- Email: [email protected]
Age-associated telomere shortening in Thoroughbred horses
Experimental Gerontology 127 (2019) 110718
Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Age-associated telomere shortening in Thoroughbred horses a,⁎
b
Joshua Denham , Kim Stevenson , Michele M. Denham a b c
T
c
Discipline of Exercise Science, School of Health and Biomedical Sciences, Bundoora West Campus, RMIT University, Bundoora, VIC 3083, Australia Discipline of Biosciences and Food Technology, School of Science, Bundoora West Campus, RMIT University, Bundoora, VIC 3083, Australia Jubilee Stud, Mount Duneed Road, Freshwater Creek, VIC 3217, Australia
A R T I C LE I N FO
A B S T R A C T
Section Editor: Richard Aspinall
Telomeres are genetically conserved repetitive terminal DNA that protect against genomic instability and shorten with ageing. Here, we reveal the leukocyte telomere length of Equus caballus by measuring terminal restriction fragments (TRFs) using Southern Blot analysis in a cohort of 43 Thoroughbred horses (age: 24 h–25 years). Heterogeneous TRFs were observed in each animal and large inter-animal variation in mean TRF was observed (range: 10.5–18.7 kbp). Mean TRFs were inversely correlated with age (r = −0.47). The estimated yearly rate of telomere attrition was 134 bp. Horses should be considered as an alternative animal model to investigate environmental and lifestyle factors that regulate telomeres and promote healthy ageing.
Keywords: Equus caballus Racehorse Ageing Telomeres Biological ageing
1. Introduction Telomeres are genetically conserved DNA at the distal ends of linear chromosomes that maintain genomic integrity. Given most healthy somatic cells in humans have very low levels or are devoid of telomerase activity (Burger et al., 1997), telomeres shorten with progressive cellular divisions which is accelerated by environmental insults (e.g. oxidative stress and inflammation) (Prasad et al., 2017). Although the telomere sequence is conserved amongst mammals (5′-TTAGGG-3′), there are large inter-species differences in the actual lengths and rate of shortening. Telomere shortening has a role in ageing, as excessive telomere attrition leads to premature ageing and serious life-threatening diseases, called telomere syndromes, in both humans and animal models (Armanios and Blackburn, 2012). The telomere length differences and rate of telomere shortening likely have implications for the evolution of the protection against cancer and longevity (Tian et al., 2018). The Thoroughbred horse (Equus caballus) is an athletic mammal, due to hundreds of years of artificial selection for traits important for racing performance. Indeed, typical Thoroughbred racehorses possess maximal aerobic fitness levels of 158 ml·kg−1·min−1 (Young et al., 2002), 2–3 times the average trained human athlete. Despite the years of selection for racing performance, there is also a high rate of wastage in the Thoroughbred horse racing industry due to the rigours associated with training. For instance, high rates of musculoskeletal injuries, psychological stress and illness have been reported (Wilsher et al.,
2006). Concerns have directed effort towards identifying potential markers of overtraining and illness in equine athletes (de GraafRoelfsema et al., 2007; McGowan and Whitworth, 2008), yet none can quantify cumulative animal welfare. Leukocyte telomere dynamics (i.e. length or rate of attrition) are proposed markers of cumulative welfare of other animals (Bateson, 2016; Seeker et al., 2018). Therefore, the analysis of telomere dynamics could be particularly useful for monitoring equine athletes to ensure they are coping with the demands of training in high performance environments. Interestingly, maximal aerobic fitness is typically positively correlated to telomere length in humans (Denham et al., 2016) and exercise training appears to buffer the harmful effects of psychological stress on telomere shortening and could regulate telomere maintenance (Denham et al., 2016; Ludlow et al., 2013; Puterman et al., 2010). As such, Thoroughbred horses could be an alternative animal model to investigate the influence of exercise and psychological stress on telomere biology. We previously found a negative correlation between age and relative leukocyte telomere length measured by qPCR in a cohort of Thoroughbred horses (Denham and Denham, 2018). To extend our previous work, we measured terminal restriction fragments (TRF) using Southern Blot analysis, to determine the length of leukocyte telomeres in a subset (N = 43) of horses. 2. Materials and methods Animal ethics clearance, consent, the blood collection, DNA
⁎ Corresponding author at: School of Health and Biomedical Sciences, Bundoora West Campus, RMIT University, Room 53, Level 4, Building 202, Bundoora, VIC 3083, Australia. E-mail address: [email protected] (J. Denham).
