Japanese Black Beef Research Articles

Folic acid supplementation during fattening period affects growth and nutritional metabolism in Japanese Black beef cattle

The folate requirements for beef cattle have not been established. Therefore, we investigated whether rumen-unprotected folic acid supplementation during the fattening period affects carcass traits and nutritional metabolism in Japanese Black beef cattle. Eighteen Beef cattle aged 16 months were divided into three groups: control, low folic acid supplementation (0.43 g DM/day), and high folic acid supplementation (0.86 g DM/day). Treatment was administered for 12 months. Folic acid supplementation dose-dependently increased serum folate levels, suggesting that supplemental folic acid can be absorbed into the body. Folic acid supplementation dose-dependently decreased serum vitamin B12 levels, while plasma total homocysteine and methylmalonic acid levels—markers for deficiency of folate and/or vitamin B12—were unaffected. Thus, the treatment did not clearly affect the nutritional status of these vitamins. Supplementation increased body weight, with no negative effects on other carcass traits. The levels of insulin-like growth factor 1, retinol, albumin, and some amino acids in serum or plasma were affected by supplementation. These results suggest that rumen-unprotected folic acid supplementation during the fattening period could increase the body weight of Japanese Black beef cattle and the mechanism of action may be related to growth-related hormones and/or the metabolism of some nutrients, including folate.

Proposal of Lactobacillus amylovorus subsp. animalis subsp. nov. and an emended description of Lactobacillus amylovorus.

Seven novel lactic acid bacterial strains (BF125T, BF186, TKL145, YK3, YK6, YK10 and NSK) were isolated from the fresh faeces of Japanese black beef cattle and weanling piglets, spent mushroom substrates, or steeping water of a corn starch production plant. These strains are rod-shaped, Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, cytochrome oxidase-negative, facultatively anaerobic, and homofermentative. Strain BF125T did not produce any gas from glucose; both d- and l-lactate were produced as end-products of glucose (D/L, 40 : 60). Growth occurred at 30-45 °C (optimum, 37 °C), pH 5.0-8.0 (optimum, pH 6.0), and with NaCl concentration of 1.0-3.0% (w/v). The G+C content of genomic DNA of strain BF125T was 37.8 mol% (whole-genome analysis). The major fatty acids were C16 : 0, C18 : 1 ω9c, C19 cyclopropane 9, 10, and summed feature 10. The 16S rRNA gene in strain BF125T showed high similarity to that of the type strain of Lactobacillus amylovorus (99.93%), and the other isolates were also identified as L. amylovorus based on these similarities. A phylogenetic tree based on the core genomes of L. amylovorus strains (n=54), including the seven isolates, showed that they could be divided into two clusters. Strains YK3, YK6, YK10, and NSK were in the first cluster, along with the type strain DSM 20531T, while the second cluster included isolates BF125T, BF186, TKL145, and other strains isolated from various animal origins. Phenotypic differences in fermentability were observed for lactose, salicin, and gentiobiose between these two groups. The intergroup digital DNA-DNA hybridization values (72.9-78.6%) and intergroup average nucleotide identity values (95.64-96.92%) were comparable to values calculated using datasets of other valid subspecies of the genus (ex-) Lactobacillus. In light of the physiological, genotypic, and phylogenetic evidence, we propose a novel subspecies of L. amylovorus, named Lactobacillus amylovorus subsp. animalis subsp. nov. (type strain BF125T=MAFF 212522T=DSM 115528T). Our findings also led to the automatic creation of Lactobacillus amylovorus subsp. amylovorus subsp. nov. and an emended description of the species L. amylovorus.

Epididymitis with Pasteurella multocida isolated from two male Japanese Black beef calves.

Two male Japanese Black calves developed an enlarged scrotum and testis. Orchiectomy was performed and pus was collected during surgery. After removal of the testis, bacteriological and histopathological examinations were conducted to investigate the cause and confirm the diagnosis. Based on the results obtained, both cases were diagnosed with epididymitis caused by an infection with Pasteurella multocida. This is the first study to show that P. multocida causes epididymitis in male calves. Further studies are required to clarify the details underlying the infection of calves with P. multocida.

Image analysis traits of multiple muscles and intermuscular/subcutaneous fat influence Japanese Black beef carcass price and genetic parameters.

