Blood 25-hydroxyvitamin D3 concentration of Japanese Black cattle at two farms with different rearing environments, Kagoshima prefecture, Japan.

Forty-two Japanese Black cattle on two farms in Kagoshima prefecture, Japan were used in this study. The rearing style of farm A was in a dark barn with a large roof to block sunlight (n=21). The rearing style of farm B was grazing, and exposed to direct sunlight (n=21). Blood sampling was performed twice on the same cattle in August 2022 (summer) and February 2023 (winter). As the results, the serum 25(OH)D3 concentrations were significantly lower in cattle of farm A than in cattle of farm B (P<0.01), and were significantly lower in winter season than in summer season (P<0.01). These results showed that there were differences in blood 25(OH)D3 concentrations between the farms or seasons.

Concentrations of antimicrobial components in milk of Japanese Black cows and their differences from dairy cows.

The components of milk from beef cows remain to be elucidated. This study examined the differences in the antimicrobial components of milk between dairy and beef cows. Quarter milk was collected from both Japanese Black (beef type) and Holstein (dairy type) cows to compare the concentrations of antimicrobial components. The concentration of lingual antimicrobial peptide (LAP) was higher, whereas that of the other antimicrobial components (lactoferrin, S100A7, and S100A8) was lower in beef cows than in dairy cows. Overall, these results indicate that the differences in antimicrobial components between beef and dairy cows may be associated with the difference in the prevalence of mastitis between them.

1,25-Dihydroxyvitamin D3 potentiates the innate immune response of peripheral blood mononuclear cells from Japanese Black cattle.

1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ), a bioactive vitamin D, is known to regulate immune responses in mammals. However, its impact on the innate immune responses of Japanese Black cattle, which are beef cattle endemic to Japan, remains unknown. Thus, in this study, we investigated the effect of 1,25(OH)2 D3 on the immune responses of peripheral blood mononuclear cells from Japanese Black cattle. As a result, the treatment of 1,25(OH)2 D3 upregulated the expression of antibacterial peptides, bovine beta-defensin 10 (DEFB10), and lingual antimicrobial peptide (LAP), in the presence and absence of lipopolysaccharide (LPS) stimulation. Moreover, 1,25(OH)2 D3 enhanced the inflammatory responses, including C-X-C motif ligand 8 (CXCL8) and nitric oxide synthase (NOS2), while reducing the expression of anti-inflammatory cytokine IL10, leading to an inflammatory phenotype. However, in contrast to humans and mice, 1,25(OH)2 D3 did not alter the expression of tumor necrosis factor (TNF) and downregulated triggering receptor expressed on myeloid cell 1 (TREM1) with LPS treatment. These results suggest that 1,25(OH)2 D3 potentiates the innate immune responses of Japanese Black cattle, albeit with different effects and mechanisms as compared to humans and mice.

Evaluation of antibody response to inactivated bovine ephemeral fever virus vaccine for Japanese Black calves in the field.

The purpose of this study was to investigate the antibody response to the bovine ephemeral fever virus (BEFV) vaccine in Japanese Black calves. Twenty-eight Japanese Black calves, which were raised on an ordinal farm, were divided into two groups. Fifteen calves received the inactivated BEFV vaccine at 12 and 16 weeks of age (vaccination group), and 13 calves did not receive the vaccine (non-vaccination group). Blood samples were obtained at 0, 4, 8, 12, 16, 20, 24, 28, and 32 weeks of age. As the results, in the vaccination group, the antibody titers at 16, 20, 24, 28, and 32 weeks of age were significantly higher than those at 0, 4, 8, and 12 weeks of age (p < 0.01). Additionally, antibody titer in the vaccination group increased after 16 weeks of age and showed a significantly higher level than that in the non-vaccination group throughout the remaining experimental period (p < 0.01). These results might be helpful in establishing a vaccination program against BEFV in calves.

A field study on the effects of inactivated bacteria vaccine for respiratory diseases in Japanese Black calves.

This study evaluated the effects of vaccination for Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in young Japanese Black calves at an ordinal farm, where respiratory diseases frequently occur at a young age. In total, 105 calves were divided into the vaccination group (n = 52), which received inactivated combined vaccine at 0 and 2 weeks of age, and the control group (n = 53), which received no vaccine. From both groups, eight calves each were randomly selected to determine the antibody titers. And, the incidence of respiratory disease and medical costs (treatment plus vaccination costs) were recorded for each group from birth to 16 weeks of age. In the vaccination group, the antibody titers against P. multocida, M. haemolytica, and H. somni were significantly higher than those in the control group after 8, 12, and 4 weeks of age, respectively (p < 0.05). The incidence of respiratory disease was significantly lower in the vaccination group compared to the control group (p < 0.01), and the medical costs per calf in the vaccination group were 46.4% lower than in the control group. These results might contribute to establishing an effective vaccination program against respiratory diseases in calves at each farm.

