Evaluation of Lipopolysaccharide and Interleukin-6 as Useful Screening Tool for Chronic Endometritis.

Universal diagnostic criteria for chronic endometritis (CE) have not been established due to differences in study design among researchers and a lack of typical clinical cases. Lipopolysaccharides (LPSs) have been reported to cause inflammation in the reproductive systems of several animals. This study aimed to elucidate the influence of LPS in the pathogenesis of CE in humans. We investigated whether LPS affected cytokine production and cell proliferation in the endometrium using in vivo and in vitro experiments. LPS concentrations were analyzed between control and CE patients using endometrial tissues. LPS administration stimulated the proliferation of EM-E6/E7 cells derived from human endometrial cells. High LPS concentrations were detected in CE patients. LPS concentration was found to correlate with IL-6 gene expression in the endometrium. Inflammation signaling evoked by LPS led to the onset of CE, since LPS stimulates inflammatory responses and cell cycles in the endometrium. We identified LPS and IL-6 as suitable candidate markers for the diagnosis of CE.

Quantitative analysis of the skull in the Japanese wolf (Canis lupus hodophilax) using CT.

In this study using computed tomography (CT), the volumes of the internal cranial cavities, such as the braincase, frontal sinus and tympanic cavity, and the ratio of the volume of each cavity to the skull volume in Japanese wolves were quantified, and CT images of the frontal sinus were observed. The results were then compared with those of other wolf subspecies, including Akita, a dog breed, to clarify the characteristics of the internal cranial cavities in Japanese wolves. The present study revealed that the Japanese wolf had a relatively larger braincase volume and a relatively smaller frontal sinus volume than the wolf ssp. (a group of wild wolf subspecies except the Japanese wolf) and Akita. Moreover, the relative and absolute tympanic cavity volumes of the Japanese wolf and Akita were significantly smaller than those of the wolf ssp. In the CT image or macroscopic observations, the frontal sinuses of the wolf ssp. and Akita were relatively well developed to the caudal and dorsal directions, respectively, compared with that of the Japanese wolf, and the tympanic cavity of the wolf ssp. was more largely swelled ventrally and medially than that of other groups.

Ultrashort Echo Time Quantitative Magnetic Resonance Imaging of the Cruciate Ligaments in Normal Beagles.

OBJECTIVE: The aim of this study was to provide normative ultrashort echo time magnetic resonance imaging (UTE MRI) data of the patellar ligament (PL), cranial cruciate ligament (CrCL) and caudal cruciate ligament (CdCL) in non-lame Beagles. STUDY DESIGN: Eight stifles from four subjects obtained immediately postmortem were imaged using UTE MRI in the true sagittal plane. Regions of interest were drawn manually and the total (T2*), short T2* (T2*S) and long T2* (T2*L) values of the signal decay were calculated to evaluate the bound and free water components of the tendon. The T2*S, T2*L and T2* values were compared between the PL, CrCL and CdCL RESULTS: The mean and standard deviation of T2*S, T2*L and T2* were as follows: 0.54 ± 0.13, 4.65 ± 1.08 and 8.35 ± 0.82 ms for the PL; 0.46 ± 0.14, 5.99 ± 0.52 and 8.88 ± 0.4 ms for the CrCL and 0.41 ± 0.13, 7.06 ± 0.57 and 9.26 ± 0.18 ms for the CdCL. Significant differences were found between the T2*L component of the PL and each CrCL/CdCL and a smaller difference was noted between the T2*L of the CrCL and CdCL (p = 0.05). No difference of the T2*S value was found between any of the ligaments. CONCLUSION: Establishing normative UTE data of the canine stifle is valuable for comparison in future studies in which normal and damaged ligaments may be evaluated, particularly in those affected limbs in which no instability is identified on physical examination in which normal and damaged ligaments may be evaluated.

Pathogen and severity-dependent immune responses in bovine mastitis: highlight the dynamics of differential somatic cell count.

