The ability of the enzyme-linked immunosorbent assay to detect antibody against Staphylococcus aureus in milk following experimental intramammary infection

Changes in the milk antibody levels against Staphylococcus aureus were measured at the start of an experimental intramammary instillation of either S. aureus (Study I) or Staphylococcus hyicus (Study II). A commercial enzyme-linked immunosorbent assay system was used. Twenty-one Holstein cows were enrolled in Study I and 15 Holstein cows were used in Study II. Pathogen instillation began 21 days before the start of the non-lactating period. Cows received intramammary antibiotic treatment in all quarters immediately after the last milking, the start of the non-lactating period. Lacteal secretions were collected before the start of the non-lactating period, and during the immediate postpartum period in both studies, and during the non-lactating period in Study I. Milk was cultured for mastitis pathogens and S. aureus antibody levels and somatic cell counts were determined from all samples. There was an approximate 2-week delay in the elevation in antibody levels in response to the instillation of S. aureus. Antibody levels remained elevated in cows with S. aureus intramammary infections postpartum, but were below threshold in cows where intramammary infections were cured during the non-lactating period. Antibody levels were elevated by S. hyicus intramammary infections, remained elevated for the first 12 days postpartum, but were below threshold by day 21 postpartum. Cows with incipient intramammary S. aureus infections might be misclassified as false negatives by the antibody test. However, results suggest that cows with S. hyicus intramammary infections that were not cured would not be misclassified if milk is withheld from test for the first 30 days postpartum, as recommended by the manufacturer of the test.     

Epidemiological Study of Non‐contagious Intramammary Infections in Nine Commercial Dairy Herds following a Staphylococcus aureus Control Programme

Changes in prevalence in intramammary infection, by pathogen type, in herds applying a stringent contagious mastitis control programme was studied. Enrollment of 1651 lactating cows and collection of milk samples was made in this ancillary study to a cohort study of the dynamics of mastitis prevalence after adoption of a strict contagious mastitis control programme that targeted the elimination of mastitis caused by Staphylococcus aureus. Nine commercial dairies in Italy were used. Aseptic collection of milk samples from all lactating cows was performed at the time of enrollment, from all cows within 7–14 days of entering the lactating herd after the date of enrollment, and from all lactating cows at 2, 4, 7, 10 and 14 months after the date of enrollment. Prevalence of intramammary infection by pathogen type was determined from culture of milk samples. Application of the strict contagious mastitis programme did not lead to an increased risk of non‐contagious mastitis. The risk of coliform, environmental streptococcal and coagulase‐negative staphylococcal intramammary infections decreased after adoption of the programme. The data reported herein indicate that the overall risk for any intramammary infections decreases with adoption of a strict contagious mastitis programme, and that such a programme therefore does not necessarily lead to an increase in environmental mastitis.     

Intramammary Infusion of β1,3‐glucan for Prevention and Treatment of Staphylococcus aureus Mastitis

Udder health problems associated with Staphylococcus aureus infections in dairy cows are difficult to control and antibiotics have limited effects. Lately, more interest has been directed towards ways to stimulate the innate immune mechanisms of the animal for better prevention and treatment of mastitis. The objectives of this study were to investigate if intramammary infusion at drying off with the immune modulator β1,3‐glucan can make the udder more resistant to experimental intra mammary S. aureus infection at this time, and to study if intramammary infusion of β1,3‐glucan into lactating udder quarters with chronic subclinical S. aureus infection can stimulate the clearing of the infection. Another aim was to evaluate the effect of β1,3‐glucan on the expression of major histocompatibility complex (MHC class II) on mammary leucocytes, measured by flow cytometry, during these circumstances. The results indicated a slight, but not statistically significant, positive effect of β1,3‐glucan at drying off on the clinical and anti‐bacterial response to S. aureus infection, but no therapeutic effect of β1,3‐glucan treatment of udder quarters with chronic subclinical S. aureus mastitis. However, the proportion of MHCII+ milk lymphocytes tended to increase after glucan infusion in those udder quarters indicating a stimulation of the antigen presenting ability. To further evaluate a possible preventive effect of β1,3‐glucan infusion at drying off more studies are needed involving a larger number of animals.     

