Tim Thesis

THE USE OF ELECTRICAL IMPEDANCE TO RAPIDLY

PREDICT BEEF TENDERNESS.

BY

TIMOTHY MARK NATH

A thesis submitted in partial fulfillment of the requirements for the

Master of Science

Major in Animal Science

South Dakota State University

2008

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This thesis is approved as a creditable and independent investigation by a

candidate for the Master of Science degree and is acceptable for meeting the thesis

requirements for this degree. Acceptance of this thesis does not imply that the

conclusions reached by the candidate are necessarily the conclusions of the major

department.

Dr. Duane Wulf Thesis Advisor Date Dr. Robert Thaler Head, Animal & Range Sciences Date

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Acknowledgements

I would like to thank several people for their help throughout graduate school and

the completion of this thesis. First of all, I am sincerely grateful for all the help and

advice I received from my advisor Dr. Wulf. I appreciate all the time and effort Dr. Wulf

put into teaching and guiding me through my research. I would also like to thank him for

allowing me to work in the meat lab. The skills I have learned working in the meat lab

will help guide me through my career. I would also like to thank Dr. Weaver for all of her

advice and support. She has been great help with my lab work and the writing of this

thesis. I would like to thank Dr. Thaler and Dr. Cartrette for serving on my committee. I

would also like to thank Michaeal Singer for providing the electrical impedance

instrument and his support through my research. I would like to especially thank Deon

Simon for all her technical help with lab work. A huge thank you goes to Adam Rhody

and his capable meat lab crew for all their help with shear force for my project. I can’t

thank the meat science graduate students enough for their help, but a very sincere thank

you goes to Tanner Machado, Dustin Mohrhauser, Sarah Wells, Andrew Everts, and

Amanda Everts. I would also like to thank my parents Mike and Janet Nath and the rest

of my family for being supportive and encouraging me throughout my education

experience. Most of all I would like to thank my wife Carissa for her help and support

through my graduate career. To our beautiful daughter Madison for giving me even more

reasons to enjoy life. Lastly, I would like to thank God for allowing me to live my life

each and every day.

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ABSTRACT



Timothy M. Nath

December 2008

A three-phase study was conducted to determine the usefulness of electrical

impedance to rapidly predict beef tenderness. Objectives of phase I were to determine

optimal measurement location and impedance’s potential to predict tenderness.

Measurements of the ribeye were the most accurate at predicting tenderness and

impedance had potential to predict beef tenderness. Objective of phase II was to compare

impedance to existing technology (BeefCam and NIR) to predict beef tenderness.

Impedance was the most effective technology at sorting out very tender carcasses.

Instruments were additive when combined to predict beef tenderness. Objectives of phase

III were to determine the effect of breed type, suspension method, and aging on

impedance measurements. Impedance decreased slightly from d 1 to d 7 postmortem.

Impedance was related to sarcomere length, proteolysis and Warner-Bratzler shear force.

In conclusion, electrical impedance was weakly related to beef tenderness, but may be

useful to sort out very tender beef carcasses and predict postmortem aging.

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Table of Contents

Page

Abstract………………………………………………………………………..................iv

List of Tables……………...………………………………………………………….….vii

List of Figures……………...………………………………………………………….…ix

Appendixes……………………………………………………………………………….x

Chapter 1. Review of Literature

Introduction…………………………………………………………………….....1

Beef Tenderness…………………………………………………………………...3

Proteolysis …………………………………….…………………………..6

Sarcomere Length …………………………….……………………….…..7

Connective Tissue………………………….………………………….…...8

Tenderness Prediction……………………………………………………………..9

Tendertec…………………………………………………………………10

BeefCam...…………………………………….………….………………11

NIR………………………………………………….………………........12

Slice Shear Force………………………………………………………...12

Electrical Impedance… …………………………….…………………..13

Literature Cited…………………………………………………………………..15

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Chapter 2. The use of electrical impedance to rapidly predict beef tenderness

Introduction…………………………………………………….............................20

Materials and Methods……………………………………………………………22

Results and Discussion……………………………………………………....…....34

Implications………………………………………………………………...……..43

Literature Cited…………………………………………………………...………64

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List of Tables

Table Page

2.1 Simple statistics for carcass and muscle traits for Phase I………………… ……44

2.2 The ability of electrical impedance measurements from various electrode

depth and placement combinations to predict Warner-Bratzler shear force

(average of d4 and d14)…...……………………….……….…......……………...45

2.3 Evaluating the ability of electrical impedance from tagged (carcass side

sample was removed) and untagged (carcass side sample was not removed)

sides to predict Warner-Bratzler shear force (average of d5 and d14)……....…..46

2.4 Predicting d5 and d14 Warner-Bratzler shear force using electrical

impedance measurements from d4, d5, d14……………………...……………...47

2.5 R-squares for various prediction models to predict Warner-Bratzler

shear force (d14)…………………...………..…………………………….…......48

2.6 Simple statistics for carcass and muscle traits for Phase II……………………...49

2.7 R-squares for various prediction models to predict Warner-Bratzler

shear force (d14)....................................................................................................50

2.8 Percentage of tough (>49.0 N) carcasses by certification rate and

instrument………………………………………………………………………..51

2.9 Percentage of very tender (<34.3 N) carcasses by certification rate and

instrument………………………………………………………………………..52

2.10 Average Warner-Bratzler shear force (N) for sort groups (fifths) by

instrument………………………………………………………………………..53

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2.11 Effect of day postmortem on impedance measurements (n = 100)….………......54

2.12 Simple statistics for carcass and muscle traits for Phase III…………...………..55

2.13 Least square means of Warner-Bratzler shear force, sarcomere length,

troponin T degradation, total collagen, resistance, reactance, and phase

angle (averaged across all aging periods)..............................................................56

2.14 Effect of day on Warner-Bratzler shear force, sarcomere length, and

troponin T degradation………….………………………………..…………...…57

2.15 Correlations of Warner-Bratzler shear force, sarcomere length, proteolysis,

total collagen, resistance, reactance, and phase angle (*P < 0.05)………….......58

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List of Figures

Figure Page

2.1 Electrode arrangement and penetrating depths…………………………………..59

2.2 The effect of breed type on resistance, reactance, and phase angle from

1 to 7 days postmortem (*P < 0.05)……......................................…….….....…...60

2.3 The effect of suspension on resistance, reactance, and phase angle from

1 to 7 days postmortem (*P < 0.05) ………………………..….…...……………61

2.4 Representative micrographs of myofibrils with normal (1.82 µm, left)

and hip suspension (2.43 µm, right) sarcomeres of longissimus muscle

(Magnification 400X)…………………………………………………………...62

2.5 Representative western blot of whole muscle protein extracts from

bovine longissimus muscle with normal (NS) and hip (HS) suspension

and postmortem aging periods were 1, 4, 7, and 10 days (d0 was used for

quantification)……………………………………………………………...….....63

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Appendix

Appendix Page

A. The effect of breed type on resistance, reactance, and phase angle impedance

measurements of longissimus lumborum steaks from d 1 to d 21 postmortem.....67

B. The effect of suspension on resistance, reactance, and phase angle impedance

measurements of longissimus lumborum steaks from d 1 to d 21 postmortem….68

C. The effect of breed type on resistance, reactance, and phase angle impedance

measurements of semitendinosus steaks from d 1 to d 10 postmortem………….69

D. The effect of suspension on resistance, reactance, and phase angle impedance

measurements of semitendinosus steaks from d 1 to d 10 postmortem……...…..70

E. The effect of breed type on resistance, reactance, and phase angle impedance

measurements of psoas major steaks from d 1 to d 10 postmortem……………..71

F. The effect of suspension on resistance, reactance, and phase angle impedance

measurements of psoas major steaks from d 1 to d 10 postmortem……………..72

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CHAPTER I

Review of Literature

Currently, the beef industry segregates carcasses into palatability groups

according to the amount of marbling in the ribeye and the percentage of ossification in

the thoracic buttons. However, marbling has been reported to be a poor predictor of

tenderness and can explain no more than 5% of the variation in palatability traits

(Morgan, et al., 1991; Wheeler, Cundiff, & Koch, 1994). Consumers are dissatisfied

when purchasing steaks from the same quality grade that vary greatly in tenderness.

Introduction

Palatability of meat consists of three main components: flavor, juiciness, and

tenderness. The National Beef Tenderness Survey reported tenderness is the single most

important factor affecting consumers’ perception of taste (Morgan et al., 1991).

However, tenderness is a leading cause of consumer dissatisfaction, due to the variation

of tenderness and inability to predict this variation among steaks. Many factors affect

meat tenderness including: genetics of the animal, gender, and physiological maturity

(Wulf, Tatum, Green, Morgan, Golden, & Smith, 1996; Huff-Lonergan, Parrish, &

Robson, 1995; Purslow, 1985). Additionally, postmortem muscle characteristics known

to influence meat tenderness include: proteolysis of myofibril proteins, sarcomere length,

and connective tissue (Koohmaraie, Kent, Shackelford, Veiseth, & Wheeler, 2002).

These numerous factors affecting tenderness make this a difficult palatability trait to

predict on-line at packing plant speeds.

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Studies have shown that consumers can detect the difference in beef tenderness and are

willing to pay a premium for guaranteed tender beef (Boleman et al., 1997; Shackelford,

Wheeler, Meade, Reagan, Byrnes, & Koohmaraie, 2001; Miller, Carr, Ramsey, Crockett,

& Hoover, 2001). The most accurate technique to predict tenderness and satisfy

consumers is the Warner-Bratzler shear force method (Bratzler, 1932; Warner, 1952).

However, this machine is not practical for use in the industry due to time constraints and

cost. Many researchers have tried to develop a machine or method that will predict beef

tenderness. However, most of these machines or methods are unable to provide sufficient

information at commercial packing-plant chain speeds to predict beef tenderness

accurately. Therefore the meat industry is looking for a simple, fast, noninvasive way to

predict beef tenderness and sort carcasses into palatability groups (NCA, 1994).

Providing a value associated with tenderness will provide an economic incentive for the

beef industry to market beef more efficiently to consumers.

