Effects of pre-slaughter administration of oral calcium gel to beef cattle on tenderness

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1 Effects of pre-slaughter administration of oral calcium gel to beef cattle on tenderness S. K. Duckett 1,3, J. G. Andrae 1, G. T. Pritchard 1, T. A. Skow 1, S. L. Cuvala 1, J. H. Thorngate 2, and W. K. Sanchez 1 1 Department of Animal and Veterinary Science and 2 Department of Food Science and Toxicology, University of Idaho, Moscow, ID Idaho Agricultural Experiment Station Number 98A18, received 28 February 2000, accepted 11 September Can. J. Anim. Sci. Downloaded from by on 02/06/18 Duckett, S. K., Andrae, J. G., Pritchard, G. T., Skow, T. A., Cuvala, S. L., Thorngate, J. H. and Sanchez, W. K Effects of pre-slaughter administration of oral calcium gel to beef cattle on tenderness. Can. J. Anim. Sci. 81: The effects of preharvest administration of oral calcium gel to beef cattle on longissimus ph, calpain activity, and tenderness were examined. Thirty steers (539 kg) were randomly assigned to one of the following treatments: control (CON, n = 15) and calcium gel administered (CA, n = 15). At 3 to 6 h prior to slaughter, the calcium gel steers were dosed via a rumen tube with calcium propionate (150 g of calcium and 630 g propionate) and propylene glycol (600 g) in a liquid suspension. Calcium gel administration prior to slaughter increased (P < 0.05) µ-calpain and m-calpain activity (caseinolytic activity per gram of muscle) with no change (P > 0.05) in calpastatin activity. Total mineral (%) and calcium (µg g 1 ) contents of the longissimus muscle were greater (P < 0.05) for CA than CON. Steaks from CA steers had Warner-Bratzler shear force (WBS) values lower (P < 0.05) than CON after 4 and 7 d of aging. No differences (P > 0.05) were noted in WBS values at days 2, 14, or 28 of aging between the treatments. Sensory panel ratings were higher (P < 0.05) for tenderness of CA steaks than CON after aging for 7 d, and juiciness and flavor ratings were similar (P > 0.05). Pre-harvest calcium gel administration elevated longissimus muscle calcium content, increased calpain activity, and accelerated postmortem aging to improve tenderness. Key words: Beef, calcium, tenderness, calpain Duckett, S. K., Andrae, J. G., Pritchard, G. T., Skow, T. A., Cuvala, S. L., Thorngate, J. H. et Sanchez, W. K L administration d un gel de calcium avant l abattage et ses effets sur la tendreté de la viande des bovins de boucherie. Can. J. Anim. Sci. 81: Les auteurs se sont intéressés à l administration d un gel de calcium aux bovins de boucherie par voie orale, avant l abattage, et à ses effets sur le ph du longissimus, sur l activité de la calpaïne et sur la tendreté de la viande. Trente bouvillons (539 kg) ont été répartis au hasard entre les traitements que voici : témoins (TÉ; n = 15) et administration du gel de calcium (CA; n = 15). De 3 à 6 h avant l abattage, on a administré une suspension liquide de propionate de calcium (150 g de calcium et 630 g de propionate) et de propylège glycol (600 g) aux bouvillons du groupe CA au moyen d une sonde enfoncée dans le rumen. L administration de gel de calcium avant l abattage augmente (P < 0,05) l activité de la µ-calpaïne et de la m-calpaïne (activité caséinolytique par g de muscle) sans modifier (P > 0,05) l activité de la calpastatine. La concentration totale de minéraux (%) et de calcium (µg g 1 ) dans le longissimus était plus importante (P < 0,05) pour chez les bouvillons CA que chez les témoins. Les biftecks des bouvillons CA présentaient une résistance au cisaillement Warner-Bratzler (RCWB) inférieure (P < 0.05) à celle relevée chez les bouvillons TÉ après 4 et 7 j de maturation. La valeur de la RCWB des deux groupes ne présentait pas d écart (P > 0,05) après 2, 14 ou 28 j de maturation. Selon la cote organoleptique les biftecks CA étaient plus tendre (P < 0,05) que les biftecks TÉ après 7 j de maturation, mais aussi juteux et savoureux (P > 0,05). L administration de gel de calcium avant l abattage augmente la teneur en calcium du longissimus, accroît l activité de la calpaïne et accélère la maturation post mortem de la viande, ce qui en améliore la tendreté. Tenderness