Molecular characterization of GPR84 in domestic cats.

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83-89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein-protein interaction with FFAR2/GPR43 on the cell membrane.

Effect of acupuncture on the energy metabolism of dogs with intervertebral disk disease and cervical disk herniation: A pilot study.

Thirteen dogs with intervertebral disk disease (IVDD) and 3 dogs with cervical disk herniation (CVDH) were examined to determine the effects of acupuncture on energy metabolism. Acupuncture points GV14, GV20-1, BL18, BL23, BL26, GB30, and ST36 were selected for IVDD, while GV14, GV20-1, GB20, and BL23 were selected for CVDH. All dogs except no.13 did not receive medication during acupuncture treatment. Acupuncture effects were evaluated based on the IVDD/CVDH evaluation scales in Oji 2015 and Tanaka and Nakayama 2015. Blood samples were taken before and 30 min after acupuncture treatment. Pyruvate and lactate concentrations, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, the MDH/LDH ratio (M/L ratio), and LDH isozyme electrophoretic patterns served as energy metabolism markers. In IVDD/CVDH dogs that showed improvements, plasma pyruvate concentrations significantly decreased, the M/L ratio increased, and the plasma LDH isozyme pattern changed from predominantly LDH5 to predominantly LDH1. These data suggest that local redox potential is improved and energy metabolism is increased in dogs with IVDD/CVDH after acupuncture treatment. Acupuncture treatments may activate the citric acid cycle and increase ATP production, followed by improvement of the disease. Future studies with a large sample size are needed to clarify this hypothesis.

Molecular characterization of free fatty acid receptors FFAR2 and FFAR3 in the domestic cat.

G protein-coupled receptors 41 and 43 were identified and characterized as free fatty acid receptors (FFAR) 3 and 2, respectively. FFAR2 and FFAR3 mediate short-chain fatty acids (SCFAs) as signalling molecules. The present study aimed to give molecular characterization of FFAR2 and FFAR3 in the domestic cat. High homology with that in other mammals was revealed by cDNA cloning of cat FFAR2 FFAR3. We analyzed the tissue distribution of cat FFAR2 and FFAR3 mRNA using quantitative polymerase chain reaction. The inhibition of intracellular cAMP concentrations was observed in cells transfected with cat FFAR2 or FFAR3 and treated with SCFAs. The activation of nuclear factor of activated T cells-luciferase reporter was only observed in cat FFAR2 transfected cells but not in FFAR3. Split luciferase assay (NanoLuc Binary Technology; NanoBiT) for FFAR2 or FFAR3 and Arrestin-3/ß-arrestin-2 revealed acetate-/propionate-induced recruitment to cat FFAR2 or FFAR3 in CHO-K1 cells. Our results indicate that FFAR2 and FFAR3 are functional receptor proteins that are expressed in cat tissues and show differential distribution patterns.

Effects of Age on Inflammatory Profiles and Nutrition/Energy Metabolism in Domestic Cats.

BACKGROUND: Animals tend to increase in body weight and body condition score (BCS) with aging. Serum diagnostic markers related to energy metabolism may show changes even in healthy cats with aging. MATERIALS AND METHODS: Seventy domestic cats were recruited for this study. Based upon the modified AAFP-AAHA Feline Life Stage Guidelines, animals were divided into six groups: Junior (7 months-2 years), Prime (3 -6 years), Mature (7-10 years), Senior (11-14 years), Geriatric-obese (15 years ≤) and Geriatric-thin (15 years ≤). Their body condition scores (BCS) ranged from 3/9 to 9/9. Changes in metabolites, inflammatory markers, hormone concentrations and enzyme activities related to energy metabolism were investigated in serum of 70 domestic cats of various ages. RESULTS: Serum glucose (GLU) concentrations in the Mature, Senior, and Geriatric-obese groups were significantly higher than those in the Junior group. Serum amyloid A (SAA) concentrations in the Geriatric-thin group were significantly increased compared with the Junior group. SAA concentrations in the Geriatric-obese group tended to increase although there were no statistically significant differences. In the Mature, Senior, Geriatric-obese and Geriatric-thin groups, malate dehydrogenase/lactate dehydrogenase (M/L) ratio, an energy metabolic indicator, tended to decrease compared with the Junior group. In the Senior group, triglyceride (TG) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese and Geriatric-thin groups, blood urea nitrogen (BUN) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese group, albumin (ALB) concentrations were decreased compared with the Junior group. CONCLUSION: Aged domestic cats tend to increase in body weight and BCS. In addition, serum GLU, TG, SAA, and BUN concentrations increased and serum ALB concentrations and M/L ratio decreased. These diagnostic markers may be useful to detect small changes related to energy metabolism with aging that may cause obesity with light inflammation in healthy cats.

