Nutritional value of acid stabilised silage and liquefied fish protein

Fish silage made by a conventional method (pH 4.0) contained a variety of active enzyme systems which not only hydrolysed fish proteins to short peptides and free amino acids, but also caused the degradation of free amino acids to ammonia and other metabolic products. Acid-stabilised fish silage (pH 2.0) contained only those polypeptides released by endogenous pepsins with only slight accumulation of free amino acids and degradation products. Liquefied fish which was autolysed for less than 1 h at 60°C at physiological pH (6.2–6.6) and then pasteurised and acidified to pH 4.0 contained polypeptides of intermediate length which remained stable. These processes provide a means for adjusting the peptide and free amino acid content of autolysed fish protein to suit the nutritional requirements of a variety of aquatic and terrestrial species.     

The nutritional quality of two types of fish silage for broiler chickens

Fish waste was ensiled either by acidification with formic acid or by fermentation with a bacterial starter culture and molasses. The resulting liquids were mixed with wheat bran (85:15 w/w liquid: bran) and dried (70°C) to produce acid silage meal (ASM) and fermented silage meal (FSM). ASM and FSM were incorporated into wheat-based diets at 25, 50 and 100 g kg^?1 at the expense of soya bean meal. There were two control diets, one which contained soya bean meal as the predominant protein supplement and a second in which fish meal (50 g kg^?1) was added at the expense of some of the soya bean meal. Starter diets (13.25 MJ ME kg^?1, 12 g kg^?1 lysine) were fed from 1-day-old to 21-days-old and finisher diets (13.25 MJ ME kg^?1, 9.5 g kg^?1 lysine) were fed from 22 to 42-days-old to six replicates each of five birds. Birds were reared in raised-wire cages, and feed intake, liveweight and mortality were recorded. There were no significant effects of dietary inclusion of either ASM and FSM on the performance of broiler chickens relative to those fed on control diets. FSM contained less crude protein and amino acids than ASM. The recovery of amino acids relative to the total crude protein content from FSM was only 78.7%, presumably as a result of formation of Maillard reaction products during drying.     

The production of naturally fermented fish silage using various lactobacilli and different carbohydrate sources

The suitability for fish silage-making of eight different Lactobacillus cultures was investigated. The industrial by-products whey powder and molasses, as well as refined sugar, were tested as fermentation substrates. Using the information obtained two batches of fish silage were made at local white fish processing plants on a semi-commercial basis and subsequently successfully used on local cattle and pig farms.     

Electrical conductivity as a tool for identification of metal contaminated fish protein

Fish acts as a good bioindicator for metal contamination. The contaminated metal ions are bonded into the protein biologically. The doping concentration and its properties were analyzed by the tools of electrical conductivity. For the analysis of fish species, Clarius batrachus was selected, subjected to metal-dissolved solution and studied for its muscle conductivity in both raw and pelletised forms. The various parameters analyzed were electrical conductivity and temperature dependent electrical conductivity on fish muscle protein in its unpelletised (natural) and pelletised form, when metals like copper, cadmium and lead were doped to the muscle protein. The studies carried out were atomic absorption spectroscopy (AAS), thermo gravimetric analysis (TGA) and electron paramagnetic resonance (EPR). The results showed that conductivity in muscle as well as its pelletised temperature dependent conductivity is a very good mechanism for the identification of cadmium, lead and copper contamination level.     

Performance of lactating dairy cows fed ryegrass silage and corn silage with ground corn, steam-flaked corn, or hominy feed

