Introduction
The quality of meat has become an important issue in recent years. Consumers
often understand quality as visual and sensory properties as well as the microbial
safety of meat. Nevertheless, meat quality can be determined in a more
unbiased way by evaluating such properties of meat as chemical composition,
pH value, tenderness, water-holding capacity, and color. Those properties
can, however, be affected by genetic and environmental factors
(Ramírez-Retamal et al., 2014).
One of the factors that determine the quality of lamb meat is the type of
feed administered to animals. The nutritional characteristics of a diet
(contents of energy, protein, fatty acids, vitamins, and minerals) affect
value-added properties of meat and, thereby, its quality (Luciano et al.,
2009b; Aouadi et al., 2014; Schiller et al., 2015). Recently, special
attention has been paid to lamb diet supplementation with phenolic compounds
(especially carnosic acid) and seleno-compounds that have the ability to
modify the ruminal microbiota profile and, hence, fatty acid metabolism in
the rumen (Del Razo-Rodriguez et al., 2013; Jordan et al., 2013;
Kišidayová et al., 2014; Cobellis et al., 2016). Recent studies have
shown a positive effect of carnosic acid (CA; a constituent of rosemary) on
the improvement of meat quality, including either extension of the shelf
life of lamb meat or slowing down meat discoloration (Morán et al.,
2012b, 2013). Dietary CA has a significant effect upon the biosynthesis of
ruminal volatile fatty acids (Miltko et al., 2016) and may beneficially
modify the profile of fatty acids (FAs) in lambs' muscles. Due to its
antioxidant properties, it may protect polyunsaturated FAs (PUFAs),
especially long-chain PUFAs (LPUFAs), from lipid peroxidation in muscles
(Morán et al., 2012a, b, 2013) and stabilize the oxidative status of
edible parts of farm animals (Ponnampalam et al., 2012; Aouadi et al., 2014).
Sparse research is, however, available regarding the effect of CA on
proteolysis of muscle protein in lamb meat during ageing. However,
the influence of Se supplementation on lamb meat quality was only determined
based on changes of pH, drip and cooking losses, meat color, and oxidative
stability (Vignolla et al., 2009), as well as shelf life of packaged meat
(Ripoll et al., 2011). To our knowledge there are no publications that
discuss the coupled dietary effect of fish oil (FO), CA, and Se on meat
quality characteristics.
Having considered the above facts, we expected that dietary CA, selenate
(SeVI), and selenized yeast (SeY) would change the biohydrogenation yield of
unsaturated FAs (UFAs) in the rumen and consequently the accumulation of
PUFAs, particularly conjugated isomers of linoleic acid (CLA) and their
precursors and metabolites, in lamb muscles. Moreover, considering our
previous studies (Wąsowska et al., 2006), we expected that dietary FO
would reduce the biohydrogenation of UFAs in the rumen and consequently
would increase the concentration of UFAs in muscles. The concentration
changes of UFAs in muscles will exert influence on physicochemical
characteristics of lamb muscles.
Therefore, the first aim of our study was to compare effects of 1 % FO
(rich in pro-oxidizing LPUFAs) added to the diet including 2 % rapeseed
oil (RO) (the experimental diet) with the control diet containing 3 % RO
on the protein profile and physicochemical characteristics of the
longissimus muscle of lambs. In our investigation, the diet with only 3 % RO was
considered as the control diet as it does not contain pro-oxidizing LPUFAs
as well as antioxidants (i.e., CA, SeY or SeVI).
The second objective of our study was to investigate effects of the addition
of CA with or without different chemical forms of Se (as SeY or SeVI) to the
diet including RO and FO on the protein profile and physicochemical
characteristics of the longissimus muscle of lambs. The effects of these experimental
diets were compared with effects of the FO-supplemented diet containing RO
as well as with the diet with only RO (the control diet). The last objective
of our study was to compare the influence of the addition of SeY to the diet
containing RO, FO, and CA with the addition of SeVI to the diet including RO,
FO, and CA on the protein profile and physicochemical characteristics of the
longissimus muscle of lambs.
Material and methods
Animals, housing, diets, and experimental design
Thirty male Corriedale lambs with an average body weight (BW) of 30.5 ± 2.6 kg
at the beginning of the experiment were individually penned and
divided into five dietary groups (groups I–V) of six animals each according to
their initial BW, so that the average initial BWs of lambs were similar
between the groups. The study protocol was approved by the Third Local
Commission of Animal Experiment Ethics at the University of Life Sciences,
Ciszewskiego 8, 02–786 Warsaw (Poland). During a 3-week preliminary period,
the animals were given free access to the chosen feed (the standard concentrate–hay
diet with vitamins and mineral premix) (Jaworska et al., 2016). In the
3-week preliminary period, the lambs from group I (the control group) were
fed the diet enriched with 3 % RO (the control diet), whereas the lambs
from groups II–V (the experimental groups) were fed the diet enriched with
2 % RO and 1 % odorless fish oil (FO) (FO diet) (Jaworska et al., 2016).