Immune responses in bovine clinical mastitis (CM) probably differ depending on the causative pathogen and disease severity. The observational study aimed to investigate whether both factors are associated with the dynamics of immune indicators, including somatic cell score (SCS), white blood cell count (WBC), serum albumin/globulin (A/G) ratio, and differential somatic cell count (DSCC). We collected blood and milk samples 0, 3, 5, 7, 14, and 21 days after CM occurred in 38 cows, and grouped the cases (n=49) by disease severity and pathogen. We analyzed data using a linear mixed model considering the effects of pathogens and severity, calculated estimated-marginal means for indicators at each time point, and compared the means between groups. The dynamics of WBC varied depending on both pathogen and severity. WBC changed drastically in either severe or coliform-caused CM, slightly elevated in streptococcal mastitis, but unchanged in staphylococcal mastitis. This possibly relates to the deficiency in innate immune response toward staphylococci. The A/G ratio also changed depending on severity, as it dropped sharply only in severe CM. We observed a non-linear relationship between DSCC and SCS, possibly due to mammary epithelial cells shedding in milk when CM occurred. When cows recovering from Streptococcus dysgalatiae mastitis, DSCC decreased while SCS remained high, suggesting a healing process requiring more macrophages. Our results demonstrate that both the severity and pathogen are associated with immune responses in CM, providing insights into mastitis pathogenesis.

Seasonal Variation and Factors Affecting Trypanosoma theileri Infection in Wild Sika Deer (Ezo Sika Deer Cervus nippon yesoensis) in Eastern Hokkaido.

Trypanosoma (Megatrypanum) spp. are isolated from domestic and wild ruminants, including deer, worldwide. The prevalence of trypanosomes in mammals is influenced by a number of factors such as host age and vector abundance. However, the seasonal variation of and factors affecting trypanosome infection in the wild deer population remain elusive. In this study, we analyzed the seasonal variation in trypanosome prevalence and the factors that affect Trypanosoma theileri Laveran, 1902, infection in wild sika deer (Ezo sika deer) Cervus nippon yesoensis (Heude, 1884) in Eastern Hokkaido through a two-year survey. Seasonal variation in the prevalence of trypanosome infection in the deer population ranged from 0 to 41% as per hematocrit concentration and 17 to 89% as per PCR results. In general, the prevalence of T. theileri by PCR in 2020 was higher than that in 2019. Moreover, the prevalence was significantly higher in the aged population than among the younger population. These findings may explain why individual conditions and sampling season were associated with trypanosome prevalence. This is the first study to investigate the seasonal variation in and risk factors affecting trypanosome infection in wild deer.

LPS Administration during Fertilization Affects Epigenetic Inheritance during Embryonic Development.

Intrauterine inflammation can cause infertility by disrupting reproductive function. The pathogenesis underlying this process may primarily involve endotoxins from lipopolysaccharides (LPS), which are produced by Gram-negative bacteria. However, the long-term effects of endotoxins in mammalian pregnancy following LPS exposure during fertilization have not been clarified. In this study, we performed experiments to analyze the influence of LPS on early embryonic development and fetal development in mice. Mice uteruses were examined for the expression of genes related to the inflammatory response. The expression of Il-1ß and Il-6 increased following the administration of 200 and 1000 µg/kg LPS. Exposure to LPS using in vitro fertilization (IVF) significantly decreased the embryonic developmental rate. A concentration of 100 µg/kg LPS significantly increased the placental weight and fetal crown -rump length (CRL), whereas a concentration of 200 µg/kg LPS significantly decreased the placenta weight and fetal weight in vivo. These findings indicate that maternal LPS during fertilization affects fetal development until the late stage of pregnancy. Thus, maternal endotoxins may affect epigenetic inheritance during embryonic development from the early to late stages of pregnancy.

Risk factors for equine trypanosomosis and hematological analysis of horses in Paraguay.

Animal trypanosomosis, caused by Trypanozoon trypanosomes (Trypanosoma evansi and T. equiperdum), and Trypanosoma vivax, is endemic to South American countries and has a negative impact on the livestock industry. However, the risk factors for trypanosomosis in Paraguay remain unknown. This study aimed to determine the risk factors for equine trypanosomosis in Paraguay based on a PCR-based molecular survey and individual horse sampling data. In this study, 739 blood samples were collected from horses in 16 departments of Paraguay between August 2019 and November 2020. To elucidate the risk factors for trypanosome infection, the relationship between trypanosome infection status detected by PCR and the location, sex, age, breed of horses, and season of sample collection was analyzed. There were no significant differences in trypanosome prevalence in horses between the eastern and western regions, ages, or breeds of horses in Paraguay. Sex and season were identified as risk factors for trypanosome infection in horses in Paraguay in the current study. These results suggest that the rainy-summer season, when vectors increase in number and their blood-sucking activity, could be the most important risk factor for trypanosome infection in Paraguay horses. Preventive measures and treatments should be developed to address these factors.