DNA Methylation of Genes Participating in Hepatic Metabolisms and Function in Fetal Calf Liver Is Altered by Maternal Undernutrition during Gestation.

This study aimed to elucidate the effects of maternal undernutrition (MUN) on epigenetic modification of hepatic genes in Japanese Black fetal calves during gestation. Using a previously established experimental design feeding the dams with 60% (LN) or 120% (HN) of their global nutritional requirements during the 8.5-month gestational period, DNA methylation in the fetal liver was analyzed with reduced representation bisulfite sequencing (RRBS). The promoters and gene bodies in the LN fetuses were hypomethylated compared to HN fetuses. Pathway analysis showed that the genes with DMR in the exon/intron in the LN group were associated with pathways involved in Cushing syndrome, gastric acid secretion, and aldosterone synthesis and secretion. Promoter hypomethylation in the LN group was frequently observed in genes participating in various signaling pathways (thyroid hormone, Ras/Rap1, PIK3-Akt, cAMP), fatty acid metabolism, and cholesterol metabolism. The promoter hypomethylated genes ALPL and GNAS were upregulated in the LN group, whereas the promoter hypermethylated genes GRB10 and POR were downregulated. The intron/exon hypomethylated genes IGF2, IGF2R, ACAD8, TAT, RARB, PINK1, and SOAT2 were downregulated, whereas the hypermethylated genes IGF2BP2, NOS3, and NR2F1 were upregulated. Collectively, MUN alters the promoter and gene body methylation of genes associated with hepatic metabolisms (energy, cholesterol, mitochondria) and function, suggesting an impact of altered gene methylation on the dysregulation of gene expression in the fetal liver.

Effects of Vitamin E Supplementation on Antioxidant, Inflammatory Biomarker, and Cell Viability of Peripheral Blood Mononuclear Cells from Japanese Black Calves with or without Lipopolysaccharide Stimulation.

The purpose of this study was to determine the effects of vitamin E supplementation on antioxidant, inflammatory status, and cell viability of peripheral blood mononuclear cells. Eighteen clinically healthy Japanese Black calves were used in this study. The peripheral blood mononuclear cells (PBMCs) were isolated from their venous blood. PBMCs were cultured with vitamin E (vitamin E group) or without vitamin E (control group), and stimulated with or without lipopolysaccharide (LPS). As a result, the total antioxidant capacities, which are the reducing power of components from Fe3+ to Fe 2+, in the cell culture supernatant with or without LPS stimulation were significantly higher in the vitamin E group than that in the control group (p<0.05 or p<0.01, respectively). Tumor necrosis factor alpha in the cell culture supernatant with LPS stimulation was significantly lower in the vitamin E group than in the control group (p<0.01). The viability of cells cultured with LPS stimulation was significantly higher in the vitamin E group than in the control group (p<0.05). These results suggested that vitamin E might be related to the antioxidant, anti-inflammatory, and cell viability of PBMCs obtained from calves.

Effects of vitamin E supplementation on serum oxidative stress biomarkers, antibody titer after live bovine respiratory syncytial virus vaccination, as well as serum and fecal immunoglobulin A in weaned Japanese Black calves.

The purpose of this study was to determine the effects of vitamin E supplementation on blood oxidative stress biomarker in weaned calves. Thirty clinically healthy 12 weeks of age Japanese Black calves were randomly assigned to two groups: 15 calves received 300 IU of vitamin E daily from 12 to 18 weeks of age (VE group), and the other 15 calves did not receive the vitamin E (control group). Blood samples were taken at 12, 14, 16, 18, and 20 weeks of age. The concentration of serum reactive oxygen metabolites at 20 weeks of age were significantly lower in the VE group than those in the control group. Vitamin E supplementation to weaned calves might affect blood oxidative stress.

Field Trial of Antibody Response To Inactivated Bacterial Vaccine in Young Holstein Calves: Influence of Animal Health Status.

Introduction: 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. Material and Methods: 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 Histophilus somni, Pasteurella multocida and Mannheimia haemolytica. 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. Results: 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. Conclusion: 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.

Effects of Vitamin C Supplementation on The Blood Oxidative Stress and Antibody Titre Against Histophilus Somni Vaccination in Calves.

