Glen A. Broderick

Determination of protein degradation rates using a rumen in vitro system containing inhibitors of microbial nitrogen metabolism.

1. A previously reported rumen in vitro system (Broderick, 1978) was modified to include chloramphenicol (CAP) with hydrazine sulphate (HS) to give quantitative recovery of protein breakdown products. Degradation rates were determined by regression v. time of log proportion remaining undegraded (computed by subtracting from added nitrogen the amount of N recovered as ammonia and amino acids). Concentrations of reagents giving optimal N recoveries and estimated degradation rates for casein and expeller soya-bean-meal (SBM) were: 1.0 mM-HS, 30 micrograms CAP/ml, 2.0 mM-mercaptoethanol, 3.3 mg maltose/ml, when protein was added at 0.125 mg N/ml. 2. Digestion of azo-casein and azo-albumin, solubilization of radioactivity from 14C-labelled casein, ovalbumin and bovine serum albumin (BSA), and hydrolysis of benzoyl-L-tyrosine p-nitroanilide and benzoyl-L-arginine p-nitroanilide were not significantly decreased by HS and CAP. This suggests that the inhibitors did not reduce microbial proteolysis. 3. Mean fractional degradation rates (/h) were: 0.395 casein, 0.135 BSA, 0.159 solvent-SBM, 0.045 expeller-SBM, 0.061 meat meal, 0.070 lucerne (Medicago sativa) hay. Extents of protein escape, estimated assuming rumen passage of 0.06/h, were 13, 28, 56 and 40% for casein, solvent-SBM, expeller-SBM and lucerne hay respectively. This method appears more reliable for assessing rumen degradability than buffer N solubility and protein digestibility with ficin protease. 4. Azo-dye treatment slowed the rate of casein degradation, measured by ammonia plus amino acid release, but did not alter digestion of BSA. 5. The validity of the inhibitor in vitro method for estimating protein degradability, as well as potential problems in its application, are discussed. The complete procedure may be limited to laboratories with automated analytical equipment, but a simplified version of the method may be more generally applicable.

Rumen protein degradation rates estimated by non-linear regression analysis of Michaelis-Menten in vitro data

An in vitro method applying Michaelis-Menten saturation kinetics was developed as an alternative approach for estimating protein degradation rates in the rumen. Non-linear regression (NLR) analysis of the integrated Michaelis-Menten equation yielded fractional degradation rates, kd, from direct estimates of the maximum velocity: Michaelis constant ratio (kd = Vmax:Km). Degradation rates obtained using data from a series of 2 h inhibitor in vitro incubations were respectively 0.989, 0.134, and 0.037/h for casein, solvent soya-bean meal (SSBM) and expeller soya-bean meal (ESBM). Degradation rates obtained from 2 h incubations had lower standard errors than those obtained using 1 h incubations; 2 h rates were not significantly different from 1 h rates, suggesting end-product inhibition was not significant at 2 h. The NLR Michaelis-Menten method was used to determine degradation rates for twelve protein sources: casein, bovine serum albumin, two samples of lucerne (Medicago sativa) hay, and four samples each of SSBM and ESBM. Statistical analysis of NLR results revealed significant differences among the twelve protein sources. Casein was degraded most rapidly (0.827/h), and the four ESBM samples most slowly (0.050-0.098/h). Degradation rate for serum albumin was 0.135/h; rates for SSBM and lucerne hays ranged from 0.160 to 0.208/h. Degradation rates estimated using the NLR method were more rapid than those obtained with a limited substrate approach; NLR rates were more consistent with in vivo estimates of rumen protein escape. Greater concentrations of slowly degraded proteins were needed with the NLR method to define curvilinearity of the degradation curve more accurately.

Replacing dietary soybean meal with canola meal improves production and efficiency of lactating dairy cows1