https://doi.org/10.1016/j.exger.2019.110718 Received 2 April 2019; Received in revised form 13 June 2019; Accepted 30 August 2019 Available online 31 August 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
Experimental Gerontology 127 (2019) 110718
J. Denham, et al.
extraction and assessment procedures have been outlined previously (Denham and Denham, 2018). Blood samples were collected from Thoroughbred horses from farms located around the Geelong region (Victoria, Australia). Aside from the newborn foals and their mothers, most of the horses included in the analysis were not directly genetically related. The horses in this study were not housed in stables, rather they were in small to moderate-sized paddocks. Blood sample collection occurred in 2016 and was finalised midway through 2017. DNA was extracted from buffy coat leukocytes during March 2017 and was completed in August 2017. DNA samples were stored at −20 °C until TRF experiments completed in January 2019. To ensure the integrity and suitability of frozen DNA samples for Southern Blot, four random samples were thawed and checked for signs of degradation using SYBR safe stained, 0.8% agarose gel electrophoresis and imaged on the Gel Documentation System (Biorad). No signs of degradation were observed (data not shown). TRFs were assayed using the TeloTAGGG Telomere Length Kit (Sigma-Aldrich) as per the manufacturer's instruction, but with some minor adjustments. Briefly, 2 μg of DNA was digested for 2 h at 37 °C with restriction enzymes, Hinf I and Rsa I, and DNA fragments separated by gel electrophoresis (ethidium bromide stained 0.8% agarose gel) for 19 h at 30 V. DNA was visualised using the Gel Documentation System (Biorad), before being prepared for transfer to a nylon membrane by Southern blotting overnight (20.5 h). DNA was UVcross linked to the membrane, washed, then exposed hybridized with a DIG-specific antibody coupled to alkaline phosphatase, washed, then exposed to a chemiluminescent substrate for alkaline phosphatase (CDP-Star) before imaging on the ChemiDoc Touch Gel Imaging System (Biorad). TRFs were compared against biotinylated DNA ladders of known lengths and mean TRFs were calculated using the ImageJ (Schneider et al., 2012) and TeloRun (Baur et al., 2004) software. The TRFs for each individual horse was measured once. 3. Results Leukocyte TRFs were heterogeneous within individual animals (Fig. 1). Large inter-animal variations in mean telomere length were observed (mean TRF range: 10.5–18.7 kbp). Leukocyte telomere length was inversely correlated to age (r = −0.47, p = .001). Linear regression analysis indicated that with each year of ageing, on average, leukocyte telomeres shorten by 134 bp (Fig. 2A). An ANOVA revealed a statistically significant main effect of age quartile on telomere length (F3, 39 = 4.26, p = .01, Fig. 2B). Compared to the newborn foals, the 7–11 year and 12–25 year old horses had much shorter telomeres (p < .01), which survived Bonferroni adjustment (both p < .05).
