Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Effects of Alfaxalone on the Induction and Maintenance of Total Intravenous Anesthesia in Gentoo Penguins (Pygoscelis papua).

Systemic anesthesia in penguins is often achieved using inhalation anesthetic agents alone, and information on injectable drugs for systemic anesthesia is limited. General anesthesia with a minimal effect on circulatory dynamics is necessary to perform noninvasive examinations and treatments in animals, including penguins. In this study, alfaxalone (ALFX), an injectable anesthetic agent, was examined to establish the optimal anesthetic method for gentoo penguins (Pygoscelis papua). Alfaxalone was administered intravenously through the metatarsal vein, and anesthesia was maintained by a constant rate infusion (CRI). A biological monitor was used to record numerous clinical indices, and the anesthetic depth was evaluated every 5 minutes during anesthesia; the CRI was adjusted until the optimal anesthetic depth was obtained. Anesthesia depth was assessed, and the CRI rate was adjusted. The CRI was stopped, and the time until recovery was recorded. Blood samples were collected to analyze plasma concentrations of ALFX. The mean total dose of ALFX required for anesthetic induction was 9 ± 1.9 mg/kg, the intubation time was 126 ± 21 seconds, and the maintenance infusion rate of ALFX was 0.3 ± 0.08 mg/kg/min. The time from discontinuation of anesthesia to extubation was 42 ± 23 minutes, and the time to recovery was 90 ± 33 minutes. Significant changes in the heart rate and blood pressure were not observed during the anesthetic events. The plasma concentration of ALFX under stable anesthesia was 6734 ± 4386 ng/mL (range, 3315-14 326 ng/mL). Although anesthesia using ALFX tended to result in a prolonged time to recovery in gentoo penguins, rapid induction of anesthesia and stable hemodynamics during anesthetic maintenance were achieved. Therefore, ALFX may be considered a suitable anesthetic method for noninvasive examinations and treatments in penguins.

The first genetic characterization of Setaria marshalli (Nematoda, Spirurida) with reliable DNA barcoding based on a mitochondrial genetic marker.

Setaria marshalli is a mosquito-borne filarial nematode that causes infection in calves younger than two years old. In the present study, nematodes were obtained from a calf in Japan and morphologically identified as S. marshalli. Additionally, the partial cytochrome oxidase subunit I (COI) region (596 bp) was analyzed for the first time to establish a reliable DNA barcode. Nucleotide sequences of COI were identical among the seven worms obtained. The COI region can be a useful marker for species discrimination in the case of S. marshalli since nucleotide variations observed between the closest congener, Setaria cervi (51/596 bp), were sufficient to allow species discrimination. However, the phylogenetic relationship of S. marshalli with its congeners was unclear in a maximum likelihood tree. We found that the partial COI sequence of S. marshalli analyzed in the present study matched a relevant section of the complete mitochondrial genome of S. labiatopapillosa that was deposited in the International Nucleotide Sequence Database. This finding suggests that S. marshalli was misdiagnosed as S. labiatopapillosa in a previous study. It is crucial to conduct accurate morphological analyses to obtain reliable molecular information regarding Setaria nematodes.

Title: Première caractérisation génétique de Setaria marshalli (Nematoda, Spirurida) avec un code-barres ADN fiable basé sur un marqueur génétique mitochondrial. Abstract: Setaria marshalli est une filaire transmise par les moustiques qui provoque une infection chez les veaux de moins de deux ans. Dans la présente étude, les nématodes ont été obtenus à partir d'un veau au Japon et identifiés morphologiquement comme S. marshalli. De plus, la région partielle de la sous-unité I (COI) de la cytochrome oxydase (596 pb) a été analysée pour la première fois afin d'établir un code-barres ADN fiable. Les séquences nucléotidiques de COI étaient identiques parmi les sept vers obtenus. La région COI peut être un marqueur utile pour la discrimination des espèces dans le cas de S. marshalli puisque les variations de nucléotides observées avec le congénère le plus proche, Setaria cervi (51/596 pb) étaient suffisantes pour permettre la discrimination des espèces. Cependant, la relation phylogénétique de S. marshalli avec ses congénères n'était pas claire dans un arbre à maximum de vraisemblance. Nous avons constaté que la séquence COI partielle de S. marshalli analysée dans la présente étude correspondait à une section pertinente du génome mitochondrial complet de S. labiatopapillosa qui a été déposée dans la base de données internationale de séquences de nucléotides. Cette découverte suggère que S. marshalli a été diagnostiqué à tort comme S. labiatopapillosa dans une étude précédente. Il est crucial de mener des analyses morphologiques précises pour obtenir des informations moléculaires fiables concernant les nématodes du genre Setaria.