Effects of Supplementation with Anti-Inflammatory Compound Extracted from Herbs in Healthy and Obese Cats.

BACKGROUND: Obesity has become a serious public health problem all over the world, and prevalence of obesity has increased in cats. Obesity is characterized by continuous low-grade inflammation based on oxidative stress by excessively produced reactive oxygen species (ROS). Supplementation with anti-oxidant and anti-inflammatory compounds is very effective to relieve the obesity condition. A plant extract mixture containing Rhus verniciflua and some other herbs, Rv-PEM01-99, shows anti-oxidant and anti-inflammatory effects in animals. The aim of this study was to evaluate the effects of supplementation with Rv-PEM01-99 as an anti-inflammatory compound in healthy and obese cats. MATERIALS AND METHODS: Ten healthy mix breed cats and four obesity disease cats were used. The healthy cats were randomly divided into control and test groups. Anti-inflammatory compound, Rv-PEM01-99, in which quercetin derivative is the main component, was supplemented to the healthy test group and the obesity disease cats at the dose of 100-120 mg/kg/day (2.5-3.0 mg/kg/day as quercetin) for 4 weeks. Metabolites, hormones and enzymes were measured before and after the compound supplementation. RESULTS: The anti-inflammatory compound supplementation decreased serum amyloid A (SAA) concentrations as inflammatory markers in both healthy and obesity disease cats. In obesity disease cats, plasma total cholesterol concentrations and AST and ALT activities decreased significantly after the compound supplementation. CONCLUSION: Quercetin derivative seems to have strong anti-inflammatory activities. In the healthy cats, anti-inflammatory compound supplementation decreased plasma NEFA and SAA concentrations. In the obesity disease cats, the compound supplementation may have alleviated obesity disease by relieving inflammation and improvement of lipid metabolism in livers.

Diagnostic Criteria for Obesity Disease in Cats.

Accumulated visceral and subcutaneous fat masses were measured with computed tomography (CT) in cats with various body condition scores (BCS) from 5/9 to 9/9. BCS does not always reflect visceral fat accumulation which induces pro-inflammatory reactions. Obese cats with accumulated visceral fat showed low plasma adiponectin and high serum amyloid A (SAA) concentrations, an inflammatory marker. Based on the above results, new diagnostic criteria for obesity disease were established as follows. For overweight cats with high BCS of >7/9, showing two or more of the following three symptoms, low adiponectin concentrations, hyperlipidemia, and high SAA concentrations, categorizes them as having obesity disease. Cats with BCS 6/9-9/9, without inflammatory reactions, were classified as simple obesity, which is similar to metabolically healthy obesity (MHO) defined in human medicine. Simple obesity group showed significantly higher adiponectin concentrations than those in control group. The obesity disease group showed significantly higher plasma triglyceride (TG) and SAA concentrations and lower concentrations of adiponectin than the control group. Moreover, plasma glucose and malondialdehyde (MDA) concentrations in the obesity disease group were higher than those in healthy control group, although the differences were not statistically significant. Establishing criteria for obesity disease based on visceral fat accumulation and inflammation markers levels contributes to early and correct diagnosis of obesity in cats.

Evaluation of blood adiponectin levels as an index for subacute ruminal acidosis in cows: a preliminary study.

