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Genome Wide Association Study For Susceptibility Of Horses For In Vitro Infection With Equine Arteritis Vrus
244
Abstracts / Journal of Equine Veterinary Science 31 (2011) 230-356
Table 1 Estimates of genetic parameters of 4 foal inspection scores; heritabilities are in bold on the diagonal, genetic correlations are above diagonal, and phenotypic correlations are below the diagonal Trait
TC
TS AM OD TS
.45 .51 .86 .87
AM
.05 .01 .00 .00
.80 .47 .72 .83
OD
.05 .05 .01 .01
.98 .88 .49 .87
TS
.01 .03 .05 .01
.96 .93 .00 .55
.01 .02 .01 .05
between sexes were minimal in later years. Number of premium foals also increased from 2001 to 2007, before decreasing in 2008 when the criteria changed. There were more premium females in 2001 and 2002 (P .05). Differences between sex, birth year, and dam breed effects were significant for all traits (P .05). Heritabilities for TC, AM, OD, and TS were high, and genetic and phenotypic correlations were high and favorable (Table 1). Heritability of PS was 0.32 on the binary scale and 0.51 when transformed to the underlying normal scale. High heritabilities for all traits indicate potential for genetic improvement through selection. High genetic correlations suggest improvement in one trait should yield genetic gains in all other traits. Estimates were higher than those from previous studies in foals, though there is a paucity in the literature [35]. Although PS is technically a function of TC, AM, OD, and TS, it is possible for evaluators to subjectively score PS prior to assigning numerical scores to composite variables. Over or underestimation of parameters due to assortative mating, evaluator bias, abbreviated use of scoring scale, and limitations in obtaining relevant fixed effects is possible. Additionally, foal conformation is dynamic, and is a function of age and stage of growth. Foal age at inspection is assumed to have a significant effect on conformation and gait scores, leading many registries to opt not to score foal parameters. However, favorable genetic correlations among foal inspection scores and scores at studbook inspection and mare performance tests have been found [3]. If these correlations can be confirmed by further research, foal inspection scores can be accurately used to select future breeding animals, thereby shortening the generation interval. Further, favorable genetic correlations between foal scores and performance results would indicate potential for early selection of competition horses. Foal scores may be more useful as a reflection of the breeding merit of the parents in a type of progeny testing, rather than as criteria for direct selection. Evaluating foals would also increase the percentage of animals evaluated, thereby increasing the amount of data for the population as a whole and increasing accuracy of selection. Conclusion: High heritabilities of foal inspection traits for ISR/ OLDNA suggest that careful selection practices can yield genetic gain in conformation and gait traits of sporthorses. With further research, foal scores could be utilized to strengthen the breeding program of ISR/OLDNA by improving accuracy of selection and reducing the generation interval to allow for increased genetic gain.
References [1] Hellsten ET, Viklund Å Koenen EPC, Ricard A, Bruns E, Philipsson J. Review of genetic parameters estimated at stallion and young horse performance tests and their correlations with later results in dressage and show-jumping competition. Livest Sci 2006;103:1-12. [2] Gilmour A, Gogel B, Cullis B, Thompson R. ASReml user guide 3.0; 2009. [3] Bösch M, Reinecke S, Röhe R, Kalm E. Genetische analyse von merkmalen in der reitpferdezucht. Züchtungskunde 2000;72:161-71. [4] Kühl K, Preisinger R, Kalm E. Analyse von leistungsprüfungen und entwicklung eines gesamtzuchtwertes für die reitpferdezucht. Züchtungskunde 1994;66:1-13. [5] Preisinger R, Wilkens J, Kalm E. Estimation of genetic parameters and breeding values for conformation traits for foals and mares in the Trakehner population and their practical implications. Lives Prod Sci 1991;29:77-86.
