In vitro study on the effects of condensed tannins of different molecular weights on bovine rumen fungal population and diversity

Figures & data

Table 1. Effects of unfractionated CTs F0 and CT fractions of different MWs on the biomass of rumen fungi at 24 h of incubation using in vitro gas production method, estimated using real-time PCR.

F0 (Unfractionated CTs) and CT fractions (30 mg g^−1 DM)^¶
Treatments^‡	0 mg (Control)	F0	F1	F2	F3	F4	F5	SEM^†	p-Value
Rumen fungal biomass (µg mL^−1) *Estimated using real-time PCR	43.1^a	39.1^abc	35.3^bc	34.6^c	36.1^bc	39.3^abc	40.3^ab	0.152	<.05

CT: condensed tannin.

Means with different superscripts within a row are significantly different (p < .05).

¶ Data are means of three replications.

† Standard error of the mean.

‡ F0, unfractionated CTs; F1, fraction F1; F2, fraction F2; F3, fraction F3; F4, fraction F4; F5, fraction F5.

Figure 1. Rarefaction curves of rumen fungal communities based on the ITS-1 gene sequences from the different treatment groups. Control (without CTs), (F0 = unfractioned CTs, F1 = Fractrion 1, F2 = Fraction 2, F3 = Fraction 3, F4 = Fraction 4 and F5 = Fraction 5) examined at a 0.03 distance level. The operational taxonomic units (OTUs) were defined by the average neighbour algorithms with 3% dissimilarity using Mothur.

Table 2. Summary of the Illumina MiSeq sequences data and statistical analysis of rumen fungi diversity in bovine rumen fluid without (control) and with unfractionated CTs F0 and CT fractions of different MWs weights from LLR.

F0 (unfractionated CTs) and CT fractions (30 mg g^−^1 DM)
Treatments^‡	Control	F0	F1	F2	F3	F4	F5	p Value
Number of sequences	80,400	108,918	112,426	114,440	133,041	93,131	121,980	ND
*OTUs	36	38	22	26	35	31	32	ND
^†Goods coverage	0.999	0.999	0.999	0.999	0.999	0.999	0.999	ND
*Chao1 (Richness)	152.2^a	132.5^ab	90.0^bc	108.5^b	152.0^a	155.4^a	169.3^a	<.05
*Shannon (Diversity)	0.245^a	0.195^bc	0.152^c	0.175^c	0.194^bc	0.221^ab	0.211^ab	<.05
ACE	158.4	152.7	159.3	206.8	180.5	182.1	201.6	.307

CT: condensed tannin; OTU: operation taxonomy units; MW: molecular weights.

Means with different superscripts within a row are significantly different (p < .05).

† Good’s coverage at a distance of 0.03.

* Diversity indices of the fungal communities were calculated using MOTHUR programme based on the ITS1 gene sequences.

‡ F0, unfractionated CTs; F1, Fraction F1; F2, Fraction F2; F3, Fraction F3; F4, Fraction F4; F5, Fraction F5.

ACE: Abundance-based coverage estimators.

ND: Not determined.

Table 3. Relative abundance (% of total sequences) of fungal genera in bovine rumen samples without (control) and with unfractionated CTs F0 and CT fractions of different MWs weights from LLR.

Treatments^‡	Relative abundance (%)^†
	F0 (unfractionated CTs) and CT fractions (30 mg g^−1 DM)
	Control	F0	F1	F2	F3	F4	F5
Unclassified Neocallimastigales	0.93	0.27*	0.05*	0.06*	0.11*	0.15*	0.09*
Unclassified Neocallimastigaceae	29.84	7.92*	1.04*	0.80*	1.79*	5.87*	7.08*
Piromyces 5	0.01	0.01	0.02	0.01	0.02	0.02	0.02
Piromyces 2	0.08	0.11	0.11	0.09	0.11	0.11	0.15*
Anaeromyces mucronatus YE505	0.01	0.01	0.01	0.01	0.01	0.02	0.01
Piromyces 4	69.12	91.67*	98.76*	99.00*	97.95*	93.83*	92.64*
Anaeromyces 1	0.01	0.01	0.01	0.02	0.01	0.01	0.01

CT: condensed tannin; LLR: Leucaena leucocephala hybrid-Rendang; MW: molecular weights.

Value with superscript ‘*’ within a row indicates significant difference (p < .05) in comparison to control (without CTs).

^† STAMP comparisons of metagenomes: the results were tested using Fisher’s exact test, two sided, 95% confidence interval and CI method (difference between proportions): Newcombe–Wilson with Bonferroni correction.

^‡ F0, unfractionated CTs; F1, Fraction F1; F2, Fraction F2; F3, Fraction F3; F4, Fraction F4; F5, Fraction F5.

Figure 2. Hierarchical clustering of fungal communities using the Bray-Curtis similarity index based on the un-weighted UniFrac method. Control (without CTs), (F0 = unfractioned CTs, F1 = Fractrion 1, F2 = Fraction 2, F3 = Fraction 3, F4 = Fraction 4 and F5 = Fraction 5). The scale represents the un-weighted UniFrac distances.

Figure 3. Principal component analysis (PCoA) of fungal ITS-1 gene sequence tags generated from an Illumina MiSeq sequencing run (F0 = unfractionated CT, F1= Fraction F1, F2= Fraction F2, F3= Fraction F3, F4= Fraction F4, and F5= Fraction F5). Percentage of the diversity distribution explained by each axis is indicated on the figure. CT: condensed tannin.

Figure 4. Distance dendogram highlighting the phylogenetic affiliation of anaerobic rumen fungal sequences. Sequences used in the tree construction include available ITS-1 reference gene sequences from GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and representative OTUs affiliated with known anaerobic fungal genera encountered in this study. The tree was constructed using neighbour-joining algorithm with the Hasegawa–Kishino–Yano (HKY) substitution model and a γ-shaped distribution of 2.0. Bootstrap values are based on 1000 replicates and are shown for branches with >50% bootstrap support. The corresponding ITS-1 region of the ascomycetous yeast Issatchenikia orientalis was used as an outgroup.

Aerts RJ, Barry TN, McNabb WC. 1999. Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agric Ecosyst Environ. 75:1–12.

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