Previous research suggested that crude protein (CP) from canola meal (CM) was used more efficiently than CP from solvent soybean meal (SBM) by lactating dairy cows. We tested whether dietary CP content influenced relative effectiveness of equal supplemental CP from either CM or SBM. Fifty lactating Holstein cows were blocked by parity and days in milk into 10 squares (2 squares with ruminal cannulas) in a replicated 5×5 Latin square trial. Five squares were fed: (1) low (14.5-14.8%) CP with SBM, (2) low CP with CM, (3) low CP with SBM plus CM, (4) high (16.4-16.7%) CP with SBM, and (5) high CP with CM; the other 5 squares were fed the same diets except with rumen-protected Met plus Lys (RPML) added as Mepron (Degussa Corp., Kennesaw, GA) and AminoShure-L (Balchem Corp., New Hampton, NY), which were assumed to provide 8g/d of absorbed dl-Met and 12g/d of absorbed l-Lys. Diets contained [dry matter (DM) basis] 40% corn silage, 26% alfalfa silage, 14 to 23% corn grain, 2.4% mineral-vitamin premixes, and 29 to 33% neutral detergent fiber. Periods were 3wk (total 15wk), and data from the last week of each period were analyzed using the Mixed procedures of SAS (SAS Institute Inc., Cary, NC). The only effects of RPML were increased DM intake and milk urea N (MUN) and urinary N excretion and trends for decreased milk lactose and solids-not-fat concentrations and milk-N:N intake; no significant RPML × protein source interactions were detected. Higher dietary CP increased milk fat yield and tended to increase milk yield but also elevated MUN, urine volume, urinary N excretion, ruminal concentrations of ammonia and branched-chain volatile fatty acids (VFA), lowered milk lactose concentration and milk-N:N intake, and had no effect on milk true protein yield. Feeding CM instead of SBM increased feed intake, yields of milk, energy-corrected milk, and true protein, and milk-N:N intake, tended to increase fat and lactose yields, and reduced MUN, urine volume, and urinary N excretion. At low CP, MUN was lower and intake tended to be greater on SBM plus CM versus SBM alone, but MUN and N excretion were not reduced to the same degree as on CM alone. Interactions of parity × protein source and parity × CP concentration indicated that primiparous cows were more responsive than multiparous cows to improved supply of metabolizable protein. Replacing SBM with CM reduced ruminal ammonia and branched-chain VFA concentrations, indicating lower ruminal degradation of CM protein. Replacing SBM with CM improved milk and protein yield and N-utilization in lactating cows fed both low- and high-CP diets.

Evaluation of Camelina sativa (L.) Crantz meal as an alternative protein source in ruminant rations

BACKGROUND Camelina sativa (CS) is an oilseed crop used for biofuel production. By-products from oil extraction are high in protein and can be used in ruminant rations; more information about their nutritive value is required also considering the antinutrional factor content of the by-products. The aim of this study was to evaluate the nutritive value of CS meal genotypes in comparison with canola. RESULTS Ten CS genotypes and one canola cultivar were evaluated. Meals were obtained from seeds after solvent oil extraction. CS average crude protein (CP) content (g kg⁻¹ dry matter) was 457. Numerical differences in lysine and sulfur amino acid content were observed among CS genotypes. Glucosinolate (mmol kg⁻¹) content was higher for CS (23.1) than canola (7.2). Sinapine content (g kg⁻¹) was lower for CS (2.79) than for canola (4.32). Differences were observed among CS genotypes for rumen undegraded protein (RUP). Average RUP (g kg⁻¹ CP) was 316 for CS and 275 for canola. CONCLUSIONS CS meal has potential for use in ruminant rations as a high-quality protein source. In vivo studies are needed to compare CS with other protein sources used in cattle rations. Implementation of breeding programs for improved meal quality is recommend.

Effects of feeding lauric acid on ruminal protozoa numbers, fermentation, and digestion and on milk production in dairy cows

The objectives of this study were 1) to determine the level of lauric acid (LA) addition to the diet necessary to effectively suppress ruminal protozoa (RP) to the extent observed when a single dose was given directly into the rumen, 2) to assess LA effects on production and ruminal metabolism, and 3) to determine the time needed for RP to reestablish themselves after LA is withdrawn from the diet of lactating dairy cows. In Exp. 1, 2 Holstein cows fitted with ruminal cannulae were used in a split-plot design pilot study. Both cows consumed the same level of LA, starting with 0 g/d and increasing to 129, 270, and 438 g/d mixed into the diet. Diets were fed as total mixed ration (TMR) and contained (DM basis) 30% corn silage, 30% alfalfa silage, and 40% concentrate. Lauric acid intake linearly decreased DMI (P = 0.03), RP numbers (P < 0.01), ruminal acetate molar proportion (P = 0.03), and ruminal ammonia concentration (P = 0.03). Lauric acid intake linearly increased ruminal valerate molar proportion (P = 0.02). A quadratic response of LA consumption was observed on total ruminal VFA concentration (P < 0.01) and propionate molar proportion (P < 0.01), with maximum responses at 270 g/d of LA intake. A quadratic response of LA consumption was also observed on total ruminal free amino acid (TAA) concentration (P < 0.01), with minimum concentration at 270 g/d of LA intake. After withdrawing the greatest LA dose from the diet, RP returned to their original numbers in 12 d. In Exp. 2, 48 multiparous Holstein cows (8 with ruminal cannulae) were blocked by days in milk into 12 blocks of 4 cows (2 blocks of cannulated cows) and randomly assigned within replicated 4 × 4 Latin squares to balanced dietary treatment sequences. Diets were fed as TMR and contained (DM basis) 36% corn silage, 29% alfalfa silage, and 35% concentrate, and LA intake levels were 0, 220, 404, and 543 g/d mixed in the TMR. In Exp. 2, LA linearly reduced RP (P < 0.01), ruminal ammonia (P < 0.01), and total free AA concentration (P < 0.01); however, dietary LA also linearly decreased DM intake (P < 0.01). Intake of LA linearly reduced ruminal total VFA concentration (P < 0.01); DM, OM, NDF, and CP digestibility (P < 0.01); and milk production and milk components (P < 0.01). Therefore, LA does not appear to be a feasible RP suppressant for feeding in practical diets.

