding protein (4.26 vs. 2.74 µg/mL) compared with FR only on d 1 of the challenge. Resistant starch also increased Hp (1.52 vs. 0.48 µg/mL) compared with CTR, but only on d 5 of the challenge. However, neither RS or FR affected concentrations of serum amyloid A, IL1ß, or circulating endotoxin compared with CTR. The lack of consistent responses in inflammatory biomarkers suggests that FR and RS did not meaningfully affect intestinal barrier function. Thus, future research evaluating the effects of hindgut acidosis and FR using more intense insults and direct metrics of intestinal barrier function is warranted.

Effects of dietary antioxidant supplementation on metabolism and inflammatory biomarkers in heat-stressed dairy cows.

Heat-stress-induced inflammation may be ameliorated by antioxidant supplementation due to the purported effects of increased production of reactive oxygen species or oxidative stress on the gastrointestinal tract barrier. Thus, study objectives were to evaluate whether antioxidant supplementation [AGRADO Plus 2.0 (AP); EW Nutrition] affects metabolism and inflammatory biomarkers in heat-stressed lactating dairy cows. Thirty-two mid-lactation multiparous Holstein cows were assigned to 1 of 4 dietary-environmental treatments: (1) thermoneutral (TN) conditions and fed a control diet (TN-CON; n = 8), (2) TN and fed a diet with AP (10 g antioxidant; n = 8), (3) heat stress (HS) and fed a control diet (HS-CON; n = 8), or (4) HS and fed a diet with AP (HS-AP; n = 8). The trial consisted of a 23-d prefeeding phase and 2 experimental periods (P). Respective dietary treatments were top-dressed starting on d 1 of the prefeeding period and continued daily throughout the duration of the experiment. During P1 (4 d), baseline data were collected. During P2 (7 d), HS was artificially induced using an electric heat blanket (Thermotex Therapy Systems Ltd.). During P2, the effects of treatment, day, and treatment-by-day interaction were assessed using PROC MIXED of SAS (SAS Institute Inc.). Heat stress (treatments 3 and 4) increased rectal, vaginal, and skin temperatures (1.2°C, 1.1°C, and 2.0°C, respectively) and respiration rate (33 breaths per minute) relative to TN cows. As expected, HS decreased dry matter intake, milk yield, and energy-corrected milk yield (32%, 28%, and 28% from d 4 to 7, respectively) relative to TN. There were no effects of AP on body temperature indices or production. Milk fat, protein, and lactose concentrations remained unaltered by HS or AP; however, milk urea nitrogen was increased during HS regardless of AP supplementation (26% relative to TN). Circulating glucose remained unchanged by HS, AP, or time. Additionally, HS decreased circulating glucagon (29% from d 3 to 7 relative to TN), but there was no additional effect of AP. There was a tendency for nonesterified fatty acid concentrations to be increased in HS-AP cows throughout P2 (60% relative to TN-CON), whereas it remained similar in all other treatments. Blood urea nitrogen increased for both HS treatments from d 1 to 3 before steadily decreasing from d 5 to 7, with the overall increase being most pronounced in HS-CON cows (27% relative to TN-CON). Further, supplementing AP decreased blood urea nitrogen in HS-AP on d 3 relative to HS-CON (15%). Circulating serum amyloid A tended to be and lipopolysaccharide binding protein was increased by HS, but neither acute-phase protein was affected by AP. Overall, AP supplementation appeared to marginally alter metabolism but did not meaningfully alter inflammation during HS.

Occult tension pneumothorax discovered following imaging for adult trauma patients in the modern major trauma system: a multicentre observational study.

BACKGROUND: Tension pneumothorax following trauma is a life-threatening emergency and radiological investigation is normally discouraged prior to treatment in traditional trauma doctrines such as ATLS. Some trauma patients may be physiologically stable enough for diagnostic imaging and occult tension pneumothorax is discovered radiologically. We assessed the outcomes of these patients and compared them with those with clinical diagnosis of tension pneumothorax prior to imaging. METHODS: A multicentre civilian-military collaborative network of six major trauma centres in the UK collected observational data from adult patients who had a diagnosis of traumatic tension pneumothorax during a 33-month period. Patients were divided into 'radiological' (diagnosis following CT/CXR) or 'clinical' (no prior CT/CXR) groups. The effect of radiological diagnosis on survival was analysed using multivariable logistic regression that included the covariates of age, gender, comorbidities and Injury Severity Score. RESULTS: There were 133 patients, with a median age of 41 (IQR 24-61); 108 (81%) were male. Survivors included 49 of 59 (83%) in the radiological group and 59 of 74 (80%) in the clinical group (p=0.487). Multivariable logistic regression showed no significant association between radiological diagnosis and survival (OR 2.40, 95% CI 0.80 to 7.95; p=0.130). There was no significant difference in mortality between the groups. CONCLUSION: Radiological imaging may be appropriate for selected trauma patients at risk of tension pneumothorax if they are considered haemodynamically stable. Trauma patients may be physiologically stable enough for radiological imaging but have occult tension pneumothorax because they did not have the typical clinical presentation. The historical dogma of the 'forbidden scan' no longer applies to such patients.