The energy content of FO and RO was 36.8 and 37.0 MJ kg-1 oil, respectively.
The fatty acid profiles of dosed FO and RO as well as other ingredients in
the diet were presented in a previous publication (Rozbicka-Wieczorek et
al., 2016). The diet allowance was changed weekly according to body weight
of lambs and supplied as two equal meals at 07:30 and 16:00 CET (central European time; UTC + 01) each day to
ensure free access to the feed. Whole served portions of meals were eaten by
the animals. Fresh drinking water was available ad libitum. The lambs were slaughtered
at the end of the 35-day experiment (Jaworska et al., 2016). After slaughter
the carcasses were chilled in a cooling room and held in the cold storage at
4 ∘C. Longissimus muscle was removed and weighed; all muscle samples were
stored in sealed tubes at -32 ∘C until analysis.
Selenized yeast (Se-Saccharomyces cerevisiae) was donated by Sel-Plex (Alltech In., Nicholasville,
KY, USA). About 83 % of the Se content of selenized yeast (SeY)
represents Se in the form of seleno-methionine incorporated into the
proteins of Saccharomyces cerevisiae; the chemical composition of SeY was presented in a previous
publication (Czauderna et al., 2009). SeVI as sodium selenate
(Na2SeO4) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
CA was purchased from Hunan Geneham Biomedical Technology Ltd. (Changsha,
Hunan, China). RO and odorless FO were supplied by AGROSOL
(Pacanów, Poland).
Meat quality traits and biochemical analysis
Meat quality parameters were evaluated in the samples taken from the
longissimus muscle. The samples were collected at the height of the last rib after the
carcasses had been cooled for 24 h after the slaughter. At the meat
processing plants, the pH value was measured 3 (pH3), 24 (pH24),
and 48 h (pH48) after slaughter with a WTW 330i pH meter (Weilheim,
Germany) with special electrodes (SenTix® SP
number 103645), enabling pH measurement directly in meat. The samples were
transported in ice-chilled polystyrene refrigerators at 4 ∘C. All
remaining analyses were conducted at the laboratory.
Meat color was measured 48 h post mortem according to the CIE L∗a∗b∗ system using a
CR310 Minolta Chroma meter with a D65 light source (Osaka, Japan). Loin
chops (length of 2 cm) were cut and bloomed for 1 h at 4 ∘C with
no surface covering prior to color measurements (in triplicate).
The drip loss percentage was determined 24 and 48 h after slaughter
according to Prange et al. (1977). During that time, longissimus muscles were placed in
a plastic bag and kept at 4 ∘C for the drip loss to appear. The drip
loss was collected into Eppendorf tubes for glucose and lactate
measurements. Muscle glucose (mmol) was determined with an Accu-Check
Active® glucometer (Accu-Check Sensor Comfort®,
Roche, Germany), which is normally employed for measuring blood glucose in
humans. Reactive strips were used for the quantitative determination of
glucose with the glucometer in the value range between 0.6 and 33.3 mmol.
The test results were obtained 60 s after placing a drop with an
approximate volume of 20 µL of drip loss on a reactive strip.
Lactic acid (mmol) was measured with the strip method using
Accutrend®
lactate type 3012522 strips (Roche, Mannheim, Germany). Samples were diluted
with distilled water (1:10) to reach lactate concentration in the following
range: 8.7 to 26 mM. The test results were also obtained 60 s after
placing a drop with an approximate volume of 20 µL of diluted drip
loss on a reactive strip. Then, the results were converted into the
concentration in undiluted drip loss. The measurements were made twice.
Muscle glycolytic potential (GP) was calculated according to the formula by
Monin and Sellier (1985) summing glucose and lactic acid and expressed as
mmol lactic acid L-1 fresh (hot) muscle tissue.
Drip and meat samples were frozen at -80 ∘C until subsequent
analysis.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
SDS-PAGE of drip loss and muscular tissue was performed according to the
method of Bollag and Edelstein (1999) using the STANDARD system (Kucharczyk
TE, Poland). Proteins were resolved on a 12 % separation gel and 5 % stacking gel. Myofibrillar proteins were extracted from 20 mg of muscle,
homogenized with 800 µL of a Tris-HCl buffer (pH 6.8) containing
0.375 M 2-mercaptoethanol, 3 % SDS, 8 M urea, and 2 M thiourea. Muscle
protein content was determined as total nitrogen using the AOAC method
number 928.08 (1974). The content of soluble proteins in the drip loss was
determined using the Biuret procedure (Gornall et al., 1949). Myofibrillar
and soluble proteins from the drip loss were dissolved (1/1, v/v) in
Tris-HCl sample buffer (pH 6.8) containing 0.375 M 2-mercaptoethanol, 3 %
SDS, 8 M urea, 2 M thiourea, and 0.05 % bromophenol blue. The mixture was
then heated for 3 min at 95 ∘C and 10 µL of the mixture was
then placed in each well. Protein bands were stained with Coomassie
brilliant blue R250 (Sigma-Aldrich, St. Louis, Missouri, USA) dissolved in
water, methanol, and acetic acid (4:5:1, v/v/v) and destained using a
solution of methanol, water, and acetic acid (1:8:1, v/v/v). Image analysis
and quantification were performed using GelScan v. 1.45 software (Kucharczyk
TE, Poland).
