Effect of supplementing a Bacillus subtilis-based probiotic on performance, intestinal integrity, and serum antioxidant capacity and metabolites concentrations of heat-stressed growing pigs.

Exposing pigs to heat stress (HS) seems to modify the intestinal microbiota which may compromise the integrity of the small intestine epithelia. Probiotics, live microorganisms, can help pigs to maintain a healthy intestinal environment. Eighty pigs (21.6 ±â€…3.4 kg body weight) exposed to HS or thermal neutral (TN) conditions were used to evaluate the effect of a Bacillus subtilis-based probiotic on performance, body temperature, and intestinal integrity. Treatments were: TN pigs fed a control diet without (TN-C) or with 1 × 106 CFU probiotic/g of feed (TN-P), and HS pigs fed a control without (HS-C) or with probiotic (HS-P). The control diet was formulated with wheat, soybean meal, and free amino acids (AA). Feed and water were freely available during the 21-d study. At completion, samples from duodenum, jejunum, and ileum were collected to analyze epithelial histology and tight junction protein expression; antioxidant activity, and free AA and metabolites in serum. Relative abundance of Lactobacillus, Bifidobacterium, Escherichia coli, and Bacillus in ileal content was analyzed. Ambient temperature in the TN room ranged from 19 to 25 °C, and in HS room from 30 to 38.5 °C. Intestinal temperature in HS-P pigs was lower than in HS-C pigs. Weight gain and feed intake reduced, but feed:gain and respiration rate increased in HS compared to TN pigs, regardless of diet (P < 0.01). Probiotic increased weight gain and improved feed:gain (P < 0.05) in both TN and HS pigs, but feed intake did not differ. Heat stress decreased villi height in jejunum and villi height:crypt depth in duodenum and jejunum (P < 0.05). Probiotic increased villi height in duodenum and ileum, and villi height:crypt depth in all small intestine segments (P < 0.05). Relative abundance of Lactobacillus and Bifidobacterium tended to reduce, and E. coli tended to increase (P < 0.10) in ileal content of HS-C pigs. Ileal relative abundance of Bacillus was higher (P < 0.01) in HS-P pigs than in HS-C and TN-C pigs. Cystathionine, homocysteine, hydroxylysine, α-amino-adipic acid, citrulline, α-amino-n-butyric acid, P-Ser, and taurine were higher in HS than in TN pigs (P < 0.05). These data confirm the negative effect of HS on performance, body temperature, and intestinal integrity of pigs. These data suggest that supplementing 1 × 106 CFU probiotic/g of feed based on Bacillus subtilis DSM 32540 may help to counteract the negative effects of HS on the performance and intestinal integrity of pigs.

High ambient temperature provokes heat stress in animals, and pigs are highly affected showing low performance, and intestinal integrity and microbiota compromised. Probiotics could help pigs to maintain a healthy intestinal environment. We analyzed the effect of a probiotic based on Bacillus subtilis on growth performance, intestinal integrity of small intestine, intestinal microbiota, antioxidant activity, and serum concentration of amino acids in pigs under heat stress (HS) and thermal neutral (TN) conditions. Ambient temperature under TN and HS conditions was 19­25 and 30­38.5 °C, respectively. Weight gain and feed intake reduced in HS pigs compared to TN pigs, but probiotic increased weight gain in both TN and HS pigs. Heat stress decreased villi size in duodenum and jejunum, and probiotic restored them. Relative abundance of Lactobacillus and Bifidobacterium reduced and Escherichia coli increased in the small intestine content of HS pigs; probiotic increased the abundance of Bacillus. The activity of antioxidant enzymes increased in HS pigs fed the probiotic diet. Serum concentration of amino acid metabolites was affected by HS. These data suggest that supplementing a Bacillus subtilis-based probiotic may help to counteract the negative effects of HS on the performance and intestinal integrity of pigs.

Effects of dietary supplementation with L-arginine on the endogenous losses of amino acids in growing pigs exposed to heat stress.

Exposing pigs to heat stress (HS) provokes higher death of intestinal cells, resulting in elevated endogenous intestinal losses (EIL) of amino acids (AA) and damage to intestinal epithelia. Arginine (Arg) is precursor for the synthesis of polyamines, which are involved in proliferation of intestinal cells and restoration of the intestinal epithelia. Thus the effect of adding L-Arg to diets for HS pigs on the EIL of AA was analyzed. Twelve pigs (23.1 ± 1.1 kg body weight) implanted with T-type cannulas at the end of ileum were individually housed and allowed 15-days for surgery recovery under thermoneutral (TN) conditions (22 ± 2 °C). Following, the pigs were randomly assigned to one of three treatments: TN pigs fed a semi-purified, corn starch-3% casein basal diet (TN-B); HS pigs with the basal diet (HS-B); HS pigs consuming the basal diet supplemented with 0.20% L-Arg (HS-Arg). The experiment consisted of two 9-day periods; each period included 7-days of adaptation to their respective diet, followed by a 2-day ileal digesta collection period. Digesta was collected during 12 consecutive hours each day. The pigs were fed twice a-day. Ambient temperature (AT) inside the TN and HS rooms ranged from 18.6 to 27.6 °C and from 29.5 to 40.7 °C, respectively. Body temperature followed a pattern similar to that of AT. The daily EIL of indispensable AA increased (P < 0.01) in the HS-B pigs compared to both the TN-B and the HS-Arg pigs, however, there was no EIL difference between the TN-B and the HS-Arg pigs (P > 0.05). Likewise, with the exception of serine, daily losses of endogenous dispensable AA in the HS-B pigs were higher (P < 0.01) in comparison with those of TN-B and HS-Arg pigs. In summary, HS exposure compared to TN conditions increases the loss of endogenous AA, but dietary supplementation with L-Arg helped to counteract the negative HS effect.