The objective of this study was to evaluate blood levels of various hormones and compounds related to energy metabolism in cows with subacute ruminal acidosis (SARA). We investigated 11 lactating cows presumed to have SARA based on duration of ruminal pH <5.6 and reticulum pH <6.3 in 2015-2016. Kraft pulp (KP) was used to supplement feed of 7 of the cows studied in an effort to reduce SARA. We continuously monitored ruminal pH and measured blood concentrations of hormones and metabolites related to energy metabolism. Blood measurements included glucose (GLU), total cholesterol (TC), free fatty acid (FFA), insulin, adiponectin (ADN), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH). Additionally, we analyzed milk data (milk yield, milk fat percentage, milk protein percentage, milk urea nitrogen, and protein fat ratio) and reproduction data. The results demonstrated that ADN levels at 4 weeks post-parturition correlated with the total amount of time that the ruminal or reticulum fluid pH was under the threshold during 1 week post-parturition, as well as the numbers of days the cows were diagnosed with SARA (SARA-positive days) up to 30 days post-parturition. SARA-positive days in 2016 were higher than those in 2015. In both years, numbers of SARA-positive days for cows supplemented with KP were lower than those for cows without KP. Increased ADN levels may be a compensatory reaction to frequent SARA which modulates the inflammatory response against high LPS levels and improves insulin resistance caused by LPS. ADN may serve as an estimative index for SARA.

Effects of astaxanthin supplementation in healthy and obese dogs.

BACKGROUND: Since astaxanthin (ASX) has potent anti-oxidative effects with inhibitory action of lipid peroxidation and singlet oxygen quenching activity, it is widely used as a functional food for keeping good health in human. Obesity is a risk factor for various metabolic disorders. It is characterized by low-grade chronic inflammation based on oxidative stress by excessively produced ROS. From the point of preventive medicine, natural compounds have been proposed as potential therapeutic agents in the prevention of metabolic disorder in companion animals. The purpose of this study is to evaluate the effects of ASX supplementation in healthy and obese dogs. MATERIALS AND METHODS: Ten healthy beagle dogs and 5 clinically obese dogs were used in this study. The healthy beagle dogs were randomly divided into 2 groups as follows: control and test groups. The test group dogs received ASX supplementation mixed with the food for 6 weeks. Five clinically obese dogs received ASX supplementation for 8 weeks. Metabolites, hormones and enzymes were measured before and after ASX supplementation. RESULTS: In the healthy dog groups, after 6 weeks, plasma triglyceride (TG) and malondialdehyde concentrations and lactate dehydrogenase (LDH) values significantly decreased in the test group. There was no significant difference in the control group. In clinically obese dogs, plasma TG concentration decreased after 8 weeks of ASX supplementation. Plasma alanine aminotransferase and LDH values clearly decreased in all 5 dogs and 4 dogs out of 5 dogs, respectively. CONCLUSION: ASX supplementation (0.3 mg/kg body weight/day) for 6 weeks in healthy dogs and 8 weeks in obese dogs induced the elevation of antioxidant function and of liver function by ameliorating lipid metabolism.

Changes in Plasma Metabolites Concentrations in Obese Dogs Supplemented With Anti-oxidant Compound.

The aim of this study is to discuss the effect of anti-oxidant supplement (Rv-PEM01-99, Kibun Foods, Inc., Tokyo, Japan) on changes in energy metabolism in obese dogs. 200 mg/kg/day of Rv-PEM01-99 (equivalent to 5 mg kg/day of quercetin derivative) were applied for 6 weeks to the Beagle dogs fed high fat diet (HFD) or control diet (CD). In the present study, body weight (BW) decreasing effect of Rv-PEM 01-99 in obese dogs was not clear. However, plasma alkaline phosphatase (ALP) activities at the end of experiment were significantly decreased compared to those at the start of experiment in obese dogs supplemented with Rv-PEM 01-99 (paired-t test, p < 0.05). In control dogs supplemented with Rv-PEM 01-99, Plasma malondialdehyde (MDA), and triglycerides (TG) levels and lactate dehydrogenase (LDH) activities were significantly decreased compared to those at the start of experiment (paired-t test, p < 0.05). From these findings, Rv-PEM 01-99 seems to be not harmful for dogs. Anti-lipid peroxide effect and liver function improvement are expected in the dogs supplemented with Rv-PEM 01-99.

