Feeding high levels of vitamin D3 does not improve tenderness of callipyge lamb loin chops.

Abstract

The objective of this study was to determine whether feeding high doses of vitamin D3 7 d before slaughter would increase muscle Ca++ levels and result in more tender loin chops. Market lambs (n = 4 callipyge and 4 normal in Exp. 1, and n = 16 calipyge and 16 normal in Exp. 2) were randomly and equally assigned to feeding groups based on callipyge genotype and experimental diet, (vitamin D3 or control). Serum Ca++, muscle Ca++, Warner-Bratzler shear force, and troponin-T degradation data were analyzed. In Exp. 1, vitamin D3 was supplemented at 1 or 2 x 10(6) IU/d. The 2 x 10(6) IU dose resulted in the greatest serum Ca++ reponse and was chosen for Exp. 2. In Exp. 2, serum Ca++ concentration was higher (P < 0.05) for normal and callipyge lambs fed the vitamin D3 diet than for the control diet fed lambs. Muscle Ca++ concentrations, however, were not higher (P = 0.28) for the vitamin D3-fed lambs. Warner-Bratzler shear values were higher (P < 0.05) for callipyge than for normal lambs, but no differences were observed with vitamin D3 supplementation. These data were supported by results from Western blot analysis of troponin-T degradation, in which no differences were observed for vitamin D3 vs control diet lambs at 14 d postmortem. This experiment showed that feeding 2 x 10(6) IU/d of vitamin D3 to market lambs, callipyge or normal, raised serum Ca++ concentration, but did not increase muscle Ca++ concentration. This lack of response in muscle Ca++ was likely the reason that no differences were observed for Warner-Bratzler shear force values or troponin-T degradation data between the vitamin D3 and control loin chops. A higher dose of vitamin D3 may be required to improve tenderness.

Full text

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B. R. Wiegand, F. C. Parrish, Jr, D. G. Morrical and E. Huff-Lonerga
chops
Feeding high levels of vitamin D3 does not improve tenderness of callipyge lamb loin
2001, 79:2086-2091.
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Feeding high levels of vitamin D3does not improve tenderness
of callipyge lamb loin chops1
B. R. Wiegand, F. C. Parrish, Jr.,2D. G. Morrical, and E. Huff-Lonergan
Iowa State University, Ames 50011
ABSTRACT:
mine whether feeding high doses of vitamin D37 d
before slaughter would increase muscle Ca++levels and
result in more tender loin chops. Market lambs (n = 4
callipyge and 4 normal in Exp. 1, and n = 16 calipyge
and 16 normal in Exp. 2) were randomly and equally
assigned to feeding groups based on callipyge genotype
and experimental diet, (vitamin D3or control). Serum
Ca++, muscle Ca++, Warner-Bratzler shear force, and
troponin-T degradation data were analyzed. In Exp. 1,
vitamin D3was supplemented at 1 or 2 × 106IU/d.
The 2 106IU dose resulted in the greatest serum Ca++
reponse and was chosen for Exp. 2. In Exp. 2, serum
Ca++concentration was higher (P < 0.05) for normal
and callipyge lambs fed the vitamin D3diet than for
the control diet fed lambs. Muscle Ca++concentrations,
The objective of this study was to deter-
Key Words: Sheep Breeds, Tenderness, Vitamin D3
2001 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2001. 79:2086–2091
Introduction
Recent research has focused on improving lean yield
and consumer acceptability of market lambs in the
UnitedStates.Lambsexhibitingthecallipygecondition
express extreme muscle hypertrophy (Carpenter et al.,
1996) and higher retail yield compared with their nor-
mally muscled contemporaries (Koohmaraie et al.,
1995). Callipyge lambs also have increased feed:gain
ratios and increased average daily gains than normally
muscled lambs resulting in improved performance
(Jackson and Green, 1993). These advantages, how-
ever, are offset by consistently higher shear force of the
longissimusmuscle(Shackelfordetal.,1997).Thus,the
challenge is to take advantage of the increased perfor-
1Journal Paper No. 18719 of the Iowa Agric. and Home Econ. Exp.
St., Ames, Project No. 3680, and was supported in part by a grant
from the Iowa Sheep and Wool Promotion Board.
2Correspondence:215MeatLaboratory(phone:515294-3280;Fax:
515 294-5066; E-mail: fparrish@iastate.edu).
Received July 7, 2000.
Accepted March 28, 2001.
