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Some postmortem aspects of broiler breast muscle Wood, Darrell Fenwick 1973

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SOME POSTMORTEM ASPECTS OF BROILER BREAST MUSCLE \Gl by DARRELL FENWICK WOOD B.Sc.(Agr.), McGill University, 1963 M.Sc., McGill University, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Food Science We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA February, 1973 In presenting this thesis in part ia l fulfilment of the requirements for an advanced degree at the University of Br i t i sh Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for f inancial gain shall not be allowed without my written permission. Department of Food S c i e n c e The University of Br i t i sh Columbia Vancouver 8, Canada Date March 30, 1973. - i i i -ABSTRACT The a b i l i t y of broiler Pj^ major muscle to develop postmortem isometric tension was studied under a variety of conditions. Muscle strips developed and released tension while suspended in phosphate buffer pH 7.2. .The addition of calcium or magnesium to the buffer enhanced tension release, as did the presence of either of these two ions and EDTA. Different rates of tension release v/ere evident from bird to bird and the rate of release was fastest in birds which required the shortest time to reach maximum tension. The " proportion of tension released within one hour of reaching maxi-mum tension correlated significantly with subsequent proportions from 2 through 12 hours after maximum tension, making i t possible to predict, with reasonable accuracy, tension release from 1 hour values. The one hour release values had a significant relation-ship with time to maximum tension but no relationship with tenderness suggesting that tension release, from bird to bird, i s not indicative of tenderness. Free struggle at slaughter, hot water scalding and mechanical plucking were shown to have an additive effect on tension parameters and tenderness of broiler P.major. Pre-slaughter epinephrine injection was shown to deplete muscle glycogen levels within 8 - 1 2 hours post-injection. ATP levels remained high resulting in considerable tension development. It appears that such tension development is sufficient to account for the subsequent toughening observed - i v -i n the muscle. B r o i l e r s were s u b j e c t e d t o three d i f f e r e n t s t r e s s s i t u a t i o n s (commercial h a n d l i n g , c o l d and h e a t ) . No s i g n i f i -cant changes i n postmortem muscle q u a l i t y were observed as a r e s u l t o f these treatments. B r o i l e r s appeared to respond d i f f e r e n t l y under the d i f f e r e n t c o n d i t i o n s w i t h heat s t r e s s s h o r t e n i n g the time t o reach maximum t e n s i o n and commercial and c o l d s t r e s s e s l e n g t h e n i n g the time t o maximum t e n s i o n . A c o l d s h o r t e n i n g e f f e c t v;as observed d u r i n g the study o f the e f f e c t of temperature on t e n s i o n p a t t e r n . The amount of c o l d s h o r t e n i n g observed was i n c r e a s e d as tempera-t u r e was lowered from 5°C to 0°C. At 2°C, the i n i t i a l c o l d s h o r t e n i n g o c c u r r e d and no f u r t h e r t e n s i o n development was observed even a f t e r 36 hours at t h i s temperature. Removal of s t r i p s from 2°C to room temperature a f t e r 12 and 24 hours r e s u l t e d i n some f u r t h e r t e n s i o n development but e s s e n t i a l l y no t e n s i o n was observed when 36 hour s t r i p s were brought t o room temperature. The c o l d s h o r t e n i n g d i d not s i g n i f i c a n t l y lower muscle ATP and c r e a t i n e phosphate l e v e l s a lthough a decrease was observed. - V -Abstract Li s t of Tables List of Figures Acknov.'ledgements INTRODUCTION LITERATURE REVIEW 3 General Muscle Tenderness 3 Antemortem Tenderness Related Factors 4 Breed and Strain 4 Sex and Age 5 Ration 6 Muscle Type 6 Slaughter and Processing Tenderness Related Factors 7 Humane Slaughter 7 Scalding 8 Beating-Picking 9 Hot Cutting 10 Pre-Rigor Tenderness Related Factors 10 Chemical Changes 11 Physical Changes 13 Post-Rigor Tenderness Related Factors 15 Resolution of Rigor 15 Aging and Frozen Storage 16 TABLE OF CONTENTS Page i i i v i i i x xi - v i -Page Methods of Measuring Muscle Tenderness 17 Isometric Tension Measurement 18 Antemortem Stress and Postmortem Muscle Quality 20 MATERIALS AND METHODS 22 Isometric Tension Experiments 22 Muscle Source 22 Isometric Tension Measurement 22 The Effect of Environment on Tension Pattern 25 The Effect of pH on Tension Pattern 25 The Effect of Calcium, Magnesium and EDTA on Tension Pattern 2 5 The Effect of Temperature on Tension Pattern 26 Effect of Processing Techniques on Tension Pattern and Tenderness 26 Epinephrine Experiments 28 Preliminary Experiment 28 Experiment 1 28 Experiment 2 29 Sample Preparation for Metabolite Assay 29 pH 29 Metabolite /analyses 30 Stress Experiments 30 Commercial Stress 30 Heat Stress 31 - v i i -Page Cold Stress - 31 Cold Shortening Experiments 32 Experiment 1 32 Experiment 2 3 3 RESULTS AND DISCUSSION 34 Isometric Tension Experiments 34 The Effect of Environment on Tension Pattern 34 The Effect of pH on Tension Pattern 36 The Effect of Temperature on Tension Pattern 40 The Effect of Calcium, Magnesium and EDTA on Tension Pattern 44 The Effect of Processing Techniques on Tension Pattern and Tenderness 55 Segregation of Broiler Controls on the Basis of Time to Reach Maximum Tension 58 The Relation Between One Hour Tension Release, Time to Maximum Tension and Shear Value 64 Epinephrine Experiments 66 Preliminary Experiment ' 66 Epinephrine Experiment 1 6 8 Epinephrine Experiment 2 71 Stress Experiments 81 Cold Shortening Studies 88 SUMMARY AND CONCLUSIONS 95 LITERATURE CITED 99 - v i i i -LIST OF TABLES Table Page I Means and Standard Errors of Time and Tension Development for Strips of Broiler major Muscle Run in Phosphate Buffer at Four Different pH Levels. 38 II Means and Standard Errors of Time and Tension Development for Strips of Broiler Pj. major Muscle Run in Buffers at Various Temperatures. 41 III Means and Standard Errors of Time and Tension Development for Strips of Broiler and Fowl P^ major Muscle Run in Phosphate Buffer and Buffer Contain-ing Three Levels of Calcium, 4 5 IV Means and Standard Errors of Time and Tension Development for Strips of Broiler P_a. major Muscle in Phosphate Buffer and Buffer Containing Mg++, EDTA, C a + + + EDTA and Mg + + + EDTA, 53 V Means and Standard Errors of Tension Parameters for Inner and Outer Strips of Broiler major Muscle Subjected to Various Post-Slaughter Treatment. 56 VI Means and Standard Errors of Pooled Tension Parameters and Shear Value Data for Inner and Outer Strips of Broiler Pj_ major Subjected to Various Post-Slaughter Treatments, 57 VII Time and Tension Means and Standard Deviations for Three Eroiler Groups Segregated on the Basis of Time to Reach Maximum Tension, 59 VIII Regression Line Parameters for Tension Release (Independent Variable) Versus Time (Dependent Vari-able) from Three Groups of Broiler Controls and for the Pooled Groups, 61 IX Simple Correlations of One Hour Tension Release Values with Subsequent Hourly Values from Three Groups of Control Broilers and for the Pooled Data from the Three Groups, 62 X Simple Correlations of Two Hour Tension Release Values with Subsequent Hourly Values from Three Groups of Control Broilers and for the Pooled Data from the Three Groups, 63 - ix -Table Page XI Tension Parameters and Shear Values for Pj_ major Muscle from Broilers Injected with Epinephrine at Various Times Pre-Slaughter. 67 XII Analysis of Variance for Parameters Studied in Epinephrine Experiment 1. 6 8 XIII Duncan's New Multiple Range Test on Significant Treatment Means from Epinephrine Experiment 1. 69 XIV Correlation Matrix for Parameters Studied in Epinephrine Experiment 1. 70 XV Analysis of Variance of Parameters Studied in Epinephrine Experiment 2, 72 XVI Duncan's New Multiple Range Test on Significant Treatment Means from Epinephrine Experiment 2. 73 XVII Correlation Matrix for Parameters Studied in Epinephrine Experiment 2. 78 XVIII Means and Standard Errors of Parameters of major Muscle from Broilers in the Commercial Stress Experiment. 82 XIX Means and Standard Errors of Parameters of P_j_ major Muscle from Broilers in the Heat Stress Experiment. 84 XX Means and Standard Errors of Parameters of Pj, major Muscle from Broilers in the Cold Stress Experiment. 85 XXI Means and Standard Errors of Tension Parameters of Broiler Pj. major Muscle Subjected to Various Post-Slaughter Temperature Treatments. 89 XXII Means and Standard Errors of ATP, HMP and CP Content of Broiler P_j_ major Muscle Subjected to Various Temperature Treatments. 92 - x -LIST OF FIGURES Figure Page 1 E f f e c t of d i f f e r e n t e x t r a c e l l u l a r incubation media on isometric tension pattern of b r o i l e r P_^  major muscle. 35 2 E f f e c t of e x t r a c e l l u l a r pK on isometric tension pattern of b r o i l e r Pj^ ma j or muscle. 37 3 E f f e c t of 1Q~3 i-l calcium on tension delcine i n b r o i l e r P^ major muscle, 47 4 E f f e c t of 10"*"* M calcium on tension decline i n fowl p^ major muscle, 48 5 E f f e c t of 10"3 M magnesium, EDTA and calcium-EDTA on tension decline i n b r o i l e r P_j_ major muscle. 52 6 Isometric tension decline i n three groups of b r o i l e r s separated on the basis of time required to reach maximum tension, 60 7 Relation between tension, time to maximum tension and shear value i n P_j_ major muscle from epinephrine treated b r o i l e r s . 75 8 Relation between muscle glycogen and ATP l e v e l s and shear value i n Pj^ major from epinephrine treated b r o i l e r s , 76 9 Relation between pH, muscle lactate and shear value i n P^ major from epinephrine treated b r o i l e r s , 77 - x i -ACKNOWLEDGE MENTS The author wishes to express h i s s i n c e r e a p p r e c i a t i o n to h i s a d v i s o r , Dr. J . F . R i c h a r d s , A s s o c i a t e P r o f e s s o r , Depart-ment of Food S c i e n c e f o r h i s guidance and encouragement d u r i n g the course of t h i s study. He i s a l s o t h a n k f u l t o the members of h i s graduate committee: Dr. S. Nak a i , Department of Food S c i e n c e Dr, W.D. Powrie, Department of Food Science Dr. C.W. Roberts, Department of P o u l t r y S c i e n c e P r o f e s s o r L.M. S t a l e y , Department of A g r i c u l t u r a l E n g i n e e r i n g Dr. M.A. Tung, Department of Food S c i e n c e . f o r t h e i r encouragement and co n t i n u e d i n t e r e s t i n the r e s e a r c h and f o r the review o f t h i s t h e s i s and a s p e c i a l note o f thanks to Dr. Tung f o r computer a s s i s t a n c e . The author i s g r a t e f u l t o the Department of P o u l t r y S c i e n c e f o r the use of p o u l t r y farm f a c i l i t i e s and f o r s u p p l y i n g some of the b r o i l e r s used i n t h i s study. He i s a l s o g r a t e f u l t o Mr. Garth Sundeen f o r t e c h n i c a l a s s i s t a n c e ) to- Miss Lynne Robinson f o r computer a s s i s t a n c e and p r e p a r a t i o n of the f i g u r e s and to the l a t e Mr, W. Gleave f o r c o n s t r u c t i o n of the chambers used i n the i s o m e t r i c t e n s i o n s t u d i e s . F i n a n c i a l support of the U n i v e r s i t y of B r i t i s h Columbia through a post-graduate F e l l o w s h i p i s g r a t e f u l l y acknowledged, A s p e c i a l note o f g r a t i t u d e i s a l s o expressed t o my w i f e , C a r o l , f o r her encouragement and understanding d u r i n g the course o f t h i s study and f o r the t y p i n g of t h i s m anuscript. INTRODUCTION U n t i l r e c e n t l y , muscle b i o l o g y has been d i v i d e d i n t o the study of muscle and the study of meat. The complementary nature of these two areas of study i s obvious and was s t r e s s e d by Dr. B.B. Marsh i n the i n t r o d u c t i o n to the Second Symposium on the P h y s i o l o g y and B i o c h e m i s t r y of Muscle as a Food (1970), i n which he s t a t e d : "In our study of postmortem muscle we must c o n s i d e r more and more the f a c t o r s which determine energy p r o d u c t i o n and u t i l i z a t i o n i n the l i v i n g t i s s u e s the purpose which the muscle s e r v e s , the speed a t which i t moves, the d u r a t i o n of i t s p e r i o d s of continuous use, the age, s p e c i e s , and degree of domes-t i c a t i o n of the animal, the s t r e s s e s (both l o n g - and short-term) to which i t has been exposed. Only when the f a c t s of l i f e i n muscle are known can, we expect t o i n f l u e n c e i t s q u a l i t i e s i n death," Two c l o s e l y r e l a t e d p h y s i c a l changes occur i n p r e -r i g o r , postmortem muscle. These are a s h o r t e n i n g or c o n t r a c -t i o n and a l o s s of e x t e n s i b i l i t y . The s h o r t e n i n g phase, on the animal c a r c a s s , i s c h a r a c t e r i z e d by an i s o m e t r i c t e n s i o n development which r e s u l t s i n the s o - c a l l e d r i g o r s t i f f e n i n g . T h i s phenomenon and a s s o c i a t e d chemical changes have been r e p e a t e d l y shown to i n f l u e n c e the u l t i m a t e a c c e p t a b i l i t y of many muscle systems as food. However, only a meagre amount of i n f o r m a t i o n on these r e l a t i o n s h i p s i s a v a i l a b l e f o r p o u l t r y muscle systems. A study of s i m u l a t e d i s o m e t r i c t e n s i o n development and i t s r e l a t i o n t o tenderness i n b r o i l e r P e c t o r a l i s major muscle i s r e p o r t e d i n t h i s t h e s i s . B r o i l e r s were s u b j e c t e d to some stressors v.'hich may be encountered p r i o r to slaughter under commercial conditions and the e f f e c t of these stressors on postmortem muscle q u a l i t y i s also reported. - 3 -LITERATURE REVIEW General Muscle Tenderness Tenderness, from the consumer viewpoint, i s high on the l i s t of factors determining the acceptability of muscle as a food. In view of this fact, food scientists have devoted considerable time and effort to the study of meat tenderness. One of the greatest concerns in studying tenderness is i t s v a r i a b i l i t y . Paul et a l , (1959)compared cooking losses and tenderness of chickens hatched from eggs laid by the same hen with those of chickens from eggs of different hens in the same flock. The use of half-sibs did not reduce variation although cooking loss and tenderness were similar for both groups. Tenderness varies from muscle to muscle within an animal and from animals of the same or differest species and this variation may be influenced by both ante- and postmortem events. A symposium in 1963 (Campbell Soup Company, 1963) dealt with many of the factors then thought to influence muscle tenderness. Antemortem factors such as breed, sex and management practices and postmortem factors such as aging and cooking method were discussed. This chronological sequence of events overlooked one relatively short, but highly significant, time period — that from slaughter to rigor onset. This period 4 -varies from 2-4.5 hours in poultry and up to 18 - 20 hours in beef and i t s importance had been demonstrated by several workers prior to the symposium (Ramsbottom and Strandine, 1949; Koonz et a l . , 1954; deFremery and Pool, 1 9 6 0 ; Locker, 1960). A review of the factors influencing avian tender-ness was published by Marion (1967) , Two basic physical changes occur in pre-rigor post-mortem muscle, a shortening or tendency to contract and a loss of extensibility. Factors which affect these changes and their relation to tenderness have been reviewed by Marsh (1972) and Newbold and Harris (1972). Both are excellent reviews of pertinent literature in this area. Since shortening or the tendency of the muscle to contract affects eventual muscle tenderness, i t follows in the sequence of events that the post-rigor resolution of these para-meters must also be important. Goll (196 8) reviewed the present literature in this area dealing, in particular, with the evidence which clearly indicates that a resolution of rigor actually occurs in postmortem muscle. The author