Effect of Oxidative Status on the Occurrence of Haemolactia in Dairy Cows after Calving.

Introduction: Dairy cows may infrequently give milk tinged with blood after calving, which is a condition termed haemolactia. Economic losses for dairy farmers are caused by cases of haemolactia because of the condemnation of such milk, potential contamination of good bulk tank milk with haemolactic milk, and need for veterinarian intervention. This study was performed to elucidate the oxidative status of dairy cows with haemolactia during the peripartum period. Material and Methods: Plasma glutathione peroxidase, malondialdehyde (MDA) and superoxide dismutase concentrations along with serum vitamin A, C and E concentrations were determined as indices of oxidative stress. The sampled dairy cows comprised two haemolactic (n = 11 and n = 6) and two non-haemolactic (n = 11 and n = 6) groups. Results: On the first day when haemolactia was identified in colostrum (at mean 2.1 days after parturition), a significantly increased concentration of plasma MDA was noted in the haemolactic group. During the prepartum period, low levels of serum vitamin E were continuously observed from prepartum week 4 to the parturition day but only in the haemolactic group. Conclusion: These results demonstrate that continuous low levels of serum vitamin E in the prepartum period may play a pivotal role as a requisite factor in the onset of haemolactia after calving.

Comparative Evaluation of Three Commercial Quantitative Real-Time PCRs Used in Japan for Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is an oncogenic virus belonging to the genus Deltaretrovirus and is the causative agent of enzootic bovine leukosis. Proviral load (PVL) determined by real-time quantitative PCR (qPCR) is now widely used as an indicator of not only BLV infection, but also BLV disease progression. To interpret PVLs determined by different qPCRs used in Japan, we compared a chimeric cycling probe-based qPCR, CY415, targeting the BLV tax region; a TaqMan probe-based qPCR, RC202, targeting the BLV pol region; and a TaqMan probe-based qPCR, CoCoMo, targeting the BLV long terminal repeat (LTR) region. Whole-blood samples collected from 317 naturally BLV-infected cattle (165 Holstein-Friesian and 152 Japanese Black) and tumor tissue samples collected from 32 cattle at a meat inspection center were used. The PVLs determined by each qPCR were strongly correlated. However, the PVL and the proportion of BLV-infected cells determined by RC202 or CoCoMo were significantly higher than those determined by CY415. Genetic analysis of three tumor tissue samples revealed that LTR region mutations or a deletion affected the PVL determined by CoCoMo. These results suggest that the TaqMan-based RC202 or CoCoMo qPCR is better than CY415 for BLV PVL analysis. However, qPCR target region mutations were not rare in tumors and could hamper PVL analysis by using qPCR.

Effect of vitamin K3 supplementation on immunoglobulin G concentration in colostrum of periparturient Holstein dairy cows.

This study was to examine the effects of dietary vitamin K (VK) 3 supplementation on immune-related substances in milk, oxidative stress indices in plasma and VK1, and menaquinone 4 (MK-4) in plasma and milk in periparturient dairy cows. Forty healthy perinatal Holstein-Friesian dairy cows were used in this study. Twenty-one animals were randomly selected and categorized into the VK3 supplemented (50 mg/day/head as VK3) group; the remaining 19 were categorized into the control group. On day 3 after calving, blood and milk were sampled, and their chemical components were determined. The VK3 supplemented group had significantly higher menaquinone 4 levels in plasma and milk on day 3 postpartum than the control group. In addition, there was a significant increase in the immunoglobulin G (IgG) level in milk. VK3 may be absorbed from the gastrointestinal tract and converted to MK-4, the biologically active form of VK, in the mammary gland and other tissues. It was thought that the increase in MK-4 level in plasma and milk induced an increase in the concentration of IgG in milk. VK3 supplementation to periparturient dairy cows may contribute to the production of colostrum with high concentrations of IgG and MK-4.