Effects of dietary protein content and crystalline amino acid supplementation patterns in low protein diets on intestinal bacteria and their metabolites in weaned pigs raised under Different sanitary conditions.

The objective of this experiment was to investigate the effects of dietary crude protein (CP) content and crystalline amino acids (CAA) supplementation patterns in low CP (LCP) diets on intestinal bacteria and their metabolites in weaned pigs raised under clean (CSC) or unclean sanitary conditions (USC). One hundred forty-four piglets (6.35 ±â€…0.63 kg) were assigned to one of six treatments in a 3 × 2 factorial arrangement based on CP content and sanitary conditions in a randomized complete block design to give eight replicates with three pigs per pen over a 21-d period. Diets consisted of a high CP (HCP; 21%) and two LCP (18%) diets supplemented with 9 CAA (Lys, Met, Thr, Trp, Val, Ile, Leu, His, and Phe) or only six CAA (Lys, Met, Thr, Trp, Val, and Ile) to meet the requirements. The CSC room was washed weekly, whereas the USC room had sow manure spread in the pens from the beginning of the study and was not washed throughout the experiment. Jejunum and colon digesta were sampled on day 21. Both jejunum and colon digesta were analyzed for ammonia nitrogen, short-chain fatty acids, and biogenic amines but only colon digesta was analyzed for microbiome composition (16s rRNA sequencing on MiSeq). Data were analyzed using R software for 16S rRNA and the MIXED procedure of SAS for microbial metabolites. Sanitation, CP content, and CAA supplementation patterns did not affect the diversity of colonic bacterial composition in weaned pigs. Pigs raised under USC had greater (P < 0.05) jejunal ammonia nitrogen concentration than those raised under CSC. Pigs fed LCP diets had reduced (P < 0.05) jejunal ammonia nitrogen concentration compared to those fed the HCP diet. Interactions between sanitation and dietary CP content were observed (P < 0.05) for: (1) jejunal acetate and (2) colonic spermidine and spermine, whereby (1) acetate concentrations decreased from NCP to LCP in pigs raised under the CSC but those concentrations increased under the USC, and (2) spermidine and spermine concentrations increased in LCP diets compared to HCP diet under USC, unlike CSC which did not show any difference between HCP and LCP. In conclusion, reducing dietary CP lowered ammonia nitrogen content regardless of sanitation and increased microbial metabolites in weaned pigs raised under USC. However, LCP diets with different CAA supplementation patterns did not affect bacterial diversity in weaned pigs, regardless of the hygienic conditions where the animals were housed.

Lowering dietary crude protein concentration by 3% to 4% units has been used as one of the strategies to promote growth and improve the gut health of weaned pigs. Undigested and endogenous protein could be available for microbial fermentation, and protein fermentation is considered detrimental to the gut health of the host animal. The unclean sanitary condition model mimics commercial raising conditions and stimulates a low-grade inflammatory and immune response. Ammonia nitrogen is one of the harmful metabolites derived by protein fermentation and pigs fed low-protein diets had decreased ammonia nitrogen than those fed high-protein diets. Also, pigs raised under unclean sanitation had greater ammonia nitrogen than those raised under clean sanitation. However, sanitation, protein content, and crystalline amino acids supplementation patterns did not affect the diversity of colonic bacterial composition in weaned pigs. The results obtained from the present study showed that a low protein diet could be used to improve gut health in weaned pigs.

Effect of dl-methionine supplementation above requirement on performance; intestinal morphology, antioxidant activity, and gene expression; and serum concentration of amino acids in heat stressed pigs.

The intestinal morphology and function can be compromised in pigs exposed to heat stress (HS), partly due to increased production of reactive-oxygen species. Because methionine (Met) functions as intracellular antioxidant, the requirement of Met may be increased in HS-pigs. The effect of dietary supplementation with dl-Met above requirement on performance, small intestine morphology, antioxidant enzymes activity, amino acid transporters expression, and serum concentration (SC) of free AA in HS-pigs was evaluated. A basal wheat-soybean meal diet was formulated to meet 100% Met requirement with the other indispensable AA exceeding at least 20% their requirement. Sixty individually housed pigs (23.0 ± 2.4 kg BW, 12 pigs per treatment) were randomly assigned to five treatments: TN100, thermal-neutral (22.7 °C) housed pigs fed the basal diet; HS100, HS120, HS140, HS160; HS-pigs (29.6 °C to 39.4 °C) fed the basal diet supplemented with dl-Met to contain 0%, 20%, 40%, and 60% dl-Met above the requirement, respectively. Pigs had free access to feed and water during the 21-d trial. Blood samples were collected on day 18 to analyze the absorptive AA-SC. The effect of ambient temperature (HS100 vs. TN100), as well as the linear and quadratic effects of increasing Met levels in the diets for HS-pigs were analyzed. The HS100 pigs gained less weight than TN100 and HS120 pigs (P < 0.01); gain:feed was also higher in HS120 pigs than in HS100 pigs (P ≤ 0.05). Feed intake of TN100 pigs was higher than that of HS-pigs fed the dl-Met supplemented diets (P < 0.05). Villi height reduced in pigs HS, but Met supplementation quadratically increased it (P < 0.05). Superoxide dismutase and catalase activities, reduced glutathione concentration, and relative expression of B0AT2 in ileum decreased (P < 0.05), but glutathione peroxidase activity increased in HS-pigs. dl-Met supplementation linearly affected catalase and glutathione peroxidase activities, as well as the relative expression of b0,+AT in jejunum (P < 0.05) of HS-pigs. The SC of Ile, Leu, Lys, Phe, and Val were higher in HS100 pigs than in TN100 pigs (P < 0.05). Graded levels of supplemental dl-Met in diets for HS-pigs linearly decreased SC of Ile, Leu, and Val (P < 0.05), tended to decrease His, Lys, and Thr (P < 0.10), and increased Met (P < 0.01). In conclusion, HS had negative effect on weight gain and intestinal morpho-physiology; however, it was ameliorated by adding 20% Met above the requirement in diets for growing pigs.