Postruminal protein supply upregulates hepatic lysine oxidation and ornithine transcarbamoylase in lactating dairy cattle.

Metabolizable protein supply is a limiting factor for milk production in dairy cows, and the availability of AA is a function of the quantity of the metabolizable protein available and of hepatic AA catabolism. This study aimed to evaluate the effect of postruminal protein infusion on key genes for ureagenesis and AA catabolism. Six multiparous Holstein cows in early lactation were used in a replicated crossover design. Cows were fed a TMR and infused postruminally with either 0 or 600 g/d of milk protein isolate. Periods were 21 d long, consisting of 14 d of adjustment to surroundings, followed by 7 d of protein infusion. On the last day of each infusion, liver samples were collected for mRNA analysis and explant culture, milk samples were collected for mRNA analysis, and blood samples were collected for plasma metabolite analysis. Postruminal infusion of protein increased milk yield by 10.5%, milk fat yield by 12.5%, milk protein yield by 20%, milk lactose yield by 11%, and total solids yield by 15.5%. Postruminal infusion of protein increased milk urea N by 23.5%, blood urea N by 18.6%, and the abundance of hepatic ornithine transcarbamoylase mRNA by 52.8%. Postruminal infusion of protein did not alter the mRNA abundance of hepatic argininosuccinate synthase, α-aminoadipate semialdehyde synthase, cysteine sulfinic acid decarboxylase, or cystathionase. The abundance of RNA for milk proteins was unchanged with postruminal protein infusion. Metabolism of l-[U 14C] Lys to CO2 was increased by 127% (0.143 vs. 0.063 ± 0.04 nmol product·mg tissue-1·h-1), and the metabolism of l-[U 14C] Ala to CO2 increased by 40.5% (0.52 vs. 0.37 ± 0.06 nmol product·mg tissue-1·h-1) with postruminal protein infusion. The rate of l-[1-14C] Met oxidation did not differ. These data indicate increased ureagenesis matched by upregulation of nonessential AA catabolism and a disproportional increase in Lys oxidation in response to increased postruminal protein infusion.

Mammary transcriptome reveals cell maintenance and protein turnover support milk synthesis in early-lactation cows.

A more complete understanding of the molecular mechanisms that support milk synthesis is needed to develop strategies to efficiently and sustainably meet the growing global demand for dairy products. With the postulate that coding gene transcript abundance reflects relative importance in supporting milk synthesis, we analyzed the global transcriptome of early lactation cows across magnitudes of normalized RNA-Seq read counts. Total RNA was isolated from milk samples collected from early-lactation cows (n = 6) following two treatment periods of postruminal lysine infusion of 0 or 63 g/day. Twelve libraries were prepared and sequenced on an Illumina NovaSeq6000 platform using paired end reads. Normalized read counts were averaged across both treatments, because EBseq analysis found no significant effect of lysine infusion. Approximately 10% of the total reads corresponded to 12,730 protein coding transcripts with a normalized read count mean ≥5. For functional annotation analysis, the protein coding transcripts were divided into nine categories by magnitude of reads. The 13 most abundant transcripts (≥50K reads) accounted for 67% of the 23M coding reads and included casein and whey proteins, regulators of fat synthesis and secretion, a ubiquitinating protein, and a tRNA transporter. Mammalian target of rapamycin, JAK/STAT, peroxisome proliferator-activated receptor alpha, and ubiquitin proteasome pathways were enriched with normalized reads ≥100 counts. Genes with ≤100 reads regulated tissue homeostasis and immune response. Enrichment in ontologies that reflect maintenance of translation, protein turnover, and amino acid recycling indicated that proteostatic mechanisms are central to supporting mammary function and primary milk component synthesis.

Survival of aging CD264+ and CD264- populations of human bone marrow mesenchymal stem cells is independent of colony-forming efficiency.

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264+ hBM-MSCs from two age-matched donors have elevated ß-galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264- hBM-MSCs. Counterintuitive to their aging phenotype, CD264+ hBM-MSCs exhibited comparable survival to matched CD264- hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264+ cells in hBM-MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.

