Outline of the Completed Research

Equine Science

Computed tomographic investigation of developmental orthopedic disease in young horses (2014−2015)

 We investigated that the use of computed tomography (CT) combined with computer-aided diagnosis (CAD) for the diagnosis of developmental orthopedic disease in young horses. In an experimental study, 5 Thoroughbred foals underwent CT myelography, and the ratio of stenosis in the cervical cord was quantitatively evaluated. The sites of stenosis were consistent with the sites of primary lesions as determined by histopathological examination. In clinical cases, the presence of fragments, bone remodeling, and sclerosis was confirmed by CT combined with CAD, despite the absence of these features on radiographs. In conclusion, CT combined with CAD is useful for diagnosing developmental orthopedic disease in horses.

Endocrine function of racehorses reared in cold climates: comparison of pituitary, thyroid, and adrenal functions in horses reared in Hidaka and Miyazaki, Japan (2012-2014)

 To determine the influence of cold climates on equine growth and endocrine function, Thoroughbred colts and fillies were reared at the Miyazaki Yearling Training Farm (Miyazaki; warm climate) and the Hidaka Training and Research Centers (Hidaka; cold climate) of the Japan Racing Association (JRA). In addition, light treatment was given to Thoroughbreds colts and fillies at the two farms and its effects on growth, gonadal function, and endocrine changes were compared. At both sites, a 100-W white lamp was set in the ceilings of the stalls from December 20 to April 10 and light treatment (14.5-h light; 9.5-h dark) was performed. Plasma concentrations of prolactin, luteinizing hormone, follicle-stimulating hormone, insulin like growth factor, testosterone, estradiol-17β, and progesterone were measured monthly. Body weight and height, girth, and cannon circumference were also measured monthly. Development of the body and gonads was faster in Miyazaki than in Hidaka in both colts and fillies. Light treatment promoted growth and activated gonadal function in both colts and fillies reared in Hidaka. In horses reared in Miyazaki, growth promotion and early activation of ovarian function by light treatment were observed in fillies, but no effect was noted in colts. In addition, light treatment increased circulating prolactin levels in horses reared in either Miyazaki or Hidaka, suggesting that prolactin plays an important role in expression of the effect of light treatment in yearlings. In summary, growth of Thoroughbred yearlings reared under natural light in Miyazaki (southern area) was superior to that in Hidaka (northern area). However, it may be possible to promote growth in Hidaka to levels close to those in Miyazaki by extending the hours of sunlight by light treatment.

A study on pregnancy and luteal function in mares: isolation and culture of equine luteal cells (2011-2013) (Joint research conducted with Okayama University)

 The corpus luteum (CL) is a mammalian endocrine organ which is transiently formed in the ovary after ovulation. The CL produces progesterone which is an essential hormone for the establishment and maintenance of pregnancy. It is clarified in several animals that the luteal function is regulated not only by pituitary hormones but also by local regulatory factors. However, the regulatory mechanism of the equine luteal function is unclear because a method of isolation and culture of equine luteal cells has not been established yet. The purpose of this study was to establish the method of isolation and culture of the equine luteal cells for elucidating the regulatory mechanisms of the equine luteal function in vitro. Experiments were conducted during reproductive season of mares, from April until the end of August. In the first year, we tried to isolate the equine luteal cells by a method of isolation of bovine luteal cells, in which the ovary is perfused with enzyme solution. In the second year, luteal tissues were cut into small pieces and digested with enzyme solution supplemented with glucocorticoid, insulin and ascorbic acid. Both methods resulted in a small number of isolated cells and low cell viability, which are insufficient for in vitro experiments. In the third year, the CL was perfused with enzyme solution through small blood vessels neighboring the CL. As a result, the number of isolated cells and cell viability were improved. Then, we sought for the optimized culture condition for isolated cells. The cells were successfully cultured at a density of 10,000 cells/cm2 in William's E medium on a culture plate coated with FNC Coating Mix. The cells expressed 3β-HSD mRNA, which is a marker of steroidogenic luteal cells. In conclusion, the method of isolation and culture of the equine luteal cells was successfully established.

