Application of MinION Amplicon Sequencing to Buccal Swab Samples for Improving Resolution and Throughput of Rumen Microbiota Analysis.

The Illumina MiSeq platform has been widely used as a standard method for studying the rumen microbiota. However, the low resolution of taxonomic identification is the only disadvantage of MiSeq amplicon sequencing, as it targets a part of the 16S rRNA gene. In the present study, we performed three experiments to establish a high-resolution and high-throughput rumen microbial profiling approach using a combination of MinION platform and buccal swab sample, which is a proxy for rumen contents. In experiment 1, rumen contents and buccal swab samples were collected simultaneously from cannulated cattle (n = 6) and used for microbiota analysis using three different analytical workflows: amplicon sequencing of the V3-V4 region of the 16S rRNA gene using MiSeq and amplicon sequencing of near full-length 16S rRNA gene using MinION or PacBio Sequel II. All reads derived from the MinION and PacBio platforms were classified at the species-level. In experiment 2, rumen fluid samples were collected from beef cattle (n = 28) and used for 16S rRNA gene amplicon sequencing using the MinION platform to evaluate this sequencing platform for rumen microbiota analysis. We confirmed that the MinION platform allowed species-level taxa assignment for the predominant bacterial groups, which were previously identified at the family- and genus-level using the MiSeq platform. In experiment 3, buccal swab samples were collected from beef cattle (n = 30) and used for 16S rRNA gene amplicon sequencing using the MinION platform to validate the applicability of a combination of the MinION platform and buccal swab samples for rumen microbiota analysis. The distribution of predominant bacterial taxa in the buccal swab samples was similar to that in the rumen samples observed in experiment 2. Based on these results, we concluded that the combination of the MinION platform and buccal swab samples may be potentially applied for rumen microbial analysis in large-scale studies.

A study of lipolysis induced by adjuncts from edible Aspergillus sp. solid culture products on ripened semi-hard cheese.

BACKGROUND: Aspergillus sp. has been used in traditional Japanese fermented foods. Protease-containing culture products of A. oryzae have been applied as the adjunct enzyme source to enrich the flavor in ripened cheese. Although proteolysis was stimulated, the increase of free fatty acids (FFA) was recognized in some products. Since an excess amount of FFA accumulation can cause rancidity in cheese products, the assessment of lipase activity was considered to be essential for the cheese adjunct preparation. RESULTS: Although an equal lipase activity from the adjunct materials of A. kawachii NBRC 4308, A. luchuensis RIB 2604 and A. oryzae AHU 7139 was applied to semi-hard cheese, the FFA level was significantly higher in A. oryzae cheese than in the others. Furthermore, the profiles of volatile components were different in experimental cheeses. An in vitro study with experimental curds demonstrated that the high FFA might not depend on the lipase retainability on curds. On the contrary, the pronounced activation of the lipases occurred in A. oryzae after incubation with the curds. Moreover, incubation of the insoluble lipase that had been attached to the cells with skim milk curd extracts allowed the release of lipases from the cells into the medium with remarkable activation. CONCLUSION: A. oryzae AHU 7139 possessed a complex lipolytic system comprising extracellular and cell-binding lipases that were attributed to the increase in FFA in A. oryzae cheese. © 2022 Society of Chemical Industry.

Effects of grazing adaptation on herbage intake, milk production, and body weight change in lactating dairy cows after turning out to pasture in early spring.

The present study investigated the effects of adaptation to grazing in early spring on the herbage intake, milk production, and body weight changes in lactating dairy cows. The experiment included 12 Holstein lactating cows during early spring. Six cows were allowed to gradually adapt to grazing (ADP) over 10 days. They were allowed to graze on a pasture for 4 h during the first week and for 19 h during the remaining 3 days (adaptation period). During the 10-day period, the remaining six cows were housed in a barn (CON). Cows in both groups received adequate silage, hay, and concentrate during the adaptation period. Subsequently, cows in both groups were allowed to graze together for 19 h for 4 weeks (experimental period). No cow received supplements during the experimental period. In the first week of the experimental period, the ADP cows showed a higher herbage intake than the CON cows. During the subsequent weeks, there were no differences in herbage intake between the two groups. At the start of the experimental period, milk production and body weight of the ADP cows were higher than those of the CON cows, and this difference lasted until the end of the experiment.

