Effects Of Dietary Fiber On Skatole Levels In Swine Body And The Underlying Regulatory Mechanisms

Skatole (3-methylindole) produced by the microbial degradation of L-tryptophan in pig large intestine, is one of the compounds responsible for boar taint. It can easily accumulate in adipose tissue. High deposition of skatole has negative effects on pork flavor. The developmental changes and spatial variation of skatole deposition in swine body were firstly investigated as basis in the present work (PartⅠ). Then, the influence of differing dietary fiber sources on in vitro L-tryptophan metabolism by porcine fecal microbiota was conducted (PartⅡ). Thereafter, the effect of dietary fiber on skatole levels in intestinal digesta and various tissues in pigs was further evaluated by animal experiment (PartⅢ). The molecular mechanisms of dietary fiber on pig skatole levels were revealed from the point of intestinal microbial composition and hepatic CYP2E1 and CYP2A6 gene expression, respectively.PartⅠ:The developmental changes and spatial variation of skatole deposition in swine bodyExperiment 1:The objective of this experiment was to study the developmental changes of skatole levels in pigs. Nine intact male Landrace pigs were slaughtered at d30,90 and 150. Samples were taken from blood, backfat, abdomen fat and liver and analyzed for skatole levels. Expression of CYP2E1 and CYP2A6 mRNA and their activities were evaluated for liver samples. Hepatic and plasma skatole contents elevated significantly with the increasing age (P<0.05). From d 30 to d 90, the backfat skatole levels tended to increase (P=0.061), while skatole levels in abdominal fat were increased significantly (P<0.05). Similarly, indole levels in plasma, liver and fat were increased as pig grows. Except for the plasma, indole levels in liver and fat were increased significantly from d 30 to d 150 (P<0.05). Levels of CYP2E1 and CYP2A6 mRNA expression decreased first and then increased; mRNA abundance at d 150 was significantly higher than that at d 30 and d 90. No significant difference was observed in CYP2E1 and CYP2A6 enzyme activities between the growing stages. The results indicated that the expression of CYP2E1 and CYP2A6 gene may be not related in skatole metabolism in prepubescent pigs.Experiment 2:Spatial variation of intestinal skatole production and microbial community in local and exotic pigs was evaluated. Intact male pigs of three purebred Jinhua and three Landrace at d 150, with 37.3 and 82.5 kg BW, respectively, were slaughtered. The pigs'hindgut were separated into cecum (Ce), proximal colon (PC), distal colon (DC) and rectum (Re) and sampled for analysis of skatole and volatile fatty acids (VFA). The mitosis and apoptosis of PC were determined by immunocytochemistry. Denaturing gradient gel electrophoresis (DGGE) and gene sequencing were adopted to study the microbial community from the intestinal contents. For both breeds, skatole concentration increased towards the rectum and segments showed significant effects on skatole (P<0.001). The Ce skatole level in Landrace was significantly lower than that in PC, DC and Re (P<0.05). Compared with Jinhua pigs, the rectal skatole and proximal and distal colonic indole levels were significantly higher in Landrace (P<0.05). The VFAs were significantly affected by breed except for isobutyrate and isovalerate (P<0.05), with higher butyrate level and lower pH in Landrace pigs (P<0.05). No significant differences were found in mitosis and apoptosis between the two breeds. The special band appeared in the Jinhua PC, DC and Re samples in which skatole concentrations were higher matched with Clostridium aminophium. The results suggested that swine genotype plays a role in the establishment of the host-specific microbiota and that the variations of fermentation patterns are more likely to explain differences in intestinal skatole production between swine breeds. The bacteria C. aminophium may be related to skatole production.Part II:Influence of differing dietary fiber sources on in vitro L-tryptophan metabolism by porcine fecal microbiotaExperiment 3:Four types of carbohydrate (CHO) sources, sugar beet pulp (SBP), rye grass hay (RYE), alfalfa hay (ALF) and fructooligosaccharides (FOS) were used to investigate their effects on the metabolism of L-tryptophan to skatole and indole by a mixed bacterial population from pig large intestines. Microbial suspensions were anaerobically incubated at 38℃, and indole compounds, pH and VFA were analyzed after 24 h fermentation. Effects of CHO sources on the microbial diversity were analyzed using DGGE,16S rDNA cloning and sequencing. Addition of SBP showed decreasing effect on skatole concentration and decreased the relative rate of skatole production significantly, whereas RYE and ALF treatments showed opposite effects. The rate of tryptophan degradation was reduced by addition of ALF. For FOS, both skatole concentration and relative rate of skatole production were significantly reduced. Total and individual VFAs (acetate, propionate, butyrate and valerate) were significantly higher in the four CHO-added groups than in the none-added. Additions of SBP, RYE, ALF and FOS in pig fecal slurries with L-tryptophan markedly decreased the culture medium pH values. The DGGE profiles showed differences between cultures with different CHO substrates, though some bands were common to all cultures. Fermentation with SBP showed a higher bacterial diversity than with others, with three distinct bands appeared. Sequences of the corresponding 16S rRNA of two bands were related to Eubacterium rectale and Clostridium sp., respectively. Three bands were present only in SBP, RYE