Genetic and seasonal variations of Trypanosoma theileri and the association of Trypanosoma theileri infection with dairy cattle productivity in Northern Japan.

Trypanosoma theileri is considered a non- or low-pathogenic trypanosome that generally causes latent infection in apparently healthy cattle; however, T. theileri propagates in the bloodstream and may cause clinical disease in pregnant animals or co-infection with bovine leukemia virus or Theileria orientalis. In the current study, a monthly survey of T. theileri infection over one year was carried out in a research dairy farm in Hokkaido, Japan to determine the 1) seasonal variations in the prevalence, 2) genetic characterization of T. theileri, and 3) associations of milk and blood parameters in dairy cattle with T. theileri infection, including data of metabolic profile tests and dairy herd performance tests, using linear mixed models. We found that 1) the prevalence of T. theileri infection was significantly higher in summer and winter than in other seasons; 2) T. theileri possibly showed genetic diversity in Eastern Hokkaido; and 3) T. theileri infection was associated with significantly lower levels of blood urea nitrogen, milk protein, and solids-not-fat, which are caused by a low rumen fermentation level. This is the first study to report the negative impact of T. theileri infection in dairy cattle, and our study indicates that control of T. theileri infection can improve the productivity of dairy cattle.

Effect of Stem Positioning on Biomechanical Performance of a Novel Cementless Short-Stem Canine Total Hip Implant.

OBJECTIVE: The aim of this study was to evaluate the effect of stem positioning on the biomechanical performance of a novel, collared, short-stem total hip implant under compression and torsion ex vivo. STUDY DESIGN: Six canine cadaveric femurs were implanted with a collared short-stem femoral implant. Canal flare index (CFI), stem angle, absolute and relative cut heights and relative size were measured radiographically and used as independent variables. Biomechanical performance of the construct was evaluated using physiologic loading (loading) and supraphysiologic loading (failure) protocols. RESULTS: During loading protocols, compressive stiffness was influenced by absolute cut height (p = 0.018). During failure protocols, peak torque was influenced by CFI (p = 0.004) and craniocaudal relative size (p = 0.005). Peak load and torsional stiffness were not impacted by any of the radiographic variables (p > 0.05). Three of six femurs developed longitudinal fractures originating at the medial calcar at the time of failure. CONCLUSION: The biomechanical performance of the collared short-stem implant was positively impacted by preserving more of the femoral neck, having a higher CFI and using a smaller implant size relative to the femoral neck isthmus.

Is Salmonella enterica shared between wildlife and cattle in cattle farming areas? An 11-year retrospective study in Tokachi district, Hokkaido, Japan.

BACKGROUND: Salmonella enterica in cattle has long been problematic and suspected to be transmitted by wildlife in Tokachi, Hokkaido, a major cattle farming area in Japan. Understanding the role of wildlife in S. enterica transmission would be helpful for developing control strategies of bovine salmonellosis. OBJECTIVES: We aimed to elucidate the possibility of S. enterica transmission between sympatric wildlife, including raccoons and crows and cattle, in Tokachi from 2008 to 2018 by analysing S. enterica detection records, and the genetic relatedness of serotypes shared between wildlife and cattle. METHODS: S. enterica detection records were based on the results of a field survey and existing cattle records at relevant organisations, including clinical reports, a monitoring survey and quarantine for introduced calves at growing farms and public calving farms. S. enterica was identified by polymerase chain reaction assay and serotyped by agglutination assay. The detection records were organised chronologically to investigate whether common serotypes in wildlife and cattle were detected in the same year. The isolates corresponding to detection records were assessed for their genetic patterns by pulsed-field gel electrophoresis. RESULTS: The prevalence of S. enterica in raccoons and crows was 10.7% (17/159) and 5.7% (55/967), respectively. The following serotypes were detected from both wildlife and cattle: Braenderup, Dublin, Infantis, Mbandaka, Montevideo, 4,[5],12:i:- and Typhimurium. Genetically similar isolates for S. Braenderup, S. Dublin, S. Montevideo and S. 4,[5],12:i:- were detected from both species in the same year. CONCLUSIONS: Our long-term retrospective observations supported that S. enterica was shared between wildlife and cattle. Wildlife invasions should be controlled at farms to prevent inter-species transmission of S. enterica from livestock farms.

Fertility risk factors in transferring Japanese Black embryos into dairy heifers: An epidemiological study.

