Environmental Factors and Their Role in Calf Health

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CHAPTER 3: THE SYNERGISTIC RELATIONSHIP OF LIPOPOLYSACCHARIDE AND BUTYRATE STIMULATING RUMEN DEVELOPMENT

ABSTRACT

The objective was to investigate the effects of orally dosed lipopolysaccharide (LPS) and sodium butyrate on rumen cell proliferation in Holstein dairy calves. The hypothesis was that LPS and butyrate synergize to promote rumen epithelial cell proliferation via the binding of LPS to toll-like receptor 4 and mobilization of volatile fatty acids (VFA) through the monocarboxylic transporter (MCT) pathways. Twenty-four single-sourced purchased bull calves arrived to the Virginia Tech (VT) dairy in one of two arrival groups, spaced 2 wk apart, and enrolled on the study for 56 d. Within each group, calves were assigned to one of four treatments: control (CON; n=5), butyrate (BUTY; n=5), LPS only (LPS-O) (n=6), or LPS plus butyrate (LPSB; n=6). All treatments were administered orally twice daily at least 30 minutes following the morning meal and consisted of : 0.9% saline (CON); 11mM sodium butyrate (BUTY); LPS ranging from 2.5 to 40 µg/kg metabolic body weight (MBW) (BW^0.75, LPS), or both butyrate and LPS (LPSB). Treatment dosage volumes increased as the study progressed, ranging from 10 to 40 mL per dose. Calves were fed milk replacer (22% CP, 20% fat, as-fed) and starter (20% CP, 3% fat, as-fed) twice daily (0600 and 1800) based MBW. Feed intake, fecal and respiratory scores, and rectal temperature were recorded daily. Calf BW, hip height, jugular blood samples, and rumen content samples (oroesophageal route) were collected weekly, 2 h after morning feeding. Calves were weaned at 6 wk of age and euthanized at 8 wk of age, whereupon ruminal weights and ruminal samples for papillae area and epithelial thickness were collected. Blood and rumen samples were analyzed for blood metabolites (BHBA, glucose, LPS-binding protein) and VFA concentrations, respectively. Data were analyzed as a 2×2 factorial with the repeated effect of week. Three non-orthogonal contrasts (CON versus the average of all other treatments; LPS-O versus LPSB, and LPSB versus BUTY) were investigated. Feed intake, health measures, and blood metabolites did not differ by treatment. Calf BW increased by week (P < 0.0001). Irrespective of week, LPSB calves weighed more than BUTY calves (LPSB, 66.9 ± 0.68 kg; BUTY, 64.0 ± 0.75 kg; P = 0.02; Table 3.5). Irrespective of week, withers heights of calves in treatments that contained LPS had an overall average of 87.7 ± 0.21 cm and overall withers height for calves on treatments that did not contain LPS were 86.8 ± 0.22 cm (P = 0.006; Table 3.6). Rumen pH and rumen VFA concentrations did not differ by treatment but did decrease and increase, respectively, with week in conjunction with increased starter intake (P < 0.0001; Table 3.8). Total empty stomach (LPSB, 2.87 ± 0.076 kg; BUTY, 2.48 ± 0.076 kg; P = 0.01; Table 3.6) and reticulorumen weights (LPSB, 1.76 ± 0.065 kg; 1.44 ± 0.065 kg; P = 0.01; Table 3.6) were higher in LPSB calves when compared to BUTY calves. No treatment differences were detected in transporter intensity, BrdU cell counts, or LPS binding protein (Table 3.9). Proteins EGFR, MCT1 and MCT4 were affected by location (P < 0.0001) and LPS binding protein was affected by week (P < 0.0001). The lack of effects observed in this study could be indicative of many possibilities including: LPS and/or sodium butyrate did not breech gastrointestinal barriers, calves became tolerant to orally dosed LPS, or the sodium butyrate was so quickly metabolized and therefore not detected in blood and rumen samples 3h after feeding. Future studies are necessary to build on the findings in this paper for a more complete understanding of the role LPS plays in the rumen of young dairy calves. Key words: dairy calf, rumen development, lipopolysaccharide