2086
however, were not higher (P = 0.28) for the vitamin D3-
fed lambs. Warner-Bratzler shear values were higher
(P < 0.05) for callipyge than for normal lambs, but no
differences were observed with vitamin D3supplemen-
tation.ThesedataweresupportedbyresultsfromWest-
ern blot analysis of troponin-T degradation, in which
no differences were observed for vitamin D3vs control
dietlambsat14dpostmortem.Thisexperimentshowed
that feeding 2 × 106IU/dof vitamin D3to market lambs,
callipyge or normal, raised serum Ca++concentration,
but did not increase muscle Ca++concentration. This
lack of response in muscle Ca++was likely the reason
that no differences were observed for Warner-Bratzler
shear force values or troponin-T degradation data be-
tween the vitamin D3and control loin chops. A higher
dose of vitamin D3may be required to improve ten-
derness.
mance and retail product yield from callipyge lambs
while marketing a product with acceptable tenderness.
Evidence supports the use of CaCl2infusion or injec-
tion for increasing the tenderness of fresh meat prod-
ucts (Koohmaraie et al., 1990; Whipple and Koohmar-
aie, 1992), presumably by increasing calcium availabil-
ity and stimulating calpain activity. If one could
increase free calcium in the muscle, the possibility ex-
ists that calpain activity and subsequent proteolysis
and tenderness would be accelerated. One way to in-
crease circulating Ca++in the animal and in muscle
(Jones et al., 1998) is by feeding vitamin D3in excess
of nutritional requirements. The biologically active
form of vitamin D, 1,25-(OH)2vitamin D, increases se-
rum calcium concentrations in the body by liberating
calcium stores from the skeleton and by promoting in-
testinal absorption of dietary calcium (Jones et al.,
1998). Consequently, the hypothesis to be tested was
that vitamin D3would increase calcium concentrations
in the muscle, and consequently enhance postmortem
calpain activity and improve tenderness. The objective
of this study was to determine whether feeding high
levels of vitamin D3for 7 d before slaughter improves
tenderness.
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Vitamin D3effects on callipyge tenderness
2087
Materials and Methods
The project consisted of two experiments and was
carried out under the guidelines of the Iowa State Uni-
versity Animal Care and Use Committee. All market
lambs were obtained from the McNay Research Unit,
McNay, IA. All parent and progeny genotypes were in-
ferred with a gene marker method at Utah State Uni-
versity, Logan (Cockett et al., 1996), to detect the pres-
ence or absence of the callipyge genotype. Lambs were
sorted into feeding groups according to genotype.
Diets
All control lambs were limit-fed 1.14 kg of shelled
corn and a commercial pelleted protein supplement
each day. Lambs on vitamin D3-treated diets received
0.11 kg of a corn-based, ground vitamin D3supplement
with theappropriate levelof D3.The vitaminD3supple-
ment replaced corn by weight in the control diet and
was administered as a top dressing to the control diet.
Limit feeding was utilized to ensure intake of the
treated diet.
Exp. 1
An initial experiment was done in order to determine
a dose-time relationship for significantly increasing se-
rum Ca++concentrations with vitamin D3. Market
lambs (n = 8) averaging 47.7 kg of body weight were
used. Normal (n = 4) and callipyge (n = 4) lambs were
individually fed. Two lambs from each genotype re-
ceived 1 × 106IU of vitamin D3and two lambs received
2 × 106IU of vitamin D3. Lambs were fed for 2 d in
order to determine maximum vitamin D3response at
each dose. A blood sample was collected on each of the
3 d preceding the feeding trial to determine a baseline
serum Ca++concentration for each lamb. Blood samples
were then drawn from each lamb at 12, 24, and 48 h
during the feeding trial. Based on the most consistent
increase in serum Ca++response, the level of 2 × 106IU
ofvitaminD3/perdaywasselectedasthedoseforExp.2.
Exp. 2
Market lambs (n = 32) were penned in groups of eight
lambs. Pens included eight normal lambs fed a control
diet, eight normal lambs fed a control diet plus 2 × 106
IU of vitamin D3, eight callipyge lambs fed a control
diet, and eight callipyge lambs fed a control diet plus
2 × 106IU vitamin D3. Lambs were fed their respective
dietsfor7d.Bloodsampleswerecollectedeachmorning
before feeding at d 1, 3, and 5 of the trial. At d 7,
lambs were transported to Iowa Lamb Corporation in
Hawarden, IA, for processing. Hot carcass weight and
dressing percentage were calculated. After a 24-h chill,
all carcasses were ribbed between the 12th and 13th
ribs, and ribeye area and 12th-rib fat were recorded.