Genome Wide Association Study For Susceptibility Of Horses For In Vitro Infection With Equine Arteritis Vrus Y.Y. Go, U. Balasuriya, and E. Bailey University of Kentucky, Lexington, KY, USA Introduction: Equine Arteritis Virus (EAV) is a RNA virus of the family Arteriviridae which is the cause of Equine Viral Arteritis (EVA). EVA often causes asymptomatic infections, but in some cases is manifest with influenza-like symptoms, abortion in pregnant mares or pneumonia in neonatal foals [1]. Determining the cause for differences in the course of infection will be valuable for development of treatments and vaccines. Previously, differences were observed between horses for the infectivity of CD3+ T lymphocytes in vitro [2]. The differences may also have a genetic basis. To investigate the possibility of a hereditary aspect for this trait, a genome wide association study (GWAS) was conducted using the Illumina Equine SNP50 chip to compare positive and negative Thoroughbred horses. Following initial screening, a candidate chromosome regions was selected and tested with the Sequenom Iplex tool to verify the association and to investigate the presence of the haplotype among horses of other breeds. Materials and Methods: Virus Tests: Peripheral blood mononuclear cells were infected, in vitro, and the infectivity and cell type determined by dual color immunofluorescence staining coupled with flow cytometry. Horses were classified as positive or negative for infection of their CD3+ T lymphocytes. Horses: DNA was isolated from 61 American Saddlebred horses, 60 Standardbred horses, 57 Thoroughbred horses, 53 Quarterhorses and the 37 Thoroughbred horses tested in the original Illumina assay (referred to here as Illumina Thoroughbreds). DNA Genotyping: DNA for the Illumina (San Diego) Equine SNP50 assay were conducted at the core facility for the Mayo Clinic in Rochester, MN. The Illumina Equine SNP50 bead chip tests up to 55,000 SNPs. Genotyping with the Sequenom (San Diego) Iplex Assay was conducted by Geneseek, Inc (Lincoln, Nebraska). Analyses: Data from both assays were analyzed using the program, PLINK [3]. For the Illumina assay, following filtering for minor allele frequency (excluding SNPs with minor allele frequencies less than .05) and genotyping (excluding SNPs with genotypes for less than 90% of the horses) 43, 277 SNPs were evaluated. Data from both studies were evaluated using the association (chi-square) and haplotyping programs. Results and Discussion: Initial testing implicated ECA11 as being associated with this trait. SNPs from this region were selected from the EquCab 2.0 SNP database and tested with additional horses, as described above. The results confirmed the association. Haplotype analyses of this region using 6 SNPs demonstrated the presence of 6 haplotypes among the 37 horses, one of which was present in every one of the 16 affected horses but only 2 of the 21 unaffected horses. The Sequenom assay was designed to further investigate this observation. Those SNPs used in both assays produced identical results with the Thoroughbred horses tested in both assays (Illumina Thoroughbreds). Differences were observed between breeds for the prevalence of infectivity of lymphocytes in vitro, ranging from 28% among Thoroughbred horses to 95% among Standardbred horses. The strongest association for Thoroughbred horses was, again, for a SNP on ECA11 with a P value of 9.39E-05. That SNP was significantly associated with the trait among Saddlebreds but not for Standardbred or Quarter Horses. The Hap-phase program was run using PLINK to investigate haplotype associations based on 6 contiguous SNPs. A unique and common haplotype was found for all breeds, with the exception of Standardbred horses. This haplotype was present at a higher frequency among horses which were positive for in vitro infection
Abstracts / Journal of Equine Veterinary Science 31 (2011) 230-356
of lymphocytes. Combining data for the two groups of Thoroughbreds, the haplotype was present among 27 of 30 positive horses but only 5 of 64 negative horses. A similar result was observed for other breeds, namely a decrease of this haplotype among negative horses. Discussion: The results confirmed the results for the SNPs on ECA11 but did not confirm the results for the other set of SNPs. The results for ECA11 were further evaluated to determine whether the effect extended to other breeds and to determine the mode of inheritance. The SNP showing the highest association in the initial assay with Illumina Thoroughbred horses, also showed the highest association with the newly selected Thoroughbred horses. However, this SNP did not show statistical significance with Quarterhorse or Standardbred groups. This was understandable for the Standardbred horse group, since only 3 of 60 horses were refractory to the infection. The breed showed little phenotypic variation and this is consistent with the lack of genetic variation. However, the Quarterhorse group exhibited the second highest level of phenotypic variation. Therefore, the scores for other SNPs were evaluated to determine if this region had an influence on the trait in other breeds and a common haplotype identified. The conservation and increased prevalence of this same haplotype among positive horses of all breeds, Thoroughbred, Standardbred, Saddlebred and Quarterhorse, indicates the same genetic basis for this phenotype, e.g., derived from a common ancestor. Conclusion: 1) There is a strong genetic association for this region of ECA11. 