Forty-eight mid-lactation Holstein cows were used in a 6-wk completely randomized block design trial with a 4 × 3 factorial arrangement of treatments to determine the effects of feeding different proportions of corn silage and ryegrass silage with supplemental ground corn (GC), steam-flaked corn (SFC), and hominy feed (HF) on the performance of lactating dairy cows. Forage provided 49% of the dietary dry matter in the experimental diets, which were formulated to meet National Research Council requirements. Ryegrass silage provided 100, 75, 50, or 25% of the total forage dry matter, with corn silage supplying the remainder. There were no interactions between the proportion of forage provided by ryegrass silage and energy supplement. Dry matter intake and milk protein percentage decreased linearly with increasing proportions of ryegrass silage, but milk protein yield was similar among forage treatments. There were no differences among forage treatments in milk yield, milk fat percentage and yield, and energy-corrected milk yield. Dry matter intake was higher and there was a tendency for increased milk fat percentage for GC compared with SFC or HF. No other differences were observed in milk yield or composition among energy supplements. Plasma urea nitrogen and glucose concentrations were similar among treatments. Under the conditions of this trial, our results indicate that feeding a combination of corn silage and ryegrass silage is more desirable than feeding ryegrass silage alone, whereas supplementation with GC, SFC, or HF supports similar levels of milk production.     

Effects of tallow in diets based on corn silage or alfalfa silage on digestion and nutrient use by lactating dairy cows

Six multiparous Holstein cows (average 31 days in milk; 36.3 kg/d of milk) fitted with ruminal cannulas were used in a 6 x 6 Latin square with 21-d periods to investigate the effects of diets that varied in forage source and amount of supplemental tallow. Isonitrogenous diets in a 2 x 3 factorial arrangement were based on either high corn silage (40:10 corn silage to alfalfa silage, % of dry matter) or high alfalfa silage (10:40 corn silage to alfalfa silage, % of dry matter) and contained 0, 2, or 4% tallow. Intakes of dry matter and total fatty acids were lower when cows were fed the high corn silage diet. Tallow supplementation linearly decreased dry matter intake. Milk yield was unaffected by diet; yields of milk fat and 3.5% fat-corrected milk were higher for the high alfalfa silage diet but were unaffected by tallow. Milk fat percentage was higher for the high alfalfa silage and tended to decrease when tallow was added to the high corn silage diet. Contents of trans-C18:1 isomers in milk fat were increased by high corn silage and tallow, and tended to be increased more when tallow was fed in the high corn silage diet. Ruminal pH and acetate:propionate were lower when high corn silage was fed. Ruminal acetate:propionate decreased linearly as tallow increased; the molar proportion of acetate was decreased more when tallow was added to the high corn silage diet. Ruminal liquid dilution rates were higher for the alfalfa silage diet; ruminal volume and solid passage rates were similar among diets. Total tract apparent digestibilities of dry matter, organic matter, crude protein, starch, energy, and total fatty acids were unaffected by diet. Digestibilities of neutral detergent fiber, acid detergent fiber, hemicellulose, and cellulose were lower when high corn silage was fed. The high alfalfa silage diet increased intakes of metabolizable energy and N, and increased milk energy and productive N. Tallow decreased the amount of N absorbed but had few other effects on utilization of energy or N. Tallow linearly increased concentrations of nonesterified fatty acids and cholesterol in plasma; cholesterol was increased by high alfalfa silage. Overall, forage source had more pronounced effects on production and metabolism than did tallow supplementation. Few interactions between forage source and tallow supplementation were detected except that ruminal fermentation and milk fat content were affected more negatively when tallow was fed in the high corn silage diet.     

A novel process for microencapsulation of fish oil with barley protein

This research represents the first scientific publication reporting barley protein as a wall material to encapsulate fish oil. Solid microcapsules were able to form in aqueous solution by pre-emulsifying barley proteins with a homogenizer followed a microfluidizer system. No organic solvent or cross-linking reagents were used in the preparation process. The wet status microcapsules were converted into free-flowing powder (dry status microcapsules) by a spray-drying process. The optimum microcapsule preparation conditions were 15% protein, oil/protein ratio = 1.0, and an inlet temperature of 150 °C. These microcapsules exhibited high encapsulation efficiency (EE: 92.9–100.2%), loading efficiency (LE: 46.5–50.1%) and low moisture content (0.75–0.90%). The oxidative stability of microencapsulated fish oil was tested at both dry status and in aqueous solutions (pH 7.0 and pH 2.0) in an accelerated storage test (40 °C, 8 weeks). These barley protein microcapsules possessed a strong ability to protect fish oil against oxidation, making them ideally suited for use in liquid/semi-liquid food systems. Food formulation tests confirmed their successful application in milk and yogurt for their respective shelf lives.     