of beef improves with postmortem aging due in part to the activity of the calpain proteolytic system (Taylor et al. 1995). The calpain proteolytic system consists of µ-calpain and m-calpain, and calpastatin, their endogenous inhibitor. Both µ-calpain and m-calpain are calcium-dependent proteases that require calcium at 5 50 and µm, respectively, for half-maximal activity (Goll 1991). Skeletal muscle has a relatively low concentration of calcium (1.35 µm; Lawrie 1998) Mots clés: Bœuf, calcium, tendreté, calpaïne indicating that the activity of these proteases may be limited. Increasing muscle calcium concentration through postmortem infusion (Koohmaraie et al. 1990) or injection (Wheeler et al. 1992) of calcium chloride solution accelerates the aging process and reduces the length of postmortem aging. However, postmortem injection of CaCl 2 results in increased surface discoloration (Wheeler et al. 1996) and promotes off-flavors at high concentrations (Morgan et al. 1991; Wheeler et al. 1993). 3 To whom correspondence should be addressed: University of Georgia, Animal and Dairy Science Complex, Athens, GA , USA ( sduckett@arches.uga.edu). 33 Abbreviations: CA, calcium gel administered; CON, control; WBS, Warner-Bratzler shear force

2 Can. J. Anim. Sci. Downloaded from by on 02/06/18 34 CANADIAN JOURNAL OF ANIMAL SCIENCE Dairy producers routinely administer oral calcium propionate/propylene glycol gels to increase blood calcium levels in cows with milk fever. Ionized blood calcium levels are increased (P < 0.05) by 24 to 29% 30 min after administration of calcium propionate or calcium chloride to non-lactating Holstein cows (Goff and Horst 1993; Sanchez et al. 1997). No information is available on the effect of altering blood calcium levels with calcium gel administration on changes in skeletal muscle calcium levels. These calcium products may provide the beef industry with a pre-slaughter application to increase muscle calcium and accelerate postmortem aging. The objectives of this research were: 1) to determine if oral administration of a calcium propionate gel prior to slaughter improves meat tenderness and 2) to assess the effect of calcium propionate administration on muscle calcium levels, calpain activity, and postmortem aging. MATERIALS AND METHODS Animals The University of Idaho Animal Care and Use Committee approved the experimental protocols and the use of animals in this study. Thirty crossbred steers (539 kg; Hereford- Simmental-Angus Salers; 18 mo of age) of similar background were obtained from a feedlot (80 d on finishing ration), transported to the University of Idaho (74 km), penned together, and fed a finishing diet (85% barley and 15% small grain silage, 12% crude protein; ad libitum) for 14 d prior to the start of the trial. Steers were randomly assigned to one of the following treatments: control (CON) or calcium gel [CA; 1.5 L of Dr. Larson s Up and Over 1000, which contained 150 g of calcium, 630 g propionate, and 600 g propylene glycol; Mark Anderson Inc. (MAi), Spring Valley, WI]. Meat from the calcium gel treated animals used in this study was approved for human food use by Food and Drug Administration (Investigational New Animal Drug 10213). Six steers (3 = CON; 3 = CA) were slaughtered each day, once per week for a 5-wk period in October and November (avg. ambient temperature = 7 C). Twenty-four hours before each group was slaughtered, they were removed from the common group and held without feed but had access to water. On the morning of slaughter, all six steers were moved through a processing facility and restrained for approximately 3 min. The CA steers were given the calcium gel via a tube inserted into the rumen. The steers were allowed a 3-h rest period and then transported (< 100 m) to the holding pens at the meats laboratory. The steers were randomly moved into the University of Idaho Meats Laboratory and slaughtered alternately by treatment (slaughter room temperature = 21 C). Because of the limitation of slaughter facility capacity, there was a range of 3 to 6 h between administration of calcium gel and slaughter. One control steer was removed from the study after slaughter due to the exhibition