After carcass measurements were made, the wholesale
loin was removed from the carcass and vacuum pack-
aged. The loin section was transported back to the ISU
Meat Laboratory, where the boneless loin was removed
and 2.54-cm chops were cut from each loin. Chops were
packaged in pairs and held for 1, 7, 14, and 21 d of
aging at 2°C. At the appropriate day of storage, chops
were frozen until needed for Warner-Bratzler shear de-
termination. A 1.27-cm chop was removed from each
loinforSDS-PAGEandWesternblottingdetermination
of troponin-T protein degradation. Blood samples were
collected via jugular puncture, transferred to heparin-
ized collection tubes, and placed in an ice-filled cooler.
Samples were transported to the lab and spun down in
a clinical centrifuge (Model CL IEC/Damon, Needham,
MA) at 1,500 × g for 15 min. Serum was pipetted into
glass vials and frozen at −30°C. At time of serum cal-
cium analysis, samples were thawed in a warm water
bath (Isotemp, Fisher Scientific, Chicago, IL) and vor-
texed to ensure a homogeneous sample. One hundred
microliters of blood serum was pipetted into a cuvette
with 4 mL of lanthium oxide (Sigma-Aldrich Co., St.
Louis, MO). This sample was measured with an atomic
absorption spectrophotometer (Analyst 100, Perkin El-
mer, Foster City, CA) at 422.7 nm. Muscle calcium was
determined by pulverizing 5-g samples of longissimus
muscle from the 12th to 13th rib section in liquid nitro-
gen. The samples were then ashed in a muffle furnace
and resuspended in 6 N HCl to dissociate the bound
Ca++. The Ca++concentrations were determined with
the same procedure as the serum samples. Serum and
muscle calcium concentrations are reported as milli-
grams of Ca++per 100 mL.
Warner-Bratzler Shear Force
At the appropriate day of postmortem aging, 2.54-cm
chops were cooked to an internal temperature of 35°C
and then turned and cooked to a final internal tempera-
ture of 71°C in a broiler oven set at 176.7°C. Chops
were cooled at 4°C overnight, and three 1.27-cm round
coreswereremovedfromeachchopforWarner-Bratzler
shear determination using an Instron Universal Test-
ing Machine (Model 4502, Canton, MA). Cores were
sheared perpendicular to the muscle fibers at a cross-
head speed of 250 mm/min.
Troponin-T Degradation Determination
Sample Preparation. Whole muscle samples were pre-
paredaccordingto Huff-Lonerganetal.(1996b). A0.2-g
sample from the longissimus muscle was homogenized
with 10 mL of a solution containing 2% (wt/vol) SDS
and 10 mM sodium phosphate buffer, pH 7.0. The sam-
plewascentrifugedat1,500×gfor15mintoprecipitate
insoluble components within the sample. The protein
concentration of the supernate was determined by us-
ingaDC ProteinAssaykit,(Bio-Rad Laboratories,Her-
cules, CA) (Lowry et al., 1951). Samples were diluted
with water to 6.4 mg/mL and prepared for SDS-PAGE.
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Wiegand et al.
2088
Table 1. Serum Ca++concentration(mg/100 mL) over feeding time
for Exp. 1 (dose-time relationship)
Dose% change
over time Genotype (IU × 106)Baselinea
12 h24 h 48 h
Normal
Normal
Callipyge
Callipyge
Normal
Normal
Callipyge
Callipyge
1
1
1
1
2
2
2
2
9.75
9.11
9.22
9.42
9.72
10.20
9.03
9.03
9.56
9.51
9.50
9.07
9.53
10.22
9.24
9.20
9.63
9.06
9.86
9.60
9.47
10.37
9.27
9.60
9.81
9.95
9.77
9.34
9.96
10.46
9.62
9.21
+0.6
+9.2
+5.9
−0.8
+2.4
+2.5
+6.5
+2.0
aMean serum Ca++3 d before feeding trial.
One volume of tracking dye (60 mM Tris-HCl [pH 6.8],
30% glycerol, 2% SDS, 1% bromophenol blue) and 0.1
vol 2-mercaptoethanol was added to each sample (1 vol
= 1 mL of protein, 1 mL of tracking dye, and 0.1 mL of
mercaptoethanol). Final concentration of the sample
was 4 mg/mL. Samples were heated at 50°C for 20 min.
