Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Nov 23:14:1282767.
doi: 10.3389/fmicb.2023.1282767. eCollection 2023.

Comparison of the effects of rumen-protected and unprotected L-leucine on fermentation parameters, bacterial composition, and amino acids metabolism in in vitro rumen batch cultures

Jishan An #  1 , Weijun Shen #  1 , Hu Liu  1 , Chen Yang  1 , Kemeng Chen  1 , Qiongwen Yuan  1 , Zhiqing Li  1 , Dingfu Xiao  1 , Zuo Wang  1 , Xinyi Lan  1 , Lei Liu  2 , Fachun Wan  1
Affiliations

Comparison of the effects of rumen-protected and unprotected L-leucine on fermentation parameters, bacterial composition, and amino acids metabolism in in vitro rumen batch cultures

Jishan An et al. Front Microbiol. .

Abstract

This study was conducted to compare the effects of rumen-protected (RP-Leu) and unprotected L-leucine (RU-Leu) on the fermentation parameters, bacterial composition, and amino acid metabolism in vitro rumen batch incubation. The 5.00 g RP-Leu or RU-Leu products were incubated in situ in the rumen of four beef cattle (Bos taurus) and removed after 0, 2, 4, 6, 12, 16, and 24 h to determine the rumen protection rate. In in vitro incubation, both RP-Leu and RU-Leu were supplemented 1.5 mmol/bottle (L-leucine HCl), and incubated after 0, 2, 4, 6, 8, 12, and 16 h to measure gas production (GP), nutrient degradability, fermentation parameters, bacterial composition, and amino acids metabolism. Results from both in vitro and in situ experiments confirmed that the rumen protection rate was greater (p < 0.01) in RP-Leu than in RU-Leu, whereas the latter was slow (p < 0.05) degraded within incubation 8 h. Free leucine from RP-Leu and RU-Leu reached a peak at incubation 6 h (p < 0.01). RU-Leu supplementation increased (p < 0.05) gas production, microbial crude protein, branched-chain AAs, propionate and branched-chain VFAs concentrations, and Shannon and Sobs index in comparison to the control and RP-Leu supplementation. RU-Leu and RP-Leu supplementation decreased (p < 0.05) the relative abundance of Bacteroidota, which Firmicutes increased (p < 0.05). Correlation analysis indicated that there are 5 bacteria at the genus level that may be positively correlated with MCP and propionate (p < 0.05). Based on the result, we found that RP-Leu was more stable than RU-Leu in rumen fluid, but RU-Leu also does not exhibit rapid degradation by ruminal microbes for a short time. The RU-Leu was more beneficial in terms of regulating rumen fermentation pattern, microbial crude protein synthesis, and branched-chain VFAs production than RP-Leu in vitro rumen conditions.

Keywords: L-leucine; bacterial community; beef cattle; in vitro technique; rumen fermentation; rumen protection rate.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A) The dynamic rumen protection rate of RU-Leu and RP-Leu in situ. (B) The dynamic rumen degradation rate of RU-Leu and RP-Leu in vitro. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine.
Figure 2
Figure 2
The effect of RU-Leu and RP-Leu on gas production (A) and DM degradability (B) of fermentable substrates. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine; DM, dry matter. a,bDifferent letters represent significantly different values (p < 0.05, n = 5).
Figure 3
Figure 3
The effect of RU-Leu and RP-Leu on concentrations of free leucine amino acids (A), free EAAs and NEAAs (B), free total EAAs (C), free total BCAAs (D), and free total AAs (E) at 6 h in vitro. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine; EAA, essential amino acids; BCAA, branched-chain amino acids. a,bDifferent letters represent significantly different values (p < 0.05, n = 5).
Figure 4
Figure 4
The effect of RU-Leu and RP-Leu on the composition of the microbiota. (A) OUT distribution across different treatment groups at 6 h in vitro. (B) PCoA score plot. (C–F) Alpha diversity evaluation of colon microbial richness and evenness by measuring Sobs, Shannon, ACE, and Chao diversity indexes. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine. a,bDifferent letters represent significantly different values (p < 0.05, n = 5).
Figure 5
Figure 5
The effect of RU-Leu and RP-Leu on the composition of the microbiota at 6 h in vitro. (A) Relative abundance (%) of bacteria at the phylum level in the three groups (mean of each group). (B) The effect of Leu treatment on relative abundances of Bacteroidota and Firmicutes. (C) Relative abundances (%) of bacteria at the genus level in the three groups (mean of each group). (D) The effect of Leu treatment on relative abundances of Rikenellaceae_RC9_gut_group and Christensenellaceae_R-7_group. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine. Data were shown as means (n = 5). Significant (p < 0.05) statistical differences were represented by (*).
Figure 6
Figure 6
(A) Linear discriminant analysis effect size (LEfSe) was performed to identify the bacteria that are differentially represented between the different groups. (B) Cladogram reported. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine. Data were shown as means (n = 5). Significant (p < 0.05) statistical differences were represented by (*).
Figure 7
Figure 7
Spearman’s rank correlation analysis between bacteria (76.65% of the total bacterial relative abundance) at the genus level and rumen fermentation parameters, including pH, microbial protein (MCP), ammonia-N, T-VFAs (total volatile fatty acids), acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, acetate: propionate (A:P), and branched chain volatile fatty acids (BCVFA). RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine. Data were shown as means (n = 5). Significant (p < 0.05) or (p < 0.01) statistical differences were represented by (*) or (**).
Figure 8
Figure 8
(A–F) Significant differences in the functional pathways among different treatment groups. RU-Leu, rumen-unprotected L-leucine; RP-Leu, rumen-protected L-leucine. a,bDifferent letters represent significantly different values (p < 0.05, n = 5).
See this image and copyright information in PMC

References

    1. Allison M. J. (1969). Biosynthesis of amino acids by rumen microorganisms. J. Anim. Sci. 29, 797–807. doi: 10.2527/jas1969.295797x, PMID: - DOI - PubMed
    1. Allison M. J., Bryant M. P. (1963). Biosynthesis of branched-chain amino acids from branched-chain fatty acids by rumen bacteria. Arch. Biochem. Biophys. 101, 269–277. doi: 10.1016/S0003-9861(63)80012-0, PMID: - DOI - PubMed
    1. Allison M. J., Bryant M. P., Doetsch R. N. (1958). Volatile fatty acid growth factor for cellulolytic cocci of bovine rumen. Science 128, 474–475. doi: 10.1126/science.128.3322.474, PMID: - DOI - PubMed
    1. Allison M. J., Bucklin J. A., Robinson I. M. (1966). Importance of the Isovalerate carboxylation pathway of leucine biosynthesis in the rumen. Appl. Microbiol. 14, 807–814. doi: 10.1128/am.14.5.807-814.1966, PMID: - DOI - PMC - PubMed
    1. AOAC (1995). Official methods of analysis. Association of Official Analytical Chemists: Arlington, VA.