The aims of this study were 1) to summarize the current status of Japanese Black (JB) embryo transfer into Holstein heifers, which is carried out on a commercial basis in Japan, and 2) to reveal fertility risk factors, including those from the environment (year and season of transfer), recipient (age, number of transfers, clinical status of the ovaries) and embryo (quality, stage, state, genetic background). We used data from 4467 JB fresh or frozen embryo transfers into Holstein heifers conducted by Zen-noh Embryo Transfer Center during 2016-2018, and the differences in fertility risk due to factors related to the environment, recipient, and embryo were statistically evaluated. Differences in fertility risk due to each variable were observed, leading to significant differences in fertility with respect to year of transfer, embryo quality, embryo state, and embryo breed. These results suggest that the fertility of JB embryos might depend on differences in genetic background. There have been no previous reports of differences in embryo fertility due to the differences among JB's bloodline combinations. In the future, overall reproductive efficiency must be monitored, including the effects of different bloodline combinations on the success of embryo recovery and transfer.

Effects of the formation and regression of accessory corpus lutea during pregnancy on plasma progesterone concentration and pregnancy status in cross-bred beef heifers.

There was examination of effects of accessory corpus lutea (CLs) formation and regression during pregnancy on circulating progesterone (P4) concentrations and pregnancy maintenance in beef heifers. Heifers (Experiment 1, n = 75; Experiment 2, n = 496) were randomly assigned to either a human chorionic gonadotropin (hCG) treatment or untreated group 5 days post-estrus, followed by embryo transfer (ET) on Days 6-8 (Day 0 = Estrus). In Experiment 1, blood samples were collected from pregnant heifers on Days 33, 40, and 47 for conducting P4 assays. Plasma P4 concentrations were greater in hCG-treated heifers than in untreated heifers on Day 33. In hCG-treated heifers with accessory CL regression between Days 33 and 47, plasma P4 decreased to concentrations similar to those of untreated heifers after Day 40. In hCG-treated pregnant heifers in Experiment 2, CL regression by Day 50 of gestation was more frequent when CLs were contralateral (49.3 %) rather than ipsilateral (4.4 %, P < 0.001) to the original CL. The hCG treatment resulted in a greater pregnancy percentages on Days 30 (80.5 % and 68.6 %, P = 0.002) and 50 (76.2 % and 65.3 %, P = 0.007) compared with untreated heifers. There, however, were no differences in either pregnancy percentages on Days 30 and 50 or pregnancy losses between hCG-treated heifers with ipsilateral and contralateral accessory CLs. These results indicate accessory CL formation improves pregnancy percentages resulting from ET. Furthermore, plasma P4 decreases associated with accessory CL regression does not affect pregnancy loss in beef heifers.

Mobility of the forearm skeleton in the Asiatic black (Ursus thibetanus), brown (U. arctos) and polar (U. maritimus) bears.

In several primates and carnivores, pronation/supination angles of the forearm skeleton were examined, and it is thought that a larger angle is useful to acquire dexterous behaviors in feeding and/or life style, including climbing. In this study, the pronation/supination angles in Asiatic black, brown and polar bears were nondestructively examined. These specimens were classified as adult or non-adult. Three or four carcasses of each group of Asiatic black and brown bears were used for CT analysis, whereas only one adult polar bear was used. The forearms were positioned within the gantry of a CT scanner in both maximally supinated and pronated states. Extracted cross-sectional CT images of two positions were superimposed by overlapping the outlines of each ulna. The centroids of the radii were detected, and then the centroid of each radius and the midpoint of a line which connects between both ends of the surface of each radius facing the ulna, were connected by lines to measure the angle of rotation as an index of pronation/supination. In adult brown and polar bears, the angles were smaller as compared with the other groups (Asiatic black and non-adult brown bears). Asiatic black and non-adult brown bears can climb trees, whereas adult brown bears and polar bears cannot. This suggests that the pronation/supination angle is related to arboreal activity in Ursidae.

Associations between plasma testosterone levels and season, nutritional status, age, and body size in free-ranging male Asian black bears (Ursus thibetanus) in central Honshu, Japan.