Gonadal and adrenal functions in the equine fetus: Secretion and possible roles of the TGF beta superfamily. Fundamental research for preventing abortion in pregnant mares (2010–2012)

 Purpose Previous studies have suggested that preventing pregnancy loss is important for improving foaling rates in equine reproduction. Nevertheless, details of the secretion, localization, and action of protein hormones such as the TGF beta superfamily members activin and anti-mu¨llerian hormone (AMH) remain unknown. In addition, although we partly understand the role of gonadal enlargement, we have not identified the activator of this process. In our previous study, activin beta A subunit was localized mainly in the epithelium of the uterine gland of the mare, suggesting that activin or other members of the TGF beta superfamily play a physiologically important role in maintaining pregnancy. To investigate the endocrinology of equine pregnancy, we collaborated with visiting researchers to localize activin and AMH in the equine fetoplacental unit and fetal gonads. Furthermore, we used ultrasonography to investigate the unique profiles of the equine fetal gonads.

Materials and Methods
 Fetal testes and ovaries at different stages of pregnancy (days 110 to 314) and the utero-placental tissues of the respective mares were analyzed immunohistochemically for activins and their receptors, intracellular Smad mediators, and AMH. We used placental and endometrial homogenates, together with weekly plasma samples taken from five pregnant Thoroughbred mares until foal heat, for activin A measurement by sandwich ELISA (Quantikine, R&D Systems, Minneapolis, USA). The equine fetal organs during pregnancy were investigated by using transrectal or transabdominal ultrasonography, or both (α7, Aloka, Tokyo, Japan).

Results and Discussion
1) The interstitial and germ cells of the fetal testis and ovary, as well as the trophoblast and endometrial cells of the utero-placenta, immunostained positively for activin type IIA/B and IA/B receptors. Smad proteins 2, 3, and 4, which are intracellular mediators of activin signaling, were also immunolocalized to all of these organs.
2) Plasma activin A levels remained low until about day 50, when they started to increase, peaking first on about day 100. They remained steady and then increased again on the day of parturition. 3) The seminiferous tubules of the fetal testis stained positively for AMH.
4) Ultrasonographically determined changes in the size of the equine fetal gonads paralleled the changes in plasma levels of estradiol in mares during the second half of gestation in mares.

 To our knowledge, these results are the first to demonstrate the possible actions of activin and AMH during pregnancy in Thoroughbred mares. Furthermore, our results reveal the biological machinery required for activin signaling in the equine fetal gonads and utero-placental tissues during several of the stages of pregnancy.

Study on improvement of growth and pre-training management for young horses (2007-2011)

 For the further "making of strong horse", we investigated new technical management for young horses and solve various problems during breeding and pre-training period in Thoroughbreds, including the total events "from fetus in the mare's uterus to racing horse". Through this study, we made prevalence of research findings by giving lectures to the owners and breeders as well as submitting our research papers to scientific journals.

The researches for foals

 Actual conditions on the club foot onset of 130 foals were investigated in Hidaka Training and Research Center and in 3 neighboring farms, suggesting that onset of club foot was associated with 1)foal's gender, 2)birth month, 3)farm, 4)increasing body weight, and 5)position of forefoot in their pasture field. These results might help breeders as useful information for the management for young horses in Hidaka, Japan.

 Foal behavior within 2 weeks after birth was investigated by using both GPS analysis and behavior observation. As a results, total track distance, track distance in trot/canter, laying time were statistically higher in foals which were born after May than in foals born before March. These result provided evidence that foals born in Hidaka were less active in winter rather than in spring, provably due to cold weather, hard soil, land covered with ice or snow, low photoperiod, and so on.

 As other research, we examined 1)effect of the endocrine function in yearling horses kept outside field in cold winter, 2) situation of potential infection of Rhodococcus equi in foals, 3) occurrence of DDSP in foals, 4)CT diagnosis of OCD in hock-joint, 5)occurrence of sesamoid born fracture in foals, 6)analysis of cross section of suspensory ligament in foals, 7)prediction of parturition by pH in milk, 8)induction of lactation in nonparturient mare.

The researches for yearlings
 Effect of extended photoperiod on reproductive endocrinology and body composition in 2 years horses Thoroughbred under pre-training were investigated. Extended photoperiod (14.5 h light:9.5 h dark) were initiated from winter solstice to April 15th, with 100 W bulb light in their stable, while control horse were kept under natural light. Stimulating effect were observed by the long day light treatment for increasing secretion of pituitary and gonadal hormones, muscle volume and molting of winter hair in horse pre-training. Light control method may be useful to improve not only endocrine events but also body composition in pre-training horses.