2) The gene exhibits a dominant mode of inheritance, e.g., a single copy of the gene is all that is needed to create this phenotype. 3) The same haplotype, hence the same gene derived from some common ancestor. probably before the formation of the Thoroughbred breed
References [1] Timoney PJ, McCollum WH. Equine viral arteritis. Vet. Clin. North Am. Equine Pract 1993;9:295-309. [2] Go YY, Zhang J, Timoney PJ, Cook F, Horohov DW, Balasuriya UBR. Complex Interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes. J Virol 2010;84:4898-911. [3] Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P. de Bakker, PIW, Daly MJ, Sham PC. PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet 2007;81:559-75.
The influence of exercise on entrainment of skeletal muscle transcription in the horse O.F. McGlynn, J.A. Browne, C.M. Blake, and B.A. Murphy University College Dublin, Belfield, Dublin, Ireland Introduction: Circadian rhythms are innate 24-h cycles in behavioural and biochemical processes that permit physiological anticipation of daily environmental cycles. Up to 10% of all transcribed genes in peripheral tissues exhibit circadian rhythmicity. Recently we provided evidence of a functional endogenous molecular clockwork mechanism in equine skeletal muscle [1]. Human athletic performance varies over time of day with reports of robust alterations in muscle strength, reaction times and mental alertness [2]. As physical exercise acts as a powerful synchronizer of circadian rhythms, it is hypothesised that performance is enhanced when training and competition times coincide. Here we investigate whether synchronization of muscle-specific performance-related genes occurs in equine skeletal muscle in response to a set training time.
245
Materials and Methods: Six 3-4 year old Thoroughbred mares accustomed to a sedentary lifestyle were used for collection of midgluteal muscle biopsies at 4-h intervals over a 24-h period. Muscle biopsies were obtained using a 6 mm diameter modified Bergstrom biopsy needle 15 cm caudodorsal to the tuber coxae on an imaginary line drawn from the tuber coxae to the head of the tail and at a depth of 80 mm. 100mg of tissue was cleaned by multiple washes in sterile RNAlaterÒ (Ambion Inc), and immediately preserved in RNAlaterÒ for 24 h at 4 C, followed by storage at 20 C. The mares then underwent an 8 week mid-morning training programme consisting of warm up, trot and cool down with speeds of up to 12km/h maintained for up to 30 minutes using an automated horse-exerciser. The intensity of exercise gradually increased each week. Weight was monitored throughout. At the end of the 8 week regime muscle biopsies were again obtained from each animal over a 24-h period. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), subsequently DNAse-treated with the RNAse-free DNAse Set (Qiagen, Hilden, Germany), and was purified with the RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. RNA was quantified using a NanoDropÒ ND1000 spectrophotometer V 3.5.2 (NanoDrop Technologies, Wilmington, DE) and RNA integrity analysed using the Agilent 20100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). 500 ng of RNA from each sample was converted to complementary (c) DNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) prior to realtime quantitative reverse transcription PCR (qRT-PCR). Assays were performed using the ABI Real-Time PCR 7500 System and Fast SYBRÒ Green Master Mix (Applied Biosystems). Oligonucleotide primers were designed for a panel of 13 candidate genes that included core clock genes, previously identified circadianly regulated genes and exercise associated genes in the horse. Results and Discussion: Two-way repeated measures ANOVA revealed a significant (a¼ P <.05,b¼ P <.01, c¼P <.001, d¼ P <.0001) interaction between circadian time (CT) and exercise for the muscle metabolism genes; MYF6a, UCP3a, MYOD1b and PDK4a. A main effect of time was observed for the clock genes ARNTLd, PER2d, NR1D1d and DBPd, as well as muscle-specific genes FOXO1d, PGC-1aa, PGC-1ba and FBXO32d. In addition, a significant effect of exercise existed for VEGFAc, MYOD1cand PGC-1ba. A one-way ANOVA revealed a significant