INTRODUCTION

Lipopolysaccharide (LPS) is a chemical of interest because of its role within the calf when subacute ruminal acidosis (SARA) is induced. This disease, SARA, occurs when rumen pH drops due to an increase of rapidly fermentable feedstuff in the rumen (Kleen et al., 2003). The threshold for rumen pH is 5.5 and anything below that can be considered SARA in mature dairy cattle (Kleen et al., 2003; Laarman and Oba, 2011). It is fairly common for calves that are going through weaning to have rumen pH below 5.5 (Suarez et al., 2006; Kim et al., 2016). Under such conditions, LPS can translocate to the bloodstream, causing life-threatening scenarios for lactating dairy cows (Gozho et al., 2005). Nagaraja et al. (1978) demonstrated that steers fed a higher grain-based diet have elevated levels of LPS in the rumen. Gozho et al. (2005) went one step further to confirm that LPS levels in the bloodstream are indeed higher when SARA is induced, but additionally showed an increased inflammatory response by looking at acute phase protein markers in the bloodstream. Considering LPS is linked with grain consumption (Gozho et al., 2005), and grain consumption is linked with rumen epithelial development in calves (Stobo et al., 1966) there are gaps in our knowledge regarding how LPS may be affecting the rumen on a mechanistic level, which is what we sought out to investigate. Toll-like receptor 4 (TLR4) is the receptor that mediates LPS signaling in many cell types across species (Nagai et al., 2002). The receptor is a class one transmembrane receptor that is expressed on the cell surface (Chaturvedi and Pierce, 2009). Harris et al. (2006) demonstrated that TLR4 is present in the GI tract of humans. Additionally, monocarboxylate transporters (MCT) are located along the GI tract of ruminants, which are responsible for transporting volatile fatty acids (VFA) from the lumen to the blood (Graham et al., 2006). Because MCT and TLR4 function on the same epidermal sheet, it is for this reason we proposed there exists a synergistic relationship between VFA and LPS (Figure 3.1). When the calf increases grain intake around weaning, butyric, propionic, and acetic acid concentrations increase. Because MCT are responsible for the transport of VFA (Kirat et al., 2007), we expect the intensity of MCT and TLR4 to be more abundant when larger amounts of VFA and LPS are present in the rumen lumen, respectively. Further, we expect these differences to coincide with measures of increased rumen growth. Stefanska et al. (2018) established that TLR4 is more abundant in SARA induced versus healthy dairy cows. Sodium butyrate is important for rumen cell proliferation (Sakata and Tamate, 1978). It is with this knowledge that we formulated our hypothesis that twice daily oral doses of either butyrate, LPS, or both will affect abundance of MCT, TLR4, and rumen cell proliferation with overall effects on somatic and rumen growth in young dairy calves. To our knowledge there are no studies to date that present research on the TLR4 pathway with regards to the synergistic relationship between LPS and VFA in dairy cattle. Previous studies have performed LPS challenges both intravenously and orally in calves and lactating cattle respectively, however, those studies investigated the immune response and not the synergistic mechanism we are proposing here (Anderson et al., 1996; Eicher et al., 2006; Ametaj et al., 2012). We hypothesize: 1) that LPS will further enhance rumen cell proliferation when present in larger quantities and 2) rumen cell proliferation will be even further enhanced when LPS and butyrate are both present in higher concentrations. With a better understanding of the mechanisms behind LPS transport in the rumen, further conclusions can be made to improve performance and health of the calf.