Effect of Intramammary Injection of RbIL‐8 on Milk Levels of Somatic Cell Count,Chemiluminescence Activity and Shedding Patterns of Total Bacteria and S. aureus in Holstein Cows with Naturally Infected‐subclinical Mastitis

The effect of intramammary injection of recombinant bovine interleukin‐8 (rbIL‐8, 1 mg/10 ml of saline) on quarter milk levels of somatic cell count (SCC), chemiluminescence (CL) activity and counts of total bacteria and Staphylococcus aureus (S. aureus) was investigated, using 10 Holstein cows with an early stage or a late stage of subclinical mastitis naturally infected with S. aureus. In the late‐stage group, milk SCC and CL activity had significant rises with maximum levels at 6 h, following maintained high levels thereafter post‐cytokine injection. The counts in milk total bacteria and S. aureus were insignificantly decreased, being increased back on day 7 post‐cytokine injection. Thus, the cytokine was inefficient for the late‐stage subclinical mastitis. However, in the early‐stage group milk SCC and CL activity declined to under pre‐injection levels on day 7 after marked and significant rises at 6 h and day 1 post‐cytokine injection. The milk total bacterial count decreased significantly on days 0.25 and 2. Furthermore, the milk S. aureus count was decreased significantly on days 1, 2, 3 and 7 by the cytokine injection. These results suggest that the rbIL‐8 has a potential as a therapeutic agent of the subclinical mastitis of dairy cows, if the cytokine is applied at an initial stage of infection.     

Cheyletiella parasitivorax infestation of cats associated with skin lesions of man

Two dry-cow therapy products were evaluated in seven factory-supply dairy herds in the Waikato area. A product containing neomycin sulphate and the benzathine salt of penicillin (Neopen D.C. White; Smith-Biolab) was used in five herds, and one containing benzathine cloxacillin (Orbenin, Beecham) was used in two herds. Non-treated control cows were included in each herd. Both products were effective in eliminating intramammary infections with Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. Efficacy of dry-cow therapy against S. aureus was 83.8% and 85.2% respectively. Spontaneous cure rate among controls was 30.8% for S. aureus during the dry period. Spontaneous cure rate for Str. uberis was 50%, while dry-cow therapy eliminated 100% and 77.8%, respectively, for the two products. Dry-cow therapy with either product eliminated more than 90% of Str. agalactiae infections while spontaneous cure rate was only 28.6%. These results further support the effectiveness of dry-cow therapy in reducing the level of subclinical mastitis in dairy herds by shortening the duration of intramammary infections.     

Application of the California mastitis test in intramammary Streptococcus agalactiae and Staphylococcus aureus infections of camels (Camelus dromedarius) in Kenya

A study was conducted on 207 lactating camels in six herds in Kenya to evaluate the California mastitis test (CMT) for the detection of intramammary infections (IMIs) caused by Streptococcus agalactiae and Staphylococcus aureus and to investigate the prevalence of both the pathogens in the camel udder. IMI with S. agalactiae was found in 12% of all camels sampled. IMI with S. aureus was present in 11% of all camels sampled. The herd-level prevalence of IMI varied between 0 and 50% for S. agalactiae and between 0 and 13% for S. aureus. Longitudinal observations over 10–12 months confirmed persistent infections for both pathogens. Observations in one herd suggested that camel pox was a contributing factor in spreading and exacerbating S. agalactiae udder infections.The CMT had quarter-level sensitivities of 77 and 68% for S. agalactiae and S. aureus in camels, respectively. The CMT specificities were 91% for both the pathogens.     