Regional differences in the fatty acid composition, and vitamin and carotenoid concentrations in farm bulk milk in Hokkaido, Japan.

In this study, we investigated the regional differences in the composition of farm bulk milk produced at three different dairy areas in Hokkaido, Japan. A field survey was conducted at Central, Tokachi, and North areas of Hokkaido, three or four times a year. At each farm, an interview questionnaire for farm basal data was conducted, and 500 ml of bulk tank milk sample was obtained. Fatty acid composition, and vitamin and carotenoid concentrations in the milk samples were determined. In Central and Tokachi areas, corn silage was used as the main forage. In North area, fresh herbage was the dominant feed in the summer season, and grass hay was the main feed in the winter season. Discriminant analysis revealed that the composition of milk samples differed among the areas and seasons. Milk from Central and Tokachi areas contained a higher ratio of linoleic acid compared with that from North area, but there were only slight differences in the composition of milk between Central and Tokachi areas. The concentrations of carotenoids and α-tocopherol were higher in samples from North area and the ratios of trans-vaccenic acid and conjugated linoleic acid were higher in the summer season than in the indoor season.

Emergent surgical embolectomy for middle cerebral artery occlusion related to cerebral angiography followed by neck clipping for an unruptured aneurysm in the anterior communicating artery.

BACKGROUND: Intracranial embolism related to cerebral angiography is rare but one of the complications of the procedure. However, the standard management of acute intracranial embolism for this etiology has not been established, and there have been very few reports in the past. CASE DESCRIPTION: A 68-year-old male was incidentally found to have an unruptured aneurysm of anterior communicating artery (ACoA). Immediately after the cerebral angiography for the purpose of detailed examination of the aneurysm, the right partial hemiparalysis and mild aphasia developed. Magnetic resonance imaging/angiography (MRI/A) revealed an occlusion in the peripheral part of the left middle cerebral artery (MCA). Due to the existence of magnetic resonance angiography-diffusion mismatch, emergent craniotomy was immediately performed to remove intra-arterial thrombus. We also performed clipping for an unruptured ACoA aneurysm with this approach. Postoperative MRI/A showed that the occluded artery was recanalized and a slight infarction was observed in the left cerebral hemisphere. The patient was discharged on foot and followed at outpatient clinic over 4 years without no neurological deficit. CONCLUSION: Emergent surgical embolectomy for distal MCA occlusion related to cerebral angiography followed by neck clipping for an unruptured aneurysm of the ACoA was successful in treating acute occlusion of the peripheral part of the MCA in a patient with an unruptured aneurysm. As there are few similar cases, there is controversy about the best management, but this surgical method can be a safe and effective treatment.

Effects of grazing adaptation on intake, ruminal fermentation, blood metabolites, and body weight change in dairy cows after turning out to pasture in early spring.

This study investigated the effect of adaptation to grazing in early spring on herbage intake, ruminal fermentation parameters, blood metabolite concentrations, and body weight change in dairy cows. The experiment was conducted on eight rumen-cannulated non-lactating cows in the early spring period. Four cows were adapted to grazing by stocking for 4 hr for 1 week (ADP group). The other cows were kept in a barn during the period (CON group). Then, both groups of cows were stocked together throughout a day on a 1 ha pasture for 3 weeks (experimental period). In the first week of the experimental period, compared to the CON group, the ADP group had a higher herbage intake, ruminal NH3 -N and total VFA concentration, and blood urea concentration, but the NEFA concentration was lower in the ADP group (p < .01). During the subsequent weeks, there were little differences in ruminal fermentation parameters and blood metabolites. Cows in the ADP group maintained their body weight, but cows in the CON group lost 60 kg of body weight in the first week of the experimental period.