Electrical impedance is a simple, fast, noninvasive technology currently used in

the medical field that utilizes phase angle to predict the life expectancy of a person dying

from a terminal illness such as cancer (Gupta et al., 2004). Phase angle is a calculated

value from the two components of electrical impedance: resistance (related to water loss)

and reactance (related to cell membrane breakdown) (Lukaski, 1996). As the value of

phase angle decreases over time the life expectancy shortens for that person (Gupta et al.,

2004). In terms of predicting beef tenderness, it is known that as a beef animal ages and

proceeds through proteolysis there is a loss of water and a breakdown of cell membranes.

Lepetit, Salè, Favier, and Dalle (2002) have shown that electrical impedance could be

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related to meat aging. Therefore, the objective of this research was to determine the

usefulness of electrical impedance to rapidly predict beef tenderness.

Tenderness as defined by Takahashi (1996) is the sum of the total of the

mechanical strength of skeletal muscle tissue and its weakening during post-mortem

ageing of meat. The National Beef Tenderness Survey revealed tenderness is the single

most important factor affecting consumers’ perception of taste (Morgan et al., 1991).

However, tenderness is the leading cause of consumer dissatisfaction, due to the variation

of tenderness and inability to measure this variation among steaks. Currently the beef

industry segregates carcasses into palatability groups based on the amount of marbling in

the ribeye and the ossification of the thoracic buttons. Morgan and associates revealed

that the current USDA grading system does a poor job at segregating carcasses into

tenderness groups (1991). In addition, Wheeler and associates have stated “marbling

explained at most 5% of the variation in palatability traits,” (1994). These authors

concluded that there is not a lack of tender steaks, but rather an inconsistency of

tenderness among steaks with similar quality grades. Thus, the National Cattlemen’s

Association (NCA, 1994) listed “development of an instrument or procedure that can

adequately measure quality, cutability and tenderness in beef carcasses in modern

packing plants” as a top priority of the beef industry. There is an incentive for the beef

industry to market tender beef based upon the increase in branded beef programs and the

Beef Tenderness

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data that demonstrates a significant segment of consumers that are willing to pay a

premium for guaranteed tender beef.

Boleman and associates (1997) were the first to show that consumers can detect

differences in beef tenderness and are willing to pay a premium for guaranteed tender

beef. In this study, beef strip steaks of known shear force values were placed into three

color-coded categories [2.27 to 3.58 kg (Red), 4.08 to 5.40 kg (White), and 5.90 to 7.21

kg (Blue)] with a premium of $1.10 per kg placed between each category. The color-

coded streaks were offered to randomly-selected consumers and after sampling

consumers purchased 94.6, 3.6, and 1.8 percent of the Red, White, and Blue steaks,

respectively. In addition, Shackelford and associates (2001) reported that 50% of

consumers would be willing to pay a $1.10 per kg premium for guaranteed tender USDA

Select loin steaks. In a study conducted by Miller and associates (2001), 78% of

consumers were willing to pay more for steaks that the retailer guaranteed as tender.

These results indicate the importance to the beef industry to be able to segregate between

tender and tough steaks and to market accordingly.

Genetic differences in tenderness have been found both among (Crouse, Cundiff,

Koch, Koohmaraie, & Seideman, 1989) and within (Shackelford, Koohmaraie, Cundiff,

Gregory, Rohrer, & Savell, 1994) breeds of cattle. Wulf and associates (1996) reported

genetic differences existing in tenderness within and between the Charolais and Limousin

breeds. In addition, breed type greatly affects beef tenderness; where Brahman

influenced cattle tend to create tougher meat due to a higher level of calpastatin than non-

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Brahman influenced cattle (Whipple, Koohmaraie, Dikeman, Crouse, Hunt, & Klemm,

1990; O’Connor, Tatum, Wulf, Green, & Smith, 1997). A method for improving meat

from Brahman influenced cattle would be aging the meat for longer periods of time

postmortem (O’Connor et al., 1997).

Physiological maturity of the animal affects tenderness in that as cattle mature,

intramuscular collagen solubility decreases, resulting in tougher beef (Purslow, 1985).

Results from Huff-Lonergan and associates (1995), reported older cattle to be tougher

than younger cattle due to an increased level of connective tissue, and bulls to be tougher

than steers due to an increased level of calpastatin. Animal’s genetics, physiological

maturity, and gender are just a few of the factors that can affect meat tenderness ante-

mortem. There are also many factors that affect meat tenderness postmortem.

Research has shown the three main factors that affect meat tenderness

postmortem are: proteolysis of myofibril proteins, sarcomere length, and connective

tissue (Koohmaraie et al., 2002). Connective tissue content increases with animal age

and is related to the workload for each muscle (Purslow, 1985). Shorter sarcomere length

leads to an increase in toughness (Herring, Cassens, & Briskey, 1965; Hostetler,

Landman, Link, & Fitzhugh, 1970). Proteolysis is the breakdown of proteins by calpains

(Huff-Lonergan, Mitsuhashi, Beckman, Parrish, Olson, & Robson, 1996). Research has

shown meat tenderizes when held at refrigerated temperatures during postmortem aging

(Taylor, Geesink, Thompson, Koohmaraie, & Goll, 1995). Since sarcomere length and

connective tissue do not change during normal refrigerated temperature, proteolysis of

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myofibril proteins is the major mechanism behind the tenderization of meat during

postmortem aging.

Proteolysis

Proteolysis is the degradation of structural proteins by cellular enzymes called

proteases (Tornberg, 1996). These proteolytic enzymes do not degrade the major

filaments of myosin and actin, but rather the structural proteins (titin, nebulin, desmin,

tropomyosin, and troponin) that regulate the integrity of the sarcomere (Goll, 1991, Huff-

Lonergan et al., 1996). Koohmaraie has shown that the degradation of these proteins

improves tenderness by reducing the organization of the filamentous structure within the

sarcomere (1996). The calpain protease system has been shown by many researchers to

be responsible for the improved tenderness during postmortem storage (Goll 1991; Huff-

Lonergan et al., 1996).

The calpain system includes µ-calpain and m-calpain (Koohmaraie and Geesink,

2006). The calcium concentration required is different for each enzyme as µ-calpain

requires micromolar calcium concentrations and m-calpain requires millimolar calcium

concentrations (Goll, 1991). Calpains are responsible for the degradation of titin,

nebulin, tropomyosin, and troponin (Goll, 1991). The protease µ-calpain is thought to be

the primary protease responsible for the breakdown of these proteins postmortem

(Koohmaraie, 1996; Geesink, Kuchay, Chrishti, & Koohmaraie, 2006).

Calpastatin is the specific inhibitor of µ-calpain and m-calpain, and thus inhibits

the rate and extent of postmortem proteolysis (Geesink and Koohmaraie, 1999). Bos

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indicus cattle have an increased calpastatin level and therefore do not go through this

process of proteolysis as rapidly as Bos taurus cattle, and is one reason for the increased

toughness in Bos indicus (Whipple et al., 1990). Shackelford and associates (1994)

reported that the heritability of calpastatin activity was high for Bos indicus and

accounted for much of the genetic variation in Warner-Bratzler shear force values. The

effect of calpains has been well documented by many researchers to affect meat

tenderness, but there may be other enzymes that could also affect proteolysis. The rate of

proteolysis is highly variable and could be a factor in the inconsistency of beef

tenderness, meaning that some beef may take a few days and others take weeks to

complete proteolysis.

Sarcomere Length

A sarcomere is defined as a segment between two neighboring z-lines that is also

the basic unit where muscle contraction and relaxation occur (Aberle, Forrest, Gerrard, &

Mills, 2001). Many studies have shown that sarcomere length is correlated with

tenderness, with a longer sarcomere having less resistance to shearing than a shorter

sarcomere (Hostetler et al., 1970; Rhee, Wheeler, Shackelford, & Koohmaraie, 2004).

During normal postmortem aging conditions, sarcomere length does not change due to

the permanent formation of actomyosin cross-bridges which locks the thick and thin

filaments in place (Savell, Mueller, & Baird, 2005). Sarcomere length can be affected by

variations in hanging of the carcass pre-rigor to alter the sarcomere lengths of different

muscles.

8

Hip suspension also known as “Tenderstretch” is a method of hanging the carcass

from the aitch bone pre-rigor, allowing the weight of the hind limb to stretch muscles in

the round and loin. Hip suspension is utilized to increase the sarcomere length of several

muscles such as the longissimus and semitendinosus; however this method shortens

sarcomeres in other muscles such as the psoas major. This method would improve the

tenderness of the longissimus and semitendinosus muscles, but would cause detrimental

effects on the already tender psoas major muscle (Hostetler et al., 1970). Tenderstretch

is not practiced in commercial packing plants due the detrimental effects of the psoas

major as well as space limitations within the plant.

Tendercut™ is another prerigor treatment which subjects muscles to more tension

by severing the bones, minor muscles, and connective tissues in the loin and(or) round

area 45 to 90 min postmortem (Wang, Claus, & Marriot, 1996). This method increases

the sarcomere length of the following muscles: longissimus, biceps femoris,

semitendinosus, and semimembranosus, (Wang et al, 1996; Ludwig, Claus, Marriott,

Johnson, & Wang, 1997; Beaty, Apple, Rakes, & Kreider, 1999). Inceasing the

sarcomere length of these muscles would result in an improved tenderness. Tendercut™

could be used in the industry as this method requires no new equipment, and it is

adaptable to the current design of existing plants.

Connective Tissue

Connective tissue is a measure of collagen cross-linking. The number of cross-

linked chains explains a large amount of the tenderness variation, and varies greatly

9

between muscles, species, breeds, and with animal age (Judge & Aberle, 1982; Purslow,

1985; Lepetit, 2007). As collagen cross-linking increases with animal age, the tougher

and less palatable the muscle becomes (Judge and Aberle, 1982). Unlike some

myofibrillar proteins, collagen is not degraded postmortem which contributes to a fixed

amount of background toughness. Perimysium is a sheath of connective tissue that

groups individual muscle fibers into bundles. Perimysium remains intact during cooking

and is the primary source of cooked meat toughness. Connective tissue content must be

addressed either ante-mortem via genetics and physiological age at slaughter, or

postmortem via cooking (Purslow, 1985).

Tenderness Prediction

Past surveys have revealed the inability of the current USDA beef quality grading

system to accurately segregate carcasses into palatability groups (Morgan et al., 1991).