The purposes of this study were to investigate the relationship between carcass unit price per 1 kg (UP) and multiple muscles and intermuscular fat (IF)/subcutaneous fat of beef carcasses using image analysis of cross-section images for Wagyu beef cattle in Japan, and to estimate their genetic parameters. The carcasses used in this study were 1,807 Japanese Black (Wagyu) cattle (1,216 steers and 591 heifers). An analysis of variance was conducted with UP as the dependent variable and market date, age in months, sex, and image analysis traits (IAT) as fixed effects, and standard partial regression coefficients were calculated for each IAT on UP. Also, the heritability of each IAT that affected UP and genetic correlation among IAT vs carcass grading traits were estimated. Not only IAT related to carcass grading traits, M. trapezius dorsi, M. latissimus dorsi, and IF traits were significant differences in UP (p<0.05). The heritability of IAT associated with UP was estimated at 0.38 to 0.85. The genetic correlations between the area and thickness of M. trapezius dorsi and M. latissimus dorsi vs rib eye area (REA) were estimated to be moderately positive (0.53 to 0.66), while the genetic correlations between the IF area percentage vs carcass weight, REA, and yield score were estimated to be negative (-0.40, -0.56, and -0.34). UP was influenced by various traits, including M. trapezius dorsi, M. latissimus dorsi, and IF traits, as well as image analysis associated with carcass grading traits. Since these IAT associated with UP had hereditary and desirable genetic correlations with carcass grading traits, these traits were also important for genetic improvement.

211 Postponed in vitro fertilization timing improves cleavage competence in oocytes with slow-predicted nuclear maturation speed in Japanese Black beef heifers

211 Postponed in vitro fertilization timing improves cleavage competence in oocytes with slow-predicted nuclear maturation speed in Japanese Black beef heifers

Age-related changes in circulating INSL3 concentrations and their associations with ovarian conditions in Japanese Black beef cattle

Information on circulating levels of insulin-like peptide 3 (INSL3) in female domesticated animals is limited, as their concentrations are significantly lower than in males. The objectives of the present study were to 1) develop a sandwich time-resolved fluorescence immunoassay (TRFIA) with higher detectability to measure blood INSL3 concentrations in female cattle, 2) determine INSL3 concentrations in female cattle among age groups and reproductive conditions, and 3) explore associations between INSL3 levels and ultrasonographic ovarian measurements. Blood was collected repeatedly from Japanese Black beef female calves (n = 12; 0－8 mo), heifers (n = 10; 10－26 mo), and cows (n = 20; 27－200 mo). Blood was taken from the cows (n = 13) at follicular, post-ovulatory, and luteal phases, and from cows with follicular cysts (n = 12). Ultrasonography of ovaries was conducted in the calves (n = 12) and the cows without ovarian diseases (n = 9). The ovarian area, as well as the number and diameters of antral follicles ≥ 2 mm, were determined in each ovary. The proposed method detected a difference in plasma INSL3 between calves (0.01 ng/mL) and heifers (0.18 ng/mL). However, the conventional assay showed similar levels for calves and heifers (1.82 vs 2.07 ng/mL). Plasma INSL3 and testosterone concentrations increased from calves to heifers (P < 0.0001), but only INSL3 rose from heifers to cows (P < 0.0001). INSL3 and testosterone concentrations did not change across the estrus cycle in cows, and the levels of both hormones in follicular cystic cows did not differ from those in the follicular phase. Ovarian area, maximal and average follicular diameters, and total volume of all follicles per animal were higher in cows than calves (P < 0.001). Plasma INSL3 concentrations correlated positively with the total volumes of all follicles in calves (P < 0.05) and cows (P < 0.05), whereas testosterone concentrations did not correlate with ovarian follicular measurements. In conclusion, plasma INSL3 concentrations measured by the proposed sandwich TRFIA showed a clear increase from female calves to cows in beef cattle. These results suggest that circulating levels of INSL3, but not of testosterone, are associated with the total volume of all antral follicles in both ovaries per animal in female cattle.

Predicting nuclear maturation speed of oocytes from Japanese Black beef heifers through non-invasive observations during IVM: An attempt using machine learning algorithms