 In addition, we performed research from an evaluation of the horse training degree of progress using a scientific index including the V200 measurement and the treadmill exercise testing, relationship between the sesamoid radiolucency grade and DOD of the 1-2 years old horse, the preventive effect of the astaxanthin on exertional rhabdomyolysis, analysis of myostatin gene polymorphism and its association with the horse body measurement such as body height and body weight, and various studies from various angles.

 Throughout this research for 5 years, we have presented various results to breeders in training course and to scientist at domestic and international meetings; 81 presentations at the educational meeting for breeders and veterinarians, 15 papers in scientific journal, 39 articles in spread magazines, 73 lectures for breeders, owners, and training course students, 17 papers orally presented at JRA investigation meeting.

Decision on BCAA requirement for Thoroughbreds using the IAAO method (2007-2008)

 Branched chain amino acids (BCAA: valine, leucine and isoleucine) are essential amino acids that play an important role in the metabolism of muscle protein. However, the respective requirements of these are not specified in Japanese light breed horse feeding standards or in the 2007 NRC Feeding Standards published in the USA. Conversely, a method of measuring the BCAA requirement in humans has already been established using the indicator amino acid oxidation (IAAO) method. Therefore, this IAAO method was used to investigate the BCAA requirement in horses. As a result, it became clear that the BCAA requirement for horses when at rest is 179mg/kg/day.

Evaluation and improvement of vitamin K nutrition status associated with growth in light breed horses: Preparation of guidelines on vitamin K requirement needed for bone formation (2006-2008)
(Commissioned research conducted by Kyoto University)

 Vitamin K (particularly menaquinone-4) is an essential vitamin for healthy bones, but the concentration of VK homologues in equine plasma needed to evaluate the vitamin K nutrition status has not previously been measured. In this study, the concentration of VK homologies in plasma was measured and a method of vitamin K supplementation was studied. Phylloquinone, menaquinone-4, menaquinone-7 and menadione supplementation tests were carried out on adult horses, whereupon it became clear that only menadione at 0.12 μmol/kg body weight/day, far less than the toxic dose, can efficiently raise the menaquinone-4 concentration in plasma. Also, supplementing mothers feed with menadione raised the menaquinone-4 concentration in plasma of nursing foals. It was also suggested that the nutritional significance of menaquinones synthesized in the digestive tract is small.

Search for pregnancy marker substances in mares (2006-2008)
(Commissioned research conducted by University of Tokyo)

 Pregnancy marker substances have yet to be identified in mares. In this study, the following four phenomena were posited as implantation phenomena: (1) embryo mobility and suppression of regression of the corpus luteum, (2) attachment of the embryo to the uterus wall, (3) regulation of calcium concentration in the uterus after capsule loss, and (4) preparation of an immune environment for the uterus. Next, using subtraction and the DNA microarray method, factors related to the four implantation phenomena were identified, and their localization, expression control and functions were analyzed. Finally, to develop a method of controlling these factors, a system for in vitro tissue and cell culture analysis was also developed.

Research on the content of water-soluble carbohydrates in pasture grasses used for light breed horses (2007-2008)
(Commissioned research conducted by Obihiro University of Agriculture and Veterinary Medicine)

 Causes of fluctuation in the content of water-soluble carbohydrates in pasture grasses used for light breed horses were studied, together with the influence of this content on the properties of horse feces in open pastures. The content of water-soluble carbohydrates in pasture grasses fluctuated not only in accordance with the type and growth stage of the grass, but also between different times of day (it was higher in the evening than in the morning). The pH of horse feces in open pastures was lower in spring than in summer and autumn, and in open pastures where supplementary feed was not given, the pH of feces was 6.0 or less. The pH of feces was lower in spring than in summer and autumn, falling below 6.0 at one point, and it was surmised that in spring, acidosis in the hind gut is more prone to occur than in summer and autumn. Nevertheless, the content of water-soluble carbohydrates in open pasture grasses was in some cases not so high in spring as in summer and autumn. It was therefore suggested that, besides the content of water-soluble carbohydrates in pasture grasses, there may be other factors that change the fermentation properties inside the hind gut.