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Comparative analysis of knowledge on schistosomiasis japonica in the local people in the former endemic area in Yamanashi Prefecture, Japan: comparisons among the background of age and occupation.

Eradication of schistosomiasis japonica in Yamanashi Prefecture was officially declared in 1996, and all surveillance and health campaign were finished by the end of 2001. Schistosomiasis control had been carried out by strong collaboration among local Government, local people and academia, thought which knowledge and experiences of the disease control were accumulated among the local people. It is 20th anniversary of the disease eradication in Yamanashi. We planned to analyze the current situation whether the local people still keep the knowledge of schistosomiasis or not. There was no more knowledge kept in the middle school students, to whom the local Government did not educate about schistosomiasis. Among adult individuals, elderly people who have experienced endemic condition still know about it at almost comparable level as in the past. However, younger people who learned it in the primary school but no experience of the disease transmission started losing the knowledge. Those situations of the adult local people were compared with the veterinarian group. It was confirmed that the veterinarian group had correct and proper knowledge of schistosomiasis japonica not only in elderly groups, but also in younger age group. Considering that methods for control and prevention of infectious diseases are common to some extent, their knowledge and experiences would be applicable for other infectious diseases in future. Therefore, the knowledge would be worth keeping in the local people. Taken together, individuals with specialty, such as veterinarians, are expected to play roles in public health for promotion of health and welfare.

Correction: Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident.

[This corrects the article DOI: 10.1371/journal.pone.0155069.].

Effectiveness of feline body mass index (fBMI) as new diagnostic tool for obesity.

Feline body mass index (fBMI), BW/PCL, length from top of patella to end of calcaneus, was developed as a new diagnostic tool for obesity in cats. To evaluate the effectiveness of fBMI for obese cats in short-term, 6 cats were induced weight gain by over-feeding with high fat diet and then they were induced weight reduction by restrict-feeding with low fat diet to measure changes in fBMI and plasma metabolite concentrations and enzyme activities. BCS 3 is correlated with fBMI 24.6-32.0, BCS 4 is correlated with fBMI 33.1-37.1 and BCS 5 is correlated with fBMI 29.9-40.3, respectively. On the correlation coefficient analysis by Pearson's method (P < 0.05), positive correlations (r = 0.403) were seen between the fBMI and plasma triglyceride (TG) levels. From these findings, fBMI seems to be more sensitive and useful indicator for obesity diagnosis in cats.

Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident.

The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 µGy/day (ranging from 1.6 to 33.9 µGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 µGy/day (6.0-133.4 µGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 µGy/day (9.1-155.1 µGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

Comparison of plasma lipoprotein profiles and malondialdehyde between hyperlipidemia dogs with/without treatment.

BACKGROUND: The aim of this study is to compare metabolic parameters, malondialdehyde as a lipid oxidation marker, and lipid profiles between dogs with untreated hyperlipidemia and hyperlipidemia with treatment, in order to examine the usefulness of malondialdehyde and lipid profiles as diagnostic parameters at early stages of hyperlipidemia. RESULTS: Dog samples were collected from four different veterinary clinics across Japan from March to June 2013. They were separated into three groups: control, untreated hyperlipidemia based on temporally screening, and hyperlipidemia with current anti-hyperlipidemic (statins and fibrates) treatment. Triglyceride levels of untreated hyperlipidemia dogs were significantly higher than those of control dogs. ALT levels of hyperlipidemic dogs with treatment were the highest among three groups. VLDL and LDL of both cholesterol and triglyceride of untreated hyperlipidemia dogs were the highest among three groups. HDL1 levels in triglyceride of hyperlipidemia dogs with treatment were significantly higher than those of control and untreated hyperlipidemia dog. Malondialdehyde concentrations of untreated hyperlipidemia dogs were significantly higher than those of control and hyperlipidemic dogs with treatment. CONCLUSIONS: In this study, dogs with untreated hyperlipidemia clearly showed abnormal lipid status, whereas hyperlipidemic dogs under anti-hyperlipidemia treatment showed more normal lipid status suggesting the effectiveness of the therapy. Anti-hyperlipidemics (statins and fibrates) for dogs are also effective in relieving elevated levels of lipids and lipid oxidation. Plasma lipid (triglyceride and cholesterol) profiles and malondialdehyde are useful diagnostic tools for identifying early stages of untreatment hyperlipidemia in dogs.