Nuclear maturation is an essential process in which oocytes acquire the competence to develop further. However, the time required for nuclear maturation during IVM varies among oocytes. Therefore, predicting nuclear maturation speed (NMS) could help identify the optimal timing for IVF and maximize the developmental competence of each oocyte. This study aimed to establish machine learning-based prediction models for NMS using non-invasive indicators during the individual IVM of Japanese Black (JB) beef heifer oocytes. We collected ovaries from abattoirs and aspirated cumulus-oocyte complexes (COCs) from follicles with diameters ranging between 2 and 8 mm. The COCs were matured individually for 18 h, and photographs of each COC were taken at the beginning and every 3 h from 12 h to the end of maturation. After IVM culture, we denuded COCs and stained oocytes to confirm the progression of meiosis. Only oocytes that reached the metaphase II (MII) stage were considered to have a fast NMS. Morphological features, including COC area, cumulus expansion ratio, expansion rate per hour, and expansion pattern, were extracted from the recorded photos and applied to develop prediction models for NMS using machine learning algorithms. The MII rates of oocytes with fast- and slow-predicted NMS differed when the decision tree (DT) and random forest (RF) models were employed (P < 0.05). To evaluate the relationship between predicted NMS by DT and RF models and fertilization dynamics during individual IVF, sperm penetration and pronuclear formation were evaluated at 3, 6, 9, and 12 h after IVF start, following 24 h of IVM. The association between predicted NMS and embryo development was investigated by performing IVC for seven days using microwell culture dishes after 24 h of IVM and 6 h of IVF. Predicted NMS did not show a significant association with fertilization dynamics. However, oocytes with fast-predicted NMS by the RF model exhibited a tendency towards a higher cleavage rate 48 h after IVF start (P = 0.08); no other relationship was found between predicted NMS and embryo development. These findings demonstrate the feasibility of using non-invasive indicators during IVM to develop prediction models for NMS of JB beef heifer oocytes. Although the effect of predicted NMS on embryo development remains unclear, customized treatments based on NMS predictions have the potential to improve the efficiency of in vitro embryo production following individual IVM culture.

142 Estimating the stage of the oestrous cycle in Japanese black beef cattle based on the number of large follicles

142 Estimating the stage of the oestrous cycle in Japanese black beef cattle based on the number of large follicles

Relationship between GnRH-induced LH increase profiles in the serum and vaginal mucus of Japanese Black beef cows.

This study aimed to investigate the relationship between increases in the luteinizing hormone (LH) profiles in the serum and vaginal mucus of cows induced by gonadotropin-releasing hormone (GnRH). Samples for LH determination were collected from Japanese Black beef cows during estrus, which was induced with a controlled internal progesterone-releasing device and the administration of cloprostenol immediately before GnRH administration and every 30 min from the start of GnRH administration until 6.5 h. The peak serum LH concentration was clearly identified at 2.5 h post-GnRH administration, with serum concentrations returning to near-pre-GnRH-administration values after 6.5 h, whereas the peak vaginal mucus LH concentration was identified 4.5 h after GnRH administration. These results indicate that the LH secretion peak in vaginal mucus appeared about 2 h after peak LH secretion in the serum.

Metabolomic profiling of postmortem aged muscle in Japanese Brown beef cattle revealed an interbreed difference from Japanese Black beef.

Japanese Brown (JBR) cattle, especially the Kochi (Tosa) pedigree (JBRT), is a local breed of moderately marbled beef. Despite the increasing demand, the interbreed differences in muscle metabolites from the highly marbled Japanese Black (JBL) beef remain poorly understood. We aimed to determine flavor-related metabolites and postmortem metabolisms characteristic to JBRT beef in comparison with JBL beef. Lean portions of the longissimus thoracis (loin) muscle from four JBRT cattle were collected at 0, 1, and 14 d postmortem. The muscle metabolomic profiles were analyzed using capillary electrophoresis time-of-flight mass spectrometry. The difference in postmortem metabolisms and aged muscle metabolites were analyzed by statistical and bioinformatic analyses between JBRT (n = 12) and JBL cattle (n = 6). A total of 240 metabolite annotations were obtained from the detected signals of the JBRT muscle samples. Principal component analysis separated the beef samples into three different aging point groups. According to metabolite set enrichment analysis, postmortem metabolic changes were associated with the metabolism of pyrimidine, nicotinate and nicotinamide, purine, pyruvate, thiamine, amino sugar, and fatty acid; citric acid cycle; and pentose phosphate pathway as well as various amino acids and mitochondrial fatty acid metabolism. The aged JBRT beef showed higher ultimate pH and lower lactate content than aged JBL beef, suggesting the lower glycolytic activity in postmortem JBRT muscle. JBRT beef was distinguished from JBL beef by significantly different compounds, including choline, amino acids, uridine monophosphate, inosine 5'-monophosphate, fructose 1,6-diphosphate, and betaine, suggesting interbreed differences in the accumulation of nucleotide monophosphate, glutathione metabolism, and phospholipid metabolism. Glycolysis, purine metabolism, fatty acid catabolism, and protein degradation were the most common pathways in beef during postmortem aging. The differentially expressed metabolites and the relevant metabolisms in JBRT beef may contribute to the development of a characteristic flavor.

