Effects of branched-chain amino acids on immune status of young racing horses.

High-intensity exercise and competition are associated with depressed immune function. Young horses, which participate in high-intensity exercise and competitions, are at increased risk for the development of infectious disease due to depression of immune function. The effects of branched-chain amino acid (BCAA) supplementation on the immune status of young racing horses were evaluated, determining whether BCAA might help to avoid or reduce immune suppression during exercise and competitions. Twenty horses (10 male and 10 female) were treated with BCAA supplementation; another twenty untreated horses (10 male and 10 female) constituted control group. Peripheral blood was collected from each animal and evaluated for lymphocyte subsets, phagocytosis analysis of monocytes and granulocytes, lymphocyte proliferative response, and expression of cytokine-encoding messenger ribonucleic acids (mRNAs). The numbers of CD4+, CD8+, and major histocompatibility complex (MHC) class II+ cells in females of the treated group were significantly higher than those in females of the control group. The lymphocyte proliferative response in female of the treated group also was significantly higher than that in females of the control group. In addition, expression of mRNAs encoding interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ) in females of the treated group was significantly higher than that in females of the control group. There were no significant differences between males of the treated and control groups. The results of this study indicated the positive effects of BCAA supplementation in counteracting immunosuppression in young female racing horses during and following high-intensity exercise.

Antibody Responses to a Reverse Genetics-Derived Bivalent Inactivated Equine Influenza Vaccine in Thoroughbred Horses.

Updating vaccine strains is important to control equine influenza (EI). Previously, we reported that a monovalent inactivated EI vaccine derived from a virus generated by reverse genetics (RG) elicited immunogenicity in horses. In the present study, we compared antibody responses to a bivalent inactivated EI vaccine generated by RG and a commercially available bivalent inactivated EI (CO) vaccine derived from wild-type equine influenza viruses in Thoroughbred horses. The CO vaccine contained A/equine/Ibaraki/1/2007 (Florida sub-lineage clade 1) and A/equine/Yokohama/aq13/2010 (Florida sub-lineage clade 2) as vaccine strains. We generated two RG viruses possessing the hemagglutinin and neuraminidase genes from A/equine/Ibaraki/1/2007 or A/equine/Yokohama/aq13/2010. These viruses were inactivated by formalin, and the hemagglutinin titer of the RG vaccine was adjusted to be the same as that of the CO vaccine. Sixteen unvaccinated yearlings (7 for the RG vaccine group and 9 for the CO vaccine group) received two doses of a primary vaccination course four weeks apart. Thirty-two vaccinated adult horses (18 in the RG-vaccinated group and 14 in the CO vaccine group) received a single dose of a booster vaccination. The patterns of hemagglutination inhibition antibody response to the primary and booster vaccinations were similar for the RG and CO groups in unvaccinated yearlings and vaccinated adult horses. These results suggest that a bivalent vaccine derived from RG viruses elicits equivalent immunogenicity to that elicited by a CO vaccine derived from wild-type viruses. RG viruses can, therefore, be used in multivalent as well as monovalent vaccines for horses.

Effects of pre-shipping enrofloxacin administration on fever and blood properties in adult Thoroughbred racehorses transported a long distance.

To evaluate the effects of single-dose enrofloxacin (ERFX) on fever and blood properties in 68 Thoroughbred racehorses after long-distance transportation, horses were assigned to receive ERFX (5 mg/kg, IV; ERFX group; n=52) or saline (0.9% NaCl) solution (50 ml, IV; control group; n=16) ≤1 hr before transportation. Horses were transported 1,122 km using commercial vans over the course of approximately 21 hr. Clinical examinations and hematologic analyses were performed before and after transportation. Rectal temperatures, white blood cell counts and serum amyloid A concentration of ERFX group were significantly lower than control group (P<0.01, P<0.01 and P<0.05, respectively). In conclusion, these results show ERFX administration just before transportation is effective at preventing transportation-associated fever in adult Thoroughbred racehorses.

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.

A study of the distribution of color Doppler flows in the superficial digital flexor tendon of young Thoroughbreds during their training periods.

Aim of this study was to evaluate the relationships of exercise and tendon injury with Doppler flows appearing in the superficial digital flexor tendon (SDFT) of young Thoroughbreds during training periods. The forelimb SDFTs of 24 one- to two-year-old Thoroughbreds clinically free of any orthopaedic disorders were evaluated using grey-scale (GS) and color Doppler (CD) images during two training periods between December 2013 to April 2015. Twelve horses per year were examined in December, February, and April in training periods that began in September and ended in April. The SDFT was evaluated in 3 longitudinal images of equal lengths (labelled 1, 2, 3 in order from proximal to distal), and 6 transversal images separated by equal lengths (labelled 1A, 1B, 2A, 2B, 3A and 3B in order from proximal to distal) of the metacarpus using both GS and CD. The running (canter and gallop) distance for 1 month before the date of the ultrasonographic examinations was increased in December, February, and April in both of the two training periods. CD flows defined as rhythmically blinking or pulsatory colored signals were found in 56 of 864 (6.4%) transversal CD images, in 28, 12, 13, and 3 images of 1A, 1B, 2A and 2B, respectively, and in 7, 14, and 35 images captured in December, February, and April, respectively. There were no longitudinal or transversal GS images indicating injury in the SDFTs in either of the two training periods. The increase of CD flows in the proximal regions of the SDFT are possibly related to the increase of the running distance during the training periods of the one- to two-year-old Thoroughbreds. Because no injury was diagnosed in the SDFTs by GS images during the training periods, the increase of CD flows in the proximal parts of SDFT is not necessarily predictive of tendon injury in the near future during the training period of young Thoroughbreds.