change in weightd over the 8 week period. There was an initial drop in weight at week 3 followed by weight gain as fat was replaced with increasing muscle mass. MYF6 is involved in myogenesis and we find that the peak expression of this circadianly regulated gene occurs during the night, corresponding with a time of regeneration and repair in a diurnal animal. MYOD1 is a member of the myogenic regulatory transcription factor (MRF) family and showed a dramatic change in its temporal expression pattern in post-exercise samples. The timing of the peak in its oscillatory expression corresponded with the time of day following the exercise period thus supporting its role in the daily maintenance of muscle phenotype [3]. UCP3 plays a role in the protection of muscle from reactive oxidative species (ROS) during oxidative stress. It is thought that a rise in UCP3 could act as an antioxidant defence mechanism to protect skeletal muscle mitochondria from exercise-induced oxidative insult [4]. We observe a peak in the circadian profile of UCP3 expression at 07:00, prior to the daily scheduled exercise which suggests an anticipatory muscle defence mechanism. PDK4 is stimulated in response to a deficiency in whole body glucose and ensures that glucose entering the cell is preferentially used for muscle glycogen re-synthesis following exercise. PDK4 is also highly associated with elite racing performance [5] thus with the knowledge that it is entrained to exercise, verification of its peak expression in competitive equine athletes could point to the most favourable time of day for training and competition. Conclusion: In summary, our study provides clear evidence that exercise regimes entrain skeletal muscle function in the horse and
Abstracts / Journal of Equine Veterinary Science 31 (2011) 230-356
Table 1 Estimates of genetic parameters of 4 foal inspection scores; heritabilities are in bold on the diagonal, genetic correlations are above diagonal, and phenotypic correlations are below the diagonal Trait
TC
TS AM OD TS
.45 .51 .86 .87
AM
.05 .01 .00 .00
.80 .47 .72 .83
OD
.05 .05 .01 .01
.98 .88 .49 .87
TS
.01 .03 .05 .01
.96 .93 .00 .55
.01 .02 .01 .05
between sexes were minimal in later years. Number of premium foals also increased from 2001 to 2007, before decreasing in 2008 when the criteria changed. There were more premium females in 2001 and 2002 (P .05). Differences between sex, birth year, and dam breed effects were significant for all traits (P .05). Heritabilities for TC, AM, OD, and TS were high, and genetic and phenotypic correlations were high and favorable (Table 1). Heritability of PS was 0.32 on the binary scale and 0.51 when transformed to the underlying normal scale. High heritabilities for all traits indicate potential for genetic improvement through selection. High genetic correlations suggest improvement in one trait should yield genetic gains in all other traits. Estimates were higher than those from previous studies in foals, though there is a paucity in the literature [35]. Although PS is technically a function of TC, AM, OD, and TS, it is possible for evaluators to subjectively score PS prior to assigning numerical scores to composite variables. Over or underestimation of parameters due to assortative mating, evaluator bias, abbreviated use of scoring scale, and limitations in obtaining relevant fixed effects is possible. Additionally, foal conformation is dynamic, and is a function of age and stage of growth. Foal age at inspection is assumed to have a significant effect on conformation and gait scores, leading many registries to opt not to score foal parameters. However, favorable genetic correlations among foal inspection scores and scores at studbook inspection and mare performance tests have been found [3]. If these correlations can be confirmed by further research, foal inspection scores can be accurately used to select future breeding animals, thereby shortening the generation interval. Further, favorable genetic correlations between foal scores and performance results would indicate potential for early selection of competition horses. Foal scores may be more useful as a reflection of the breeding merit of the parents in a type of progeny testing, rather than as criteria for direct selection. Evaluating foals would also increase the percentage of animals evaluated, thereby increasing the amount of data for the population as a whole and increasing accuracy of selection. Conclusion: High heritabilities of foal inspection traits for ISR/ OLDNA suggest that careful selection practices can yield genetic gain in conformation and gait traits of sporthorses. With further research, foal scores could be utilized to strengthen the breeding program of ISR/OLDNA by improving accuracy of selection and reducing the generation interval to allow for increased genetic gain.