Fig. 1. A representative analysis of Thoroughbred horse terminal restriction fragment (TRF) lengths. Horse donors are numbered 1–9 and the TRFs are measured in kilo-base pairs (kbp). Age (years).
than humans, yet age approximately three times quicker. The equine and human telomeric proteins, namely shelterin, are comparable enough to elicit telomere extension and immortalization in equine fibroblasts when introduced with the two major protein components of human telomerase, hTERT and hTERC in vitro (Vidale et al., 2012). Our study confirms the inverse correlation between age and telomere length, and has characterised the length of leukocyte telomeres in Thoroughbred horses. Given the much shorter lifespan and higher annual rate of telomere attrition in horses – estimated from our data (134 bp) – compared to humans (31 bp) (Chen et al., 2011), horses could be an alternative animal model of accelerated ageing to determine the influence of disease and environmental factors (e.g. psychological stress and exercise) on telomere biology. Future work should investigate the rate of telomere length change with ageing, traits and environmental factors correlated to telomere length and the molecular mechanisms governing telomere regulation in the horse.
4. Discussion and conclusions Previous investigations have analysed leukocyte telomere length in context with age and different traits in other equine breeds (e.g. donkeys) (Argyle et al., 2003) or have measured them in arbitrary units (Denham and Denham, 2018; Katepalli et al., 2008). Unlike donkeys, Thoroughbred horses are a much more athletic breed of equids and until now the actual leukocyte telomere length were unknown. The TRFs of Thoroughbred horses appear to be a similar length to that of donkeys (Argyle et al., 2003) and breeds of zebra (Vidale et al., 2012). However, there seems to be much more variation amongst newborn Thoroughbred foals, which is consistent with our previous investigation (Denham and Denham, 2018). Considering the evidence that suggests early life telomere length predicts life-span of animals (Eastwood et al., 2018; Heidinger et al., 2012) and that telomere length is linked to immune function (Weng, 2012), the large inter-animal variation in leukocyte telomere length warrants future in-depth analyses. Such studies could have implications for animal welfare and particularly relevant for breeders and Thoroughbred racehorse trainers in the industry. Thoroughbred horses have average telomeres ~5–10 kbp longer
Declaration of competing interest MD owns and operates a Thoroughbred horse stud.
2
Experimental Gerontology 127 (2019) 110718
J. Denham, et al.
research project. References Argyle, D., Ellsmore, V., Gault, E.A., Munro, A.F., Nasir, L., 2003. Equine telomeres and telomerase in cellular immortalisation and ageing. Mech. Ageing Dev. 124, 759–764. Armanios, M., Blackburn, E.H., 2012. The telomere syndromes. Nat. Rev. Genet. 13, 693–704. Bateson, M., 2016. Cumulative stress in research animals: telomere attrition as a biomarker in a welfare context? Bioessays 38, 201–212. Baur, J.A., Wright, W.E., Shay, J.W., 2004. Analysis of mammalian telomere position effect. Methods Mol. Biol. 287, 121–136. Burger, A.M., Bibby, M.C., Double, J.A., 1997. Telomerase activity in normal and malignant mammalian tissues: feasibility of telomerase as a target for cancer chemotherapy. Br. J. Cancer 75, 516–522. Chen, W., Kimura, M., Kim, S., Cao, X., Srinivasan, S.R., Berenson, G.S., Kark, J.D., Aviv, A., 2011. Longitudinal versus cross-sectional evaluations of leukocyte telomere length dynamics: age-dependent telomere shortening is the rule. J. Gerontol. A Biol. Sci. Med. Sci. 66, 312–319. de Graaf-Roelfsema, E., Keizer, H.A., van Breda, E., Wijnberg, I.D., van der Kolk, J.H., 2007. Hormonal responses to acute exercise, training and overtraining. A review with emphasis on the horse. Vet. Q. 29, 82–101. Denham, J., Denham, M.M., 2018. Leukocyte telomere length in the Thoroughbred racehorse. Anim. Genet. 