Cyclical heat stress during lactation influences the microstructure of the bovine mammary gland.

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.

Relationships between gastrointestinal permeability, heat stress, and milk production in lactating dairy cows.

Heat stress (HS) is a global issue that decreases farm profits and compromises animal welfare. To distinguish between the direct and indirect effects of HS, 16 multiparous Holstein cows approximately 100 DIM were assigned to one of 2 treatments: pair fed to match HS cow intake, housed in thermoneutral conditions (PFTN, n = 8) or cyclical HS (n = 8). All cows were subjected to 2 experimental periods. Period 1 consisted of a 4 d thermoneutral period with ad libitum intake. During period 2 (P2), the HS cows were housed in cyclical HS conditions with a temperature-humidity index (THI) ranging from 76 to 80 and the PFTN cows were exposed to a constant THI of 64 for 4 d. Dry matter intake of the PFTN cows was intake matched to the HS cows. Milk yield, milk composition, rectal temperature, and respiration rate were recorded twice daily, blood was collected daily via a jugular catheter, and cows were fed twice daily. On d 3 of each period, Cr-EDTA and sucralose were orally administered and recovered via 24 h total urine collection to assess gastrointestinal permeability. All data were analyzed using the GLIMMIX procedure in SAS. The daily data collected in P1 was averaged and used as a covariate if deemed significant in the model. Heat stress decreased voluntary feed intake by 35% and increased rectal temperature and respiration rate (38.4°C vs. 39.4°C and 40 vs. 71 respirations/min, respectively). Heat stress reduced DMI by 35%, which accounted for 66% of the decrease in milk yield. The yields, and not concentrations, of milk protein, fat, and other solids were lower in the HS cows on d 4 of P2. Milk urea nitrogen was higher and plasma urea nitrogen tended to be higher on d 3 and d 4 of HS. Glucose was 7% lower in the HS cows and insulin was 71% higher in the HS cows than the PFTN cows on d 4 of P2. No difference in lipopolysaccharide-binding protein was observed. Heat stress cows produced 7 L/d more urine than PFTN cows. No differences were detected in the urine concentration or percentage of the oral dose recovered for Cr-EDTA or sucralose. In conclusion, HS was responsible for 34% of the reduction of milk yield. The elevated MUN and the tendency for elevated plasma urea nitrogen indicate a whole-body shift in nitrogen metabolism. No differences in gastrointestinal permeability or lipopolysaccharide-binding protein were observed. These results indicate that, under the conditions of this experiment, activation of the immune system by gut-derived lipopolysaccharide was not responsible for the decreased milk yield observed during HS.

Depression, anxiety and PTSD symptoms before and during the COVID-19 pandemic in the UK.

BACKGROUND: The impact of the coronavirus disease 2019 (COVID-19) pandemic on mental health is still being unravelled. It is important to identify which individuals are at greatest risk of worsening symptoms. This study aimed to examine changes in depression, anxiety and post-traumatic stress disorder (PTSD) symptoms using prospective and retrospective symptom change assessments, and to find and examine the effect of key risk factors. METHOD: Online questionnaires were administered to 34 465 individuals (aged 16 years or above) in April/May 2020 in the UK, recruited from existing cohorts or via social media. Around one-third (n = 12 718) of included participants had prior diagnoses of depression or anxiety and had completed pre-pandemic mental health assessments (between September 2018 and February 2020), allowing prospective investigation of symptom change. RESULTS: Prospective symptom analyses showed small decreases in depression (PHQ-9: -0.43 points) and anxiety [generalised anxiety disorder scale - 7 items (GAD)-7: -0.33 points] and increases in PTSD (PCL-6: 0.22 points). Conversely, retrospective symptom analyses demonstrated significant large increases (PHQ-9: 2.40; GAD-7 = 1.97), with 55% reported worsening mental health since the beginning of the pandemic on a global change rating. Across both prospective and retrospective measures of symptom change, worsening depression, anxiety and PTSD symptoms were associated with prior mental health diagnoses, female gender, young age and unemployed/student status. CONCLUSIONS: We highlight the effect of prior mental health diagnoses on worsening mental health during the pandemic and confirm previously reported sociodemographic risk factors. Discrepancies between prospective and retrospective measures of changes in mental health may be related to recall bias-related underestimation of prior symptom severity.

Assessment of Activity Profiles in Older Adults and Lower Limb Bone Parameters: Observations from the Hertfordshire Cohort Study.