References [1] Hellsten ET, Viklund Å Koenen EPC, Ricard A, Bruns E, Philipsson J. Review of genetic parameters estimated at stallion and young horse performance tests and their correlations with later results in dressage and show-jumping competition. Livest Sci 2006;103:1-12. [2] Gilmour A, Gogel B, Cullis B, Thompson R. ASReml user guide 3.0; 2009. [3] Bösch M, Reinecke S, Röhe R, Kalm E. Genetische analyse von merkmalen in der reitpferdezucht. Züchtungskunde 2000;72:161-71. [4] Kühl K, Preisinger R, Kalm E. Analyse von leistungsprüfungen und entwicklung eines gesamtzuchtwertes für die reitpferdezucht. Züchtungskunde 1994;66:1-13. [5] Preisinger R, Wilkens J, Kalm E. Estimation of genetic parameters and breeding values for conformation traits for foals and mares in the Trakehner population and their practical implications. Lives Prod Sci 1991;29:77-86.
Genome Wide Association Study For Susceptibility Of Horses For In Vitro Infection With Equine Arteritis Vrus Y.Y. Go, U. Balasuriya, and E. Bailey University of Kentucky, Lexington, KY, USA Introduction: Equine Arteritis Virus (EAV) is a RNA virus of the family Arteriviridae which is the cause of Equine Viral Arteritis (EVA). EVA often causes asymptomatic infections, but in some cases is manifest with influenza-like symptoms, abortion in pregnant mares or pneumonia in neonatal foals [1]. Determining the cause for differences in the course of infection will be valuable for development of treatments and vaccines. Previously, differences were observed between horses for the infectivity of CD3+ T lymphocytes in vitro [2]. The differences may also have a genetic basis. To investigate the possibility of a hereditary aspect for this trait, a genome wide association study (GWAS) was conducted using the Illumina Equine SNP50 chip to compare positive and negative Thoroughbred horses. Following initial screening, a candidate chromosome regions was selected and tested with the Sequenom Iplex tool to verify the association and to investigate the presence of the haplotype among horses of other breeds. Materials and Methods: Virus Tests: Peripheral blood mononuclear cells were infected, in vitro, and the infectivity and cell type determined by dual color immunofluorescence staining coupled with flow cytometry. Horses were classified as positive or negative for infection of their CD3+ T lymphocytes. Horses: DNA was isolated from 61 American Saddlebred horses, 60 Standardbred horses, 57 Thoroughbred horses, 53 Quarterhorses and the 37 Thoroughbred horses tested in the original Illumina assay (referred to here as Illumina Thoroughbreds). DNA Genotyping: DNA for the Illumina (San Diego) Equine SNP50 assay were conducted at the core facility for the Mayo Clinic in Rochester, MN. The Illumina Equine SNP50 bead chip tests up to 55,000 SNPs. Genotyping with the Sequenom (San Diego) Iplex Assay was conducted by Geneseek, Inc (Lincoln, Nebraska). Analyses: Data from both assays were analyzed using the program, PLINK [3]. For the Illumina assay, following filtering for minor allele frequency (excluding SNPs with minor allele frequencies less than .05) and genotyping (excluding SNPs with genotypes for less than 90% of the horses) 43, 277 SNPs were evaluated. Data from both studies were evaluated using the association (chi-square) and haplotyping programs. Results and Discussion: Initial testing implicated ECA11 as being associated with this trait. SNPs from this region were selected from the EquCab 2.0 SNP database and tested with additional horses, as described above. The results confirmed the association. Haplotype analyses of this region using 6 SNPs demonstrated the presence of 6 haplotypes among the 37 horses, one of which was present in every one of the 16 affected horses but only 2 of the 21 unaffected horses. The Sequenom assay was designed to further investigate this observation. Those SNPs used in both assays produced identical results with the Thoroughbred horses tested in both assays (Illumina Thoroughbreds). Differences were observed between breeds for the prevalence of