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 (EAA1; incubations 1 and 2), and Arg, Asp, Asn, Cys, Glu, Gln, Gly, Ile, Pro, Ser, Thr, and Tyr (EAA2 and NEAA; incubations 3 and 4). Each incubation included treatments with ammonium sulfate (AS; 101 mg/L) as a negative control and hydrolyzed CN (Amicase; 177 mg/L) as a positive control. Incubation medium samples were collected at 24 h and analyzed for VFA and ammonia-N concentrations, as well as 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: EAA1 (incubations 1 and 2), EAA2 and NEAA (incubations 3 and 4) versus Amicase and AS. Additionally, responses to Amicase and individual AA treatments were compared expressed as the percent difference from AS (negative control). In incubations 3 and 4, compared with AS and EAA2 treatment groups, Amicase and NEAA had a greater total gas production. Total VFA was greatest for Amicase, while the molar proportion of acetate and acetate-to-propionate ratio were lowest and isovalerate was greatest for the EAA1 group. On an individual AA basis and relative to AS (negative control) acetate-to-propionate ratio decreased for Cys, Asp, Tyr, Gln, Met, Asn, Ile, and Thr by 4.5% to 12.5%. Furthermore, the isobutyrate molar proportion increased by 98.2% with Val, 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 incubations 1 and 2 was greater for the EAA1 group compared with AS and Amicase, and the incorporation of ammonia-N into bacterial-N was greater for the EAA1 group, and tended to be greater for Amicase, versus AS. Incorporation of ammonia-N into bacterial-N was greater for Leu, Tyr, Met, Ile, Val, Trp, 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 proportions of their respective branched-chain VFA. Data demonstrated that 9 EAA and 3 NEAA supplemented individually improved the incorporation of ammonia-N into bacterial-N, indicating that some AA can enhance N utilization in the rumen.