Betaine Modulates Rumen Archaeal Community and Functioning during Heat and Osmotic Stress Conditions In Vitro

Abstract

Rumen archaea play an important role in scavenging ruminal hydrogen (H₂) and thus facilitate rumen fermentation. They require optimum temperature and osmolality for their growth and metabolism; however, a number of external factors may put archaea under heat and osmotic stress. Betaine is an osmolyte, molecular chaperone, and antioxidant; therefore, it bears potential to combat against these stressors. In this in vitro study, three betaine levels, namely, 0 (control), 51 (low), and 286 (high) ppm, were used. Each of these was subjected to two temperatures (39.5 and 42°C) and two osmolality conditions (295 and 420 mOsmol kg^-1) with n = 6 per treatment. Sequencing analyses of the solid phase (which use solid materials containing primarily fibrous materials of low-density feed particles) and the liquid phase (rumen fermenter liquid) using 16S rRNA revealed that more than 99.8% of the ruminal archaea in fermenters belong to the phylum Euryarchaeota. At the genus level, Methanobrevibacter was the most prevalent in both phases, and Methanosaeta was only detected in the liquid phase. The genera Methanobrevibacter and Methanobacterium both showed a positive correlation with methane (CH₄) formation in the liquid and solid phases, respectively (P < 0.05). Heat stress increased the relative abundance of genus Methanimicrococcus at the expense of candidate archaeal genus Vadin CA11 (P < 0.05). In the solid phase, osmotic stress significantly reduced the Shannon and Simpson indices of diversity, and relative abundance was higher for Methanobrevibacter at the expense of Methanimicrococcus. In the liquid phase, osmotic stress increased not only the abundance-based coverage estimator (ACE) and singles parameters of diversity but also the relative abundances of Methanosphaera and Methanobacterium. The overall decrease in all gas parameters and estimated metabolic hydrogen ([2H]) utilization was observed during osmotic stress conditions (P < 0.05). Betaine enhanced the diversity of solid phase archaea as indicated by the increase in ACE and singles during heat stress, and only a high dose improved all diversity parameters in the liquid phase during osmotic stress (P < 0.05). Thus, betaine alleviates the effects of heat stress and osmotic stress on the archaea community.

Figure 1

Principle coordinate analysis (PCoA) for beta-diversity of ruminal archaea in the liquid phase of the incubation content of fermenters, under the effects of (a) temperature (red squares: 39.5°C; blue circles: 42°C), (b) osmolality¹ (blue circles: normal; red squares: osmotic stress), and (c) betaine supplementation² (red squares: control; orange triangles: low; blue circles: high). The first 3 components were plotted and in total principle component explained 69.74% of the total variation (PC1 = 43.07, PC2 = 16.45, and PC3 = 10.22%, respectively). ¹Normal osmolality ~ 295 mOsmol kg⁻¹; hyperosmolality ~ 420 mOsmol kg⁻¹. ²Betaine levels: control (0), low (51), and high (286) ppm.

Figure 2

Diversity parameters of ruminal archaea in liquid phase as affected by betaine addition¹ and osmolality². Different superscripts on hyperosmolality bars represent significant difference (P < 0.05). ^∗P < 0.05. ¹Betaine levels: control (0), low (51), and high (286) ppm. ²Normal osmolality ~ 295 mOsmol kg⁻¹; hyperosmolality ~ 420 mOsmol kg⁻¹.

Figure 3

Principle coordinate analysis (PCoA) for beta-diversity of ruminal archaea in the solid phase of the incubation content of fermenters, under the effects of (a) temperature (red squares: 39.5°C; blue circles: 42°C), (b) osmolality¹ (blue circles: normal; red squares: osmotic stress), and (c) betaine supplementation² (red squares: control; orange triangles: low; blue circles: high). The first 3 components were plotted and in total principle component explained 77.61% of the total variation (PC1 = 47.78, PC2 = 21.46, and PC3 = 8.37%, respectively). ¹Normal osmolality ~ 295 mOsmol kg⁻¹; hyperosmolality ~ 420 mOsmol kg⁻¹. ²Betaine levels: control (0), low (51), and high (286) ppm.

Figure 4

Diversity parameters of ruminal archaea in solid phase as affected by betaine addition¹ and temperature. Different superscripts on heat stress (42°C) bars represent significant difference (P < 0.05). ^∗P < 0.05. ¹Betaine levels: control (0), low (51), and high (286) ppm.