Testosterone is an important regulator of male reproduction in terms of spermatogenesis and physiological changes in the accessory reproductive organs. Despite the important role of the testosterone level in male reproduction, testosterone levels vary among male bears even during the breeding season, and the causative underlying factors remain unknown. We postulated that testosterone levels are higher during the breeding season than during other seasons in free-ranging male bears, and that testosterone levels increase with advancing age, larger body size, and better nutritional status. We assessed potential factors (season, time of sampling, body condition index, head circumference, and age) associated with the testosterone level in 80 blood samples collected from free-ranging Asian black bears (Ursus thibetanus) captured in barrel traps in the Ashio-Nikko Mountains and Okutama Mountains in central Honshu Island, Japan. The plasma testosterone level was higher during the breeding season (May-July) than during the non-breeding season (August-November). The body condition index was significantly and positively associated with the plasma testosterone level. None of the other factors were significantly associated with the plasma testosterone level. Therefore, the body condition index may be essential for maintaining high plasma testosterone levels. These findings imply that testosterone secretion might be activated in free-ranging males during the breeding season, and animals with good nutritional status may be able to maintain high plasma testosterone levels. This study uncovered an association between nutritional conditions and reproductive activity in male bears.

Age-Related Arteriolar Changes With Lipid and Amyloid Deposits in the Gonads of Dogs.

Arteriolar lesions with lipid and/or amyloid deposits are frequently detected in canine gonads by routine histopathologic examination; however, they have never been examined in detail. In the present study, a total of 139 testes/epididymides and 200 ovaries from 72 male (4 months to 14 years old) and 105 female (7 months to 16 years old) dogs were examined for arteriolar lesions. Arteriolar lesions were detected in 21 of 72 male dogs (29%) and 54 of 105 female dogs (51%). These lesions were histologically classified into 4 types: "fibromuscular hypertrophy," characterized by thickening of the tunica intima; "focal vasculitis," characterized by mononuclear cell infiltration; "vacuolar change," consisting of lipid accumulation and infiltration of foamy cells; and "hyalinosis," characterized by irregular thickening with amyloid deposits. In the lesions of vacuolar change and hyalinosis, lipid deposition and infiltration of α-SMA-positive cells and Iba-1-positive cells were also observed. Foamy cells and amyloid deposits were immunopositive for apolipoproteins and oxidized low-density lipoprotein-related proteins. These results indicate that vacuolar change is possibly an early stage of atherosclerosis, and that amyloid may deposit as a consequence of the microenvironment associated with atherogenesis. Logistic regression analysis revealed that arteriolar lesions with lipid deposits were associated with age and interstitial cell tumors in male dogs, and with age in female dogs. Aging is likely an important risk factor of arteriolar lesions with lipid deposits of the canine gonads.

Radiographic and Biomechanical Assessment of Three Implant Designs for Canine Cementless Total Hip Replacement.

OBJECTIVE: The aim of this study was to evaluate the relationship between radiographic fit/fill measurements and biomechanical performance of three canine cementless total hip implant designs using an in vitro biomechanical testing protocol that replicates compression and torsion. STUDY DESIGN: Eighteen (six/group) canine cadaveric femurs were implanted with one of three cementless total hip implant designs: (1) collarless, (2) collared or (3) lateral bolt stems. Femoral length, canal flare index (CFI), canal fill, stem fit, stem level and stem angle were measured as independent variables. Biomechanical performance was tested using physiological, non-destructive gait loading (loading protocols) and destructive testing (failure protocols). RESULTS: During loading protocols, compressive stiffness was influenced by stem level (p < 0.05) and torsional stiffness was influenced by stem level and CFI for collarless stems (p < 0.05). During failure protocols, peak load was influenced by mediolateral (ML) stem angle (p < 0.05) and CFI (p < 0.01) for collarless stems and CFI for lateral bolt stems (p < 0.05). Peak torque was influenced by ML stem angle, craniocaudal stem angle and CFI for collarless stems (p < 0.05) and average ML fill for collared stems (p < 0.05). CONCLUSION: Biomechanical performance of collarless stems in cementless hip arthroplasty is more impacted by radiographic fit/fill than lateral bolt and collared stems. As a result, collarless stems may be more dependent on preoperative fit and intraoperative precision.

Behavioral effects of scents from male mature Rathke glands on juvenile green sea turtles (Chelonia mydas).

Sea turtles can detect airborne and waterborne odors, but whether they recognize scents from the same species and if so, how they affect their behavior remains unknown. The present study evaluated the behavioral effects of odorants on juvenile green sea turtles (Chelonia mydas). The odorants were derived from Rathke glands (external scent glands) of mature male green sea turtles, and from two types of food. The activity of the juveniles increased when exposed to food scents, and significantly decreased compared with controls when exposed to scents from Rathke glands. These findings indicated that scents from the same species affect behavior, and that chemical communication via olfaction has important outcomes for sea turtles.

