Effects of Essential Fatty Acid Supplementation on in vitro Fermentation Indices, Greenhouse Gas, Microbes, and Fatty Acid Profiles in the Rumen

Abstract

This study estimated the effect of essential fatty acid (FA) supplementation on fermentation indices, greenhouse gases, microbes, and FA profiles in the rumen. The treatments used pure FAs consisting of C18:2n-6 FA (LA), C18:3n-3 FA (LNA), or a mixture of these FAs at 1:1 ratio (Combo). In vitro rumen incubation was performed in 50 mL glass serum bottles containing 2 mg of pure FAs, 15 mL of rumen buffer (rumen fluid+anaerobe culture medium = 1:2), and 150 mg of synthetic diet (411 g cellulose, 411 g starch, and 178 g casein/kg dry matter) at 39°C for 8 h with five replications and three blanks. In rumen fermentation indices, LA exhibited highest (P < 0.05) ammonia-N and total gas volume after 8 h of incubation. Furthermore, LA presented lower (P < 0.05) pH with higher (P < 0.05) total volatile fatty acid (P = 0.034) than Combo, while LNA was not different compared with those in the other treatments. Additionally, Combo produced highest (P < 0.05) CO₂ with lowest (P < 0.05) CH₄. In the early hours of incubation, LA improved (P < 0.005) Fibrobacter succinogenes and Ruminococcus flavefaciens, while LNA improved (P < 0.005) Ruminococcus albus. After 8 h of incubation, LNA had lower (P < 0.05) methanogenic archaea than LA and Combo but had higher (P < 0.05) rumen ciliates than LA. R. albus was higher (P < 0.05) in LA than in LNA and Combo. It was observed that the rate of biohydrogenation of n-6 and n-3 FAs was comparatively lowest (P < 0.05) in Combo, characterized by higher C18:2n-6 and/or C18:3n-3 FA and polyunsaturated FA (PUFA) concentrations with lower (P < 0.05) concentrations of C18:0 and saturated FA and the ratio of saturated FAs to PUFAs. Therefore, this study concluded that dietary C18:2n-6 could improve populations of fibrolytic bacteria and rumen fermentation indices, but dietary mixture of pure C18:2n-6 and C18:3n-3 is recommended because it is effective in reducing enteric methane emissions and resisting biohydrogenation in the rumen with less effect on rumen microbes.

Keywords: biohydrogenation; fatty acid; methane emission; rumen fermentation; rumen microbes.

FIGURE 1

Changes in rumen pH (A) and concentrations of NH₃-N (B), acetate (C), and propionate (D) during rumen incubation with FAs for 8 h. Diet with C18:2n-6, LA (filled circles); diet with C18:3n-3, LNA (filled triangles); and diet with the mixture of C18:2n-6 and C18:3n-3 at a ratio of 1:1 (filled squares). The respective significance levels of the supplementary treatment, incubation period, supplementary treatment × incubation period, and SEM for pH, NH₃-N, acetate, and propionate are P < 0.001, P = 0.054, P = 0.041, and SEM = 0.057; P < 0.001, P = 0.115, P < 0.001, and SEM = 1.064; P < 0.001, P = 0.071, P < 0.001, and SEM = 0.715; and P < 0.001, P = 0.037, P ≤ 0.001, and SEM = 0.487. Different symbols at the same hour differ significantly. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 2

Changes in CH₄ (A) and CO₂ (B) emissions during rumen incubation with FAs for 8 h. Diet with C18:2n-6, LA (filled circles); diet with C18:3n-3, LNA (filled triangles); and diet with the mixture of C18:2n-6 and C18:3n-3 at a ratio of 1:1 (filled squares). The respective significance levels of the supplementary treatment, incubation period, supplementary treatment × incubation period, and SEM for CH₄ and CO₂ are P < 0.001, P = 0.001, P < 0.001, and SEM = 3.021 and P < 0.001, P < 0.001, P = 0.005, and SEM = 91.69. Different symbols at the same hour differ significantly. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 3

Fold change in methanogenic archaea (A), rumen ciliate (B), F. succinogenes (C), R. flavefaciens (D), and R. albus (E) compared with that of the blank during rumen incubation with FAs for 8 h. Diet with C18:2n-6, LA (filled circles); diet with C18:3n-3, LNA (filled triangles); and diet with the mixture of C18:2n-6 and C18:3n-3 at a ratio of 1:1 (filled squares). The respective significance levels of the supplementary treatment, incubation period, supplementary treatment × incubation period, and SEM for methanogenic archaea, rumen ciliates, F. succinogenes, R. flavefaciens, and R. albus are P = 0.255, P = 0.157, P = 0.492, and SEM = 2.046; P < 0.001, P = 0.458, P = 0.019, and SEM = 0.344; P < 0.001, P < 0.001, P = 0.003, and SEM = 0.124; P < 0.001, P = 0.032, P = 0.301, and SEM = 0.168; and P < 0.001; P = 0.001, P < 0.001, and SEM = 0.099. Different symbols at the same hour differ significantly. ^∗P < 0.05; ^∗∗P < 0.01.

FIGURE 4

Changes in C18:2n-6 (A), C18:3n-3 (B), C18:1cis-9 (C), and C18:0 (D) concentrations (%, FA) during rumen incubation with FAs for 8 h. Diet with C18:2n-6, LA (filled circles); diet with C18:3n-3, LNA (filled triangles); and diet with the mixture of C18:2n-6 and C18:3n-3 at a ratio of 1:1 (filled squares). The respective significance levels of the supplementary treatment, incubation period, supplementary treatment × incubation period, and SEM for concentrations of C18:2n-6, C18:3n-3, C18:1n-9, and C18:0 are P < 0.001, P < 0.001, P < 0.001, and SEM = 1.915; P < 0.001, P < 0.001, P < 0.001, and SEM = 1.111; P < 0.001, P = 0.001, P = 0.218, and SEM = 0.306; and P < 0.001, P < 0.001, P = 0.036, and SEM = 1.741. Different symbols at the same hour differ significantly. ^∗P < 0.05; ^∗∗P < 0.01.