Effects Of Dietary Pectin On Microbial Structure And Microbial Protein Synthesis In The Rumen

Pectin is a natural complex polysaccharides and one of the major components of the primary cell wall of plants, thus it is a structural carbohydrate present in plant feedstuffs of ruminants. Pectins commonly exist in fiber feeds such as alfalfa hays, Chinese wild rye, corn stover and rice straw, with alfalfa especially rich in pectin content. Pectin and starch belong to non-fibrous carbohydrate but with remarkable different fermentation characteristics. Up to date, there seems to be very few studies focused on the effect of pectin on ruminal microbial structure and the knowledge of pectinolytic bacteria in the rumen is very limited. Therefore, the objectives of this study were to evaluate effects of pectin on ruminal fermentation and microbial protein pply and especially, to explore the microbial species and group associated with pectin-rich diets. This study contained two parts. In the first part, specific bacterial species or group associated with pectin-rich alfalfa diet compared in the rumen of (?)y cows was explored and quantitative assay method of specific pectinolytic species was developed. In the second part, the effects of pectin supplementation on rumen fermentation, microbial protein supply and microbial structure in the rumen of Hu sheep was systematically investigated.1. Effects of dietary pectin on ruminal bacteria and detection of pectinolytic bacteria1.1Effects of dietary pectin content on bacterial group in the rumen of dairy cows. The yields of chemical-extracted pectin from alfalfa hay, corn stover and (?)se wild rye were determined to be9.3,2.8and3.7%, respectively. With454pyrosequencing method, the abundance of Selenomonas, Treponema and T. saccharophilum were found to be significant higher in the rumen of dairy cows fed on alfalfa hay as main forage source compared to those fed on corn stover. Pure culture based studies demonstrated that these species are soluble carbohydrates fermenters.1.2Effects of combination of pectin with different feeds on the distribution of ruminal bacteria. To evaluate whether Selenomonas and Treponema are pectinolytic bacteria, in vitro rumen fermentation was conducted using alfalfa, corn stover, pectin, corn starch or their combination as the substrates. The cluster analysis of DGGE banding patterns showed that combination of corn stover with pectin as the substrate resulted in the band patterns being closely grouped with those of alfalfa. On the contrary, combinations of corn stover with starch diverted the pattern away from alfalfa. It is indicated from a microbial ecological aspect that pectin is one of the important nutritional differences that exist between corn stover and alfalfa. The specific bands associated with pectin-rich treatments were identified to be dominated by the members of Treponema genus, with one clone showing99%similarity with T. saccharophilum. Real-time PCR analysis demonstrated that the growth of Treponema genus was remarkably supported by inclusion of pectin but not starch, highlighting their specific ability to degrade pectin. However, the relative abundances of S. ruminantium were not significantly different between the fermentation fluids with pectin and starch as substrate.1.3Development a real-time PCR assay targeting at the ruminal pectinolytic T. saccharophilum to access its abundance in both in vitro and in vivo. Due to lack of a reliable and accurate detection method, the abundance of T. saccharophilum in the rumen has not been well determined. Thus, the specific primers targeting at the16S rRNA gene of T. saccharophilum was developed in the present study and the specificity and accuracy of the primers was confirmed, which might also be used in further ruminal ecological studies. The results obtained in the present study clearly demonstrate that quantification of T. saccharophilum in vivo and in vitro is possible by real-time PCR using our designed primers. In vitro results showed that the population of T. saccharophilum was significantly higher with alfalfa or pectin as substrates than with corn stover or Chinese wild rye (P<0.05). However, addition of starch did not support the growth of T. saccharophilum. Similarly, the in vivo results also showed that the relative abundance of T. saccharophilum in the rumen of cows fed alfalfa hay diet was significantly higher (P<0.05) than those in the rumen of cows fed the diet containing Chinese wild rye, corn stover or rice straw. The population of T. saccharophilum species, particularly with alfalfa diet, is comparable or even higher than those of other common rumen bacterial species. The relatively high proportion of T. saccharophilum in the rumen might expand our knowledge of the importance of this bacterium in the rumen, especially for pectin digestion.2. Effects of pectin on production performance, rumen fermentation and microbial structure in the rumen of Hu sheep2.1Effects of pectin on rumen fermentation parameters and microbial protein synthesis in vitro. An in vitro fermentation was conducted to investigate the effects of pectin and starch on ruminal fermentation. With700mg corn stover and150mg soybean meal as basic substrates, the following treatments were used:(1)150mg corn starch+0mg pectin (PeO),(2)100mg corn starch+50mg pectin (Pe50),(3)50mg corn starch+100mg pectin (Pe100), and (4)0mg corn starch+150mg pectin (Pel50). The rate of gas production increased with the increasing ratio of pectin, with highest value in Pe100and Pe150and lowest in Pe0(P<0.05), especially during the early fermentation before6h. Total gas production in24h was significantly higher with Pe100and Pe150compared to PeO and Pe50. Substituting pectin for starch linearly increased the ratios of acetate to propionate. The pH of fermentation fluid increased with the increasing pectin ratio, significantly lower in PeO and Pe50than in e100or Pe150(P<0.05). The MCP production was highest in Pe150and lowest in Pe0(P<0.05). The results obtained here indicated that inclusion of pectin optimized the fermentation rate, environmental pH and further promoted the MCP production.2.2Effects of pectin on production performance and rumen fermentation of Hu sheep. To evaluate the effects of pectin on animal growth,45male sheep were divided into five groups of nine animals each, and randomly assigned to five dietary treatments with supplementation of0,2.5and5%pectin or corn starch, respectively, to a basal diet consisted of corn stover and concentrate mixture (50:50, DM basis). Dry matter intake was increased quadratically or decreased linearly with the increasing level of pectin or starch, respectively. Increasing level of pectin led to a linear increase (P<0.05) in microbial protein synthesis and daily weight gain, with highest values at5%pectin among all treatments (P<0.05). The TG levels in the serum of sheep fed with pectin diets were significant lower compared to control diet and starch diets (P<0.05).2.3Effects of pectin on the bacterial structure in the rumen of Hu sheep. To obtain a deep understanding of ruminal bacteria involved in pectin digestion, Illumina MiSeq sequencing platform were used to generate large-scale16S rRNA gene sequencing data from genomic DNA of ruminal bacteria associated with the above experimental diets in section2.2. Multi-comparison on phylum, family, genus and OTU level demonstrated that the distribution patterns of bacteria among diets Ctrl, Pe and St were significantly different. Real-time PCR quantification showed that, besides Treponema genus and T. saccharophilum, most of typical rumen bacteria were higher when starch was supplemented. However, with high-throughput sequencing technology, it is found that the majority of bacteria groups that enriched by pectin were remain uncultured or not well studied, most of them belonging to Firmicutes, including Mogibacteriaceae, Lachnospiraceae and Erysipelotrichi. Genus such as Butyrivibrio, Copromccus, Moryella, Holdemania, L7A_E11and Bulleidia were all associated with pectin digestion to a different extent.In summary, compared to starch, pectin is a more desirable carbohydrate, which could promote the rumen MCP synthesis and thus improve the growth performance of ruminants. The growth of ruminal bacteria Treponema and Firmicutes were enhanced by pectin, indicating that they may play an important role in microbial protein synthesis under a pectin-rich diet. A better understanding of microbial populations that accompany dietary differences will expand our knowledge of ecological importance of bacteria in the rumen and may further lead to beneficial strategies to improve ruminant production performance.