emphasized the need for further research on the effects of lysosomes and pH changes on postmortem myofibillar proteins in order to better under-stand what causes the resolution of rigor. Antemortem Tenderness Related Factors Breed and Strain The effect of breed and strain on tenderness has been - 5 -d i f f i c u l t to establish. Shrimpton and Miller (1960) com-pared two strains of chickens and found male Leghorns to be less tender than White Rock males and females. Goodwin (1966) compared six strains of Broad Breasted Bronze turkeys and reported that strain did not significantly influence shear values. Larmond et a_l. (1968) found that the effect of breed on goose quality was negative for the genotypes studied. Moran et a l . (1970) and Larmond et a l . (1970) reported that a Cornish male - White Rock female cross gave better carcass quality than the pure strains or reciprocal crosses but no flavor or shear differences were evident between sexes or crosses. Sex and Age It has been generally established that the older the poultry at slaughter the less tender the meat. May et a l . (1962) found that 72 - week-old chickens were less tender than 10 - week-old chickens regardless of the time analyzed postmortem or the aging temperature. Goodwin et al.. (1969) studied 12 strains of birds grown to 8 weeks of age before processing. No difference was found between commercial strains in the shear values of breast and thigh. There was, however, a slight difference in tenderness of the sexes with males having lower shear values for both breast and thigh muscle. A sex re l a t e d tenderness difference i n b r o i l e r s was also demonstrated by Larmond et a l . (1969), but a study of Large White turkeys revealed no s i g n i f i c a n t sex differences i n eating q u a l i t y (Larmond ejt a l . , 1971) . These authors also found that tenderness decreased with age at slaughter. Ration The composition of the d i e t has very l i t t l e or no e f f e c t on tenderness as long as the d i e t provides for optimum growth rate (Marsden et aJU, 1957 a, b; Goerty et aJL,, 1961). Varying the energy l e v e l , however, has been shown to a l t e r the body composition, b a s i c a l l y through protein and f a t changes (Donaldson e_t al_., 1956; Summers et a l . , 1965; Marion et a l . , 1967; Goodwin et. a l . , 1969). Shrimpton and M i l l e r (1960) showed that birds kept on f u l l feed were more tender than birds kept on a r e s t r i c t e d d i e t . Muscle Type In most reports shear values for breast muscle (light) are higher than those for thigh muscle (dark). Peterson et a l . (1959) found that l i g h t muscle i n young birds was s i g n i f i c a n t l y tougher than dark but i n older birds the reverse was found, van den Berg et, a l . (1963, 1964). found that tenderization i n breast muscle was e s s e n t i a l l y complete aft e r 1 - 2 days post-mortem, whereas in leg muscle a second tenderization period occured 2 - 5 days postmortem. Slaughter and Processing Tenderness Related Factors Humane Slaughter Normal slaughtering procedure for poultry consists of suspending birds by their feet, brief e l e c t r i c a l stunning and exsanguination by an external throat cut. This procedure can result in a great deal of struggle before e l e c t r i c a l stunning and a considerable lapse of time before the bird becomes unconscious. deFremery and Pool (1958) found that a relationship existed between struggle at slaughter and tenderness whereas Lineweaver (1959) and Dodge and Stadelman (1960) found no relationship between struggling and postmortem tenderization• Anesthetization, by sodium pentobarbital (Nembutal) has been used in an attempt to find a more humane method of slaughter (May and Huston, 1959; Goodwin et a l . , 1961; Stadelman and Wise, 1961; deFremery, 1965), Goodwin et a l . (1961) found that the method of slaughter had no significant effect on breast muscle tenderness but the use of Nembutal produced some detrimental effects on tenderness. Stadelman and Wise (1961) found that anesthetization with Nembutal leng-thened the period of maximum toughness in chickens. This was later verified by deFremery (1965) but his tenderness findings were contrary to those of Goodwin et a l . (1961), Landes et a l . - 8 -(1971) also found that turkeys anesthetized with Nembutal were more tender than nonanesthetized controls. Immobilization of turkeys and chickens using carbon dioxide has been demonstrated (Drewniak et a l . , 1955; Kotula et al.,1957, 1961). A 75 second exposure to 33 - 36 percent carbon dioxide concentration was shown to keep broilers uncon-scious, but alive, during shackling, sticking and bleeding (Kotula et a l . , 1961). Scalding The adverse effects of high scalding temperatures on poultry tenderness have long been knov/n. Present scalding techniques vary but in general f a l l within the range of 125 -140°F for times of from 30 - 150 seconds. Shannon et a l . (1957) studied 6 levels of scald temperatures for 6 different times at each level. The study showed that time of scald and temperature of scald s i g n i f i -cantly reduced tenderness and time had a greater effect than temperature. These findings were verified on turkeys by Klose et al. (1959) and chickens by Pool et a l , (1959). Wise and Stadelman (1959) investigated the effect of scald time and temperature on tenderness at various depths within chicken Pectoralis major muscle. The authors found that the toughening effect of high temperature-long time scalding was related to the depth to which the scald heat penetrated the muscle. These same authors (Wise and Stadelman, - 9 -1961) suggested t h a t two aspects may be important i n d e s c r i b i n g the s c a l d i n g e f f e c t on tenderness. F i r s t l y , p r o t e i n denatura-t i o n at e l e v a t e d temperatures and secondly, an undefined e f f e c t caused by h o l d i n g the carcass at temperatures i n excess of normal body temperature. Klose e t a l . (1971a) and Kaufman et a l . (1972) e x p e r i -mented w i t h a technique u s i n g a chamber and subatmospheric steam i n an e f f o r t t o e l i m i n a t e immersion s c a l d i n g . The t e c h n i -que was e f f e c t i v e i n reducing p o l l u t i o n and m i n i m i z i n g water requirements but the end product had about the same s h e l f l i f e and degree of tenderness as immersion scalded b i r d s . B e a t i n g - P i c k i n g P i c k i n g machines, which have sto u t rubber f i n g e r s on a r a p i d l y r e v o l v i n g drum, have been shown to produce muscle which i s s i g n i f i c a n t l y tougher than hand picked c o n t r o l s (Wise and Stadelman, 1957; P o o l e t al.,1959; Klose et a l . , 1959) . deFremery and Pool (1960) showed t h a t severe mechanical h a n d l i n g of f r e s h chicken muscle caused r a p i d l o s s of ATP and a r a p i d drop i n muscle pH. Sayre (1969, 1970) a l s o showed t h a t mechanical p i c k i n g l e d t o a r a p i d drop i n pH and subsequent toughness i n the muscle. The author report e d t h a t a combination of s c a l d i n g and b e a t i n g produced postmortem changes s i m i l a r t o those produced by s c a l d i n g and p i c k i n g alone. - 10 -Hot C u t t i n g Lowe (1948) found induced toughness i n b r e a s t muscle which was cut w i t h i n 1 hour of s l a u g h t e r . T h i s toughness p e r -s i s t e d even a f t e r 24 hours of aging and subsequent c o o l i n g . Toughening by p r e - r i g o r c u t t i n g has a l s o been r e p o r t e d by Koonz et a l . (1954) , P o o l e t al. (1959) and Nixon and M i l l e r (1967) . The above r e p o r t s d i d not g i v e p a r t i c u l a r a t t e n t i o n to the type of c u t t i n g . K l o s e e t a l . (197lb)have e x t e n s i v e l y s t u d i e d the e f f e c t of c u t t i n g p o u l t r y c a r c a s s e s at s p e c i f i c times p o s t - s l a u g h t e r , the t y p e s - o f c u t s made and the s p e c i f i c muscles a f f e c t e d by the cut made. They found t h a t k n i f e c u t -t i n g the wings a t the shoulder j o i n t and f l a t t e n i n g the b r e a s t a t 20, 60 and 120 minutes postmortem gave shear v a l u e s t w i c e t h a t o b t a i n e d f o l l o w i n g the same procedure a t 22 hours p o s t -mortem. On the o t h e r hand, i f the wing was sawed o f f a t a p o i n t beyond the b r e a s t muscle i n s e r t i o n , thereby l e a v i n g the wing stub a t t a c h e d to the b r e a s t s e c t i o n , no p r e - r i g o r toughen-i n g e f f e c t was o b t a i n e d . The authors thus concluded t h a t f o r optimum tenderness i n cut-up p o u l t r y c o n s i d e r a t i o n should be g i v e n t o time postmortem and l o c a t i o n of cut i n r e l a t i o n t o b r e a s t muscle. P r e - R i g o r Tenderness R e l a t e d F a c t o r s The b i o p h y s i c a l and b i o c h e m i c a l changes which take p l a c e i n postmortem mammalian muscle have been the s u b j e c t o f - 11 -e x t e n s i v e r e s e a r c h . The changes which occur i n postmortem a v i a n muscle ar e , i n g e n e r a l , the same as those i n the mammal-i a n s p e c i e s . The most obvious change i s the s t i f f e n i n g of the muscle as i t passes i n t o r i g o r m o r t i s . T h i s phenomenon i s accompanied by s e v e r a l b a s i c chemical changes i n c l u d i n g the disappearance of glycogen, ATP and N - p h o s p h o r y l c r e a t i n e ; the appearance of ammonia and i n o s i n i c a c i d from the deamination of a d e n y l i c a c i d ; and the accumulation of l a c t i c a c i d through a n e r o b i c g l y c o l y s i s of muscle glycogen s t o r e s (deFremery, 1966 a ) . Reviews on p r e - r i g o r changes (Newbold and H a r r i s , 1972) and p o s t - r i g o r r e s o l u t i o n ( G o l l , 196 8) have covered many of the p e r t i n e n t r e f e r e n c e s i n these a r e a s . Chemical Changes One of the most important f a c t o r s i n the r e l a t i o n s h i p between postmortem chemical changes and tenderness i s the r a t e and e x t e n t of the pH change i n the muscle. Treatments which i n c r e a s e the r a t e of pH drop b r i n g about r e s i d u a l toughness i n the muscle (deFremery and P o o l , 1960; Khan and Nakamura, 1970, 1971). The e f f e c t of pH change on tenderness has been d e t e r -mined b a s i c a l l y through a c c e l e r a t i o n , r e t a r d a t i o n and blockage or p r e v e n t i o n of postmortem g l y c o l y s i s . A c c e l e r a t i o n o f g l y c o l y s i s i s u s u a l l y accomplished through f r e e s t r u g g l e a t death, s c a l d i n g or mechanical p i c k i n g . These e f f e c t s have been d i s c u s s e d i n p r e v i o u s s e c t i o n s . - 12 -Retardation of glycolysis in poultry muscle has been effected by e l e c t r i c a l stunning or injection of Nembutal prior to exsanguination (deFremery, 1965; Sayre, 1969, 1970). Both treatments result in high pH values post-slaughter and a sub-sequent slow decline of muscle pH. The blockage of glycolysis may be accomplished by antemortem injection of sodium iodoacetate (deFremery and Pool, 1963; Sayre 1969, 1970) to inhibit the enzyme phosphoglyceralde-hyde dehydrogenase. Antemortem epinephrine injections, to e l i -minate muscle glycogen, has been widely used as a means of eliminating glycolysis (deFremery and Pool, 1963; deFremery, 1965; Khan and Nakamura, 1970; Sayre, 1969, 1970). These authors found minimized postmortem glycolysis and increased tenderness through injection of epinephrine. Klose et a l , (1970) found that muscle depleted of glycogen by antemortem epinephrine injections did not have lower shear values than normal muscle. The authors were unable to explain reasons for this discrepancy but did question the overall effectiveness of epinephrine injections for lowering muscle glycogen. The decrease of N-phosphorylcreatine (PC) and ATP in muscle are closely related since the immediate postmortem source of ATP occurs through dephosphorylation of PC and phosphorylation of adenosine diphosphate (ADP). The PC in chicken breast muscle i s very lab i l e (deFremery, 1965, 1966a) - 13 -and the t r a n s i t o r y pH in c r e a s e i n p o u l t r y muscle immediately postmortem has been a t t r i b u t e d t o the f r e e c r e a t i n e l i b e r a t e d a t s l a u g h t e r (Dodge and P e t e r s , 1960) . Under normal c o n d i t i o n s the l e v e l of ATP i n chicken b r e a s t muscle i s approximately 10 u moles/g f r e s h t i s s u e . This l e v e l remains r e l a t i v e l y high f o r the f i r s t 1 - 3 hours postmortem then drops r a p i d l y and the muscle passes i n t o r i g o r m ortis when the ATP l e v e l has f a l l e n t o about 30 percent of i t s i n i t i a l l e v e l (deFremery, 1966a). P h y s i c a l Changes Muscle i n the l i v i n g animal i s s o f t , p l a s t i c and e x t e n s i b l e , but i n r i g o r i t becomes r i g i d and r e l a t i v e l y i n e x t e n s i b l e . I n a d d i t i o n to l o s i n g e x t e n s i b i l i t y u n r e s t r a i n e d muscle shortens d u r i n g r i g o r development. Newbold and H a r r i s (1972) have e x t e n s i v e l y reviewed the aspects o f pre-r i g o r s h o r t e n i n g . This shortening i s g r e a t l y dependent on temperature but not a l l muscles show the same extent of sho r t e n i n g . Locker and Hagyard (1963) d e f i n e d a " c o l d shortening" phenomenon i n ox neck muscle and s i n c e t h a t r e p o r t ovine (Cook and Langsworth, 1966), p o r c i n e (Galloway and G o l l , 1967), Hendricks e t a l . , 1971) and avian muscles (Smith e t a l . , 1969) have been reporte d to " c o l d shorten ". Jungk and Marion (1970) reported t h a t no " c o l d shortening" was e v i d e n t i n turkey b r e a s t muscle but t h i g h - 14 -muscle did exhibit "cold shortening" (Marion, 1971), Marsh and Thompson (1958), Locker and Hagyard (1963) and Marsh and Leet (1966) reported that the amount of "cold shortening" decreased as the period between slaughter and exposure to cold increased. Interesting effects on tenderness of beef and lamb carcasses have been effected by changing postmortem hanging practices (Herring et a l . , 1965 a, b; Bouton and Harris, 1972b). Different points of attachment prevent different muscles from shortening leading to an improved tenderness in these muscles. No work of this nature has been done with avian species. How-ever, Hegarty and Allen (1972) found that stretching pre-rigor turkey muscles, which had been excised from the carcass, did not significantly lower shear values and in some cases the stretched muscles were significantly tougher than unstretched controls. Klose et a l , , (1970) studied the effect of pre-rigor contraction on the tenderness of postmortem chicken muscle. E l e c t r i c a l stimulation, beating, freeze-thawing or heating reduced muscle length, in most cases, to betv/een 40 and 50 percent of the original rest length. Subsequent shear values for the contracted, cooked muscles were found to be about one-half those for uncontracted controls. The authors speculated that the extreme state of contraction effected changes at the sarcomere level which resulted in the myofibrils being more susceptible to a shearing stress. - 15 -P o s t - R i g o r Tenderness R e l a t e d F a c t o r s  R e s o l u t i o n of Rigor P o s t - r i g o r t e n d e r i z a t i o n i n muscle i n v o l v e s a r e s o -l u t i o n of r i g o r m o r t i s . The evidence r e l a t i n g t o the a c t u a l r e s o l u t i o n o f r i g o r was reviewed by G o l l (196 8 ) . Two l i n e s o f evidence were d i s c u s s e d . F i r s t l y , muscle h e l d i s o m e t r i c a l l y b e g i n s t o develop t e n s i o n immediately a f t e r death and i n c r e a s e s t o a maximum at v a r y i n g times postmortem. Regardless of s p e c i e s , the a b i l i t y t o m a i n t a i n i s o m e t r i c t e n s i o n s l o w l y de-c l i n e s a f t e r the p o i n t of maximum t e n s i o n has been reached. The second l i n e of evidence s u g g e s t i n g t h a t a r e s o l u t i o n o f r i g o r o c c u r s i n postmortem muscle i s the o b s e r v a t i o n t h a t s a r -comeres which have undergone e x t e n s i v e postmortem s h o