Posology and Field Efficacy Study with Novobiocin for Intramammary Infusion in Nonlactating Dairy Cows

Four dose levels of novobiocin (50, 200, 400, 600 mg) were compared with no drug for the intramammary treatment of Staphylococcus aureus, Streptococcus agalactiae and other streptococcal infections present in the udder of dairy cows at the initiation of the dry period. Treatment success was evaluated by comparing the microbiological status of duplicate pretreatment quarter milk samples collected at drying off with the microbiological status of duplicate quarter milk samples collected four to ten days postcalving. Infection status of 1318 cows in 75 herds in five geographic locations was determined. Treatment effects on infected cows were evaluated by least squares analysis of variance with treatment, herd, lactation number, days dry and milk production at drying off considered as variables. The dose of 400 mg novobiocin per quarter was demonstrated to be significantly more effective (P < 0.05) than no drug and significantly better than (P < 0.05) or equal to the other doses for curing infections caused by S. aureus, S. agalactiae and other streptococci. A significant reduction (P < 0.05) in the overall rate of new udder infections acquired during the dry period was observed in cows treated with ≥ 200 mg novobiocin at drying off. The data supported the conclusion that the cow rather than the quarter is the appropriate experimental unit in the evaluation of intramammary mastitis treatments. Herd and lactation number were the most significant variables affecting cures.     

Pre-parturition staphylococcal mastitis in primiparous replacement goats: persistence over lactation and sources of infection

This investigation reported for the first time the occurrence of intramammary infections caused by Staphylococcus in primiparous replacement goats before parturition and the persistence of clinical Staphylococcus aureus infection during the lactation period. Subclinical infections, mainly caused by coagulase negative staphylococci (CoNS), did not persist during lactation. Genotyping analysis indicated that environment seems to play a moderate role as source of intramammary infections to goats before parturition, but causative agents of mastitis in lactating animals are not genotypically related to environmental staphylococci. The occurrence and persistence of intramammary infections in replacement goats demonstrate the need to consider those animals as potential sources of infections in dairy goat herds.     

Efficacy of extended intramammary ceftiofur therapy against mild to moderate clinical mastitis in Holstein dairy cows: A randomized clinical trial

Few studies have investigated the efficacy of extended ceftiofur therapy and none have focused on extended therapy for naturally occurring clinical mastitis. The objective of this study was to compare the efficacy of extended intramammary ceftiofur therapy of 8 d duration with a standard 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows. Holstein cows from 22 dairy herds (n = 241) were randomly allocated to the 2 treatment groups. For each case of mastitis, 125 mg of ceftiofur hydrochloride was administered intramammary once a day for 2 or 8 d. Clinical cure, 21 d after the last treatment, was 89% (98/110) in each group. Bacteriological cure 21 d after the last treatment for the 2- and 8-day regimens were 32% (15/47) and 61% (25/41), respectively, for all bacteria (P = 0.007), 64% (9/14) and 82% (9/11), respectively, for streptococci (P = 0.50), and 0% (0/20) and 47% (9/19), respectively, for Staphylococcus aureus (P = 0.0004). There were no statistical differences between groups for new intramammary infections. Overall, ceftiofur extended therapy increased cure when compared to a 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows.     

Inflammatory and metabolic responses to an intramammary lipopolysaccharide challenge in early lactating cows supplemented with conjugated linoleic acid

