Prediction of the Spontaneous Estrus Expression Period Based on Large (≥10 mm) Follicle Numbers in Lactating Holstein Dairy Cows.

The objectives of this study were (1) to investigate the distribution of large (≥10 mm) follicle numbers during the estrous cycle and (2) to compare the timing of the estrus expression period after the ovarian examination between cows with one large follicle (1F) and two or more large follicles (2F) with functional corpus luteum (CL) at the ovarian examination in lactating Holstein dairy cows. In experiment 1, we performed 393 ovarian examinations by ultrasonography, addressed the existence of CL (≥20 mm) and large follicle numbers, and classified cows into 1F (n = 229) and 2F (n = 164) groups. The 1F appearance rates were beyond 75% each day during 3 to 12 d after estrus. However, 2F appearance rates were beyond 75% each day during 15 to 24 d after estrus. In experiment 2, we performed 302 ovarian examinations by ultrasonography and classified cows into the 1F (n = 168) and 2F (n = 134) groups. Estrus detection was performed for 24 d after the ovarian examination in each cow. In the 2F group, 75% of estrus occurred within 9 d of the ovarian examination. However, 75% of estrus occurred 10 d after the ovarian examination in 1F. Days from the ovarian examination to estrus were significantly shorter in the 2F (6.0 d; median, 7.2 ± 4.0 d; mean ± SD) than in the 1F (13 d, 12.4 ± 4.3 d) group. In conclusion, focusing on ≥10 mm follicle numbers with CL could be useful for predicting the estrus expression period.

Evaluation of Prolonged Endometrial Inflammation Associated with the Periparturient Metabolic State in Dairy Cows.

The objectives of this study were to assess the sequential dynamics of the endometrial polymorphonuclear cells (PMN) after calving by endometrial cytology, and clarify the factors that cause prolonged endometrial inflammation in lactating dairy cows. A total of 33 lactating Holstein dairy cows were used from -4 to 8 wk relative to calving (0 wk: the calving week). Endometrial samples were obtained sequentially from 2 to 8 wk. Body condition score and backfat thickness were obtained weekly from -4 to 8 wk. Blood samples collected from -4 to 8 wk were analyzed for indicators of energy status, hepatic function, systemic inflammation, and calcium. Blood amino acids were measured at 2 wk. Daily milk production was determined between 5 and 65 d postpartum. Based on the sequential cytological analysis, the endometrial inflammation threshold was set at ≥5.0% PMN, and the median wk of PMN% lower than 5.0% was 4.5 wk in this study; therefore, we classified the cows into the early group (cows with endometrial inflammation converged within 4 wk: n = 17) and the late group (cows with endometrial inflammation converged at or after 5 wk: n = 16). There were no differences in daily milk production, energy status, hepatic function, blood calcium concentration, and systemic inflammatory response. The late group had lower body condition scores and backfat thickness during the experimental period, and a higher blood concentration of 3-methyl histidine, indicating muscle breakdown, was observed in the late group at 2 wk. Our findings indicated that the lack of body fat reservation during the peripartum period and the increased muscle breakdown after calving were risk factors for prolonged endometrial inflammation.

Bovine endometrium-derived cultured cells are suitable for lipofection.

Bovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.

Metabolomic profiles of plasma and uterine luminal fluids from healthy and repeat breeder Holstein cows.

BACKGROUND: Repeat breeding is a critical reproductive disorder in cattle. The problem of repeat breeder cattle remains largely unmanageable due to a lack of informative biomarkers. Here, we utilized metabolomic profiling in an attempt to identify metabolites in the blood plasma and uterine luminal fluids. We collected blood and uterine fluid from repeat breeder and healthy cows on day 7 of the estrous cycle. RESULTS: Metabolomic analysis identified 17 plasma metabolites detected at concentrations that distinguished between the two groups, including decreased various bile acids among the repeat breeders. However, no metabolites that varied significantly were detected in the uterine luminal fluids between two groups. Among the plasma samples, kynurenine was identified as undergoing the most significant variation. Kynurenine is a metabolite produced from tryptophan via the actions of indoleamine 2,3-dioxygenase (IDO). As IDO is key for maternal immune tolerance and induced in response to interferon tau (IFNT, ruminant maternal recognition of pregnancy factor), we examined the responsiveness to IFNT on peripheral blood mononuclear cells (PBMC) isolated from healthy and repeat breeder cows. The mRNA expression of IFNT-response makers (ISG15 and MX2) were significantly increased by IFNT treatment in a dose-dependent manner in both groups. Although treatment with IFNT promoted the expression of IDO in PBMCs from both groups, it did so at a substantially reduced rate among the repeat breeder cows, suggesting that decreased levels of kynurenine may relate to the reduced IDO expression in repeat breeder cows. CONCLUSIONS: These findings provide valuable information towards the identification of critical biomarkers for repeat breeding syndrome in cattle.

Analysis of lipoprotein profiles of healthy cats by gel-permeation high-performance liquid chromatography.