r t e n i n g w i l l , a f t e r s e v e r a l days, lengthen a g a i n (Gothard e t a l . , 1966; Stromer and G o l l , 1967; Takahashi e t a l . , 1967). One p o s s i b l e e x p l a n a t i o n f o r the observed r e s o l u t i o n o f r i g o r i s l o s s of Z - l i n e s t r u c t u r e and weakening and e v e n t u a l r u p t u r e of the bonds between the I and Z f i l a m e n t s . Henderson et a l . (1970) showed t h a t the Z - l i n e o f bo v i n e , p o r c i n e and r a b b i t muscle l o s t i t s i n t e g r i t y d u r i n g postmortem s t o r a g e . S i m i l a r f i n d i n g s have been r e p o r t e d i n c h i c k e n muscle by Takahashi e t a l . (1967) b ut Sayre (1969, 1970) found I - Z weakening t o be more prominent than Z - l i n e d e g r a d a t i o n i n postmortem c h i c k e n muscle. - 16 A second possible cause of the resolution of rigor i s the weakening of the actin-myosin interaction. Such weak-ening may be effected by a very specific and limited proteolysis of myosin, actin and/or one of the regulatory proteins (Goll, 1968) , Evidence of such a weakening was f i r s t reported by Fujimaki et. a l . (1965) . Aging and Frozen Storage The aging period i s considered the time when a pro-cessor can most influence the tenderness of chicken and turkey muscle. The different aging requirements for red and white muscle (van den Berg et a l . , 1964) must be taken into consider-ation, when considering an adequate aging period. Marion and Goodman (1967) and Welbourn et a l , (1968) have studied turkey quality in relation to aging, time and ch i l l i n g treatment prior to freezing. Larger turkeys were found to require a shorter aging period prior to freezing sug-gesting that additional tenderization occurs in large turkeys during freezing and thawing (Marion and Goodman, 1967), Several chemical changes have been shown to occur in frozen poultry muscle that may relate to tenderness (Khan et a l . , 1963; Khan, 1964). The change in protein and nonprotein con-stituents of chicken breast muscle was followed during frozen storage. Nonprotein constituents increased with frozen storage indicating that some proteolysis had occurred. This proteolysis could affect the solubility and ion-binding properties of the - 17 -protein, and thus affect tenderness, loss of juiciness and subsequent development of dryness in the meat. Methods of Measuring Muscle Tenderness One of the greatest d i f f i c u l t i e s in comparing avail-able data on avian tenderness i s the tremendous variation in procedures used to determine tenderness. Marion (1967) emphasized the need to standardize mechanical and sensory methodology so that individual researchers may be able to determine how their data compares with researchers in other laboratories. Pearson (1963) presented a review of objective and subjective methods for measuring meat tenderness and pointed out the advantages and limitations of each method. Most of the objective methods of determining meat tenderness use some form of a shearing device and record the force required to shear or compress a sample of standard size. The relationship between instrument tenderness values and sensory panels has been widely studied over the last two decades (Deatherage and Garnatz, 1952; Klose et a l , , 1961; White et a l . , 196 4; Pangborn et a l . , 1965; Sharrah et a l . , 1965 a, b; Pool and Klose, 1969, Szczesniak et a l . , 1970; Larmond and Petrasovits, 1972). A comparison of objective methods has been published by Bouton and Harris (1972a). Some of the more recent studies have raised objections on both the theoretical and practical aspects of the shear type method - 18 -f o r determining meat tenderness (Sharrah et a l . , 1965 b; Pool and K l o s e , 1969; Szczesniak e t a l , , 1970). A d i f f e r e n t approach to the measurement of meat tenderness has been t r i e d by some workers. Nakamura (1972) measured t e n s i l e s t r e n g t h of muscle f i b e r s i n order to study postmortem aging of chicken b r e a s t muscle. Stanley et. a l . (1972) performed two b a s i c types of o b j e c t i v e measurements on raw porcine muscle shearing and break s t r e n g t h t e s t s ; and sarcomere l e n g t h , e l a s t i c i t y , s t r e s s r e l a x a t i o n and break e l o n g a t i o n . The r e s u l t s of these t e s t s were compared w i t h t a s t e panel e v a l u a t i o n s of tenderness, e l a s t i c i t y and chew count on cooked meat. T e x t u r i z e d vegetable p r o t e i n was used as a reference by the panel and proved u s e f u l i n reducing v a r i a -t i o n s between chew count and o b j e c t i v e e v a l u a t i o n . The authors concluded t h a t there are two major s t r u c t u r a l c o n t r i b u t i o n s of raw muscle to cooked meat tenderness, a connective t i s s u e f a c t o r and a c o n t r a c t i o n f a c t o r , and d i f f e r e n t o b j e c t i v e methods should be used f o r t h e i r e v a l u a t i o n . Zachariah et a l . (1971) have a l s o t e s t e d a method f o r p r e d i c t i n g tenderness on raw muscle. E l e c t r i c a l impedance measurements made on p o u l t r y tended to i n d i c a t e t h a t h i g h impedance was a s s o c i a t e d w i t h tender b i r d s and low impedance w i t h tough b i r d s . I s o m e t r i c Tension Measurement The f i r s t s t u d i e s on the use of i s o m e t r i c t e n s i o n - a d -measurements to f o l l o w the time-course of r i g o r m o r t i s i n bovine and r a b b i t muscle were reported by Busch e_t a l . (1967) and Jungk et a l . (1967), P r i o r to t h i s time e x t e n s i b i l i t y measurements were used i n order t o q u a n t a t i v e l y f o l l o w r i g o r m o r t i s . The measurement of i s o m e t r i c t e n s i o n provides s e v e r a l unique advantages over e x t e n s i b i l i t y measurements (Busch e t a l . / 1972). The most important advantage i s i t s a b i l i t y t o detect changes which correspond to not only onset of r i g o r but res o -l u t i o n of r i g o r as w e l l . Schmidt e t a l . (1968) d e s c r i b e the development of an i s o t o n i c and i s o m e t r i c rigorometer which allowed measurement of i s o m e t r i c t e n s i o n (shortening) and i s o t o n i c t e n s i o n ( l o s s of e x t e n s i b i l i t y ) simultaneously. These same authors (Schmidt et a l . , 1970 a, b) have used the rigorometer t o study some f a c t o r s a f f e c t i n g the time-course of r i g o r m o r t i s i n porcine muscle. Jungk and Marion (1970) have demonstrated i s o m e t r i c t e n s i o n development and d e c l i n e i n turkey muscle and have e s t a b l i s h e d a l i n e a r r e l a t i o n s h i p between temperature and t e n s i o n development i n br e a s t muscle. Busch et a l . (1972a)published some improvements i n the procedure f o r measuring postmortem i s o m e t r i c t e n s i o n and have reported e x t e n s i v e data on t e n s i o n development from three mammalian species (porcine, bovine, r a b b i t ) . - 20 -Antemortem Stress and Postmortem Muscle Quality Animals are exposed to many forms of stress during growth and in particular during shipment to market and slaughter. These stresses increase the need for hormones which are produced, by the adrenal gland. Selye (1956) defines stress as: "the state manifested by a specific syndrone which consists of a l l the non-specifically induced changes within a biological system". In his early work (Selye, 1950) noted that animals exposed to a number of stress-producing factors, such as emotional excitement, fatigue, cold and inanition, always reacted with an increased secretion of hormones from the adrenal medulla and adrenal cortex. One effect of these hormones i s alteration of li v e r and muscle glycogen levels and changes in the latter are of particular importance in relation to postmortem muscle quality. The effect of stress on postmortem muscle quality i s exemplified by the Poland China breed of pigs. These animals have been shown to be extremely susceptible to antemortem stress and yield a high incidence of pale soft exudative (PSE) muscle (Sayre et a l . , 1963 a, b; Briskey, 1964; Kastenschmidt et a l . , 1966, 1968; Lister et a l , , 1970; Sair et al.,1970). In beef animals, stress has been implicated in the incidence of dark cutting beef (Lawrie, 1958, 1966 a,b). To date, no such stress effect has been demonstrated in poultry muscle although the - 21 -tremendous v a r i a t i o n i n tenderness may, i n some way, be re-lated to va r i a t i o n s i n stress s u s c e p t i b i l i t y i n the avian species. The influence of stress on bovine, porcine and ovine meat q u a l i t y has been reviewed by Hedrick (1965) and Judge (1969) and on growth and performance by Wilson (1971). Ringer (1971) and Siegel (1971) have reviewed the l i t e r a t u r e on poultry adaptation to confinement rearing systems and stress and environment re s p e c t i v e l y . However, a study of the e f f e c t s of antemortem stress on postmortem muscle q u a l i t y i n the avian species i s lacking. Lack of.information i n the above mentioned area and the need f o r a comprehensive study of the r e l a t i o n s h i p between isometric tension development and decline and b r o i l e r tender-ness prompted the research which i s presented i n t h i s t h e s i s . - 22 -MATERIALS AND METHODS Isometric Tension Experiments Muscle Source The laying hens used in this study were obtained from the Department of Poultry Science at U.B.C. The birds were approximately 2 year old New Hampshires, The broilers were obtained from a local processing plant, transported to the U.B.C. poultry farm and kept in a range house for 7 - 1 0 days prior to use to allow for adjustment to the new sur-roundings. The broilers were maintained on a 20 percent broiler grower ration and were slaughtered at 8 - 12 weeks of age. For slaughter, birds were placed in a metal funnel, exsanguinated by an outside neck cut and allowed to bleed for about 2 minutes. The wings and legs were manually restrained during slaughter in addition to restr i c -tion provided by the funnel. Isometric Tension Measurement Isometric tension development and decline were measured using an E & M 6-channel physiograph f i t t e d with isometric transducers. The physiograph was obtained from Narco-Bio-Systems Inc. formerly E & M Instrument Co., Houston, Texas. - 23 -Muscle for tension measurement was obtained immed-iately after exsanguination by cutting the breast skin and excising a 1 cm wide strip of muscle tissue from the anterior portion of the Pectoralis major. The strip was cut parallel to the direction of the muscle fibers and care was taken to prevent stretching of the muscle during excision and subse-quent strip preparation. A regression line was prepared by carefully measuring the weight and cross-sectional area of 2 several muscle strips 5 cm in length and 0.1 - 1.0 cm in cross section. A l l strips used in subsequent studies were cut to 5 cm in length, weighed and the cross-sectional area determined from the regression line. Most strips ranged 2 between 0.15 - 0.25 cm since this size best f i t t e d the clamping system used. One end of a muscle strip was clamped in a battery cable clamp (Mueller No. 48B) and the other end was attached to a second clamp. This second clamp was fixed on a plexi-glass rod within a plexiglass cylinder, 9.5 cm in diameter and 20 cm high. The rod was fixed about 4 cm above the chamber bottom in order to permit the use of magnetic s t i r -ring. The chamber was f i l l e d with enough buffer to cover the muscle strip and top clamp and the muscle strip was attached to an isometric transducer by means of 6 lb test monofilament fishing line tied to the free clamp. Approxi-2 mately 5.0 g/cm tension was applied to each strip in order to attain some measure of uniformity of starting conditions. - 24 The p h y s i o g r a p h was c a l i b r a t e d so t h a t a 1 cm pen d e f l e c t i o n was e q u i v a l e n t t o 5 g t e n s i o n . Up t o 6 s t r i p s c o u l d be s t u d i e d from each b i r d . The maximum ti m e l a p s e from e x s a n -g u i n a t i o n t o attachment o f s t r i p s from one b i r d was 20 m i n u t e s . The e n t i r e system used i n t h i s and subsequent s t u d i e s i s shown i n P l a t e 1. - 25 -The Effect of Environment on Tension Pattern Four different systems were studied in order to select the appropriate chamber media for the muscle strips. System 1 consisted of a humid chamber produced by lining the chambers with moistened chromatography paper, placing about 3 cm of salt solution in the bottom of the chamber and covering the top of the chamber with Saran wrap allowing a small space for the attachment of the strip to the trans-ducer. The other 3 systems consisted of f i l l i n g the chamber with one of d i s t i l l e d water, phosphate buffer pH 7.2, ionic strength 0.15 or Tris-acetate buffer pH 7.1, ionic strength 0.22 (Goll et a l . , 1970). The Effect of pH on Tension Pattern Phosphate buffers of pH 5.8, 6.3, 6.7 and 7.2 were prepared as described by Gomori (1955) . Six broilers were used in this study and four strips were cut from each bird. One strip was run at each pH and tension measurements were made at room temperature (22 - 25°C). The length of measurements varied between 16 - 20 hours. The Effect of Calcium, Magnesium and EDTA on Tension Pattern Calcium chloride, magnesium chloride and EDTA were - 26 -added to the pH 7.2 phosphate buffer. Solutions were prepared to contain 10"^, 10""*, 10-"*^  M calcium, lo""* M magnesium, 10""* M EDTA, 10 M calcium + 10 J M EDTA and 10 M magnesium + 10~^ H EDTA. Several strips from broilers were run in each solution and strips from 6 laying hens were run in the buffer _3 plus 10 M calcium. The Effect of | Temperature on Tension Pattern Muscle strips were run in phosphate buffer ranging from 0 - 60°C. The cold temperature strips were run in cold-rooms at about 2 and 5°C and the 0°C readings were obtained by pre-cooling the buffer to 0°C in a freezer. Temperatures above room temperature were attained by placing small, 50 watt aquarium heaters in the buffer chambers and s t i r r i n g magnetically to maintain even heat distribution. The heaters were able to maintain preset temperatures within + 1°C. Temp-eratures studied were 0, 2, 5, 23 1, 30, 37, 43, 50 and 60°C. Effect of Processing Techniques on Tension Pattern and  Tenderness Five different treatments were studied within this experiment. 