MATERIALS AND METHODS

Animals, Housing, and Treatments

Animals. All experimental procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee (protocol #17-040). Twenty-four Holstein bull calves sourced from a single farm located 232 km from Virginia Tech were used. Calves were transported via trailer and arrived in one of two groups of 12 calves each. The first group (5d ± 1d of age; mean ± standard deviation) was on site from May 29, 2018 to July 25, 2018. The second group (6d ± 2d of age; mean ± standard deviation) was on site from June 12, 2018 to August 8, 2018. We verified that farm staff at the farm of origin fed colostrum to all calves by quantifying serum immunoglobulin G (Radial Immunodiffusion Plates, Triple J Farms, Bellingham, WA) of each calf upon arrival to Virginia Tech. Serum immunoglobulin G averaged 2,086 mg/dL ± 354 mg/dL; (mean ± standard deviation); no calf had failure of passive transfer of immunity (range: 1,378 to 2,661 mg/dL IgG, with failure of passive transfer cutoff of 800 mg/dL). Twice-daily meals after colostrum feeding at the farm of origin consisted of milk replacer (20% CP (as-fed), 20% fat (as-fed); 454 g powder (as-fed)/calf per day). No starter was offered at the farm of origin. Each calf received the following vaccinations and injections at the farm of origin:1mL multi-min subcutaneously and 2mL enforce 3 nasally. Upon arrival to Virginia Tech, all calves were screened for bovine viral diarrhea virus by ear notch test (Snap BVDV Antigen Test, IDEXX, Westbrook, MN); all calves tested negative. Further, body weight (BW), withers height (WH), and hip height (HH) were recorded for each calf. All calves received 1.94 L of oral electrolytes (50g powder; Diaque, Boehringer Ingelheim Vetmedica, INC., Duluth, GA) within 1 h of arrival to Virginia Tech. Housing. Calves were individually housed in and fed from hutches for the duration of the experiment. Hutches were naturally ventilated and bedded with fresh sawdust on a weekly basis. Nose-to-nose contact was prevented by placing hutches approximately 1m apart. Calves were bottle-fed milk replacer until they were bucket trained; thereafter milk replacer was fed from open 9.5 L buckets. Calves were fed twice daily (600 and 1800h). Prior to weaning, calves were fed at 32% MBW (BW^0.75), or 12% BW in two equally sized meals. Amount of MR was adjusted weekly. The MR was mixed at 13% solids and fed at 20 g powder DM per kg of MBW and contained 22.8% CP, 20.7% fat DM basis (Performance, Purina Animal Nutrition LLC, Shoreview, MN). Calves had ad libitum access to drinking water and were fed a common medicated calf starter (20% CP, 2% fat, 50 g/ton monensin sodium, 9.1 g/ton diflubenzuron (as-fed); Ampli-Calf, Purina Animal Nutrition LLC, Shoreview, MN) in increasing amounts each week based on MBW. After weaning calves were offered a total of 3% BW of the starter. The MR, water, and starter intakes were recorded daily for each calf by weighing back any refusals. Calves were weaned starting on d 42 of the experiment (6 wk) with MR reduced to once daily feeding (50% volume reduction) at 1800 h for 5 d. Fresh MR and starter samples were collected weekly for analysis. Weekly samples were pooled into one composite sample for analysis; pooling was deemed appropriate because all MR and starter were from the same manufactured lots. Samples of starter refusals were also collected on a daily basis and pooled into one composite sample per week to ensure the feed composition did not vary between fresh and refused starter samples. All samples were stored at -20°C and underwent analysis at Cumberland Valley Analytical Services (Waynesboro, PA) using the relative feed value package with additional starch, ether extract, and ash options (Table 3.1). Dry matter percentages of refused starter were determined by heating the composited sample to 100°C for 24 h. This information was used to correct starter intake data. Treatments. Within 24 h of arrival, calves were randomly allocated to one of four oral treatments: control (CON), lipopolysaccharide only (LPS-O), butyrate (BUTY), or LPS and butyrate (LPSB). The CON consisted of 0.9% sodium chloride (Fisher Chemical, Fair Lawn, NJ) dissolved in distilled water. The LPS consisted of increasing weekly concentrations of LPS from Escherichia coli O55:B5 (catalog no. L2880; Sigma Aldrich Saint Louis, MO) dissolved in distilled water, as described further below. The BUTY consisted of 11 mM of sodium butyrate (Alfa Aesar, Ward Hill, MA) dissolved in distilled water. The LPSB consisted of a combination of the LPS and BUTY solutions based on metabolic BW of the individual calf, as described below. LPS concentrations were selected based on results from a pilot trial in our laboratory (Daniels et al., unpublished) and a study by Iqbal and others (2013) where oral dosages of LPS were administered to lactating dairy cattle. All solutions were mixed and stored (4°C) in non-pyrogenic glassware. Each calf had its own stock vial of its assigned treatment, which was mixed fresh at least once per week. In the first 2 wk of the experiment, calves assigned to a treatment containing LPS received 2.5 μg LPS/kg BW^0.75. Thereafter, LPS dose doubled every 2 wk so that calves assigned to a treatment containing LPS received a dosage of 20 μg/kg BW^0.75 in week 7 of the experiment. In the final week of the experiment (wk 8), LPS dosage was doubled again resulting in a dosage of 40 μg LPS/kg BW^0.75. All treatments were orally administered twice daily; eqivolume dosages were used in the following manner. In weeks 1 and 2, dosing volume for all treatments was 10 mL. In wk 3 and 4, dosing volume was 20 mL. In weeks 5 and 6, dosing volume was 30 mL. In weeks 7 and 8, dosing volume was 40 mL. In all cases, treatments were administered at least 30 min after feeding (but before 1h after feeding) through 50 mL dosing syringes (Agri-Pro Dosing Syringe; Valley Vet Supply Marysville, KS) equipped with 0.7 cm tips. For treatment administration, a member of the research team donned a pair of new nitrile gloves, entered the calf’s hutch, manually restrained the calf, placed the dosing syringe in the calf’s mouth, and manually emptied the complete contents of the dosing syringe. Average time to perform this entire procedure was 1 min per calf. Each calf was observed momentarily afterward to ensure that it swallowed its treatment. The size and style of the dosing syringes allowed us to administer treatments at the juncture between the oral cavity and esophagus. We chose to administer treatments at least 30 min after feeding in hopes that calves’ reticular grooves would be open (Wise et al., 1984) due to them no longer anticipating a liquid meal. Admittedly, we did not verify this; nonetheless, we assume that nearly all of each dose went into the rumen rather than the abomasum. Final number of calves for each treatment were: CON, n=5; LPS-O, n=6; BUTY, n=5; LPSB, n=6. During the first 3 wk of the experiment one CON calf and one BUTY calf were euthanized due to poor prognosis. The BUTY calf had a suspected umbilical mycoplasma infection in its umbilicus that spread to the calf’s hind joints. The CON calf presented with severe diarrhea and signs of respiratory infection; it was euthanized upon suspicion of peritonitis. These deaths are not thought to be treatment related. Data from these calves are not reported.