Studies on training and management of yearling horses (2002-2006)

 To study new guidelines for training techniques based on scientific indicators of aerobic exercise ability and bone metabolism.

 1. Evaluation of V200 values among JRA yearlings in Hidaka and Miyazaki Traing Fram:
 We measured the V200 of JRA yearlings in Hidaka and Miyazaki that were sold between 2002 and 2006 (192 colts, 155 fillies) in February and April of the 2nd year. As a result, V200 rose significantly between February and April in both yearling farms, although the values for Miyazaki tended to be higher than those for Hidaka. To study the relationship between aerobic exercise ability and exercise intensity of yearlings in more detail, moreover, we investigated the V200 of horses sold between 1999 and 2006 in February and April of the 2nd year. As a result, we were able to conveniently classify the period from 1999 to 2002 as one in which gentle exercise load was imposed in the first half of the training and the exercise load was intensified in the finishing stages ("The type of higher exercise intensity in the latter half during the yearling's training"), and the period from 2003 to 2006 as one in which heavy exercise load was imposed from the first half to improve physical fitness ("The type of higher exercise intensity in the first half during the yearling's training"). In fact, yearlings subjected to the latter of these two training patterns tended to enter races at an earlier age. From this it was thought that this pattern makes it possible to mitigate the increase in exercise intensity in the second half of the training, making yearlings more easily adaptable to training after entering the Training Centers.

 2. Changes in bone mineral content associated with training progress:
 On investigating changes in bone mineral content associated with training progress in private yearling farms, no difference was found in bone mineral content in June of the 2nd year between farms that imposed heavy exercise and moderate exercise load. However, in December of the 1st year, when the study was started, there were some farms in which bone mineral content was larger than in other yearling farms.
In a separate study, night and day grazing before training by riding in the field was effective in improving aerobic exercise ability. Therefore, taking this into account at the same time, exercise in the appropriate period was thought important for bone growth and building up fitness in the yearling phase.

 3. The impact of treadmill exercise on yearlings:
 Since high intensity exercise in winter can easily cause injuries to the locomotive organs of mares, we used research horses for training by riding in the field, in conjunction with high intensity exercise using a treadmill (TM) to avoid the load associated with field riding (once a week from February of the 2nd year, intensity close to all-out), and investigated the impact. As a result, the V200 of research horses in April of the 2nd year was significantly higher than that of JRA yearlings trained only by riding in the field. From this, we understood that TM exercise in the training phase is effective in improving aerobic exercise ability, and that the aerobic exercise ability obtained from conventional training by riding in the field alone does not necessarily reach its maximum potential in the training phase. On the other hand, TM exercise given to yearlings was thought to entail a number of problems, such as the possibility that it could have an adverse effect on building up running form.

 4. Relationship between the V200 and fat-free mass of horses in the training phase:
 A significantly positive correlation was seen between V200 and fat-free mass in April of the 2nd year, and we found that fat-free mass is valid as a new method for estimating aerobic exercise ability.

 1. Increasing physical fitness from the first half of the training phase makes it easier to adapt to high intensity exercise in the second half (is effective in building up physical fitness).
 2. For bone growth and improved fitness in the training phase, it is important to apply rearing management before training by riding in the field.
 3. By using TM and designing methods of rearing management, it is possible to impose exercise load that better improves aerobic exercise ability.
 4. We found that fat-free mass is valid as a new method for estimating aerobic exercise ability.

Studies on correct feeding for yearlings ; Supplementing nutrients with a view to enhancing muscle volume and strength and preventing bone disease (2002-2006)

 Increasing muscle volume is important in order to improve running ability, while strong bones are necessary to prevent injuries during exercise. In research until now, we have clarified the impact on body condition score due to changes in blood amino acid concentration following protein ingestion after exercise, as well as the impact of exercise on mineral and bone metabolism, among others. In this research, with a view to creating strong muscle and bones, we measured 1) the rate of synthesis and degradation of muscle protein, and investigated the impact of exercise and post-exercise nutrient ingestion on these. 2) Using a simple method of evaluating the bone mineral content of horses, we investigated changes in standard bone mineral content accompanying the growth of horses in the training phase in Japan, while also studying the impact of various types of exercise and nutrient conditions on bone mineral content.