1. Exsanguination + restricted struggle - control This Temperature was selected as representative of room temperature which varied between 22 - 25°C. - 27 -2, Exsanguination + restricted struggle + hot V7ater scald 3, Exsanguination + free struggle + hot water scald 4, Exsanguination + restricted struggle + pluck 5, Exsanguination + free struggle + hot water scald + pluck Exsanguination with restricted struggle was done as previously described. Simulation of processing plant conditions was done by suspending birds by their feet and allowing them to struggle freely both before and after exsanguination. Scalding con-sisted of 90 second submersion in water at 60°C and plucking was done on a rotary picker, f i t t e d with pliable rubber fingers, for 60 seconds. Six muscle strips were taken immediately from one side of the breast for tension measurement. Three strips were taken from muscle close to the skin and three from muscle near the breast bone in order to study an "inner" and "outer" effect in the muscle. The exposed breast muscle was covered with Saran wrap, packed in drained crushed ice and aged for 24 hours at 2°C after which tenderness measurements were performed in a manner similar to that reported by deFremery and Pool (1960). The excised breast muscle was clamped between two 1/8 inch aluminum plates f i t t e d with metal spacers so that cooked muscle approximately 0.7 cm in thickness was obtained. The muscle was cooked in boiling water for 30 minutes and - 28 -cooled in running tap water for 5 minutes. Strips of parall e l fibres, 1.5 cm wide were prepared and sheared using an Allo-Kramer shear press. A single blade shear c e l l , 250 lb ring and 9 cm/min cross head speed were used for a l l shears. A minimum of 10 shears per bird was obtained and in most cases attenuation was set at the 5 percent level. Epinephrine Experiments Preliminary Experiment The equivalent of 4 mg epinephrine/kg (Sigma Chemical Co., St. Louis, Missouri) body weight was injected into the breast muscle of 6 broilers which were k i l l e d at 3, 6, 9, 12, 15 and 18 hours post-injection. Tension and tender-ness measurements were made as previously described. Two control birds were also run. Experiment 1 A total of 18 male and 18 female broilers were used in this experiment. A 2 x 3 x 6 randomized complete block design (Cochran and Cox, 196 4) was used. Treatments consisted of unin-jected controls and exsanguination at 2,4, 8, 12 and 16 hours post-injection. The experiment was run over a 6 day period and males and females were k i l l e d on separate days. Injection was done intramuscularly into the thigh and dosage was as used - 29 in the preliminary experiment. Blood was collected at death for blood lactate analysis according to the method of Hadjwassiliou and Rieder (196 8). Tension and tenderness were also measured. Experiment 2 The procedure and design of experiment 1 were duplicated except that post-injection slaughter times of 4, 8, 10, 12 and 24 hours were used. Muscle samples were taken within 2 minutes after exsanguination, frozen in liquid nitrogen (LN2) and stored in aluminum f o i l envelopes under for subsequent metabolite analysis. Sample Preparation for Metabolite Assay The samples stored under- LN2 were powdered by the method of Borchert and Briskey (1965) as modified by Vanderstoep (1971) . The frozen samples v/ere pulverized in a macro-model V i r t i s homogenizer for 1.5 minutes at approximately 11,000 rpm. The powdered sample was replaced in aluminum f o i l envelopes and stored under pH of the muscle samples was determined by modifying the method of Cassens and Newbold (1967). 1 - 2 g powdered sample was homogenized in 10 ml neutralized 0.005 M sodium - 30 -iodoacetate at 2°C. The slurry was allowed to warm to room temperature and pK was measured using a Corning Model 10 pH meter. Metabolite Analyses ATP was determined by the method of Lamprecht and Trautschold (1963) with two modifications. Two grams of previously powdered sample were added to 7.5 ml instead of 6.5 ml perchloric acid and 1 cm cuvettes were used instead of 2 cm c e l l s . This latter change necessitated alteration of the quantities of intermediates, cofactors and enzymes used. Lactate was determined by the method of Hohorst (19G3) and glycogen by the method of Pfleiderer (1963). Optical density measurements were made using a Unicam SP 800 recording spectrophotometer. A l l chemicals were of reagent grade, made up in glass d i s t i l l e d water. Enzymes, cofactors and intermediates used were obtained from Sigma Chemical Co., St. Louis, Missouri. Stress Experiments Commercial Stress Twenty-eight broilers were used in this experiment. One male and one female was obtained from a local processing plant just prior to slaughter, transported to the Food Science - 31 -laboratory at U.B.C. and k i l l e d immediately. This procedure was repeated on seven different days. One control male and female, from a supply maintained at U.B.C, was k i l l e d on each of these days. Immediately after exsanguination, 15 - 20 g breast muscle was frozen in LN 2 and subsequently analysed for pH and ATP as previously described. Tension and tenderness were also measured as previously described. Keat Stress Twenty female broilers were used in this study. Fourteen birds were placed in hot air at 110°F for 3 hours prior to exsanguination. Seven of these birds were k i l l e d with restricted struggle and seven were allowed to struggle freely. Six birds were k i l l e d as controls. Samples of muscle were excised immediately post-slaughter and frozen in LN 2 for "0" hour pH and ATP analysis. Tension and tenderness were measured and 24 hour pH was also determined. Cold Stress Twenty-seven female broilers were studied in this experiment. Eighteen birds were placed in a 2°C coldroom. Nine were exsanguinated after 2 hours and 9 after 6 hours. The remaining nine birds were k i l l e d as controls. - 32 -Samples of muscle were excised immediately post-slaughter and frozen in for subsequent ATP, glycogen and hexose monophosphate analysis and tension and tenderness were measured. Cold Shortening Experiments Experiment 1 Broilers, of mixed sex, were used in this experiment. The birds were exsanguinated with restricted struggle and 6 muscle strips were prepared from each bird for tension mea-surement. Two strips were run in buffer at room temperature, 4 strips were attached to transducers in a 2°C coldroom then pre-cooled buffer at 2°C was added to the chambers. These four strips were kept in the coldroom in order to observe ten-sion development at this temperature. Two strips were removed from the coldroom at 12, 24 and 36 hours post-maximum tension. The time of maximum tension was determined from the two control strips run at room temperature. The 2°C buffer was exchanged for room temperature buffer and the strips were attached to isometric transducers at room temperature to observe the a b i l i t y of the strips to develop tension. A similar series of tests was done in which strips were attached to transducers at room temperature, 2°C buffer was added and after the i n i t i a l "cold shortening" occured the buffer was replaced with room temperature buffer for further observation of tension pattern. - 33 -These strips were designated as "0" time samples. A total of 15 birds v;as used so that each treatment contained data from 6 different birds and 12 different muscle strips. Experiment 2 Six birds were used in this experiment. Birds were exsanguinated with restricted struggle and a sample of muscle was immediately frozen in LN2» Twelve strips of muscle, proportional in size to strips used for tension measurement, were then prepared. Eight strips were placed in phosphate buffer at 2°C and after 3 minutes, four were removed, dried rapidly on a paper towel and frozen in EN2» T h e o t n e r four were removed after 7 minutes and similarily frozen. The remaining 4 strips were placed in buffer at room temperature at the same time as the eight strips were placed in the cold buffer. These strips were removed after 7 minutes and similarly frozen, ATP,creatine phosphate and hexose monophos-phate analyses were subsequently performed on the muscle samples according to the method of Lamprecht and Stein (1963). - 34 -RESULTS AND DISCUSSION Isometric Tension Experiments. The Effect of Environment on Tension Pattern Ideally, isometric tension pattern should be studied on strips suspended in a i r , thereby minimizing external effects. The isometric tension pattern of several strips was studied by suspending strips in chambers maintained at a high relative humidity. These conditions, however, did not prevent the sur-face of the muscle strips from drying and spurious tension patterns were obtained. These findings are similar to those of Busch et_ a l . (1972 a) who found that occasionally, surface dehydration occurred even at 95-98 percent relative humidity. These same authors also found that the isometric tension pattern of rabbit psoas muscle was identical whether the strips were suspended in air or in a saline buffer. On the basis of this finding, two different buffer systems and d i s t i l l e d water were used as liquid media and their effect on broiler muscle tension pattern was studied. The tension patterns obtained in the Tris-acetate buffer were almost identical to those obtained in the phosphate buffer (Figure 1). Tension maximum was reached in 4.3 hours in both buffers compared to 8,3 hours in the d i s t i l l e d water. The main difference between the two buffer systems was in the tension decline. After 16 hours postmortem, strips in the Tris-acetate buffer had declined to about 60 percent of the TIME POST MORTEM (Hours) Figure 1. E f f e c t of d i f f e r e n t e x t r a c e l l u l a r incubation media on isometric tension pattern of b r o i l e r P. major muscle. - 36 -maximum tension and were declining slowly. Strips in phosphate buffer had declined to about 45 percent and were continuing to show a rapid rate of decline. The Tris-acetate buffer appeared cloudy after 16 hours, whether or not sodium azide was added to the buffer, suggesting that some form of exchange may have occurred between the buffer and the muscle. This may explain why the rate of tension decline was levelling off at this point in time. In contrast, the phosphate buffer remained clear. The tension pattern obtained in d i s t i l l e d water, as expected, was quite different from the patterns obtained in the two buffers. The tension developed much slower and after peak-ing, tension declined slowly u n t i l about 85 percent maximum tension was reached. Very l i t t l e decline was observed beyond this point. This i s probably due to a flow of soluble materials, in particular salt ions, from inside the muscle into the lower ionic strength water. On the basis of the above findings, phosphate buffer was selected for use in a l l subsequent studies. Other points considered in making this selection were i t s ease and simpli-city of preparation and i t s previous use by Jungk and Marion (1970) and Marion (1971) for tension studies on turkey muscle. The Effect of pH on Tension Pattern Average tension patterns for strips maintained in buffers of varying pH are reported in Figure 2. The values Figure 2. Effect of extracellular pK on isometric tension pattern of broiler major muscle. were obtained by averaging the hourly values for tension and time to maximum tension of 6 broilers.. The patterns do not diff e r substantially in rate of tension development or decline. This method of obtaining a tension pattern does/however, give misleading values for both time to maximum tension (referred to as time henceforth) and the actual maximum tension developed. This arises through the fact that a l l birds do not reach maxi-mum tension at the same time. The true mean values for tension and time are shown in Table 1. These values were obtained by averaging the actual values for tension and for time for the strips from the broilers. Paired comparison t-tests were done by pairing the pH 5.8, 6.3 and 6.8 strips individually, with the control (pH 7.2) strips. TABLE I. MEANS AND STANDARD ERRORS OF TIME AND TENSION DEVELOPMENT FOR STRIPS OF BROILER P^ MAJOR MUSCLE RUN IN PHOSPHATE BUFFER AT FOUR DIFFERENT pH LEVELS. 2 pH Time, hr Tension, g/cm 7.2 4.50 + 0.61 40.26 + 2.80 6.7 3.90 + 0.63* 34.75 + 3.92 6.3 3.60 + 0.39* 27.73 + 1.51 ** 5.8 3.33 + 0.22* 28.15 + 2.08 * * Significantly different from pH 7.2 (p<0.05). ** p<0.01 39 -Time values f o r pH 6.7 and 7.2 are both approximately 4,3 hours when taken from the Figu r e 2. This compares t o t r u e values of 3,9 and 4,5 f o r pH 6.7 and 7.2 r e s p e c t i v e l y . S i m i -l a r l y , peak t e n s i o n values f o r pH 6.7 and 7.2 are 31 and 34 2 g/cm from F i g u r e 2 versus 34.75 and 40.26 r e s p e c t i v e l y from Table 1. A d e f i n i t e t rend i s evident from the pH data. As pH decreased so d i d the time r e q u i r e d t o reach maximum t e n s i o n and amount of t e n s i o n . The times f o r pH 5.8, 6.3 and 6.7 are s i g n i f i c a n t l y lower (p<0.05) than f o r the c o n t r o l , pH 7.2. The average t e n s i o n of s t r i p s a t pH 5.8 or 6.3 was s i g n i f i -c a n t l y lower than at pH 7.2. The r e s u l t s obtained here d i f f e r somewhat from those obtained by Busch e t a!L. (1972a) who reported no change i n the amount of t e n s i o n i n r a b b i t psoas muscle between pH 5.5 - 7.0. However, a t pH 5.0, t e n s i o n appeared t o decrease. They a l s o found a decrease i n time r e q u i r e d t o reach maximum t e n s i o n as the pH decreased from 7.0 to 5.0. These data were obtained a t 37°C. When the experiment was conducted at 2°C, the opposite r e s u l t was obtained i e . decreasing pH from 7,0 t o 5.0 in c r e a s e d the time r e q u i r e d f o r maximum t e n s i o n t o develop. The f a c t t h a t the above data were obtained u s i n g r a b b i t muscle and t h a t d i f f e r e n t temperatures gave d i f f e r e n t r e s u l t s make i t d i f f i c u l t t o draw analogies to the present data on b r o i l e r muscle which were obtained at approximately 25°C. - 40 -One similarity does exist between the two sets of data. Both show that decreasing the extracellular pH does not hinder the ab i l i t y of muscle to develop or release isometric tension. The fact that time and tension are altered by de-creasing extracellular pH i s d i f f i c u l t to explain, Ap r i l et a l , (1968) and Rome (1968) have shown that extracellular pH does not alter intracellular pH to any great extent but does change the selective permeability of the sarcolemma. This may or may not be the case in broiler muscle as i t appears that in the lower pH buffers, anerobic glycolysis i s halted earlier than in control buffers, thus resulting in less tension and shorter time to maximum tension. If the observed phenomena were due to a change in sarcolemmal permeability then a greater change in the overall tension pattern would be expected,due to loss of ions and/or metabolites and co-factors v i t a l to the contrac-tion and relaxation phases of the tension pattern. The Effect of Temperature on Tension Pattern During post-slaughter handling poultry muscle en-counters temperatures ranging from 60°C in scald tanks down to near 0°C during ice-slush cooling and aging. Because of this a study was initiated to determine the effect of temperatures within this range on postmortem isometric tension pattern. Temperatures between 5°C and 20°C were not studied because broilers are not exposed to this range during the pre-rigor - 41 -p e r i o d and pass through i t q u i t e r a p i d l y on subsequent c o o l i n g . The r e s u l t s of these s t u d i e s on temperature e f f e c t s are pre-sented i n Table I I . The time values are reported as minutes i n s t e a d of hours because of the r a p i d t e n s i o n development i n some of the treatments. TABLE I I . MEANS AND STANDARD ERRORS OF TIME AND TENSION DEVELOPMENT FOR STRIPS OF BROILER P^ MAJOR MUSCLE RUN IN BUFFERS AT VARIOUS TEMPERATURES. Temperature, °C Time, min Tension, g/cirr 0 3.1 + 0.2 ** 65.37 + 6.5 ** 2 3.3 + 0.2 ** 38.23 + 3.6 ** 5 2.2 + 0.2 17.24 + 2.3 ** S i g n i f i c a n t l y d i f f e r e n t from 5°C (p<0.01) 23 276.7 + 22.1 46.40 + mm 2.0 30 217.8 + 82.0 49.73 + 6.5 37 116.5 + 44.4 ** 65.46 + 11.7 43 49.3 + 15.6 ** 73.75 + 9.4 ** 50 8.1 + 1.2 ** 316.89 + 19.3 ** 60 0.5 + ** 290.18 + 29.2 ** ** S i g n i f i c a n t l y d i f f e r e n t from 23°C (p<0.01) - 42 -The data for 0°, 2° and 5°C show d e f i n i t e l y , that cold shortening ^ occurs i n b r o i l e r P_j_ ma j or muscle. In f a c t , the increasing e f f e c t of cold shortening i s demonstrated with-i n the temperature range from 0 - 5°C, deFremery and Pool (1960) found that the rate of ATP decline i n postmortem b r o i l e r muscle was faster at 0°C than at 10°C and minimal between 10 -20°C. Smith et a l . (1969) f i r s t demonstrated a cold shortening e f f e c t i n avian muscle. They found that shortening at 0°C was s i g n i f i c a n t l y greater than i n the 12 - 18°C range. The above r e s u l t s are i n close accord with the present findings. The present data, however, demonstrated a more dramatic cold shortening e f f e c t that was reported by Smith et a l . (1969). These authors found that shortening was e s s e n t i a l l y complete af t e r 3 hours i n b r o i l e r s whereas the present study shows that shortening i s v i r t u a l l y instantaneous and the tension developed i s e s s e n t i a l l y a l l abated within 15 - 30 minutes a f t e r maximum development. The difference between these data probably r e f l e c t the fact that isometric tension as determined i n the present experiment, i s a much more sensitive technique than the mea-surement of length determined by Smith e± a l . (1969). The f a c t that the time and tension are smallest at 5°C suggests also that a point of minimal tension development Cold shortening i n the l i t e r a t u r e r e f e r s to the actual length change which