The exposure of pigs to ambient temperature above their comfort zone affects several functions of the small intestine, especially those related with digestion of feed and absorption of nutrients, which in turn reduces the availability of nutrients for growth. Amino acids such as methionine are involved in multiple functions of intestinal cells. Thus, methionine supplementation may help pigs to overcome the negative impact of their exposure to high ambient temperature. Indeed, methionine supplementation to the diet increased growth rate and feed efficiency of pigs housed under heat stress, which was presumably associated with an improvement in the utilization of the absorbed amino acids.

Apparent and standardised ileal amino acid digestibilities in heat-stressed pigs fed wheat-soybean meal diets supplemented with l-arginine and dl-methionine.

Heat stress (HS) exposure may damage the small intestine epithelia of pigs affecting the digestibility and absorption of amino acids (AA). Arg and Met can enhance antioxidant and intestinal cell proliferation activity, thus supplementing them in diets might alleviate epithelial damage and correct the reduced AA digestibility. The effect of adding extra l-Arg and dl-Met to diets on the apparent (AID) and standardised ileal digestibility (SID) of AA was analysed in a 10-day experiment conducted with 10 ileal-cannulated HS pigs (25.3 ± 2.4 kg body weight). The pigs were divided into two treatments: Control, wheat-soybean meal diet supplemented with l-Lys, l-Thr, dl-Met and l-Trp; and control diet plus 0.20% l-Arg and 0.20% dl-Met (Arg + Met). Following an 8-day diet adaptation period, ileal digesta was continuously collected for 12 h, starting at 0700, on Days 9 and 10. All pigs were daily exposed to 29.6-36.1°C; ileal temperature was continuously monitored at 15-min intervals. Feed was provided twice a day. The ileal temperature ranged from 40.3 to 41.5°C. Daily ileal outflow of His, Ile, Leu, Phe, Thr, Ser and Tyr decreased (p < 0.05), and that of Arg, Val and Pro tended to decrease (p < 0.10) in the Arg + Met pigs. The AID of Arg, His, Met, Thr and Tyr, and the SID of His, Met, and Thr increased in pigs fed the Arg + Met diet (p < 0.05). Thr and Val had the lowest AID values whereas Arg, Met, and Lys had the highest values. Arg (r = 0.64) and Met (r = 0.84) intake were highly correlated with their AID values; Met intake was highly correlated with its SID value (r = 0.72). Valine and Arg had the lowest whereas Arg had the highest SID values. In conclusion, supplementing l-Arg and dl-Met above the requirement decreases the ileal outflow of several AA, and increases the AID and SID of some essential AA in HS pigs.

Ileal alkaline phosphatase is upregulated following functional amino acid supplementation in Salmonella Typhimurium-challenged pigs.

We recently showed that functional amino acid (FAA) supplementation improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs, which was further improved by a longer adaptation period. It is expected that the effects are associated with increased activity of intestinal alkaline phosphatase (IAP). The objective of this study was to evaluate the effects of FAA supplementation and adaptation period on the ileal, cecal, and colonic activity of IAP in weaned pigs challenged with ST. In experiment 1, a total of 32 mixed-sex weanling pigs were randomly assigned to dietary treatments in a 2 × 2 factorial arrangement with low (LP) or high protein (HP) content and basal (FAA-) or FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements) as factors. In experiment 2, a total of 32 mixed-sex weanling pigs were randomly assigned to one of four dietary treatments, being FAA- fed throughout the experimental period (FAA-) or an FAA profile fed only in the post-inoculation (FAA + 0), for 1 wk pre- and post-inoculation (FAA + 1), or throughout the experimental period (FAA + 2). In experiments 1 and 2, after a 7- and 14-d adaptation period, respectively, pigs were inoculated with saline solution containing ST (3.3 and 2.2 × 109 CFU/mL, respectively). Plasma alkaline phosphatase was measured on days 0 and 7 post-inoculation in experiment 1, and IAP (ileum, cecum, and colon) was measured in experiments 1 and 2. Correlations among ileal IAP and serum albumin and haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and reduced:oxidized glutathione, ileal myeloperoxidase, ST shedding and ileal colonization, and post-inoculation average daily gain, feed intake (ADFI), and gain:feed were also analyzed. In experiment 1, plasma alkaline phosphatase was decreased with ST inoculation and the overall content was increased in LP-FAA+ compared with LP-FAA- (P < 0.05). Moreover, ileal IAP was increased in FAA+ compared with FAA- pigs in both studies (P < 0.05) regardless of adaptation time (P > 0.05). IAP was positively correlated with MDA and ADFI and negatively correlated with SOD and ST shedding in experiment 1 (P < 0.05). These results demonstrate a positive effect of FAA supplementation, but not adaptation period, on ileal alkaline phosphatase activity in Salmonella-challenged pigs, which may be associated with improvements in antioxidant balance.

Functional amino acid (FAA) supplementation has been shown to improve gut health and antioxidant defense in weaned piglets challenged with Salmonella Typhimurium (ST), regardless of dietary protein content. The beneficial effects were further improved when pigs were adapted to FAA for 2 wk prior to the ST challenge. Recent evidence has shown that intestinal alkaline phosphatase (IAP), which may be influenced by nutritional factors, attenuates intestinal inflammation, possibly due to gut microbiota modulation. This study is the first to identify that ileal IAP activity is increased following FAA supplementation in ST-challenged pigs, regardless of adaptation period. Moreover, ileal IAP activity correlated with systemic markers of antioxidant defense, which highlights the enzyme's role in attenuating systemic infection. Overall, the development of feeding strategies with positive effects on IAP activity is of interest, due to the enzyme's central role on the gut and whole-body homeostasis and health.

