Effects of Supplemental Citrulline on Intestinal Neutrophil Infiltration during Heat Stress and Nutrient Restriction in Growing Pigs

Heat stress (HS) and nutrient restriction impair intestinal barrier function and allow passage of endotoxin into portal and ultimately systemic circulation. Earlier data from the present study indicated citrulline supplementation improves intestinal morphology in the ileum during HS. Current objectives were to determine how citrulline supplement affects neutrophil infiltration in intestinal mucosa during HS. Supplements were fed twice daily at 0600 and 1800 h and consisted of 20 g of cookie dough without citrulline (CON) or with 0.13 g/kg BW L-citrulline (CIT; 99.3% purity; MP Biomedicals, Santa Ana, CA). Forty crossbred gilts (30 ± 2 kg) were assigned to 1 of 5 supplemental-environmental treatments: 1) thermoneutral (TN; 23.6 ± 0.1°C) ad libitum feed (AL) with CON (TNAL; n=8), 2) TN pair-fed (PF) with CON (PF-CON; n=8), 3) TN PF with CIT (PF-CIT; n=8), 4) HS AL with CON (HS-CON; n=8), and 5) HS AL with CIT (HS-CIT; n=8). Acclimation lasted 4 d and all pigs received the CON supplement. During period 1 (P1; 7 d), pigs were kept in TN, fed AL, and fed their respective supplemental treatments. During period 2 (P2; 60 h), HS-CON and HS-CIT animals were fed AL and exposed to cyclical HS (33.6 to 38.3°C) while TNAL, PF-CON, and PF-CIT remained in TN and were fed either AL or PF to their HS counterparts to negate the effect of dissimilar nutrient intake. Animals were sacrificed following P2 and segments of jejunum, ileum, and colon were collected and analyzed for morphology and were stained for myeloperoxidase (a measure of neutrophil infiltration). There were no treatment differences on neutrophil infiltration in the jejunum. Pigs exposed to HS tended to have increased ileum neutrophil infiltration relative to TNAL controls (30%; P=0.09), but did not differ from PF animals. Interestingly, PF-CON pigs had increased ileum neutrophil infiltration relative to TNAL and PF-CIT pigs (49 and 63%, respectively; P≤0.02). In the colon, HS increased neutrophil infiltration 62% (P=0.02) relative to TNAL controls, but this did not differ from PF-CON pigs as colon neutrophil infiltration was also increased in PF-CON relative to TNAL and PF-CIT pigs (64 and 108%, respectively; P≤0.03). There was no effect of citrulline supplementation in HS animals in any intestinal segment measured. In summary, HS and nutrient restriction increase neutrophil infiltration in the distal gastrointestinal tract. Supplemental citrulline does not diminish this immune response during HS but does ameliorate it under nutrient-restriction conditions.