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

This study aimed to evaluate the impact 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 d in milk housed in climate-controlled rooms were paired by body weight 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 total mixed ration 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 to 80), while PFTN cows remained at 64 THI and daily dry matter intake was matched to HS. 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 vs. 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. Total number of nucleated MEC per area were greater in HS (389 ± 1.05) 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 compared with PFTN. Immunoblotting analyses showed a decrease abundance for phosphorylated STAT5 and S6K1, and an increase in LC3 II in HS compared with PFTN. 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, while an upregulation of proteins associated with autophagy in HS compared with PFTN.

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 d in milk 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. P1 consisted of a 4 d thermoneutral period with ad libitum intake. During 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. DMI of the PFTN cow 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 (GIP). 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. HS decreased voluntary intake by 35% and increased rectal temperature and respiration rate (38.4 vs 39.4°C and 40 vs 71 respirations/min, respectively). HS reduced dry matter intake (DMI) by 35% which accounted for 66% of the decrease in milk yield. The yield, and not concentration, of milk protein, fat, and other solids were lower in the HS cows on d 4 of P2. Milk urea nitrogen (MUN) was higher and plasma urea nitrogen (PUN) 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 (LBP) was observed. HS 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 PUN indicate a whole-body shift in nitrogen metabolism. No differences in GIP or LBP were observed. These results indicate that, under conditions of this experiment, activation of the immune system by gut derived lipopolysaccharide was not responsible for the decreased milk yield observed during HS.

Persistence of sugars used for intestinal permeability measures in an in vitro rumen environment.

Orally administered synthetic sugars are routinely used as markers of intestinal permeability in nonruminants and young calves, but not adult ruminants, likely because of uncertainty surrounding degradation of such sugar markers (e.g., d-mannitol, sucralose, lactulose) in the rumen. The objective was to evaluate persistence of d-mannitol, sucralose, and lactulose in a closed in vitro rumen fermentation system over 48 h. The null hypothesis was that sugar concentration would not be affected by time. Rumen contents were collected and processed under anerobic conditions a total of 12 times from a ruminally cannulated lactating Holstein cow. These 12 rumen samplings reflect 4 in vitro experiments (d-mannitol, sucralose, lactulose, and d-glucose as a positive methodological control), each replicated 3 times. For each replication, filtered rumen contents and rumen buffer (1:3; vol/vol) were added to a series of six 500-mL flasks, each containing 3 filter bags. Each filter bag contained 500 mg of ground total mixed ration (94.2% dry matter; 15.2% crude protein, 40.9% neutral detergent fiber, 3.9% fat, and 6.2% ash, dry matter basis) and three 5-mm glass beads. The 6 flasks represented 0, 6, 12, 24, and 48 h time points, and a 48-h negative control flask. A single sugar was tested during each experimental replicate. Final flask concentrations of each sugar were 4.07 mg/mL d-glucose, 1.99 mg/mL d-mannitol, 2.17 mg/mL sucralose, or 3.10 mg/mL lactulose. Flasks were incubated under anerobic conditions at 39°C where they remained undisturbed until the designated time of removal (0, 6, 12, 24, or 48 h). At removal, an aliquot of each flask was removed and sugar concentration was quantified by HPLC-mass spectrometry. Data for each experiment were analyzed using an ANOVA model that included the single fixed effect of time (0, 6, 12, 24, or 48 h); flask within replicate was the random term. Lactulose was not resolved in any samples due to interfering components within the sample matrix; no lactulose data are presented. As expected, positive methodological control of glucose decreased to negligible concentrations by 6 h of in vitro incubation. d-Mannitol followed the same pattern as glucose, which was different from our hypothesis. The interpretation is that d-mannitol is degraded in the in vitro rumen culture system and, by extension, is therefore not a viable choice to use in in vivo intestinal permeability tests in adult ruminants when dosed orally. As hypothesized, sucralose concentration did not change over 48 h of incubation in a closed in vitro rumen fermentation system. This suggests feasibility of orally dosed sucralose in adult ruminants as a rumen-inert marker of intestinal permeability with subsequent analysis of biological samples (e.g., urine, blood) by HPLC-mass spectrometry.

Algorithm development for individualized precision feeding of supplemental top dresses to influence feed efficiency of dairy cattle.