Characterization of microRNA expression in B cells derived from Japanese black cattle naturally infected with bovine leukemia virus by deep sequencing.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a malignant B cell lymphoma. However, the mechanisms of BLV-associated lymphomagenesis remain poorly understood. Here, after deep sequencing, we performed comparative analyses of B cell microRNAs (miRNAs) in cattle infected with BLV and those without BLV. In BLV-infected cattle, BLV-derived miRNAs (blv-miRNAs) accounted for 38% of all miRNAs in B cells. Four of these blv-miRNAs (blv-miR-B1-5p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p) had highly significant positive correlations with BLV proviral load (PVL). The read counts of 90 host-derived miRNAs (bta-miRNAs) were significantly down-regulated in BLV-infected cattle compared to those in uninfected cattle. Only bta-miR-375 had a positive correlation with PVL in BLV-infected cattle and was highly expressed in the B cell lymphoma tissue of EBL cattle. There were a few bta-miRNAs that correlated with BLV tax/rex gene expression; however, BLV AS1 expression had a significant negative correlation with many of the down-regulated bta-miRNAs that are important for tumor development and/or tumor suppression. These results suggest that BLV promotes lymphomagenesis via AS1 and blv-miRNAs, rather than tax/rex, by down-regulating the expression of bta-miRNAs that have a tumor-suppressing function, and this downregulation is linked to increased PVL.

Increased T-cell responses that control bovine leukemia virus proviral load in beef cattle under dietary vitamin A restriction for marbling.

Bovine leukemia virus (BLV) proviral load is controlled by T-cell responses, which require vitamin A (VA) derived from food. However, whether dietary VA restriction for marbling impairs the T-cell responses that control BLV proviral load in beef cattle is unknown. We assessed T-cell subsets, interferon (IFN)-γ gene expression, and BLV proviral load in naturally BLV-infected Japanese Black cattle that were fed a diet with decreased VA levels. We found that the percentage of CD4+ T cells increased over time during dietary VA restriction. In addition, BLV proviral load was negatively correlated with the percentage of CD4+ T cells and with the level of IFN-γ gene expression. These observations suggest that dietary VA restriction for marbling enhances T-cell responses that control BLV proviral load and thus does not promote leukemogenesis in fattening beef cattle.

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.

Effectiveness of on-farm continuous flow high-temperature short-time pasteurization for inactivation of bovine leukemia virus in milk.

The effectiveness of on-farm continuous flow high-temperature short-time (HTST) pasteurization (i.e., 72°C for 15 s) for the inactivation of bovine leukemia virus (BLV) in milk was investigated with a sheep bioassay. Four sheep that had been inoculated with completely pasteurized milk containing approximately 3.4 × 107 BLV-infected peripheral blood mononuclear cells (PBMC) and treated by either HTST pasteurization or laboratory-scale low-temperature long-time (LTLT) pasteurization (i.e., 60°C for 30 min), remained negative for BLV for at least 17 weeks after inoculation. In contrast, all sheep inoculated with unpasteurized or inadequately pasteurized milk containing the same number of BLV-infected PBMC were tested positive for BLV and anti-BLV antibodies within 3 weeks after inoculation. These results suggest that on-farm continuous flow HTST pasteurization was equivalent value with inactivated BLV on the LTLT procedure and can effectively inactivate BLV in the milk. Therefore, on-farm HTST pasteurization of the pooled colostrum or milk used in automated feeding systems is likely to protect group-housed preweaned calves from BLV infection, thereby improving animal health on dairy farms.

Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

Calving-related intradural avulsion injuries of the thoracolumbar spinal nerve roots in a calf.