Studies have shown that consumers can detect the difference in beef tenderness and are

willing to pay a premium for guaranteed tender beef (Boleman et al., 1997; Miller et al.,

2001; Shackelford et al., 2001). Thus, the National Cattlemen’s Association (NCA,

1994) listed “development of an instrument or procedure that can adequately measure

quality, cutability and tenderness in beef carcasses in modern packing plants” as a top

priority of the beef industry. There have been many attempts to identify instrumental

methods for predicting meat tenderness. Most of these were intended for laboratory

research tools and varied widely in their efficacies.

10

In more recent investigations of objective predictions of meat tenderness, the goal

has been to develop on-line systems for grading carcasses based on tenderness. The ideal

system would involve an objective, noninvasive, tamper-proof, accurate technology. Past

technologies evaluated for their potential as on-line tenderness grading tools include

Tendertec (Ferguson, 1993), BeefCam (Wyle, Cannell, Belk, Goldberg, Riffle, & Smith,

1998), near-infrared spectroscopy (Park, Chen, Hruschka, Shackelford, & Koohmaraie,

1998), and slice shear force (Shackelford, Wheeler, & Koohmaraie, 1999). Many

instruments have been developed to predict tenderness, but few are as accurate as

Warner-Bratzler shear force (Bratzler, 1932; Warner, 1952). Warner-Bratzler shear force

is the most commonly used objective method to measure tenderness, but is costly, time

consuming, and difficult to fit into industry operations because it must be done on cooked

steaks. Therefore, the industry is looking for a machine that would provide sufficient

information at commercial packing-plant chain speeds to accurately predict beef

tenderness.

Tendertec

Initial studies performed by Ferguson (1993) showed promise for the Tendertec to

predict beef tenderness. The Tendertec is an instrument equipped with a 14-cm piston

that encountered two decleration stops occurring at 2 and 6 cm and an overall

predetermined depth of 8 cm. The probe tip is inserted perpendicularly between the

dorsal spinous processes of thoracic and lumbar vertebra through the multifidus dorsi and

into the longissimus lumborum. George and associates (1997) reported that the Tendertec

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probe detected some differences in connective tissue, however it was not better than

USDA quality grade at segmenting A-maturity carcasses into tenderness categories. Belk

and associates (2001) concluded that Tendertec was able to sort carcasses of older,

mature cattle based on tenderness, but failed to consistently detect the differences in

steaks derived from youthful carcasses. In addition, the Tendertec did not meet the

demands of a noninvasive system as this machine would puncture holes into the meat.

BeefCam

Researchers have reported that objective color measurements of beef longissimus

to be related to tenderness and can sort carcasses into palatability groups using lean color

(Wulf, O’Connor, Tatum, & Smith, 1997). These findings led researchers at Colorado

State University and Hunter Associates Laboratory (Reston, VA) to develop a prototype

video imaging system (prototype BeefCam) that would be able predict beef tenderness.

The prototype BeefCam instrument would quickly provide a noninvasive visual image of

the ribeye.

Pilot studies performed by Wyle and associates (1998) indicated that the

prototype BeefCam instrument could quickly identify carcasses likely to produce steaks

that were tender, based on Warner-Bratzler shear force values, after a 14 day aging

period. However, the prototype BeefCam instrument did have limitations that prevented

its use in a commercial setting. According to the National Beef Instrument Assessment

Planning Symposium (NLSMB, 1994), for an instrument to be successful, it must be

tested under real-world conditions.

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Vote, Belk, Tatum, Scanga, & Smith (2003) reported that the Computer Vision

System equipped with a BeefCam module was able to capture and segment video images

at commercial packing-plant chain speeds and produce information useful in explaining

observed variation in Warner-Bratzler shear force values of steaks. This information

could be used to sort carcasses according to expected palatability differences, even in

carcasses with similar marbling scores. BeefCam measurements could aid in the

selection of tender or tough steaks and therefore improve consumer satisfaction.

Near-Infrared

Near-infrared reflectance (NIR) spectroscopy is a rapid, nondestructive system

that gathers information from samples through measurements of reflected light. The light

reflected back through the NIR system contains information about properties associated

with meat tenderness (Park et al., 1998). Shackelford, Wheeler and Koohmaraie (2004)

developed a commercially available tenderness prediction system based on visible and

NIR spectroscopy that could be used on-line. Rust and associates (2008) reported that

the NIR system was able to successfully sort tough from tender longissimus lumborum

samples to 70% certification levels. These authors concluded that NIR scanning offers an

in-plant opportunity to sort carcasses into tenderness outcome groups and more

importantly guaranteed-tendered branded beef programs (Rust et al., 2008).

Slice Shear Force

Shackelford and associates (1999) developed a system for classifying beef

tenderness based on a rapid, simple method of measuring cooked longissimus shear force.

13

Longissimus steaks (2.54 cm thick) were trimmed free of subcutaneous fat and bone then

rapidly cooked using a belt grill. A 1-cm-thick, 5-cm-long slice was removed from the

cooked longissimus parallel with the muscle fibers to measure shear force. Slices were

sheared with a flat, blunt-end blade using an electronic testing machine. The entire

process was completed in less than 10 min. Therefore, in commercial application, this

process could be completed during the 10- to 15-min period that carcasses are normally

held to allow the ribeye to bloom for quality grading. Wheeler and associates (2002)

concluded that slice shear force, not a prototype BeefCam or colorimeter systems,

accurately identified “tender” beef. However, the industry is reluctant to implement this

system because of cost and a loss of product.

Electrical Impedance

Electrical impedance is a simple, fast, noninvasive technology currently used in

the medical field that utilizes phase angle to predict the life expectancy of a person dying

from a terminal illness such as cancer (Gupta et al., 2004). According to Foster and

Lukaski (1996), electrical impedance is measured by introducing a small alternating

current into the body and measuring the potential difference that results. Electrical

impedance utilizes two components, resistance and reactance. Resistance is the pure

opposition of a biological conductor to the flow of an alternating current. Reactance is

the voltage stored by a condenser for a brief period of time. Phase angle is a calculated

value from resistance and reactance (Lukaski, 1996). As the value of phase angle

decreases, the life expectancy shortens for that person (Gupta et al., 2004).

14

In terms of predicting beef tenderness, it is known that as a beef animal ages and

proceeds through proteolysis, there is a change from bound water to free water and a

breakdown of membranes. Past researchers have shown that electrical impedance could

be related to tenderness (Byrne, Troy, & Buckley, 2000). Lepetit and associates (2002)

showed a decrease in electrical impedance values during postmortem aging and

concluded that electrical impedance could be related to meat aging. Based on these

results, we hypothesized that electrical impedance will predict beef tenderness through

the resistance, reactance, and phase angle.

This research was conducted in three phases and the objectives for each phase

were to: (1) determine optimal electrode arrangement and anatomical measurement

location and determine if electrical impedance had potential to predict beef tenderness,

(2) compare electrical impedance to existing technology (BeefCam and NIR) to predict

beef tenderness, and (3) determine the effect of breed type (Bos taurus vs. Bos indicus),

suspension method (traditional vs. hip), and postmortem aging on electrical impedance

measurements.

15

Literature Cited

Aberle, E. D., J. C. Forrest, D. E. Gerrard, and E. W. Mills. (2001). Principles of Meat Science. 4th ed. Kendall/Hunt Dubuque, IA. pp. 13-14.

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Koohmaraie, M. and G. H. Geesink. (2006). Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system. Meat Science, 74, 34-43. Lepetit, J., P. Salè, R. Favier, and R. Dalle. (2002). Electrical impedance and tenderization in bovine meat. Meat Science, 60, 51-62. Lepetit, J. (2007). A theoretical approach of the relationship between collagen content, collage cross-links and meat tenderness. Meat Science, 76, 147-159. Ludwig, C. J., J. R. Claus, N. G. Marriott, J. Johnson, and H. Wang. (1997). Skeletal alterations to improve beef longissimus muscle tenderness. Journal of Animal Science, 75, 2404-2410. Lukaski, H. C. (1996). Biological indexes considered in the derivation of the bioelectrical impedance analysis. American Journal of Clinical Nutrition, 64, 397S-404S. Miller, M. F., M. A. Carr, C. B. Ramsey, K. L. Crockett, and L. C. Hoover. (2001). Consumer thresholds for establishing the value of beef tenderness. Journal of Animal Science, 79, 3062–3068. Morgan, J. B., J. W. Savell, D. S. Hale, R. K. Miller, D. B. Griffin, H. R. Cross, and S. D. Shackelford. (1991). National Beef Tenderness Survey. Journal of Animal Science, 69, 3274-3283. NCA. (1994). National Beef Tenderness Conference: Executive Summary. National Cattlemen’s Association, Englewood, CO. NLSMB. (1994). National beef instrument assessment plan. National Live Stock and Meat Board, Chicago, IL. O’Connor, S. F., J. D. Tatum, D. M. Wulf, R. D. Green, and G. C. Smith. (1997). Genetic effects on beef tenderness in Bos indicus composite and Bos taurus cattle. Journal of Animal Science, 75, 1822-1830. Park, B., Y. R. Chen, W. R. Hruschka, S. D. Shackelford, and M. Koohmaraie. (1998). Near-infrared reflectance analysis for predicting beef longissimus tenderness. Journal of Animal Science, 76, 2115-2120. Purslow, P. P. (1985). The physical basis of meat texture: Observations on the fracture

behaviour of cooked bovine M. Semitendinosus. Meat Science, 12, 39-60.