Individualized, precision feeding of dairy cattle may contribute to profitable and sustainable dairy production. Feeding strategies targeted at optimizing efficiency of individual cows, rather than groups of animals with similar characteristics, is a logical goal of individualized precision feeding. However, algorithms designed to make feeding recommendations for specific animals are scarce. The objective of this study was to develop and test 2 algorithms designed to improve feed efficiency of individual cows by supplementing total mixed rations (TMR) with varying types and amounts of top-dressed feedstuffs. Twenty-four Holstein dairy cows were assigned to 1 of 3 treatment groups as follows: a control group fed a common TMR ad libitum, a group fed individually according to algorithm 1, and a group fed individually according to algorithm 2. Algorithm 1 used a mixed-model approach with feed efficiency as the response variable and automated measurements of production parameters and top-dress type as dependent variables. Cow was treated as a random effect, and cow by top-dress interactions were included if significant. Algorithm 2 grouped cows based on top-dress response efficiency structure using a principal components and k-means clustering. Both algorithms were trained over a 36-d experimental period immediately before testing, and were updated weekly during the 35-d testing period. Production performance responses for dry matter intake (DMI), milk yield, milk fat percentage and yield, milk protein percentage and yield, and feed efficiency were analyzed using a mixed-effects model with fixed effects for feeding algorithm, top dress, week, and the 2- and 3-way interactions among these variables. Milk protein percentage and feed efficiency were significantly affected by the 3-way interaction of top dress, algorithm, and week, and DMI tended to be affected by this 3-way interaction. Feeding algorithm did not affect milk yield, milk fat yield, or milk protein yield. However, feeding costs were reduced, and hence milk revenue increased on the algorithm-fed cows. The efficacy of feeding algorithms differed by top dress and time, and largely relied on DMI shifts to modulate feed efficiency. The net result, for the cumulative feeding groups, was that cows in the algorithm 1 and 2 groups earned over $0.45 and $0.70 more per head per day in comparison to cows on the TMR control, respectively. This study yielded 2 candidate approaches for efficiency-focused, individualized feeding recommendations. Refinement of algorithm selection, development, and training approaches are needed to maximize production parameters through individualized feeding.

Effects of sodium salicylate and time postpartum on mammary tissue proliferation, gene transcript profile, and DNA methylation.

Previous studies have demonstrated nonsteroidal antiinflammatory drug treatment in early lactation had a positive impact on whole-lactation milk production in older cows. The objective of this study was to evaluate proliferative, transcriptional, and epigenetic changes in the mammary gland that could explain increased production responses due to nonsteroidal antiinflammatory drug treatment. Sodium salicylate (SAL; 125 g/d) or water (CON) were administered via oral drench to multiparous Holstein cows (n = 8/treatment) once daily for 3 d beginning approximately 24 h after parturition, and mammary tissue was collected on d 1, 4, and 45 postpartum. Day 1 tissue was collected immediately preceding the initial drench, and d 4 tissue was collected 24 h following the final drench. Blood was collected twice weekly and analyzed for plasma glucose, insulin, ß-hydroxybutyrate, free fatty acids, and prolactin. Cows were milked twice daily until d 7 of lactation, and thrice daily for the remainder of the study. Total RNA extracted from tissue was deep-sequenced and analyzed for differential gene expression using DESeq2. We detected no treatment effect on milk yield or plasma metabolites through 45 d of lactation; additionally, no change in mammary epithelial cell proliferation was detected when assessed by Ki67 labeling. Comparison of SAL versus CON revealed that only 16 of 18,286 genes were differentially expressed (false discovery rate <0.1) in mammary tissue collected on d 45, whereas no differentially expressed genes due to treatment were detected on d 1 or 4. Analysis of transcriptional differences over time showed downregulation of pathways related to immune cell recruitment and differentiation, and extensive overlap with pathways related to cholesterol synthesis and liver X receptor signaling. Global DNA methylation of mammary tissue was decreased for CON compared with SAL. Transcriptome analysis emphasized extensive involvement of immune-related signaling pathways in the switch from lactogenesis to galactopoiesis, and changes in methylation with SAL treatment merit future investigation into epigenetic effects on milk production.

Rumen volatile fatty acid molar proportions, rumen epithelial gene expression, and blood metabolite concentration responses to ruminally degradable starch and fiber supplies.

