Branched-chain amino acids (BCAA; valine, isoleucine, and leucine) can be deaminated by many amylolytic bacteria to branched-chain volatile fatty acids (BCVFA, isobutyrate, 2-methylbutyrate, and isovalerate), which are growth factors for some cellulolytic bacteria. Many cellulolytic bacteria cannot uptake BCAA or decarboxylate them, thus depending on cross feeding for BCVFA precursors for carboxylation to BCAA or synthesis of branched-chain fatty acids (BCFA; iso even-chain, iso odd-chain, and anteiso odd-chain) and branched-chain aldehydes (BCALD) found in bacterial phospholipid and plasmalogens, respectively.
Supplemental BCVFA and valerate, a straight-chain volatile fatty acid (VFA) that is also a growth factor for some rumen bacteria, have been previously supplemented together in a combination commonly referred to as ‘isoacids’. However, prior in vivo studies have either provided only one BCVFA individually or all BCVFA and valerate but not different combinations of isoacids. Our objective in the first study was to determine an optimal combination of isoacids. Sixty (28 primiparous and 32 multiparous) lactating Jersey cows (106 ± 54 days in milk) were blocked and randomly assigned to either a control (CON) treatment without any isoacids, 2-methylburtyate (MB, 12.3 mmol/kg DM), 2-methylbutyrate and isobutyrate (MB + IB, 7.7 and 12.6 mmol/kg DM of MB and IB), or all 4 isoacids (ISO, 6.2, 7.3, 4.2, and 5.1 mmol/kg DM of MB, IB, isovalerate, and valerate, respectively). The CON diet was fed for 2-wk covariate period, then cattle were fed their assigned treatment for the 8-wk sampling period (n=15). Daily intake and milk yield were recorded and samples from 4 consecutive milkings a week were analyzed for milk components. The milk fatty acid profile was analyzed on wk 5 and wk 9. Treatment tended to interact with parity for both fat and protein concentrations. Primiparous cows did not differ by treatment, whereas multiparous cows supplemented with MB + IB tended to have greater protein concentration compared to CON and MB treatments and greater fat concentration compared to all other treatments in multiparous cows. Though milk yield and dry matter intake (DMI) did not change with treatment, there was an interaction with week for lactation efficiency (measured as milk energy/DMI). Supplementation of MB + IB tended to increase lactation efficiency compared with CON during first interval (wk 3 and 4) and third interval (wk 7 and 8). However, cows fed MB alone had the numerically lowest lactation efficiency. The differences were greater during the earlier weeks of the study and decreased as cows entered late lactation. The percentage of 15:0 anteiso FA of milk fat was highest with cows fed MB, was greater than CON or MB + IB, but did not differ from ISO. In our study MB + IB and ISO both improved feed efficiency and not at the expense of average daily can, but MB + IB appeared to be the optimal combination.
Further research was needed for evaluating how BCVFA benefits vary under differing dietary conditions. The next study was an incomplete block design with 8 continuous cultures used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were: high (HF, 67% forage) or low forage (LF, 33% forage), without or with supplemental corn oil (CO; 3% dry matter), and without or with 2.15 mmol/d each of isovalerate, isobutyrate, and 2-methylbutyrate. From each BCVFA, 5 mg/d of 13C was dosed into each vessel. The isonitrogenous diets consisted of 33: 67 alfalfa:orchardgrass pellet, which replaced a concentrate pellet that mainly consisted of ground corn, soybean meal, and soybean hulls. The pH of the vessels was managed to minimize pH differences by diet.
The effects of BCVFA supplementation on nutrient degradation, ruminal fermentation, and prokaryotic profile were determined. Supplementing BCVFA increased neutral detergent fiber (NDF) degradability by 7.6% and improved bacterial N synthesis by 6.6% for organic matter truly degraded and by 6.5% for truly degraded N. The prokaryotic profile shifted mainly by diet. The relative sequence abundance decreased with LF for Fibrobacter succinogenes, Ruminococcus flavefaciens, and genus Butyrivibrio. Recovery of the 13C dosed as BCVFA decreased by 31% with LF relative to HF. Even though more label was recovered with HF, both NDF degradability and efficiency of bacterial N synthesis improved under all dietary conditions. Therefore, BCVFA supplementation could improve feed efficiency in dairy cows under diverse dietary conditions, even those that can inhibit cellulolytic bacteria.
To explore the effects of varied diets and BCVFA supplementation on bacterial lipid metabolism, total fatty acid (FA), bacterial FA, and bacterial aldehydes (ALD) flow from the vessels were collected and analyzed. The ALD are recovered after vinyl ether lipids are hydrolyzed from plasmalogen phospholipids. Supplementation of BCVFA did not influence biohydrogenation index but increased total FA flow leaving the vessels by 13.5%. The percent of BCFA in the bacterial FA profile decreased from 9.46% with HF to 7.06% with LF, and the percent of BCALD in the bacterial ALD profile decreased from 55.4% with HF to 51.4% with LF. Supplemental CO specifically decreased iso even-chain FA and iso even-chain ALD, indicating isobutyrate incorporation decreased in bacterial lipids. Dose recovery in bacterial lipids, BCFA, and BCALD decreased by 42.2%, 48.3%, and 29.0%, respectively, with LF versus HF. There was no appreciable label outside of branched-chain lipids. Although ALD were less than 6% of total bacterial lipids, they accounted for 26.0% of 13C recovered in lipids. The recovery of the label in iso odd-chain FA was greater than iso even-chain FA, whereas recovery of iso even-chain ALD was greater than recovery in iso odd-chain ALD. However, most of the label was recovered with anteiso odd-chain FA and anteiso odd-chain ALD, indicating that 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Both BCFA and BCALD are important for function and growth because membrane homeostasis is necessary to adapt membrane fluidity under different conditions. Therefore, BCVFA supplementation can provide the primers necessary for bacterial structure and can support the rumen microbial consortium.
Finally, the influence of dietary conditions and BCVFA supplementation on bacterial protein metabolism was explored. Lower forage in the diet increased bacterial BCAA flow by 9.12% and BCAA percentage in bacterial AA profile by 1.99%. Supplementation of BCVFA increased total AA flow by 13.0% and bacterial BCAA flow by 10.7%. Recovery of 13C in bacterial AA C was higher with HF 96.3 µg/ mg compared to 66.1 µg/ mg with LF, despite greater bacterial BCAA flow with LF. The greater outflow of total AA and bacterial BCAA would have post-ruminal effects, which would potentially explain prior post-absorptive responses from BCVFA supplementation to dairy cattle. The recovery was greatest with leucine, followed by isoleucine, and finally valine. Although isotope recovery in bacteria was greater with HF, BCVFA supplementation increased total AA and bacterial BCAA flow under all dietary conditions. Both increases could potentially explain post-absorptive responses from BCVFA supplementation.
In conclusion, 2-methylbutyrate is the BCVFA most generally needed by bacteria, supporting previous research in our lab. Isovalerate is generally used for leucine synthesis but much less so for lipids, also supporting our previous research showing it the least likely BCVFA to benefit bacteria. Although isobutyrate had the least carboxylation to its parent BCAA (valine), it seems to have a major role in synthesis of iso even-chain FA that are converted to vinyl ethers in plasmalogens. Further research is needed to understand this role, but the results do support why its combination with 2-methylbutyrate provided the best response in the lactation study.