of dark, firm and dry condition as verified by a longissimus muscle ph of 6.3. Hot carcass weight was obtained for each carcass before chilling at 0 C. Calpain/Calpastatin Activity About 45 min postmortem (after completion of the slaughter process and before chilling), a 10-g sample of pre-rigor longissimus muscle from the right side was obtained for determination of µ- and m-calpain and calpastatin activities (Koohmaraie 1990) using ion exchange chromatography. Calpain activity is reported as the caseinolytic activity that increases absorbance one unit at 278 nm after a 60 min incubation at 25 C. One unit of calpastatin activity is defined as the amount that inhibited one unit of m-calpain activity. Carcass Data/Sample Collection At 45 min and 48 h postmortem, ph of the right longissimus muscle at the 3rd lumbar vertebra was determined using a spear tip electrode (Model 8163BN, Orion, Beverly, MA) connected to a portable ph meter (Model , Cole- Parmer, Vernon Hills, IL). Carcasses will typically reach a temperature of 4 C in h after slaughter in our current chilling facility at the University of Idaho. At 48 h postmortem, fat thickness, kidney, pelvic and heart fat, ribeye area, marbling score (US Department of Agriculture 1997), and yield grade (US Department of Agriculture 1997) were measured on each carcass and recorded. Eleven steaks (seven at 2.54 cm thick; four at 1.27 cm thick) were removed from the right longissimus between the first and sixth lumbar vertebrae for determination of moisture, ash and total calcium content (Association of Official Analytical Chemists 1990), Warner-Bratzler shear force (American Meat Science Association 1995) at five postmortem aging times (2, 4, 7, 14, and 28 d), sensory panel analysis at 7 d postmortem, and postmortem proteolysis of troponin-t (1.27 cm thick) at four postmortem aging times (2, 4, 7, 14 d). Steaks were trimmed of subcutaneous fat and bone, individually vacuum packaged, randomly assigned to the aging treatments, held at 2 C for their appropriate age, and frozen at 20 C for subsequent measurements. Warner-Bratzler Shear Force Steaks (2.54 cm thick) were thawed for 18 h at 2 C and then broiled on Farberware Open Hearth Grills (Model R4550; Farberware, Bronx, NY) to an internal temperature of 71 C (American Meat Science Association 1995) as monitored using copper constantan thermocouples and a Digi-Sense temperature logger (Cole Parmer, Niles, IL). After cooking, the steaks were cooled at 22 C for 4 h and six 1.27 cm cores were removed parallel to the muscle fiber with a mechanical coring device. Cores were sheared using a Texture Analyzer (TA-XT2; Texture Technologies Corp., Scarsdale, NY) equipped with a Warner-Bratzler knife, and peak shear force recorded. Crosshead speed was set at 20 cm min 1. Sensory Panel An eight-person sensory panel (five males, three females, aged 20 to 45 yr, students and faculty from the University of Idaho) was trained according to American Meat Science Association Guidelines (American Meat Science Association 1995). Loin steaks (2.54 cm thick; aged for 7 d) were cooked to an internal temperature of 71 C, cut into 1-cm 1-cm 2.54-cm cubes using a plexiglass grid (14 cm long 12 cm wide 4 cm deep, with slots spaced 1.25 cm apart) and served immediately to each sensory panel member. Sensory panelists marked a 10-cm line scale for tenderness, juiciness,

3 DUCKETT ET AL. PRE-SLAUGHTER CALCIUM AND TENDERNESS 35 Can. J. Anim. Sci. Downloaded from by on 02/06/18 and flavor for each sample (1 = extremely tough, dry, bland; 10 = extremely tender, juicy, or intense). Postmortem Proteolysis Myofibrils from longissimus of five animals per treatment aged at 2 C for 2, 4, 7, and 14 d were purified at 2 C according to the myofibril preparation purification of Huff-Lonergan et al. (1994). Protein concentrations were determined using a commercial bicinchoninic acid procedure (Pierce, Rockford, IL). The purified myofibrils were diluted to 1.33 mg ml 1 and three volumes of each sample were immediately combined with one volume of 25 C sample buffer/tracking dye solution. Samples were heated at 50 C for 20 min before loading onto polyacrylamide gels. A 12% polyacrylamide [acrylamide/bisacrylamide = 37.5:1 (wt/wt)] slab separating gel with a 5% polyacrylamide stacking gel was used to examine changes in troponin-t. Fifty micrograms of protein from the myofibril preparation were loaded into each lane of the gel. Gels (14 cm 16 cm 1.5 mm thick) were run in a Hoefer SE 600 Standard Dual Cooled Gel Electrophoresis Unit (Amersham Pharmacia Biotech, San Francisco, CA) in duplicate at a constant voltage setting of 60 V for h. Gels used for transfer were equilibrated for 15 min. at 22 C in a transfer buffer containing 25 mm Tris, 192 mm glycine, 2 mm EDTA, 15% (vol/vol) methanol and 0.1% (wt/vol) sodium dodecyl sulfate. Samples were blotted onto a nitrocellulose membrane using a constant current setting of 150 ma for 75 min (Hoefer TE70, SemiPhor Semi-Dry Transfer Unit, Amersham Pharmacia Biotech, San Francisco, CA). After transfer, the membranes were incubated for 1 h at 25 C in blocking solution (5% nonfat dry milk, 0.1% Tween 20 in phosphate buffered saline). Blots were incubated for 30 min at 25 C in solutions containing dilutions of primary antibodies in a PBS-Tween solution identical to the blocking solution. The primary antibody used in the Western blotting procedure was monoclonal anti-troponin-t (JLT-12, Sigma Chemical Company, St. Louis, MO) diluted to 1: and the secondary antibody was anti-mouse IgG peroxidase conjugate diluted to 1: (A-4416, Sigma Chemical Co., St. Louis, MO). Membranes were subjected to four 5-min washes in PBS, 0.1% Tween 20 following each antibody incubation. An enhanced chemiluminescent detection substrate (Supersignal, Pierce, Rockford, IL) was used to detect the presence of troponin-t and its degradation products. Figure 3 is a representative western blot of the differences between N and CA for all animals analyzed. Statistical Analysis Differences due to calcium gel administration for carcass, longissimus ph, moisture, ash, calpain/calpastatin, and sensory data were analyzed using the student-t test procedure of SAS (SAS Institute, Inc., Cary, NC). Differences in Warner- Bratzler shear force due to calcium gel administration across postmortem aging time were assessed using the General Linear Model procedure (SAS Institute, Inc., Cary, NC) with treatment, aging time and the two-way interaction in the model. Initial models included slaughter day as a main effect; however, this effect was not significant (P > 0.05) and was removed from the final models. Shear force means Table 1. Carcass and longissimus characteristics of calcium gel (CA) and control (CON) treatments CON CA SEM Hot carcass weight (kg) Adjusted fat thickness (cm) Ribeye area (cm 2 ) Kidney, pelvic and heart fat (%) Yield grade Marbling score z Longissimus muscle ph at 45 min ph at 48 h Moisture (%) Ash (%) 1.06a 1.10b 0.01 Calcium (µg g 1 ) 78.36a 92.37b 4.50 z Marbling score: = slight, = small (US Department of Agriculture, 1997). a,b Means in the same row with different letters differ (P < 0.05). were separated using a protected least significant difference test. Equations for the change in Warner-Bratzler shear force across postmortem age were developed using linear regression and break point analysis procedures to minimize standard error of prediction. Dummy variable regression was used to test differences in slopes and intercepts of these regression lines due to treatment. RESULTS AND DISCUSSION The CA steers were dosed with 1.5 L of calcium propionate/propylene glycol gel at 3 to 6 h before slaughter. This time range was targeted based on blood calcium response for non-lactating, pregnant dairy cows given 1 L of calcium propionate/propylene glycol gel (Sanchez et al. 1997). This work showed that serum ionized and total calcium values were greater than pre-dose levels for 6 h after oral dosing. In more recent work with feedlot steers, serum normalized calcium values were greater than pre-dose levels for 6 h after drenching (Duckett et al. 1999). Drenching with water (Duckett et al. 1999) or propylene glycol (Sanchez et al. 1997) had no effect on serum calcium levels. The authors recognize the limitations of determining dose time based on blood calcium responses; however, no data are available on the change in muscle