Effects of Beef Breed Type, Residual Feed Intake and Collagen Heat Solubility on Meat Quality, Connective Tissue Characteristics and Expression of Collagen-Related Genes of Semimembranosus Muscle by Rabaa Hamed A thesis submitted in partial fulfillment of the requirements for the degree of Master Science in Animal Science Department of Agricultural, Food and Nutritional Science University of Alberta ©Rabaa Hamed, 2020 Abstract Cattle breed, residual feed intake and genetics are factors that affect beef quality. Genotype can influence meat toughness and approximately 46% of beef quality variation can be attributed to the variation between animals. Calpain (CAPN1) and calpastatin (CAST) genes are associated with myofibrillar protein degradation early post mortem and are correlated with variation in beef toughness but the relationships between genes controlling collagen synthesis and degradation and beef toughness have not yet been examined. The objectives of this research were to: (1) determine the effect of breed and residual feed intake on beef quality and collagen characteristics; and (2) determine how the expression of genes involved in collagen synthesis and degradation were related to meat quality and collagen characteristics of the semimembranosus, a muscle from the inside round. Seventy-one (71) carcasses were harvested from purebred Angus (n = 23) and Charolais (n = 24) and Angus crossbred (Kinsella composite, n = 24) steers of high (n = 35) and low (n = 36) residual feed intake status. The m. semimembranosus muscle (inside round) was removed from the right side of each carcass, and steaks from each muscle were aged for 3 and 13 days. Breed and residual feed intake did not affect meat quality and collagen characteristics however post mortem aging decreased Warner-Bratzler shear force and increased collagen heat solubility. From the data set, twelve steers with low [10.66 + 2.99 (standard deviation, SD) %] and twelve with high [20.61 + 7.51 (SD)%] intramuscular collagen heat solubility at day 3 post mortem were selected within each breed. The expression of 14 candidate genes in the m. semimembranosus was evaluated using quantitative real time polymerase chain reaction (RT-qPCR) using gene specific primer pairs relative to 18S RNA expression. Breed type significantly (P<0.05) affected the mean ∆Ct of CTGF, FOXO1, P4HA1 and SMAD2. Collagen heat solubility did not affect the expression level of the genes; however, there was an interaction between breed type and collagen heat solubility in the ∆Ct mean for CTGF. Sarcomere length was negatively correlated (P<0.001) with COL3A1 and COL5A2 and P4HA1 (P<0.05) gene expression. Warner-Bratzler shear force at 3 days post-mortem was negative correlated with COL3A1 (P<0.05) and FOXO1 (P<0.01) but was positively correlated with CTGF, IGF-1 while SMAD2. COL5A2 and MMP2 were positively correlated (P<0.05) with heat-soluble collagen at 3 days post-mortem. The concentration of pyridinoline cross links (mol PYR/mol collagen) was negatively correlated (P<0.05) with COL3A1 and COL5A2 while positively correlated with COL5A1. Expression of LOX was positively ii correlated (P<0.01) with the concentration of pyridinoline (nmol PYR/ g raw meat). Results indicated that expression of genes related to collagen synthesis and degradation were related to increased beef toughness. Overall, this study indicated that selection for low RFI cattle will not compromise m. semimembranosus meat quality, and that the contribution of collagen to beef toughness can be influenced through breed type selection. iii To my husband Mutaz Mukhtar iv Acknowledgements I would like to express my sincere thanks and deepest gratitude to my supervisor Dr. Heather Bruce for her continuous advice, fruitful suggestions, and encouragement throughout my master program. I would like to also thank my supervisory committee members, Dr. Leluo Guan and Dr. Jonathan Curtis for helpful discussions and monitoring the progress of my projects. Thank you Yanhong Chen for all the help and support you gave me during the gene expression study. My thanks extend also to Dr. Bimol Roy for his constant help and unlimited support. Special thanks are due to all my lab-mates especially, Dr. Shahid Mahmood, Dr. Huaigang Lei, Maidah Khaliq, Zhiqiang Jiu, Esther Ijiwade, and Prince Opoku for their unlimited help, motivation and encouragement. Special thanks to my Father Ahmed Hamed and my mother Mariam Khaleel, and my sisters and brothers who have been always there for me with their love, support and motivation. Special thanks to my husband Mutaz Muhktar, my daughter Minna and my son Faris for their patience, encouragement, and support during this long time towards completing my degree. My thanks are also extending to Eiman Osman, who became like a family member here in Canada, and I would like to thank her for unlimited support in all life matters. Finally, I would like to thank the Islamic Development Bank scholarship for giving me this chance to study at the University of Alberta. My thanks also extend to Alberta Innovates Bio Solutions, Alberta Livestock and Meat Agency, the Beef Cattle Research Council and the NSERC CREATE for Assuring Meat Safety and Quality for providing funding for my research and my stipend. v Table of Content Contents Abstract ........................................................................................................................................... ii Acknowledgements ......................................................................................................................... v Table of Content ............................................................................................................................ vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................ xi Abbreviations ................................................................................................................................ xii Chapter 1: Introduction and literature review ................................................................................. 1 1.1 Beef quality and how it is measured ................................................................................................... 2 1.1.1. Meat technological measurements ...................................................................................... 3 1.1.2. Sensory measurements ......................................................................................................... 3 1.1.3. Collagen quality measurement ............................................................................................. 5 1.2 Factors affecting beef quality ............................................................................................................ 6 1.2.1 Beef breed .................................................................................................................................... 6 1.2.2 Residual feed intake ..................................................................................................................... 8 1.2 3 Animals age and pre-slaughter conditions................................................................................... 9 1.2.4 Sarcomere length ....................................................................................................................... 10 1.2.5 Post-mortem aging .................................................................................................................... 12 1.2.6 Calpain and calpastatin activities ............................................................................................... 14 1.2 7 Connective tissue characteristics ............................................................................................... 15 1.2.7.1 Collagen structure and classification ................................................................................. 16 1.2.7.2 Collagen biosynthesis .......................................................................................................... 17 1.2.7.3 Collagen cross-linking formation ........................................................................................ 19 1.2.7.4 Collagen degradation .......................................................................................................... 20 1.2.7.5 Total collagen content, heat soluble collagen and meat quality ....................................... 22 1.3 Gene expression and meat quality .................................................................................................. 23 1.4 Collagen related genes ................................................................................................................ 24 1.4.1 Collagen Type III Alpha 1 Chain (COL3A1) ................................................................................. 25 1.4.2 Collagen Type V ( COL V A1 and COLV A2) ................................................................................ 25 1.4.3 Connective tissue growth factor (CTGF) .................................................................................... 25 1.4.4 Insulin-like growth factor-1 (IGF-1) ............................................................................................ 26 1.4.5 Lysyl Oxidase (LOX) ..................................................................................................................