Effects Of Tea Seed Saponin On The Community Composition Of Microorganism And Methanogenesis In Rumen Of Beef Cattle

Rumen is an important digestive organ of ruminants and an important place for producing methane. The variation in the community composition of rumen flora will affect the environment stability of the rumen, the digestion and utilization of feed and methane emissions. And there are close relationships among rumen protozoa, bacteria, methanogens, including interspecies hydrogen transfer and symbiotic association, so the variations in the community compositions of ruminal ciliates may affect the change in the community compositions of rumen bacteria and methanogens. Recently, tea seed saponins (TSS) has been found to be used as a regulatory agent for rumen fermentation due to its specific biological activity. Therefore this study was carried out to investigate the effects of TSS on fermentation pattern, methane production, and variations in abundance and community compositions of ruminal ciliates, bacteria and methanogens at the taxonomy level using the qPCR and high-throughput sequencing. This study will ascertain the associations between the community compositions of rumen ciliates, bacteria and methanogens with rumen fermentation and methanogenesis, and the microbiological mechanisms in the regulatory effect of TSS on rumen fermentation and methanogenesis.Exp.1 The effect of tea seed saponin on communities composition of ruminal ciliates and methanogenesis in steersSix Belmont Red composite steers (363 ± 8.5 kg, live weight) were selected to investigate the effects of TSS supplementation on the abundance and communities composition of ruminal ciliates, and methane production of beef steers. According to the feeding way of fattening steers fed on high concentrate based diet in the production, grazing steers were allowed to adjust to the basal diet (concentrate:forage,77:23, DM basis) for 56 days, and fed the same basal diet to steers for 14d more as the pre-control period (BD period). Supplementation with TSS in the BD in the AM feeding diet was for 20 days (TSS period), followed by suspension of the TSS supplement for another 14 days (post-control period, BDP). Methane emissions were detected and rumen fluid samples were collected in the last two days of each period. Real-time qPCR and Illumina high-throughput sequencing were used to study the variations in the abundance and community composition of rumen ciliate protozoa.The results showedthat: 1)Entodinium, Metadinium, Eudiplodinium, Polyplastron and Isotricha were the dominant genera in the rumen of all steers, and TSS addition significantly reduced the amount of the amylolytic protozoa (Entodinium) (P<0.05), while increasing the proportion of cellulolytic protozoa(Polyplastron and Eudiplodinium) (P<0.05) compared to BD steers.2) TSS supplementation reduced the proportion of Entodinium andIsotrichidae OTUs, while increased the proportion of Polyplastron and Eudiplodinium OTUs. And the proportion of Metadinium OTUs increased in BDP steers.3) TSS addition increased the abundance of ruminal ciliates(P<0.05), however, which was decreased to 85% of protozoa amount of BD steers when TSS addition were ceased.4) The BDP steers had a lower proportion of the glycolytic Isotricha compared to BD and TSS steers (P<0.05), and a higher proportion of the fibrolytic Polyplastron compared with BD steers.5) CH4 emissions were not affected by the addition of TSS, but compared to BD and TSS steers, daily CH4 emissions (g) and yield (g CH4/kg DMI) were lower in BDP steers (P<0.05).6) Correlation analyses showed that CH4 emissions was positively related with the abundance of Isotricha genus, but negatively related with he abundance ofMetadinium genus. To conclude, TSS supplementation changed ruminal protozoal community by inhibiting the growth of the amylolytic protozoa (Entodinium), and promoting the growth of the cellulolytic protozoa (Polyplastron and Eudiplodinium). What's more, CH4 emissions was positively related with Isotricha and negatively related with Metadinium, so decreasing the proportion of Isotricha or increasing the amount of Metadinium maybe as an effective strategy to reduce methane emission from ruminants.Exp.2 The effect of tea seed saponin on the community composition of rumen bacteria and rumen fermentation pattern in steersThe relationships between rumen ciliated protozoa and bacteria include predator & prey, symbiosis or parasitic relation. Based on figuring out the the regulatory effect of TSS on the community composition of rumen protozoa, Real-time qPCR and Illumina high-throughput sequencing were used to study the variations in the abundance and community composition of rumen bacteria as well as rumen fermentation. The results showed that:1)The Firmicutes (46.1%), Bacteroidetes (44.4%), Prevotellaceae (32.1%), Acidaminococcaceae (19.9%), Ruminococcaceae (11.3%), Prevotella (24.3%) and Succiniclasticum (18.7%) were the predominant bacterial phyla, families and genera in the rumen of cattle, respectively.2)TSS supplementation significantly reduced the relative abundance of Firmicutes and increased the proportion of Proteobacteria and Fibrobacteres at the phylum level(P<0.05); and reduced the relative abundance of the bacterium converting succinate to propionate (Succiniclasticum), while increased the proportion of fibrolytic bacteria (Ruminococcus and Butyrivibrio) at the genus level (P<0.05).3) TSS supplementation reduced the proportion of Firmicutes OTUs, while increased the proportion of Bacteroidetes OTUsand Proteobacteria OTUs. 4)TSSregulated selectively the ruminal fibrolytic bacteria by decreasing the relative abundance of R. flavefaciens (P<0.05), while increasing the proportion of R. albus and F. succinogenes(P<0.05).5) TSS supplementation changed rumen fermentation pattern by significantly inhibiting the