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Rhee, M. S., T. L. Wheeler, S. D. Shackelford, and M. Koohmaraie. (2004). Variation in palatability and biochemical traits within and among eleven beef muscles. Journal of Animal Science, 82, 534-550. Rust, S. R., D. M. Price, J. Subbiah, G. Kranzler, G. G. Hiton, D. L. Vanoverbeke, and J. B. Morgan. (2008). Predicting beef tenderness using near-infrared spectroscopy. Journal of Animal Science, 86, 211-219. Savell. J. W., S. L. Mueller, and B. E. Baird. (2005). The chilling of carcasses. Meat Science, 70, 449-459. Shackelford, S. D., M. Koohmaraie, L. V. Cundiff, K. E. Gregory, G. A. Rohrer, and J. W. Savell. (1994). Heritabilities and phenotypic and genetic correlations for bovine postrigor calpastatin activity, intramuscular fat content, Warner-Bratzler shear force, retail product yield, and growth rate. Journal of Animal Science, 72, 857-863. Shackelford, S. D., T. L.Wheeler, and M. Koohmaraie. (1999). Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness. Journal of Animal Science, 77, 2693–2699. Shackelford, S. D., T. L. Wheeler, M. K. Meade, J. O. Reagan, B. L. Byrnes, and M. Koohmaraie. (2001). Consumer impressions of Tender Select beef. Journal of Animal Science, 79, 2605-2614. Shackelford, S. D., T. L. Wheeler, and M. Koohmaraie. (2004). Development of optimal protocol for visible and near-infrared reflectance spectroscopic evaluation of meat quality. Meat Science, 68, 371-381. Takahashi K. (1996). Structural weakening of skeletal muscle tissue during postmortem ageing of meat: The non-enzymatic mechanism of meat tenderization. Meat Science, 43, S67-S80. Taylor, R. G., G. H. Geesink, V. F. Thompson, M. Koohmaraie, and D. E. Goll. (1995). Is z-disk degradation responsible for postmortem tenderization? Journal of Animal Science, 73, 1351-1367. Tornberg, E. (1996). Biophysical aspects of meat tenderness. Meat Science, 43, S175-

S191. Vote, D. J., K. E. Belk, J. D. Tatum, J. A. Scanga, and G. C. Smith. (2003). Online prediction of beef tenderness using a computer vision system equipped with a BeefCam module. Journal of Animal Science, 81, 457-465.

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Wang, H., J. R. Claus, and N. G. Marriott. (1996). Prerigor treatment and endpoint

temperature effects on U.S. Choice beef tenderness. Journal of Muscle Foods, 7, 45-54.

Warner, K. F. (1952). Adventures in testing meat for tenderness. Proceedings Reciprocal Meat Conference, 5, 156–160. Wheeler, T. L., L. V. Cundiff, and R. M. Koch. (1994). Effect of marbling degree on beef palatability in Bos taurus and Bos indicus cattle. Journal of Animal Science, 72, 3145-3151. Wheeler, T. L., D. Vote, J. M. Leheska, S. D. Shackelford, K. E. Belk, D. M. Wulf, B. L. Gwartney, and M. Koohmaraie. (2002). The efficacy of three objective systems for identifying beef cuts that can be guaranteed tender. Journal of Animal Science, 80, 3315-3327. Whipple, G., M. Koohmaraie, M. E. Dikeman, J. D. Crouse, M. C. Hunt and R. D. Klemm. (1990). Evaluation of attributes that affect longissimus muscle tenderness in Bos taurus and Bos indicus cattle. Journal of Animal Science, 68, 2716-2728. Wulf, D. M., J. D. Tatum, R. D. Green, J. B. Morgan, B. L. Golden, and G. C. Smith. (1996). Genetic influences on beef longissimus palatability in Charolais- and Limousin-Sired steers and heifers. Journal of Animal Science, 74, 2394-2405. Wulf, D. M., S. F. O’Connor, J. D. Tatum, and G. C. Smith. (1997). Using objective measures of color to predict beef longissimus tenderness. Journal of Animal Science, 75, 684–692. Wulf, D. M. and J. K. Page. (2000). Using measurements of muscle color, pH, and electrical impedance to augment the current USDA beef quality grading standards and improve the accuracy and precision of sorting carcasses into palatability groups. Journal of Animal Science, 78, 2595–2607. Wyle, A. M., R. C. Cannell, K. E. Belk, M. Goldberg, R. Riffle, and G. C. Smith. (1998). An evaluation of the portable HunterLab Video Imaging System (BeefCam) as a tool to predict tenderness of beef carcasses using objective measures of lean and fat color. Final Report to the National. Cattlemen’s Beef Association, Englewood, CO. Department of Animal Science, Colorado State University, Fort Collins.

20

CHAPTER II



Timothy M. Nath

Department of Animal & Range Sciences

South Dakota State University, Brookings 57007

Introduction

Palatability of meat consists of three main components: flavor, juiciness, and

tenderness. The National Beef Tenderness Survey revealed tenderness as the single most

important factor affecting consumers’ perception of taste (Morgan et al., 1991).

However, tenderness is a leading cause of consumer dissatisfaction, due to the variation

of tenderness and inability to predict this variation among steaks. Currently, the beef

industry segregates carcasses into palatability groups according to the amount of

marbling in the ribeye and the physiology maturity of the carcass. However, marbling

has been reported to be a poor predictor of tenderness and can explain approximately 5%

of the variation in palatability traits (Morgan, et al., 1991; Wheeler, et al., 1994).

Consumers are dissatisfied when purchasing steaks from the same quality grade that vary

greatly in tenderness. Studies have shown that consumers can detect differences in beef

tenderness and are willing to pay a premium for guaranteed tender beef (Boleman et al.,

1997; Shackelford et al., 2001; Miller et al., 2001). Therefore, the meat industry is

looking for a simple, fast, noninvasive way to predict beef tenderness and sort carcasses

into palatability groups (NCA, 1994). Electrical impedance is a simple, fast, noninvasive

21

technology currently used in the medical field that utilizes phase angle to predict the life

expectancy of a person dying from a terminal illness such as cancer (Gupta et al., 2004).

Phase angle is a calculated value from the two components of electrical impedance:

resistance (related to water loss) and reactance (related to cell membrane breakdown)

(Lukaski, 1996). In terms of predicting beef tenderness, it is known that as postmortem

beef muscle ages and proceeds through proteolysis there is a loss of water and a

breakdown of cell membranes. Therefore, we hypothesized that electrical impedance

will predict beef tenderness through the resistance, reactance, and phase angle.

22

Materials and Methods

Description of the Instrument

Electrical impedance was measured with a prototype instrument utilizing four

electrodes in a linear arrangement (outside two electrodes = electrical source; middle two

electrodes = detection) that measured reactance and resistance (Figure 2.1). Phase angle

is calculated from reactance and resistance with the following equation: [Phase Angle =

degrees(arctangent(reactance/resistance))].

Phase I

Carcass Selection & Electrical Impedance

Phase I was conducted in a commercial beef packing plant (Swift & Company,

Greeley, Colorado). Electrical impedance was measured on the exposed longissimus

muscle of 300 randomly-chosen beef carcasses to define population variation. The four

electrical impedance electrodes were attached to four separate probes in a linear

arrangement penetrated the exposed longissimus muscle 5 cm (Figure 2.1a). The same

arrangement of electrodes and probe depth was used to select carcasses (n=92) to equally

represent all quantiles of the population. These carcasses were then moved to a separate

rail for further testing. Carcasses were measured with five different electrode depth and

placement combinations. The first three combinations were all in a linear arrangement

with penetrating depths of 5 cm, 1.3 cm, or 0 cm (Figure 2.1a,b,c). Another linear

arrangement expanded 50 cm over the strip loin with the detection and electrical source

probes 2.5 cm apart on each end, with each probe penetrating the strip loin 5 cm. The

23

last combination measured from strip to round with the detection and electrical source

probes 2.5 cm apart with two probes in the strip loin and two probes in the round at 5 cm

penetration. South Dakota State University (SDSU) personnel recorded hot carcass

weight (HCW), longissimus muscle area (REA), adjusted fat thickness, estimated

percentage of kidney, pelvic and heart fat (KPH), and USDA marbling score. Yield

grades were calculated from adjusted fat thickness, HCW, REA, and KPH. In addition,

camera carcass data were collected from the BeefCam computer system. After carcass

data were collected and electrical impedance measured, a section of longissimus was

excised, vacuum-packaged, packed in coolers with ice, and transported back to SDSU

Meat Lab. Upon arrival at SDSU, two 2.5-cm thick steaks were removed from each

sample. The first steak was utilized for d 5 measurements of electrical impedance and

Warner-Bratzler shear force, and the second steak was utilized for d 14 measurements.

Electrical impedance measurements on steaks were in a linear arrangement with

penetrating depths of 1.3 cm or 0 cm (Figure 2.1b,c).

Warner-Bratzler Shear Force Determination

Fresh longissimus samples were vacuumed packaged and stored at 4°C until

appropriate aging length (d 5 or d 14). Samples were removed from vacuum packages

and cut into 2.5-cm-thick steaks. Steaks were cooked on a belt-fed impingement oven

(Model 1132-000-A, Lincoln Foodservice Products, Inc., Fort Wayne, IN). Peak internal

cooked temperature measurements were recorded for each steak using a hand held

thermometer (model 39658-K, Atkins Technical, Gainesville, FL). Cooked steaks were

cooled for 24 hours at 4°C before removing 6 to 8 cores (1.27 cm in diameter) parallel to

24

the muscle fiber orientation (AMSA, 1995). A single, peak shear force measurement was

obtained for each core using a Warner-Bratzler shear force machine (G-R Electric

Manufacturing Company, Manhattan, KS). Peak shear force values for each core were

averaged to obtain Warner-Bratzler shear force value for each steak.

Statistical Analysis

Data were analyzed using the PROC REG procedure of SAS (SAS Inst. Inc.,

Cary, NC). Warner-Bratzler shear force was predicted using the following regressor

variables; electrical impedance, marbling, carcass traits, and camera color using both

simple linear regression analysis (single regressor variable) and multiple linear regression

analysis (all combinations of regressor variables) (Meyers, 1990).

Phase II


Phase II was performed in a commercial beef packing plant (Swift & Company,

Cactus, Texas). Electrical impedance was measured on the exposed longissimus muscle

of 300 randomly-chosen beef carcasses to define population variation. The four

electrodes were attached to four separate probes that were in a linear arrangement and

penetrated the exposed longissimus muscle 5 cm (Figure 2.1a). The same arrangement of

electrodes and probe depth was used to select carcasses (n=300) to equally represent all

quantiles of the population. One hundred carcasses were selected three consecutive days

for a total of 300 carcasses. These carcasses were then moved to a separate rail for

25

further testing. Electrical impedance measurements on carcasses were measured with

either penetrating (1.3 cm) or surface (0 cm) probes in a linear arrangement (Figure

2.1b,c). Impedance measurements of carcasses 1 to100 were recorded on three

consecutive days at the plant to determine the effect of measurement day on Tenderness

prediction ability. Impedance measurements of carcasses 101 to 300 were measured only

one day at the plant. SDSU personnel recorded hot carcass weight (HCW), longissimus

muscle area (REA), adjusted fat thickness, estimated percentage of kidney, pelvic and

heart fat (KPH), and USDA marbling score. Yield grades were calculated from adjusted

fat thickness, HCW, REA, and KPH. In addition, camera carcass data were collected

from the BeefCam and NIR computer systems. After carcass data were collected and

electrical impedance measured, a section of longissimus was excised, vacuum-packaged,

packed in coolers with ice, and transported back to SDSU Meat Lab. Upon arrival at

SDSU, the samples were stored under refrigeration until d 14 postmortem. On d 14, a

2.5-cm thick steak was removed from each sample. Electrical impedance was measured

and the steaks were cooked for Warner-Bratzler shear force analysis Electrical impedance

measurements on steaks were in a linear arrangement with penetrating depths of 1.3 cm

or 0 cm (Figure 2.1b,c).