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.

Live video footage from scene to aid helicopter emergency medical service dispatch: a feasibility study.

BACKGROUND: Obtaining accurate information from a 112 caller is key to correct tasking of Helicopter Emergency Medical Services (HEMS). Being able to view the incident scene via video from a mobile phone may assist HEMS dispatch by providing more accurate information such as mechanism of injury and/or injuries sustained. The objective of this study is to describe the acceptability and feasibility of using live video footage from the mobile phone of a 112 caller as an HEMS dispatch aid. METHODS: Live footage is obtained via the 112 caller's mobile phone camera through the secure GoodSAM app's Instant-on-scene™ platform. Video footage is streamed directly to the dispatcher, and not stored. During the feasibility trial period, dispatchers noted the purpose for which they used the footage and rated ease of use and any technical- and operational issues they encountered. A subjective assessment of caller acceptance to use video was conducted. RESULTS: Video footage from scene was attempted for 21 emergency calls. The leading reasons listed by the dispatchers to use live footage were to directly assess the patient (18/21) and to obtain information about the mechanism of injury and the scene (11/21). HEMS dispatchers rated the ease of use with a 4.95 on a 5-point scale (range 4-5). All callers gave permission to stream from their telephone camera. Video footage from scene was successfully obtained in 19 calls, and was used by the dispatcher as an aid to send (5) or stand down (14) a Helicopter Emergency Medical Services team. CONCLUSION: Live video footage from a 112 caller can be used to provide dispatchers with more information from the scene of an incident and the clinical condition of the patient(s). The use of mobile phone video was readily accepted by the 112 caller and the technology robust. Further research is warranted to assess the impact video from scene could have on HEMS dispatching.

Milk fat response and milk fat and urine biomarkers of microbial nitrogen flow during supplementation with 2-hydroxy-4-(methylthio)butanoate.

2-Hydroxy-4-(methylthio)butanoate (HMTBa) is a methionine analog that has been observed to attenuate biohydrogenation (BH)-induced milk fat depression (MFD), possibly through reducing the shift to altered BH pathways. It has also been suggested that HMTBa increases microbial protein synthesis in the rumen. Our objectives were to stimulate BH-induced MFD and (1) verify HMTBa inhibition of BH-induced MFD and changes in milk fatty acids (FA) associated with altered rumen BH (i.e., trans-10 C18:1); and (2) determine the effect of HMTBa on milk fat (i.e., odd- and branched-chain FA) and urine biomarkers related to microbial N flow. Twenty-four multiparous cows (45.6 ± 8.5 kg of milk/d; mean ± standard deviation) and 12 primiparous cows (32.8 ± 3.1 kg of milk/d) were arranged in a crossover design. Treatments were unsupplemented control and HMTBa fed at 0.1% of diet dry matter intake. The experiment was 80 d and included a 10-d pretrial covariate period. Each experimental period included 2 phases that differed in risk for BH-induced MFD, including a 28-d low-risk phase (31.6% neutral detergent fiber, 21.8% starch, and no oil) and a 7-d moderate-risk phase (28.7% neutral detergent fiber, 28.1% starch, and 1.0% soybean oil). We found no interaction of treatment and parity. Milk fat yield (1.43 ± 0.51 kg/d) and milk fat trans-10 C18:1 (0.42 ± 0.08 g/100 g of FA) did not differ between treatments during the low-risk phase. However, during the moderate-risk phase, HMTBa maintained higher milk fat concentration (3.91 vs. 3.79%), tended to maintain higher milk fat yield (1.44 vs. 1.38 kg/d), and decreased milk fat trans-10 C18:1 (0.61 vs. 0.93% FA) compared with control. Additionally, HMTBa increased milk fat concentration and secretion of odd- and branched-chain FA by 5.3 and 10.2%, respectively, but urinary biomarkers of microbial N flow (i.e., purine derivatives) did not differ between treatments. However, rumen bacterial samples were not available to provide cow- or treatment-specific microbial protein-to-marker ratios, which is a critical source of variation. Additionally, transfer of odd- and branched-chain FA to milk is dependent on several factors that may affect interpretation of these biomarkers. In conclusion, HMTBa decreased absorption of alternate BH intermediates and maintained higher milk fat when feeding a diet with moderate-risk for MFD.

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.

Meta-analysis of 2-hydroxy-4-methylthio-butanoic acid supplementation on ruminal fermentation, milk production, and nutrient digestibility.