Effect of summer heat stress on gene expression in bovine uterine endometrial tissues.

Heat stress negatively affects reproductive functions in cows. Increased temperature disturbs fetal development in utero. However, the effect of heat stress on uterine endometrial tissues has not been fully examined. Using qPCR analysis, we measured the mRNA expression of various molecular markers in uterine endometrial tissue of dairy cows from Hokkaido, Japan, in winter and summer. Markers examined were heat shock proteins (HSPs), antioxidant enzymes (catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase 4), inflammatory cytokines, and interferon stimulated genes. Our results showed heat stress, body and milk temperatures were higher during summer than during winter. Expression levels of HSP27, HSP60, and HSP90 mRNA, and of catalase and copper/zinc superoxide dismutase mRNA were lower in summer than in winter. Tumor necrosis factor alpha expression was higher in summer than in winter. In conclusion, summer heat stress may reduce the expression of HSPs, affecting the levels of inflammatory cytokines in bovine uterine endometrial tissue.

Effects of ear corn silage supplementation on milk production and milk fatty acid profiles in grazing dairy farms.

This study investigated the effects of substituting ear corn silage (ECS) for commercial formula feed on milk production and milk fatty acid profiles in grazing dairy farms during the summer season. A field survey was conducted on five grazing dairy farms in every summer month of 2017, 2018, and 2019. Three of the five farms substituted fresh ECS for the commercial formula feed at a ratio of 2:1 from July of each year (ECS farms). Other farms maintained the same feeding management as before (non-ECS farms). An interview survey was conducted on each farm to calculate feed intake and milk yield per cow. Feed and milk samples were collected in each survey. Milk compositions and milk fatty acid profiles were determined. The substitution of ECS for the commercial formula feed did not affect milk yield or milk composition, but ECS farms maintained low levels of milk urea compared with non-ECS farms (p < .01). The ECS substitution also influenced some of the milk fatty acid proportions; C16:0 and C16:1 increased, and trans-11 C18:1, cis-9,trans-11 C18:2, and the sum of polyunsaturated fatty acids decreased, while these fatty acid proportions were maintained in non-ECS farms throughout the summer season (p < .05).

Highly accurate and explainable detection of specimen mix-up using a machine learning model.

Background Delta check is widely used for detecting specimen mix-ups. Owing to the inadequate specificity and sparseness of the absolute incidence of mix-ups, the positive predictive value (PPV) of delta check is considerably low as it is labor consuming to identify true mix-up errors among a large number of false alerts. To overcome this problem, we developed a new accurate detection model through machine learning. Methods Inspired by delta check, we decided to conduct comparisons with the past examinations and broaden the time range. Fifteen common items were selected from complete blood cell counts and biochemical tests. We considered examinations in which ≥11 among the 15 items were measured simultaneously in our hospital; we created individual partial time-series data of the consecutive examinations with a sliding window size of 4. The last examinations of the partial time-series data were shuffled to generate artificial mix-up cases. After splitting the dataset into development and validation sets, we allowed a gradient-boosting-decision-tree (GBDT) model to learn using the development set to detect whether the last examination results of the partial time-series data were artificial mixed-up results. The model's performance was evaluated on the validation set. Results The area under the receiver operating characteristic curve (ROC AUC) of our model was 0.9983 (bootstrap confidence interval [bsCI]: 0.9983-0.9985). Conclusions The GBDT model was more effective in detecting specimen mix-up. The improved accuracy will enable more facilities to perform more efficient and centralized mix-up detection, leading to improved patient safety.

Wall thickness control in biotubes prepared using type-C mold.