Fresh longissimus samples were vacuumed packaged and stored at 4°C until

appropriate aging length (d 14). Samples were removed from vacuum packages and cut

into 2.5-cm-thick steaks. Steaks were cooked on a belt-fed impingement oven (Model

26

1132-000-A, Lincoln Foodservice Products, Inc., Fort Wayne, IN). Peak internal cooked

temperature measurements were recorded for each steak using a hand held thermometer

(model 39658-K, Atkins Technical, Gainesville, FL). Cooked steaks were cooled for 24

hours at 4°C before removing 6 to 8 cores (1.27 cm in diameter) parallel to the muscle

fiber orientation (AMSA, 1995). A single, peak shear force measurement was obtained

for each core using a Warner-Bratzler shear force machine (G-R Electric Manufacturing

Company, Manhattan, KS). Peak shear force values for each core were averaged to

obtain Warner-Bratzler shear force value for each steak.


Data were analyzed using the PROC REG procedure of SAS. Warner-Bratzler

shear force was predicted using the following regressor variables; carcass traits, camera

color, BeefCam, NIR, and electrical impedance using both simple linear regression

analysis (single regressor variable) and multiple linear regression analysis (all

combinations of regressor variables). Accuracy was calculated of each instrument to

certify carcasses as “not tough,” where error rate equals percent of those certified

carcasses that were, in fact, tough (WBS > 49.0 N). Accuracy was calculated for each

instrument to certify carcasses as “very tender,” where accuracy rate equals percent of

those certified carcasses that were, in fact, very tender (WBS < 34.3 N). These

accuracies were calculated for various certifications rates. Pearson’s correlation

coefficients were generated using the PROC CORR procedure of SAS to examine effect

of day on impedance measurements.

27

Phase III


Phase III was performed at the SDSU Meat Lab and utilized 14 steer carcasses of

Bos taurus (n=7) and Bos indicus (n=7) breed type. Samples were removed from the

longissimus muscle approximately 30 min after death and frozen for protein degradation

analysis. After splitting the carcass down the backbone, the right side of the carcass

(n=14) was hung from the Achilles tendon (normal suspension) and the left side of the

carcass (n=14) was hung from the aitch bone (hip suspension). The front and rear limbs

of the hip suspension carcasses were tied with a rope and pulled together until the two

limbs were parallel. Carcasses were then chilled for 24 hours at 4ºC.

Following carcass chilling, SDSU personnel recorded hot carcass weight (HCW),

longissimus muscle area (REA), adjusted fat thickness, estimated percentage of kidney,

pelvic and heart fat (KPH), and USDA marbling score. Yield grades were calculated

from adjusted fat thickness, HCW, REA, and KPH. A section of longissimus was

excised, cut into 2.5-cm thick steaks, vacuum-packaged, aged for 1, 4, 7, or 10 d

postmortem and then frozen. Warner-Bratzler shear force was measured at 1 and 10 d

postmortem. Sarcomere length was measured at 1, 4, 7, or 10 d postmortem. Rate of

proteolysis was evaluated by the degradation of troponin T (TnT), at 0, 1, 4, 7, and 10 d

postmortem (day 0 samples collected at death was used to quantify the amount of TnT

degradation that occurred from 1 to 10 d postmortem). Electrical impedance was

28

measured on carcasses at the exposed longissimus from 1 to 7 d postmortem, with either

penetrating (1.3 cm) or surface (0 cm) probes in a linear arrangement (Figure 2.1b,c).


Fresh longissimus samples were cut into 2.5-cm-thick steaks, vacuumed packaged

and stored at 4°C until appropriate aging length (d 1 or d 10) then frozen. Samples were

thawed 24 hours at 4°C and cooked on an electric clam shell grill (George Forman

Indoor/Outdoor Grill, Model GGR62, Lake Forest, IL). Peak internal cooked

temperature measurements were recorded for each steak using a hand held thermometer

(model 39658-K, Atkins Technical, Gainesville, FL). Cooked steaks were cooled for 24

hours at 4°C before removing 6 to 8 cores (1.27 cm in diameter) parallel to the muscle

fiber orientation (AMSA, 1995). A single, peak shear force measurement was obtained

for each core using a WBS force machine (G-R Electric Manufacturing Company,

Manhattan, KS). Peak shear force values for each core were averaged to obtain Warner-

Bratzler shear force value for each steak.

Myofibril Preparation

Upon completion of appropriate aging lengths (1, 4, 7, or 10 d postmortem),

myofibrils were purified according to a modified procedure of Swartz, Greaser, & Marsh,

(1993). Approximately 2 g of muscle were minced with a knife and homogenized in 35

ml of rigor buffer (RB, 75 mM KCl, 10 mM imidazole, 2 mM MgCl2, 2 mM EGTA, 1

mM NaN3, pH 7.2) for two 15 sec bursts using a Ultra-Turrax T25 homogenizer (Janke

& Kunkel GmbH & Co. KG.) at medium speed. The suspension was centrifuged at 1,000

29

x g for 10 min at 4˚C. Supernatant was decanted and the remaining pellet was

homogenized in 35 ml of RB for two 15 sec bursts at medium speed. The suspension was

centrifuged again at 1,000 x g for 10 min. Again the supernatant was decanted and the

remaining pellet was re-suspended by shaking in 35 ml of RM and centrifuged at 1,000 x

g for 10 min. Final myofibril pellets were re-suspended in 20 ml of RB plus 0.1 mM

phenylmethylsulfonyl fluoride and were ready for sarcomere length determination.

Sarcomere Determination

Purified myofibrils were transferred onto a microscope slide using centrifugation

(Cytofuge 2, Model M801-22, Westwood, MA). Samples were then incubated for 60

min at 37˚C with monoclonal anti-α-actinin (sarcomeric), (A7811 Sigma, St. Louis, MO)

diluted 1:5000 in RB. Samples were washed three times in RB (2 min per wash) and then

incubated with a donkey anti-mouse IgG, FITC (fluorescein isothiocyanate) conjugated

secondary antibody diluted 1:500 in RB for 60 min at 37̊ C. Samples were washed three

times in RB (2 min per wash). Samples were mounted with 30 µl mounting media (75

mM KCl, 10 mM Tris, pH 8.5, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3

, 1 mg/ml p-

phenylenediamine, 75% v/v glycerol). Sarcomere length was measured directly using a

microscope (Olympus AX70) equipped with a fluorescence filter (FITC) at 400X

magnification. Myofibril images were captured using Olympus DP71 camera and

sarcomere length was measured using Image Analysis Softeware (Image Pro-Plus 5.1).

The average of five sarcomeres was determined across 20 myofibrils per sample.

30

Whole Muscle Protein Extraction

Upon completion of appropriate aging lengths (0, 1, 4, 7, or 10 d postmortem),

sample proteins were extracted as described by Huff-Lonergan, Mitsuhashi, Parrish, &

Robson (1996) with slight modifications. Briefly, 0.2 g of muscle were minced and

added to 25 volumes of homogenizing solution (10 mM sodium phosphate, 10% w/v

volume SDS, pH 7.0). Samples were homogenized using a motor driven Dounce

homogenizer and clarified by centrifugation at 1,500 X g for 15 min at 4°C. Protein

concentration was determined using the RC/DC Protein assay (based on the Lowry assay)

(Bio-Rad Laboratories, Hercules, CA) and samples were diluted with water to a final

concentration of 2.5 mg/ml. One volume of each sample was added to five volumes of

sample buffer (3mM EDTA, 3% w/v SDS, 30% v/v glycerol, 0.003% w/v pyronin Y, 30

mM Tris-HCl pH 8.0) and 0.1 vol of 2-mercaptoethanol for a final protein concentration

of 1.56 mg/ml. Samples were immediately heated to 100°C for 5 min and stored at -

20°C.

Gel Electrophoresis and Transfer conditions

Muscle extracts were loaded on 15% polyacrylamide resolving gels (Ready Gel,

Tris-HCl gels; Bio-Rad Laboratories, Hercules, CA). Gels were run on the Bio-Rad

Criterion Cell system (Bio-Rad Laboratories, Hercules, CA) at constant 200 V for

approximately 90 min at 4°C. The running buffer used in both the upper and lower

chambers consisted of 25 mM Tris, 192 mM glycine, 2 mM EDTA, and 0.1% w/v SDS.

Following electrophoresis, gels were equilibrated for 30 min at room temperature in

31

transfer buffer (25 mM Tris, 192 mM glycine, and 15% v/v methanol). Gels were

transferred to polyvinylidene difluoride (PVDF) membranes at 4°C using a Criterion

blotter (Bio-Rad Laboratories, Hercules, CA) at a constant 90 V for 45 min.

Western blotting

Following transfer, membranes were blocked in Odyssey Blocking Buffer (OBB)

( Licor, Lincoln, NE) for 60 min at room temperature. Blots were incubated for 60 min at

room temperature with rabbit anti-Actin (20-33, Sigma, St. Louis, MO) and monoclonal

anti-TnT (JLT-12, Sigma, St. Louis, MO), diluted 1:5000 in OBB and Phosphate

buffered saline with Tween (PBST). Blots were washed four times with PBST (5 min per

wash) and then incubated with conjugated secondary antibodies, goat anti-mouse and

goat anti-rabbit (Licor, Lincoln, NE) diluted 1:25000 in PBST for 60 min at room

temperature. Blots were washed four times in PBST and bands were visualized using a

Licor Odyssey Software (Lincoln, NE). Immunoreactive TnT was identified and the

disappearance of intact TnT was quantified using Licor Odyssey scanner (Lincoln, NE).