The objective of this work was to characterize rumen volatile fatty acid (VFA) concentrations, rumen epithelial gene expression, and blood metabolite responses to diets with different starch and fiber sources. Six ruminally cannulated yearling Holstein heifers (body weight = 330 ± 11.3 kg) were arranged in a partially replicated Latin square experiment with 4 treatments consisting of different starch [barley (BAR) or corn (CRN)] and fiber [timothy hay (TH) or beet pulp (BP)] sources. Treatments were arranged as a 2 × 2 factorial. Beet pulp and TH were used to create relative changes in apparent ruminal fiber disappearance, whereas CRN and BAR were used to create relative changes in apparent ruminal starch disappearance. Each period consisted of 3 d of diet adaptation and 15 d of dietary treatment. In situ disappearance of fiber and starch were estimated from bags incubated in the rumen from d 10 to 14. From d 15 to 17, rumen fluid was collected every hour from 0500 to 2300 h. Rumen fluid samples were pooled by animal/period and analyzed for pH and VFA concentrations. On d 18, 60 to 80 papillae were biopsied from the epithelium and preserved for gene expression analysis. On d 18, one blood sample per heifer was collected from the coccygeal vessel. In situ ruminal starch disappearance rate (7.30 to 8.72%/h for BAR vs. 7.61 to 10.5%/h for CRN) and the extent of fiber disappearance (22.2 to 33.4% of DM for TH vs. 34.4 to 38.7% of DM for BP) were affected by starch and fiber source, respectively. Analysis of VFA molar proportions showed a shift from propionate to acetate, and valerate to isovalerate on TH diets compared with BP. Corn diets favored propionate over butyrate in comparison to BAR diets. Corn diets also had higher molar proportions of valerate. Expression of 1 gene (SLC9A3) were increased in BP diets and 2 genes (BDH1 and SLC16A4) tended to be increased in TH diets. Plasma acetate demonstrated a tendency for a starch by fiber interaction with BAR-BP diets having the highest plasma acetate, but other metabolites measured were not significant. These results suggest that TH has the greatest effect on shifts in VFA molar proportions and epithelial transporters, but does not demonstrate shifts in blood metabolite concentrations.

Graduate Student Literature Review: Potential mechanisms of interaction between bacteria and the reproductive tract of dairy cattle.

Although the presence of bacteria has been characterized throughout the reproductive tracts of multiple species, how these bacteria may interact with the host has yet to be described. Previous reviews have described how pathogenic bacteria interact with the reproductive tract to cause infections such as metritis. This review aimed to summarize the knowledge related to pathogenic and nonpathogenic bacteria in various locations of the bovine reproductive tract and the possible mechanisms underlying host-microbe interactions during gametogenesis and early pregnancy. Lactic acid bacteria such as Lactobacillus seem to be beneficial in multiple areas of the reproductive tract: they have been associated with increased oocyte quality when in follicular fluid and secrete reactive oxygen species that are beneficial during placental angiogenesis. However, other bacteria, including Enterococcus, Staphylococcus, and Streptococcus, may modulate T helper cells that inhibit maternal recognition of pregnancy. Available data on the reproductive microbiome focus on variations in microbial communities and their associations with reproductive performance. However, research on these host-microbiome interactions may provide more insight on how bacteria affect fertility.

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.

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.

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.

Performance strategies affect mammary gland development in prepubertal heifers.

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.

Technical note: Mammary gland ultrasonography to evaluate mammary parenchymal composition in prepubertal heifers.

