Abstract
Obesity, a major concern for equine welfare, is highly prevalent in the leisure horse population. Skeletal-muscle and adipose tissues are important determinants of maintenance energy requirements. The myostatin and perilipin pathways play key roles in the regulation of muscle mass and lipolysis respectively and have both been associated with obesity predisposition in other mammalian species. High quality samples, suitable for molecular biology, are an essential prerequisite for detailed investigations of gene and protein expression. Hence, this study has evaluated a) the post-mortem stability of RNA extracted from skeletal-muscle and adipose-tissues collected under commercial conditions and b) the tissue-specific presence of myostatin, the moystatin receptor (activin receptor IIB, ActRIIB), follistatin and perilipin, genes and proteins across a range of equine tissues. Objectives were addressed using tissues from 7 Thoroughbred horses presented for slaughter at a commercial abattoir; a) samples were collected at 7 time-points from Masseter muscle and perirenal adipose from 5 minutes to 6 hours post-mortem. Extracted RN was appraised by Optical Density analysis and agarose-gel electrophoresis. b) Quantitative real time PCR and Western Blotting were used to evaluate gene and protein expression in anatomically-defined samples collected from 17 tissues (6 organs, 4 skeletal muscles and 7 discrete adipose depots). The results indicate that, under the present collection conditions, intact, good quality RNA could be extracted from skeletal-muscle for up to 2 hours post-mortem. However, RNA from adipose tissue may be more susceptible to degradation/contamination and samples should be collected no later than 30 minutes post-mortem. The data also show that myostatin and ActRIIB genes and proteins were almost exclusively expressed in skeletal muscle. The follistatin gene showed a more diverse gene expression profile, with expression evident in several organs, adipose tissue depots and skeletal muscles. Perilipin gene and protein were almost exclusively expressed by adipose tissue.
Highlights
Obesity, having reached epidemic proportions among horses and ponies in industrialised nations, is considered a key concern for equine welfare [1,2]
It is widely accepted that skeletal muscle and adipose tissues engage in cross-talking pathways which ensure that they work in synergy to conserve energy balance and whole body homeostasis [4,5]
By 360 minutes the average ratio had reduced to 1.42 and the variation was considerably increased (Table 4). These results show Masseter muscle is resilient to post-mortem RNA degradation, but samples should be obtained within 2 hours of death to ensure the Tissue Massater muscle
Summary
Obesity, having reached epidemic proportions among horses and ponies in industrialised nations, is considered a key concern for equine welfare [1,2]. Adipose tissue and skeletal muscle can be considered as labile reserves of energy and nutrients within the body which can be used as buffers at times of negative or positive energy balance [3]. The specific anabolic/catabolic pathways which are activated during periods of energy imbalance may be dependent on factors which regulate or modify the relative contributions of muscle or adipose tissue to whole body composition. It is widely accepted that skeletal muscle and adipose tissues engage in cross-talking pathways which ensure that they work in synergy to conserve energy balance and whole body homeostasis [4,5]. Muscle and adipose tissues act and interact dynamically to promote energy homeostasis but in states of active weight gain/ loss, homeostasis is over-ridden and the relative contributions of these tissues to body composition are altered. Two proteins have attracted increasing interest in the regulation of tissue reserves; myostatin, which regulates reserves of metabolically active muscle [6]; and perilipin, which regulates intra-cellular lipolysis [7]
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