infectivity of lymphocytes in vitro, ranging from 28% among Thoroughbred horses to 95% among Standardbred horses. The strongest association for Thoroughbred horses was, again, for a SNP on ECA11 with a P value of 9.39E-05. That SNP was significantly associated with the trait among Saddlebreds but not for Standardbred or Quarter Horses. The Hap-phase program was run using PLINK to investigate haplotype associations based on 6 contiguous SNPs. A unique and common haplotype was found for all breeds, with the exception of Standardbred horses. This haplotype was present at a higher frequency among horses which were positive for in vitro infection
Abstracts / Journal of Equine Veterinary Science 31 (2011) 230-356
of lymphocytes. Combining data for the two groups of Thoroughbreds, the haplotype was present among 27 of 30 positive horses but only 5 of 64 negative horses. A similar result was observed for other breeds, namely a decrease of this haplotype among negative horses. Discussion: The results confirmed the results for the SNPs on ECA11 but did not confirm the results for the other set of SNPs. The results for ECA11 were further evaluated to determine whether the effect extended to other breeds and to determine the mode of inheritance. The SNP showing the highest association in the initial assay with Illumina Thoroughbred horses, also showed the highest association with the newly selected Thoroughbred horses. However, this SNP did not show statistical significance with Quarterhorse or Standardbred groups. This was understandable for the Standardbred horse group, since only 3 of 60 horses were refractory to the infection. The breed showed little phenotypic variation and this is consistent with the lack of genetic variation. However, the Quarterhorse group exhibited the second highest level of phenotypic variation. Therefore, the scores for other SNPs were evaluated to determine if this region had an influence on the trait in other breeds and a common haplotype identified. The conservation and increased prevalence of this same haplotype among positive horses of all breeds, Thoroughbred, Standardbred, Saddlebred and Quarterhorse, indicates the same genetic basis for this phenotype, e.g., derived from a common ancestor. Conclusion: 1) There is a strong genetic association for this region of ECA11. 2) The gene exhibits a dominant mode of inheritance, e.g., a single copy of the gene is all that is needed to create this phenotype. 3) The same haplotype, hence the same gene derived from some common ancestor. probably before the formation of the Thoroughbred breed
References [1] Timoney PJ, McCollum WH. Equine viral arteritis. Vet. Clin. North Am. Equine Pract 1993;9:295-309. [2] Go YY, Zhang J, Timoney PJ, Cook F, Horohov DW, Balasuriya UBR. Complex Interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes. J Virol 2010;84:4898-911. [3] Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P. de Bakker, PIW, Daly MJ, Sham PC. PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet 2007;81:559-75.
The influence of exercise on entrainment of skeletal muscle transcription in the horse O.F. McGlynn, J.A. Browne, C.M. Blake, and B.A. Murphy University College Dublin, Belfield, Dublin, Ireland Introduction: Circadian rhythms are innate 24-h cycles in behavioural and biochemical processes that permit physiological anticipation of daily environmental cycles. Up to 10% of all transcribed genes in peripheral tissues exhibit circadian rhythmicity. Recently we provided evidence of a functional endogenous molecular clockwork mechanism in equine skeletal muscle [1]. Human athletic performance varies over time of day with reports of robust alterations in muscle strength, reaction times and mental alertness [2]. As physical exercise acts as a powerful synchronizer of circadian rhythms, it is hypothesised that performance is enhanced when training and competition times coincide. Here we investigate whether synchronization of muscle-specific performance-related genes occurs in equine skeletal muscle in response to a set training time.