Gel System.
Polyacrylamide
amide:N,N′-bis-methylene acrylamide of 100:1 [wt/wt])
were used. Each gel consisted of 0.1% (wt/vol) SDS,
0.67% (vol/vol) N′N′N′N′-tetramethylethylenediamine,
0.1% (wt/vol) ammonium persulfate,and 0.375 M Tris-
HCl (pH 8.8). Additionally, a 4% polyacrylamide stack-
ing gel (acrylamide:N,N′-bis-methylene acrylamide of
100:1 [wt/wt]) was used over the 15% gel. Gel dimen-
sions were 8 cm wide × 9 cm tall × 1.5 mm thick. Gels
were electrophoresed using a Hoefer SE260 unit (Phar-
macia Biotech, Piscataway, NJ). The upper and lower
chamber running buffer consisted of 25 mM Tris, 192
mM glycine, 2 mM EDTA, and 1% (wt/vol) SDS. A 60-
?g protein sample was loaded into each well and run
at a constant voltage of 120 V at 25°C. Gels were trans-
ferred by electroelution to polyvinylidene difluoride
membranes.
Transfer Conditions. Gelswere equilibratedin25mM
Tris, 192 mM glycine, and 15% (vol/vol) methanol. Gels
were transferred with a Hoefer TE22 Mighty Small
Transphorunit ata constantvoltage of90 Vfor 90min.
gels (15%acryl-
Table 2. Serum Ca++concentration(mg/100 mL)
changes over feeding time at 2 × 106IU vitamin
D3for Exp. 2 (primary feeding trial)
% change
over time
GenotypeDiet1 d 3 d5 da
Normal
Normal
Callipyge
Callipyge
SE
Control
Vitamin D3
Control
Vitamin D3
9.30
8.74
9.24
9.11
0.21
9.28
9.34
9.01
9.26
0.21
9.44b
10.27c
9.29b
9.89c
0.21
+1.5
+14.9
+0.5
+7.8
aValues within a column with different letters significant at P <
0.05.
Western Blotting
Western blotting was done with slight modifications
to the method of Huff-Lonergan et al. (1996b). The pri-
mary antibody was an anti-troponin-T antibody (JLT-
12 Sigma Chemical Co.) diluted at 1:15,000 in solution
(80mMdisodiumhydrogenorthophosphate,anhydrous,
20 mM sodium dihydrogen orthophosphate, 100 mM
sodium chloride, 0.1% [vol/vol] polyoxyethylene sorbi-
tan monolaurate [Tween-20]). The secondary antibody
was an IgG horseradish peroxidase-conjugated anti-
body (A2554, Sigma Chemical Co.), diluted 1:5,000 in
solution (Tween-20). Gels were incubated (25°C) in the
blocking solution for 1 h, in the primary antibody for 1
h, and in the secondary antibody for 1.5 h. Gels were
rinsed three times for 10 min per wash at each incuba-
tion step. Chemiluminescence was used to detect la-
beled bands according to the manufacturer’s directions
(ECL, Amersham, Arlington Heights, IL).
Statistical Analysis
Data were analyzed using the general linear model
of SAS (SAS Inst Inc., Cary, NC) in a 2 × 2 factorial
design for each experiment. The model included fixed
effects of genotype, diet, and pen, where appropriate.
Additionally, repeated measures analysis was used for
the postmortem aging portion of the study, in which
chops were removed from each loin and analyzed over
days of vacuum storage.
Table 3. Least squares means and standard errors for
longissimus muscle Ca++concentrations
Muscle Ca++
(mg/100 mL)
Standard
errorGenotype Diet
Normal
Normal
Callipyge
Callipyge
Control
Vitamin D3
Control
Vitamin D3
5.38
7.15
4.24
4.14
1.71
1.60
1.60
1.60
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Vitamin D3effects on callipyge tenderness
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Table 4. Least squares means and standard errors
for carcass values by genotype
Carcass wt,
kg
12th-rib Fat,
cm
Ribeye area,
cm2
Genotype
Normal
Callipyge
SE
P-value
26.69
31.76
0.55
0.03
0.85
0.60
0.07
0.01
16.89
23.41
2.48
0.001
Results and Discussion
In Exp. 1, serum Ca++increased by small percentages
for both callipyge and normal lambs over the 7-d trial
at both 1 and 2 × 106IU vitamin D3/d (Table 1). Mont-
gomery et al. (1997) reported a 30% increase in serum
Ca++for beef cattle when feeding at a lower dose:body
weighttreatment.Thesedifferencesmayhavebeendue
to the vehicle of delivery in each of these studies, for
whichMontgomeryetal.(1997)usedavitaminD3bolus
and our study used a vitamin D3premix in the feed.