Expression of proposed methionine transporters along the gastrointestinal tract of pigs and their regulation by dietary methionine sources.

BACKGROUND: Given the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs. METHODS: A total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of B0AT1, ATB0,+, rBAT, ASCT2, IMINO, LAT4, y+LAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of B0AT1, ASCT2, LAT2, and LAT4. RESULTS: Expression of all investigated transcripts differed significantly along the GIT. B0AT1, rBAT, y+LAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB0,+ showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (y+LAT1) intestine. Protein expression of B0AT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met (small intestine) and L-Met (small intestine, oral mucosa, and stomach). CONCLUSION: A high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues.

Changes in growth performance, plasma metabolite concentrations, and myogenic gene expression in growing pigs fed a methionine-restricted diet.

Background: Methionine (Met) is usually the second or third limiting amino acid in swine diets and plays vital roles in promoting the growth, especially, the muscle growth of pigs. This research evaluated the effects of dietary Met restriction on the growth performance, plasma metabolite concentrations, and myogenic gene expression in growing pigs. Materials and methods: Eight genes in two families (myogenic regulatory factor family and myocyte enhancer factor 2 family) were selected for the analysis. Twenty individually penned barrows (crossbred, 23.6 ± 2.4 kg) were randomly allotted to two dietary treatments (n = 10). A diet based on corn and soybean meal (Diet 1, Met-restricted) was formulated to meet or exceed the energy and nutrient requirements, except for Met. Diet 2 (Met-adequate) was formulated by adding crystalline DL-Met to Diet 1 to meet the Met requirement. During the 4-week feeding trial, average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were measured. Immediately before and after the feeding trial, blood was sampled via jugular venipuncture for plasma nutrient metabolite analysis, while Longissimus dorsi muscle were sampled via aseptic biopsy for gene expression analysis. Data were analyzed with Student t-test. Results: Pigs fed the Met-restricted diet had lower ADG and G:F (P < 0.01). Plasma Met, cysteine, and taurine concentrations were lower (P < 0.05), while glycine and histidine concentrations were higher (P < 0.05), in pigs fed the Met-restricted diet. Furthermore, the pigs fed the Met-restricted diet tended to express less myogenic factor 6 (Myf6) and myocyte enhancer factor 2D (Mef2D) mRNA in longissimus dorsi muscle (P < 0.09). Conclusion: Given the fact that Myf6, assisted by Mef2D, is involved in myocyte differentiation, this study suggests that the reduced growth performance in the Met-restricted pigs may be associated with a reduced muscle cell differentiation.

Effects of supplemental d-methionine in comparison to l-methionine on nitrogen retention, gut morphology, antioxidant status, and mRNA abundance of amino acid transporters in weanling pigs.

An N-balance experiment was conducted to test the hypothesis that d-Methionine (d-Met) has the same bioavailability and efficacy as l-Methionine (l-Met) when fed to weanling pigs. A Met-deficient basal diet containing 0.24% standardized ileal digestible (SID) Met was formulated. Six additional diets were formulated by adding 0.036%, 0.072%, or 0.108% d-Met or l-Met to the basal diet, and these diets, therefore, contained 77%, 87%, or 97% of the requirement for SID Met. Fifty-six barrows (10.53 ± 1.17 kg) were housed in metabolism crates and allotted to the seven diets with eight replicate pigs per diet. Feces and urine were collected quantitatively with 7-d adaptation and 5-d collection periods. Blood and tissue samples from pigs fed the basal diet and pigs fed diets containing 0.108% supplemental Met were collected on the last day. Results indicated that N retention (%) linearly increased (P < 0.01) as supplemental d-Met or l-Met increased in diets. Based on N retention (%) as a response, the linear slope-ratio regression estimated the bioavailability of d-Met relative to l-Met to be 101% (95% confidence interval: 57%-146%). The villus height and crypt depth in the jejunum were not affected by the Met level or Met source. Total antioxidant capacity or thiobarbituric acid reactive substance concentrations in plasma or tissue samples from pigs fed the control diet or diets containing 0.108% supplemental d-Met or l-Met were not different. Abundance of mRNA for some AA transporters analyzed in intestinal mucosa of pigs also did not differ. Therefore, it is concluded that d-Met and l-Met are equally bioavailable for weanling pigs.

Effect of arginine supplementation on the morphology and function of intestinal epithelia and serum concentrations of amino acids in pigs exposed to heat stress.

The exposure of pigs to heat stress (HS) appears to damage their intestinal epithelia, affecting the absorption of amino acids (AAs). Arg is involved in the restoration of intestinal epithelial cells but HS reduces Arg intake. The effect of dietary supplementation with Arg on the morphology of intestinal epithelia, AA transporter gene expression, and serum concentration (SC) of free AAs in HS pigs was analyzed. Twenty pigs (25.3 ± 2.4 kg body weight) were randomly assigned to two dietary treatments: Control (0.81% Arg), wheat-soybean meal diet supplemented with l-Lys, l-Thr, dl-Met, and l-Trp, and the experimental diet where 0.16% free l-Arg was supplemented to a similar Control diet (+Arg). All pigs were individually housed and exposed to HS, fed ad libitum with full access to water. The ambient temperature (AT), recorded at 15-min intervals during the 21-d trial, ranged on average from 29.6 to 39.4 °C within the same day. Blood samples were collected on day 18 at 1600 hours (AT peak); serum was separated by centrifugation. At the end of the trial, five pigs per treatment were sacrificed to collect samples of mucosa scratched from each small intestine segment. The expression of AA transporters in intestinal mucosa and the SC of AAs were analyzed. Villi height (VH) was higher (P < 0.01) in the duodenum, jejunum, and ileum but the crypt depth did not differ between the Control and the +Arg pigs. Supplementation of l-Arg increased the mRNA coding for the synthesis of the cationic AA transporter b0,+ (P < 0.01) and the neutral AA transporter B0 (P < 0.05) in the duodenum by approximately 5-fold and 3-fold, respectively, but no effect on mRNA abundance was observed in the jejunum and ileum. The supplementation of l-Arg increased serum Arg, His, Met, Thr, Trp, and urea (P < 0.05) and also tended to increase Val (P < 0.10) but did not affect Ile, Lys, Leu, and Phe. These results indicate that supplementing 0.16% l-Arg to the Control diet may help to improve the function of the small intestine epithelium, by increasing the VH, the abundance of AA transporters, and the SC of most indispensable AAs in pigs exposed to HS conditions. However, the lack of effect of supplemental Arg on both Lys SC and weight gain of pigs suggests that increasing the Lys content in the +Arg diet might be needed to improve the performance of HS pigs.