INTRODUCTION: The purpose of this study was to determine the effects of vitamin C supplementation on blood oxidative stress biomarkers and antibody response to vaccination in calves. MATERIAL AND METHODS: Thirty-four clinically healthy 2 week old Japanese Black calves were randomly assigned to two groups. Seventeen calves formed the VC group which received 1,000 mg of vitamin C daily from 2 to 8 weeks of age, and the other 17 calves of the control group did not receive supplementation. All calves received an inactivated Histophilus somni vaccine at 4 and 8 weeks of age. Blood samples were taken at 2, 4, 8 and 12 weeks of age. RESULTS: The concentration of the serum reactive oxygen metabolites (d-ROMs), and the oxidative stress index (OSI), which is calculated from the d-ROMs and biological antioxidant potential, were significantly lower at 8 weeks of age in the VC group than in the control group (P < 0.05). The antibody titres to H. somni in the VC group were significantly higher than those in the control group at 12 weeks of age after the second vaccination (P < 0.05). CONCLUSION: Vitamin C supplementation to calves may reduce oxidative stress and enhance the antibody production after vaccination with H. somni.

Maternal Nutrition During Gestation Alters Histochemical Properties, and mRNA and microRNA Expression in Adipose Tissue of Wagyu Fetuses.

We hypothesized that maternal low or high nutrition would give unique effects to morphological and molecular dynamics in adipose tissue of fetus of fatty breed Wagyu (Japanese Black) cattle which produce highly marbled beef. This study aimed to determine the effects of maternal energy intake in Wagyu cows, during gestation on fetal adipose tissue development, histochemical properties, and gene and microRNA (miRNA) expression. Cows were allocated to one of two nutritional energy groups: 120% (HIGH) or 60% nutritional requirements of (LOW). Fetuses (n = 6 per treatment) were removed from pregnant cows by cesarean section at fetal age 260 ± 8 days and euthanized. Subcutaneous adipose tissue (SAT), thoracic cavity visceral adipose tissue (TVAT), and perirenal adipose tissue (PAT) were collected for analysis. In histochemical analysis, in SAT and PAT, HIGH fetuses had greater diameter of adipocytes than LOW fetuses (P<0.05). Only in SAT, LOW fetuses had more Leptin (LEP) mRNA and tended to have more Peroxisome Proliferator-Activated Receptor gamma (PPARG) CCAAT-enhancer-binding proteins alpha (CEBPA) and Glucose transporter (GLUT) 4 mRNA(P<0.10). In all SAT, TVAT, and PAT, LOW fetuses had higher levels of the brown adipose tissue (BAT) biomarkers Uncoupling Protein (UCP) 1 and PPARG coactivator (PGC) 1α mRNA than HIGH fetuses (P<0.08). Meanwhile, in the other adipose tissue, LOW fetuses had lower PPARG, CEBPA, and Zinc Finger Protein (ZFP) 423 (in TVAT and PAT), FASN (in TVAT), LEP and GLUT4 mRNA (in PAT; P<0.10). In particular, in TVAT and PAT, LOW fetuses exhibited lower expression of WAT biomarkers (PPARG and ZFP423). Differential expression of various miRNAs related to adipogenesis between the LOW and HIGH fetuses was detected in an adipose tissue-specific manner (P<0.10). Based on adipose tissue-specific effects of maternal nutrition, these findings suggested that poor maternal nutrition in Wagyu cattle increased BAT development in SAT, TVAT and PAT, while elevated maternal nutrition stimulated fetal SAT development compared with that of TVAT and PAT.

Effects of heat-killed Enterococcus faecalis T-110 supplementation on gut immunity, gut flora, and intestinal infection in naturally aged hamsters.

Infectious diseases are a threat to elderly individuals, whose immune systems weaken with age. Among the various infectious diseases, Clostridium difficile infection is associated with a high rate of mortality in elderly individuals and is a serious health problem worldwide, owing to the increasing infection rates. Probiotic use has been proposed as an effective countermeasure for C. difficile infection. The aim of this study was to evaluate the effects of heat-killed Enterococcus faecalis T-110 on intestinal immunity, intestinal flora, and intestinal infections, especially C. difficile infections, in naturally ageing animals, for extrapolating the results to elderly human subjects. Twenty female hamsters were randomly distributed into two groups. Group 1 was fed a basal diet and group 2 was fed a basal diet supplemented with heat-killed E. faecalis for 7 days. Heat-killed E. faecalis T-110 improved the gut immunity and microflora, especially Clostridium perfringens and C. difficile, in naturally aged hamsters. Therefore, heat-killed E. faecalis T-110 use may be a countermeasure against age-related immune dysfunction and intestinal infections, especially C. difficile infection, in elderly humans. However, further investigation in this regard is needed in humans.