A type-C mold based on in-body tissue architecture was previously developed for preparing small-diameter biotube vascular grafts with a 2-mm diameter and approximately 1-mm wall thickness. In this study, the type-C mold was modified for preparing large-diameter biotubes with controlled wall thicknesses. Four types of molds were assembled by inserting silicone center rods (outer diameters 11, 13, 15, 17 mm) into stainless steel cages (inner diameter 19 mm) and surgically embedded in the abdominal subcutaneous pouches of Holstein cows. After 8-12 weeks, connective tissues occupied the rod-cage gap in the molds to form biotubes. The wall thickness of the biotubes obtained after removing the molds was approximately 1-3 mm, which corresponded to approximately 80% of each gap distance. The breaking strength almost linearly increased with the wall thickness of the biotubes. The strength of the biotubes with wall thickness over 1.5 mm was higher than that of beagle blood vessels. The thickest biotubes were as strong as bovine pericardium and can be used as an alternative trachea graft because of their adequate lumen-holding force.

Effects of the presence of grazing-experienced heifers on the development of foraging behavior at the feeding station scale for first-grazing season calves.

In this study, effects of grazing-experienced heifer presence on foraging behavior development at the feeding station (FS) scale for first-grazing season calves were determined. A group of four calves grazing alone (C), and another comprising four calves (Wc) and three grazing-experienced heifers (Wh), were alternately stocked every day for 2 h on the same pasture for 26 days. The foraging time budget for Wc was significantly longer than that for C (P < 0.05) on day 7, and was similar to that for Wh on all days. Bite rate per FS was significantly higher for Wc (15.5 bite/min) than for C (13.2 bite/min) from day 1 to 14 (33.4 vs. 29.0 bite/min, respectively; P < 0.01) and significantly lower than that for Wh on all days. The number of steps taken between adjacent FSs by Wc (4.7) was significantly lower than that for C (7.2) on days 1 and 14 (2.1 vs. 2.9 steps, respectively; P < 0.01) and was similar to Wh on all days. This suggests that grazing-experienced heifers have positive effects on the foraging behavior development of new-season grazing calves through 14 days after the start of stocking.

Effect of timing and type of supplementary grain on herbage intake, nitrogen utilization and milk production in dairy cows grazed on perennial ryegrass pasture from evening to morning.

The present study aimed to clarify the effect of timing and type of supplementary grain in grazing dairy cows on herbage dry matter intake (HDMI), nitrogen utilization and milk production. Eight lactating cows were allowed to graze from evening to morning during three seasonal periods (spring, summer, autumn). They were randomly allocated to four treatments (timing: pre- (Pre) or post-grazing (Post), for large grain allotments consisting of 75% of daily grain offered; grain type: barley or corn) in 4 × 4 Latin square designs in each period. In the spring period, HDMI was greater for cows fed corn than those fed barley (P = 0.005), whereas cows in the Pre treatment had a similar HDMI, higher (P = 0.049) urinary purine derivative concentration and greater (P = 0.004) milk yield compared with cows in the Post treatment. In the summer and autumn periods, timing treatments did not affect HDMI, nitrogen utilization or milk production, but cows supplemented with barley had higher urinary purine derivatives concentration (P < 0.05) and milk yield (P < 0.05) compared with those supplemented with corn. The results indicate that large grain allotments immediately before evening grazing during early grazing seasons increased ruminal microbial protein synthesis and milk production without reducing HDMI regardless of grain type.

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.

Effect of water-soluble carbohydrate content in orchardgrass pasture on grazing time and rumen fermentation in dairy cows.