Ozonated cotton stalks as a silage additive: Fermentation data on lucerne with particular reference to protein degradation

Fermentation patterns of lucerne silages were studied in laboratory silos. The treatments consisted of: (a) fresh (200 g kg>^?l DM) lucerne, ensiled without any treatment (L); (b) lucerne wilted to 525 g kg^?1 DM prior to ensilage (WL); (c) fresh lucerne + cotton stalks at a ratio of 60:40 on a dry matter (DM) basis (L + CS); and (d) fresh lucerne + ozone-treated cotton stalks at the same ratio as above (L+O₃). Silos were opened after 90 days and the silages analysed. The highest DM loss was found in the L silage (14·7%), whereas in the L+O₃ silage DM loss was practically nil. Both wilting and the addition of untreated cotton stalks proved to be effective in reducing DM losses during fermentation. The production of lactic acid and volatile organic acids in the L+ O₃ and WL silages was lower than in the L and L+CS silages. The poorest ability to preserve forage protein was found in the L silage, in which only 28 % of the protein was recovered after 90 days. The greatest ability to preserve protein was found in the L+O₃ silage, in which 78 % of the protein was maintained. Ammonia production followed generally similar patterns. Amino acids underwent extensive degradation in the L silage. Recovery of amino acids in the WL silage was in the range 69–93 %, and in the L + O₃ silage it was almost complete. Ozonated CS proved to be a good silage additive with respect to energy and protein preservation. Its future use in the field would allow direct ensilage of fresh leguminous material immediately after harvest, producing a high quality silage.     

Laboratory evaluation of previously fermented juice as a fermentation stimulant for lucerne silage

The potential use of forage juice anaerobically stored for 2 days (previously fermented juice, PFJ) as a silage additive was investigated. Lucerne (450 g) was ensiled in laboratory‐scale silos (900 ml capacity) with or without sucrose (20 g kg⁻¹) or PFJ (2.5 ml kg⁻¹). Silages treated with both additives were also made and fermentation quality, in vitro dry matter digestibility and in vitro ruminal NH₃ production were determined. The control silage showed very poor quality with high butyrate and NH₃‐N concentrations. Addition of sucrose significantly increased lactate but failed to inhibit the deterioration by clostridial fermentation. Use of PFJ further increased lactate and greatly reduced butyrate and NH₃‐N production during storage. The combination of sucrose and PFJ was more effective in improving fermentation than either additive on its own. In vitro digestibility appeared to be higher when lucerne was well preserved. Ruminal NH₃ production in vitro was decreased in silages with sucrose and PFJ added at ensiling. This result indicates that use of PFJ can be a promising way to stimulate lactate production in some difficult direct cut silage. The potential of improving fermentation may be comparable to conventional sugar additives. © 1999 Society of Chemical Industry     

Cellulose acetate,a new candidate feed supplement for ruminant animals: In vitro evaluations

Cellulose acetate (CA), a derivative of cellulose in which some hydroxyl groups are substituted with acetyl groups, was evaluated as a new cellulosic feed source for ruminants. In the present work, a series of in vitro studies was carried out to determine how CA supplementation affects rumen fermentation and microbiota. Batch culture studies were conducted to select the type of CA suitable for feed use and to define the optimal supplementation level. Rumen fluid from 2 Holstein cows was mixed with McDougall's buffer in test tubes into which grass hay and concentrate containing a fiber source [cellulose (control), water-soluble CA (WSCA), or insoluble CA] had been placed. Each fiber source was supplemented at 10% of total substrate. Tubes were incubated for 24 h to determine fermentation and microbial parameters. Then, the dose response of these parameters to different supplementation levels of WSCA (0, 7.5, 15, 22.5, and 30%) was tested in the same manner. We also operated a continuous culture system with WSCA supplementation and evaluated the effects on digestibility, fermentation, and microbial parameters. The supplementation level of WSCA was set at 15% of total feed. In batch culture studies, WSCA, but not insoluble CA, yielded dose-dependent increases in ruminal acetate levels. In the continuous culture system study, WSCA yielded increases in ruminal acetate levels and in the abundance of bacteria of the genus Prevotella, including Prevotella ruminicola. Dry matter digestibility and total gas production were not affected. These results suggest that WSCA supplementation at 15% of total feed yielded increased acetate levels without negatively affecting feed digestion; these effects may reflect activation of Prevotella species. As ruminal acetate is involved in milk fat synthesis, WSCA can be considered as a candidate feed additive suitable for dairy cattle.     

