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This study was conducted to investigate the relationship between serum free amino acids (FAA) and the onset of milk fever in dairy cows around the time of calving. The serum concentration of FAA was compared between multiparous cows with milk fever (MF group; n=7) and healthy multiparous cows (Control Group; N=16) kept on a Holstein dairy farm. Blood samples were obtained 3 to 4 weeks prepartum, on the day of calving, and 1 month post partum for analysis of serum amino acids and lipid parameters. The levels of all essential amino acids in the MF group were lower than those in the Control group. Significant differences between groups were observed in phenylalanine, valine, isoleucine, and leucine at 1 month post partum, and in tryptophan on the day of calving. Regarding the non essential amino acids, a significantly lower level of arginine was observed in the MF group compared to the Control group on the day of caving, as well as 1 month post partum. A significant positive correlation was detected between cholesterol and branched chain amino acids (BCAA) levels (r=0.531, P<0.01). These results showed that MF cows had lower levels in several FAA around the time of calving even before the onset of MF.

The vaccination against bacterial respiratory diseases in calves has been generally recognised as useful for the prevention of infections. Inducing an immunological response after vaccination is important for obtaining protection from infections. The aim of the study was to investigate the effects of probiotics on the immunological response to vaccination against bacterial respiratory diseases in young Japanese Black calves. Twenty-four Japanese Black calves were randomly divided into two groups (12 calves for the research group and 12 calves for the control group) on the seventh day of life (dol). The research group received 30 g per day of live bacteria mix consisting of and until the 63 dol. The control group did not receive the bacteria mix. All calves were vaccinated against bacterial respiratory diseases twice, at 21 and 42 dol. Blood samples were obtained from all calves at 7, 21, 42 (prior to the second vaccination), 45, 49 and 63 dol for determination of antibody titres, leukocyte numbers and cytokine genes. Lymphocyte counts, T cell (CD3 , CD4 and CD8 cell) counts and relative expressions of cytokine genes (interleukin (IL)-4 and IL-17A) at 45, 49 and 63 dol were significantly higher in the research group compared than in the control group. The addition of probiotics to young Japanese Black calves' feed promoted an immunological reaction to vaccination against bacterial respiratory diseases.

Early vaccination of cattle with an inactivated commercial bacterial vaccine against bovine respiratory disease has been reported to increase antibody production and can alleviate the disease. However, its dosage has been little investigated in young Holstein calves. This study addresses the need to establish guide values for vaccine dosage in these animals.Healthy calves received an inactivated vaccine for Histophilus somni, Pasteurella multocida and Mannheimia haemolytica intramuscularly at the ages of 1 and 4 weeks. Administered vaccine doses were 1.0 mL for the primary and booster vaccinations (1.0 + 1.0 group), 0.5 mL for the primary and 1.0 mL for the booster vaccination (0.5 + 1.0 group), or 0.5 mL for both vaccinations (0.5 + 0.5 group).Differences in the vaccine responses between the 1.0 + 1.0 group and 0.5 + 1.0 group were minor. However, the number of calves with a positive vaccine response to H. somni in the 0.5 + 0.5 group was less than half of that in the 1.0 + 1.0 and 0.5 + 1.0 groups. In logistic regression analysis, although the booster vaccination dose was positively correlated with seropositivity for H. somni, the primary vaccination dose was not correlated with vaccine response. The number of calves with positive vaccine responses to M. haemolytica was low even after booster vaccination regardless of the dose.The dose of 0.5 mL can be used for primary vaccinations in newborn Holstein calves, but 1.0 mL may be required for booster vaccinations.