Effect of Beta-Carotene Supplementation on the Peripheral Blood Leukocyte Population in Japanese Black Calves.

The purpose of this study was to determine the effect of beta-carotene supplementation to Japanese Black calves on the peripheral blood leukocyte population. Twenty-two Japanese Black calves were alternately assigned to two groups. Eleven calves received 20 mg/d of beta-carotene orally from 2 to 8 wk of age (BC group), and the other 11 calves did not receive (control group). The serum beta-carotene concentration in the BC group at 4, 8 and 12 wk of age were significantly higher than those in the control group (p<0.05). The numbers of CD4+ cells in the BC group were significantly higher than those in the control group at 4 wk of age (p<0.05). These results confirmed that beta-carotene supplementation to Japanese Black calves affected the peripheral blood leukocyte population.

Pharmacokinetics in plasma and alveolar regions of healthy calves subcutaneously administered a single dose of enrofloxacin.

This study aimed to analyze the pharmacokinetics of enrofloxacin (ERFX) and its metabolite ciprofloxacin (CPFX) in plasma, as well as their migration to, and retention in, the epithelial lining fluid (ELF) and alveolar cells within the bronchoalveolar fluid (BALF). Four healthy calves were subcutaneously administered a single dose of ERFX (5 mg/kg). ERFX and CPFX dynamics post-administration were analyzed via a non-compartment model, including the absorption phase. The Cmax of plasma ERFX was 1.6 ± 0.4 µg/ml at 2.3 ± 0.5 hr post-administration and gradually decreased to 0.14 ± 0.03 µg/ml at 24 hr following administration. The mean residence time between 0 and 24 hr (MRT0-24) in plasma was 6.9 ± 1.0 hr. ERFX concentrations in ELF and alveolar cells peaked at 3.0 ± 2.0 hr and 4.0 ± 2.3 hr following administration, respectively, and gradually decreased to 0.9 ± 0.8 µg/ml and 0.8 ± 0.5 µg/ml thereafter. The plasma half-life (t1/2) of ERFX was 6.5 ± 0.7 hr, while that in ELF and alveolar cells was 6.5 ± 3.6 and 7.4 ± 4.3 hr, respectively. The Cmax and the area under the concentration-time curve for 0-24 hr for ERFX were significantly higher in alveolar cells than in plasma (P<0.05). These results suggest that ERFX is distributed at high concentrations in ELF and is retained at high concentrations in alveolar cells after 24 hr in the BALF region; hence, ERFX may be an effective therapeutic agent against pneumonia.

Distribution of Cryptosporidium species isolated from diarrhoeic calves in Japan.

Cryptosporidium spp. are enteric protozoan parasites that infect a wide range of hosts including humans, and domestic and wild animals. The aim of this study was to molecularly characterize the Cryptosporidium spp. found in calf faeces in Japan. A total of 80 pre-weaned beef and dairy calves' diarrhoeic faecal specimens were collected from nine different prefectures in Japan. A nested polymerase chain reaction targeting the small subunit 18S rRNA and GP60 genes were used to detect the Cryptosporidium genotypes and subtypes. 83.8% (67 out of 80) of the specimens were positive for Cryptosporidium spp.; Cryptosporidium was found in both beef and dairy calves. Cryptosporidium parvum was the predominant species, detected in 77.5% (31/40) of beef calves and 80% (32/40) of dairy calves. Cryptosporidium bovis was also detected, 5.0% (2/40) of dairy calves, and C. ryanae was also found 2.5% (1/40) of dairy calves. One mixed-species infection, 2.5% (1/40) was detected in a beef calf having C. parvum, and C. ryanae. We detected the most common subtype of C. parvum (i.e., IIaA15G2R1), as well as other subtypes (i.e., IIaA14G3R1, IIaA14G2R1, and IIaA13G1R1) that have not previously been detected in calves in Japan. Our results demonstrate the widespread diversity of Cryptosporidium infection in calves in Japan.

Effect of combined vaccination for Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni to prevent respiratory diseases in young Japanese Black calves in the field.

To evaluate the preventive effects of combined vaccination for Pasteurella multocida, Mannheimia haemolytica and Histophilus somni on respiratory diseases in Japanese Black calves, 295 calves at one farm were alternately assigned to two groups; 147 calves received the vaccine at 4 and 8 weeks of age (vaccination group), and the other 148 calves did not receive vaccine (control group). The incidences of respiratory diseases were 25.9 and 70.9% in the vaccination and control groups, respectively, and the odds ratio for comparison between the two groups was 0.143 (95% confidence interval: 0.086-0.238). Administration of the multiple vaccine to Japanese black calves might be one of effective factor for prevention of respiratory diseases.