occurs i n excised muscles subjected to low temp-eratures. In t h i s t h e s i s , however, cold shortening i s used to describe the rapid tension development at 0 , 2 and 5°C. - 43 -i s being approached as suggested by the data of deFremery and Pool (1960) and Smith et a l . (1969). This cold temperature effect w i l l be discussed further in a subsequent section of this thesis. The time and tension data for temperatures of 23°C and above is consistent with an increase in glycolysis at higher temperatures. Though time tends to shorten and tension increase, with increasing temperature, no significant changes are apparent u n t i l 37°C i s reached. The most striking results were observed at 50 and 60°C. These temperatures were studied because the outer layers of breast muscle could easily reach temperatures in this range during scalding. In view of the previously discussed toughening which occurs due to the scald-ing procedure, these data provide an insight as to why the toughening occurs. The association between muscle contraction and ten-derness has been well established (Herring et a l . , 1965 a; Marsh and Leet, 1966; Howard and Judge, 1968). The great 2 increase in tension developed at 50 and 60°C (ca. 300 g/cm ) 2 versus 23°C (ca. 50 g/cm ) suggests that, in the outer layers of broiler P^ major, contraction would be maximal within a very short period of time. The increased glycolytic rate at these temperatures would result in a low pH, A combination of the low pH and high temperature could lead to denaturation of the highly contracted myofibrils thus preventing the normal - 44 -tenderization which occurs in postmortem muscle. Hamm (1966) showed that actomyosin solubility i s greatly decreased by heating in the range from 40 - 60°C. Khan (1971) has shown that dephosphorylation of ATP at high temperatures affects the mode or extent of stiffening of the muscular tissue thus pre-venting tenderization. The present data indicates that the altered mode of stiffening could be the result of increased tension (contraction) developed at 50 - 60°C. This increased contraction would mean an increase in actomyosin formation which upon denaturation would result in the toughening observed in scalded poultry. The combination of low pH and high temperature may also affect tenderness through alterations in connective tissue. Schaller and Powrie (1972) showed slight changes in connective tissue of broiler P^ major due to heating at 60°C. The Effect of Calcium, Magnesium and EDTA on Tension Pattern Three different calcium concentrations were prepared and used in this study on broiler and fowl muscle. The time and tension data for broilers and the time data for the fowl are presented in Table III. The presence of the 3 levels of calcium did not significantly affect the time to maximum tension for the broilers _ 3 or fowl. The 10 M level did, however, significantly affect the amount of tension developed in the broiler muscle (p<0.01). - 45 -TABLE III. MEANS AND STANDARD ERRORS OF TIME AND TENSION • DEVELOPMENT FOR STRIPS OF BROILER AND FOWL P^ MAJOR MUSCLE RUN IN PHOSPHATE BUFFER AND BUFFER CONTAINING THREE LEVELS OF CALCIUM Calcium Concentration Control 10"3M 10"4M 10'"7M Broiler Time 4.18 + 0.62 4.06 + 0.50 4.04 + 0.86 3.70 + 0.72 Tension 51.71 + 4.32 36.43 + 4.63* 45.61 + 4.09 54.89 + 7.10 Fowl Time 9.63 + 0.53 10.50 + 0.62 9.10 + 0.71 10.17 + 0.67 ** Significantly different from control (p<0.01) The most noticeable difference, with respect to broilers versus fowl, i s the difference between the time to maximum tension. The time required in fowl i s twice that of broilers which would indicate a much slower rate of anaerobic glycolysis in the post-mortem fowl or considerably higher i n i t i a l levels of glycogen and ATP. Although i t i s not possible to accurately compare tension values for fowl and broilers, i t i s possible to obtain a rough estimate. The cross-sectional area of the f i r s t 3 0 strips cut after preparing the correlation curve, when averaged, give an indication of the average size of muscle strips being prepared at that time. If this value i s applied to the average - 46 -t e n s i o n v a l u e f o r the fowl then approximately 40 g/cm t e n s i o n i s o b t a i n e d . T h i s amount i s i n l i n e with b r o i l e r c o n t r o l v a l u e s . The t e n s i o n r e l e a s e data f o r c o n t r o l and 10 M ca l c i u m s t r i p s are shown i n F i g u r e 3 f o r b r o i l e r s and F i g u r e 4 f o r f o w l . In both cases, the presence of 10 M c a l c i u m i n the e x t r a c e l l u l a r b u f f e r caused a s i g n i f i c a n t i n c r e a s e i n -4 -7 the r a t e o f t e n s i o n r e l e a s e . In b r o i l e r muscle, 10 and 10 M c a l c i u m caused a s l i g h t but n o n - s i g n i f i c a n t i n c r e a s e i n r a t e of t e n s i o n d e c l i n e . The 10""'* M l e v e l i n fowl produced a s i g n i -f i c a n t e f f e c t but 10 M was s i m i l a r to the c o n t r o l . There i s an obvious d i f f e r e n c e i n the r a t e of t e n s i o n d e c l i n e between b r o i l e r and fowl c o n t r o l s t r i p s . At 12 hours post-maximum t e n s i o n , the b r o i l e r c o n t r o l s had d e c l i n e d t o 40 p e r c e n t of the maximum t e n s i o n , whereas the fowl c o n t r o l s had d e c l i n e d t o o n l y 60 p e r c e n t . T h i s d i f f e r e n c e i n t e n s i o n r e l e a s e may be r e l a t e d t o the o b s e r v a t i o n t h a t the Z - l i n e i n o l d e r animals i s l e s s l a b i l e than the Z - l i n e i n younger animals ( G o l l , 1970). T h i s may f u r t h e r r e l a t e to the e s t a b l i s h e d f a c t t h a t o l d e r p o u l t r y are l e s s tender than b r o i l e r age p o u l t r y (May e t ajL., 1962; Larmond e t a l . , 1971). The e f f e c t o f c a l c i u m on t e n s i o n development i s not as c l e a r c u t as i t s e f f e c t on the r a t e o f t e n s i o n r e l e a s e . None of the l e v e l s t e s t e d a f f e c t e d the time t o reach maximum t e n s i o n and o n l y the 10" 3 M l e v e l decreased the amount of t e n s i o n developed i n b r o i l e r muscle. S i n c e c a l c i u m i s r e s p o n s i b l e f o r - 47 -TIME POST MAXIMUM TENSION (Hours) Figure 3. E f f e c t of 10 M calcium on tension decline i n b r o i l e r P. major muscle« C i r c l e s and bars are means ± 1 standard error. - 48 -Figure 4. E f f e c t of 10 M calcium on tension decline i n fov;l major muscle. C i r c l e s and bars are means + 1 standard error. - 49 -c o n t r a c t i o n i n v i v o (Hasselbach, 196 4 ) , one must assume t h a t the sarcolemma remains impermeable to e x t r a c e l l u l a r c a l c i u m d u r i n g most of the p r e - r i g o r p e r i o d . The observed decrease _3 i n the amount of t e n s i o n developed i n the presence o f 10 M c a l c i u m suggests t h a t a c r i t i c a l c o n c e n t r a t i o n may have been reached or exceeded thus promoting a more r a p i d p e n e t r a t i o n of the c a l c i u m i n t o the muscle. Busch e t a l . (1972 b) found t h a t 10" 3 M c a l c i u m reduced t e n s i o n development i n r a b b i t psoas muscle and suggested t h a t a c a l c i u m s t i m u l a t e d p r o c e s s caused the l o s s of i s o m e t r i c t e n s i o n and t h a t the maximum t e n s i o n developed r e p r e s e n t e d a balance between t e n s i o n development and l o s s of a b i l i t y t o m a i n t a i n t e n s i o n a t any p a r t i c u l a r time. These authors i s o l a t e d a " c a l c i u m a c t i v a t e d s a r c o p l a s m i c f a c t o r " from r a b b i t muscle and demonstrated i t s a b i l i t y to e f f e c t com-p l e t e Z - l i n e removal from r a b b i t muscle m y o f i b r i l s i n the p r e -sence of a t l e a s t 10 M c a l c i u m . T h i s c r i t i c a l c a l c i u m c o n c e n t r a t i o n i s i n a c c o r d w i t h the t e n s i o n r e l e a s e f i n d i n g s _3 which show t h a t the presence o f 10 M c a l c i u m causes a marked i n c r e a s e i n t e n s i o n r e l e a s e w h i l e 10""'* M c a l c i u m does not d i f f e r s u b s t a n t i a l l y from the c o n t r o l s . F u r t h e r data on the r o l e of c a l c i u m i n postmortem muscle has been p r o v i d e d by Davey and G i l b e r t (1969) and Haga e t a l . (1966) . Haga et. a l , (1966) found t h a t c a l c i u m i o n s promoted the e x t r a c t i o n o f a c t i n from muscle. In t h i s case, s t r u c t u r a l weakening w i t h i n muscle c o u l d occur a f t e r r i g o r m o r t i s s i n c e the s a r c o p l a s m i c r e t i c u l u m l o s e s i t s a b i l i t y t o sequester c a l c i u m a t t h i s time. - 50 -Davey and Gilbert (1969) suggested that calcium was necessary for the weakening of muscle structure since EDTA was found to stabilize muscle fine structure during aging. These findings are consistent with the enhanced tension release in _3 the presence of 10 M calcium. The events which are involved in loss of isometric tension (resolution of rigor) have been categorized (Gqll, 1968) as loss of Z-line structure which leads to eventual rupture of the bonds between the I - Z filaments, and weaken-ing of the actin-myosin interaction. Loss of Z-line structure has been demonstrated by several workers using muscle from different sources (Stromer and Goll, 1967, beef; Henderson et a l . , 1970 rabbit and porcine; Takahaski, et a l . , 1967, chicken). In most cases, however, this loss was demonstrated by blendorizing aged muscle pieces and examining the fragmented myofibrils. It i s extremely d i f f i c u l t , using this technique, to determine i f the resolution of rigor i s due to breaks at the I - Z junction or loss of Z-line structure. However, i t should be noted that loss of Z-line structure has also been demonstrated in situ ,(Henderson et a l . , 1970). Sayre (1969) found that the Z-line in chicken muscle, aged for 24 hours, was s t i l l intact. Upon blendorizing, the muscle appeared to fragment in the I-band region and not the A-band or Z-line regions. These results are in agreement with the results of Fukazawa et a l . (1963) who found that breaks in myofibrils always took place in the I-band region. These data combined with the data of Takahaski et a l . (1967) suggest that - 51 -resolution of rigor and tension release, in poultry muscle, may be due to weakening of the muscle structure at or near I - Z junction. If this junction i s structually weakened, then fragmentation procedures for myofibrillar preparation would rupture these weakened areas allowing Z-line material to diffuse away. This would give the appearance of Z-line disintegration during postmortem storage. Busch et a l . (1972 a, b) have shown that the pre-sence of lO" 3 n EDTA or EGTA (calcium chelators) in the extra-cellular buffer did not interfere with tension development in rabbit or porcine muscle. They did, however, prevent the release of tension for up to 48 hours post-maximum tension indicating that calcium had been successfully removed from i t s role in stimulating tension release. The above findings were not observed in broiler muscle. A study of the effects of magnesium (necessary in contraction process as well as calcium), EDTA and equimolar combinations of calcium-EDTA and magnesium-EDTA on tension development and release is presented in Table IV and Figure 5. The presence of the above materials in the extracellular buffer did not substantially alter the time to reach maximum tension, further suggesting that the sarcolemma remains v i r -tually impermeable to extracellular materials in early post-mortem muscle. EDTA and equimolar concentrations of EDTA and calcium or magnesium significantly lower the amount of - 52 -F i g u r e 5. E f f e c t of 10" 3 M magnesium, EDTA and calcium-EDTA on t e n s i o n d e c l i n e i n b r o i l e r P_j_ major muscle. - 53 TABLE IV. MEANS AND STANDARD ERRORS OF TIME AND TENSION DEVELOPMENT FOR STRIPS OF BROILER Pj. MAJOR MUSCLE IN PHOS-PHATE BUFFER AND BUFFER CONTAINING Hg T T, EDTA, C a + + + EDTA AND Mg + + + EDTA. Treatment Time, hr Tension, g/cm' Control 5.74 + 0.76 51.25 + 2.36 (n=ll) 10" 3M Mg 6.48 + 1.21 41.62 + 4.81 (n=5) (6.44 + 1.13)a (53.12 + 3.79) 10"3M EDTA 4.70 + 0.96 38,38 + 1.17* (n=5) (5.39 1.27) (50,12 + 3.79) 10" 3M C a + + 4.65 + 0.96 32.57 + 3.73* + 10-3M EDTA (6.18 + 1.48) (49.22 + 3.97) (n=5) 10"3M l Mg + + 4.56 + 0.95 32.90 + 0.95* 10 M EDTA (5.77 + 0.89) (56.14 + 5.40) (n=3) mmm * Significantly different from control (p<0.05) a Values within parentheses are averages for control strips run simultaneously with the various treatment strips and used in the paired comparison analyses. tension developed. Magnesium tended to lower tension somewhat but not significantly from control values. The most unexpected finding in this study was the effect of these materials on the rate of tension release. EDTA, which prevents tension release in porcine and rabbit muscles,, stimulates tension release in broiler P_j_ major muscle. When equimolar concentrations of calcium or magnesium were added fco buffer containing 10" J M EDTA an additive e f f e c t was observed on tension release (Figure 5), Only the release data for calcium-EDTA i s shown because the magnesium-EDTA data were v i r t u a l l y the same. The release data for s t r i p s run i n magnesium containing buffer, i f superimposed on the calcium data i n Figure 3, are again i d e n t i c a l to calcium. The r o l e of magnesium i n tension release, though not v e r i f i e d , i s pro-bably somewhat d i f f e r e n t than that of calcium. Magnesium ions act as a p l a s t i c i z e r i n muscle allowing a c t i n and myosin to s l i p passively past each other. I t i s therefore possible, that the magnesium stimulates the d i s s o c i a t i o n of actomyosin r e s u l t i n g i n an increased rate of tension d e c l i n e . The nature of the additive e f f e c t s of calcium-EDTA and magnesium-EDTA i s not known. Calcium and magnesium are both e f f e c t i v e l y chelated by EDTA (Blaedel and Meloche, 1963) with calcium forming a s l i g h t l y more stable complex than mag-nesium. One possible explanation f o r the additive e f f e c t on tension release may be that the complex of EDTA-Ca or EDTA-Mg, which i n t h i s form i s uncharged, may pass through the sarcolemma with greater ease than either of the ions when present singu-l a r l y . Once inside the c e l l , other ions such as zinc, lead, i r o n , which displace calcium and magnesium i n an EDTA complex, could bring about a release of calcium and magnesium ions thereby stimulating a more rapid rate of tension release. This explanation i s purely speculative and a thorough examination of muscle s t r i p s , treated i n the above manner, i s warranted at the - 55 -electron microscopic level. This study is of particular impor-tance in view of the unique transverse tubular system which has been demonstrated in chicken pectoral muscle (Mendell, 1971) . The Effect of Processing Techniques on Tension Pattern and  Tenderness Processing techniques have been shown to adversely affect poultry tenderness by several workers (Shannon et a l . 