Aim: This experiment was conducted to determine the effect of yeast cell wall (YCW) supplementation on production performances and blood biochemical indices such as liver enzyme activities, energy metabolites, and electrolyte concentrations of dairy cows in different lactation periods (LP). Materials and Methods: Thirty-two lactating Holstein cows were assigned into 2×2 factorial arrangement, in which the factors were the treatment (TM) (control [n=16] vs. YCW [n=16]) and the LP (early lactation [n=14] vs. mid-lactation [n=18]). The cows with day in milk (DIM) <120 (81±7 DIM) were defined as early lactating cows, whereas the cows with DIM >120 (179±5 DIM) were assumed as mid-lactating cows. The YCW (SafMannan; Phileo, Lesaffre Animal Care, France) was used as the dietary supplement (10 g/cow/day) in this experiment. The statistical analysis of the data was performed by the two-way analysis of variance using the general linear model procedure to determine the main effects (TM and LP) and their interaction (TM×LP) on production performances and blood biochemical parameters of experimental cows. Results: No significant effects (p>0.05) of YCW supplementation on production performances and blood biochemical indices of cows in TM groups (control vs. YCW) were observed; however, some obvious effects were detected in LP (early- and mid-lactation). Milk and milk component yield of cows in early lactation were significantly higher (p<0.05) than in mid-lactation, whereas somatic cell count and milk urea nitrogen were not different (p>0.05) with the YCW supplementation. The higher level (p<0.05) of serum albumin was found in mid-lactating cows after YCW supplementation. Before the experiment, the higher (p<0.05) non-esterified fatty acid (NEFA) and NEFA/total cholesterol (T-Cho) ratio, and the lower (p<0.05) calcium (Ca) concentration were observed in early lactating cows comparison with mid-lactating cows; however, there were not different after YCW supplementation. Conclusion: The positive effects of YCW supplementation on milk and milk component yields, energy metabolite, especially NEFA and NEFA/T-Cho ratio and Ca concentration were observed in early lactating cows rather than mid-lactating cows.

The objective of this research was to evaluate the antibody response to multiple doses of an inactivated mixed vaccine against , , and , and to investigate the influence of age at time of vaccination in the field. Healthy female Holstein calves received the vaccine at the age of 5-12 days and 2, 3, or 4 weeks later in the first experiment or at 1, 2, or 3 weeks of age and 4 weeks later in the second. Blood samples were collected at each vaccination and 3 weeks after the booster dose. Based on the antibody titres after the vaccinations, calves were divided into positive and negative groups for each of the bacteria. Calves in the control group were vaccinated only once at the age of 19-26 days. Antibody titres against and were significantly increased by the booster. After the second vaccinations, the titres against each bacterium were higher than those of the control group, and the -positive percentage in calves with high maternal antibody levels (MAL) exceeded that in calves with low MAL. In the first experiment, a majority of the -positive calves tended to have received the primary dose at seven days of age or older. A booster dose of the inactivated bacterial vaccine in young Holstein calves increased antibody production and overcame the maternal antibodies. Calves should be vaccinated first at seven days of age or older.

The conjunctiva-associated lymphoid tissue (CALT) has been used as a target site for mucosal vaccinations in several animals. In this study, we compared the morphological features of CALT in the eyelid and third eyelid between Holstein calves and adult cows. In the eyelids, CALTs in the form of diffused lymphoid tissue (DLT) and lymphatic follicles (LF) were observed, where DLTs were dominant and LFs were scarce. The CALTs of cows comprised T-, B-cells, macrophages, and antigen-presenting cells (APCs). In particular, B-cells were dominant except in the eyelids of the calves. The epithelial layer covering the CALT is often discontinuous and lacks goblet cells. Cytokeratin18 is strongly expressed in the epithelial layer covering the CALT, except in the third eyelids of adult cows. IgA-positive cells were diffusely distributed in the lamina propria of the conjunctiva of the eyelids and third eyelids. The eyelid CALT area in calves was lower than that in adult cows. Furthermore, the CALT of calves had a lower cellularity of B-cells and a higher cellularity of macrophages than that of adult cows. These histological characteristics indicate that CALT plays a role in the mucosal immune-inductive and effector sites. Furthermore, lower cellularity of B-cells in the CALT of calves indicates that the function of CALT as a mucosal immune induction site is less developed in calves than in adult cows.