CHAPTER 1: LITERATURE REVIEW 
Dairy Calf Growth and Performance
Contemporary Dairy Calf Feeding Practices
Liquid Phase
Transition Phase.
Ruminant Phase.
Morphological Rumen Development
Metabolic Rumen Development
Microbial Rumen Development
Rumen pH
Environmental Factors and Their Role in Calf Health
Lipopolysaccharide
Host-Microbe Interactions
REFERENCES
CHAPTER 2: RUMINAL, DIET, AND ENVIRONMENTAL FACTORS THAT AFFECT DAIRY CALF ERFORMANCE
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Literature Search, Inclusion Criteria, and Data Cleaning
Model Permutations and Model Derivation Procedures
RESULTS AND DISCUSSION
Rumen Response Variable Models
Performance Response Variable Models
CONCLUSIONS
REFERENCES
CHAPTER 3: THE SYNERGISTIC RELATIONSHIP OF LIPOPOLYSACCHARIDE AND BUTYRATE STIMULATING RUMEN DEVELOPMENT
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Animals, Housing, and Treatments
Experimental Measures and Procedures
Slaughter Procedure
Organ Weights and Contents
Tissue Collection and Processing
Fluorescent Immunohistological Analysis
Image Acquisition and Analysis
Statistical Analyses
RESULTS AND DISCUSSION
Feed Intake
Body and Organ Weights and Health Measures
Receptor Intensity and BrdU Labeling
Sodium Butyrate and Blood Metabolites
LPS Effects
CONCLUSIONS
REFERENCES
CHAPTER 4: CONCLUSIONS AND IMPLICATIONS
APPENDICES

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Environmental, Molecular, and Dietary Factors that Influence Rumen Development in Dairy