Density gradient ultracentrifugation (DGUC) and gel electrophoresis are conventionally used to obtain lipoprotein profiles of animals. We recently applied high-performance liquid chromatography with a gel permeation column (GP-HPLC) and an on-line dual enzymatic system to dogs for lipoprotein profile analysis. We compared the GP-HPLC with DGUC as a method to obtain a feline lipoprotein profile. The lipoprotein profiles showed large and small peaks, which corresponded to high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively, whereas very low-density lipoprotein (VLDL) and chylomicron (CM) were only marginally detected. This profile was very similar to that of dogs reported previously. Healthy cats also had a small amount of cholesterol-rich particles distinct from the normal LDL or HDL profile. There was no difference in lipoprotein profiles between the sexes, but males had a significantly larger LDL particle size (P=0.015). This study shows the feasibility of GP-HPLC for obtaining accurate lipoprotein profiles with small sample volumes and provides valuable reference data for healthy cats that should facilitate diagnoses.

Is experience on a farm an effective approach to understanding animal products and the management of dairy farming?

The understanding of animal products and dairy farming is important for the promotion of dairy farming. Thus, to examine the effects of farm experience on the understanding of animal products and the management of dairy farming, the interaction between students and dairy cows was investigated in groups of first-year veterinary nursing students in 2011 and 2012 (n = 201). These students included 181 women and 20 men. Nine items about dairy cows were presented in a questionnaire. The survey was performed before and after praxis on the educational farm attached to the authors' university. After praxis on the farm, increases occurred in the number of positive responses to the items involving the price of milk, dairy farming and the taste of milk. For these items, a significant difference (P < 0.05) was found between the scores obtained before and after training. The results of the study suggested that farm experience is useful for improving the understanding of animal products and dairy farming.

Effect of estrus synchronization treatment after luteolysis on Holstein heifers as embryo transfer recipients.

This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 µg GnRH (gonadotropin-releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 µg GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 1-3 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.

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.

Effects of vitamin E on the osteoblast differentiation.

Vitamin E is thought to affect bone formation and bone remodeling. In this study, we investigated the effects of vitamin E (alpha-tocopherol and delta-tocopherol) on the osteoblasts isolated from rat calvariae. At 4 and 7 days (Day 4 and 7) after induction of osteoblastic differentiation, treatment of alpha-tocopherol (100 and 200 microM) and delta-tocopherol (2 and 20 microM) for 3 days significantly decreased alkaline phophatase activity of the cultured osteoblasts. At Day 14, however, no significant change was detected in ALP activity and expression of bone sialoprotein mRNA in the osteoblasts treated with alpha-tocopherol or delta-tocopherol for 3 days. Expression of osteocalcin mRNA was decreased by treatment of alpha-tocopherol (100 and 200 microM) and delta-tocopherol (2 and 20 microM) at Day 4 and 7. At Day 14, expression of osteocalcin mRNA was decreased only with treatment of 200 microM alpha-tocopherol. In addition, the noncalcified nodules were decreased by treatment of alpha-tocopherol (200 microM) and delta-tocopherol (20 microM) at Day 7. However, treatment of alpha-tocopherol and delta-tocopherol showed no significant change of formation of calcified nodules at Day 14. These results indicate that vitamin E inhibits differentiation of osteoblasts especially from early stage to osteoid-producing stage.

Application of gel permeation HPLC for lipoprotein profiling in dogs.

A high performance liquid chromatography system with a gel permeation column (GP-HPLC) and an on-line dual enzymatic system was applied to lipoprotein analysis in dogs. A high density lipoprotein (HDL) fraction obtained by conventional ultracentrifugation gave a single peak at around 28-29 min. Similarly, a low density lipoprotein (LDL) fraction gave single peak at around 24-25 min. The lipoprotein profiles of healthy dogs were contained large HDL peaks and small LDL peaks, and VLDL and CM were only marginally detected. In diabetic dogs, concentrations of VLDL-triglyceride and VLDL-total-cholesterol were elevated significantly. The lipoprotein profile analysis by GP-HPLC method would be useful in explication of abnormality of lipid metabolism in dogs.

Changes in peripheral lymphocyte subsets in the type 1 diabetic dogs treated with insulin injections.

Plasma metabolites and peripheral lymphocyte subsets were measured in ten diabetic and ten control dogs to investigate their significances as indicators to evaluate immune states in the diabetic dogs. Diabetic dogs were treated with insulin injections, however their plasma glucose and fructosamine concentrations were significantly higher than those of the controls. There were no significant differences in counts of total white blood cells (WBC) and lymphocyte CD8(+) cells (cytotoxic T cells) between the control and the diabetic dogs. In the diabetic dogs, the counts of CD3(+) (T cells), CD4(+) (Helper T cells) and CD21(+) (B cells) cells and the peripheral lymphocytes CD4/CD8 ratio were significantly lower than those in the control dogs. We confirmed abnormality of lymphocyte subsets in insulin treated diabetic dogs and it may relate to depression of immunocompetence and high susceptibility to common infectious diseases.

Comparison of enzyme activities in plasma and leukocytes in dairy and beef cattle.

Concentrations of plasma glucose, immunoreactive insulin (IRI) and free fatty acid (FFA) and activities of enzymes related to energy metabolism and lactate dehydrogenase (LDH) isoenzyme pattern in plasma and leukocytes were investigated in lactating Holstein cows (dairy cattle) and fattening Japanese Black Wagyu x Holstein steers (beef cattle). IRI concentrations and LDH and malate dehydrogenase (MDH) activities in the plasma of beef cattle were significantly higher than those in dairy cattle. The cytosolic ratio of MDH/LDH activity in the leukocytes of beef cattle was significantly higher than that of dairy cattle. These findings might be associated with the different energy metabolism between dairy and beef cattle.