Distribution of marbofloxacin in the bronchoalveolar region in healthy calves.

The purpose of this study was to clarify the distribution of marbofloxacin (MBFX) within the bronchoalveolar region of calves. Four clinically healthy calves were intramuscularly injected with a single dose of MBFX (2 mg/kg). Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained for each calf at 0 (before administration), 1, 2, 6 and 24 hr after injection of MBFX. The injections and series of sample collections were conducted and repeated again after two weeks. The results show that the MBFX concentrations in the pulmonary epithelial lining fluid (ELF) were significantly higher than that in plasma and in alveolar cells at 2 hr after injection (P<0.05). For concentrations of MBFX within the ELF, the mean area under the MBFX concentration curve calculated during the 0 to 24 hr timeframe (AUC0-24) was significantly higher than the mean determined from samples collected from the plasma (P<0.05). These results suggest that intramuscularly injected MBFX was well distributed in the bronchoalveolar region.

Effect of Beta-Carotene Supplementation on the Serum Oxidative Stress Biomarker and Antibody Titer against Live Bovine Respiratory Syncytial Virus Vaccination in Japanese Black Calves.

The purpose of this study was to determine the effect of vaccination and beta-carotene supplementation on blood oxidative stress and antibody response in calves. Thirty Japanese Black calves were randomly assigned to two groups. Fifteen calves received 20 mg of beta-carotene supplemented into their daily provided rations from 2 to 8 weeks of age (BC group), and the other 15 calves did not receive the daily beta-carotene supplement (control group). All calves received a commercially available modified live bovine respiratory syncytial (RS) virus vaccine at 4 and 8 weeks of age. Blood samples were taken at 2, 4, 8, and 12 weeks of age. At 4 weeks of age, the concentration of reactive oxygen metabolites within serum were significantly lower in the BC group than the concentrations measured in the control group. Also at 4 weeks of age, the concentration of biological antioxidant capacity within serum was significantly higher in the BC group than the concentrations measured in the control group. Both groups showed a gradual decrease of antibody titers to live bovine RS virus in the samples taken from 2 to 12 weeks of age. These results confirmed that beta-carotene supplementation decreased oxidative stress. However, beta-carotene supplementation did not affect the antibody response to live bovine RS virus vaccination, perhaps due to the presence of the maternal antibody.

Seroprevalence of Cryptosporidium parvum and Neospora caninum in cattle in the southern Kyushu region of Japan.

Cryptosporidium parvum and Neospora caninum are common parasites in domesticated cattle worldwide, including in Japan. We carried out a serological survey to detect C. parvum and N. caninum infection among cattle in the southern Kyushu region of Japan-including the small islands-by indirect enzyme-linked immunosorbent assay based on recombinant antigens. We found that total seropositivity in 570 Japanese black cattle was 96.3% for C. parvum and 18.4% for N. caninum. Although seroprevalence was correlated with cattle age, differences in the seroprevalence of C. parvum among age groups were not statistically significant. On the other hand, N. caninum seroprevalence increased with age, suggesting horizontal transmission through ingestion of food or water contaminated with oocysts. These findings underscore the importance of monitoring C. parvum and N. caninum in cattle and implementing measures to prevent the spread of infection to other livestock and to humans.

Probiotic supplementation improves reproductive performance of unvaccinated farmed sows infected with porcine epidemic diarrhea virus.

We investigated if probiotic supplementation could improve the health and reproductive performance of unvaccinated lactating sows infected with porcine epidemic diarrhea (PED) virus. Twenty unvaccinated pregnant sows were equally allocated to probiotic-supplemented (P) and control (C) groups. For the experiment, 15 g/day of probiotic compound BIO-THREE PZ was given to P sows. Reproductive performance was checked daily. The number of neonates fostered by each sow was maintained at eight throughout the experiment. Individual milk production post-parturition was measured twice. Milk protein and fat ratios were determined by a milk analyzer. Total immunoglobulin (Ig) A and G concentrations were measured by ELISA. At day 7 post-parturition, the body weight of P sows was 10 kg higher than that of C sows, and at day 3 post-parturition, P sows produced more milk (+2 kg) and had a higher IgA concentration in whey than did C sows (p < .05). Finally, unlike C sows, P sows tended to return to estrus faster, and had larger piglets at birth with a lower mortality percentage during early days of suckling. In conclusion, probiotic compound BIO-THREE PZ helped strengthen the immune system of unvaccinated, PED-infected sows and improved their reproductive performance.