Methionine is considered one of the most important essential AA for milk protein synthesis in dairy cows. Supplementation of unprotected, free Met is nearly 100% degraded by ruminal microorganisms, which complicates supplementation. 2-Hydroxy-4-methylthio-butanoic acid (HMTBa) can be converted to Met in the body and is used as a Met source in dairy production. However, results of published studies assessing the effects of supplementing Met sources, including HMTBa, on performance variables are inconsistent. A meta-analysis was performed to quantitatively summarize the accumulated results of HMTBa supplementation on animal performance and nutrient digestibility. Data pertaining to HMTBa dose, dietary composition, and major performance variables (rumen volatile fatty acid composition, milk production, nutrient digestibility) were collected from 39 articles containing 169 treatment means. Publications were from scientific journals published from 1970 to 2018; 1 internal report from Novus International Inc. (St. Charles, MO) was also included. The HMTBa effects on response variables were analyzed using linear mixed models with random study effects. Other explanatory variables tested included neutral detergent fiber and crude protein percent as well as days in milk. Results showed that HMTBa supplementation increased blood Met concentration and milk fat yield but had no effect on nutrient digestibility.

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.

A Novel, Sterilized Microvascular Tissue Product Improves Healing in a Murine Pressure Ulcer Model.

BACKGROUND: Processed microvascular tissue (PMVT), a human structural allograft, is derived from lyophilized human tissue containing microcirculatory cellular components. Since PMVT serves as a source of extracellular matrix (ECM), growth factors, cytokines, and chemokines modulating angiogenesis, inflammation, apoptosis, and endogenous cell recruitment, we hypothesized its application would accelerate wound regeneration in a validated pressure ulcer (PU) model developed in C57BL/6 mice using two 24-hour cycles of skin ischemia/reperfusion created by placement and removal of external magnets. METHODS: Two identical PU injuries (n = 50 female mice) were treated with (a) topical particulate PMVT, (b) injected rehydrated PMVT, or (c) saline control injection, and assessed daily for closure rates, scab formation/removal, and temperature. A baseline control cohort (n = 5) was euthanized at day 0 and treatment group cohorts (n = 5) were killed at 3, 7, or 14 days postinjury. The PU injuries were collagenase-digested for flow cytometric analysis of inflammatory, reparative, and stem cell frequencies and analyzed by hematoxylin and eosin (H&E) histology and immunofluorescence. RESULTS: PMVT-accelerated wound closure, most notably, topical PMVT significantly increased mean closure from d5 (13% versus -9%) through d13 (92% versus 38%) compared with phosphate-buffered saline (PBS) controls (P < 0.05). PMVT also hastened scab formation/removal, significantly accelerated disappearance of inflammatory myeloid (CD11b+) cells while upregulating α-smooth muscle actin, vascular endothelial growth factor A, and placental growth factor and raised skin temperature surrounding the PU site, consistent with increased blood flow. CONCLUSIONS: These results indicate that PMVT has potential as an advanced treatment for restoring normal tissue function in ischemic wounds and merits clinical study.

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.

Decoy TRAIL receptor CD264: a cell surface marker of cellular aging for human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are a mixture of progenitors that are heterogeneous in their regenerative potential. Development of MSC therapies with consistent efficacy is hindered by the absence of an immunophenotype of MSC heterogeneity. This study evaluates decoy TRAIL receptor CD264 as potentially the first surface marker to detect cellular aging in heterogeneous MSC cultures. METHODS: CD264 surface expression, regenerative potential, and metrics of cellular aging were assessed in vitro for marrow MSCs from 12 donors ages 20-60 years old. Male and female donors were age matched. Expression of CD264 was compared with that of p16, p21, and p53 during serial passage of MSCs. RESULTS: When CD264+ cell content was 20% to 35%, MSC cultures from young (ages 20-40 years) and older (ages 45-60 years) donors proliferated rapidly and differentiated extensively. Older donor MSCs containing < 35% CD264+ cells had a small size and negligible senescence despite the donor's advanced chronological age. Above the 35% threshold, CD264 expression inversely correlated with proliferation and differentiation potential. When CD264+ cell content was 75%, MSCs were enlarged and mostly senescent with severely compromised regenerative potential. There was no correlation of the older donors' chronological age to either CD264+ cell content or the regenerative potential of the donor MSCs. CD264 was upregulated after p53 and had a similar expression profile to that of p21 during serial passage of MSCs. No sex-linked differences were detected in this study. CONCLUSIONS: These results suggest that CD264 is a surface marker of cellular age for MSCs, not the chronological age of the MSC donor. CD264 is first upregulated in MSCs at an intermediate stage of cellular aging and remains upregulated as aging progresses towards senescence. The strong inverse correlation of CD264+ cell content to the regenerative potential of MSCs has possible application to assess the therapeutic potential of patient MSCs, standardize the composition and efficacy of MSC therapies, and facilitate aging research on MSCs.