As muscle strength and function decline with age the optimal high-impact physical activity (PA) required for bone remodelling is rarely achievable in older adults. This study aimed to explore the activity profiles of community-dwelling older men and women and to assess the relationship between individual PA profiles and lower limb bone parameters. Participants from the Hertfordshire Cohort Study wore triaxial accelerometers for 7 days and counts of low (0.5-1.0 g), medium (1.0-1.5 g), and high (> 1.5 g) vertical-impact activity were calculated. Two years later, participants underwent a pQCT scan of the tibia (4% and 38% sites) to obtain measures of bone mineral density and bone geometry. Linear regression was used to quantify associations between bone and PA loading profiles adjusting for age, sex, loading category, and BMI. Results are presented as ß [95% confidence interval]. Bone and PA data were available for 82 participants. The mean (SD) age at follow-up was 81.4(2.7) years, 41.5% (n = 34) were women. The median low-impact PA count was 5281 (Inter-quartile range (IQR) 2516-12,977), compared with a median of only 189 (IQR 54-593) in medium, and 39 (IQR 9-105) in high-impact counts. Positive associations between high-impact PA and cortical area (mm2), polar SSI (mm3), and total area (mm2) at the 38% slice (6.21 [0.88, 11.54]; 61.94 [25.73, 98.14]; 10.09 [3.18, 16.99], respectively). No significant associations were found at distal tibia. These data suggest that maintaining high (> 1.5 g)-impact activity is difficult for older adults to achieve; however, even small amounts of high-impact PA are positively associated with selected cortical bone parameters 2 years later.

Determining individual trajectories of joint space loss: improved statistical methods for monitoring knee osteoarthritis disease progression.

OBJECTIVES: Knee osteoarthritis (KOA) progression is frequently monitored by calculating the change in knee joint space width (JSW) measurements. Such differences are small and sensitive to measurement error. We aimed to assess the utility of two alternative statistical modelling methods for monitoring KOA. MATERIAL AND METHODS: We used JSW on radiographs from both the control arm of the Strontium Ranelate Efficacy in Knee Osteoarthritis trial (SEKOIA), a 3-year multicentre, double-blind, placebo-controlled phase three trial, and the Osteoarthritis Initiative (OAI), an open-access longitudinal dataset from the USA comprising participants followed over 8 years. Individual estimates of annualised change obtained from frequentist linear mixed effect (LME) and Bayesian hierarchical modelling, were compared with annualised crude change, and the association of these parameters with change in WOMAC pain was examined. RESULTS: Mean annualised JSW changes were comparable for all estimates, a reduction of around 0.14 mm/y in SEKOIA and 0.08 mm/y in OAI. The standard deviation (SD) of change estimates was lower with LME and Bayesian modelling than crude change (SEKOIA SD = 0.12, 0.12 and 0.21 respectively; OAI SD = 0.08, 0.08 and 0.11 respectively). Estimates from LME and Bayesian modelling were statistically significant predictors of change in pain in SEKOIA (LME P-value = 0.04, Bayes P-value = 0.04), while crude change did not predict change in pain (P-value = 0.10). CONCLUSIONS: Implementation of LME or Bayesian modelling in clinical trials and epidemiological studies, would reduce sample sizes by enabling all study participants to be included in analysis regardless of incomplete follow up, and precision of change estimates would improve. They provide increased power to detect associations with other measures.

Infant milk feeding and bone health in later life: findings from the Hertfordshire cohort study.

Using data from the Hertfordshire cohort study, this study examined the effect of breastfeeding and bottle feeding on adult lumbar spine and femoral neck bone mineral content (BMC) and bone mineral density (BMD). The type of infant milk feeding was significantly associated with lumbar spine BMD in males. INTRODUCTION: Using data from the Hertfordshire cohort study (HCS), this study aims to examine the effect of infant milk feeding on bone health in later life by comparing the effect of breastfeeding and bottle feeding on lumbar spine and femoral neck BMC and BMD. METHODS: Information about infant milk feeding, birth weight (kg) and weight at 1 (kg) was collected by health visitors between 1931 and 1939 in Hertfordshire. BMC and BMD measurements were taken by DXA scan between 1998 and 2004. Linear regression models adjusted for conditional weight at 1, age at DXA scan, sex, adult BMI, smoking behaviour, alcohol consumption, physical activity, dietary calcium, and prudent diet score. RESULTS: Infant milk feeding was significantly associated with lumbar spine BMD (b = - 0.028; 95% CI, - 0.055; - 0.000; p value, 0.047) in males. On average, males who consumed breastmilk alternatives in infancy had lower lumbar spine BMD measurements than those who were fed only breastmilk. These associations remained significant in fully adjusted models. There were no significant associations between infant milk feeding and bone health for females. CONCLUSIONS: Significant associations between infant milk feeding and lumbar spine BMD in males indicate that breastmilk may be protective for the bone health of male babies. The evidence presented here underscores the potential lifelong benefits of breastfeeding and may highlight the differences between osteoporotic risk factors for males and females.