Bovine mammary gland development studies are often terminal or involve invasive biopsy procedures. Therefore, noninvasive means of assessing mammary development should be considered as alternative methods in live animals. The objective was to test if mammary ultrasonography can be used as a noninvasive way to estimate mammary parenchyma (PAR) composition in prepubertal dairy heifers with different average daily body weight gains. In the 84 d preceding, the ultrasound exam heifers were maintained in 1 of 3 treatment groups. Individual heifers were fed a high gain (1 kg/d; n = 6), low gain (0.5 kg/d, n = 6), or maintenance (n = 6) treatment diet. To achieve desired body weight gains, heifers were fed differing amounts of the same silage-based diet. Mammary glands of 18 crossbred heifers Holstein:Gyr underwent a single mammary ultrasound exam immediately before heifer slaughter, which took place when heifers weighed 142.0 ± 8.0 kg and were 200 d old. The 4 mammary glands of each heifer were evaluated using a real-time B-mode ultrasound machine equipped with a 6.5-MHz micro-convex transducer. Digital images (8-bit) of glands were obtained and PAR was identified within gland. Average pixel values per unit of PAR area were determined for each gland and analyzed at the level of heifer. Pixel results were interpreted on the basis that lower average pixel values reflect PAR with relatively high amounts of protein as opposed to fat. To help validate that the pixel value within PAR is associated with composition of PAR, pixel findings were compared with histological [number of adipocytes in PAR (Nad) and epithelial area in PAR (Ep)] and biochemical [percent crude protein in PAR (%CP), percent ether extract in PAR (%EE), PAR weight (WPAR), and mammary fat pad weight (WFAT)] composition of PAR in these same heifers. Within PAR, %EE and WFAT were positively correlated with pixel values, whereas %CP, Ep, and Nad were negatively correlated. Parenchyma weight did not correlate with pixel values. Regression analyses (fixed effect log-pixel value; random effect treatment) were used to estimate Nad, Ep, %CP, %EE, WPAR, and WFAT. Sensitivity analysis of regression equations revealed that accuracy of tested equations ranged from 0.77 to 0.93 and precision ranged from 0.56 to 0.82. Concordance correlation coefficients of the equations ranged from 0.41 to 0.76. In conclusion, ultrasonography of PAR can accurately measure and predict PAR composition in prepubertal dairy heifers growing at various rates of gain.

Effects of nutrient intake level on mammary parenchyma growth and gene expression in crossbred (Holstein × Gyr) prepubertal heifers.

This study investigated the effects of increased nutrient intake levels on prepubertal mammary parenchyma development in crossbreed (Holstein × Gyr) dairy heifers. Eighteen heifers age 3 to 4 mo were fed 1 of 3 nutrient intake levels (n=6 per treatment) designed to sustain an average daily gain of 0.0kg/d (maintenance, MA), 0.5kg/d (low gain, LG), or 1.0kg/d (high gain, HG). Serum blood samples collected on d 42 and 84 after a 12-h fast were analyzed for triglycerides, leptin, insulin, and insulin-like growth factor 1 (IGF-1). Liver and mammary parenchyma were biopsied on d 42 and harvested on d 84 for gene expression analysis. Parenchyma samples were also used for biochemical and histological analysis. Mammary parenchyma weight was lower in HG than in MA or LG heifers, but mammary extraparenchymal fat was greater in HG heifers than in other groups. Heifers fed the HG diet had a greater fraction of ether extract in their parenchyma than the others and a smaller fraction of crude protein in their parenchyma than MA heifers. Moreover, the HG and LG heifers had greater body fat mass than MA heifers. Nutrient intake level had no effect on the number of intraparenchymal adipocytes. Heifers fed the HG diet had greater serum IGF-1 than the others, and serum insulin was lower in the MA than the HG or LG heifers. Liver GHR, IGF1, and IGFBP3 mRNA expression was higher, but IGFBP2 mRNA was lower in HG heifers than in others. The parenchyma mRNA expression of lipogenic markers, such as CD36, ACCA, FASN, and ADIPOR1, was upregulated by nutrient intake level. Significant nutrient intake × time interactions for lipogenic genes during the experimental period indicated variable gene expression depending on the time point of prepubertal mammary gland development. Overall, our data suggest that enhancing nutrient intake increased body fat accumulation and lipogenesis in the mammary gland to the detriment of parenchyma growth. Moreover, increased lipogenesis in the parenchyma of HG heifers may indicate that fat accumulation occurred because of adipocyte hypertrophy and not differences in adipogenesis. The implications of these results for milk yield needs to be elucidated.

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.

Housing system may affect behavior and growth performance of Jersey heifer calves.

Social pressure is increasing to adopt alternative housing and management practices that allow farm animals more opportunity to exercise and demonstrate social behavior. The present study investigated the effect of pair housing on the behavior and growth performance of Jersey heifer calves. Forty female Jersey calves were allocated to individual or pair housing at birth and monitored for 9 wk. Calves were provided with a single hutch, and those allocated to the pair housing treatment were provided a pen enclosure twice the size of individually housed calves and only one hutch was provided per pair. All calves were fed milk replacer via bucket twice per day (1.89 L/feeding first 7 d; 2.27 L/feeding until weaned) and had ad libitum access to grain and water. Gradual weaning commenced on d 49 by reducing the calves' milk allowance to one feeding per day, and weaning occurred on d 56. Grain consumption was monitored daily and calves were weighed weekly. Direct behavioral observations were conducted twice per week. Calves housed in pairs tended to have greater average daily gain compared with calves housed individually (0.63 vs. 0.59 ± 0.02 kg/d, respectively). Pair housing also increased final body weight compared with individual housing (64.9 vs. 61.7 ± 0.59 kg, respectively). During observation periods, calves housed individually spent more time engaging in nonnutritive sucking than calves housed in pairs (21.5 vs. 8.15 ± 0.03% of total observations). Calves housed in pairs were observed cross sucking 13.5% of the time during observational periods. Although housing Jersey calves in pairs may increase measures of growth performance, future research should aim to reduce cross-sucking behavior within the Jersey breed through alternative feeding systems or environmental enrichment.