Effects of lauric acid on ruminal protozoal numbers and fermentation pattern and milk production in lactating dairy cows

The objectives of this study were to evaluate lauric acid (LA) as a practical ruminal protozoa-suppressing agent and assess effects of protozoal suppression on fermentation patterns and milk production in dairy cows. In a pilot study, 6 lactating Holstein cows fitted with ruminal cannulae were used in a randomized complete-block design trial. Cows were fed a basal total mixed ration (TMR) containing (DM basis) 15% alfalfa silage, 40% corn silage, 30% rolled high moisture shelled corn, and 14% solvent soybean meal, and assigned to 1 of 3 treatments: 1) control, 2) 160 g/d of LA, or 3) 222 g/d of sodium laurate, which is equimolar to 160 g/d of LA, all given as a single dose into the rumen via cannulae before feeding. Both agents showed high antiprotozoal activity when pulse dosed at these amounts via ruminal cannulae, reducing protozoa by 90% (P<0.01) within 2 d of treatment. Lauric acid reduced ruminal ammonia concentration by 60% (P<0.01) without altering DMI. Both agents reduced ruminal total free AA concentration (P<0.01) and LA did not affect ruminal pH or total VFA concentration. In a large follow-up feeding trial, 52 Holstein cows (8 with ruminal cannulae) were used in a randomized complete-block design trial. Cows were assigned to 1 of 4 diets and fed only that diet throughout the study. The TMR contained (DM basis) 29% alfalfa silage, 36% corn silage, 14% rolled high moisture shelled corn, and 8% solvent soybean meal. The 4 experimental diets were similar, except part of the finely ground dry corn was replaced with LA in stepwise increments from 0 to 0.97% of dietary DM, which provided (as consumed) 0, 83, 164, and 243 g/d of LA. Adding these amounts of LA to the TMR did not affect DMI, ruminal pH, or other ruminal traits, and milk production. However, LA consumed at 164 and 243 g/d in the TMR reduced the protozoal population by only 25% and 30% (P=0.05), respectively, showing that these levels, when added to the TMR, were not sufficient to achieve a concentration within the rumen that promoted the antiprotozoal effect of LA.

Chemical and ruminal in vitro evaluation of Canadian canola meals produced over 4 years.

To test the effects of year and processing plant on the nutritional value of canola meal (CM), 3 CM samples/yr were collected from each of 12 Canadian production plants over 4yr (total=144). Samples of CM were analyzed for differences in chemical composition and for in vitro ruminal protein degradability using the Michaelis-Menten inhibitor in vitro (MMIIV) method. In the MMIIV method, protein degradation rate (kd) was estimated by 2 methods: from net release (i.e., blank corrected) of (1) ammonia plus AA determined by o-phthaldialdehyde fluorescence (OPAF) assay or (2) ammonia, AA, plus oligopeptides determined by o-phthaldialdehyde absorbance (OPAA) assay; rumen-undegradable protein (RUP) was computed assuming passage rates of 0.16 and 0.06/h for, respectively, soluble and insoluble protein. Casein, solvent soybean meal (SSBM), and expeller soybean meal (ESBM) were included in all incubations as standard proteins. Differences among years and plants were assessed using the mixed procedures of SAS. Small but significant differences were found in CM among years for chemical composition, including N solubility; some of these differences may have been related to changes in our analytical methods over time. However, adjustment of degradation activity of individual in vitro incubations based on the mean degradation activity over all incubations yielded kd and RUP that did not differ by year using either assay. Simultaneously incubating CM samples from 2yr in the same in vitro runs confirmed that no year effects existed for kd or RUP. Differences existed in chemical composition of CM among the 12 processing plants over the 4yr of sample collection. Moreover, consistent differences in kd and RUP were observed among plants: kd ranged from 0.069 to 0.113/h (OPAA assay) and 0.075 to 0.120/h (OPAF assay), and RUP estimates ranged from 51 to 43% (OPAA assay) and 49 to 41% (OPAF assay). Regression of kd on insoluble N content of CM yielded correlation coefficients (R(2))=0.40 (OPAA assay) and 0.42 (OPAF assay), and regressions of kd on NDIN and N-fraction B3 yielded R(2)<0.02. Mean estimates from both OPAA and OPAF assays for casein, SSBM, ESBM, and CM were, respectively, kd=0.764, 0.161, 0.050, and 0.093/h and RUP=18, 33, 56, and 45%. A range of 8 percentage units from lowest to highest RUP suggests that substantial differences exist in metabolizable protein content of CM produced by different processing plants.