Evaluation of the potential use of discard species for fish silage and assessment of its oils for human consumption

Waste or by-products contains a substantial amount of nutrients. Fish silage is an alternative way for assessment of fish discards and waste generated in the fish processing industries. The aim of the study was to evaluate the potential use of discard species (Equulites klunzingeri, marine fish and Carassius gibelio, freshwater fish) for fish silage and chemical quality of fish oil extracted from fish silages prepared with acid and fermented methods. Lipid quality of fish oils extracted from fish silages with LAB strains was evaluated in terms of thiobarbituric acid (TBA), peroxide value (PV), p-Anisidine (AV), totox values and fatty acid composition. The results showed that PV, TBA, AV and totox values of all extracted fish oils were within the acceptable limits for edible oils. Recovered fish oils from silage contained high level of polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), serving as a source of good quality fish oil for human consumption.     

Protein nutrition of dairy cows receiving grass silage diets: Effects of feeding a protein supplement of unbalanced amino acid composition

Eight cows were used in a replicated 4 × 4 latin square design experiment with period lengths of 3 weeks to investigate the effects of protein supplementation of a grass silage diet on silage intake and milk production. The four treatments were (i) the basal diet, consisting of grass silage ad libitum plus 2 kg day^?1 of molassed sugar beet pulp; (ii) basal diet plus 1 kg day^?1 of fish meal (F): (iii) basal diet plus 0.8 kg day^?1 of feather meal 1 (FE1); and (iv) basal diet plus 0.8 kg day^?1 of feather meal 2 (FE2). Silage intake was significantly (P < 0.01) increased by the F supplement but was not affected by the FE supplements; values were 9.9, 10.6, 9.6 and 9.6 kg DM day^?1 for basal, F, FE1 and FE2, respectively. Relative to the basal treatment, F increased (P < 0.001) the yield of milk and milk protein but, again, these were unaffected by FE; values were, for milk yield, 13.6, 16.3, 13.7 and 13.1 kg day^?1; and, for milk protein yield, 420, 545, 429 and 414 g day^?1 for basal, F, FE1 and FE2, respectively. However, supplementation with FE produced increases in the concentration (P < 0.001) and yield (P < 0.05) of milk fat; values were 42.8, 39.9, 46.6 and 47.8 g kg^?1 and 575, 646, 628 and 618 g day^?1 respectively, for the basal, F, FE1 and FE2 treatments. The profile of amino acids in blood plasma was characterised by markedly lower (at least P < 0.01) concentrations of methionine, lysine, tryptophan and histidine for the diets containing FE compared with F. The results show pronounced effects of the amino acid composition of the undegraded protein component of the supplement on both silage intake and milk production but also draw attention to a possible relationship between amino balance and the secretion of milk fat.     