A longer adaptation period to a functional amino acid-supplemented diet improves growth performance and immune status of Salmonella Typhimurium-challenged pigs.

We recently showed that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs. It is not known if ST-challenged pigs will benefit from a longer adaptation period to FAA. The objective of this study was to evaluate the effects of different adaptation periods to diets containing FAA above requirements for growth on performance and immune response of weaned pigs subsequently challenged with ST. A total of 32 mixed-sex weanling pigs (11.6 ± 0.3 kg) were randomly assigned to 1 of 4 dietary treatments, being a basal amino acid (AA) profile fed throughout the experimental period (FAA-) or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) fed only in the postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). After a 14-d adaptation period, pigs were inoculated with ST (2.15 × 109 CFU/mL). Growth performance, body temperature, fecal score, acute-phase proteins, oxidant/antioxidant balance, score for ST shedding in feces and intestinal colonization, and fecal and digesta myeloperoxidase (MPO) were measured pre- and postinoculation. Postinoculation body temperature and fecal score, serum haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and fecal MPO were increased while serum albumin and plasma reduced glutathione (GSH):oxidized glutathione (GSSG) were reduced compared to pre-inoculation (P < 0.05). Average daily gain and G:F were greater in FAA+2 pigs compared to FAA- pigs (P < 0.05). Serum albumin was higher in FAA+2 and FAA+1 compared to FAA+0 and FAA- pigs (P < 0.05) while FAA+2 pigs had lower haptoglobin compared to FAA- (P < 0.05). Plasma SOD was increased and GSH:GSSG was decreased in FAA- pigs compared to the other treatments (P < 0.05). Score for ST shedding in feces was progressively lower from d 1 to 6 regardless of treatment (P < 0.05) and was lower in FAA+2 pigs compared to FAA- and FAA+0 (P < 0.05). Counts of ST in colon digesta were higher in FAA- and FAA+0 pigs compared to FAA+2 (P < 0.05). Fecal and colonic digesta MPO were lower in FAA+2 and FAA+1 pigs compared to FAA- (P < 0.05). These results demonstrate a positive effect of a longer adaptation period to FAA-supplemented diets on performance and immune status of weaned pigs challenged with Salmonella.

Functional amino acid supplementation, regardless of dietary protein content, improves growth performance and immune status of weaned pigs challenged with Salmonella Typhimurium.

High dietary protein may increase susceptibility of weaned pigs to enteric pathogens. Dietary supplementation with functional amino acids (FAA) may improve growth performance of pigs during disease challenge. The objective of this study was to evaluate the interactive effects of dietary protein content and FAA supplementation above requirements for growth on performance and immune response of weaned pigs challenged with Salmonella. Sixty-four mixed-sex weanling pigs (13.9 ± 0.82 kg) were randomly assigned to dietary treatments in a 2 × 2 factorial arrangement with low (LP) or high protein (HP) content and basal (AA-) or FAA profile (AA+; Thr, Met, and Trp at 120% of requirements) as factors. After a 7-d adaptation period, pigs were inoculated with either a sterile saline solution (CT) or saline solution containing Salmonella Typhimurium (ST; 3.3 × 109 CFU/mL). Growth performance, body temperature, fecal score, acute-phase proteins, oxidant/antioxidant balance, ST shedding score in feces and intestinal colonization, fecal and digesta myeloperoxidase (MPO), and plasma urea nitrogen (PUN) were measured pre- and postinoculation. There were no dietary effects on any measures pre-inoculation or post-CT inoculation (P > 0.05). Inoculation with ST increased body temperature and fecal score (P < 0.05), serum haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), PUN, and fecal MPO, and decreased serum albumin and plasma reduced glutathione (GSH):oxidized glutathione (GSSG) compared with CT pigs (P < 0.05). ST-inoculation reduced average daily gain (ADG) and feed intake (ADFI) vs. CT pigs (P < 0.05) but was increased by AA+ vs. AA- in ST pigs (P < 0.05). Serum albumin and GSH:GSSG were increased while haptoglobin and SOD were decreased in ST-inoculated pigs fed AA+ vs. AA- (P < 0.05). PUN was higher in HP vs. LP-fed pigs postinoculation (P < 0.05). Fecal ST score was increased in ST-inoculated pigs on days 1 and 2 postinoculation and declined by day 6 (P < 0.05) in all pigs while the overall score was reduced in AA+ vs. AA- pigs (P < 0.05). Cecal digesta ST score was higher in HP vs. LP-fed pigs and were lower in AA+ compared with AA- fed pigs in the colon (P < 0.05). Fecal and digesta MPO were reduced in ST pigs fed AA+ vs. AA- (P < 0.05). These results demonstrate a positive effect of FAA supplementation, with minimal effects of dietary protein, on performance and immune status in weaned pigs challenged with Salmonella.