 1. Impact of branched chain amino acids (BCAA) on inhibition of muscle protein degradation:
 Using twelve research yearlings, we administered branched chain amino acids (BCAA) immediately after exercise. This is thought to be effective in inhibiting muscle protein degradation. As a result, the blood BCAA concentration rose significantly in the BCAA administered group after exercise compared to that of the non-administered group. However, the administration of BCAA after exercise produced no marked effect on the inhibition of muscle protein degradation.

 2. Method of measuring the synthesis rate and degradation rate of muscle protein, and the effect of accelerating the synthesis rate of muscle protein:
 We established a method of measuring the rate of muscle protein synthesis and degradation in hindlimb muscles (gluteal and crus muscle groups), with stable isotope phenylalanine ( 2H5 Phe) as a marker. Using this, we studied the impact of exercise on muscle protein synthesis and degradation. As a result, when imposing approximately 90% HRmax exercise load using a treadmill, the rate of muscle protein synthesis after exercise was unchanged compared to during rest. Therefore, we studied the impact of various nutrient preparations after exercise on the rate of amino acid absorption by hindlimb muscles. The result was that 10% amino acids + glucose most significantly accelerated the rate of muscle protein synthesis, while the difference between synthesis and degradation increased. It also became clear that administrating both solutions of amino acids and glucose promotes synthesis of myogenic protein inside muscle tissue.

 3. Method of measuring bone mineral content (micro densitometry) using X-rays and preparation of a BMC growth curve:
 It became clear that the method of measuring bone mineral content using X-rays (micro densitometry) had a repeatability (CV%) of 1.0-1.6 and reproducibility of (CV%) 1.3-1.7, making it sufficiently precise for practical application. We created a BMC growth curve using this method. As a result, it became clear that bone mineral content rises while bone metabolism markers fall with increasing age.

 4. The effect of administering milk basic protein (MBP) and organic minerals as nutrients to accelerate bone metabolism in horses:
 On feeding organic minerals with high bioavailability to weaned yearlings, bone formation was accelerated and bone mineral content increased compared to feeding with inorganic minerals. Meanwhile, an increase in bone mineral content was seen when yearlings subjected to exercise load were fed with milk basic protein (MBP). This made it clear that organic minerals and MBP work to accelerate bone formation, and we are now able to recommend them as nutrients to accelerate bone metabolism in horses.

Research on an efficient method of reproduction management for broodmares: Development of a method of detecting ovarian activity in mares by measuring hormones (2004-2006)

 In racehorse breeding in recent years, there has been a tendency to produce foals earlier in the year to take maximum advantage of market prices. However, since horses are seasonally reproductive, the reality is that maiden and barren broodmares only return to estrus towards the end of April. Consequently, we are required to develop a simple method of reliably inducing estrus at an earlier stage. Moreover, in the early part of the breeding season, silent heat and prolonged estrus caused by unovulatory follicles commonly occur. As such, there is an urgent need to develop an objective method of evaluating ovarian activity that can be applied in breeding areas. The purpose of this research was to endocrinologically develop diagnostic methods for the reproductive physiology of horses, and particularly for ovarian activity, and to objectively evaluate the ovarian activity of broodmares under various rearing environments in breeding farms. At the same time, we studied the effects of light control as a method of inducing estrus in the early part of the breeding season.

 1. Development of a method to measure hormones by time-resolved fluorescent immunoassay:
 Measurements of the concentration of progesterone (P), estradiol and testosterone in the blood of horses using the time-resolved fluorescent immunoassay were good in both accuracy and sensitivity, and it became clear that these can be measured in about 3 hours without using any special facilities.

 2. Significance of the blood P concentration a weekly measurement method:
 A weekly measurement of blood P concentration is extremely useful as a method of evaluating ovarian activity in maiden (barren) mares in the early part of the breeding season, when silent heat and failure of ovulation are common. It is therefore seen as a useful means of mating these horses in a planned fashion.

