Supplemental material for In vitro assessment of the effect of free amino acids on ruminal fermentation and 15N-enrichment of ruminal nitrogen pools

The objective of this study was to evaluate the effects of individual AA on ruminal fermentation and N utilization in vitro. Four, 24-h batch culture incubations were conducted with rumen fluid from 3 (2 per incubation run) donor lactating dairy cows as inoculum and a standard TMR (16.5% CP, 30.9% NDF, and 25.5% starch of DM) as the basal substrate. Nitrogen-15 labeled ammonium sulfate (165 mg/L; 15 mg 15N/L) was used as a tracer to determine microbial ammonia-N incorporation in all treatments. Amino acids were supplemented individually (at 155 mg/L) as follows: His, Leu, Lys, Met, Phe, Trp, Val (incubation 1 and 2), and Arg, Asp, Asn, Cys, Glu, Gln, Gly, Ile, Pro, Ser, Thr, and Tyr (incubation 3 and 4). Each incubation included treatments with ammonium sulfate (AS; 101 mg/L) as a negative control and hydrolyzed casein (Amicase; 177 mg/L) as a positive control. Incubation medium samples were collected at 24 h and analyzed for VFA and ammonia-N concentrations, and NDF degradability. A subset of samples was processed for determination of 15N enrichment (i.e., atom percent excess) of 3 rumen N pools: solids, bacterial, and ammonia pools, respectively. For statistical analysis, the individual AA treatments were grouped and analyzed by set of incubations: EAA (incubation 1 and 2), and EAA and NEAA (incubation 3 and 4) vs. Amicase and AS. Additionally, responses to Amicase and individual AA treatments were compared, expressed as the percent difference from AS (negative control). In incubation 3 and 4, compared with AS and EAA treatment group, Amicase and NEAA group had a greater total gas production. Total VFA was greatest for Amicase, and the molar proportion of acetate, and acetate to propionate ratio were lowest and isovalerate was greatest for the EAA group. On an individual AA basis and relative to AS (negative control) acetate to propionate ratio decreased for Cys, Asp, Tyr, Glu, Met, Asn, Ile, and Thr by 4.5 to 12.5%. In addition, molar proportion of isobutyrate increased by 98.2% with Val supplementation, whereas Pro increased valerate by 84.1%, and Ile and Leu increased isovalerate by 71.1 and 61.1%, respectively. Nitrogen-15 enrichment of the bacterial-N pool in incubation 1 and 2 was greater for the EAA group compared with AS and Amicase, and the incorporation of ammonia-N into bacterial-N was greater for the EAA group, and tended to be greater for Amicase, vs. AS. Incorporation of ammonia-N into bacterial-N was greater for Leu, Tyr, Met, Ile, Val, Gly, Phe, His, Pro, Cys, and Arg, ranging from 9.9 to 23%, relative to AS, but the proportion of bacterial-N in solids-N (i.e., a proxy for microbial protein synthesis) was not affected. Overall, supplemental AA (supplied at a dose of 155 mg/L) had minor effects on in vitro fermentation variables and fiber degradability, but supplementation of branched-chain AA and Pro increased molar proportion of their respective branched-chain VFA. Data demonstrated that 8 EAA and 4 NEAA supplemented individually improved the incorporation of ammonia-N into bacterial-N, indicating that some AA can enhance N utilization in the rumen.