Additionally, Swanek et al. (1999) used a corn and vita-
min D3pellet for beef cattle and observed a 34% serum
Ca++increaseovera7-dfeedtrial.Onepossibleexplana-
tion is that the methods of Montgomery et al. (1997)
andSwaneketal.(1999)mayhaveresultedinimproved
intake of the vitamin D3due to more direct delivery to
the digestive system than our method.
Calcium plays an important role in the postmortem
degradation of muscle proteins (Goll et al., 1983;
Koohmaraie et al., 1988). Calcium has been shown to
activate the muscle proteases calpains, which have
been shown to degrade troponin-T, and as a result the
simultaneous production of the 30-kDa component
(Olson and Parrish., 1977). This degradation of tropo-
nin-T to the 30-kDa component has been linked to meat
tenderness (MacBride and Parrish, 1977; Huff-Loner-
gan et al., 1996a). Given the relationship between cal-
cium and meat tenderness, it seems logical that efforts
to increase cellular Ca++before slaughter might also
increase tenderness through further stimulating the
action of the calpain proteases.
Experiment 2 resulted in greater (P < 0.05) serum
Ca++increases when control and vitamin D3diets were
Table 5. Warner-Bratzler shear force values (kg) of loin chops by genotype
and diet over 21 d postmortem aging
Warner-Bratzler Shear Forcea
GenotypeDiet1 d7 d 14 d21 d
Normal
Callipyge
Normal
Callipyge
Standard Error
Control
Control
Vitamin D3
Vitamin D3
3.41bc
5.85d
3.16b
4.75cd
0.48
3.56c
5.44d
2.82b
4.63cd
0.50
3.35bc
5.07d
2.86b
4.39cd
0.46
3.08bc
4.35d
2.34b
3.70cd
0.42
a,b,c,dMeans within a column with different letters are significant at P < 0.05.
compared at 5 d (Table 2). Additionally, normal lambs
fedvitaminD3exhibitedhigher (P<0.05)serumCa++at
5dthanvitaminD3fedlambs.Althoughtheseincreases
were greater in Exp. 2 than in Exp. 1, the magnitude
of change was not nearly as great as reported for beef
cattle, in which increases of 20 to 30% were observed
(Montgomery et. al. 1997; Swanek et al., 1999). Addi-
tionally, muscle Ca++data were not different between
the genotype or diet groups (Table 3). These data sug-
gest that the high levels of vitamin D3used in this
study did not result in increases in muscle Ca++concen-
trations.
Carcass data are presented in Table 4 to characterize
the differences between the normal and callipyge car-
casses. As expected, callipyge lamb carcasses exhibited
heavier(P<0.01)hotcarcassweights,greater(P<0.03)
ribeye areas, and less (P < 0.001) 12th rib fat than
normal lamb carcasses. Similar data have been re-
ported (Jackson and Green, 1993; Koohmaraie et al.,
1995; Carpenter et al., 1996). An explanation for ribeye
area differences between callipyge and normal lambs
has been reported by Koohmaraie et al. (1995) and Car-
penter et al. (1996). They reported a significantly
greater percentage of white, fast-twitch, glycolytic fi-
bersaswell asincreasedmusclefiber diameterforcalli-
pyge longissimus muscle than for fibers from normal
lambs.
In our study, 1-d shear force values were higher (P
<0.05)forcallipygethanfornormalcontrolandvitamin
D3fed lamb longissimus, regardless of diet (Table 5).
The same differences existed at 21 d of postmortem
aging. Shear force values are higher for longissimus
muscle from callipyge lambs than those from normal
lambs (Koohmaraie et al., 1995; Wiegand et al., 1998).
Also,Shackelfordetal.(1997)reportedsignificant123%
greater shear values for longissumus muscle from calli-
pyge lambs than from normal lambs. Given these shear
force results, it seems that the 21-d aging period was
more responsible for the tenderization process than the
vitamin D3treatment.
Western blot data (Figure 1) showed evidence of tro-
ponin-T degradation for each treatment group regard-
lessofdietorgenotypeby14dofaging.Thehighestand
lowest Warner-Bratzler shear values are represented
withinFigure1foreachgenotypeanddietcombination.
The Western blot results indicated no differences in the
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