49, 452–456. Denham, J., O'Brien, B.J., Charchar, F.J., 2016. Telomere length maintenance and cardiometabolic disease prevention through exercise training. Sports Med. 46, 1213–1237. Eastwood, J.R., Hall, M.L., Teunissen, N., Kingma, S.A., Hidalgo Aranzamendi, N., Fan, M., Roast, M., Verhulst, S., Peters, A., 2018. Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird. Mol. Ecol. 28 (5), 1127–1137. Heidinger, B.J., Blount, J.D., Boner, W., Griffiths, K., Metcalfe, N.B., Monaghan, P., 2012. Telomere length in early life predicts lifespan. Proc. Natl. Acad. Sci. U. S. A. 109, 1743–1748. Katepalli, M.P., Adams, A.A., Lear, T.L., Horohov, D.W., 2008. The effect of age and telomere length on immune function in the horse. Dev. Comp. Immunol. 32, 1409–1415. Ludlow, A.T., Ludlow, L.W., Roth, S.M., 2013. Do telomeres adapt to physiological stress? Exploring the effect of exercise on telomere length and telomere-related proteins. Biomed. Res. Int. 2013, 601368. McGowan, C.M., Whitworth, D.J., 2008. Overtraining syndrome in horses. Comp. Exercise Physiol. 5, 57–65. Prasad, K.N., Wu, M., Bondy, S.C., 2017. Telomere shortening during aging: attenuation by antioxidants and anti-inflammatory agents. Mech. Ageing Dev. 164, 61–66. Puterman, E., Lin, J., Blackburn, E., O'Donovan, A., Adler, N., Epel, E., 2010. The power of exercise: buffering the effect of chronic stress on telomere length. PLoS One 5, e10837. Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. Seeker, L.A., Ilska, J.J., Psifidi, A., Wilbourn, R.V., Underwood, S.L., Fairlie, J., Holland, R., Froy, H., Salvo-Chirnside, E., Bagnall, A., Whitelaw, B., Coffey, M.P., Nussey, D.H., Banos, G., 2018. Bovine telomere dynamics and the association between telomere length and productive lifespan. Sci. Rep. 8, 12748. Tian, X., Doerig, K., Park, R., Can Ran Qin, A., Hwang, C., Neary, A., Gilbert, M., Seluanov, A., Gorbunova, V., 2018. Evolution of telomere maintenance and tumour suppressor mechanisms across mammals. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 373. Vidale, P., Magnani, E., Nergadze, S.G., Santagostino, M., Cristofari, G., Smirnova, A., Mondello, C., Giulotto, E., 2012. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts. Chromosoma 121, 475–488. Weng, N.P., 2012. Telomeres and immune competency. Curr. Opin. Immunol. 24, 470–475. Wilsher, S., Allen, W.R., Wood, J.L., 2006. Factors associated with failure of thoroughbred horses to train and race. Equine Vet. J. 38, 113–118. Young, L.E., Marlin, D.J., Deaton, C., Brown-Feltner, H., Roberts, C.A., Wood, J.L., 2002. Heart size estimated by echocardiography correlates with maximal oxygen uptake. Equine Vet. J. Suppl. 467–471.
Fig. 2. Terminal restriction fragment (TRF) analysis in Thoroughbred horses. (A) A moderate negative correlation between age and mean TRF. Based off the linear model, mean TRF = −0.1345 (95%CI: −0.213–-0.056) × age+14.57 (95%CI: 13.76–15.39). (B) Mean TRF over age (y) using a quadratic function is illustrated (mean TRF = 14.64 (95%CI: 13.68–15.6) + −0.16 (95%CI: −0.38–0.055) × age + 0.0014 (95%CI: −0.0088–0.012) × age^2). (C) Mean TRF ± SD in newborn foals (24 h old), 3–6 year (4.4 ± 1.17 y), 7–11 year (9.5 ± 1.51 y) and 12–25 year (16.42 ± 4.21 y) old horses (n = 11, 14.95 ± 1.55; n = 10, 13.80 ± 2.20; n = 10, 12.72 ± 1.15; n = 12, 12.66 ± 1.85, respectively, overall p = .01). Data generated from an ANOVA model with Fisher's LSD. **p < .01.