Growth performance and gut health of Escherichia coli-challenged weaned pigs fed diets supplemented with a Bacillus subtilis direct-fed microbial.

This study was conducted to investigate the effects of a direct-fed microbial (DFM) product (Bacillus subtilis strain DSM 32540) in weaned pigs challenged with K88 strain of Escherichia coli on growth performance and indicators of gut health. A total of 21 weaned pigs [initial body weight (BW) = 8.19 kg] were housed individually in pens and fed three diets (seven replicates per diet) for 21 d in a completely randomized design. The three diets were a corn-soybean meal-based basal diet without feed additives, a basal diet with 0.25% antibiotics (neo-Oxy 10-10; neomycin + oxytetracycline), or a basal diet with 0.05% DFM. All pigs were orally challenged with a subclinical dose (6.7 × 108 CFU/mL) of K88 strain of E. coli on day 3 of the study (3 d after weaning). Feed intake and BW data were collected on days 0, 3, 7, 14, and 21. Fecal scores were recorded daily. On day 21, pigs were sacrificed to determine various indicators of gut health. Supplementation of the basal diet with antibiotics or DFM did not affect the overall (days 0-21) growth performance of pigs. However, antibiotics or DFM supplementation increased (P = 0.010) gain:feed (G:F) of pigs during the post-E. coli challenge period (days 3-21) by 23% and 24%, respectively. The G:F for the DFM-supplemented diet did not differ from that for the antibiotics-supplemented diet. The frequency of diarrhea for pigs fed a diet with antibiotics or DFM tended to be lower (P = 0.071) than that of pigs fed the basal diet. The jejunal villous height (VH) and the VH to crypt depth ratio (VH:CD) were increased (P < 0.001) by 33% and 35%, respectively, due to the inclusion of antibiotics in the basal diet and by 43% and 41%, respectively due to the inclusion of DFM in the basal diet. The VH and VH:CD for the DFM-supplemented diet were greater (P < 0.05) than those for the antibiotics-supplemented diet. Ileal VH was increased (P < 0.05) by 46% due to the inclusion of DFM in the basal diet. The empty weight of small intestine, cecum, or colon relative to live BW was unaffected by dietary antibiotics or DFM supplementation. In conclusion, the addition of DFM to the basal diet improved the feed efficiency of E. coli-challenged weaned pigs to a value similar to that of the antibiotics-supplemented diet and increased jejunal VH and VH:CD ratio to values greater than those for the antibiotics-supplemented diet. Thus, under E. coli challenge, the test DFM product may replace the use of antibiotics as a growth promoter in diets for weaned pigs to improve feed efficiency and gut integrity.

Bioavailability of the calcium salt of dl-methionine hydroxy analog compared with dl-methionine for nitrogen retention and the preference of nursery pigs for diets based on the 2 forms of methionine.

Experiments were conducted to determine the relative bioavailability (RBV) of the calcium salt of the hydroxy analog of dl-methionine (MHA-Ca, 84%) to dl-methionine (dl-Met, 99%) as Met sources fed to pigs. In experiment 1, 42 crossbred barrows (initial BW of 15.0 ± 0.7 kg) were allotted to 7 treatments in an N-balance study. The basal diet (BD) was formulated to contain 15.4% CP and 0.22% Met (70% of requirement). Diets included (1) BD, (2) BD + 0.025% dl-Met, (3) BD + 0.050% dl-Met, (4) BD + 0.075% dl-Met, (5) BD + 0.038% MHA-Ca, (6) BD + 0.077% MHA-Ca, and (7) BD + 0.115% MHA-Ca. An increase in dietary inclusion rates of both Met sources linearly increased (P < 0.01) N retained (g/d) and N retention (% of intake). Using linear slope-ratio regression, the RBV value of MHA-Ca to dl-Met for N retained (g/d) was 63.0% on a product-to-product basis (75.0% on an equimolar basis). In experiment 2, 40 crossbred barrows (initial BW of 15.5 ± 1.5 kg) were allotted to 5 treatments in another N-balance study. The BD was formulated to contain 17.0% CP and 0.22% Met (70% of requirement). Diets included (1) BD, (2) BD + 0.030% dl-Met, (3) BD + 0.060% dl-Met, (4) BD + 0.046% MHA-Ca, and (5) BD + 0.092% MHA-Ca. Increasing levels of dl-Met or MHA-Ca increased N retained (g/d) and N retention (% of intake) linearly (P < 0.001) and quadratically (P < 0.05). Using linear slope-ratio regression, a product-to-product RBV value of MHA-Ca to dl-Met was 68.4% (81.4% on an equimolar basis) for N retained (g/d). In experiment 3, 276 pigs (12 barrow and 11 gilt replicates; initial BW of 7.09 ± 1.1 kg) were used in 3 diet preference studies. Pigs were randomly allotted to 1 of 3 treatment comparisons of feed choice: (1) BD (0.23% Met) or BD + 0.07% dl-Met; (2) BD or BD + 0.0825% MHA-Ca, and (3) BD + 0.07% dl-Met or BD + 0.0825% MHA-Ca. Pigs consumed a higher percentage (55 vs. 45%; P = 0.008) of their total feed intake from the diet supplemented with 0.07% dl-Met in Comparison 1, but a lower percentage (45 vs. 55%; P = 0.003) of their total feed intake from the diet supplemented with 0.0825% MHA-Ca in Comparison 2. There was no diet preference for dl-Met or MHA-Ca in Comparison 3. The observed Met source preference differences occurred in the barrow replicates but not in the gilt replicates. These results demonstrated the mean RBV of MHA-Ca to dl-Met of 65.7% on a product-to-product (wt/wt) basis or 78.2% on an equimolar basis and that a preference for Met sources was observed in barrows but not in gilts.