Hepatic expression of aminoadipate semialdehyde synthase is unchanged by postruminal lysine supply in lactating dairy cows.

Lysine supply is potentially limiting for milk production in dairy cows. The availability of Lys to the mammary gland and other tissues is a function of the quantity of metabolizable Lys supplied and Lys catabolism by the liver. Likewise, Lys catabolism may be influenced by Lys supply. This study evaluated the effect of increased postruminal Lys supply on the expression of aminoadipate semialdehyde synthase (AASS, a committing step in Lys catabolism in the liver) and ornithine transcarbamoylase and argininosuccinate synthase (key urea cycle enzymes that are responsive to protein supply). Eight multiparous peak Holstein cows were used in a replicated 4 × 4 Latin square. Cows were fed a Lys-limiting ration and infused postruminally with 0, 9, 27, or 63 g/d of Lys. The study consisted of 10 d of pretreatment followed by 10 d of Lys infusion. On the last day of each period, liver and milk samples were collected for mRNA analysis, and blood samples were collected for analysis of amino acids and Lys metabolites. Milk protein percent increased by 5.9%, plasma Lys increased by 74%, and α-aminoadipic acid increased by 51% with postruminal infusion of 63 g/d Lys compared with 0 g/d. Expression of AASS, ornithine transcarbamoylase, and argininosuccinate synthase mRNA in liver did not differ with postruminal infusion of Lys. Milk fat globule mRNA for major milk proteins and AASS were not affected by Lys infusion. Postruminal infusion of Lys resulted in an 86% greater increase in AASS mRNA in the liver compared with mammary mRNA. These changes suggest that hepatic Lys metabolism is not responsive to Lys supply at the transcription level, and that the availability of Lys to extrahepatic tissue may be determined by hepatic Lys metabolism.

Effects of supplementing methionine hydroxy analog on beef cow performance, milk production, reproduction, and preweaning calf performance.

Mature Simmental × Angus cows (214 cows; 635 ± 7 kg) were utilized to determine the effects of late gestation and early postpartum supplementation of methionine hydroxy analog (MHA) on cow BW, BCS, milk production, milk composition, reproduction, and calf performance until weaning in a fall-calving, cool-season grazing system. Cows were stratified by BW, age, AI sire, and assigned to 1 of 12 pastures (17 or 18 cows·pasture). Pastures were randomly allotted to 1 of 2 treatments: control (0.45 kg·cow·d of wheat midd-based pellets, = 6) or supplement including MHA (0.45 kg·cow·d of wheat midd-based pellets including 10 g MHA supplied as MFP (Novus International, Inc., St. Charles, MO; = 6). Treatments were fed 23 ± 7 d prepartum through 73 ± 7 d postpartum. Cow BW was collected at postcalving (27 ± 7 d postpartum), end of supplementation (73 ± 7 d postpartum), AI, pregnancy check, and end of trial (192 and 193 ± 7 d postpartum). At 73 ± 7 d postpartum, a subset of cow-calf pairs was used in a weigh-suckle-weigh procedure to determine milk production, and milk samples were taken to determine milk composition ( = 45·treatment). Serum from blood was collected at 73 ± 7 and 83 ± 7 d postpartum to determine cow cyclicity and concentrations of 2-hydroxy4-(methylthio) butanoic acid (HMTBa) and L-Methionine. After supplementation, all cow-calf pairs were managed as a common group until weaning (193 ± 7 d of age). Cows were bred via AI at 97 ± 7 d postpartum and clean-up bulls were turned out 11 d post-AI for a 55-d breeding season. Cows fed MHA had greater ( < 0.01) serum concentrations of HMTBa. Cow BW and BCS were not different ( ≥ 0.10) at any time points between treatments. There was no treatment effect ( ≥ 0.17) on calf birth BW, calf weaning BW (193 ± 7 d of age), or calf ADG. Calculated 24-h milk production, milk composition and component production did not differ ( ≥ 0.21). There were no differences ( ≥ 0.50) in percentage of cows cycling, AI conception rate, and overall pregnancy rate between treatments. Post-trial nutritional modeling suggests cows experienced several nutritional deficiencies beyond methionine (Met) that limited the response to Met supplementation. Although supplementation of MHA during late gestation through estimated peak lactation increased serum HMTBa concentrations, it did not affect cow performance, cow milk production, or calf performance when fall-calving cows grazed cool-season forages.