and ALF samples. Two of these represented to Lachnospira pectinoschiza and Clostridium disporicum DSM 5521, respectively. These results suggested that the reduced concentration of skatole observed in the presence of SBP and FOS may be caused by decreased tryptophan degradation to indolic compounds and by shifted microbial metabolism of tryptophan toward indole production at the expense of skatole, resultant from the changed microbial ecosystem. The bacteria Clostridium disporicum DSM 5521 may play a role in the production of skatole. PartⅢ:Effect of dietary fiber on skatole levels in intestinal digesta and various tissues, microbial composition and hepatic cytochrome P450 expressions in pigsExperiment 4:The experiment was conducted to monitor the time-course changes in skatole concentrations and microbial communities in feces of barrows due to the inclusion of selected fermentable fibers in diets. A total of 18 barrows (Duroc x Landrace x Yorkshire), with an initial BW of 85 kg, were randomly assigned to three dietary treatments and fed at libitum on one of the following isonitrogeneous diets: control (CON),20%inclusion of sugar beet pulp (SBP) and 20%inclusion of alfalfa meal (ALF). For 4 pigs in each group, fresh fecal samples were collected per rectum at d 1,8,15,22 and 29. The VFA profile, skatole and indole concentrations were analyzed. Microbial community was also measured by DGGE. The skatole concentrations in the feces of SBP and ALF groups increased first and then decreased. Compared to the CON, the skatole concentrations in the feces of SBP-fed pigs were significantly higher at d 15,22 and 29 of treatment (P<0.05). At all time points, the skatole levels in SBP were relatively higher, while the ALF had no significant effect on skatole. Dietary fiber had significant effect on skatole level (P<0.0001), whereas indole level was not influenced by diets. The total VFA levels were higher and the pH value was lower in pigs fed SBP at d 8,15,22 and 29. The DGGE band number and similarity were significantly higher for SBP-fed pigs at d 22. Based on DGGE fingerprint patterns from d 15,22 and 29 treatments of SBP, changes in fecal microbiota occurred as skatole production increased. Sequences of the corresponding 16S rRNA of DGGE bands indicated that the bacteria from the order of Clostridiales and the family of Lachnospiraceae were associated with skatole production.Experiment 5:The objective of this experiment was to evaluate the spatial variation of intestinal skatole production and microbial community in barrows due to dietary fiber feeding. The experimental animals and diets were the same as those in experiment 4. After 33 days of feeding, four pigs from each group were slaughtered and samples were taken for skatole, VFA and microbial community analysis in Ce, PC, DC and Re intestinal contents. Significant segments effect was observed on skatole concentration (P<0.05). The DC skatole levels were significantly higher than that in Ce for SBP-fed pigs. Skatole levels in PC, DC and Re from ALF-fed pigs were all significantly higher than that in Ce. Compared to the CON, skatole concentrations in the Ce were reduced in both SBP and ALF groups. The total VFA levels were constantly decreased from Ce to Re for all the groups, with the highest level in the SBP-fed pigs. Apoptotic activities in DC were increased by SBP feeding (P<0.05). Distinct DGGE bands were appeared in SBP group, with two bands related to Lactobacillus johnsonii and Lachnospiraceae, respectively which may be responsible for skatole formation.Experiment 6:Effects of dietary fiber sources on tissue skatole accumulation and hepatic expression of CYP2E1 and CYP2A6 in barrows were investigated in this experiment. The experimental animals and diets were the same as those in experiment4. Four pigs from each group were slaughtered and tissue samples were analyzed for skatole concentrations. Expressions of CYP2E1 and CYP2A6 genes were evaluated in liver samples by real-time PCR. Growth performance and carcass characteristics were not affected by dietary treatments, but average backfat thickness were significantly reduced by SBP or ALF feeding (P<0.05). Compared to CON, SBP feeding decreased the mesenteric and abdominal fat skatole and indole levels, but tended to increase backfat skatole concentration, whereas ALF feeding showed significant decreasing effect on skatole and indole level in adipose tissue. Real-time RT-PCR analysis revealed that SBP inclusion increased hepatic CYP2E1 mRNA levels, whereas ALF feeding increased both CYP2E1 and CYP2A6 expressions. No significant differences were observed in CYP2E1 and CYP2A6 enzyme activities between the treatments. These results indicated that skatole levels could be improved by ALF inclusion, which has better effect on skatole than SBP. The lowed tissue skatole levels in ALF-fed pigs appear to be associated with higher exressions of CYP2E1 or CYP2A6 genes.In summary, in prepubescent pigs, skatole depositions increased as pig grows and the expression of CYP2E1 and CYP2A6 genes may be not involved in skatole metabolism. The intestinal skatole concentration increased towards the rectum. Microbial communities as well as fermentation patterns varied among different pig breeds and intestinal segments, and were changed by dietary fiber inclusion and further influence skatole production. The bacteria of C. aminophium, C. disporicum, L. johnsonii and Lachnospiraceae may be related to skatole formation. Pig skatole deposition could be reduced by ALF incorporation in the diet, which has better effect on skatole than SBP. The results may provide theoretical and practical basis for the improvement of pork quality.