Efficiency of standardized ileal digestible lysine utilization for whole body protein deposition in pregnant gilts and sows during early-, mid-, and late-gestation.

The efficiency of SID Lys utilization (kSID Lys) in gilts and sows during early (days 48 to 52), mid (days 75 to 79), and late gestation (days 103 to 107) was investigated using 88 pregnant females (PIC 1050; 27 gilts, 27 parity 1 sows, 34 parity 2+ sows; 192.96 ± 22.84 kg at days 42 ±1 of gestation) and whole body nitrogen (N) retention balance studies. Females were assigned to 1 of 4 SID Lys levels ranging from 40% to 70% of the daily SID Lys requirements above maintenance for a parity 1 sow according to the NRC (2012) gestating sow model in each gestation period. Experimental diets were isocaloric (3,335 kcal ME/kg) and isoproteic (11.75 % CP) and dietary indispensable AA were set to meet or exceed 100% of AA:Lys ratios. The slope of the linear response to graded SID Lys intake was defined as kSID Lys. With the aim of increasing the accuracy of kSID Lys estimates, gilt data from the current study was combined with gilt data from a previous study conducted at the same facility using 4 SID Lys levels ranging from 60% to 90% of the daily SID Lys requirements above maintenance for gilts according to the NRC (2012) gestating sow model. Whole body Lys retention of the combined gilt data set was assessed with different broken-line and nonlinear models. The kSID Lys was 0.65, 0.38, and 0.52 for early-, mid-, and late-gestation, respectively, in gilts. A linear response to graded SID Lys intake was found in late gestation only in parity 1 and 2+ sows; kSID Lys was determined as 0.44 and 0.52 in late gestation for parity 1 and parity 2+ sows, respectively. There were no differences in kSID Lys in late gestation between parities. For the combined gilt data, the model of best performance (reduced error and greater goodness of fit) was the Hoerl model. Maximum kSID Lys (i.e., g SID Lys retention/g SID Lys intake) in gilts was 0.67, 0.54, and 0.53 in early, mid, and late gestation predicted at 7.2, 9.1, and 13.5 g of SID Lys intake/d, respectively, based on the Hoerl model. Maximum SID Lys retention in gilts was similarly predicted at 8.5, 10.5, and 20.9 g of SID Lys intake per day in early, mid, and late gestation and resultant kSID Lys of 0.61, 0.51, and 0.44, respectively. The findings of this study demonstrate that kSID Lys varies by stage of gestation and SID Lys intake level and that, at least in gilts, a dynamic kSID adjusted for daily intake more adequately reflects biological response and hence allows more precise feeding of pregnant females.

Crystalline amino acids do not influence calculated values for standardized ileal digestibility of amino acids in feed ingredients included in diets for pigs.

An experiment was conducted to test the hypothesis that addition of crystalline amino acids (AA) to diets during the adaptation or collection periods will not influence calculated values for apparent ileal digestibility (AID) or standardized ileal digestibility (SID) of AA in corn and soybean meal (SBM). Seven ileal-cannulated barrows (initial body weight: 77.9 ± 2.6 kg) were allotted to a 7 × 7 Latin Square design with 7 dietary treatments and 7 periods. Dietary treatments included feeding diets based on corn or SBM without or with crystalline AA for the entire 7-d period or with crystalline AA during the adaptation period, but without crystalline AA during the collection period. An N-free diet was also used. Each experimental period consisted of 5 d of adaptation to the diets followed by 2 d of ileal digesta collection, with digesta being collected for 9 hr/d starting after feeding the morning meal. Thus, the entire experiment lasted 49 d. The AID and SID of crude protein (CP) and AA were calculated using values determined in corn or in SBM without or with crystalline AA to determine if crystalline AA influenced calculated values for AID or SID of CP and AA. Results indicated that addition of crystalline AA to diets fed during the entire 7-d period increased (P < 0.05) AID of some AA in corn and SBM. However, no differences in SID of CP and AA were observed between pigs fed the diets without crystalline AA and pigs fed the diets with crystalline AA if dietary crystalline AA were ignored in calculations of SID values. This indicates that crystalline AA were 100% absorbed before the distal ileum and did not affect calculated values for SID of AA if calculations were based only on the AA in corn or SBM. Therefore, it is concluded that crystalline AA may be added to experimental diets in digestibility experiments before and during collection periods without affecting results, if crystalline AA are disregarded in the calculation of AID or SID of AA in ingredients.

The direct and difference procedures result in similar estimates for amino acid digestibility in feed ingredients fed to growing pigs.

An experiment was conducted to test the hypothesis that values for standardized ileal digestibility (SID) of amino acids (AA) in corn, wheat, and wheat middlings obtained using the direct procedure are not different from values obtained using the difference procedure. Sixteen ileal-cannulated barrows (initial BW: 69.5 ± 5.0 kg) were allotted to a replicated 8 × 4 Youden Square design with 8 diets and 4 periods. Each period consisted of 5 d of adaptation to the diet and 2 d of collection of ileal digesta. Four diets were based on soybean meal (SBM), corn, wheat, or wheat middlings as the only AA-containing ingredients. Three additional diets were based on a mixture of SBM and corn, SBM and wheat, or SBM and wheat middlings, and an N-free diet was also used. The apparent ileal digestibility (AID) and the SID of crude protein (CP) and AA for the 4 diets containing SBM, corn, wheat, or wheat middlings as the sole source of AA were calculated using the direct procedure. The AID and SID of CP and AA for the 3 mixed diets containing SBM and corn, wheat, or wheat middlings were also calculated and the contribution of digestible AA from SBM was subtracted from the AID or SID values for the diets. The AID or SID of AA in corn, wheat, and wheat middlings were subsequently calculated by difference. Results indicated that the AID values for a few AA were lower (P < 0.05) if the direct procedure was used instead of the difference procedure, regardless of ingredient. The AID of Trp was greater in corn and wheat middlings, and the SID of Trp in corn and wheat middlings tended to be greater, if the direct procedure rather than the difference procedure was used, but that was not the case for wheat (interaction, P < 0.05 and P < 0.10, respectively). However, for all other indispensable AA, and for most of the dispensable AA, the SID of AA in corn, wheat, and wheat middlings was not different between the difference procedure and the direct procedure. Therefore, values for SID of AA in cereal grains and fiber-rich ingredients may be determined using either the direct or the difference procedure.