Longitudinal effect of repeat breeding in Japanese Black beef cattle at a low parity on subsequent fertility in commercial cow–calf operations

Repeat breeding (RB) has been defined as failure to conceive from three or more regularly spaced services in the absence of detectable abnormalities, and cause economic loss on the farm owing to a declining conception rate. This study aimed to evaluate the reproductive characteristics of RB in Japanese Black cattle by assessing the longitudinal effect of RB at a low parity on subsequent fertility raised in commercial cow–calf operations. Data used in this study were collected from 764 farms in Miyazaki. Data from 2005 until 2014 comprised 158,945 AI records with 80,453 parity records for 22,565 cows. The effect of RB at heifers and parities 1 and 2 cows on subsequent fertility and survival on four consecutive parities was investigated. The results showed that cows with the number of inseminations to conception (INS) of ≥4 at a low parity had a higher probability of being RB again at subsequent parities compared with cattle with INS 1 or 2. Additionally, the degree of the negative effect of an INS ≥4 on fertility and survival increased as they became older. On the other hand, cows with an INS of 2 had similar fertility and survival with those with an INS of 1. In summary, this study suggests that repeat breeders at a low parity have a higher probability of RB again at subsequent parities compared with cattle with INS 1 or 2, and the negative effect of an INS ≥4 increases as parity increases.

Estimates of genetic parameters for adenosine triphosphate-related compounds at different aging periods and NT5E genotypes in Japanese Black beef.

Previous studies on Japanese Black beef showed that single nucleotide polymorphisms in the ecto-5'-nucleotidase (NT5E) gene affected the degradation rate of inosine 5'-monophosphate (IMP), which has contributed to the umami taste, especially between Postmortem Days 4 and 7. Therefore, this study estimated the genetic parameters of IMP and its degradation products on Postmortem Days 4 and 7 using the model with or without the NT5E genotype. The heritability estimates of IMP on Postmortem Days 4 and 7 were moderate by the model without the NT5E genotype (0.44 and 0.32, respectively). When the NT5E genotype was included in the model, the heritability of IMP on Postmortem Day 4 did not change, whereas that on Day 7 decreased from 0.32 to 0.08. The genetic correlation of IMP between Postmortem Days 4 and 7 was highly positive using the model with the NT5E genotype. Regarding the estimated breeding values (EBVs) of IMP, the ranking of EBVs among NT5E genotypes was not changed between Postmortem Days 4 and 7, when the model with the NT5E genotype was used. The study suggested that the model including NT5E genotype would allow for appropriate genetic parameter estimation and breeding value evaluation in adenosine triphosphate-related compounds (ATPRCs) under different aging periods.

Ultra-rapid real-time polymerase chain reaction assay for genotyping of the NT5E gene in Japanese black beef using a mobile PCR device, PCR 1100.

The concentration of inosine 5'-monophosphate (IMP) in beef is an important factor contributing to beef palatability. A previous study suggested that single nucleotide polymorphisms (SNPs) in the ecto-5'-nucleotidase (NT5E) gene strongly affect the concentration of IMP under postmortem conditions by regulating NT5E enzymatic activity in beef. Genotyping of the NT5E gene is performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or real-time PCR assay. However, these conventional laboratory assays require large installed instruments. They also involve complicated procedures and are time-consuming. Here, the PCR primers and probes for the NT5E gene (rs42508588 SNP) were designed and synthesized, and we examined the rapid genotyping of the NT5E gene using a PicoGene PCR 1100 mobile PCR device. The results showed that this system enabled rapid amplification of each allele at approximately 19.4s per cycle, with a total run time of 13 min 36 s. This device is portable and does not require a power supply, which facilitates its use not only in specific laboratories but also in meat production farms and distribution stages of beef.

Changes in immunoglobulin A and immunoglobulin G concentrations in the saliva of Japanese Black beef cows during calving: A pilot study.

