Effects of limonene on ruminal concentrations, fermentation, and lysine degradation in cattle.

Previous in vitro data showed that was inhibited by limonene. We further evaluated effects of limonene on growth of in vitro as well as on ruminal concentrations of in vivo. With in vitro cultivation in anaerobic brain-heart infusion broth, limonene decreased growth of . Thymol also reduced growth of , but it was less effective than limonene. Tylosin effectively reduced growth of in vitro. Although the response over fermentation times and concentrations of antimicrobials differed somewhat between tylosin and limonene, the 2 antimicrobial agents yielded similar inhibitory effects on growth of at concentrations ranging from 6 to 24 mg/L. The effects of limonene on ruminal concentration in vivo were tested in 7 ruminally cannulated heifers (225 kg initial BW) used in a 7 × 4 Youden square design. Treatments included: 1) control, 2) limonene at 10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, and 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples of ruminal contents were collected before treatment initiation and after 4, 7, and 10 d of treatment for measuring by the most probable number method using selective culture medium. Limonene linearly decreased ( = 0.03) ruminal concentration, with the lowest concentration achieved with 40 mg of limonene/kg dietary DM. Limonene tended ( ≤ 0.07) to linearly reduce ruminal molar proportions of propionate and valerate while tending to linearly increase ( ≤ 0.10) those of butyrate and 2-methyl butyrate. Limonene did not affect ruminal NH concentrations or degradation rates of lysine. Neither CRINA-L ( = 0.52) nor tylosin ( = 0.19) affected ruminal concentrations. CRINA-L significantly decreased ruminal concentrations of NH and molar proportions of 3-methyl butyrate, whereas tylosin significantly decreased molar proportions of propionate while increasing those of butyrate and tending to increase those of acetate. Limonene supplementation reduced ruminal concentrations of suggesting that it may have the potential to reduce the prevalence of liver abscesses, although further research is needed to assess the effect of limonene in feedlot cattle.

Efficiency of lysine utilization by growing steers.

This study evaluated the efficiency of Lys utilization by growing steers. Five ruminally cannulated Holstein steers (165 ± 8 kg) housed in metabolism crates were used in a 6 × 6 Latin square design; data from a sixth steer was excluded due to erratic feed intake. All steers were limit fed (2.46 kg DM/d), twice daily, diets low in RUP (81% soybean hulls, 8% wheat straw, 6% cane molasses, and 5% vitamins and minerals). Treatments were 0, 3, 6, 9, 12, and 15 g/d of Lys continuously abomasally infused. To prevent AA other than Lys from limiting performance, a mixture providing all essential AA to excess was continuously abomasally infused. Additional continuous infusions included 10 g urea/d, 200 g acetic acid/d, 200 g propionic acid/d, and 50 g butyric acid/d to the rumen and 300 g glucose/d to the abomasum. These infusions provided adequate ruminal ammonia and increased energy supply without increasing microbial protein supply. Each 6-d period included 2 d for adaptation and 4 d for total fecal and urinary collections for measuring N balance. Blood was collected on d 6 (10 h after feeding). Diet OM digestibility was not altered ( ≥ 0.66) by treatment and averaged 73.7%. Urinary N excretion was decreased from 32.3 to 24.3 g/d by increasing Lys supplementation to 9 g/d, with no further reduction when more than 9 g/d of Lys was supplied (linear and quadratic, < 0.01). Changes in total urinary N excretion predominantly were due to changes in urinary urea N. Increasing Lys supply from 0 to 9 g/d increased N retention from 21.4 to 30.7 g/d, with no further increase beyond 9 g/d of Lys (linear and quadratic, < 0.01). Break-point analysis estimated maximal N retention at 9 g/d supplemental Lys. Over the linear response surface of 0 to 9 g/d Lys, the efficiency of Lys utilization for protein deposition was 40%. Plasma urea N tended to be linearly decreased ( = 0.06) by Lys supplementation in agreement with the reduction in urinary urea N excretion. Plasma concentrations of Lys linearly increased ( < 0.001), but Leu, Ser, Val, and Tyr ( ≤ 0.02) were linearly reduced by Lys supplementation, likely reflecting increased uptake for protein deposition. In our model, Lys supplementation promoted significant increases in N retention and was maximized at 9 g/d supplemental Lys with an efficiency of utilization of 40%.

Effect of ruminal ammonia supply on lysine utilization by growing steers.

Six ruminally cannulated Holstein steers (202 ± 15 kg) were used to study the effects of ruminal ammonia loading on whole-body lysine (Lys) utilization. Steers were housed in metabolism crates and used in a 6 × 6 Latin square design. All steers received 2.52 kg DM/d of a diet (10.1% CP) containing 82% soybean hulls, 8% wheat straw, 5% cane molasses, and 5% vitamins and minerals, and 10 g/d of urea (considered to be part of the basal diet) was ruminally infused continuously to ensure adequate ruminal ammonia concentrations. All steers were ruminally infused continuously with 200 g/d of acetic acid, 200 g/d of propionic acid, and 50 g/d of butyric acid and abomasally infused with 300 g/d of glucose continuously to increase energy supply without increasing microbial protein supply. Steers were also abomasally infused continuously with an excess of all essential AA except Lys to ensure that Lys was the only limiting AA. Treatments were arranged as a 3 × 2 factorial with 3 levels of urea (0, 40, or 80 g/d) continuously infused ruminally to induce ammonia loading and 2 levels of Lys (0 or 6 g/d) continuously infused abomasally. Treatments did not affect fecal N output ( = 0.37). Lysine supplementation decreased ( < 0.01) urinary N excretion from 51.9 g/d to 44.3 g/d, increased ( < 0.01) retained N from 24.8 to 33.8 g/d, increased ( < 0.01) plasma Lys, and decreased ( ≤ 0.05) plasma serine, tyrosine, valine, leucine, and phenylalanine. Lysine supplementation also tended ( = 0.09) to reduce plasma urea-N. Urea infusions linearly increased ( = 0.05) retained N (27.1, 29.3, and 31.5 g/d) and also linearly increased ( < 0.01) urinary N excretion (31.8, 48.1, and 64.4 g/d), urinary urea (21.9, 37.7, and 54.3 g/d), urinary ammonia (1.1, 1.4, and 1.9 g/d), and plasma urea (2.7, 4.0, and 5.1 mM), and linearly decreased plasma alanine ( = 0.04) and plasma glycine ( < 0.01). Assuming that retained protein is 6.25 × retained N and contains 6.4% Lys, the incremental efficiencies of infused Lys utilization were 51%, 59%, and 69% for steers receiving 0, 40, and 80 g/d of urea, respectively, indicating that ruminal ammonia loads may improve the efficiency of Lys utilization. This is supported by observed increases in whole body-protein deposition in response to ammonia loading of our steers that were, by design, Lys deficient.