UC Davis

Maintenance of gastrointestinal health and function is dependent on a variety of factors. Commercially raised pigs are particularly susceptible to disruption of intestinal homeostasis during the period directly after weaning, where enterotoxigenic Escherichia (E.) coli (ETEC) infection and subsequent diarrhea pose a serious challenge. The two most prominent strains of ETEC are F4 and F18—distinguished by different fimbrial structures allowing for adherence of bacteria to the porcine small intestinal lining. The inflammatory response to ETEC begins by detection of the bacterial endotoxin, lipopolysaccharide (LPS). The production of additional toxins incites diarrhea and the inflammatory mediators produced by the host during infection can exacerbate impaired intestinal barrier integrity. In efforts to preserve efficacy of antibiotics and delay the development of antimicrobial-resistant bacterial populations, antibiotic use at subtherapeutic doses cannot be employed to address ETEC in piglets. Among other potential intervention strategies, dietary supplementation with short-chain fatty acids (SCFAs) may support piglet health under ETEC-challenge conditions due to immunomodulatory effects and/or impacts on intestinal barrier integrity. Four studies were conducted to evaluate the biological activities exerted by derivatives of two SCFAs: Butyrate (C4) and valerate (C5). The first experiment aimed to determine the effects of different butyrate-based compounds in vitro using porcine cell culture models. Butyric acid, sodium butyrate, monobutyrin, and tributyrin were evaluated for effects on transepithelial electrical resistance (TEER) in porcine jejunum epithelial cell line, IPEC-J2, and for impacts on secretion of pro-inflammatory cytokines by porcine alveolar macrophages (PAMs). All butyrate derivatives except tributyrin increased (P < 0.05) TEER in IPEC-J2 at different time points compared with control. Secretion of pro-inflammatory cytokine, TNF-α, was reduced (linear; P < 0.05) in lipopolysaccharide (LPS)-challenged PAMs treated with butyric acid or sodium butyrate. Based on results from the first experiment, different butyrate-based compounds influenced TEER and elicited anti-inflammatory effects. The second experiment aimed to determine if the SCFA, valerate, exerted impacts on TEER and/or reduced secretion of TNF-α in vitro similarly to butyrate. This experiment aimed to fill the gap in our understanding of the potential modulatory effects of valerate on the porcine host in vitro. Another porcine epithelial cell line, IPEC-1 was pre-treated with different inclusion levels of valerate glycerides, then challenged with 10 µg/mL LPS. Treatment tended (P = 0.055) to affect repeated measures of TEER at 2, 4, and 6 h post-challenge, where inclusion of valerate glycerides maintained numerically higher percentage of TEER compared to pre-challenge. The secretion of TNF-α was reduced (P < 0.05) in PAMs treated with 500 µg/mL valerate glycerides and challenged by 1 µg/mL LPS. Taken together, the results from the first two experiments led to a pilot animal trial with the hypothesis that dietary monoglycerides of butyrate and/or valerate reduce the inflammatory status and frequency of severe diarrhea in piglets under ETEC-challenge conditions. The second aim of this pilot experiment was to determine an appropriate disease challenge inoculation dose for coinfection by two strains of ETEC—F4 and F18 (5109 CFU/mL each). All piglets coinfected with ETEC F4 and F18 exhibited increased diarrhea severity scores after inoculation, and hemolytic coliforms indicative of ETEC infection were present in fecal cultures post-inoculation (PI). Both butyrate and valerate monoglyceride supplementation reduced (P < 0.05) frequency of severe diarrhea. Pigs fed valerate glycerides tended (P = 0.061) to have lower neutrophils and had significantly reduced (P < 0.05) serum TNF-α on d 4 PI. This pilot experiment established an appropriate experimental dose for an ETEC F4-ETEC F18 coinfection disease model in weaned piglets and demonstrated the beneficial, non-nutrient impacts of supplementing weaned piglet diets with SCFA glycerides. The final experiment was a full-scale animal trial in which monoglycerides and triglycerides of valerate were evaluated for their effects on diarrhea, local and systemic inflammatory status, intestinal morphology and gene expression, and serum metabolomic profiles in piglets infected with 1010 CFU/mL ETEC F18. The frequency of severe diarrhea over the entire trial period was reduced (P < 0.05) in piglets fed trivalerin. Intestinal mucosal mRNA expression of TNF-α was decreased (P < 0.05) in piglets fed trivalerin, while that of ZO1 was increased (P < 0.05) compared with control. Trivalerin supplementation also maintained reduced (P < 0.05) serum TNF-α throughout the trial period. On d 6 PI, piglets in all treatment groups exhibited reduced (P < 0.05) serum concentration of several metabolites (adenosine, inosine, and shikimic acid) compared with control. In conclusion, the collective findings demonstrate the potential beneficial impacts of using glycerides of butyrate or valerate as dietary supplements for piglets during the post-weaning period.