Immunoglobulin A (IgA) in saliva, mostly consisting of secretory IgA, plays an important role in the mucosal immune mechanism. This study evaluated changes in salivary IgA and Immunoglobulin G (IgG) concentrations in Japanese Black cows (n = 16) during calving. Individual saliva samples were collected -2, 0, and 2weeks postpartum. Immunoglobulin concentrations differed significantly among weeks (P < 0.05), but the effect of parity and week × parity was insignificant. Salivary IgA concentrations decreased drastically (P < 0.05) after calving compared with those at -2weeks postpartum and remained low until 2weeks postpartum. The salivary IgG concentrations decreased gradually during peripartum and differed at -2 and 2weeks postpartum (P < 0.05). Considering the immunoglobulin concentrations at -2weeks postpartum as the reference standard for 100%, the rates of decrease in IgA concentrations (36.7 ± 6.9%) were significantly lower (P < 0.05) than those of IgG (70.3 ± 10.1%) at calving day. To our knowledge, this is the first report indicating that salivary IgA concentrations decreased drastically after calving in Japanese Black cows. Further studies monitoring the secretory functions of IgA in the salivary gland are essential for understanding maternal immunity in cattle.

Potential impact of botanically diverse pasture on the nutritional, physiological, and immunological status of grazing cows

AbstractThe aim of this study was to reveal the potential impact of botanically diverse pasture on the nutritional, physiological, and immunological status of grazing cattle using multifaceted indices. Ten Japanese black beef cows (325.5 ± 40.6 kg of body weight [BW], 7.9 ± 3.8 years of age) were used in this experiment. Five of them grazed on a 1.8‐ha grassland (botanically diverse pasture: DP) that was composed of sown grassland and grazable forestland (approximately 34 plant species). The other five cows grazed on 1.0 ha of sown grassland with only a few plant species (botanically monotonous pasture: MP, approximately 5 plant species) for 2 months. Blood samples were collected approximately every 2 weeks. In DP cows, the hemoglobin (HGB) concentration, hematocrit (HCT) ratio, and superoxide dismutase (SOD) activity increased linearly after the start of grazing, as did plasma sodium (Na), calcium (Ca), and potassium (K) concentrations. Urea nitrogen (UN) levels were higher in DP than in MP cows throughout the grazing period, whereas in MP cows, the red blood cell (RBC) count, HGB concentration, and HCT ratio decreased quadratically after the start of grazing. The DP cows gained more BW than the MP cows throughout the grazing period. Thus, the increased intake and/or the change in the ingested plant species in DP cows might have promoted the increase in the plasma UN concentration. In summary, the present study showed that grazing in botanically diverse pasture improved the nutritional and physiological status of cows; however, it aggravated the imbalance of protein and energy intake induced by grazing within 2 months.

Verification of Expiration Date Determination of Japanese Black Beef Aimed at Chilled Shipping Export

Verification of Expiration Date Determination of Japanese Black Beef Aimed at Chilled Shipping Export

Dynamics of microbiota in Japanese Black beef stored for a long time under chilled conditions

In this study, changes in the microbiota of Japanese Black beef carcasses, which are expected to be transported for a long time in chilled temperatures, were investigated. Three Japanese Black beef samples (carcasses A, B, and C) immediately after slaughter were stored at 0 °C for 15 weeks under aerobic and vacuum conditions. The initial bacterial counts were 50 CFU/g for carcass A and less than the reliable quantitative detection limit for carcasses B and C. Under aerobic storage conditions, the bacterial count increased to 8.0 log CFU/g or higher, which is a measure of putrefaction, at 6–9 weeks. Under anaerobic storage conditions, the bacterial counts of carcasses A and C reached 3.5–6.5 log CFU/g, but carcass B showed no bacterial growth during the 15-week storage period. The predominant group was Pseudomonas spp. under aerobic conditions and Serratia spp. under anaerobic conditions. To the best of our knowledge, there is no previous study investigating the transition of microbiota when Japanese Black beef is stored at low temperatures for a long period of time, and the results of this study are considered very important findings for the expansion of international trade of Japanese Beef in the future.

Comparison of muscle fiber type and chemical composition of Japanese black beef by meat quality grade, sex, and loin site

Comparison of muscle fiber type and chemical composition of Japanese black beef by meat quality grade, sex, and loin site

Changes in Composition of Rumen Bacterial Community and Relationship to Meat Quality in Japanese Black Beef Cattle During Fattening Stages

Changes in Composition of Rumen Bacterial Community and Relationship to Meat Quality in Japanese Black Beef Cattle During Fattening Stages

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10–14, 15–22, and 23–30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, “Biosynthesis of secondary metabolites” was significantly higher in the fluid fraction than in the solid fraction. “Two-component system” in the middle stage was significantly lower and “Purine metabolism” in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as “Biosynthesis of secondary metabolites” and “Sugar metabolism.” Moreover, the solid fraction was correlated with “Purine metabolism” and “Biosynthesis of secondary metabolism”. These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.