Supplementation of dairy cows with trans‐10, cis‐12 conjugated linoleic acid (CLA) allows nutrient repartitioning despite an energy deficiency in early lactation, which might be a benefit for the immune system, too. In this study, we investigated potential nutrient sparing effects of CLA in early lactating cows with low plasma glucose concentrations exposed to an intramammary lipopolysaccharide (LPS) challenge. Fifteen multiparous Holstein cows were exposed to an intramammary LPS challenge in week 4 p.p. Eight cows (CLA) were supplemented daily with 70 g of lipid‐encapsulated CLA (6.8 g trans‐10, cis‐12 and 6.6 g of the cis‐9, trans‐11 CLA isomer; CLA) and seven cows with 56 g of control fat (CON). Blood samples were obtained every 30 min along with rectal temperature, heart and respiratory rate, and milk samples were taken hourly until 10 hr after the LPS application. Plasma was analysed for concentrations of glucose, free fatty acids, beta‐hydroxybutyrate (BHB), cortisol, insulin and glucagon. In milk, somatic cell count and activity of lactate dehydrogenase were determined. Initial plasma glucose concentration was lower in CLA than in CON. During the immunostimulation, CLA had higher glucose concentrations than CON, and BHB decreased distinctly in CLA, whereas CON cows maintained BHB concentration at a lower level. Body temperature in CLA increased earlier, the difference between peak and basal temperature was higher, and the decline thereafter occurred earlier. In conclusion, CLA supplementation of early lactating cows exposed to an intramammary LPS challenge affected local and systemic immune responses. We assume that CLA supplementation triggered glycogen storage. Cows supplemented with CLA provided more glucose and preferentially used BHB as an energy source during the immune response. The more intense metabolic and more concentrated endocrine responses support an immunomodulatory effect of CLA supplementation.     

Effects of intramammary infusion of Bifidobacterium breve on mastitis pathogens and somatic cell response in quarters from dairy cows with chronic subclinical mastitis

The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis‐causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase‐negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 10⁴ cells/ml prior to B. breve infusion that decreased to < 30 × 10⁴ cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non‐antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.     

Efficacy of vaccination and antimicrobial treatment to eliminate chronic intramammary Staphylococcus aureus infections in dairy cattle

OBJECTIVE: To determine whether a combination of vaccination and extended intramammary antimicrobial treatment would eliminate chronic intramammary Staphylococcus aureus infections in lactating dairy cows. DESIGN: Randomized controlled clinical trial. ANIMALS: 50 dairy cows with chronic mastitis caused by S aureus. PROCEDURE: Cows were identified and paired within herd on the basis of days in milk, lactation number, milk production, and numbers of quarters infected. Treated cows (n=20) received 3 doses of a polyvalent S aureus bacterin on days 1, 15, and 21 of the study along with intramammary administration of pirlimycin in all 4 quarters once daily for 5 treatments (days 16 to 20). Control cows (n=23) received no treatment. Follow-up samples for bacteriologic culture were collected for at least 3 months after treatment to determine treatment success rates. RESULTS: Significantly more S aureus infections were eliminated from treated cows (8/20 [40%]), compared with control cows (2/23 [9%]). The proportion of infected quarters that yielded negative results throughout the follow-up period was also significantly higher in treated cows (13/28 [46%]) than in control cows (2/41 [5%]). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a combination of vaccination and antimicrobial treatment can be successful in eliminating some cases of chronic intramammary S aureus infections in dairy cattle. However, it is important to consider extended treatment protocols carefully because many cows are likely to remain infected with S aureus despite treatment and vaccination.     

Effect of nutrient levels during the far‐off period on postpartum productivity in dairy cows

The present study aimed to determine the effects of differing nutrient levels during the far‐off period on postpartum metabolism and milk production in lactating cows. Twenty‐six multiparous cows were assigned to three dietary treatments in the far‐off period: a low‐energy diet (L, n = 9, 80% intake of the total digestible nutrients requirement), a moderate‐energy diet (M, n = 8, 105%) and a high‐energy diet (H, n = 9, 130%). During the close‐up period, all cows were provided with 105% intake. After parturition, all cows were fed a lactation diet. The BCS recovery was slow, and low milk yield was found in the H group. In the L group, BCS recovery was favorable after parturition, and lactation persistence was increased. The L group had low rumen endotoxin activity and a high initial ovulation rate after parturition. These findings indicate that a high‐energy diet during the far‐off period has a deleterious effect on milk production. In contrast, the restricted diet in the far‐off period increased adaptability with respect to peri‐parturition metabolic changes, improved the post‐parturition nutritional state, and increased milk production. Furthermore, it suggests that the nutrient levels in the far‐off period affect rumen endotoxin activity and reproductive function after parturition.     