Systematic screening using FRAX® leads to increased use of, and adherence to, anti-osteoporosis medications: an analysis of the UK SCOOP trial.

In the large community-based SCOOP trial, systematic fracture risk screening using FRAX® led to greater use of AOM and greater adherence, in women at high fracture risk, compared with usual care. INTRODUCTION: In the SCreening of Older wOmen for Prevention of fracture (SCOOP) trial, we investigated the effect of the screening intervention on subsequent long-term self-reported adherence to anti-osteoporosis medications (AOM). METHODS: SCOOP was a primary care-based UK multicentre trial of screening for fracture risk. A total of 12,483 women (70-85 years) were randomised to either usual NHS care, or assessment using the FRAX® tool ± dual-energy X-ray absorptiometry (DXA), with medication recommended for those found to be at high risk of hip fracture. Self-reported AOM use was obtained by postal questionnaires at 6, 12, 24, 36, 48 and 60 months. Analysis was limited to those who initiated AOM during follow-up. Logistic regression was used to explore baseline determinants of adherence (good ≥ 80%; poor < 80%). RESULTS: The mean (SD) age of participants was 75.6 (4.2) years, with 6233 randomised to screening and 6250 to the control group. Of those participants identified at high fracture risk in the screening group, 38.2% of those on treatment at 6 months were still treated at 60 months, whereas the corresponding figure for the control group was 21.6%. Older age was associated with poorer adherence (OR per year increase in age 0.96 [95% CI 0.93, 0.99], p = 0.01), whereas history of parental hip fracture was associated with greater rate adherence (OR 1.67 [95% CI 1.23, 2.26], p < 0.01). CONCLUSIONS: Systematic fracture risk screening using FRAX® leads to greater use of AOM and greater adherence, in women at high fracture risk, compared with usual care.

Jumping Joints: The Complex Relationship Between Osteoarthritis and Jumping Mechanography.

We investigated the relationship between lower limb osteoarthritis (OA) and muscle strength and power (assessed by jumping mechanography) in UK community-dwelling older adults. We recruited 249 older adults (144 males, 105 females). OA was assessed clinically at the knee according to ACR criteria and radiographically, at the knee and hip, using Kellgren and Lawrence grading. Two-footed jumping tests were performed using a Leonardo Mechanography Ground Reaction Force Platform to assess maximum muscle force, power and Esslinger Fitness Index. Linear regression was used to assess the relationship between OA and jumping outcomes. Results are presented as ß (95% confidence interval). The mean age of participants was 75.2 years (SD 2.6). Males had a significantly higher maximum relative power during lift off (mean 25.7 W/kg vs. 19.9 W/kg) and maximum total force during lift off (mean 21.0 N/kg vs. 19.1 N/kg) than females. In adjusted models, we found significant associations in males between clinical knee OA and maximum relative power [- 6.00 (CI - 9.10, - 2.94)] and Esslinger Fitness Index [- 19.3 (- 29.0, - 9.7)]. In females, radiographic knee OA was associated with total maximum power [- 2.0 (- 3.9, - 0.1)] and Esslinger Fitness Index [- 8.2 (- 15.9, - 0.4)]. No significant associations were observed for maximum total force. We observed significant negative associations between maximum relative power and Esslinger Fitness Index and clinical knee OA in males and radiographic knee OA in females. We have used novel methodology to demonstrate relationships between muscle function and OA in older adults.

The relationship of embolic particle size to patient outcomes in prostate artery embolisation for benign prostatic hyperplasia: a systematic review and meta-regression.