calcium in relation to increasing serum calcium levels by oral calcium administration. Hansard et al. (1952) noted that calcium ions appeared rapidly interchangeable between blood and soft tissues when using Ca 45 as a tracer. Hot carcass weight, adjusted fat thickness, ribeye area, kidney, pelvic and heart fat, yield grade, and marbling score did not differ (P > 0.05; Table 1) between treatments. The ph of the longissimus at 45 min after exsanguination tended to be lower (P = 0.06) for CA than CON. However at 48 h postmortem, ph values of the longissimus were similar (P > 0.05) between treatments. Lawrie (1998) reported that pre-slaughter intravenous administration of calcium salts accelerates the rate of postmortem glycolysis. Moisture content in the longissimus was similar (P = 0.21) between treatments. The percent ash content in the longissimus was 4% higher (P = 0.01) for CA than CON. Total

4 Can. J. Anim. Sci. Downloaded from by on 02/06/18 36 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 2. Effect of pre-harvest calcium gel administration (CA) on longissimus calpain and calpastatin activities CON CA SEM Calpastatin activity z µ-calpain activity y 0.96a 1.15b 0.06 m-calpain activity y 1.08a 1.30b 0.07 z One unit of calpastatin activity is defined as the amount that inhibited one unit of m-calpain activity. y Calpain activity is reported as the caseinolytic activity that increases absorbance one unit at 278 nm after a 60 m incubation at 25 C. a,b Means in the same row with different letters differ (P < 0.05). calcium content (µg g 1 ) of the longissimus was 11% higher (P = 0.04) for CA than CON. Goff and Horst (1993) have shown that administering calcium gel (50 g Ca 2+ as CaCl 2 in 250 ml of water) via a rumen tube increased total blood calcium at 30 min after dosing by about 18%. Hansard et al. (1952) found that calcium 45 administered intravenously comes to equilibrium between blood and soft tissue in about 1 h and remains at equilibrium for at least 8 h after infusion. Calpastatin activity (Table 2) did not differ (P > 0.05) between CON and CA treatments. The activity of µ- and m- calpain was 20% higher (P < 0.01) for CA than CON in prerigor muscle at 45 min. post-slaughter. The higher activities of µ- and m-calpain in at-death muscle suggests that the total amount or percentage of calpain present was in the active form, possibly due to changes in calcium concentrations and/or gene expression. Others have shown reductions in the activity of µ- and m-calpain, and calpastatin after calcium chloride was injected into the muscle postmortem and activity measured at 24 h postmortem (Koohmaraie et al. 1990; Wheeler et al. 1992). The reduction in activity after calcium chloride injection is believed to be the result of autolysis of the calpains and calpastatin in the excess calcium (Koohmaraie et al. 1990; Koohmaraie 1992). However, Geesink and Goll (1995) and Boehm et al. (1998) have found that µ-calpain becomes tightly associated with the myofibrils during postmortem storage resulting in an incomplete extraction and underestimation of its activity. Calcium gel steaks had 14 and 18% lower (P < 0.05) WBS values at 4 and 7 d of postmortem aging, respectively, than CON (Fig. 1). Shear force values did not differ (P > 0.05) between treatments at 2, 14 or 28 d of aging. The reductions in shear force at early postmortem ages (day 7) but not at advanced postmortem ages (days 7 to 28) agree with those reported for feeding high levels of vitamin D 3 to steers for 5 10 d prior to slaughter (Montgomery et al. 1997; Swanek et al. 1999). Both vitamin D 3 and calcium propionate gel administration increase intestinal calcium absorption; however, calcium gel presumably acts by increasing passive diffusion of calcium from the lumen to extracellular fluids when lumenal concentration is elevated whereas vitamin D 3 stimulates active transport of calcium across intestinal Fig. 1. Rate of postmortem tenderization for control (CON) and calcium gel (CA). Equations for WBS across postmortem aging are: CA WBS for days 2 7, kg = age, d, r 2 = 0.35, SEp = 1.20; N-WBS, days 2 14, kg = age, d, r 2 = 0.34, SEp = Slopes for the two lines differ (P = 0.01).