Enhancing in vitro degradation of alfalfa hay and corn silage using feed enzymes

A series of in vitro fermentation experiments was performed to assess the effects of 4 feed enzyme products (FE) that varied in enzymatic activities on the degradation of alfalfa hay and corn silage. The FE contained a range of endoglucanase, exoglucanase, xylanase, and protease activities, and a range of dose rates (DR) was used. The objective of the study was to identify effective formulations and optimum DR, and to establish if combining FE would further improve fiber degradation. For alfalfa hay, quadratic increases in gas production and degradation of dry matter (DM) and fiber were observed for all FE, with maximum responses at low to medium DR. For corn silage, none of the FE increased gas production or DM degradation, but all FE increased NDF degradation, with optimum DR in the low to medium range. The proteolytic enzyme papain improved fiber degradation of alfalfa hay and corn silage in a manner similar to that observed for polysaccharidase FE. Among the polysaccharidase FE, added activities of endoglucanase and exoglucanase were positively correlated with improvement in neutral detergent fiber (NDF) degradability of corn silage, whereas only added endoglucanase activity tended to be correlated with improvement in NDF degradability of alfalfa hay. Combining effective polysaccharidase FE further improved fiber degradation of both forages, with greater improvements for corn silage. Combining polysaccharidase and proteolytic FE further improved NDF degradation of corn silage, but not alfalfa hay. Combination treatments generally resulted in additive effects with increases in fiber degradation equal to the sum of the improvements for the individual enzyme components. Improved fiber degradation of corn silage was associated with decreased acetate to propionate ratios. Enzyme products that improve in vitro degradation of forages may have the potential to improve lactational performance of dairy cows.     

Determination of the first-limiting amino acid for milk production in dairy cows consuming a diet of grass silage and a cereal-based supplement containing feather meal

Lactating dairy cows consuming a diet of grass silage and a cereal-based supplement containing feather meal were given intravenous infusions of amino acids to determine the first-limiting amino acid for milk production, methionine having been shown to be not-limiting in a previous experiment. The three infusion treatments were a mixture of methionine, lysine, histidine and tryptophan (4AA); the mixture without lysine (-Lys); and the mixture without histidine (-His). The 4AA treatment markedly increased the yield of milk protein by about 18% and this response was not diminished by omission of lysine. However, exclusion of histidine produced no response over basal, confirming histidine as the first-limiting amino acid. In a second experiment, lactating cows receiving a similar basal diet were used to examine the effects on milk production of progressively substituting avian blood meal (rich in histidine and poor in methionine) for part of the feather meal. Blood meal was substituted for 0, 0.10, 0.20 and 0.40 of the feather meal in the supplement. The yield of milk protein was increased by about 15% by the first level of inclusion of blood meal, but there was no further response beyond the first level of inclusion. The results of the feeding trial confirm that dietary addition of protein rich in histidine and of low ruminal degradability substantially increased milk production with this basal diet, although it should be noted that the calculated supply of all the essential amino acids were also increased, by varying degrees, by substitution of blood meal. The results of the two experiments are discussed in relation to the likely importance of histidine as a limiting amino acid in dairy cows consuming diets typical of those used in practice. © 1999 Society of Chemical Industry     

Substitution of fish silage protein and a free amino acid mixture for fish meal protein in a chicken diet

White Leghorn chickens were fed a basal diet with about 550 g kg^?1 of the protein from fish meal. Graded amounts of fish silage protein or an amino acid mixture simulating fish meal protein were substituted for parts of the dietary fish meal protein. In two experiments 150 and 300 g kg^?1 of fish meal protein were replaced by fish silage protein or by an amino acid mixture simulating fish meal. In one experiment graded amounts of 0, 50, 100, 200, 300 and 400 g kg^?1 of fish meal protein were replaced by fish silage protein. Growth and feed efficiency were the same or better when some of the protein was from fish silage. The results showed that replacement of dietary fish meal with fish silage did not reduce the dietary quality of the feed for young growing chickens. It is concluded that it might be advantageous to substitute fish silage for some of the fish meal under the experimental conditions used.     

Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 10⁵ cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 10⁶ cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 10⁶ cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = ?0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = ?0.51), Pantoea (r = ?0.46), Pseudomonas (r = ?0.45), and Stenotrophomonas (r = ?0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = ?0.59), Sphingomonas (r = ?0.66), Pantoea (r = ?0.69), Pseudomonas (r = ?0.69), and Stenotrophomonas (r = ?0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.     