To account for potential variation in protein loading, intensities of protein bands were

expressed relative to the total intensity of actin bands within the lane. The relative

intensity of bands was then normalized to the relative intensity of intact TnT at 0 h. The

percent degradation of the first three bands of TnT was calculated as percent TnT

degraded from day 0.

32

Total Collagen Content

Total collagen content was calculated from hydroxyproline quantification similar

to the methods by Bergman and Loxley (1961) and Hill (1966) with slight modifications.

Approximately, 2 g of powdered meat was placed into a 50-mL centrifuge tube and 12 ml

of ¼ strength Ringer’s solution was added. Tubes were heated for 63 min at 77°C in a

shaking water bath. Tubes were then centrifuged at 3000 x g for 5 min. After

centrifugation supernatant was transferred to a flat bottom flask for analysis of soluble

collagen. The residue in the tube was washed with 8 mL of Ringer’s solution and

centrifugation was performed again. After centrifugation supernatant was transferred to

the same flat bottom flask. Soluble collagen was then hydrolyzed in 20 mL of 12N HCl.

Pellet remaining in the tube was transferred using 40 mL of 6N HCL (four 10 mL rinses)

to the flat bottom flask for analysis of insoluble collagen. Flat bottom flasks were placed

under condensers and refluxed in a heated sand bath for 16 hours. After refluxing, pH of

the samples were adjusted to 6.2 with 12 M NaOH. Sample and 1 g of charcoal were

transferred to a 250 mL volumetric flask and brought up to volume with double-distilled

H20. After filtration, 1 mL of sample solution was pipetted into a 25 mL test tube. Two

mL of isopropanol were added, vortexed and then immediately 1 mL of oxidant solution

was added, vortexed and reacted for 4 min. Thirteen mL of Erhlich’s solution were

added to the tubes, vortexed and heated for 25 min at 60°C in a water bath. Tubes were

cooled for five min in cold water. Absorbance was read at 558 nm with a

spectrophotometer (Shimudzu UV 2101 PC). Hydroxyproline was converted to collagen

by multiplying by 7.25 (Goll et al., 1963). Values form soluble and insoluble collagen

33

were added together to obtain of total collagen. Data are expressed as percent of collagen

per gram of raw muscle.


Warner-Bratzler shear force, sarcomere length, troponin T degradation, total

collagen, resistance, reactance, and phase angle were analyzed using the PROC GLM

procedure of SAS, with animal as the experimental unit. Least squares means were

calculated and separated using the PDIFF option. Fixed main effects in the model

included breed (B), suspension (S), and day postmortem (D) and their interactions (B x S,

S x D, B x D, B x S x D). Pearson’s correlation coefficients were generated using the

PROC CORR procedure of SAS to examine relationship among dependent variables.

34

Results and Discussion

Phase I

Carcass Traits

Means and standard deviations for hot carcass weight (HCW), adjusted fat

thickness, ribeye area, calculated USDA yield grade, marbling score, and Warner-

Bratzler shear (WBS) are presented in Table 2.1. Carcasses sampled in phase I had 11 kg

higher HCW, 0.2 cm less adjusted fat thickness and 6.5 cm2

Electrical impedance measurements on the exposed longissimus using 5 cm

penetration depth on d 4 were more accurate at predicting WBS than measurements at

other anatomical locations or using other electrode designs on d 4 (Table 2.2). In

addition, impedance measures on steaks (d 5 & d 14) tended to be better predictors than

measures on carcasses (d 4). Impedance measures from tagged (carcass side that sample

was removed) and untagged sides (carcass side that sample was not removed) were

similar in their ability to predict WBS force, and using the average of two measurements

did not significantly improve accuracy compared to using only the tagged side (Table

larger ribeye areas when

compared to the average industry carcass (NBQA, 2005). Consequently, calculated

USDA yield grade was 0.2 lower than industry average, but marbling score was similar.

Overall, carcasses sampled were similar to the average industry carcass (NBQA, 2005).

Warner-Bratzler shear force decreased 13.5% from d 5 to d 14 postmortem.


35

2.3). Therefore, impedance could be measured on either side of the carcass without

altering impedance’s ability to predict beef tenderness. Day 4 impedance measurements

on the carcass were better at predicting d 14 WBS force than d 5 WBS force (Table 2.4).

In addition, d 5 impedance was better at predicting d 5 WBS force versus d 14 WBS

force, but d 14 impedance was similar at predicting both d 5 and d 14 WBS force.

Marbling was the poorest predictor of WBS (Table 2.5), explaining only 4% of

the variation in WBS force, similar to Wheeler, Cundiff, and Koch (1994) who reported

marbling explained, at most, 5% of the variation in palatability traits, but different from

results by Wulf and Page (2000) who concluded marbling score, by itself, explained 12%

of the variation in beef palatability. In addition, marbling added little to nothing when

combined with other predictors. Carcass traits, color, and impedance were similar in

their prediction capability, and when added together were quite additive suggesting that

each is explaining a different component of tenderness. Conclusion from phase I, was

that electrical impedance was able to predict beef tenderness.

Phase II

Carcass Traits


thickness, ribeye area, calculated USDA yield grade, marbling score, and Warner-

Bratzler shear (WBS) are presented in Table 2.6. Carcasses sampled in phase II had 13

kg higher HCW with similar adjusted fat thickness and 3.3 cm2 larger ribeye areas when

compared to the average industry carcass (NBQA, 2005). Calculated USDA yield grade

36

was similar, but marbling score was 33 points lower than industry average. Overall,

carcasses sampled were similar to the average industry carcass (NBQA, 2005).


By themselves, NIR, Impedance, and BeefCam explained 7%, 5%, and 3% of the

variation in 14 d WBS force, respectively (Table 2.7). When used together, NIR and

Impedance explained 12% of the variation. Because they were additive, it appears that

NIR and Impedance were measuring two different components of beef tenderness. In

order for an instrument to “eliminate the tough cattle”, it must be able to certify a large

percentage of the population while minimizing the percentage tough cattle within the

certified group. The 70 to 90% certification rates would be of the most interest in this

“eliminate-the-tough-cattle” scenario. The NIR system was the most effective instrument

at eliminating the tough carcasses (Table 2.8). Neither marbling nor impedance were

useful in trying to sort off the tough carcasses and certify a large percentage of the

population. An instrument could be used to select a premium group of exceptional beef,

if it were able to “identify the very tender cattle”. The 20 to 40% certification rates

would be of the most interest in this “identify-the-very-tender-cattle” scenario. The

Impedance system was the most effective instrument at identifying the very tender

carcasses (Table 2.9). The NIR system was not useful in trying to sort off the very tender

carcasses. Therefore, it appears that the reason that NIR and Impedance appeared

additive in Table 2.7 was because NIR was the most effective at identifying the tough

carcasses and Impedance was the most effective at identifying the very tender carcasses.

37

Another way to compare the technologies is to look at their effectiveness to sort

into tenderness groups. Table 2.10 shows average d 14 WBS force for each fifth of the

sample population sorted by predicted tenderness by each instrument. Each fifth contains

56 to 60 carcasses. The 1st fifth was predicted to be the most tender and the 5th


thickness, ribeye area, calculated USDA yield grade, marbling score, Warner-Bratzler

shear (WBS), and electrical impedance are presented in Table 2.12. Carcasses sampled

in phase III had 25 kg lower HCW, 0.4 cm less adjusted fat thickness with similar ribeye

areas when compared to the average industry carcass (NBQA, 2005). Consequently,

calculated USDA yield grade was 0.4 lower along with marbling score 67 points lower

than industry average. Overall, carcasses sampled were slightly smaller and leaner than

fifth was

predicted to be the least tender. Similar to the results in Tables 2.8 and 2.9, Impedance

was the most effective technology at identifying the most tender carcasses, whereas NIR

was the most effective technology at identifying the toughest carcasses.

Impedance measurements on d 1 postmortem were not highly correlated with

measurements on d 2 and d 3 postmortem (Table 2.11), however d 2 impedance

measurements were highly correlated with d 3 impedance measurements. Therefore,

impedance is a more accurate predictor of beef tenderness the more beef ages.

Phase III

Carcass Traits

38

the average industry carcass (NBQA, 2005). Warner-Bratzler shear force decreased

21.1% from d 1 to d 10 postmortem.

Warner-Bratzler Shear Force

As expected, longissimus WBS values were lower for Bos taurus (43.6 N) than

Bos indicus (57.0 N) (P < 0.01, Table 2.13) and similar to values reported by Crouse et

al. (1989) for beef longissimus at d 7 postmortem. Additionally, HS resulted in lower

WBS values (46.4 N) than NS (54.3 N) (P < 0.0001, Table 2.13). Previous research by

Herring et al. (1965) and Hostetler et al. (1972), reported longissimus samples with

longer sarcomeres via hip suspension resulted in lower shear force values. Mean WBS

values decreased significantly (P < 0.0001) over the aging period from 56.9 N at d 1 to

43.7 N by d 10 postmortem (Table 2.14), indicating a significant improvement in

tenderness. There was a significant breed x day interaction for WBS (P < 0.05, Table

2.14). At d 10 postmortem WBS values were lower in both Bos taurus and Bos indicus

than at d 1 postmortem, but Bos taurus decreased WBS to a greater extent than Bos

indicus (P < 0.05).

Sarcomere Length

Representative micrographs of myofibrils with normal (1.82 µm) and hip

suspension (2.43 µm) sarcomeres are presented in Figure 2.4. Sarcomere length was

longer for Bos taurus (2.19 µm) than for Bos indicus (2.06 µm) (P < 0.05, Table 2.14),

which is different from previous research by Whipple et al., (1990) and Stolowski et al.,

(2006) who found no difference in sarcomere length between Bos taurus and Bos indicus.

39

Longer sarcomeres found in Bos taurus was a result of hip suspension as there was no

difference in carcasses from normal suspension. As expected, HS resulted in longer

sarcomeres than NS (P < 0.0001, Table 2.13) and is in agreement with previous research

by Herring et al. (1965) and Hostetler et al. (1972). There was a significant breed x

suspension interaction for sarcomere length (P < 0.001, Table 2.13). Hip suspension

increased sarcomere length of both Bos taurus and Bos indicus, but HS improved

sarcomere length of Bos taurus to a greater extent than Bos indicus (P < 0.001). This

interaction could be a result of different skeletal structure found between the two species

resulting in longer sarcomeres in the HS Bos taurus.