Calving difficulty may lead to traumatic peripheral nerve injury. A male, 8-mo-old, Japanese Black calf with a history of secondary dystocia as a result of fetal gigantism had lameness and gait disturbance. At autopsy, multifocal dural thickening with adhesions to the adjacent spinal cord was observed at T12-13 and L4-5 vertebral levels. Microscopically, numerous traumatic neuroma-like fascicles of nerve twigs were embedded in the dura mater with abundant collagenous connective tissue. By immunohistochemistry, axons and Schwann cells were confirmed in each nerve fascicle. Our observations suggest that avulsion injuries in the preganglionic fibers of the spinal nerve roots, and secondary spinal cord compression, resulted in the development of neurologic signs.

Changes in oxidative stress parameters in healthy and diseased Holstein cows during the transition period in Yamagata Prefecture, Japan.

We investigated changes in oxidative stress markers during the transition period in healthy Holstein cows and those with postpartum diseases. Transition control (TC) Holstein cows (n=9) were evaluated for longitudinal changes during the transition period and postpartum diseased (PD) cows with ketosis (n=10), abomasal displacement (n=9), and acute mastitis (n=10) were evaluated in comparison to control cows (n=10). In the TC group, blood samples were collected at 2 weeks prepartum and at 1, 2, 4, 6, and 8 weeks postpartum. Milk yield and composition were measured at 2 and 4 weeks postpartum. In the PD group, blood samples were collected at the first day of examination during the 60 days postpartum. Peripheral oxidative stress parameters (malondialdehyde, MDA; potential antioxidant capacity, PAO; and glutathione peroxidase) were measured, and biochemical analyses were performed. In the TC group, MDA increased significantly postpartum and was correlated with milk yield, blood glucose (Glu), free fatty acid (FFA), ß-hydroxybutyric acid (BHB), and aspartate aminotransferase. Compared to the control cows, PD cows with ketosis had significantly higher MDA and significantly lower PAO. Moreover, MDA was significantly correlated with Glu, FFA, and BHB. Postpartum increase in MDA might interact with milk yield and Glu, FFA, and BHB in the TC cows, and postpartum diseases, especially ketosis, might signify its increase and interaction with Glu, FFA, and BHB.

Effects of feeding management on disease incidence and blood metabolites in dairy herds in Iwate Prefecture, Japan.

The objective of the present study was to identify the effect of feeding management on disease incidence and blood metabolite levels in dairy herds in Iwate Prefecture, Japan. A generalized linear model approach was used to identify the risk factors for ketosis and displaced abomasum (DA) in dairy herds (n=30), and metabolic profile test (MPT) results were compared to verify the involvement of the factors. Consequently, the proportion of corn silage (CS) with ≥30% of dry matter (DM) fed to cows during the lactation period was confirmed as the most reliable risk factor for ketosis, while no risk factor was identified for DA. Meanwhile, the incidence rates of ketosis and DA were significantly (P<0.05) higher in the herds that were fed CS (n=20) than in those fed a non-CS diet (n=10). When the MPT results of the herds fed with CS containing ≥30% of DM (HCS group, n=4; 76 cows), with CS containing <30% of DM (LCS group, n=14; 285 cows), and a non-CS diet (NCS group, n=12; 236 cows) were compared, the HCS group showed higher beta-hydroxybutyric and lower blood urea nitrogen concentrations for until 49 days after parturition. Overall, feeding cows with CS diets containing over 30% of DM might increase their risk of developing negative energy and protein balances, thereby resulting in increasing incidences of ketosis in the Iwate Prefecture.

Effects of an increased concentrate diet on rumen pH and the bacterial community in Japanese Black beef cattle at different fattening stages.

This study evaluated the effects of increasing the proportion of concentrate in the diet on the rumen pH and bacterial community in Japanese Black beef cattle at different fattening stages. Six rumen-cannulated beef cattle were studied in the middle (Mid group, n=3, age 21-22 months) and late (Late group, n=3, age 31 months) fattening stages. The cattle were fed rice straw with control (CON period) or high-concentrate (HC period) diets for 14 consecutive days in each period. Rumen pH was measured continuously and the rumen fluids were collected on the last day of each period. The 24-hr mean and minimum rumen pH in the Mid group were significantly (P<0.05) lower during the HC period compared with the CON period, whereas those in the Late group were continuously low during both periods. In the Late group, the ruminal volatile fatty acid and lactic acid concentrations were significantly (P<0.05) higher during the HC period. During the HC period, the proportions of Prevotella and Caloramator were significantly (P<0.05) higher and lower, respectively, in the Mid group. From these findings, significant changes in the rumen pH and bacterial community induced by dietary changes were mainly observed in the Mid group. Therefore, the ruminal fermentative function in response to a higher concentrate diet might adapt differently in Japanese Black beef cattle at the two different fattening stages.