Bovine respiratory disease (BRD) is one of the primary causes of death in young calves. Vaccination against infection by the common bacteria causing BRD is possible; however, the physical condition of the young calves that enables antibody production when stimulated by early immunisation remains to be elucidated. Healthy young female Holstein calves on a commercial dairy farm were fed a colostrum replacer and administered primary and booster immunisations with an inactivated vaccine against the bacterial pneumonia agents , and . At each immunisation, the body weight and height at the withers were measured and the body mass index (BMI) was calculated. Blood was sampled immediately before immunisation and 3 weeks following the booster. The calves were divided into positive and negative groups based on the antibody titre at the final blood sampling. Maternal antibody titres at the primary immunisation and BMI, nutritional status and oxidative stress at both immunisations were compared between the two groups. Antibody titre at the primary and BMI at both immunisations were significantly higher in the positive than in the negative group (P < 0.05). Additionally, serum gamma globulin was significantly higher in the positive group (P < 0.05), indicating a strong correlation between maternal antibody and serum gamma globulin levels. Elevated maternal antibody titre and higher BMI are positive factors for successful early immunisation, for which suitable colostrum may also be fundamental in young calves administered inactivated vaccines.

Lactoferrin (Lf) and transferrin (Tf) are iron-binding proteins that can bind various metal ions. This study demonstrates the heme-binding activity of bovine Lf and Tf using biotinylated hemin. When both proteins were coated on separate plate wells, each directly bound biotinylated hemin. On the other hand, when biotinylated hemin was immobilized on an avidin-coated plate, soluble native Lf bound to the immobilized biotinylated hemin whereas native Tf did not, suggesting that a conformational change triggered by coating on the plate allows the binding of denatured Tf with hemin. Incubation of Lf with hemin-agarose resulted in negligible binding of Lf with biotinylated hemin. Lf in bovine milk also bound to immobilized biotinylated hemin. These results demonstrate that bovine Lf has specific heme-binding activity, which is different from Tf, suggesting that either Tf lost heme-binding activity during its evolution or that Lf evolved heme-binding activity from its Tf ancestral gene. Additionally, Lf in bovine milk may bind heme directly, but may also bind heme indirectly by interaction with other milk iron- and/or heme-binding proteins.

The expressions of cytokines mRNA, including interleukin-4 (IL-4), interleukin- 17A (IL-17A) and interferon-gamma (IFN-γ), their master regulatory transcription factors, and signal transducers and activator of transcription (STAT) stimulated in vitro with Pasteurella (P.) multocida soluble antigen were examined in peripheral blood mononuclear cells (PBMC) from Holstein calves. The healthy Holstein calves were divided into three groups; 2 weeks old (2W Group, N=8), 6 weeks old (6W Group, N=8), and 10 weeks old (10W Group, N=8). PBMC were stimulated in vitro by soluble antigen of P. multocida. There were significantly lower expressions of IFN-γ, IL-4, and STAT-6 mRNA of PBMC stimulated with P. multocida soluble antigen in the 2W Group compared to that in the 10W Group. Expression of IL-17A and IFN-γ in PBMC stimulated with P. multocida soluble antigen were significantly higher compared with the PBMC without stimulation in the 6W groups. The results of the present study demonstrated that 2W old calves had decreased cytokine expression of PBMC when in vitro stimulated with P. multocida soluble antigen in vitro.

The characteristics of immune factors in somatic cells from lactating dairy cows and their association with commensal bacteria in normal milk have not been clarified. This study investigated the relationship between the pathogenic bacteria in milk and somatic cell immune factors in healthy lactating cows. In total 44 healthy Holstein cows were studied on one farm. Milk samples were collected aseptically using a cannula and these samples were cultured for detection of bacteria and analysis of mRNA of immune factors expressed by somatic cells. Cows were divided into two groups based on the microbial status of their milk samples: 12 cows showed bacteria in cultures (positive group), and the other 32 cows did not (negative group). The mRNA levels of IL-6, lactotransferrin, and cathelicidin expressed by somatic cells after milking decreased significantly compared to those before milking in both groups (P < 0.05). There were significantly lower mRNA levels of IL-6 and cathelicidin in the positive group compared to those in the negative group before milking. These results suggest that mRNA levels of IL-6 and cathelicidin expressed by the somatic cells may be affected by the presence of bacteria in healthy lactating dairy cows.