Postmortem Proteolysis

A representative Western blot of whole muscle protein extracts from bovine

longissimus with normal and hip suspension is presented in Figure 2.5. Degradation of

troponin T was utilized in this study to measure the rate of postmortem proteolysis.

Troponin T is a regulatory protein not typically involved in the tenderization of meat, but

has been considered a good marker for proteolysis (Negishi, Yamamoto, and Kuwata,

1996; Penny & Dransfiled, 1979). Bos taurus had more breakdown of troponin T than

Bos indicus (P > 0.05, Table 2.13) and is agreement with past research by O’Connor et

al., (1997) that reported Bos indicus cattle have an increased level of calpastatin and

therefore aged at slower rate than Bos taurus. Hip suspension resulted in more

breakdown of troponin T than NS, (P < 0.0001, Table 2.13) and confirmed past research

by Weaver, Bowker, and Gerrard, (2008) that concluded troponin T proteolysis is

40

sarcomere length-dependent and an increase in sarcomere length resulted in more rapid

protein degradation. As expected, there was increased degradation of intact troponin T

during aging from d 1 to d 10 postmortem (P < 0.0001, Table 2.14).

Collagen Content

Collagen (soluble, insoluble, and total) was measured to determine the amount of

connective tissue in each longissimus sample. The was no difference between Bos taurus

and Bos indicus for collagen values (Table 2.13). Therefore, breed type had no effect on

the amount of connective tissue found in the longissimus muscle in this study.


Electrical impedance values, reactance and phase angle, were lower for Bos

taurus than Bos indicus (P < 0.05, Table 2.13), indicating reactance and phase angle

could be related to the level of calpastatin. Hip suspension resulted in increased

resistance values than NS (P < 0.0001, Table 2.13), indicating that resistance was

dependent upon sarcomere length. Hip suspension resulted in lower phase angle values

than NS (P < 0.0001, Table 2.13), possibly because an increase in sarcomere length

resulted in a weakened structure. The breed x suspension interaction was significant for

resistance (P < 0.001, Table 2.13). Hip suspension resulted in higher resistance values

than NS, but Bos indicus resulted in a greater increase in resistance values than Bos

taurus, (P < 0.001). The breed x suspension interaction was significant for reactance (P

< 0.0001, Table 2.13). Hip suspension had lower reactance values than NS for Bos

taurus, but HS had higher reactance values than NS for Bos indicus, (P < 0.0001). The

41

breed x suspension interaction was significant for phase angle (P < 0.0001, Table 2.13).

Hip suspension resulted in lower phase angle values than NS for Bos taurus, but no

difference was found in phase angle for Bos indicus for suspension method, indicating

sarcomere length did not influence phase angle values of Bos indicus cattle (P < 0.0001).

Phase angle decreased from d 1 to d 10 postmortem (P < 0.01, Figure 2.2), indicating

phase angle may be inversely related to postmortem proteolysis. Previous work by

Lepetit et al., (2002) and Damez, Clerjon, Abouelkaram, and Lepetit, (2007) reported a

decrease in electrical impedance during aging and suggested electrical impedance could

potentially evaluate meat aging. In addition, Byrne et al., (2000) reported electrical

measurements changed significantly between d 1 and d 14 postmortem and were

significantly correlated to WBS.

Correlations

Correlations are presented in Table 2.15. Sarcomere length was negatively

related to WBS (r = -0.38), indicating longer sarcomeres resulted in a lower WBS values.

In addition, sarcomere length was related to total collagen (r = 0.57), indicating longer

sarcomeres had more connective tissue. Postmortem proteolysis was inversely related to

WBS (r = -0.59), indicating proteolysis of myofibril proteins is the reason meat

tenderizes during postmortem aging. Reactance and sarcomere length were negatively

correlated (r = -0.24), indicating increased sarcomere length resulted in greater

breakdown of membranes. Phase angle was negatively correlated to sarcomere length (r

= -0.38), and postmortem proteolysis (r = -0.25), confirming phase angle is inversely

42

related to postmortem proteolysis. In addition, phase angle was related to WBS (r =

0.51), indicating phase angle can predict beef tenderness.

43

Implications

In conclusion, electrical impedance is weakly related to beef tenderness, but may

be useful to sort out very tender beef carcasses. Phase angle was inversely related to

extent of postmortem proteolysis. Therefore, electrical impedance may be useful to

predict beef aging.

44

Table 2.1. Simple statistics for carcass and muscle traits for Phase I

Trait n Mean SD Minimum Maximum Hot carcass weight, kg 92 371 39 203 462 Adjusted fat thickness, cm 92 1.10 0.48 0.30 2.54 Ribeye area, cm 92 2 92.9 12.9 46.5 124.5 Calculated USDA yield grade 92 2.7 0.9 0.8 4.4 Marbling score 92 a 431 115 250 960 Warner-Bratzler shear, N Longissimus, d 5 postmortem 92 43.1 10.8 19.6 83.4 Longissimus, d 14 postmortem 92 37.3 8.2 14.7 66.7 a300 = "Slight00," 400 = "Small00," 500 = "Modest00," 600 = "Moderate00 ."

45

Table 2.2. The ability of electrical impedance measurements from various electrode depth and placement combinations to predict shear force (average of d4 and d14) Measurement R-square R-square

(impedance only) (with other carcass traits)None 0.00 0.34Carcass Measurements (day 4) Ribeye 5 cm puncturea 0.14 0.42 Ribeye 1.3 cm puncturea 0.06 0.40 Ribeye surfacea 0.08 0.38 Strip loin 5 cm punctureb 0.05 0.37 Strip to round 5 cm puncturec 0.07 0.38Steak Measurements (day 5) Ribeye 1.3 cm puncturea 0.19 0.45 Ribeye surfacea 0.20 0.45Steak Measurements (day 14) Ribeye 1.3 cm puncturea 0.28 0.49 Ribeye surfacea 0.27 0.48

aElectrodes in a linear arrangement with 5 cm between each electrode bElectrodes in a linear arrangement with 5 cm between detection and electrical source over a 50 cm field cElectrodes placed in the strip loin and round with 5 cm between detection and electrical source

46

Table 2.3. Evaluating the ability of electrical impedance measurements from tagged (carcass side sample was removed) and untagged (carcass side sample was not removed) sides to predict shear force (average of d5 and d14) Measurement R-square R-square

(impedance only) (with other carcass traits)None 0.00 0.34Ribeye 5 cm puncturea

Tagged side 0.14 0.42 Untagged side 0.12 0.41 Average of both 0.15 0.42Ribeye 1.3 cm puncturea

Tagged side 0.06 0.40 Untagged side 0.05 0.37 Average of both 0.05 0.39Ribeye surfacea

Tagged side 0.08 0.38 Untagged side 0.08 0.36 Average of both 0.11 0.37

aElectrodes in a linear arrangement with 5 cm between each electrode

47

Table 2.4. Predicting d5 and d14 shear force using electrical impedance measurements from d4, d5, and d14

Measurement R-square R-square R-square R-square (impedance only)(with other traits) (impedance only)(with other traits)

None 0.00 0.34 0.00 0.30Day 4 Ribeye 5 cm puncturea 0.10 0.39 0.15 0.38 Ribeye 1.3 cm puncturea 0.03 0.39 0.08 0.35 Ribeye surfacea 0.07 0.38 0.10 0.34Day 5 Ribeye 1.3 cm puncturea 0.23 0.45 0.11 0.37 Ribeye surfacea 0.22 0.44 0.13 0.38Day 14 Ribeye 1.3 cm puncturea 0.26 0.44 0.25 0.47 Ribeye surfacea 0.22 0.42 0.26 0.47

d 5 WBS force d 14 WBS force

aElectrodes in a linear arrangement with 5 cm between each electrode

48

Table 2.5. R-squares for various prediction models to predict shear force (d14) Variables used in the prediction model R-squareMarbling 0.04Carcass traits (other than marbling) 0.19Color (from camera) 0.14Impedance (ribeye 5 cm puncture – day 4) 0.15Marbling + Carcass traits 0.22Marbling + Color 0.15Marbling + Impedance 0.15Carcass traits + Color 0.28Carcass traits + Impedance 0.26Color + Impedance 0.29Marbling + Carcass traits + Color 0.30Marbling + Carcass traits + Impedance 0.27Marbling + Color + Impedance 0.29Carcass traits + Color + Impedance 0.37Marbling + Carcass traits + Color + Impedance 0.38

49

Table 2.6. Simple statistics for carcass and muscle traits for Phase II Trait n Mean SD Minimum Maximum Hot carcass weight, kg 300 373 33 269 476

Adjusted fat thickness, cm 300 1.27 0.42 0.41 3.56 Ribeye area, cm 300 2 89.7 9.7 69.0 131.0 Calculated USDA yield grade 300 3.0 0.7 1.0 5.9 Marbling score 300 a 399 61 290 640 Warner-Bratzler shear, N Longissimus, d 14 postmortem 300 33.3 7.8 20.6 71.6 a300 = "Slight00," 400 = "Small00," 500 = "Modest00," 600 = "Moderate00."