Two experiments were conducted to clarify the effect of water-soluble carbohydrate (WSC) content in orchardgrass pasture on the diurnal distribution of grazing time. Six ruminally cannulated, non-lactating dairy cows were grazed on either of two pastures with different orchardgrass cultivars containing low WSC (LWSC; cultivar: 'Hokkai 28') or high WSC (HWSC; cultivar: 'Harunemidori'). The cows were grazed in morning and evening sessions in experiment 1, whereas the cows were grazed throughout the day in experiment 2. In experiment 1, grazing time of the cows on HWSC was longer than that of the cows on LWSC (P < 0.01). This difference was larger in the morning session than in the evening session (pasture × grazing session: P < 0.05). Effects on herbage intake were similar to those on grazing time. In experiment 2, daily total grazing time was longer for the cows on HWSC than for those on LWSC (P < 0.05). The cows on HWSC spent a longer time grazing than those on LWSC in the morning between 03.00 and 09.00 hours (P < 0.01). The results indicated that prolonged grazing time in the period between dawn and early morning could increase daily herbage intake in cows grazed on pastures of orchardgrass cultivars with high-WSC content.

Herbage intake and ruminal digestion of dairy cows grazed on perennial ryegrass pasture either in the morning or evening.

This study aimed to clarify diurnal fluctuations of herbage intake, ruminal fermentation of herbage carbohydrates and proteins, and digesta particulate weight in the rumen of grazing dairy cows. Six ruminally cannulated, non-lactating dairy cows were grazed on perennial ryegrass/white clover pasture either in the morning (04.00 to 08.00 hours) or the evening (16.00 to 20.00 hours). Cows grazed in the evening spent more time (P < 0.01) and consumed more herbage (P < 0.01) compared with cows grazed in the morning. Higher (P < 0.05) daily mean concentrations of total volatile fatty acid, propionate and n-butyrate in rumen fluid were observed for cows grazed in the evening compared with cows grazed in the morning. Although cows grazed in the evening ingested more crude protein compared with cows grazed in the morning, no significant difference in NH3 -N concentration in rumen fluid was observed between them. The ratio of purine-derivative concentration to creatinine concentrations was higher (P < 0.01) in the urine of cows grazed in the evening than in cows grazed in the morning. These results clearly indicated that evening grazing was advantageous for dairy cows compared with morning grazing, in terms of ruminal fermentable energy intake and nitrogen utilization efficiency.

Relationship of rumen fill and fermentation to diurnal and seasonal variation of herbage intake in dairy cows grazed on perennial ryegrass pasture.

To clarify the effect of digesta weight in the reticulorumen on diurnal and seasonal fluctuations in herbage intake, six ruminally cannulated, non-lactating dairy cows were grazed on perennial ryegrass/white clover pasture during morning and evening sessions in spring and autumn. The digesta weight of fresh matter, dry matter (DM) and fiber in the reticulorumen at the beginning and the end of each grazing session was lower in spring than in autumn (P < 0.01). Although the digesta weight was similar between the sessions at the beginning of grazing, it was greater for the evening than for the morning at the end of grazing (P < 0.01). The large particles proportion in the digesta was lower for the morning than the evening (P < 0.01), and it was lower in spring than in autumn (P < 0.01). The concentrations of volatile fatty acids in rumen fluid were generally higher in spring compared with autumn. The herbage DM intake during the evening was greater compared with the morning in both seasons (P < 0.01). However, there was no difference in herbage DM intake between seasons. The results showed that the rumen digesta fill was not the sole factor explaining diurnal and seasonal variation of herbage intake in grazing dairy cows.

Discrimination of "grazing milk" using milk fatty acid profile in the grassland dairy area in Hokkaido.

Milk produced by the grazing system, referred to as "grazing milk" contains many components required for human health. The milk fatty acid (FA) profile is strongly associated with the diet on the farms. In the present study, based on the FA profile of farmer's bulk milk, we determined how to discriminate between milk produced on grazing and on a confinement system. A field survey was conducted four times (grazing and confinement season) in the Konsen (29 farms) and Okhotsk (25 farms) area in Hokkaido. Farmer's bulk milk samples and details of feeding management were collected and the FA profile of milk was measured. Milk produced during the grazing season contained less C16:0 and cis-9 C16:0, and more C18:0, cis-9 C18:1, trans-11 C18:1, cis-9,12 C18:2, cis-9,trans-11 C18:2 and cis-9,12,15 C18:3 than milk produced during the confinement season. Discrimination analysis using 16 FA revealed that almost all milk samples were discriminated correctly (confinement season: 90% correct and 10% borderline, grazing season: 88% correct, 9% borderline and 3% incorrect). For farmers that were categorized incorrectly and were considered borderline in the grazing season, the dependency on pasture was low compared with that for farmers correctly discriminated. Therefore, to claim "grazing milk", a high dependency on pasture is required for grazing dairy farmers.