The effect of dietary starch on the digestion by sheep of cell wall monosaccharide residues in maize silage

The digestibility of individual cell wall monosaccharide residues from maize silage (MS) and from maize silage + concentrate (MS+con) diets, was examined in sheep equipped with rumen and duodenal cannulas. Dry matter digestibility was 73.8 and 86.6% in the MS and MS+con treatments, respectively. The overall digestibility of cell walls (CW) and CW monosaccharide residues was not depressed in the MS+con treatment. The values for total CW, CW glucose, CW uronic acid and for total xylose were 68.3, 77.0, 71.6 and 65.2 for MS and 69.4, 80.3, 75.3 and 63.7 for MS+con, respectively. However, the digestion of CW and CW monosaccharide residues in the rumen of the MS+con sheep was reduced and the contribution of the lower tract to the digestion of the potentially digestible CW-sugar residues was in the range of 15 to 20%. Digestibility of the soluble uronic acid was adversely affected by the concentrate, its digestibility being reduced from 88.6% in the MS to 68.7% in the MS+con treatment. Based on compositional considerations, it is envisaged that the CW structural unit of the ensiled maize plant is made of cellulose fibres enveloped by a thin, poorly lignified and penetrable matrix. The degree of branching of the matrix polysaccharides is low compared with other forage plants. It is suggested that the above mentioned properties significantly contribute to the high quality of the ensiled maize plant.     

Effect of feeding a corn hybrid selected for leafiness as silage or grain to lactating dairy cattle

A leafy corn hybrid was compared to a grain corn hybrid as silage and high moisture grain to evaluate dry matter intake, milk yield, and milk composition. Sixteen multiparous Holstein cows averaging 97 DIM were used in a feeding trial based on 4 x 4 Latin squares with 21-d periods. Each of four diets contained (dry basis) 8% chopped hay, 42% corn silage, 11% high moisture corn grain, 10% whole, fuzzy cottonseed, and 29% protein concentrate. One diet used leafy corn as both high moisture grain and silage. A second diet contained grain corn hybrid (control) as both high moisture grain and silage. A third diet contained leafy corn for high moisture grain and control corn for silage and the fourth diet used control corn for high moisture grain and leafy corn for silage. Cows fed diets containing leafy silage produced more milk and milk protein and ate more DM than cows fed control silage. The corn hybrid used for high moisture grain did not influence milk yield or composition. Dry matter intake was greater for cows fed the diet containing both leafy high moisture grain and leafy silage than for cows fed both control high moisture grain and control silage, but milk yield and composition were not different. When fed as silage, the leafy corn hybrid used in this experiment supported greater DMI as well as higher milk and protein yields when compared to the grain corn hybrid.     

Variability in the ranking of the three most‐limiting amino acids for milk protein production in dairy cows consuming grass silage and a cereal‐based supplement containing feather meal

Previous experiments showed that histidine was clearly first‐limiting for milk protein production in cows consuming grass silage and a cereal‐based supplement containing feather meal. Two experiments were carried out to investigate further the responses of milk production to the intravenous infusion of amino acids in dairy cows consuming this basal diet. In Experiment 1, four cows received the following treatments: (1) the basal diet (Basal); (2) Basal plus jugular infusion of 114 g d⁻¹ of essential amino acids containing 6 g d⁻¹ of histidine (EAA); (3) Basal plus histidine, methionine and lysine in the amounts supplied by EAA (3AA); and (4) Basal plus 6 g d⁻¹ of histidine (His). The EAA and 3AA treatments increased (P < 0.05) the yield of milk protein by 106 g d⁻¹; the corresponding increase (P < 0.05) for the His treatment was 60 g d⁻¹. This result confirmed those of earlier experiments in showing histidine to be clearly first‐limiting. In Experiment 2, five cows received the following treatments: (1) basal diet (Basal); (2) Basal plus jugular infusion of (g d⁻¹) histidine, 9.0, methionine, 10.0, lysine, 25.5, and tryptophan, 4.8 (4AA); (3) Basal plus 4AA minus methionine (−Met); (4) Basal plus 4AA minus lysine (−Lys); and (5) Basal plus 4AA minus tryptophan (−Trp). Milk protein yield was increased (P < 0.05) only by the 4AA and −Trp treatments; the increases were 101 and 126 g d⁻¹ respectively. These results indicate that methionine and lysine were also limiting. Taking the results of these experiments, together with those published previously, suggests that, despite all reasonable attempts to maintain a constant diet composition, the ranking of the three most‐limiting amino acids varied between experiments. © 2000 Society of Chemical Industry