Growth, ruminal measurements, and health characteristics of Holstein bull calves fed an Aspergillus oryzae fermentation extract.

A fermentation extract of the fungus Aspergillus oryzae can be used as a prebiotic. The objective was to determine if dietary inclusion of a fermentation extract of A. oryzae as well as calf age would alter growth, health, performance parameters, and the growth and development of the rumen in Holstein calves from birth thru 1 wk postweaning; it was hypothesized that it would. Purchased bull calves (n=52) that originated from 1 of 13 farms were used in this experiment. All calves had serum IgG greater than 10 mg/mL. Calves were randomly assigned to a slaughter age, 4 (n=16) or 8 wk (n=36), and treatment, control (n=27) or fermentation extract of A. oryzae (AMF; n=25). Calves were housed and fed individually; no bedding was used and no forage was fed. Calves assigned to AMF were fed 2 g of AMF daily. Liquid AMF was delivered in milk replacer for the first 4 wk of the study; solid AMF was top-dressed on texturized starter thereafter. Calves were fed nonmedicated milk replacer twice daily (22.0% crude protein, 20.0% fat, dry matter basis; 680 g/d) and were weaned upon consumption of 0.91 kg of starter (20% crude protein, 2.0% fat; medicated with decoquinate) for 3 consecutive days or on d 45 of the study, whichever came first. Calves had ad libitum access to starter and water throughout the study. Feed intake as well as fecal and respiratory scores were recorded daily; body weight, withers height, and hip height were recorded weekly. Gross rumen measurements and rumen samples for future gross and histological analyses were taken at 4 and 8 wk. All calves grew similarly; weaning age averaged 40.39±0.77 d. Lifetime average daily gain was 0.60±0.05 kg/d and lifetime gain-to-feed ratio was 0.56±0.05. Milk replacer, starter, total dry matter intake, gross and histological rumen measurements, rumen pH, fecal and respiratory scores, and total medical costs were not affected by treatment. Despite total medical costs not differing by treatment, a lower percentage of AMF calves were treated for respiratory ailments and respiratory treatment cost was lower for AMF compared with control. Several factors may have contributed to an overall lack of observed treatment effect in this study, including include the use of a low-forage (compared with concentrate) diet, immunocompetent calves, and the selected product dose and routes of delivery. Dietary inclusion (2 g/d) of an extract of A. oryzae did not affect calf growth, intake, and ruminal or health measurements.

Examination of weekly mammary parenchymal area by ultrasound, mammary mass, and composition in Holstein heifers reared on 1 of 3 diets from birth to 2 months of age.

Monitoring in vivo growth of mammary parenchyma (PAR) has historically been difficult, necessitating slaughter studies to measure PAR quantity. Advances in ultrasound (US) technology warrant revisiting its use as a noninvasive tool to monitor PAR growth in vivo. The level of nutrient intake during the first 2mo of life may affect measures of mammary growth and composition. Objectives were to examine the utility of US as an in vivo tool to quantify PAR cross-sectional area in Holstein heifers reared on 1 of 3 diets from birth to 2mo of age, assessing potential dietary effects; assess the relationships between weekly US measurements, teat length, manual palpation of PAR scores, and PAR mass at 2mo of age; and examine mammary composition in experimental animals. Holstein heifers (n=24; 41±1kg of initial body weight) from a single farm were randomly assigned to 1 of 3 milk replacers that differed in source and amount of fat. Milk replacer was fed at 660g of dry matter/d until weaning at 42 d. Heifers had ad libitum access to a common calf starter (20% crude protein) and water for the duration of the 56-d trial. Teat length and palpation scores were obtained weekly. A real-time B-mode US with a 7.5-MHz convex probe was used to examine 2-dimensional PAR area in all 4 glands of heifers once weekly from 2 to 3 d of age to harvest at 56 d. The left front and left rear glands were also examined by US 24h postharvest to validate final US measurements, and then bisected to produce a sagittal plane view of PAR for comparison with US images. Mass and composition of mammary gland tissue were determined at 8 wk using standard methodology. Over the course of this 8-wk trial, average teat length increased from 11 to 17mm. The PAR area started small (6.6±3.2mm(2) per gland) and increased to 42.1±2.5mm(2) per gland by the end of the trial. As anticipated, based on measurements obtained at slaughter, US measurements were more related to amount of PAR (r=0.74) than either teat length (r=0.34) or palpation scoring (r=0.63). Importantly, US is quantitative, whereas palpation scoring is subjective. Diet did not affect mass or composition of PAR in young heifers; total udder PAR mass averaged 1.40±0.80g. In conclusion, we showed that in heifers younger than 2mo of age, obtaining weekly PAR measurements via ultrasound is an effective quantitative tool for measuring changes in PAR area in vivo. Future studies may incorporate and expand upon the methods developed here to determine what quantitative evaluation of PAR in young heifers can reveal about milk production capacity.