Effects of autogenous toxoid-bacterin in lactating cows with Staphylococcus aureus subclinical mastitis

To evaluate clinical effects of autogenous toxoid-bacterin treatment for Staphylococcus aureus subclinical mastitis in lactating cows, 22 cows which had at least one S. aureus infected quarter were selected from among cows at a S. aureus prevalent dairy farm. Eleven cows were injected with their own autogenous toxoid-bacterin and the others were maintained as non-injected control. In the toxoid-bacterin injected group, 27% of infected quarters were cured during the 12-week trial, compared to 5% in the control group. New intramammary infections with S. aureus were only detected in 3 quarters of the control group. Mean IgG antibody titer against S. aureus somatic antigens and alpha-toxin in serum and milk were significantly increased in the toxoid-bacterin injected group (p<0.05) and remained higher than those of the control group which showed no significant changes (p<0.05). In contrast to the control group, from 3 weeks after the second injection of the toxoid-bacterin injected group, mean S. aureus cfu/ml in milk samples from injected quarters with S. aureus was significantly decreased until the end of the study (p<0.05). In the toxoid-bacterin injected group, significant decreases of mean SCC were detected from milk samples from infected quarters with S. aureus from week 7 to week 10 (p<0.05). These data show that autogenous toxoid-bacterin treatment against S. aureus subclinical mastitis in lactating cows may increase the cure rate of the infections, reduce the severity of the infections and also prevent occurrence of the new infections.     

Microbiologic investigation of an epizootic of mastitis caused by Serratia marcescens in a dairy herd.

An epizootic of subclinical and clinical mastitis caused by Serratia marcescens was investigated in a 1,000-cow dairy farm in California. Serratia marcescens was isolated from 13 to 18% of composite milk samples obtained from lactating dairy cows. During monthly milk sampling performed during a 4-month period, S marcescens was isolated from 38.8 to 62.3% of composite milk samples obtained from cows from which S marcescens was previously isolated. Few cows infected with S marcescens had evidence of clinical mastitis. Somatic cell count value was associated with isolation of S marcescens. Cows with somatic cell counts greater than 500,000 were 5.48 times as likely to have intramammary infections with S marcescens, compared with cows with somatic cell count less than or equal to 500,000. Lactation number also was associated with S marcescens intramammary infection. After adjusting for the effect of lactation number, cows with high somatic cell count values were 2.98 times as likely to have intramammary infection with S marcescens, compared with cows with low somatic cell counts. Infection with S marcescens was independent of days in lactation, production string, and daily milk production. Eleven months after the beginning of the epizootic, S marcescens was isolated from organic bedding samples obtained from the dairy. Despite numerous attempts, other sources of S marcescens could not be identified on this dairy.     

Pathogen group specific risk factors at herd, heifer and quarter levels for intramammary infections in early lactating dairy heifers

Risk factors for intramammary infections caused by coagulase-negative staphylococci, contagious major pathogens and environmental major pathogens in early lactating heifers were evaluated at the herd, heifer and quarter levels. In total, 764 quarters of 191 dairy heifers in 20 randomly selected farms in Flanders (Belgium) were sampled. Quarter milk samples were collected between 1 and 4 days in milk and between 5 and 8 days in milk for bacteriological culture. Data were analyzed using multivariable, multilevel logistic regression analysis. Higher average herd milk somatic cell count (>200,000 cells/mL), not having an effective fly control strategy, contact with lactating cows prior to calving and moderate to severe udder edema prior to calving increased the odds of intramammary infections caused by contagious major pathogens. Poor heifer hygiene and lack of mineral/vitamin supplementation prior to calving were risk factors for intramammary infection caused by environmental major pathogens. Teat apex colonization with coagulase-negative staphylococci prior to calving seemed to protect quarters against intramammary infections caused by major pathogens. Poor heifer hygiene before calving, a non-clipped udder and not practicing of teat dipping prior to calving increased the odds of intramammary infection with coagulase-negative staphylococci. Although management is important in the prevention and control of intramammary infections in early lactating heifers, most variation in the prevalence of intramammary infections resided at the heifer and quarter levels, indicating that the susceptibility for intramammary infections around calving is mainly determined by heifer and quarter characteristics.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.     