Effects of active dried Saccharomyces cerevisiae on ruminal fermentation and bacterial community during the short-term ruminal acidosis challenge model in Holstein calves.

We investigated the effects of active dried Saccharomyces cerevisiae (ADSC) on ruminal pH, fermentation, and the fluid bacterial community during the short-term ruminal acidosis challenge. Five rumen-fistulated male Holstein calves (147.0 ± 5.8 kg of body weight; 3.6 ± 0.2 mo of age) were used in a crossover design, and 0 g (control group, n = 5) or 2 g (SC group, n = 5) of ADSC (1 × 1010 cfu/g) was administered twice daily for 21 consecutive days. Calves were fed a high-forage diet during the first 15 d (d -14 to d 0; prechallenge), a high-grain diet for 2 d (d 1 and 2; ruminal acidosis challenge), and a high-forage diet for 4 d (d 3 to 6; postchallenge). Ruminal pH was measured continuously. Rumen fluid samples were collected once daily (0800 h) on d 0, 3, 4, and 6 and twice daily (0800 and 1100 h) on d 1 and 2. Bacterial DNA was extracted from fluid samples collected on d 0 and 3. The 24-h and 1-h mean ruminal pH was significantly depressed during the ruminal acidosis challenge in each group, although the changes were more severe in the SC group, consistent with a significant increase in lactic acid on d 2 (1100 h) compared with d 0 and a significantly higher proportion of butyric acid on d 2 (1100 h) compared with the control group. Feeding a high-grain diet caused a decrease in bacterial diversity due to high acidity in both groups. The relative abundances of the genus Bifidobacterium and operational taxonomic unit (OTU) 3 (Bifidobacterium species) increased significantly in both groups but were higher in the SC group. Correlation analyses indicated that OTU3 (Bifidobacterium species) were positively correlated with lactic acid concentration and that OTU1 (Prevotella species) and OTU5 (Succinivibrio species) were correlated with the proportion of butyric acid. These results suggest that ADSC supplementation induced the intense decreases in ruminal pH by increased butyric and lactic acid production through a high-grain diet fermentation by rumen fluid bacterial species during the short-term ruminal acidosis challenge in Holstein calves after weaning.

Changes in ruminal and reticular pH and bacterial communities in Holstein cattle fed a high-grain diet.

BACKGROUND: Subacute ruminal acidosis (SARA) is characterized by a ruminal pH depression, and microbiota can also be affected by a higher acidity and/or dietary changes. Previous studies have revealed similar patterns in pH reduction in the rumen and reticulum, whereas changes in reticular pH and bacterial community following a high-grain diet are not fully understood. The aim of this study was to investigate the changes in reticular pH and bacterial community structure following a high-grain diet simultaneously with those in the rumen. RESULTS: SARA was diagnosed when ruminal and reticular pH remained under 5.6 for 350 ± 14 and 312 ± 24 min/d, respectively, on the last day of the CON period. During the CON period, lower proportion of acetic acid and higher proportion of butyric acid were observed compared with the HAY period. The proportions of acetic acid and propionic acid were lower and higher, respectively, in the rumen compared with the reticulum. From 454 pyrosequencing analysis, the relative abundance of several genera differed significantly between the two periods and the two locations. During the HAY period, higher relative abundances of Prevotella, Eubacterium, Oscillibacter, and Succiniclasticum and lower relative abundances of Ruminococcus, Clostridium, and Olsenella were identified compared with the CON period. Furthermore, the relative abundance of Eubacterium was lower in the rumen compared with the reticulum. Bacterial diversity indices were significantly different between the HAY and CON periods, being higher in the HAY period. The quantitative real-time PCR showed that the copy numbers of several cellulolytic bacteria (Fibrobacter succinogenes and Ruminococcus albus) were higher during the HAY period. CONCLUSION: A high-grain diet showed similar impacts on the pH, fermentation, and bacterial community structure in the rumen and reticulum. During the CON period, ruminal and reticular pH decreased following the high-grain challenge, and lower bacterial diversity and changes in the bacterial composition, similarity, and bacterial copy numbers were observed due to a higher acidity and dietary changes compared with the HAY period. These changes may influence the fermentative ability of the rumen and reticulum.