Acknowledgements We thank the owners/trainers for enrolling their animals in this
3
Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Short report
Age-associated telomere shortening in Thoroughbred horses a,⁎
b
Joshua Denham , Kim Stevenson , Michele M. Denham a b c
T
c
Discipline of Exercise Science, School of Health and Biomedical Sciences, Bundoora West Campus, RMIT University, Bundoora, VIC 3083, Australia Discipline of Biosciences and Food Technology, School of Science, Bundoora West Campus, RMIT University, Bundoora, VIC 3083, Australia Jubilee Stud, Mount Duneed Road, Freshwater Creek, VIC 3217, Australia
A R T I C LE I N FO
A B S T R A C T
Section Editor: Richard Aspinall
Telomeres are genetically conserved repetitive terminal DNA that protect against genomic instability and shorten with ageing. Here, we reveal the leukocyte telomere length of Equus caballus by measuring terminal restriction fragments (TRFs) using Southern Blot analysis in a cohort of 43 Thoroughbred horses (age: 24 h–25 years). Heterogeneous TRFs were observed in each animal and large inter-animal variation in mean TRF was observed (range: 10.5–18.7 kbp). Mean TRFs were inversely correlated with age (r = −0.47). The estimated yearly rate of telomere attrition was 134 bp. Horses should be considered as an alternative animal model to investigate environmental and lifestyle factors that regulate telomeres and promote healthy ageing.
Keywords: Equus caballus Racehorse Ageing Telomeres Biological ageing
1. Introduction Telomeres are genetically conserved DNA at the distal ends of linear chromosomes that maintain genomic integrity. Given most healthy somatic cells in humans have very low levels or are devoid of telomerase activity (Burger et al., 1997), telomeres shorten with progressive cellular divisions which is accelerated by environmental insults (e.g. oxidative stress and inflammation) (Prasad et al., 2017). Although the telomere sequence is conserved amongst mammals (5′-TTAGGG-3′), there are large inter-species differences in the actual lengths and rate of shortening. Telomere shortening has a role in ageing, as excessive telomere attrition leads to premature ageing and serious life-threatening diseases, called telomere syndromes, in both humans and animal models (Armanios and Blackburn, 2012). The telomere length differences and rate of telomere shortening likely have implications for the evolution of the protection against cancer and longevity (Tian et al., 2018). The Thoroughbred horse (Equus caballus) is an athletic mammal, due to hundreds of years of artificial selection for traits important for racing performance. Indeed, typical Thoroughbred racehorses possess maximal aerobic fitness levels of 158 ml·kg−1·min−1 (Young et al., 2002), 2–3 times the average trained human athlete. Despite the years of selection for racing performance, there is also a high rate of wastage in the Thoroughbred horse racing industry due to the rigours associated with training. For instance, high rates of musculoskeletal injuries, psychological stress and illness have been reported (Wilsher et al.,
2006). Concerns have directed effort towards identifying potential markers of overtraining and illness in equine athletes (de GraafRoelfsema et al., 2007; McGowan and Whitworth, 2008), yet none can quantify cumulative animal welfare. Leukocyte telomere dynamics (i.e. length or rate of attrition) are proposed markers of cumulative welfare of other animals (Bateson, 2016; Seeker et al., 2018). Therefore, the analysis of telomere dynamics could be particularly useful for monitoring equine athletes to ensure they are coping with the demands of training in high performance environments. Interestingly, maximal aerobic fitness is typically positively correlated to telomere length in humans (Denham et al., 2016) and exercise training appears to buffer the harmful effects of psychological stress on telomere shortening and could regulate telomere maintenance (Denham et al., 2016; Ludlow et al., 2013; Puterman et al., 2010). As such, Thoroughbred horses could be an alternative animal model to investigate the influence of exercise and psychological stress on telomere biology. We previously found a negative correlation between age and relative leukocyte telomere length measured by qPCR in a cohort of Thoroughbred horses (Denham and Denham, 2018). To extend our previous work, we measured terminal restriction fragments (TRF) using Southern Blot analysis, to determine the length of leukocyte telomeres in a subset (N = 43) of horses. 2. Materials and methods Animal ethics clearance, consent, the blood collection, DNA
⁎ Corresponding author at: School of Health and Biomedical Sciences, Bundoora West Campus, RMIT University, Room 53, Level 4, Building 202, Bundoora, VIC 3083, Australia. E-mail address: [email protected] (J. Denham).