Age effects on plasma cholesterol and triglyceride profiles and metabolite concentrations in dogs.

BACKGROUND: In dogs, occurrence of lipid metabolism disorders such as obesity and diabetes mellitus has increased markedly in recent years. Hyperlipidemia has been regarded as a common characteristic for obese animals and hyperlipidemic condition may be associated with inflammation, oxidative stress and lipid composition changes. In this study, we investigated the changes in plasma cholesterol and triglyceride (TG) profiles and metabolite concentrations in 24 dogs (young group: 0-7 years old, n = 12, aged group: 8-13 years old, n = 12). RESULTS: Plasma adiponectin (ADN) concentrations were significantly lower in aged dogs than those in young dogs (mean ± SD, 17.2 ± 10.0 µg mL-1 vs 29.3 ± 12.5 µg mL-1, respectively; P <0.05). Although there were no significant differences statistically, aged dogs showed significantly higher plasma alpha1- acid glycoprotein (alpah1-AG) levels compared to those in young dogs. Plasma cholesterol lipoprotein and TG lipoprotein were divided into four fractions by biphasic agarose gel electrophoresis technique. The levels of the third TG-lipoprotein fraction from the positive pole (TG Fraction 3) were significantly higher in aged dogs than in young dogs (mean ± SD, 143.0 ± 109.3 mg dL-1 vs 55.2 ± 31.3 mg dL-1, respectively; P <0.05). On the correlation coefficient analysis by Peason's method, moderate positive correlations were seen between the age and TG (r = 0.446, P = 0.029), TG Fraction 3 (r = 0.516, P = 0.010), malondialdehyde (r = 0.146, P = 0.043), alpha-1 AG (r = 0.448, P = 0.028) levels, respectively. Moderate negative correlations were seen the age and total cholesterol (TC) Fraction 2 (r = -0.446, P = 0.029), glucose (r = -0.637, P = 0.001), ADN (r = -0.408, P = 0.048), respectively. CONCLUSIONS: Present data suggest biochemical characteristics of lipid metabolism disorder may be affected by aging in dogs.

Parabacteroides chinchillae sp. nov., isolated from chinchilla (Chincilla lanigera) faeces.

Strains of Gram-stain-negative, anaerobic, rod-shaped bacteria were isolated from chinchilla (Chinchilla lanigera) faeces, and strain ST166(T) was investigated taxonomically. Phylogenetic analyses of 16S rRNA gene sequences revealed that strain ST166(T) belonged to the genus Parabacteroides. Strain ST166(T) formed a distinct line of descent, and the highest sequence similarity to ST166(T) was found with Parabacteroides merdae JCM 9497(T) (95.6%) and Parabacteroides johnsonii JCM 13406(T) (95.0%). Analysis of hsp60 gene sequences also supported these relationships. Based on the phenotypic and phylogenetic characteristics, the novel species Parabacteroides chinchillae sp. nov. is proposed. The type strain of P. chinchillae sp. nov. is ST166(T) ( = JCM 17104(T) =CCUG 62154(T)).

Contents of various elements in the organs of seabirds killed by an oil spill around Tsushima Island, Japan.