1957; Pool et al.,1959; Wise and Stadelman, 1959, 1961). In view of these findings, various processing techniques, singu-l a r l y and in combination, were applied to broilers postmortem in order to study tension parameters in relation to tenderness (shear value). The effects of the various treatments on tension parameters from the two different levels of P^ major muscle are shown in Table V. Only the combination treatment of pre-slaughter struggle, scalding and plucking produced a s i g n i f i -cant difference in tension parameters between outside and inside breast muscle. Wise and Stadelman (1959) found that shear was significantly related to the depth at which samples were taken, to the temperature of the scald water and to the duration of scald. Shear value were not determined for different muscle depths, but based on the significant decrease in time to maxi-mum tension, i t i s possible that treatment 5 could result in significant differences between inner and outer layers. - 56 -TABLE V . MEANS AND STANDARD ERRORS OF TENSION PARAMETERS FOR INNER AND OUTER STRIPS OF BROILER P*. M A J O R MUSCLE SUB-JECTED TO VARIOUS POST-SLAUGHTER TREATMENTS, Treatment 3 Time, hr Tension, g/cm2 Tension release b at one hour, % 1 outer 3.9 + 0.6 33.1 + 1.7 21.3 + 3.5 inner 3.5 + 0.6 35.6 + 1.7 26.1 + 3.7 2 outer 2.7 + 0.5 67.7 + 6.7 25.0 + 1.7 inner 3.0 + 0.4 56.1 + 2.7 20.7 + 1.7 3 outer 2.7 + 0.5 43.9 + 4.4 21.4 + 5.1 inner 3.1 + 0.4 53.4 + 3.7 19.0 + 3.7 4 outer 2.2 + 0.4 41.8 + 4.1 28.1 + 3.2 inner 2.1 + 0.3 47.0 + 3.9 28.4 + 3.2 5 outer 1.9 + 0.4* 65.6 +10.5 29.5 + 5.1* inner 3.0 + 0.3 55.0 + 9.6 16.3 + 2.5 treatments NO. 1 : Control - r e s t r i c t e d struggle (n=15) 2 : Struggle, pluck (n=5) 3 : Restricted struggle, scald (n=6) 4 : Struggle, scald (n=7) 5 : Struggle, scald, pluck (n=8) * S i g n i f i c a n t l y d i f f e r e n t from inner s t r i p s (p<0.05) k Amount of tension released at one hour post maximum tension. - 57 -The data for inner and outer strips combined and the shear values are presented in Table VI. On an overall basis, only treatment 4 produced a significant change in time to maximum tension. Treatments 2, 4 and 5 significantly increased the amount of tension developed compared to the con-t r o l . The shear values indicate an additive response to the various processing techniques. The procedures which seem to have the greatest affect on the tenderness are free struggle at slaughter and scalding. TABLE VI. MEANS AND STANDARD ERRORS OF POOLED TENSION PARA-METERS AND SHEAR VALUE DATA FOR INNER AND OUTER STRIPS OF BROILER P. MAJOR MUSCLE SUBJECTED TO VARIOUS POST-SLAUGHTER TREATMENTS. 3. 2 b Treatment Time,hr Tension, g/cm Tension Release Shear at one hour,% Value,lbs 1 3.7 + 0.6 36.8 + 1.5 23.7 + 3.3 4.5 + 0.3 2 2.8 + 0.4 61.9 + 4.6** 22.8 + 2.3 5.6 + 0.3* 3 2.9 + 0.5 48.6 + 3.7** 20.2 + 4.3 6.3 + 0.6* 4 2.2 + 0.3* 44.4 + 3.9 28.3 + 2.9 9.0 + 0.5** 5 2.5 + 0.6 59.8 + 6.2** 22.9 + 3.5 10.5 + 0.9** a For treatment breakdown see Table V. k Amount of tension released at one hour post maximum tension. * Significantly different from control (p < 0.05) ** P < 0.01 The tenderness data are in accord with results found by other workers (Shannon et a l . , 1957; Pool et a l . , 1959; Wise and - 58 -Stadelman, 1959, 1961). The data also show that tension re-lease does not correlate with shear value. A l l treatments showed f i r s t hour tension release values in the range of 20 -28 percent and there were no significant differences between treatments and controls. The fact that maximum toughening was observed for treatments involving scalding relates to the findings of the temperature studies discussed earlier. The tension values did not approach those found at 50 and 60°C because the outer layers of breast muscle, which may reach these temperatures, had a pa r t i a l l y cooked appearance and were discarded. It was noted during shear measurement that a sharp break in shear peak occurred after the blade had passed through the outer layer of the breast muscle demonstrating the existence of a toughened shell around the outer areas of breast muscle probably caused by high temperature-low pH denaturation. Segregation of Broiler Controls on the Basis of Time to Reach  Maximum Tension It was noted during the course of the tension experi-ments that muscle strips in control buffer varied considerably in rate of tension release. There was a definite pattern be-tween time to reach maximum tension and the proportion of tension released within 12 hours post-maximum tension. The data for control strips from 35 birds were segregated into three groups, on the basis of time required to reach maximum tension. The - 59 -means and standard deviations for the tension parameters of these three groups and for the pooled birds are presented in Table VII. TABLE VII. TIME AND TENSION MEANS .AND STANDARD DEVIATIONS FOR THREE BROILER GROUPS SEGREGATED ON THE BASIS OF TIME TO REACH MAXIMUM TENSION. Group a Time, min s d b Tension, g/cm"^  sd I (n=10) 147.2** 30.3 47.74 15.65 II (n=15) 234.7 40.3 46.09 8.94 III (n=10) 473.1** 74.1 51.88 12.32 • Pooled (n=35) 299.3 134.7 48.50 12.60 aTension maximum for the three groups was observed between: Group I 0 - 3 hours postmortem Group II - 3 - 6 hours postmortem Group III - } 6 hours postmortem sd Standard Deviation ** Significantly different from Group II (p<0.01) Segregation on the above time basis gave three groups of birds which did not differ with regard to amount of tension developed but differed significantly from each other on the basis of the time required to reach maximum tension. The ten-sion release data for the three groups are presented in Figure 6 and the regression line parameters for each Group are presented - 60 Figure 6, Isometric tension decline i n three groups of b r o i l e r s separated on the basis of time required to reach maximum tension. - 61 -in Table VIII. It can be seen that the Group I birds, which reached maximum tension in less than 3 hours, released tension much more rapidly than did Groups II or III birds. S t a t i s t i c a l t-test analysis of difference of means showed that Group I birds released tension at a faster rate (p<0.01) than Groups II and III birds. Groups II and III differed significantly from each other (p<0,05) only during the f i r s t 5 hours post-maximum tension. TABLE VIII. REGRESSION LINE PARAMETERS FOR TENSION RELEASE (INDEPENDENT VARIABLE) VERSUS TIME (DEPENDENT VARIABLE) FROM THREE GROUPS OF BROILER CONTROLS AND FOR THE POOLED GROUPS. Groupa Intercept Slope R2 I (n=10) 80.10 -5.33 0.711 II (n=15) 92.60 -4.56 0.776 III (n=10) 97.66 -4.54 0.849 Pooled (n=35) 90.47 -4.77 0.642 a See footnote for Table VII. The tension pattern for Group I birds i s almost identical to the release _3 pattern observed when 10 M . calcium was added to the buffer. It is possible that the rapid onset of rigor, as demonstrated by the short time to maximum tension, stimu-lated a more rapid or greater release of calcium from the sarcoplasmic reticulum once rigor had occurred. The difference in release of tension was obvious at 1 hour post-maximum tension and i t appeared possible to predict, - 62 -with some degree of accuracy, the 12 hour release from the 1 hour value. In order to test this hypothesis, the 1 hour release values for each group were, correlated with the values for subsequent hourly values. The groups were analyzed i n d i -vidually and then the data were pooled and a general relation-ship was established. The results of these analyses are presented in Table IX. The data show that, for Group I birds, T7-J3LE IX. SIMPLE CORRELATIONS OF ONE HOUR TENSION RELEASE VALUES WITH SUBSEQUENT HOURLY VALUES FROM THREE GROUPS OF CONTROL BROILERS AND FOR THE POOLED DATA FROM THE THREE GROUPS. Time, hr Group I a (n=10) Group II (n=15 Group III (n=10) Pooled (n=35) 2 .958** .825** .564* .950** 4 .904** .596* .475 .850** 6 .838** .456 .480 .789** 8 .859** .474 .224 .775** 10 .359** .367 -.006 .731** 12 .901** .306 -.088 .699** See footnote for Table VII. * p<0.05 ** p<0.01 there i s a significant correlation (p<0.01) between 1 hour ten-sion release and subsequent hourly values up to 12 hours post-maximum tension. The relationship i s less pronounced for Groups II and III. This i s probably due to the larger standard deviations - 63 -in time to maximum tension as demonstrated in Table VII. The correlation between 1 hour release and subsequent hourly values is significant (p<0.01) when the data for the 35 birds i s pooled. This shows that one could obtain a reasonably accurate pattern of tension release by measuring only the 1 hour tension release. The data presented in Table X shew that the relation-ships between 2 hour values and subsequent hourly values are generally stronger than for the 1 hour data. The r values for Groups II and III, in particular, are improved considerably as are the r values for the pooled data. TABLE X. SIMPLE CORRELATIONS OF TWO HOUR TENSION RELEASE VALUES WITH SUBSEQUENT HOURLY VALUES FROM THREE GROUPS OF CON-TROL BROILERS AND FOR THE POOLED DATA FROM THE THREE GROUPS. Time, hr Group I a Group II Group III Pooled (n-10) (n=15) (n=10) (n=35) 4 .965** .914** .866** .951** 6 .889** .818** .767** .899** 8 .873** .828** .689** .883** . 10 .845** .749** .506 .841** 12 .828** .695** .414 .798** a See footnote for Table VII. * p<0.05 ** p(0.01 - 64 -T h e Relation Between O n e Hour Tension Release, Tine to Maximum  Tension and Shear Value Analyses were performed to establish i f rate of ten-sion release was significantly correlated to the time to reach maximum tension and i f so to establish the relationship. The data from 1 5 0 strips were f i t t e d to linear, logarithmic and hyperbolic models in order to determine the most suitable relationship for the data. It was found that the time to maxi-mum tension and 1 hour tension release were linearly related. With percent relative tension (% RT) as the dependent variable and time as the independent variable, the following equation for the regression line was obtained: % RT = 6 1 . 7 4 + , 4 0 8 Time, n« 1 5 0 , R2 = . 4 6 2 ( p < 0 . 0 1 ) . Since tension release was more rapid when time to maximum tension was shortest and since 1 hour tension release was significantly related to release for subsequent hours (up to 12 hours post-maximum tension), i t was decided to determine the relation between 1 hour tension release and the eventual tenderness observed in broilers. Linear, logarithmic and hyperbolic models were applied to the data for 1 hour tension release and shear values from 1 0 0 broilers. One hour tension release values ranged from 0 - 4 8 percent and shear values ranged from 2 . 5 to 1 4 . 3 pounds. No significant relationship was observed for any of the models studied, suggesting that the tension release observed in individual birds i s not indicative of tenderness. - 65 -deFremery and Pool (1963) established that treatments which accelerate r i g o r mortis r e s u l t i n increased toughness (shear value) i n b r o i l e r breast muscle. The present data on the r e l a t i o n s h i p between shear value, time to maximum tension and one hour tension release raises some i n t e r e s t i n g questions with regard to the r e l a t i o n s h i p between tension release i n b r o i l e r P. major muscle and tenderness of the muscle. Birds which e x h i b i t the most rapid rate of tension development also show the most rapid rate of decline even though, i n most cases, the amount of tension developed does not d i f f e r s i g n i f i c a n t l y . The rapid tension development indicates accelerated r i g o r mortis which should lead to toughness according to deFremery and Pool (1963). This however, i s not the case since a p o s i t i v e r e l a t i o n -ship e x i s t s between tension development and release and not between release and shear value. Three possible reasons for the lack of a s i g n i f i c a n t r e l a t i o n s h i p between shear value and tension release are: sample error, the s u i t a b i l i t y of shear measurement for assessing o v e r a l l muscle tenderness and the determination of shear values was done only at 24 hours postmortem. This 24 hour post-mortem time may be long enough to allow a greater release of tension i n the Group II and III birds thus bringing them more i n l i n e with Group I b i r d s . I t would be of i n t e r e s t to study the t e n s i l e and break strength parameters of uncooked muscle i n r e l a t i o n to tension parameters and to study these parameters at a f i x e d time post-maximum tension for each b i r d . Perhaps i n - 66 -this way a better idea of the relationship between tension release, the resolution of rigor mortis and tenderization may be obtained. On the basis of the present data, one can conclude that variations in tension release, from bird to bird, are not indicative of the tenderness of the individual birds at 24 hours postmortem. The overall tension pattern for a pooled group of broilers i s , however, indicative of the observed tenderization process in broilers. Most tenderization in P^ major muscle of broilers occurs within a few hours post-rigor and during this period 50 percent or more of the isometric tension i s re-leased. The tenderization phenomenon in broilers is much more rapid than in pork and beef muscle, which show a slower tension development and decline. It may be concluded that the tension pattern i s indicative of the tenderization phenomenon of a species but tension patterns for individuals within a species, may or may not relate to the actual tenderness of the i n d i v i -dual animal or muscle. Epinephrine Experiments  Preliminary Experiment This experiment was conducted to determine the approx-imate time when the effect of epinephrine injection was maximal in the broilers being used. deFremery and Pool (1963) and Sayre (1969, 1970) used a pre-slaughter injection time of 16 hours to - 67 -effect glycogen depletion in broilers, whereas deFremery (1966b) used 18 hours and Klose et, a l . (1970) 15 hours. Khan and Nakaraura (1970) found that the epinephrine effect was maximal at 12 hours post-injection. The data obtained in this prelimi-nary study are presented in Table XI. The data, though limited to one bird per time, suggest that the maximum effect of epine-phrine occurs between 9 and 12 hours post-injection. TABLE XI. TENSION PARAMETERS AND SHEAR VALUES FOR P^ . MAJOR MUSCLE FROM BROILERS INJECTED WITH EPINEPHRINE AT VARIOUS TIMES PRE-SLAUGHTER. Pre-slaughter Time, Tension, Shear Injection Time, min g/cm^ Value, hr lbs Control 399 54.85 4.06 3 213 41.96 4.15 6 38 94.90 6.58 9 27 157.70 7.01 12 43 96.72 7.40 15 92 99.36 4 .62 18 49 37.93 6.53 The injections in this prelimanary experiment were done intramuscularly into the P_j_ major muscle. It was noted during subsequent tenderness measurements that shear values for muscle near the site of injection were negligible while values for areas away from the site of injection were generally higher - 6 3 -than control birds. In viev; of this localized epinephrine effect, injections for the subsequent experiments were done intramuscularly into the thigh muscle. Epinephrine Experiment 1 The analysis of variance showed that there was a significant treatment effect for a l l parameters studied (Table XII). Treatment means and the results of Duncan's new multiple range test are presented in Table XIII. The tension and time data show an inverse relationship with maximum tension being developed in the shortest time. These values also indicate the effect of the epinephrine injections was maximal at 8 hours post-injection. This is somev/hat earlier than the 12 hour value found by Khan and Nakamura (1970) . TABLE XII. ANALYSIS OF VARIANCE FOR PARAMETERS STUDIED IN EPINEPHRINE EXPERIMENT 1. Source df Mean Squares Tension Time Blood Lactate Shear Value Sex 1 2505.7 180.0 0.085** 9.28 Treatment 5 11918.4** 32077.0** 0.044** 9.96* SxT 5 461.0 7149.4 0.011 1.18 Error 24 682.9 7389.5 0.011 3.23 Total 35 * p<0.05 ** p<0.01 69 TABLE XIII. DUNCAN'S NEW MULTIPLE RANGE TEST ON SIGNIFICANT TREATMENT MEANS FROM EPINEPHRINE EXPERIMENT 1. Post-Injecrtion Slaughter Time, hours Control 12 16 Tension g/cm Time, min Blood Lactate, mM/lOOml Shear Value, lbs 43.4a* 49.5a 107,9b 161.4 119.2b 103.6b 234.5a 166.5ab 65.4bc 32.3c 107.3bc 93.5bc 0.33abc 0.51a 0.45ab 0.26c 0.36bc 0.41ab 2.6a 3.2ab 5.0bc 5.6c 5.5bc 5.3bc *Means in the same row with similar superscripts do not dif f e r significantly (p<0.05) A slight increase was found in blood lactate concen-tration in birds k i l l e d 2 hours after injection. This is pro-bably due to the rapid breakdown of glycogen to lac t i c acid which then diffuses out of the muscle into the blood stream. The lowest level of blood lactate was found in the 8 hour group where tension was maximum and time minimum. Results of shear value analysis were contrary to most findings in that significant toughening was found instead of a tenderization. Khan and Nakamura (1970) found that muscle from broilers, injected with epinephrine 2 or 6 hours before slaughter , was more tender than control muscle after 24 hours of postmortem - 70 -storage. The present data shows no significant difference between control and 2 hour samples and significant toughening in 4, 8, 12 and 16 hour samples. deFremery and Pool (1963) and deFremery (1966b)found that elimination of postmortem glycolysis by epinephrine injec-tions