https://doi.org/10.1016/j.exger.2019.110718 Received 2 April 2019; Received in revised form 13 June 2019; Accepted 30 August 2019 Available online 31 August 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.
Experimental Gerontology 127 (2019) 110718
J. Denham, et al.
extraction and assessment procedures have been outlined previously (Denham and Denham, 2018). Blood samples were collected from Thoroughbred horses from farms located around the Geelong region (Victoria, Australia). Aside from the newborn foals and their mothers, most of the horses included in the analysis were not directly genetically related. The horses in this study were not housed in stables, rather they were in small to moderate-sized paddocks. Blood sample collection occurred in 2016 and was finalised midway through 2017. DNA was extracted from buffy coat leukocytes during March 2017 and was completed in August 2017. DNA samples were stored at −20 °C until TRF experiments completed in January 2019. To ensure the integrity and suitability of frozen DNA samples for Southern Blot, four random samples were thawed and checked for signs of degradation using SYBR safe stained, 0.8% agarose gel electrophoresis and imaged on the Gel Documentation System (Biorad). No signs of degradation were observed (data not shown). TRFs were assayed using the TeloTAGGG Telomere Length Kit (Sigma-Aldrich) as per the manufacturer's instruction, but with some minor adjustments. Briefly, 2 μg of DNA was digested for 2 h at 37 °C with restriction enzymes, Hinf I and Rsa I, and DNA fragments separated by gel electrophoresis (ethidium bromide stained 0.8% agarose gel) for 19 h at 30 V. DNA was visualised using the Gel Documentation System (Biorad), before being prepared for transfer to a nylon membrane by Southern blotting overnight (20.5 h). DNA was UVcross linked to the membrane, washed, then exposed hybridized with a DIG-specific antibody coupled to alkaline phosphatase, washed, then exposed to a chemiluminescent substrate for alkaline phosphatase (CDP-Star) before imaging on the ChemiDoc Touch Gel Imaging System (Biorad). TRFs were compared against biotinylated DNA ladders of known lengths and mean TRFs were calculated using the ImageJ (Schneider et al., 2012) and TeloRun (Baur et al., 2004) software. The TRFs for each individual horse was measured once. 3. Results Leukocyte TRFs were heterogeneous within individual animals (Fig. 1). Large inter-animal variations in mean telomere length were observed (mean TRF range: 10.5–18.7 kbp). Leukocyte telomere length was inversely correlated to age (r = −0.47, p = .001). Linear regression analysis indicated that with each year of ageing, on average, leukocyte telomeres shorten by 134 bp (Fig. 2A). An ANOVA revealed a statistically significant main effect of age quartile on telomere length (F3, 39 = 4.26, p = .01, Fig. 2B). Compared to the newborn foals, the 7–11 year and 12–25 year old horses had much shorter telomeres (p < .01), which survived Bonferroni adjustment (both p < .05).
Fig. 1. A representative analysis of Thoroughbred horse terminal restriction fragment (TRF) lengths. Horse donors are numbered 1–9 and the TRFs are measured in kilo-base pairs (kbp). Age (years).
than humans, yet age approximately three times quicker. The equine and human telomeric proteins, namely shelterin, are comparable enough to elicit telomere extension and immortalization in equine fibroblasts when introduced with the two major protein components of human telomerase, hTERT and hTERC in vitro (Vidale et al., 2012). Our study confirms the inverse correlation between age and telomere length, and has characterised the length of leukocyte telomeres in Thoroughbred horses. Given the much shorter lifespan and higher annual rate of telomere attrition in horses – estimated from our data (134 bp) – compared to humans (31 bp) (Chen et al., 2011), horses could be an alternative animal model of accelerated ageing to determine the influence of disease and environmental factors (e.g. psychological stress and exercise) on telomere biology. Future work should investigate the rate of telomere length change with ageing, traits and environmental factors correlated to telomere length and the molecular mechanisms governing telomere regulation in the horse.