Cold exposure increases slow-type myosin heavy chain 1 (MyHC1) composition of soleus muscle in rats.

The aim of this study was to examine the effects of cold exposure on rat skeletal muscle fiber type, according to myosin heavy chain (MyHC) isoform and metabolism-related factors. Male Wistar rats (7 weeks old) were housed individually at 4 ± 2°C as a cold-exposed group or at room temperature (22 ± 2°C) as a control group for 4 weeks. We found that cold exposure significantly increased the slow-type MyHC1 content in the soleus muscle (a typical slow-type fiber), while the intermediate-type MyHC2A content was significantly decreased. In contrast to soleus, MyHC composition of extensor digitorum longus (EDL, a typical fast-type fiber) and gastrocnemius (a mix of slow-type and fast-type fibers) muscle did not change from cold exposure. Cold exposure increased mRNA expression of mitochondrial uncoupling protein 3 (UCP3) in both the soleus and EDL. Cold exposure also increased mRNA expression of myoglobin, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) and forkhead box O1 (FOXO1) in the soleus. Upregulation of UCP3 and PGC1α proteins were observed with Western blotting in the gastrocnemius. Thus, cold exposure increased metabolism-related factors in all muscle types that were tested, but MyHC isoforms changed only in the soleus.

Dietary fat influences the expression of contractile and metabolic genes in rat skeletal muscle.

Dietary fat plays a major role in obesity, lipid metabolism, and cardiovascular diseases. To determine whether the intake of different types of dietary fats affect the muscle fiber types that govern the metabolic and contractile properties of the skeletal muscle, we fed male Wistar rats with a 15% fat diet derived from different fat sources. Diets composed of soybean oil (n-6 polyunsaturated fatty acids (PUFA)-rich), fish oil (n-3 PUFA-rich), or lard (low in PUFAs) were administered to the rats for 4 weeks. Myosin heavy chain (MyHC) isoforms were used as biomarkers to delineate the skeletal muscle fiber types. Compared with soybean oil intake, fish oil intake showed significantly lower levels of the fast-type MyHC2B and higher levels of the intermediate-type MyHC2X composition in the extensor digitorum longus (EDL) muscle, which is a fast-type dominant muscle. Concomitantly, MyHC2X mRNA levels in fish oil-fed rats were significantly higher than those observed in the soybean oil-fed rats. The MyHC isoform composition in the lard-fed rats was an intermediate between that of the fish oil and soybean oil-fed rats. Mitochondrial uncoupling protein 3, pyruvate dehydrogenase kinase 4, and porin mRNA showed significantly upregulated levels in the EDL of fish oil-fed rats compared to those observed in soybean oil-fed and lard-fed rats, implying an activation of oxidative metabolism. In contrast, no changes in the composition of MyHC isoforms was observed in the soleus muscle, which is a slow-type dominant muscle. Fatty acid composition in the serum and the muscle was significantly influenced by the type of dietary fat consumed. In conclusion, dietary fat affects the expression of genes related to the contractile and metabolic properties in the fast-type dominant skeletal muscle, where the activation of oxidative metabolism is more pronounced after fish oil intake than that after soybean oil intake.

Effect of 48-h food deprivation on the expressions of myosin heavy-chain isoforms and fiber type-related factors in rats.

The primary aim of this study was to examine the effects of 48-h food deprivation on rat skeletal muscle fiber type, according to myosin heavy-chain (MyHC) isoform composition and some metabolism-related factors in both slow-type dominant and fast-type dominant muscle tissues. Male Wistar rats (7 wk old) were treated with 48-h food deprivation or ad libitum feeding as control. After the treatment, the soleus muscle (slow-type dominant) and the extensor digitorum longus (EDL, fast-type dominant) were excised. We found that 48-h food deprivation did not affect MyHC composition in either the soleus or EDL, compared with fed rats by electrophoretic separation of MyHC isoforms. However, 48-h food deprivation significantly increased the mRNA expression of fast-type MyHC2B in the EDL muscle. Moreover, food deprivation increased fatty acid metabolism, as shown by elevated levels of related serum energy substrates and mRNA expression of mitochondrial uncoupling protein (UCP) 3 and lipoprotein lipase (LPL) in both the soleus and EDL. UCP3 and LPL are generally expressed at higher levels in slow-type fibers. Furthermore, we found that food deprivation significantly decreased the protein amounts of PGC1α and phosphorylated FOXO1, which are known as skeletal muscle fiber type regulators. In conclusion, 48-h food deprivation increased mRNA expression of fast-type MyHC isoform and oxidative metabolism-related factors in EDL, whereas MyHC composition at the protein level did not change in either the soleus or EDL.