gave chicken meat that was tender immediately post-slaugh-ter. These authors, however, did not compare the effect on tenderness after 24 hours of aging. In order to determine the relationship between tension, time, blood lactate and shear value, a simple correlation analysis was run on the data. The correlation analysis i s presented in Table XIV. TABLE XIV. CORRELATION MATRIX FOR PARAMETERS STUDIED IN EPINEPHRINE EXPERIMENT 1. Tension Time Blood Lactate Time .711** Blood Lactate .315 .196 Shear .611** .454** .331* *p<0.05 ** p<0.01 As expected, the regression analysis showed s i g n i f i -cant relationships between the tension developed, the time to maximum tension and shear values. Blood lactate level was significantly related to shear value (p<0.05). - 71 -The analysis of variance (Table XII) showed that a l l parameters, except blood l a c t a t e , f a i l e d to show a s i g n i f i c a n t sex e f f e c t . The male broilers,were s i g n i f i c a n t l y higher than females with control l e v e l s being 0.44 and 0.32 m Holes/lOOmls respe c t i v e l y . The e f f e c t was p a r t i c u l a r i l y noticeable i n the 2 hour treatments where males averaged 0,63 m Holes/lOOmls versus 0,39 m Moles/10Omls for females. On the basis of experiment, i t was decided to repeat the experiment and i n addition, c o l l e c t data f o r muscle ATP, glycogen, pH and l a c t a t e . The times of sampling were changed somewhat to t r y to better define the time of maximal epinephrine e f f e c t . Epinephrine Experiment 2 The analysis of variance i s presented i n Table XV and treatment means and the r e s u l t of Duncan's new multiple range test are presented i n Table XVI. As i n Experiment 1, the 8 hour pre-slaughter i n j e c t i o n time gave the maximal e f f e c t . How-ever, i n a l l cases, there were no s i g n i f i c a n t differences between 8, 10 and 12 hour pre-slaughter i n j e c t i o n s . I t v/ould appear from t h i s that the time of maximum e f f e c t extends over at least a 4 hour range and that the number of birds used i n the t e s t was not s u f f i c i e n t to e s t a b l i s h a more exact time. Muscle glycogen l e v e l , which indicates the extent of the epinephrine e f f e c t , was minimum at 8 hours and s l i g h t l y higher at TABLE XV. ANALYSIS OF VARIANCE OF PARAMETERS STUDIED IN EPINEPHRINE EXPERIMENT 2 . Source df Mean Squares Tension Time ATP Glycogen Blood Lactate Tissue Lactate pH Shear Value Sex 1 953 .3 9184.0 24.1* 13.3** 0.006 34.0 0.06 24.6** Treatment 5 7920.8** 43361.0** 12.8* 10.7** 0.015 ** 539.9 * * 0.71 9.7** SxT 5 692.3 682.1 1.9 3.8 0.027 124.5 0.09 2.2 Error 24 1122.2 4070.6 4.7 1.1 0.033 66.2 0.07 2.2 Total 35 * p<0.05 ** p<0.01 - 73 -TABLE XVI. DUNCAN'S NEW MULTIPLE RANGE TEST ON SIGNIFICANT TREATMENT MEANS FROM EPINEPHRINE EXPERIMENT 2. Post-Injection Slauahter Tine, hours 4 8 10 12 24 Tension, 40.19a* 78.47a 128.35b 123.15b 127.20b 76.44a g/crrr^  Time, 265.2 87.5a 45.2a 46.1a 50.5a 119.0a rain ATP 5.79ab 5.56ab 3.67b 3.76b 3.10b 6.76a u Moles/g Glycogen, 3.38 1.13a 0.27a 0.45a 0.67a 1.52a mg/g Slaughter 6.04a 6.19a 6.77b 6.79b 6,62b 6.11a pH Tissue 45.0a 45.2a 28.2b 24.6b 30.8b 44.2a Lactate, /u Moles/g Shear 4.57a 4.69a 6.51b 7.27b 6.84b 4.53a Value, lbs *Means in the same row with similar superscripts do not di f f e r significantly (p<0.05) 10 and 12 hours and by 24 hours had recovered to one half of the control level. It i s of interest to note that the ATP level re-mained quite high throughout the entire time period studied. Since A.TP i s needed for shortening, this observation i s consis-tent with the development of considerable tension in the 8, 10 and 12 hour muscle strips. There was very l i t t l e glycogen pre-sent in the muscles at these time periods, therefore, l i t t l e - 74 postmortem glycolysis. Consequently, the time to maximum ten-i sion was very short even though tension was high. The pH and tissue lactate values follow a pattern •i similar to glycogen and time to maximum tension. The control, 4 and 24 hour levels were similar and lactate dropped while pH rose in the 8, 10 and 12 hour samples. The relationship between shear values and the signi-ficant treatment parameters is shown in Figures 7, 8 and 9. Shear value has a positive relationship with tension and pH and negative with glycogen, time to maximum tension, tissue lactate and ATP. It i s of interest to note that no significant blood lactate effect was obtained in this experiment. A simple correlation analysis was performed on the data from the second experiment and the correlation matrix i s presented in Table XVII. It can be seen that there are signi-ficant correlations between a l l parameters except blood lactate. It is d i f f i c u l t to explain the difference between blood lactate values from experiments 1 and 2. The analysis of variance (Table XV) shows that, similar to experiment 1, there was no sex difference for tension and time. Sex differences were observed for glycogen, ATP and shear value. The significant sex effect for shear value was not observed in experiment 1, however, shear values for females were consistently higher than for males. The shear value average for control males was 4.4 pounds versus 4.8 pounds for females. In the 10 hour group where shear values were maximum, the males averaged 5.8 pounds to 8.8 pounds for the females. - 75 -Figure 7 . Relation betv/een tension, time to maximum tension and shear value in major muscle from epine-phrine treated b r o i l e r s . - 76 -Figure 8. Relation between muscle glycogen and ATP l e v e l s and shear value i n major from epinephrine treated - 77 -6 n! 1 * 1 1 1 1 ° 0 4 8 12 16 20 24 P O S T I N J E C T I O N S L A U G H T E R T I M E ( H o u r s ) Figure 9. Relation betv/een pH, muscle lactate and shear value i n P. major muscle from epinephrine treated b r o i l e r s . TABLE XVII. CORRELATION MATRIX FOR PARAMETERS STUDIED IN EPINEPHRINE EXPERIMENT 2. Tension Time ATP Glycogen pH Blood Tissue Lactate Lactate Time .781** ATP .705** .587** Glycogen .542** .689** .437** pH .731** .598** .631** .475* Blood Lactate .068 .010 .100 .115 .049 Tissue Lactate .610** .466** .479** ,404* .816** .149 Shear Value .762** .570** .826** .454** .770** .020 .647** * p<0.05 ** p<0.01 - 79 -The greatest sex difference in glycogen levels was found i n the control birds where values of 6.07 and 1.70 mg/g tissue were found for males and females respectively. In spite of this great difference in control levels, both males and females showed minimum glycogen levels (0.32 and 0.23mg/g tissue respectively) at 8 hours. The ATP levels for females remained lower than for males throughout a l l of the treatment times. Both levels did, however, approach equality at 8 hours where the males averaged 3.8 and females 3.5 u Moles/g tissue. After 8 hours the ATP level in males began rising while the level in females continued to drop reaching a minimum of 1.6 p Moles/g tissue at 12 hours. In general the sex differences seem to be related to differences in levels present in control birds. It also appears as i f females may react somewhat differently to epinephrine than do males. This i s particularly noticeable in the ATP and ten-derness data. The data from a l l the epinephrine experiments, i n -cluding the preliminary experiment, show that depletion of glycogen induces toughness in broilers examined at 24 hours postmortem. This i s in contrast to the data of Khan and Kakamura (19 70) but the results obtained for the various para-meters show why toughness may be expected. Sayre (1969) found that shear values from chickens, injected pre-slaughter with epinephrine, did not change appre-- S o -ciably during the aging period. He suggested that this muscle becomes inextensible quickly due to lack of ATP, but due to the high pH and possible integrity of the endoplasmic reticulum, there may be no great stimulus for contraction or tension development. The present data shows that this i s not so. There is ample ATP remaining in the muscle, even when glycogen has been essentially depleted, and considerable tension is developed. The fact that the muscle becomes inextensible quickly is verified by the present data which demonstrated that the time required to reach maximum tension decreased to 45 min-utes when the epinephrine effect was maximal. The fact that the epinephrine injected birds develop a great deal of tension post-mortem may explain why the toughening was observed. This a b i l i t y to develop tension may also explain the observed tenderness i f epinephrine treated birds are cooked without aging. Marsh and Leet (1966) found in beef neck muscle that 20 percent contrac-tion was associated with a"fair" degree of tenderness; between 20 - 40 percent contraction was associated with a rapid decrease in tenderness to a minimum and between 40 - 60 percent contrac-tion gave increased tenderness approximating that observed at 20 percent contraction. /vlthough most workers have found an increase in ten-derness in epinephrine treated birds, Klose et_ a l . (1970) did not find the expected lower shear values. They questioned the a b i l i t y of epinephrine to deplete muscle glycogen but Cori - B l -and Cori (1928) have shown that under proper conditions, this technique depletes muscle glycogen prior to slaughter. Further-more, the present data and that of Khan and Makamura (1970) show clearly that glycogen levels are depleted by pre-slaughter epinephrine injections. The epinephrine experiments were carried out to study the response of the broiler under a severe stress situation. An apparent adverse reaction, in the form of tougher muscle, resulted from these studies and because females appeared to react more adversely than males a l l subsequent stress experi-ments except for the "commercial stress", were applied to females only. Stress Experiments The results from the study on broilers, exposed to antemortem commercial handling,(Table XVIII) show that there are no significant differences between "stressed" and control birds for any of the parameters studied except time required to reach maximum tension. The "stressed" females required signi-ficantly longer (p<0.05) than their control counterparts to reach maximum tension. Though males showed the same trend, the difference was not significant. On a combined sex basis "stressed" birds were significantly different from controls (p<0.05). For the most part, the differences between control and "stressed" males are negligable. Although only the time difference i s significant in the females, most other parameters TABLE XVIII. MEANS AND STANDARD ERRORS OF PARAMETERS OF P. MAJOR MUSCLE FROM BROILERS IN THE COMMERCIAL STRESS EXPERIMENT Female Male Combined Sexes Stress Control Stress Control Stress Control Time, hr 4.81+0.7* 3.02+0.7 6.25+1.2 4.49+0.4 5.53+0.7* 3.76+0.4 Tension, g/cm2 44.3 +3.4 35.3 +2.8 39.8 + 1.3 40.7 +5.3 42.0 +1.8 38.0 +3.0 Shear Value, lbs 4.60+0.4 4.05+0.3 4.05+0.3 4.88+0.3 4.33+0.3 3.97+0.2 ATP, u Moles/g 7.98+0.5 5.92+0.8 8.48+0.9 8.46+0.9 8.23+0.4 7.19+0.7 pH 5.99+0.05 5.77+0.09 5.92+0.07 5.93+0.05 5.95+0.04 5.85+0.06 * S i g n i f i c a n t l y d i f f e r e n t from corresponding control (p<0.05) approached significance at the p<0.0 5 level. The most striking of these differences i s the difference in muscle ATP levels. These data suggest a possible sex related difference in resis-tance or adaptation to commercial handling conditions. The results for female broilers subjected to a thermal stress prior to slaughter (Table XIX) suggest that the birds were affected by the heat treatment and that the effect was intensi-fied by allowing the birds to struggle freely during slaughter. The time to maximum tension was not shortened significantly by the heat treatment alone but the free struggle at slaughter apparently accelerated glycolysis so that the time difference v/as significant (p<0,05). The amount of tension was increased significantly by both "stress" treatments. The significant differences between "stres5ed"-free struggle birds and control birds for time to maximum tension and 1 hour tension release are consistent with data reported earlier on the effects of commercial processing techniques on tension parameters. They also confirm the lack of relationship between tension release and tenderness within the range of parameters studied. The results for the "cold stress" experiment are presented in Table XX. As in the commercial stress experiment, there v/as a lengthening of the time required to reach maximum tension. This effect was accompanied by a tendency for tension, shear value, ATP and glycogen concentration to be high and hexose monophosphate to be lower in "cold-stressed" birds. - 34 -TABLE XIX. MEANS AND STANDARD ERRORS OF PARAMETERS OF P^ MAJOR MUSCLE FROM BROILERS IN THE HEAT STRESS EXPERIMENT. Parameter Control, RSa Stres !3, RS Stre: S3, S b Time, hr 4.77 + 1.1 3.12 + 0.6 2.14 + 0.2* Tension, g/cm^ 38.2 ; t 1 .7 48.1 + 4.1* 52.6 + 4.2** Shear Value, lbs 5.06 + 0.4 5.50 + 0.5 5.10 + 0.4 ATP, u Moles/g 7.26 + 1.0 6.11 + 0.6 5.69 + 0.7 pH, "0" hr 6.14 + 0.04 6.05 i 0.05 5.96 + 0.03* pH, "24" hr 5.64 + 0.03 5.61 + 0.02 5.68 + 0.03 1 Hour b Tension Release, % 22.6 + 2.3 33.4 + 4.0 38.5 + 2.3* aRS - Restricted struggle at slaughter S - Free struggle at slaughter * Significantly different from control (p^O.05) ** p<0.01 Amount of tension released at one hour post maximum tension. The birds subjected to 2°C for 2 hours were signi-ficantly tougher (p<0.05) than control birds. Although the values do not dif f e r significantly in most cases, the cold stress effect appeared to be maximal at 2 hours. - 85 -TABLE XX. MEANS AND STANDARD ERRORS OF PARAMETERS OF P. MAJOR MUSCLE FROM BROILERS III THE COLD STRESS EXPERIMENT. Parameter Control Stress 2 hr at 2°C 6 hr at 2°C Time, hr 5.59 + 0.5 7.73 + 0.8* 8.29 + 1.2 Tension, g/cm2 49.06 + 3.3 51.99 + 3.2 46.86 + 4.4 Shear Value, lbs 5.91 + 0.3 6.97 + 0.4* 6.11 + 0.3 ATP, u Holes/g 6.08 + 0.9 6.66 + 0.4 6.34 + 0.5 Glycogen, rcg/g 4.53 + 0.4 5.60 + 0.6 5.04 + 0.7 HMPa 6 .96 + 0.3 6.12 + 0.5 6.42 + 0.5 cl HMP - Hexose Monophosphate * Significantly different from control (p<0.05) The most surprising effect in the "cold stress" study was the effect on time to maximum tension. The 6 hour birds had the longest time to maximum tension but since the variation was large, the values did not differ significantly from the controls. Three of the nine birds in this group, however, took from 11 - 12 hours to reach maximum tension. This i s a relatively high incidence when compared to the fact that only 1 broiler, in over 100 control broilers studied in this project, - 86 -required as long as 12 hours to reach maximum tension. The most important agents which have been proven to be stressors to the fowl are temperature extremes, handling, shaking, food and/or water deprivation and debeaking, (Freeman, 1971). In the i n i t i a l "alarm phase", which results after ex-posure to a stressor, adrenalin or epinephrine i s released from the adrenal medulla causing the passage of potassium from the muscles to the blood and the breakdown of li v e r and muscle glycogens to glucose and la c t i c acid (Lawrie, 1966). If the effect of the stressor were severe enough, one would expect alterations in postmortem muscle due to depletion of muscle glycogen. The results from the epinephrine experi-ments, reported earlier in the thesis, verify this effect. The results from the stress experiments, however, are somewhat ambiguous and varied. This may be due, in part, to the fact that none of the stressors studied was severe enough to e l i c i t the response observed by the actual injection of epinephrine. The data from the "commercial stress" and "cold stress" experiments show a different trend than the data from the "heat stress" experiment. These findings are in accord with Seigel (1971) who found that different environmental stimuli produced evidence of "stress response" in birds but often several c r i t e r i a of response had to be evaluated since responses to a particular stimulus may contradict or mask a single response. Such masking or contradiction of responses - 87 -may be of p a r t i c u l a r importance i n studying a so-called "commercial stress" which involves several p o t e n t i a l stressors (heat, cold, handling, crowding). The