The fundamental aim of chronobiological analysis methods in the field of sport physiology in the horse is to establish the time interval in which the organism gives the best performance. Since it is well known that numerous physiological parameters (electrocardiographic, haemogasanalytic and haemodynamic) vary during physical exercise and influence athletic potential considerably, we studied the periodic variations in the following haematological parameters at a different frequency in 12 horses (6 athletes and 6 sedentary): erythrocytes (RBC), total leukocyte count (WBC), haemoglobin (Hb), packed cell volume (PCV), platelets (PLT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Some of the parameters studied showed periodicity at different frequencies both in athletic and sedentary horses: in athletic horses -infradian (RBC, PCV), circadian (RBC, WBC, PCV), ultradian (RBC, WBC, Hb); in sedentary horses-infradian (RBC, Hb, PLT), circadian (RBC, PCV), ultradian (RBC, Hb). The results obtained, therefore, can suggest that physical exercise is an exogenous synchronizer which modulates the periodicity of the haematopoietic system and of the functions of the organs involved in athletic performance.

Knowledge about changes in the osmotic fragility of erythrocytes during exercise in athletic horses which is introduced in this review can be summarized as follows; 1) Changes in the osmotic fragility of equine erythrocytes during exercise are an important indicator of intravascular haemolysis. 2) The sensitivity of the regulatory volume is decreased by a K-Cl cotransporter, lactate and/or pH susceptibility, and the osmotic fragility of erythroid membrane due to exercise stress increases with the repeated accumulation of red cells in the spleen. 3) The increase in blood flow and the release of erythrocytes from the spleen into the circulation have little effect on the changes in the osmotic fragility of erythrocytes during exercise. 4) Increases in blood pH and temperature caused by aerobic exercises cause osmotic resistance in erythrocytes. 5) The decrease in pH and increase in blood lactate and peroxide caused by anaerobic exercises promotes the osmotic fragility of erythrocytes. 6) The osmotic fragility of erythrocytes could be detected by the modification, degeneration, decomposition and oxidation-reduction in cell membrane lipids and proteins, and cell volume regulation, changes which follow pH and temperature changes during exercise. 7) The K-Cl cotransporter which controls the intracellular ionic homeostasis and regulatory volume decrease has a functional interrelation with the fragility of erythrocytes. The osmotic fragility of erythrocytes in athletic horses is therefore an indicator of the effects of training and/or athletic performance and stress.

To clarify the endocrinological characteristics of mares with granulosa-theca cell tumor (GTCT), 2 mares with GTCT and a long estrous period were investigated. Before removal of the GTCT in both cases, the concentrations of testosterone, immunoreactive (ir-) inhibin and follicle stimulating hormone (FSH) in the peripheral blood were higher than those of normal mares at the follicular phase, whereas the plasma concentrations of progesterone were constantly low. In Case 1 mare, the plasma concentrations of testosterone, ir-inhibin and follicle stimulating hormone (FSH) changed to significantly high levels. In Case 2 mare, plasma concentrations of all hormones except for progesterone changed to significantly high levels. After surgical removal of the GTCT, the circulating concentration of ir-inhibin decreased rapidly to the basal level. The ir-inhibin levels in GTCT tumor fluid were similar to the follicular fluid in healthy follicles 3.5-5.0 cm in diameter. Concentrations of estradiol in the GTCT tumor fluid were low, but testosterone levels were higher than those in healthy follicles 3.5-5.0 cm in diameter. These results suggest that high levels of circulating inhibin in addition to high levels of testosterone and low levels of progesterone may be used as a clinical sign in the diagnosis of GTCT in mares.

Complementary DNA (cDNA) clones encoding equine testicular steroidogenic enzyme 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) have been isolated from equine testicular cDNA library. Polymerase chain reaction (PCR) with 3β-HSD specific primers was performed with equine testicular cDNA, and PCR fragment was used as a probe for screening of equine testicular λ ZAP II phage cDNA library. Positive clones were excised into plasmid vector pBluescript and determined their nucleotide sequences. Total 1651 bp of the cDNA sequence was consisted with 112 bp of 5' flanking untranslated sequence, 1122 bp open reading frame encodes 373 amino acids, and putative poly-adenylation signal “AATAAA” at 385 bp downstream of the stop codon. The nucleotide and the deduced amino acid sequence of equine 3β-HSD were very similar to the known mammalian 3β-HSD sequences. From the deduced amino acid sequence, equine testicular 3β-HSD may be concluded to catalyze 3β-dehydrogenation and Δ-isomerization, but 3-ketoreduction as well as murine isoforms. As the clone was used in Northern blot analysis of equine tissues, the expression of equine 3β-HSD mRNA was prominent in the classical steroidogenic tissues including placenta.