AIM: To explore the relationship of embolic particle size used in prostate artery embolisation (PAE) to patient outcomes. MATERIALS AND METHODS: A systematic review of PubMed, EMBASE, and the Cochrane database was undertaken to identify all existing studies using PAE for benign prostatic hyperplasia (BPH). Inclusion criteria included prospective studies reporting baseline and 12-month International Prostate Symptom Score (IPSS) and particle size. Exclusion criteria were overlapping studies, commentaries, abstracts, and letters. Data extraction from eligible studies included the size of embolic particle, particle material, and baseline and 12-month values for the following patient outcomes: IPSS, IPSS quality of life, urinary flow rate (Q-max), prostate volume, prostate specific antigen, and post-void residual volume. A meta-regression analysis was then undertaken to examine the relationship of particle size to patient outcome measures. RESULTS: Six studies with a total of 687 patients were identified. Meta-regression analysis demonstrated particle size as a statistically significant (p<0.001) moderator of 12-month IPSS change following PAE. No statistically significant relationships were identified with other patient outcome measures. CONCLUSION: Smaller embolic particle size is associated with a greater reduction in IPSS following PAE.

COVID-19 and associations with frailty and multimorbidity: a prospective analysis of UK Biobank participants.

BACKGROUND: Frailty and multimorbidity have been suggested as risk factors for severe COVID-19 disease. AIMS: We investigated, in the UK Biobank, whether frailty and multimorbidity were associated with risk of hospitalisation with COVID-19. METHODS: 502,640 participants aged 40-69 years at baseline (54-79 years at COVID-19 testing) were recruited across UK during 2006-10. A modified assessment of frailty using Fried's classification was generated from baseline data. COVID-19 test results (England) were available for 16/03/2020-01/06/2020, mostly taken in hospital settings. Logistic regression was used to discern associations between frailty, multimorbidity and COVID-19 diagnoses, after adjusting for sex, age, BMI, ethnicity, education, smoking and number of comorbidity groupings, comparing COVID-19 positive, COVID-19 negative and non-tested groups. RESULTS: 4510 participants were tested for COVID-19 (positive = 1326, negative = 3184). 497,996 participants were not tested. Compared to the non-tested group, after adjustment, COVID-19 positive participants were more likely to be frail (OR = 1.4 [95%CI = 1.1, 1.8]), report slow walking speed (OR = 1.3 [1.1, 1.6]), report two or more falls in the past year (OR = 1.3 [1.0, 1.5]) and be multimorbid (≥ 4 comorbidity groupings vs 0-1: OR = 1.9 [1.5, 2.3]). However, similar strength of associations were apparent when comparing COVID-19 negative and non-tested groups. However, frailty and multimorbidity were not associated with COVID-19 diagnoses, when comparing COVID-19 positive and COVID-19 negative participants. DISCUSSION AND CONCLUSIONS: Frailty and multimorbidity do not appear to aid risk stratification, in terms of positive versus negative results of COVID-19 testing. Investigation of the prognostic value of these markers for adverse clinical sequelae following COVID-19 disease is urgently needed.

Role of urinary cations in the etiology of interstitial cystitis: A multisite study.

OBJECTIVE: To determine whether patients with interstitial cystitis have elevated levels of toxic urinary cations, to identify and quantify these cationic metabolites, and to assess their cytotoxicity. METHODS: Isolation of cationic fraction was achieved by solid phase extraction using an Oasis MCX cartridge on urine specimens from interstitial cystitis patients and controls. C18 reverse phase high-performance liquid chromatography was used to profile cationic metabolites, and they were quantified by the area under the peaks and normalized to creatinine. Major cationic fraction peaks were identified by reverse phase high-performance liquid chromatography and liquid chromatography-mass spectrometry. HTB-4 urothelial cells were used to determine the cytotoxicity of cationic fraction and of individual metabolites. RESULTS: The reverse phase high-performance liquid chromatography analysis was carried out on cationic fraction metabolites isolated from urine samples of 70 interstitial cystitis patients and 34 controls. The mean for controls versus patients was 3.84 (standard error of the mean 0.20) versus 6.71 (0.37) mAU*min/µg creatinine, respectively (P = 0.0001). The cationic fraction cytotoxicity normalized to creatinine for controls versus patients in mean percentage was -7.79% (standard error of the mean 3.32%) versus 20.03% (standard error of the mean 2.75%; P < 0.0005). The major toxic cations were 1-methyladenosine, 1-methylguanine, N2 ,N2 -dimethylguanosine and L-tryptophan. CONCLUSIONS: These data confirm significant elevation of toxic cations in the urine of interstitial cystitis patients. These toxic cations likely represent a primary cause of interstitial cystitis, as they can injure the bladder mucus and initiate an epithelial leak.