Large-Scale Glycomics of Livestock: Discovery of Highly Sensitive Serum Biomarkers Indicating an Environmental Stress Affecting Immune Responses and Productivity of Holstein Dairy Cows.

Because various stresses strongly influence the food productivity of livestock, biomarkers to indicate unmeasurable environmental stress in domestic animals are of increasing importance. Thermal comfort is one of the basic principles of dairy cow welfare that enhances productivity. To discover sensitive biomarkers that monitor such environmental stresses in dairy cows, we herein performed, for the first time, large-scale glycomics on 336 lactating Holstein cow serum samples over 9 months between February and October. Glycoblotting combined with MALDI-TOF/MS and DMB/HPLC allowed for comprehensive glycomics of whole serum glycoproteins. The results obtained revealed seasonal alterations in serum N-glycan levels and their structural characteristics, such as an increase in high-mannose type N-glycans in spring, the occurrence of di/triantennary complex type N-glycans terminating with two or three Neu5Gc residues in summer and autumn, and N-glycans in winter dominantly displaying Neu5Ac. A multivariate analysis revealed a correlation between the serum expression levels of these season-specific glycoforms and productivity.

Long-term oral feeding of lutein-fortified milk increases voluntary running distance in rats.

To evaluate the effects of lutein-fortified milk administration on running exercise, a voluntary wheel-running model was performed in rats. Four-week-old F344 rats were administered test milk (10 mL/kg) daily following a 4-h fasting period, and their running distances were measured each day for a 9-week period. Total weekly running distance significantly increased from the sixth week until the end of the test period in lutein-supplemented rats (lutein-fortified milk administered) compared with control rats (vehicle administered). This increase was not apparent in rats administered lutein alone. In the lutein-fortified-milk exercise group compared with the sedentary control group, carnitine palitroyltransferase 1 (CPT-1), total AMP-activated protein kinase (tAMPK), and phosphorylated AMP-activated protein kinase (pAMPK) contents were significantly increased in the gastrocnemius muscle, with a concomitant decrease in triglyceride and total cholesterol levels in the blood and liver. Furthermore, the lutein level in blood of lutein-administered rats significantly decreased with exercise. These results suggest that lutein-fortified milk may enhance the effect of exercise by effective utilization of lipids when combined with voluntary running.

Effect of feeding non-fibrous carbohydrate before grazing on intake and nitrogen utilization in dairy cows throughout the grazing season.

Ten lactating cows were used to determine the effect of feeding non-fibrous carbohydrate (NFC) supplement before grazing on feed intake and nitrogen (N) utilization throughout a grazing season. The experiment was conducted from June to September. Cows grazed twice a day (2.5 h × 2) under a set stocking system and were fed NFC supplement (1 kg/4 kg of milk yield) 2 h before grazing (PRE) or immediately after grazing (POST). Cows were also fed a grass and corn silage mixture ad libitum. Herbage dry matter intake (DMI) was greater for PRE than for POST throughout the experiment and decreased from June to September. Conversely, silage DMI was less for PRE than for POST throughout the experiment and increased over the grazing season. Consequently, total DMI for PRE did not differ from that for POST. Milk urea-N concentration and urinary urea-N excretion in June did not differ between the treatments, whereas that after July was higher for PRE than for POST. Proportion of urinary N excretion to absorbed N intake in June was lower for PRE than for POST, but that after July was higher for PRE than for POST. Feeding NFC supplement before grazing would improve N utilization when cows eat large amounts of herbage high in N.