Nationwide serological surveillance of non-tsetse-transmitted horse trypanosomoses in Mongolia.

In Mongolia, horses play important roles, not only in livestock production, but also in terms of culture, tradition, and Mongolian beliefs. Although the presence of non-tsetse-transmitted horse trypanosomoses, which are caused by infections with Trypanosoma evansi (surra) and T. equiperdum (dourine), has been reported in the country, whether there is a nationwide epidemic of these infectious diseases is unknown. In the present study, a nationwide surveillance of horse trypanosomoses was performed. The sample sizes for each province, the whole country, and male and female horses were, respectively, 96, 2,400, and 316 and 306. In total, 3,641 samples of horse sera were collected by simple random sampling. The rTeGM6-4r-based ELISA, which was applied for surra against cattle and water buffalo and dourine against horse, revealed that the overall sero-prevalence of the diseases in Mongolia was 4.8%. Among them, high sero-prevalences were observed in the central provinces (5.2-11.0%, p < 0.05) of the country. The sero-prevalence was significantly higher in females than in males (6.0% and 4.0%, p < 0.05, respectively) and in non-castrated males (8.4%, p < 0.01) compared with castrated males (3.0%). These results suggested that currently, horse trypanosomoses are a nationwide endemic problem in Mongolia. Knowledge of the nationwide endemic status of non-tsetse-transmitted horse trypanosomoses in Mongolia will be useful to prevent these diseases.

Effects of hCG administration on corpus luteum development and plasma sex steroid hormone concentration in beef heifers differ according to the locational relationships of the original corpus luteum and the first-wave dominant follicle.

The aims of this study were to compare the effects of an intramuscular human chorionic gonadotropin (hCG) administration on corpus luteum (CL) development, plasma progesterone (P4) and estradiol (E2) concentration in ipsilateral (first-wave dominant follicle [W1DF] in the same ovary as the CL) and contralateral (W1DF and CL in opposite ovaries) cattle. Cross-bred beef heifers (Holstein × Japanese black, n=83) with synchronized ovulation were randomly assigned to either treatment with 1,500 IU hCG or no treatment on day 5 post-ovulation and were subdivided into ipsilateral (hCG treatment, n=21; no treatment, n=23) or contralateral (hCG treatment, n=17; no treatment, n=17) groups. Five heifers were excluded from the study, as they presented with double ovulation in response to hCG treatment. The effects of hCG treatment, location (ipsilateral and contralateral), and the number of days post-ovulation (days 5, 7, and 14) were analyzed using three-way ANOVA. hCG treatment significantly increased CL diameter on day 7 and plasma P4 concentration on days 7 and 14 in the contralateral group, but not the ipsilateral group. In contrast, hCG treatment decreased plasma E2 concentration on days 7 and 14 in both groups. In summary, our results indicate that the hCG treatment more significantly promoted CL development and increased plasma P4 concentration in the contralateral than in the ipsilateral group.

Blood markers of osteoclastic differentiation in periparturient dairy cows at different parities, with and without milk fever.

Osteoprotegerin (OPG) inhibits osteoclast (OC) differentiation. TRAP5b (tartrate-resistant acid phosphatase 5b) secreted by OCs reflects the numbers of mature OCs. This study assessed these OC-related markers around parturition in cows of different parities and in cows with milk fever (MF). The blood OPG and TRAP5b concentrations, as well as the ratio of OPG to TRAP5b (O/T), were measured beginning 3 weeks before (-21 d) and over a few days after calving in 49 Holstein Friesian cows at first (n = 8), second (n = 17), third (n = 12), and fourth or greater (n = 12) parities. The ratio of O/T at -21 d to O/T at calving (PreCOT) was also calculated. In the third and greater parities, seven cows developed MF (non-MF, n = 17). Regardless of the development of MF, the serum OPG started to decline in the last week of gestation only in the cows entering the second lactation, while the blood TRAP5b increased at calving in all cows. O/T decreased toward parturition only in multiparous cows. The decrease in O/T at caving was less pronounced in MF cows. PreCOT was negatively correlated with lactation number only in multiparous cows (n = 41, ρ = -0.50, P < .01). This study implied that OC differentiation toward calving was tapered in cows with advanced parities, and these indexes predict the risk of MF.