Adaptation to various sources of dietary sulfur by ruminants.

The objective of these experiments was to determine adaptation by ruminants to dietary sulfur. In Exp. 1, lambs (n = 54; BW = 33.6 ± 0.4 kg) were allotted to 3 treatments: 1) 0% added dietary S (0%S), 2) 0.2% added dietary S (0.2%S), or 3) 0.4% added dietary S (0.4%S). Sulfur was added to the diet as Na(2)SO(4). Lambs fed the 0.2%S and 0.4%S diets had greater (P < 0.01) ADG and G:F compared to those fed the 0%S diet. There was time × diet interaction (P < 0.01) on ruminal hydrogen sulfide gas (H(2)S) concentrations. Ruminal H(2)S was not detected in lambs fed 0%S at any time. Ruminal H2S were not affected (P > 0.19) by diet on d 1 or 8; however, H(2)S were greater (P < 0.01) for lambs fed 0.2%S and 0.4%S than for lambs fed 0%S on d 15, 22, and 29 (0.2% was 931, 846, and 1,131 mg/L and 0.4% was 975, 737, and 1,495 mg/L on d 15, 22, and 29, respectively). These data suggest it takes at least 29 d for peak ruminal H(2)S to occur after exposure to Na(2)SO(4). In Exp. 2, lambs (n = 66; BW = 51.1 ± 0.4 kg) were allotted to 3 treatments: 1) 60% dried distillers grains with solubles (DDGS), 2) corn-based diet with Na(2)SO(4), or 3) corn-based diet with H(2)SO(4). All diets were formulated to contain 0.4%S. Lambs fed Na(2)SO(4) had greater (P < 0.05) ADG, DMI, and G:F than those fed H(2)SO(4) or 60% DDGS. A time × diet interaction occurred (P < 0.01) for ruminal H(2)S. There was no difference (P = 0.82) in H(2)S of lambs on d 1. However, at d 14 and 27 lambs fed supplemental Na(2)SO(4) had the lowest H(2)S concentrations while lambs fed 60% DDGS had the greatest (P < 0.01 on both d); lambs fed H(2)SO(4) were intermediate and different than both. These data suggest that at the same dietary S concentration, acidic S sources increased H(2)S and decreased DMI and ADG. In Exp. 3, Angus cross calves (n = 72; average initial BW = 324 ± 3 kg) were allotted to 3 treatments: 1) corn-based control d 0 through 85 (0%DDGS), 2) gradual step up to 60% DDGS diet (20% DDGS d 0 to 6, 40% DDGS d 7 to 13, 50% DDGS d 14 to 20, and 60% DDGS d 21 to 85; Step-up), or 3) 60% DDGS d 0 to 85 (60%DDGS). Overall, cattle fed 0%DDGS had increased (P < 0.05) DMI and ADG compared with those fed 60%DDGS or Step-up, and G:F was not affected (P = 0.42) by dietary treatment. On d 14, ruminal H(2)S concentrations were greater (P < 0.01) for cattle fed 60%DDGS and Step-up than for those fed 0%DDGS, and they did not differ (P ≥ 0.22) between DDGS-containing diets. These data illustrate that source of S impacts ruminal S metabolism and that S from DDGS is more readily reduced than S from Na(2)SO(4) or H(2)SO(4).