 3. Changes in blood P concentration in normally pregnant mares and standard values for the gestation period:
 We traced changes in the blood P concentration of 20 normally pregnant mares, and computed standard values for the gestation period. On comparing these to abnormal cases, we proved that, in cases of early embryonic loss or miscarriage, blood P concentration shows low values throughout the gestation period, indicating the importance of endocrinological diagnosis in the gestation period.

 4. Usefulness of the Light Control method:
 1) As one method of stimulating ovarian activity, we studied the usefulness of the Light Control method (LC method, creating an illuminated environment of around 100 lux for 14.5 hours in the daytime and 9.5 hours at night) from the winter solstice, using 94 maiden and barren broodmares from neighboring farms. As a result, cases of confirmed first ovulation in the LC group were 51.9 % by the final week of the February and 80.5 % by the final week of March. We also succeeded in advancing the timing of ovulation by maiden and barren broodmares by about 1.5 months using the LC method.
 2) Between one month before and one month after foaling, we studied the usefulness of the LC method for broodmares due to give birth in January and February. As a result, normal first ovulation was confirmed in 45 out of 46 mares (97.8%). On the other hand, in pregnant mares due to give birth in the same period without any special measures as a control, clear ovarian inactivity and failure of ovulation were observed in 3 out of 9 mares. This proved that the method is also useful for broodmares due to give birth in midwinter.

 We endocrinologically studied efficient management methods for broodmares, and indicated guidelines for this. Namely, 1) we established a quick and simple method of measuring blood progesterone (P) in horses. 2) A weekly blood P concentration measurement was useful for estimating the estrus cycles of broodmare and evaluating the luteal functions of pregnant mares. 3) We established and evaluated a Light Control method in Hokkaido, with the finding that it is useful for barren mares and pregnant mares due to give birth in January and February. From these results, it was suggested that, by introducing objective diagnostic methods and the Light Control method, we will be able to make further progress in improving the reproductive efficiency of light horses.

Establishment of objective standards for the recovery of uterine functions in the puerperal period of light horses (2004-2006)

 Our aim was to establish objective diagnostic standards whereby veterinarians in sites of breeding could apply a shared understanding when evaluating postpartum uterine functions and deciding the propriety of mating. Besides this, we set out to ascertain rearing management methods and blood properties in broodmares before and after parturition, and to study the correlation between pregnancy rates and early pregnancy loss.

 1. Diagnostic standards for uterine function recovery:
 We periodically measured the resistance index (RI), pulsatility index (PI) and time-averaged velocity (evaluation markers for blood flow) of uterine arteries from before pregnancy to one month postpartum, using Color Doppler Ultrasonography. As a result, both RI and PI rose significantly at 3 days and 8 days postpartum, compared to the values before parturition. Uterine arteries were easily visualized in all mares during the late stage of pregnancy using Color Doppler Ultrasonography. Mean PI and RI values were 0.77-0.84 and 0.52-0.53 during late gestation. They then showed an abrupt increase to 1.73 and 0.74, respectively, after parturition. In addition, the mean time-averaged velocity was over 70 cm/s during the last three months of gestation, followed by an abrupt decrease to approximately 20 cm/s at both 3 and 8 days after parturition. A significant rise in PI and a decrease in time-averaged velocity were shown 30 days after parturition, compared with the 8th day postpartum.
 From these results, it was strongly suggested that the blood flow in uterine arteries, which transport oxygen and nutrients to the fetus, increases gradually during the gestation period, then falls sharply with parturition. Judging from changes in blood flow, meanwhile, the possibility was suggested that physiological uterine recovery is still incomplete at the first estrus after parturition.

 2. Breeders questionnaire survey on parturition as well as mating and conception at first estrus after parturition:
 We carried out multivariate studies on the results of a questionnaire survey aimed at breeders, concerning more than 1,300 cases of parturition as well as mating and conception at first estrus after parturition. As a result, we proved that there is a major correlation between conception in postpartum estrus, on the one hand, and the age of the mare and date of the previous parturition, on the other.

 3. Impact of energy requirement and feed intake level on postpartum broodmares:
 In farms where feed intake levels did not meet the energy requirement of postpartum broodmares, problems such as a decrease in pregnancy rates at first estrus after parturition were observed. Also, from biochemical tests, the possibility was suggested that decreases in lipids, protein concentrations and other properties of blood could act as markers for the nutritional management of horses.