Twenty nine oil-soaked birds were collected from around the Coast of Tsushima Island. The contents of eight elements in the livers and kidneys of the birds were investigated. Statistically higher concentrations of vanadium and thallium in the liver and of titanium in the kidney were found in the birds that were found dead compared with those that died after rescued. A significant correlation (r=0.695, P<0.01) was observed only for the molybdenum content between the kidneys and livers from the birds found dead. Although the controls of the eight elements of birds investigated in the present study remain unexplained, some of lower concentration in rescued birds can be blamed on a decrease in food intake of birds. The relation between oil contamination and concentration of elements need to be further explored.

Relation of reproductive performances and rectal palpation for luteum function of heifers 7 days after estrus.

Diagnosis of corpus luteum (CL) function by rectal palpation (RP) has been widely used for recipient selection of embryo transfer (ET), a technology essential for genetic improvements in cattle. To examine the accuracy of RP diagnosis method, the relationship between RP-based CL function and reproductive performance was compared in this study. In Experiment 1, CL of Holstein heifers on day 7 after estrus was classified into functional or hypoplastic by RP, and the results were compared with ultrasonographic (US) images and plasma progesterone (P4) levels. As a result, heifers with functional CL judged by RP had a mean maximum CL diameter of 20.1 ± 3.1 mm on US and a mean P4 concentration of 8.1 ± 2.3 ng/mL. These values were significantly greater than those of heifers with hypoplastic CL (12.4 ± 5.4 mm, 4.0 ± 2.8 ng/mL) (P < 0.001). In Experiment 2, the length of the estrus cycle was examined between functional CL and hypoplastic CL. The rate of heifers with a normal estrus cycle length with 18-25 days was significantly lower with hypoplastic CL than with functional CL (16/24 vs. 43/46, P < 0.01). In Experiment 3, 543 inseminated heifers were similarly classified by CL function by RP 7 days after estrus. The heifers with functional CL showed higher pregnancy rate compared with the heifers with hypoplastic CL (75.2 vs. 47.9%, P < 0.0001). Finally, the CL function of 66 heifers was examined by RP on day 7 post-estrus, and ET was performed in 49 (74.2%) heifers with functional CL. As a result, 27 (55.1%) of them became pregnant. Taken together, these results reconfirm that RP on day 7 after estrus is useful for selection of heifers with functional CL.

Bacteroides stercorirosoris sp. nov. and Bacteroides faecichinchillae sp. nov., isolated from chinchilla (Chinchilla lanigera) faeces.

Strains of gram-negative anaerobic rods were isolated from chinchilla (Chinchilla lanigera) faeces, and three strains, ST161(T), ST33 and ST37(T), were investigated taxonomically. Based on phylogenetic analyses and specific phenotypic characteristics, the three strains were allocated to the genus Bacteroides. Phylogenetic analyses of their 16S rRNA gene sequences revealed that strain ST161(T) formed a distinct line of descent, with highest sequence similarity to strain ST33 (98.7 %) and Bacteroides oleiciplenus JCM 16102(T) (97.7 %). High levels of DNA-DNA relatedness (79-89 %) were found between strains ST161(T) and ST33, but low levels were found between strain ST161(T) and B. oleiciplenus JCM 16102(T) (33-37 %) and between strain ST33 and B. oleiciplenus JCM 16102(T) (33-37 %). These data clearly indicated that strains ST161(T) and ST33 represent a single novel species. 16S rRNA gene sequence analyses showed that strain ST37(T) also formed a distinct line of descent, with highest sequence similarity to Bacteroides acidifaciens JCM 10556(T) (96.5 %) and Bacteroides caccae JCM 9498(T) (95.6 %). Analysis of hsp60 gene sequences also supported these relationships. Based on phenotypic and phylogenetic characteristics, two novel species, Bacteroides stercorirosoris sp. nov. and Bacteroides faecichinchillae sp. nov., are thus proposed. The type strains of B. stercorirosoris and B. faecichinchillae are ST161(T) ( = JCM 17103(T) = CCUG 60872(T)) and ST37(T) ( = JCM 17102(T) = CCUG 60873(T)), respectively. The DNA G+C contents of strains ST161(T) and ST37(T) were 45.7 and 41.0 mol%, respectively.