Kenji Korosue

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

Summary The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.

Three-dimensional ultrasound imaging of the equine fetus

The objective was to assess the optimal procedure for real-time, three-dimensional (3D) ultrasound (US) imaging for assessing the equine fetus during the first half of gestation and the possibility of using 3D US imaging of the equine fetus in clinical applications. Seventeen pregnant mares were examined by 3D US between Days 35 and 180 of gestation. Abdominal and endo-vaginal real-time 3D transducers used in human medicine were used for transrectal and transvaginal examinations, respectively. Images were recorded by both 3D stationary and real-time movies. In a comparison of four methods, transrectal examination with a bulb-shaped abdominal 3D transducer enabled the equine fetus to be clearly visualized, and did not require sedation of the mare. Therefore, this approach was the most suitable procedure for examining equine fetuses during the first half of gestation. Each scan required only a few seconds and an entire examination took <10 min in total. The 3D volume image was easy to restore after the examination and could be rotated to any angle the examiner desired. Fetal surface structures, including the head, body, limbs, and genital tubercle, were observed as 3D images which enabled fetal development to be characterized. For early (Days 60-70), but not later (Days 90-150) periods, 3D ultrasonography was not able to evaluate fetal structure in detail as well as conventional 2D ultrasonography. In conclusion, 3D ultrasonography of the equine fetus was a valuable adjunct to 2D ultrasonography and a convenient modality for more detailed assessment of fetal structures.

Comparison of growth and endocrine changes in Thoroughbred colts and fillies reared under different climate conditions

ABSTRACT Development and endocrine changes in Thoroughbreds colts and fillies were compared between those reared at two facilities of the Japan Racing Association, the Hidaka Training and Research Center (Hidaka) and Miyazaki Yearling Training Farm (Miyazaki). Thoroughbred colts and fillies born in Japan between 2003 and 2010 were used. Each colt group and filly group was divided into 2 groups, respectively, and raised in Hidaka or Miyazaki for 7 months from September at 1 year old to April at 2 years old. For the growth parameters, the body weight, height at withers, and girth and cannon circumferences were measured once a month. For parameters of endocrine function, circulating prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin-like growth factor-1 (IGF-1), testosterone, progesterone, and estradiol-17β levels were measured. Regarding growth, the rate of increase over the 7-month period was significantly higher in both colts and fillies raised in Miyazaki than in Hidaka in all 4 parameters: body weight, height at withers, and girth and cannon circumferences. The endocrine changes of the colts and fillies born in 2007 were as follows. In colts, although circulating prolactin tended to be higher in colts reared in Hidaka from October to April, circulating LH, FSH, testosterone, estradiol-17β and IGF-1 tended to be higher in colts reared in Miyazaki than in Hidaka, suggesting that the gonadotropin-releasing hormone-LH/FSH system and the growth hormone-IGF-1 system were more active in colts reared in Miyazaki as compared with those reared in Hidaka. In fillies, circulating prolactin tended to be higher in fillies reared in Hidaka in February and March, but no significant difference was noted in the serum LH, FSH, IGF-1, or progesterone level between the 2 groups. Circulating estradiol-17β tended to be higher in fillies reared in Miyazaki than in Hidaka in October and November. Regarding ovarian function, the initial ovulation occurred by the end of March in 2 (16.7%) of 12 fillies reared in Hidaka and 7 (38.9%) of 18 fillies reared in Miyazaki, suggesting that the ovarian function was more active in fillies reared in Miyazaki as compared with those reared in Hidaka. Based on these findings, it was clarified that development of the body and gonads was faster in Miyazaki compared with Hidaka in both colts and fillies.

Comparison of pH and refractometry index with calcium concentrations in preparturient mammary gland secretions of mares

OBJECTIVE To test the usefulness of measuring pH and refractometry index, compared with measuring calcium carbonate concentration, of preparturient mammary gland secretions for predicting parturition in mares. DESIGN Evaluation study. ANIMALS 27 pregnant Thoroughbred mares. PROCEDURES Preparturient mammary gland secretion samples were obtained once or twice daily 10 days prior to foaling until parturition. The samples were analyzed for calcium carbonate concentration with a water hardness kit (151 samples), pH with pH test paper (222 samples), and refractometry index with a Brix refractometer (214 samples). The sensitivity, specificity, and positive and negative predictive values for each test were calculated for evaluation of predicting parturition. RESULTS The PPV within 72 hours and the NPV within 24 hours for calcium carbonate concentration determination (standard value set to 400 μg/g) were 93.8% and 98.3%, respectively. The PPV within 72 hours and the NPV within 24 hours for the pH test (standard value set at 6.4) were 97.9% and 99.4%, respectively. The PPV within 72 hours and the NPV within 24 hours for the Brix test (standard value set to 20%) were 73.2% and 96.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the pH test with the standard value set at a pH of 6.4 would be useful in the management of preparturient mares by predicting when mares are not ready to foal. This was accomplished with equal effectiveness of measuring calcium carbonate concentration with a water hardness kit.