5 DUCKETT ET AL. PRE-SLAUGHTER CALCIUM AND TENDERNESS 37 Can. J. Anim. Sci. Downloaded from by on 02/06/18 Fig. 2. Effect of pre-harvest calcium gel administration on sensory panel tenderness, juiciness and flavor ratings (1 = extremely tough, dry, and bland; 10 = extremely tender, juicy, and intense). Fig. 3. Postmortem proteolytic degradation of troponin-t (TNT) in myofibrils isolated from bovine longissimus of control (CON) or calcium gel (CA) treatments aged for 2, 4, 7 and 14 d postmortem. epithelium (Goff and Horst 1993). The completion of postmortem aging determined using break point analysis was earlier (P > 0.05) for calcium gel than controls (7 vs. 14 d). The rate of postmortem tenderization was also faster (P < 0.01; 0.42 vs kg d 1 ) for CA compared with CON. The intercepts of the lines for both treatments were similar (P = 0.70) indicating that both treatments had similar shear force values at the start of aging. Infusion of 0.3 M calcium chloride into the carotid artery of lamb carcasses after slaughter elevated water-extractable calcium levels to 1346 µg g 1 and accelerated postmortem tenderization aging time to 1 d (Koohmaraie et al. 1990). Thus, pre-slaughter (calcium gel or vitamin D) and postmortem treatments (calcium chloride infusion or injection) that increase muscle calcium levels appear to accelerate the postmortem aging rate so that tenderness is greater at early postmortem times ( 7 d) for these treatments; however, the extent of aging does not appear to be affected. The rate of acceleration in postmortem aging also appears dependent upon the increase in muscle calcium levels observed with pre-slaughter or postmortem treatment. Average sensory panel ratings for tenderness, juiciness, and flavor of the control and calcium gel steaks after 7 d of postmortem aging are presented in Fig. 2. Steaks from the calcium gel treatment had greater (P = 0.02) tenderness ratings than controls. Sensory juiciness and flavor ratings were similar (P = 0.28 and 0.34) between treatments. Sensory panelists were able to detect differences in tenderness between treatments at day 7, which is in agreement with the observed differences in WBS values. Swanek et al. (1999) reported similar increase in sensory tenderness at day 7 for vitamin D supplementation. Troponin-T (TNT) isoforms (Clarke et al. 1976) were present in both the CON and CA myofibrils at 2 d postmortem (Fig. 3). In the CON myofibrils, TNT bands were present at day 4 and day 7 postmortem. In the CA myofibrils, the TNT bands were almost completely degraded at day 7. At day 14, no TNT bands were visible for either CON or CA. The disappearance of TNT bands coincided with the completion of aging as determined by the lack of a change in WBS at day 7 in CA and day 14 in N. Others (Olson et al. 1977; Koohmaraie et al. 1984; Ho et al. 1994) have shown that the disappearance of the TNT bands is associated with the extent of postmortem aging. Steaks with a higher shear force value have been shown to exhibit a slower degradation of TNT (Huff-Lonergan et al. 1996). Huff-Lonergan et al. (1996) have also shown that incubation of myofibrils with µ-calpain will result in the disappearance of TNT and the appearance of degradation products similar to the response observed with aging.

6 Can. J. Anim. Sci. Downloaded from by on 02/06/18 38 CANADIAN JOURNAL OF ANIMAL SCIENCE In summary, pre-slaughter administration of calcium propionate/propylene glycol gel to steers at 3 to 6 h prior to slaughter increased muscle calcium levels and calpain activity. These changes resulted in a faster rate of postmortem tenderization and an increase in tenderness at early postmortem aging times (4 and 7 d). Further studies are needed to address dose, time, and other routes of calcium gel administration before the application is ready for commercial usage. ACKNOWLEDGMENTS Appreciation is expressed to Kara Watson, Travis Skow, Rory Johnson, Cindy Hooper and Vandal Brand Meats for assistance in data collection, and to Sue Pritchard for technical assistance. Research supported by the Idaho Beef Council. Calcium propionate/propylene glycol gel donated by MAi, Spring Valley, WI. 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