Plasma adrenaline (Ad), noradrenaline (NA), adrenocorticotropin (ACTH) and cortisol responses to exhaustive incremental treadmill exercise were investigated in seven Thoroughbreds. Blood lactate (LA), heart rate (HR) and oxygen uptake (VO2) were measured and their correlations with plasma hormone levels were examined. Although the magnitude of plasma Ad, NA and ACTH response to the exercise was great, that of cortisol was very slight. Plasma Ad and NA levels reached peaks at exhaustion in all Thoroughbreds. On the other hand, plasma ACTH levels reached peaks at a different time in each Thoroughbred, followed by a 10-15 min delayed peak of plasma cortisol levels. The maximal levels of plasma Ad, NA, ACTH and cortisol were significantly 300, 150, 70 and 1.8 fold higher, respectively, than the pre-exercise levels. Plasma Ad, NA and ACTH during exercise were highly and significantly correlated to each other. The noticeable increase in plasma Ad levels during exercise was characteristic of Thoroughbreds. Plasma Ad, NA and ACTH responses during exercise were significantly correlated with treadmill velocity, blood LA, HR and VO2 in an exponential manner. The threshold for blood LA was significantly lower than the threshold for Ad, NA and ACTH. In conclusion, the present data on plasma CA and ACTH responses to exercise suggest that the sympathoadrenal axis and the pituitary gland are highly activated by exercise in Thoroughbreds. In addition, it is suggested that plasma CA and ACTH levels as well as LA and HR may be useful indicators for estimating exercise stress and physical fitness in the Thoroughbred.

Various clinical and tissue materials derived from an experimentally infected mare were examined for the equine arteritis virus (EAV) by the RT-PCR method. For the reverse transcriptional step, a specific primer (R1 primer) and random (nonspecific) 9-mers primer were used. The specific 292 bp band could be detected clearly with the random primer, but not the specific primer from clinical and various autopsy samples of the pregnant mare and aborted fetus. The sensitivity to detect 7 strains of EAV by RT-PCR with the random primer was tested. The detection limit of Bibuna and Vienna strains of EAV was about 100 PFU, although that of other strains, Bucyrus, modified Bucyrus, 84KY-A1, Red Mile and Wroclaw ranged from 1 to 10 PFU. By our RT-PCR method, EAV could be detected within 1 day and could be obtained directly from a urine sample, from which the virus was difficult to isolate without pretreatment due to toxicity in cell culture.

Endoscopic, cytologic, bacteriologic and histologic examinations of the endometrium were performed on 34 subfertile Turkaman cross-bred mares to evaluate the significance of the hysteroscopic method and biopsy as a gyneacological evaluation. Fifty percent of the mares showed pathological changes on endoscopy, including adhesions, fluid accumulation, endometrial cysts and atrophy of the endometrial folds. Three, 23 and 8 cases classified as grades II A, II B and III, respectively, in biopsy samples base on the Kenney and Doig classification [11]. Multiple endometrial cysts occurred in 12 mares (35.3%) and 2 of them were younger than 10 years. Most of the large lymphatic cysts were situated near the bifurcation of the uterine body. Adhesions were found only in one mare. Only 4 of the 17 cases in which endometritis was clearly diagnosed were identified by endoscopy, but none of the endometrial cysts or intraluminal adhesions which were identified by endoscopy were determined by uterine biopsy, cytology and culture. In view of the results of this experiment, it can be concluded that in diagnosing endometritis, biopsy, cytology and culture of the endometrium is more diagnostic than endoscopy, but in differentiating gross pathological changes in the uterus such as intrauterine adhesions and endometrial cysts, endoscopy is more efficient.

We examined nerve growth factor (NGF) activity in sera obtained from 5 Anglo-Arab and 6 Thoroughbred horses after long-distance truck transportation. NGF activity was evaluated by neurite outgrowth from rat pheochromocytoma cells that respond to NGF. Five of 11 horses had mild or severe fever after the truck shipping; significant NGF activity was detected in serum samples collected from 4 of the fevered horses. The addition of polyclonal antibodies to murine 2.5S NGF completely abrogated the neurotrophic activity detected in the samples. These results suggested a possible role of NGF in the pathogenesis of equine shipping fever associated with continuous stress by truck transportation.

A rapid colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was applied to evaluate equine lymphocyte blastogenic response to mitogens. The optimum concentrations of concanavalin A (Con A), phytohemagglutinin-P (PHA) and pokeweed mitogen (PWM) were 5 μg/ml, 2.5 μg/ml, and a 1:80 dilution, respectively, when lymphocytes were cultured at a concentration of 1 × 106 cells/ml for 5 days in a 96-well microplate. Significant correlations between the results obtained by the MTT assay and the 3H-thymidine incorporation assay were observed. The correlation coefficients were 0.727 (n=68), 0.692 (n=57), and 0.681 (n=44), when lymphocytes were stimulated with Con A, PHA, and PWM, respectively. The MTT assay may be simple and reliable for studying equine cellular immune functions and a suitable alternative to conventional assay systems using radioactive materials.