Subclinical Infection of the Silicone Breast Implant Surface as a Possible Cause of Capsular Contracture.

In order to reexamine the possible association between bacterial presence and capsular contracture, 55 silicone devices (mammary implants or tissue expanders) were cultured at the time of their removal from 40 patients. Special culture techniques were used in an attempt to recover bacteria adhering to the smooth-surfaced implant and encased in glycocalyx biofilm. Bacteria were detected on 56% (15 of 27) of implants surrounded by contracted capsules and on 18% (5 of 28) of those without capsular contracture (p < 0.05). Only three implants tested positive using routine plating techniques. The predominant isolate was Staphylococcus epidermidis. The concept that capsular contracture is associated with subclinical infection of silicone implants is supported by this study. With changes in the microbiological technique, bacterial recovery and growth occurs at a frequency greater than previously thought.

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.

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.

Diet Quality and Bone Measurements Using HRpQCT and pQCT in Older Community-Dwelling Adults from the Hertfordshire Cohort Study.

There are few data describing associations between dietary patterns and bone microarchitecture. This study investigated the relationship between diet quality and HRpQCT and pQCT measures in older adults. Data were available for 184 men and 166 women. Dietary data were collected at baseline (1998-2003) using an administered food frequency questionnaire. A 'prudent' diet score (PDS) was identified using principal component analysis and used as an indicator of dietary quality. HRpQCT and pQCT images were acquired at follow-up in 2012, from the non-dominant distal radius and tibia using Scanco XtremeCT and Stratec XCT2000 instrument scanners, respectively. The mean (SD) PDS was - 0.24 (1.23) for men and 0.62 (1.14) for women. In women, a significant positive relationship was found between baseline dietary pattern and total and trabecular area at both the radius and the tibia, measured by HRpQCT. Similar trends were observed with pQCT parameters. Positive associations were observed for tibia total area (38% slice). At the radius, significant positive associations were found for total area (4% slice) and polar strength strain index (33% slice). All relationships remained robust to adjustment. For men, although patterns were similar, there were no significant associations for HRpQCT outcomes. Significant associations were observed for baseline PDS and polar strength strain and total area (66% slice) at the radius, measured by pQCT. Our data suggest that diets high in fruit, vegetables, oily fish and whole grain cereals in early old age are associated with greater bone size but not volumetric bone density or microarchitecture in later life in women.

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.

Technical note: Infusion, sampling, and vacuum-assisted collection devices for use in ruminally cannulated calves.

Calves can be ruminally cannulated at young ages, but equipment size limitations preclude use of an infusion and sampling device in these small animals. Likewise, a procedure to easily evacuate rumen contents in young calves has not been described. Overcoming these technical complications related to assessment of ruminal passage kinetics, nutrient digestion, and volatile fatty acid absorption would aid in future studies advancing our knowledge of dairy calf nutrition. The first objective was to design and fabricate 2 devices (one device for infusion and sampling, and another for vacuum-assisted collection) suitable for use in young ruminally cannulated dairy calves. The second objective was to test the utility of these tools when performing procedures commonly used in ruminant nutrition research. A single weaned 62-d-old ruminally cannulated calf was used to evaluate the ability to infuse a solution of LiCoEDTA and sample rumen contents through the cannula cap over a period of 2 h to assess the rumen liquid passage rate (procedure 1). The device was capable of infusing the LiCoEDTA and sampling the rumen fluid, as evidenced by the presence of elevated Co concentrations in the sampled rumen fluid. Using the fluid samples obtained, liquid passage rate within the calf was estimated to be 40.2% of ruminal fluid/h. The second procedure tested the vacuum-assisted collection device and consisted of evacuating and weighing the rumen contents, which is considered a key preparatory step in washed reticulorumen technique experiments that aim to measure nutrient absorption. In agreement with existing literature, evacuated rumen contents represented approximately 4% of the calf's body weight. In conclusion, custom-built devices for infusion, sampling, and vacuum-assisted collection were efficacious when tested in a 62-d-old ruminally cannulated calf fed a diet of 100% texturized starter (18% crude protein, as-fed). Fellow scientists may employ and further modify these techniques to suit their needs when assessing passage kinetics, nutrient digestion, and volatile fatty acid absorption in calves.

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.