apparent lengthening of the time to reach maxi-mum tension observed i n the "commercial and cold stress" experiments suggests that glycogen l e v e l s have not been d i s -turbed and that the period of postmortem g l y c o l y s i s has, i n some manner, been lengthened. This e f f e c t has not been pre-vio u s l y observed i n b r o i l e r s and i s d i f f i c u l t to explain. Lawrie (1966) suggested that the difference i n bovine response to pre-slaughter f a s t i n g or exercise, as compared to other' species, may r e l a t e to the greater capacity of the ruminant muscle to gain energy by d i r e c t catabolism of f a t t y acids, thereby conserving carbohydrate. The p o s s i b i l i t y that poultry may be able to e l i c i t t h i s response, under c e r t a i n stress s i t u a t i o n s , cannot be overlooked. The commercial implications of such a phenomenon i s obvious. The study on commercial processing techniques, reported e a r l i e r , showed that postmortem muscle parameters were s i g n i f i c a n t l y altered by the various procedures. I t i s therefore evident, that any change which affected postmortem g l y c o l y s i s would be magnified by the processing techniques. In the case of the "cold and commercial stressed" b i r d s , the apparent increase i n time to maximum tension should have a b e n e f i c i a l e f f e c t on the ultimate muscle q u a l i t y . Conversely, - 33 the shortened time to maximum tension, observed in "heat stressed" birds would be magnified by the processing proce-dures and the muscle quality should be adversely affected. Bovine, ovine and porcine species when subjected to stressors prior to slaughter, usually have lower muscle glycogen, higher postmorten pH, increased water holding capacity and improved tenderness and juiciness (Hedrick, 1965), However, under certain conditions postmortem quality i s adversely affected in bovine and porcine muscles. This i s evidenced by the occurence of "dark cutting" beef and "pale, soft, exudative" pork which have been studied by several workers (Lawrie, 1958, 1966 a, b; Sayre et a l . , 1963 a b; Lister et a l , , 1970) The only adverse quality attribute observed during the study of avian muscle was the slight increase in shear value in the 2 hour cold stressed birds. This may be related to the fixed 24 hour aging period given a l l birds post-slaughter rather than the actual cold treatment. Control birds reached maximum tension at 5,6 hours post-slaughter whereas the 2 hour birds peaked at 7,7 hours post-slaughter. This shows that the 2 hour birds would have, on the average, a shorter aging time than the control birds. Cold Shortening Studies During the study of the effects of temperature on isometric tension pattern, a cold shortening phenomenon was observed which appeared to increase in intensity at 5°C down - 89 -to 0°C, The e f f e c t o f t h i s s h o r t e n i n g on the a b i l i t y of muscle s t r i p s t o develop f u r t h e r t e n s i o n i s demonstrated i n T a b l e XXI. The 15 b r o i l e r s s t u d i e d i n t h i s experiment developed an average t e n s i o n o f 30.9 g/cm-' w i t h i n 3 minutes at 2°C. Mien t h i s t e n -s i o n was developed through c o l d s h o r t e n i n g , i t v/as r e l e a s e d almost as r a p i d l y as i t developed so t h a t i n most cases the s t r i p s had r e t u r n e d to the o r i g i n a l s t a r t i n g p o i n t w i t h i n 15 minutes a f t e r attachment t o the t r a n s d u c e r s . TABLE XXI. MEANS AND STANDARDS ERROR OF TENSION PARAMETERS OF BROILER Pj. MAJOR MUSCLE SUBJECTED TO VARIOUS POST-SLAUGHTER TEMPERATURE TREATMENTS. Treatment Tension, g/cm 2 Time, min Room Temperature, 61.31 + 2.7 331.67 + 23.5 c o n t r o l C o l d S h o r t e n i n g , 2°C 30.94 + 5.9 2.93 + 0.3 "0" t i m e a 43.57 + 8.1 261.33 + 42.0 12 hours PMT b 12.62 + 0.03 63.16 + 11.8 2 4 hours PMT 7.93 + 0.03 102.67 + 35.5 36 hours PMT 1.77 + 0.8 40.5 + 10.3 a " 0 " time r e f e r s to s t r i p s p l a c e d i n b u f f e r at 2°C to observe the c o l d s h o r t e n i n g e f f e c t then immediately s u b j e c t e d t o b u f f e r a t room temperature t o observe f u r t h e r t e n s i o n development. These t h r e e times r e f e r to muscle s t r i p s h e l d i n b u f f e r at 2°C f o r v a r i o u s l e n g t h s o f time post-maximum t e n s i o n (PMT), then p l a c e d i n b u f f e r at room temperature t o observe t e n s i o n develop-ment. The time to maximum t e n s i o n f o r these s t r i p s v/as d e t e r -mined from c o r r e s p o n d i n g c o n t r o l s t r i p s run a t room temperature. - 90 -When strips were allowed to shorten at 2°C, then brought up to room temperature by changing the buffer, they s t i l l developed a considerable amount of tension during the course of a normal tension pattern. The time to reach maximum tension was about one hour shorter than for control strips and the amount of tension was only about two-thirds that of the controls. If the tension developed on cold shortening is added to the subsequent tension developed at room temperature, then the total exceeds that developed by controls. Holding muscle strips in buffer at 2°C for periods of 12, 24 and 36 hours demonstrated a decreasing a b i l i t y of the strips to develop isometric tension subsequently. At 3 6 hours post-maximum tension, the strips had essentially lost their a b i l i t y to develop tension. This indicates that glycolysis had continued in the strips at 2°C but the rate was probably too slow to effect a tension development, other than the i n i t i a l cold shortening tension, at this temperature. The mechanism of the cold shortening phenomenon i s somewhat obscure. Locker and Kagyard (1963) showed that cold shortening, in beef muscle, started within a few minutes of commencement of cooling and was almost complete within one hour. Smith et al_. (1969) demonstrated a cold shortening effect in turkey and chicken major and found that shortening was maxi-mal at 0°C. This shortening was essentially complete after 3 hours in chickens and 5 hours in turkeys. The variation between - 91 -these studies and the present studies on b r o i l e r P. major may be due to the use of small s t r i p s of muscle i n a buffer at 2°C as opposed to whole muscles cooled i n a i r at 0°C. The temperature equilibrium would be attained much faster i n the s t r i p s of muscle thereby accounting for the more rapid shorten-ing observed. The toughening, as a r e s u l t of cold shortening, des-cribed by Newbold and Karris (1972) and Marsh (1972) i s not l i k e l y to occur i n poultry muscle. This i s evident through the a b i l i t y of the s t r i p s to relax to i n i t i a l l e v e l s immediately a f t e r the cold shortening has reached i t s peak. In order to batter understand the nature of the cold shortening, a second study was i n i t i a t e d to measure the e f f e c t of cold shortening on some of the energy r i c h phosphate com-pounds i n muscle. The e f f e c t of cold shortening on the ATP concentration i s of p a r t i c u l a r i n t e r e s t since shortening occurs only when ATP i s present in the muscle. The r e s u l t s of t h i s study ara shown i n Table XXII. The ATP l e v e l s were lowered somewhat by subjecting s t r i p s of muscle to buffer at 2°C for 3 or 7 minutes before freezing i n l i q u i d nitrogen for analysis, A lower ATP l e v e l was also evident i n muscle s t r i p s held i n buffer at room temperature for 7 minutes. S i g n i f i c a n t differences between cold-treated and control s t r i p s were found but cold-treated s t r i p s did not d i f f e r from s t r i p s held for 7 minutes i n buffer at room tempera-ture. This lac3c of a significant decrease in ATP levels i s in accord with Busch et. a l . (1967) who observed only a small change in ATP level during large tension development at 2°C. It i s evident, however, that a significant amount of ATP need not be hydrolyzed to effect the cold shortening. This is more plausible when one considers that a normal tetanic contraction in muscle i s capable of developing 2 - 3 kg tension/cm muscle (Huxley, 1958), The 30.9 g/cm^ obtained during cold shortening represents only about 1 percent of the potential tetanus, therefore ATP levels may not change significantly. The pre-sence of adequate levels of ATP subsequent to shortening would explain why the muscle i s able to relax immediately after cold shortening since ATP also acts as a plasticizer in muscle allowing actin and myosin to slide freely past each other, TABLE XXII. MEANS AND STANDARD ERRORS OF THE ATP, HHP AND CP a CONTENT OF BROILER P. MAJOR MUSCLE SUBJECTED TO VARIOUS TEMPERATURE TREATMENTS, Treatment HMP ATP CP Control 6.95 + 0.68 9.23 + 0.64 2.02 + 0.4 3 min at 2°C 4.48 + 0 .56** 8.40 + 0.49 2.23 + 0.24 7 min at 2°C 4.17 + 0.52** 8.06 + 0.51* 2.52 + 0.25 7 min at 25°C 5.35 + 0.36 8.56 + 0.55 2.44 + 0.21 Abbreviations used are: HMP - hexose monophosphate ATP - adenosine triphosphate CP - creatine phosphate * Significantly different from control (p<0.05) ** p<0.01 - 9 3 -Harsh (1966) postulated that cold shortening, l i k e thaw r i g o r , resulted from i n a c t i v a t i o n of a relaxing factor by calcium release, t h i s being the r e s u l t of a s a l t " f l u x " . Such a s a l t " f l u x " may be explained i n terms of the r e l a t i v e rates of d i f f u s i v e and chemical processes since the temperature c o e f f i c i e n t of d i f f u s i o n i s considerably lower than that of chemical reaction, BY applying the above postulation to the cold shortening phenomenon observed i n b r o i l e r muscle, i t i s possible to suggest a sequence of events occuring i n the muscle when sub-jected to cold buffer, A s a l t " f l u x " of calcium ions, released from the sarcoplasmic reticulum (SR), would stimulate the myo-f i b r i l l a r adenosine triphosphatase (myosin) to s p l i t ATP, thereby e f f e c t i n g shortening or contraction. The amount of contraction would be proportional to the concentration cf the s a l t "flux" which apparently varies s u b s t a n t i a l l y between 5°C and 0°C. The s a l t " f l u x " may be a temporary phenomenon r e s u l t i n g from the "cold shock" and i t would appear that a f t e r having l o s t the a b i l i t y to sequester calcium, the SR again becomes operable. The calcium pump reverses and calcium i s removed from the m y o f i b r i l s and sequestered by the SR. Since there i s s t i l l a considerable amount of ATP present to act as a p l a s t i c i z e r , the muscle relaxes to i t s o r i g i n a l r e s t length. If held at or near 0°C, the s t r i p s maintain a slow rate of g l y c o l y s i s which i s not rapid enough to e f f e c t a further tension development. After 36 hours, at or near 0°C, s u f f i c i e n t g l y c o l y s i s has occurred within the muscle strips to eliminate any further a b i l i t y to develop tensipn even i f the strips are brought to room temperature. The decrease in hexose monophosphate levels may result from an increased phosphorylation of fructose-6-phosphate to fructose-1, 6-diphosphate which i s catalyzed by phosphofructo-kinase. This however, i s speculative and warrants further study as does the apparent increase in creatine phosphate levels. - 95 -SUMMARY AND CONCLUSIONS The effects of buffer, pH, temperature and various ions were studied in relation to tension development and de-cline in broiler major muscle. A tension pattern, similar to those reported for turkey, porcine, bovine and rabbit muscle, v/as observed using phosphate buffer as an incubation media. Tension maximum v/as attained in control broilers at approxi-mately 4 . 5 hours postmortem and an average of 45 g tension/cm muscle v/as obtained. Changing the pH or temperature of the buffer did not affect the a b i l i t y of the broiler muscle to develop or release tension but both factors in some instances, altered the amount of tension developed and the time required to reach maximum tension. The effect of temperature v/as found to vary consider-ably. Temperatures in the range of those used in commercial scalding were shown to cause a 6 - 7 fold increase in tension development within a very short time. Such tension develop-ment may be related to the toughening which results from scalding of broilers. - 3 The presence of 10 II calcium or magnesium ions in the incubation buffer produced a marked change in the rate of tension release. Their effect on tension development v/as minimal indicating that the sarcolemma of postmortem muscle may be impermeable to extracellular materials for a short - 96 -period of time postmortem. Chelation of the calcium or mag-nesium with EDTA produced an additive e f f e c t on tension release suggesting that non-charged complex may r e a d i l y enter the muscle and that a displacement of calcium or magnesium from the complex may then take place thereby releasing these ions to e f f e c t a more rapid tension decline. A pattern was observed i n control s t r i p s run i n phosphate buffer. Three groups of b r o i l e r s were categorized on the basis of time required to reach maximum tension. Sepa-r a t i o n on t h i s basis revealed that birds reaching maximum tension i n less than 3 hours postmortem had a s i g n i f i c a n t l y greater rate of tension release than birds i n the 3 - 6 hour or greater than 6 hour categories. The rate of release f o r s t r i p s from the 0 - 3 hour group coincided with the rate of release f o r s t r i p s run i n buffer containing 10 J M calcium, suggesting a difference i n calcium regulation i n t h i s group of b i r d s . I t was found possible to predict the proportion of maximum tension released at 12 hour post-maximum tension from the proportion released at 1 or 2 hours. The one hour value was also s i g n i f i c a n t l y correlated to the time required to reach maximum tension. Tenderness and 1 hour tension release value were not s i g n i f i c a n t l y r e l a t e d i n d i c a t i n g that the rate of tension release from b i r d to b i r d i s not i n d i c a t i v e of sub-sequent tenderness. The e f f e c t of various processing treatments on tension - 97 -parameters and broiler tenderness was studied. An additive response v/as observed for the different combinations studied with the combined commercial process of free struggle at slaughter, hot water scalding and mechanical plucking yielding the greatest changes and significantly toughening broiler breast muscle. The effect of pre-slaughter epinephrine injections on postmortem tension parameters, tenderness and various compounds related to postmortem glycolysis in muscle v/as studied. The maximum effect of epinephrine injection occurred between.8-12 hours post-injection. At this time, muscle glycogen levels were essentially depleted but muscle ATP v/as s t i l l sufficient to effect considerable tension development during the extremely short period of postmortem glycolysis. This abi l i t y to develop considerable tension appeared to be the cause of the toughening observed in the muscle from epinephrine injected birds. The effect of several antemortem stressors on post-mortem broiler muscle v/as studied. The stressors studied (commercial, cold and heat) did not significantly alter muscle quality. A difference in response to the heat stress, as com-pared to the commercial and cold stresses, v/as observed. The response of birds to a stressful situation i s characterized by release of epinephrine. It is evident that the situations studied in this thesis v/ere not severe enough to e l i c i t the magnitude of response observed from the actual injection of this hormone. - 9 8 -A cold shortening phenomenon was observed i n the range from 0 - 5°C and the magnitude of the shortening increased as the temperature decreased towards 0 °C . Subsequent studies at 2°C showed that af t e r the i n i t i a l cold shortening further tension development did not occur at t h i s temperature. Further tension development did occur i f the 2°C buffer was exchanged for buffer at room temperature. A holding period of 36 hours at 2°C was necessary to eliminate further tension development whan buffer temperature was raised to room temperature. 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