4. Discussion and conclusions Previous investigations have analysed leukocyte telomere length in context with age and different traits in other equine breeds (e.g. donkeys) (Argyle et al., 2003) or have measured them in arbitrary units (Denham and Denham, 2018; Katepalli et al., 2008). Unlike donkeys, Thoroughbred horses are a much more athletic breed of equids and until now the actual leukocyte telomere length were unknown. The TRFs of Thoroughbred horses appear to be a similar length to that of donkeys (Argyle et al., 2003) and breeds of zebra (Vidale et al., 2012). However, there seems to be much more variation amongst newborn Thoroughbred foals, which is consistent with our previous investigation (Denham and Denham, 2018). Considering the evidence that suggests early life telomere length predicts life-span of animals (Eastwood et al., 2018; Heidinger et al., 2012) and that telomere length is linked to immune function (Weng, 2012), the large inter-animal variation in leukocyte telomere length warrants future in-depth analyses. Such studies could have implications for animal welfare and particularly relevant for breeders and Thoroughbred racehorse trainers in the industry. Thoroughbred horses have average telomeres ~5–10 kbp longer
Declaration of competing interest MD owns and operates a Thoroughbred horse stud.
2
Experimental Gerontology 127 (2019) 110718
J. Denham, et al.
research project. References Argyle, D., Ellsmore, V., Gault, E.A., Munro, A.F., Nasir, L., 2003. Equine telomeres and telomerase in cellular immortalisation and ageing. Mech. Ageing Dev. 124, 759–764. Armanios, M., Blackburn, E.H., 2012. The telomere syndromes. Nat. Rev. Genet. 13, 693–704. Bateson, M., 2016. Cumulative stress in research animals: telomere attrition as a biomarker in a welfare context? Bioessays 38, 201–212. Baur, J.A., Wright, W.E., Shay, J.W., 2004. Analysis of mammalian telomere position effect. Methods Mol. Biol. 287, 121–136. Burger, A.M., Bibby, M.C., Double, J.A., 1997. Telomerase activity in normal and malignant mammalian tissues: feasibility of telomerase as a target for cancer chemotherapy. Br. J. Cancer 75, 516–522. Chen, W., Kimura, M., Kim, S., Cao, X., Srinivasan, S.R., Berenson, G.S., Kark, J.D., Aviv, A., 2011. Longitudinal versus cross-sectional evaluations of leukocyte telomere length dynamics: age-dependent telomere shortening is the rule. J. Gerontol. A Biol. Sci. Med. Sci. 66, 312–319. de Graaf-Roelfsema, E., Keizer, H.A., van Breda, E., Wijnberg, I.D., van der Kolk, J.H., 2007. Hormonal responses to acute exercise, training and overtraining. A review with emphasis on the horse. Vet. Q. 29, 82–101. Denham, J., Denham, M.M., 2018. Leukocyte telomere length in the Thoroughbred racehorse. Anim. Genet. 49, 452–456. Denham, J., O'Brien, B.J., Charchar, F.J., 2016. Telomere length maintenance and cardiometabolic disease prevention through exercise training. Sports Med. 46, 1213–1237. Eastwood, J.R., Hall, M.L., Teunissen, N., Kingma, S.A., Hidalgo Aranzamendi, N., Fan, M., Roast, M., Verhulst, S., Peters, A., 2018. Early-life telomere length predicts lifespan and lifetime reproductive success in a wild bird. Mol. Ecol. 28 (5), 1127–1137. Heidinger, B.J., Blount, J.D., Boner, W., Griffiths, K., Metcalfe, N.B., Monaghan, P., 2012. Telomere length in early life predicts lifespan. Proc. Natl. Acad. Sci. U. S. A. 109, 1743–1748. Katepalli, M.P., Adams, A.A., Lear, T.L., Horohov, D.W., 2008. The effect of age and telomere length on immune function in the horse. Dev. Comp. Immunol. 