The effect of reduced dietary glycine and serine and supplemental threonine on growth performance, protein deposition in carcass and viscera, and skin collagen abundance of nursery pigs fed low crude protein diets.

Thirty five barrows (initial body weight [BW]: 15.1 ± 1.0 kg) were used to determine the effect of partially replacing Gly + Ser with Thr in reduced crude protein (CP) diets on growth performance, protein deposition in carcass and viscera, and skin collagen abundance during the late nursery phase to 25 kg BW. Pigs were individually fed one of five iso-nitrogenous diets (n = 7) for 21 d. The basal diet met estimated essential amino acids (AA) requirements by using all essential AA plus Gly and Ser in free form (CON; 12.1% CP; as-fed, analyzed contents). The remaining four diets were formulated by reducing total Gly and Ser concentrations to 60% or 20% of the CON diet. The N removed with Gly and Ser was replaced with either crystalline Thr or Glu. Total analyzed Thr made up either 1.59% (T1; 12.5% CP) or 2.34% (T2; 12.2% CP) of the Thr-supplemented diets, and total analyzed Glu made up either 3.47% (G1; 12.7% CP) or 4.64% (G2; 12.9% CP) of the Glu-supplemented diets. Pigs were slaughtered on day 21 to determine body composition and skin collagen abundance via bright field microscopy. Overall, average daily gain (ADG) and G:F and final carcass weights were greater for pigs fed diets supplemented with Glu (G1 + G2) vs. those fed diets supplemented with Thr (T1 + T2; P < 0.05, P = 0.060, and P = 0.050 for ADG, G:F, and final carcass weight, respectively); intermediate values were observed for CON. Nitrogen retention in carcass plus viscera and the AA profile of deposited protein in the carcass were not influenced by dietary treatment. Pigs fed the T2 and G2 diets had greater retention of Thr (vs. CON and G2) and Glu (vs. CON and T2) in the viscera protein, respectively (P < 0.05). The apparent utilization efficiency of standardized ileal digestible Thr for protein deposition in carcass plus viscera was less for pigs fed T2 (15.1%) vs. those fed CON (56.7%) or G2 (58.6% ± 2.9%) diets (P < 0.001). Only pigs fed T1 had skin collagen abundance not different from CON; pigs fed G1, G2, and T2 had reduced skin collagen abundance compared with CON and T1 (P < 0.01). Using Glu as an N source when Gly and Ser were reduced to 60% and 20% of CON in reduced CP diets maintained ADG for pigs between 15 and 25 kg BW, whereas supplying Thr as a N source reduced ADG and carcass weight. When dietary Gly and Ser were supplied at 60% of CON, only Thr supplementation rescued skin collagen abundance. Therefore, supplemental Thr at excess levels is not sufficient to replace N from Gly and Ser in reduced CP diets fed to late nursery pigs, despite supporting skin collagen abundance as a secondary indicator of Gly status.

Dietary Supplementation of dl-Methionine Potently Induces Sodium-Dependent l-Methionine Absorption in Porcine Jejunum Ex Vivo.

BACKGROUND: Methionine is an essential amino acid (AA) with many fundamental roles. Humans often supplement l-Met, whereas dl-Met and dl-2-hydroxy-4-(methylthio)butanoic acid (dl-HMTBA) are more frequently used to supplement livestock. OBJECTIVES: The study aimed to investigate whether dietary Met source alters the absorptive capacity for Met isomers in the small intestine of piglets. METHODS: A total of 27 male 10-wk-old piglets in 3 feeding groups received a diet supplemented with 0.21% dl-Met, 0.21% l-Met, or 0.31% dl-HMTBA to meet the Met + cystine requirement. After ≥10 d, absorptive fluxes of d-Met or l-Met were measured at a physiological concentration of 50 µM and a high concentration of 5 mM in duodenum, middle jejunum, and ileum ex vivo. Data were compared by 2-factor ANOVA. RESULTS: Across diets, fluxes of both Met isomers at both tested concentrations increased from duodenum to ileum by a factor of ∼2-5.5 (P < 0.05). Pigs supplemented with dl-Met had greater (P < 0.085) absorptive fluxes at 50 µM l-Met (0.50, 2.07, and 3.86 nmol · cm-2 · h-1) and d-Met (0.62, 1.41, and 1.19 nmol · cm-2 · h-1) than did pigs supplemented with dl-HMTBA (l-Met: 0.28, 0.76, and 1.08 nmol · cm-2 · h-1; d-Met: 0.34, 0.58, and 0.64 nmol · cm-2 · h-1) in duodenum, jejunum, and ileum, respectively. Only in jejunum of dl-Met-fed pigs, fluxes at 50 µM l-Met were reduced by the omission of luminal Na+ (from 3.27 to 0.86 nmol · cm-2 · h-1; P < 0.05) and by a cocktail of 22 luminal AAs (to 1.05 nmol · cm-2 · h-1; P < 0.05). CONCLUSIONS: Dietary supplementation of dl-Met increases the efficiency of l-Met and d-Met absorption at physiologically relevant luminal Met concentrations along the small intestine of pigs, including a very prominent induction of an Na+-dependent transport system with preference for l-Met in the mid-jejunum. Dietary supplementation with dl-Met could be a promising tool to improve the absorption of Met and other AAs.