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Rumen archaea play an important role in scavenging ruminal hydrogen (H 2 ) 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 (CH 4 ) 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.

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