Utilization of protein in red clover and alfalfa silages by lactating dairy cows and growing lambs

Feeding trials were conducted with lactating cows and growing lambs to quantify effects of replacing dietary alfalfa silage (AS) with red clover silage (RCS) on nutrient utilization. The lactation trial had a 2 × 4 arrangement of treatments: AS or RCS fed with no supplement, rumen-protected Met (RPM), rumen-protected Lys (RPL), or RPM plus RPL. Grass silage was fed at 13% of dry matter (DM) with AS to equalize dietary neutral detergent fiber (NDF) and crude protein contents. All diets contained (DM basis) 5% corn silage and 16% crude protein. Thirty-two multiparous (4 ruminally cannulated) plus 16 primiparous Holstein cows were blocked by parity and days in milk and fed diets as total mixed rations in an incomplete 8 × 8 Latin square trial with four 28-d periods. Production data (over the last 14 d of each period) and digestibility and excretion data (at the end of each period) were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Although DM intake was 1.2 kg/d greater on AS than RCS, milk yield and body weight gain were not different. However, yields of fat and energy-corrected milk as well as milk content of fat, true protein, and solids-not-fat were greater on AS. Relative to AS, feeding RCS increased milk and energy-corrected milk yield per unit of DM intake, milk lactose content, and apparent N efficiency and reduced milk urea. Relative to AS, apparent digestibility of DM, organic matter, NDF, and acid detergent fiber were greater on RCS, whereas apparent and estimated true N digestibility were lower. Urinary N excretion and ruminal concentrations of ammonia, total AA, and branched-chain volatile fatty acids were reduced on RCS, indicating reduced ruminal protein degradation. Supplementation of RPM increased intake, milk true protein, and solids-not-fat content and tended to increase milk fat content. There were no silage × RPM interactions, suggesting that RPM was equally limiting on both AS and RCS. Supplementation of RPL did not influence any production trait; however, a significant silage × RPL interaction was detected for intake: RPL reduced intake of AS diets but increased intake of RCS diets. Duplicated metabolism trials were conducted with lambs confined to metabolism crates and fed only silage. After adaptation, collections of silage refusals and excreta were made during ad libitum feeding followed by feeding DM restricted to 2% of body weight. Intake of DM was not different when silages were fed ad libitum. Apparent digestibility of DM, organic matter, NDF, and hemicellulose was greater in lambs fed RCS on both ad libitum and restricted intake; however, acid detergent fiber digestibility was only greater at restricted intake. Apparent and estimated true N digestibility was substantially lower, and N retention was reduced, on RCS. Results confirmed greater DM and fiber digestibility in ruminants and N efficiency in cows fed RCS. Specific loss of Lys bioavailability on RCS was not observed. Based on milk composition, Met was the first-limiting AA on both silages; however, Met was not limiting based on production and nutrient efficiency. Depressed true N digestibility suggested impaired intestinal digestibility of rumen-undegraded protein from RCS.

A 100-Year Review: Protein and amino acid nutrition in dairy cows

Considerable progress has been made in understanding the protein and amino acid (AA) nutrition of dairy cows. The chemistry of feed crude protein (CP) appears to be well understood, as is the mechanism of ruminal protein degradation by rumen bacteria and protozoa. It has been shown that ammonia released from AA degradation in the rumen is used for bacterial protein formation and that urea can be a useful N supplement when lower protein diets are fed. It is now well documented that adequate rumen ammonia levels must be maintained for maximal synthesis of microbial protein and that a deficiency of rumen-degradable protein can decrease microbial protein synthesis, fiber digestibility, and feed intake. Rumen-synthesized microbial protein accounts for most of the CP flowing to the small intestine and is considered a high-quality protein for dairy cows because of apparent high digestibility and good AA composition. Much attention has been given to evaluating different methods to quantify ruminal protein degradation and escape and for measuring ruminal outflows of microbial protein and rumen-undegraded feed protein. The methods and accompanying results are used to determine the nutritional value of protein supplements and to develop nutritional models and evaluate their predictive ability. Lysine, methionine, and histidine have been identified most often as the most-limiting amino acids, with rumen-protected forms of lysine and methionine available for ration supplementation. Guidelines for protein feeding have evolved from simple feeding standards for dietary CP to more complex nutrition models that are designed to predict supplies and requirements for rumen ammonia and peptides and intestinally absorbable AA. The industry awaits more robust and mechanistic models for predicting supplies and requirements of rumen-available N and absorbed AA. Such models will be useful in allowing for feeding lower protein diets and increased efficiency of microbial protein synthesis.