Collection and frozen storage of semen for artificial insemination in red foxes (Vulpes vulpes).

This study was conducted on red foxes to determine the appropriate voltage in electroejaculation for semen collection from stud males, and to confirm whether frozen semen with bovine semen extender can be used for artificial insemination. The proper load voltage for electroejaculation was 3-4 V based on semen collection rates and concentrations of spermatozoa. Frozen semen was prepared according to the known procedure for cows. In frozen-thawed semen, a relatively high conception rate (81.3%) was obtained in vixens, in which the optimum insemination time was detected by vaginal electrical resistance. These findings demonstrate that the restricted condition for semen collection by electroejaculation with cryopreservation of semen using bovine semen extender can be applied to artificial insemination of red foxes.

Effects of repeated subacute ruminal acidosis challenges on the adaptation of the rumen bacterial community in Holstein bulls.

We investigated the effect of repeated subacute ruminal acidosis (SARA) challenges on the pH, fermentative function, and bacterial community in the rumen. Four rumen-cannulated Holstein bulls were fed a high-forage diet for 7 d (HF period) followed by a high-grain diet for 7 d (HG period). Four SARA challenges were carried out consecutively (first, second, third, and fourth challenges). The ruminal pH was measured continuously during the experiment, and rumen fluid samples during the first to fourth challenges were collected at 0800, 1400, and 2000 h on the last days of each feeding period for analysis; volatile fatty acid components, NH3-N, and lactic acid concentrations were measured. Bacterial community structure was analyzed at 0800 h during the first and fourth challenges on the last days of each period. The 24-h mean ruminal pH was decreased during the transition from high-forage to high-grain diet and tended to differ between the HF and HG periods. During the HG period, ruminal pH <5.6 was maintained for a longer period in the first and second challenges (350 and 405 min/d, respectively) than in the third and fourth challenges (both 120 min/d). A marked increase in total volatile fatty acid and NH3-N concentrations during the HG period was observed in the later challenges. In addition, lower and higher proportions of acetic and butyric acids, respectively, were observed during the HG period than during the HF period. A total of 37 core bacterial genera were found in all samples; however, the relative abundance of several genera differed significantly between the HF and HG periods (Prevotella, Ruminococcus, Eubacterium, and Oscillibacter) and between the first and fourth challenges (Eubacterium and unclassified Clostridiaceae). During the HG period, lower relative abundances of Prevotella, Eubacterium, and Oscillibacter and higher relative abundance of Ruminococcus were detected compared with during the HF period. The relative abundances of Eubacterium and unclassified Clostridiaceae were lower in the first challenge than in the fourth challenge. Bacterial diversity was greater during the HF period than during the HG period and was greater during the fourth challenge than during the first challenge. Interestingly, diversity indices during the HG period of the fourth challenge were higher than those during the HF period of the first challenge. These results suggested that rumen fermentation in Holstein cattle can adapt to repeated SARA challenges by minimizing the adverse changes in ruminal pH. Moreover, the composition and diversity of the ruminal bacterial community may be affected by ruminal pH and vice versa.

First detection of Allobilharzia visceralis (Schistosomatidae, Trematoda) from Cygnus cygnus in Japan.

Adult schistosomes were detected in the veins or capillaries of the large intestine, mesentery, liver, and adrenal glands in eight of 13 whooper swans (Cygnus cygnus) examined in Iwate Prefecture, Japan. However, neither eggs nor severe tissue injuries were observed in any of the swans. The schistosomes were definitively identified as Allobilharzia visceralis based on the nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region. Allobilharzia visceralis infections have been reported in whooper swan in Iceland and tundra swan (Cygnus columbianus) in North America. These detections suggest that A. visceralis is distributed extensively along the swan flyways because the swans are migratory birds. To the best of our knowledge, this is the first report of A. visceralis infection in Asia.