Effects of pre-shipping marbofloxacin administration on fever and blood properties in healthy Thoroughbreds transported a long distance.

The present study evaluated the effects of single-dose marbofloxacin in protecting horses against fever associated with transportation using 48 healthy Thoroughbreds. All horses were premedicated with interferon-α (0.5 U/kg, sublingually, every 24 hr) for 2 days before transportation and on the day of transportation. Horses were randomly assigned to receive marbofloxacin (2 mg/kg, IV, once; MRFX group), enrofloxacin (5 mg/kg, IV, once; ERFX group) or saline (0.9% NaCl) solution (10 ml, IV, once; control group) ≤1 hr before being transportation. Each group contained 16 horses (8 males, 8 females). Horses were transported 1,210 km using commercial vans over the course of approximately 26 hr. Clinical examinations and hematologic analyses were performed on all horses both before and after transportation. Post-transportation neutrophil to lymphocyte ratios were significantly lower in horses in the MRFX group compared with the control horses. The serum amyloid A levels were significantly lower in horses in the MRFX group and ERFX group compared with the control horses. Regarding the post-transportation rectal temperatures, fever was detected in 0 horses and 1 horse in the MRFX and ERFX groups, respectively, whereas fevers exceeding 39.1°C were detected in 2 horses in the control group. Additionally, the number of essential post-transportation treatments provided by veterinarians was reduced 3-fold in the MRFX and ERFX groups compared with the saline group. MRFX provided ERFX-like protection against fever associated with long-distance transportation, yielding significantly better protection than saline. Administration of MRFX just before transportation deserves a further study for efficacy in preventing horse fever associated with transportation.

Effects of ozonated autohemotherapy on the antioxidant capacity of Thoroughbred horses

The performance of horses undergoing regular intense exercise is adversely affected by oxidative stress. Thus, it is important to increase antioxidant production in horses in order to reduce oxidative stress. Ozonated autohemotherapy (OAHT) reportedly promotes antioxidant production. This study aimed to evaluate the effects of OAHT on antioxidant capacity. Ten Thoroughbred horses were used in this study. After the OAHT, we collected serum samples and measured biological antioxidant potential (BAP). We found that BAP began to increase after the OAHT and was significantly higher in the OAHT group than at 3 (P<0.01) and 7 days (P<0.05) after OAHT than in the control group at 3 and 7 days after starting collection of blood samples. Therefore, it was shown that OAHT improved the antioxidant capacity of the horses.

Different effects of an extended photoperiod treatment on growth, gonadal function, and condition of hair coats in Thoroughbred yearlings reared under different climate conditions

ABSTRACT One- to two-year-old Thoroughbred colts and fillies being reared in Miyazaki (warm climate) and Hidaka (cold climate), Japan, were administered extended photoperiod (EP) treatment between December 20 and the following April 10, and its effect on growth, endocrine changes, gonadal activation, and hair coat condition was investigated. In colts reared in Miyazaki, no effect of EP treatment was noted on the growth indices, including body weight (BW), height at withers (HW), girth, and cannon circumference (CC), whereas the BWs and CCs of fillies were significantly higher in the EP treatment group than the control. In Hidaka, the BWs and HWs of colts and HWs of fillies were significantly higher in the EP treatment group. Gonadal activation characterized by an increase in circulating hormone concentrations was earlier in the EP treatment group for fillies reared in Miyazaki [luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), and estradiol-17β (E2)] and in colts (LH, testosterone, and E2) and fillies (LH, FSH, P4, and E2) reared in Hidaka. Regardless of sex and climate, prolactin was significantly higher in the EP treatment group, whereas insulin-like growth factor (IGF-I) was not. Initial ovulation occurred before April in more of the EP treatment group than the control regardless of the climate. Molting of the hair coat, examined in March, was advanced in the EP treatment group regardless of sex and climate. These results suggest that EP treatment may promote growth and gonadal activation in fillies reared in Miyazaki and in colts and fillies reared in Hidaka and that the effect may be mediated by prolactin.