50

Table 2.7. R-squares for various prediction models to predict shear force (d14)

Prediction Model R-square SDSU Carcass Data 0.07 Camera Carcass Data 0.06 Camera Color Data 0.05 BeefCam 0.03 NIR 0.07 Impedance 0.05 14-d Impedance 0.16 CamCarc + CamColor 0.11 CamCarc + BeefCam 0.07 CamCarc + NIR 0.13 CamCarc + Impedance 0.12 CamColor + BeefCam 0.05 CamColor + NIR 0.09 CamColor + Impedance 0.11 BeefCam + NIR 0.10 BeefCam + Impedance 0.07 NIR + Impedance 0.12 CamCarc + CamColor + BeefCam 0.11 CamCarc + CamColor + NIR 0.15 CamCarc + CamColor + Impedance 0.17 CamCarc + BeefCam + NIR 0.14 CamCarc + BeefCam + Impedance 0.12 CamCarc + NIR + Impedence 0.18 CamColor + BeefCam + NIR 0.11 CamColor + BeefCam + Impedance 0.12 CamColor + NIR + Impedance 0.16 BeefCam + NIR + Impedance 0.15 CamCarc + CamColor + BeefCam + NIR 0.16 CamCarc + CamColor + BeefCam + Impedance 0.17 CamCarc + CamColor + NIR + Impedance 0.21 CamCarc + BeefCam + NIR + Impedance 0.19 CamColor + BeefCam + NIR + Impedance 0.17 CamCarc + CamColor + BeefCam + NIR + Impedance 0.21

51

Table 2.8. Percentage of tough (>49.0 N) carcasses by certification rate and instrument Certification Rate, % Marbling NIR BeefCam Impedance No System Perfect System Impedance d14

10 3.3 0.0 0.0 0.0 4.3 0.0 0.020 3.3 0.0 0.0 0.0 4.3 0.0 0.030 2.2 0.0 2.4 1.1 4.3 0.0 0.040 1.7 0.0 1.8 1.7 4.3 0.0 1.750 4.0 0.7 1.4 2.7 4.3 0.0 1.360 4.4 1.7 1.8 4.4 4.3 0.0 1.770 3.8 2.4 2.5 4.8 4.3 0.0 1.480 4.6 2.1 4.0 4.2 4.3 0.0 2.990 4.8 3.4 3.6 4.4 4.3 0.0 3.0

100 4.3 4.4 4.6 4.3 4.3 4.3 4.3n 300 295 281 300 300 300 300

52 Table 2.9. Percentage of very tender (<34.3 N) carcasses by certification rate and instrument

Certification Rate, % Marbling NIR BeefCam Impedance No System Perfect System Impedance d1410 46.7 36.7 42.9 46.7 30.3 100.0 43.320 43.3 33.9 46.4 48.3 30.3 100.0 43.330 40.0 33.7 40.5 46.7 30.3 100.0 37.840 37.5 35.6 42.0 45.0 30.3 75.8 35.050 36.7 33.1 38.0 41.3 30.3 60.7 35.360 33.3 33.3 38.5 38.3 30.3 50.6 35.070 33.3 32.9 36.5 35.7 30.3 43.3 33.380 30.8 32.2 34.2 33.3 30.3 37.9 32.590 30.7 31.6 32.8 32.6 30.3 33.7 33.0100 30.3 30.5 31.7 30.3 30.3 30.3 30.3

n 300 295 281 300 300 300 300

53

Table 2.10. Average Warner-Bratzler shear force (N) for sort groups (fifths) by instrumentSort Group Marbling NIR BeefCam Impedance No System Perfect System Impedance d14

1st Fifth 31.5 31.7 31.5 30.2 33.4 24.8 30.82nd Fifth 32.9 32.0 32.5 32.2 33.4 29.1 33.23rd Fifth 35.0 33.7 32.8 35.2 33.4 32.1 32.14th Fifth 35.1 33.0 35.7 34.7 33.4 36.1 34.15th Fifth 32.9 37.1 35.2 35.2 33.4 45.2 37.2

n 300 295 281 300 300 300 300

54

Table 2.11. Effect of day postmortem on impedance measurements (n = 100)R Xc PA

MeansDay 1 47.2 31.1 33.3Day 2 46.1 30.6 33.5Day 3 42.6 27.1 32.3

CorrelationsDay 1&2 0.67 0.56 0.74Day 1&3 0.76 0.59 0.68Day 2&3 0.88 0.86 0.87

Correlation with WBSDay 1 -0.20 -0.18 -0.03Day 2 -0.05 0.03 0.09Day 3 -0.08 0.06 0.15

55

Table 2.12. Simple statistics for carcass and muscle traits for Phase III

Trait n Mean SD Minimum Maximum

Hot carcass weight, kg 14 335 21 303 384 Adjusted fat thickness, cm 14 0.91 0.50 0.20 1.83 Ribeye area, cm 14 2 83.0 8.5 67.7 99.4 Calculated USDA yield grade 14 2.5 0.7 1.5 4.2 Marbling score 14 a 365 89 280 620 Warner-Bratzler shear, N Longissimus, d 1 postmortem 28 56.9 9.8 40.2 78.5 Longissimus, d 10 postmortem 28 44.1 11.8 26.5 9.6 Sarcomere length, µm 112 2.12 0.36 1.64 3.01 Troponin T degradation, % 112 44.0 22.0 86.0 11.0 Total collagen, % 14 0.39 0.04 0.31 0.46 Electrical impedance Resistance 196 62.64 6.98 49.00 80.00 Reactance 196 37.83 7.65 21.00 63.00 Phase Angle 196 30.94 4.44 18.69 38.93 a300 = "Slight00," 400 = "Small00," 500 = "Modest00," 600 = "Moderate00."

56 Table 2.13. Least square means of WBS force, sarcomere length, troponin T degradation, collagen, resistance, reactance, and phase angle (averaged across all aging periods)

Breed Main Effects Suspension Main Effects Breed x Suspension Interaction

TA IN Pooled

TA IN SEM P < F NS HS SEM P < F NS HS NS HS SEM P < F

WBS, N 43.6 57.0 2.20 0.0010 54.3 46.4 1.10 <0.0001 47.5 39.7 61.1 53.0 1.70 0.9131

Sarcomere length, µm 2.19 2.06 0.03 0.0155 1.82 2.43 0.02 <0.0001 1.83 2.55a 1.81c 2.30a 0.03 b 0.0009

Troponin T degradation, % 46.0 42.0 1.00 0.0350 42.0 47.0 1.00 <0.0001 43.0 48.0 41.0 47.0 1.00 0.6090

Soluble Collagen, % 0.04 0.04 0.01 0.5091 - - - - - - - - - -

Insoluble Collagen, % 0.35 0.34 0.01 0.4368 - - - - - - - - - -

Total Collagen, % 0.39 0.38 0.02 0.8077 - - - - - - - - - -

Resistance 61.16 64.12 2.15 0.3498 60.59 64.69 0.38 <0.0001 60.12 62.20a 61.06b 67.18ab 0.54 c 0.0003

Reactance 33.82 41.84 1.29 0.0009 38.03 37.62 0.58 0.6190 35.90 31.73b 40.16a 43.51c 0.43 d <0.0001

Phase Angle 28.81 33.08 0.87 0.0047 31.96 29.92 0.30 <0.0001 30.69 26.92b 33.23a 32.93c 0.43 c <0.0001

a,b,c Means for the breed x suspension interaction within a row lacking a common superscript letter differ (P< 0.05).

57 Table 2.14. Effect of day shear force, sarcomere length, and troponin T degradation

Day Main Effects Breed x Day Interactions

TA IN Pooled

1 4 7 10 SEM P < F 1 4 7 10 1 4 7 10 SEM P < F

WBS, N 56.9 - - 43.7 1.10 <0.0001 52.1 - f - 35.2 61.7e - g - 52.4 1.60 f 0.0242

Sarcomere length, µm 2.13 2.14 2.11 2.12 0.03 0.8985 2.20 2.21 2.18 2.18 2.07 2.07 2.04 2.06 0.05 0.9941

Troponin T degradation, % 18.0 34.0d 51.0c 75.0b 1.00 a <0.0001 19.0 36.0 54.0 76.0 16.0 31.0 49.0 73.0 1.00 0.6912 a,b,c,d Means within a row lacking a common superscript letter differ (P< 0.05). e,f,g Means for the breed x day interaction within a row lacking a common superscript letter differ (P< 0.05).

58

Table 2.15. Correlations of Warner-Bratzler shear force, sarcomere length, proteolysis, collagen, resistance, reactance, and phase angle (* P < 0.05)

Variable SL a PROT SC IC TC R Xc PA

WBS -0.38* -0.59* -0.25 -0.25 -0.24 -0.12 0.34 0.51*

SL - 0.10 0.31 0.64* 0.57* 0.13 -0.24* 0.38*

PROT - - -0.17 0.07 -0.30 0.14 -0.14 0.25*

SC - - - 0.58* 0.83* 0.24 0.08 -0.80

IC - - - - 0.93* 0.40 -0.14 -0.44

TC - - - - - 0.37 -0.06 -0.33

R - - - - - - 0.49* -0.08

Xc - - - - - - - 0.83*

a

R = Resistance, Xc = Reactance, PA = Phase Angle

WBS = Warner-Bratzler shear, SL= Sarcomere Length, PROT = Troponin T degradation, %, SC= Soluble Collagen, %, IC=Insoluble Collagen, %, TC= Total Collagen, %,

59

Figure 2.1. Electrode arrangement and penetrating depths.

a)

b)

c)

2.5 cm 5.0 cm 2.5 cm

2.5 cm 5.0 cm 2.5 cm

2.5 cm 5.0 cm 2.5 cm

5.0 cm

1.3 cm

0 cm

60

Figure 2.2. The effect of breed type on resistance, reactance, and phase angle on the exposed longissimus muscle from d 1 to d 7 postmortem (*P < 0.05)

61

Figure 2.3. The effect of suspension on resistance, reactance, and phase angle on the exposed longissimus muscle from d 1 to d 7 postmortem (*P < 0.05)

62

Figure 2.4. Representative micrographs of myofibrils with normal (1.82 µm, left) and hip suspension (2.43 µm, right) sarcomeres of longissimus muscle (Magnification 400X)

63

Figure 2.5. Representative western blot of whole muscle protein extracts from bovine longissimus muscle with normal (NS) and hip (HS) suspension and postmortem aging periods were 1, 4, 7, and 10 days (d0 was used for quantification)

NS HS . 0 1 4 7 10 1 4 7 10 Band

} 1-3 4 & 5

6

7

64

Literature Cited

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Appendix A. The effect of breed type on resistance, reactance, and phase angle impedance measurements of longissimus lumborum steaks from d 1 to d 21 postmortem

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Appendix B. The effect of suspension on resistance, reactance, and phase angle impedance measurements of longissimus lumborum steaks from d 1 to d 21 postmortem

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Appendix C. The effect of breed type on resistance, reactance, and phase angle impedance measurements of semitendinosus steaks from d 1 to d 10 postmortem

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Appendix D. The effect of suspension on resistance, reactance, and phase angle impedance measurements of semitendinosus steaks from d 1 to d 10 postmortem

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Appendix E. The effect of breed type on resistance, reactance, and phase angle impedance measurements of psoas major steaks from d 1 to d 10 postmortem

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Appendix F. The effect of suspension on resistance, reactance, and phase angle impedance measurements of psoas major steaks from d 1 to d 10 postmortem

Tim Thesis

Education

flat bottom

proc corr

adjusted fat

adjusted fat

hot carcass

predict shear

bratzler shear

bratzler shear