245
Materials and Methods: Six 3-4 year old Thoroughbred mares accustomed to a sedentary lifestyle were used for collection of midgluteal muscle biopsies at 4-h intervals over a 24-h period. Muscle biopsies were obtained using a 6 mm diameter modified Bergstrom biopsy needle 15 cm caudodorsal to the tuber coxae on an imaginary line drawn from the tuber coxae to the head of the tail and at a depth of 80 mm. 100mg of tissue was cleaned by multiple washes in sterile RNAlaterÒ (Ambion Inc), and immediately preserved in RNAlaterÒ for 24 h at 4 C, followed by storage at 20 C. The mares then underwent an 8 week mid-morning training programme consisting of warm up, trot and cool down with speeds of up to 12km/h maintained for up to 30 minutes using an automated horse-exerciser. The intensity of exercise gradually increased each week. Weight was monitored throughout. At the end of the 8 week regime muscle biopsies were again obtained from each animal over a 24-h period. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), subsequently DNAse-treated with the RNAse-free DNAse Set (Qiagen, Hilden, Germany), and was purified with the RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. RNA was quantified using a NanoDropÒ ND1000 spectrophotometer V 3.5.2 (NanoDrop Technologies, Wilmington, DE) and RNA integrity analysed using the Agilent 20100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). 500 ng of RNA from each sample was converted to complementary (c) DNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) prior to realtime quantitative reverse transcription PCR (qRT-PCR). Assays were performed using the ABI Real-Time PCR 7500 System and Fast SYBRÒ Green Master Mix (Applied Biosystems). Oligonucleotide primers were designed for a panel of 13 candidate genes that included core clock genes, previously identified circadianly regulated genes and exercise associated genes in the horse. Results and Discussion: Two-way repeated measures ANOVA revealed a significant (a¼ P <.05,b¼ P <.01, c¼P <.001, d¼ P <.0001) interaction between circadian time (CT) and exercise for the muscle metabolism genes; MYF6a, UCP3a, MYOD1b and PDK4a. A main effect of time was observed for the clock genes ARNTLd, PER2d, NR1D1d and DBPd, as well as muscle-specific genes FOXO1d, PGC-1aa, PGC-1ba and FBXO32d. In addition, a significant effect of exercise existed for VEGFAc, MYOD1cand PGC-1ba. A one-way ANOVA revealed a significant change in weightd over the 8 week period. There was an initial drop in weight at week 3 followed by weight gain as fat was replaced with increasing muscle mass. MYF6 is involved in myogenesis and we find that the peak expression of this circadianly regulated gene occurs during the night, corresponding with a time of regeneration and repair in a diurnal animal. MYOD1 is a member of the myogenic regulatory transcription factor (MRF) family and showed a dramatic change in its temporal expression pattern in post-exercise samples. The timing of the peak in its oscillatory expression corresponded with the time of day following the exercise period thus supporting its role in the daily maintenance of muscle phenotype [3]. UCP3 plays a role in the protection of muscle from reactive oxidative species (ROS) during oxidative stress. It is thought that a rise in UCP3 could act as an antioxidant defence mechanism to protect skeletal muscle mitochondria from exercise-induced oxidative insult [4]. We observe a peak in the circadian profile of UCP3 expression at 07:00, prior to the daily scheduled exercise which suggests an anticipatory muscle defence mechanism. PDK4 is stimulated in response to a deficiency in whole body glucose and ensures that glucose entering the cell is preferentially used for muscle glycogen re-synthesis following exercise. PDK4 is also highly associated with elite racing performance [5] thus with the knowledge that it is entrained to exercise, verification of its peak expression in competitive equine athletes could point to the most favourable time of day for training and competition. Conclusion: In summary, our study provides clear evidence that exercise regimes entrain skeletal muscle function in the horse and