production of propionic acid production and increasing acetate:propionate ratio, while improving the production of isobutyric acid (P<0.05). Accordingly, the overall results suggested that TSS changed the community composition of rumen bacteria by inhibiting the growth of succinate-fermenting organism Succiniclasticum and R. flavefaciens, while promoting the growth of other fibrolytic bacteria (R. albus, F. succinogenes andButyrivibrio), which altered the pattern of rumenfermentation from propionicacidtypefermentation to acetic acidtype.Exp.3 The effect of tea seed saponin on the abundance and community composition of rumen methanogens in steersRumen protozoa and bacteria can provide the substrates (metabolic hydrogen and C1 compound) required for methanogenesis. There are close symbiotic associations between ciliated protozoa and methanogens, and competitive relationships in utilizing hydrogen between certain rumen bacteria and methanogens. Based on figuring out the regulatory effect of TSS on the community composition of rumen protozoa and bacteria, Real-time qPCR and Illumina high-throughput sequencing were used to study the variations in the abundance and community composition of methanogens. The results showed that:1) The Methanobrevibacter spp. was the most dominant genus, accounting for 95% or more, and phylogenetic analysis showed that 86% of 15 dominant OTUswere placedinMbr. SGMT clade, which were the predominant clade in the rumen of beef steers.2) TSS addition increased the absolute abundance of RCC (P<0.05), while there were no effect of on the abundance of total ruminal methanogens and Methanobrevibacter spp. in response to TSS supplementation.3) TSS supplementation reduced the abundance of methanogens belonging to Mbr. SGMT clade, while increased the density of methanogens in Mbr. RO clade.4) Phylogenetic analysis showed that the methanogens isolated from Isotricha protozoa placed in the Mbr. SGMT clade, and the methanogcns isolated from Polyplastron protozoaplaced in the Mbr. RO clade. Collectively, the overall results suggested that TSS changed the community composition of methanogens by differentiately regulating the aboundance of rumen methanogens at the species level, as TSS inhibited the growth of predominant methanogens (SGMT clade), while promoted the growth of methanogens (RO clade) with less abundance. Moreover, the symbiotic methanogens in the Isotricha protozoa mainly belong to SGMT clade.Exp.4 Comparative study of the methanogenic activity of different methanogensThis study was to compare the growth capacity and methanogenic activity Mbr. gottschalkii and Mbr. ruminantium, which are the key represent species in Mbr. SGMT clade and RO clade in the dominant Methanobrevibacter genus, respectively. The time-course experiment was conducted for the two methanogens, and designed 13 time points (0,8,12,16,20,24,30,36,48,60,72,84,96 h) for collecting growth data, methane production and the pellet of methanogen cells used for gDNA and RNA extraction. The results showed that:1) Mbr. ruminantium has a longer logarithmic growth period (40h) and plateau phase period (24h) compared to Mbr.gottschalkii,24h and 12h, respectively.2) The maximum of biomass and specific growth rate of Mbr. ruminantium were higher than that of Mbr.gottschalkii (P<0.05).3) The amounts of H2 used for methanogenesis were similar between the two methanogens(P>0.05), while the CH4 production of Mbr. ruminantium was higher than that of Mbr.gottschalkii (P<0.05). 4) The cumulative methane production of the two methanogens were positively correlated with the expression level of mcrA gene and their abundance (P< 0.05), and the abundance of methanogens was also positively correlated with the expression level of mcrA gene.5) Mbr. ruminantiumcangrow and produce a small amount of methane under low hydrogen concentration, whileMbr. gottschalkiicannotgrow and generate methane. To conclude, Mbr. ruminantium, the represent of RO clade methanogens with low abundance, has a higher methanogenic activity compared to Mbr. gottschalkii, the represent of SGMT clade methanogens with predominant abundance. Moreover, both the abundance of methanogens and the expression level of mcrA gene can be used to assess the methanogenic activity of methanogens to some extent.In conclusion, different species of ciliated protozoa, bacteria, and methanogens showed different degrees of sensitivity to tea seed saponin, and TSS supplementation in the high-concentrate diet changed the community compositions of rumen ciliates, bacteria and methanogens. As for rumen ciliates, TSS supplementation selectivelyinhibited the growth of the amylolytic protozoa (Entodiniwn), while promoted the growth of the cellulolytic protozoa (Polyplastron and Eudiplodinium).As for rumen bacteria, TSS supplementation selectively reduced the relative abundance of one bacterium genus converting succinate to propionate (Succiniclasticum) and R. flavefaciens, whileincreased the proportion of fibrolytic bacteria (Ruminococcus and Butyrivibrio), and altered the rumen fermentation pattern in the rumen of steers fed on the high concentrate diet. As for rumen methanogens, TSS supplementation mainly inhibited the growth of predominant methanogens in Mbr. SGMT clade, whileselectively promoted the growth of less abundant methanogens in Mbr. RO clade. Mbr. ruminantium, the represent of RO clade methanogens with low abundance, has a higher methanogenic activity compared to Mbr. gottschalkii, the represent of SGMT clade methanogens with predominant abundance. So TSS selectively inhibited the growth of the Isotricha protozoa and its symbiotic methanogens in Mbr. SGMT clade withdominant abundance and lower methanogenic activity, while increased methanogens in Mbr. RO clade withless abundance and higher methanogenic activity, which affected the methane production in the rumen of steers.