Epidemiology of Streptococcus uberis Intramammary Infections in a Dairy Herd

From 1987 to 1991, almost 36 000 quarter samples of mammary secretion representing 1790 lactations of 510 dairy cows from a research herd were collected for bacteriological examination. The percentage of cows infected with Streptococcus uberis ranged from 12 to 16 % of cows/year. S. uberis was isolated from 14.2 % of lactations over the 5-year period. The prevalence of S. uberis intramammary infection (IMI) was significantly higher in cows with ≥4 lactations than in cows with 3 or fewer lactations. Regardless of lactation number, the prevalence of S. uberis was highest before parturition, during early lactation and near drying off. The prevalence of S. uberis infected quarters ranged from 1.3 to 2.3 % of quarters/year; the prevalence rate for the 5-year period was 2 % of quarters. The quarter prevalence of S. uberis was lowest in cows with ≤3 lactations, increased significantly with lactation number and was highest in cows with ≥6 lactations. The percentage of quarters infected with S. uberis varied significantly by year. The majority (95 %) of S. uberis IMI were subclinical. The ratio of subclinical IMI to clinical IMI was lowest during early lactation, and increased with days in milk, and with lactation age except for cows in their 5th and 6th lactations. Results of this epidemiological investigation suggest that opportunities exist where suitable control measures could be applied to reduce the impact of S. uberis infections in the dairy herd.     

Aspects of the epidemiology of bovine staphylococcal mastitis

Abstract

Strains of Staphylococcus aureus which are not susceptible to many antibiotics are now generally regarded as the most important pathogens in bovine mastitis. More effective methods for controlling the disease must, therefore, take into account recent advances in our knowledge of the epidemiology of the disease. These include information about the principal habitats of S. aureus in the host, the minimal numbers of organisms that may establish new udder infections, which animals act as reservoirs of infection in herds, and the transmission of infection in lactating and non-lactating cows. Such matters are discussed in relation to the application of mastitis control measures involving herd management, hygiene, antibiotic therapy, culling and milking machine maintenance.     

Effect of increased concentrate allotment before evening grazing on herbage intake,nitrogen utilization and rumen fermentation in dairy cows grazed on perennial ryegrass pasture

Two experiments were conducted to elucidate the effect of increased concentrate allotment before evening grazing on herbage intake, nitrogen utilization and rumen fermentation in dairy cows. In experiment 1, nine lactating cows were grazed in the morning and evening sessions (2.5 h each). The cows were allocated to treatments of three concentrate allotment levels before the evening grazing session by altering proportions to daily total offered; 25%, 50% and 75%. Daily herbage dry matter intake quadratically decreased with increasing the concentrate allotment levels (P < 0.05). Nitrogen utilization was similar among treatments. To investigate diurnal changes in rumen fermentation, a second experiment was conducted where six ruminally cannulated non‐lactating dairy cows grazed in the morning and evening sessions (3 h each) were subjected to the same treatments as experiment 1. Total volatile fatty acid concentration in the rumen linearly increased with increasing the concentrate allotment levels throughout the pre‐evening grazing session to the post‐morning grazing session (P < 0.01). The results indicate that dairy cows reduce daily herbage intake but do not alter nitrogen utilization with increasing concentrate allotment before evening grazing. © 2016 Japanese Society of Animal Science