Abnormal pregnancies associated with deviation in progestin and estrogen profiles in late pregnant mares: A diagnostic aid

Measurement of progestines and estrogens in maternal plasma has been advocated as an indicator of foeto-placental function. The goals of the present study were as follows: to determine progestin and estrogen concentrations in mares with normal and abnormal pregnancies during late gestation, evaluate the diagnostic value of hormone concentrations, and investigate the relationship between hormone concentrations after treatment and the survival of foals. Blood samples were collected monthly from 459 pregnant mares between Day 201 of gestation and foaling. Progestin and estrogen concentrations were measured using a time-resolved fluoroimmunoassay with anti-progesterone and anti-17β-estradiol antibodies. The cutoff values of hormone concentrations that best discriminated between healthy foals and foal loss groups were determined using receiver-operating characteristic (ROC) curve analysis. Mares with compromised pregnancies are treated with progestins, tocolytics and antibiotics according to clinical signs, ultrasonographic examination and hormonal test. We investigated the relationship between the restoration of hormone concentrations after treatment and the survival rate of the foals of 62 compromised mares. Maternal serum progestin and estrogen concentrations on Days 241-320 of gestation differed significantly between healthy foals and foal loss of each group. Predictions of pregnancy outcomes were based on ROC curve analysis that established cutoff values of serum progestin and estrogen concentrations for each 20-day interval during gestation. All mares with improved concentrations of progestins or estrogens after treatment delivered healthy foals. When hormone concentrations got worse after treatment, the rate of loss was higher when treatment was administered during Days 201-290 of gestation than after Day 290. Mares with high progestin and low estrogen concentrations were likely to deliver aborted/dead foals during mid-to-late gestation. These results suggest that measurement of progestin and estrogen concentrations may be useful for diagnosing abnormal pregnancy and implementing early treatment strategies.

Isolation and characterization of equine dental pulp stem cells derived from Thoroughbred wolf teeth

Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are capable of self-renewal and differentiation into multiple cell lineages. Methods for cell therapy using MSCs have been developed in equine medicine. Recently, human dental pulp stem cells (DPSCs) have drawn much attention owing to their trophic factor producing ability and minimally invasive collection methods. However, there have been no reports on equine dental pulp-derived cells (eDPCs). Therefore, the aim of this study was to isolate and characterize the eDPCs from discarded wolf teeth. Plastic-adherent spindle-shaped cells were isolated from wolf teeth. The doubling time of the isolated eDPCs was approximately 1 day. Differentiation assays using induction medium eDPCs differentiated into osteogenic, chondrogenic and adipogenic lineages. The eDPCs expressed mesenchymal makers (CD11a/18, CD44, CD90 CD105 and MHC class I and II), but did not express hematopoietic markers (CD34 and CD45). Taken together, the results show that eDPCs can be isolated from discarded wolf teeth, and they satisfy the minimal criteria for MSCs. Thus, these eDPCs can be referred to as equine DPSCs (eDPSCs). These eDPSCs may become a new source for cell therapy.

The cross‐sectional area changes in digital flexor tendons and suspensory ligament in foals by ultrasonographic examination

REASONS FOR PERFORMING STUDY Flexural deformities are common conditions of growing horses and are suggested to have a relationship with the contraction of musculotendinous units. However, limited studies have documented the changes in each tendon and ligament in the metacarpal region with age. OBJECTIVES To investigate the changes in the cross-sectional area (CSA) of each tendon and ligament in the metacarpal region with age by ultrasonographic examination. STUDY DESIGN Longitudinal study of foals from Day 1 to age 24 months. METHODS The CSA of the superficial digital flexor tendon, deep digital flexor tendon, accessory ligament of the deep digital flexor tendon and suspensory ligament was measured by ultrasonographic examination at monthly intervals from Day 1 to age 24 months in 7 Thoroughbred foals. RESULTS Changes in superficial digital flexor CSA in all regions were larger than those of other structures from 10 months to 15 months. The suspensory ligament CSA was significantly larger than those of other structures on Day 1 in both the region of suspensory origin (RSO) and region of suspensory body (RSBO). This condition continued until 2 months in the RSO and until 5 months in the RSBO. The changes in deep digital flexor CSA were larger than those of other structures from 2 to 5 months in both the RSO and RSBO. CONCLUSIONS The rate of change in each structure varies with age. Thus, the functional adaptation with age that takes place may differ among structures because the primary function of each structure differs.