32, 1409–1415. Ludlow, A.T., Ludlow, L.W., Roth, S.M., 2013. Do telomeres adapt to physiological stress? Exploring the effect of exercise on telomere length and telomere-related proteins. Biomed. Res. Int. 2013, 601368. McGowan, C.M., Whitworth, D.J., 2008. Overtraining syndrome in horses. Comp. Exercise Physiol. 5, 57–65. Prasad, K.N., Wu, M., Bondy, S.C., 2017. Telomere shortening during aging: attenuation by antioxidants and anti-inflammatory agents. Mech. Ageing Dev. 164, 61–66. Puterman, E., Lin, J., Blackburn, E., O'Donovan, A., Adler, N., Epel, E., 2010. The power of exercise: buffering the effect of chronic stress on telomere length. PLoS One 5, e10837. Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. Seeker, L.A., Ilska, J.J., Psifidi, A., Wilbourn, R.V., Underwood, S.L., Fairlie, J., Holland, R., Froy, H., Salvo-Chirnside, E., Bagnall, A., Whitelaw, B., Coffey, M.P., Nussey, D.H., Banos, G., 2018. Bovine telomere dynamics and the association between telomere length and productive lifespan. Sci. Rep. 8, 12748. Tian, X., Doerig, K., Park, R., Can Ran Qin, A., Hwang, C., Neary, A., Gilbert, M., Seluanov, A., Gorbunova, V., 2018. Evolution of telomere maintenance and tumour suppressor mechanisms across mammals. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 373. Vidale, P., Magnani, E., Nergadze, S.G., Santagostino, M., Cristofari, G., Smirnova, A., Mondello, C., Giulotto, E., 2012. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts. Chromosoma 121, 475–488. Weng, N.P., 2012. Telomeres and immune competency. Curr. Opin. Immunol. 24, 470–475. Wilsher, S., Allen, W.R., Wood, J.L., 2006. Factors associated with failure of thoroughbred horses to train and race. Equine Vet. J. 38, 113–118. Young, L.E., Marlin, D.J., Deaton, C., Brown-Feltner, H., Roberts, C.A., Wood, J.L., 2002. Heart size estimated by echocardiography correlates with maximal oxygen uptake. Equine Vet. J. Suppl. 467–471.
Fig. 2. Terminal restriction fragment (TRF) analysis in Thoroughbred horses. (A) A moderate negative correlation between age and mean TRF. Based off the linear model, mean TRF = −0.1345 (95%CI: −0.213–-0.056) × age+14.57 (95%CI: 13.76–15.39). (B) Mean TRF over age (y) using a quadratic function is illustrated (mean TRF = 14.64 (95%CI: 13.68–15.6) + −0.16 (95%CI: −0.38–0.055) × age + 0.0014 (95%CI: −0.0088–0.012) × age^2). (C) Mean TRF ± SD in newborn foals (24 h old), 3–6 year (4.4 ± 1.17 y), 7–11 year (9.5 ± 1.51 y) and 12–25 year (16.42 ± 4.21 y) old horses (n = 11, 14.95 ± 1.55; n = 10, 13.80 ± 2.20; n = 10, 12.72 ± 1.15; n = 12, 12.66 ± 1.85, respectively, overall p = .01). Data generated from an ANOVA model with Fisher's LSD. **p < .01.
Acknowledgements We thank the owners/trainers for enrolling their animals in this
3