Chromatographic analysis of amino acids in the Longissimus
muscle
Longissimus muscle samples (about 400–500 mg fresh muscle) were hydrolyzed with 50 mL of
6 M HCl at 104 ± 2 ∘C for 20 h in sealed tubes. After cooling, the
hydrolysates were filtered through filter paper and then a filter residue
was washed three times with ∼ 10 mL of water. Hydrochloric
acid was removed from the filtrates in a vacuum rotary evaporator. Ten
milliliters of water were added to the residue and then evaporated to
dryness again in vacuum to remove residues of HCl. The evaporating procedure
was repeated twice. The residues were stored at -18 ∘C when not in use.
The residue was re-dissolved in 1 mL of 0.4 M borate buffer (pH 9.9). The
resulting solution (I) was used for the derivatization procedure as below.
Preparation of the derivatizing reagent: 75 mg of o-phthaldialdehyde was dissolved in 4.5 mL of methanol
and 0.5 mL of 0.4 M borate buffer. Next, 70 µL of ethanethiol was added
and the resulting solution was mixed. Methanol, ethanethiol,
o-phthaldialdehyde, and all amino acids used were from Sigma-Aldrich (St. Louis, MO,
USA).
Derivatization procedure: to 20 µL of the resulting solution (I), 1 mL of the derivatizing
reagent and 10 µL of 1 M NaOH were added. The contents were mixed and
reacted for 3 min at room temperature. At the end of the 3 min
derivatization period, the processed samples were injected onto the Nova Pak
C18 column (4 µm, 250 × 4.6 mm, I.D.; Milford, MA, USA). Amino
acid contents in processed muscle samples were chromatographically
determined according to Czauderna et al. (2002).
Chromatographic equipment: a Waters Alliance separation module (model 2690; Milford, MA, USA) with a
Waters fluorescence detector (model 474; Milford, MA, USA) was used for the
gradient elution systems (Czauderna et al., 2002).
Effect of different diets on quality characteristics of the longissimus muscle of lambs.
Variable
Group
I
II
III
IV
V
Diet
Control
+1 % FO
+1 % FO +0.1 % CA
+1 % FO +0.1 % CA +0.35 ppm Se as SeY
+1 % FO +0.1 % CA +0.35 ppm Se as SeVI
pH3
6.77 ± 0.04
6.73 ± 0.19
6.62 ± 0.20
6.73 ± 0.04
6.66 ± 0.25
pH24
5.47 ± 0.06
5.65 ± 0.04
5.71 ± 0.48
5.47 ± 0.07
5.63 ± 0.06
pH48
5.40a ± 0.02
5.60b ± 0.11
5.51ac ± 0.04
5.48ac ± 0.03
5.50ac ± 0.05
Meat color: L∗
43.97a ± 1.49
43.53a 1.87
46.63b ± 2.54
46.63b ± 1.26
43.26a ± 2.20
a∗
23.26a ± 1.61
19.29b ± 0.82
20.89c ± 1.34
20.18bc ± 1.34
17.59bc ± 0.81
b∗
9.55c ± 1.51
4.30a ± 0.70
7.30b ± 3.03
6.74b ± 1.98
4.17a ± 0.41
Drip loss 24 h (%)
2.57 ± 1.87
1.56 ± 0.70
1.92 ± 0.38
1.93 ± 0.40
1.92 ± 0.83
Drip loss 48 h (%)
4.23 ± 1.90
3.17 ± 1.00
3.31 ± 0.30
3.09 0.48
3.38 ± 0.81
Glucose (mmol L-1)
6.15 ± 1.37
4.73 ± 1.30
6.60 ± 1.71
6.60 ± 1.08
6.06 ± 1.35
Lactate (mmol L-1)
92.50 ± 6.37
86.50 ± 14.05
89.17 ± 9.54
87.17 ± 6.18
77.00 ± 12.30
Glycolytic potential (mmol L-1)
104.80 ± 7.12
95.95 ± 16.45
102.36 ± 11.07
100.36 ± 4.47
89.12 ± 11.94
a,b,c – means with different superscripts in the same row differ
significantly at Pα≤0.05;
FO – fish oil, CA – carnosic acid, SeY – the organic chemical form of
selenium, SeVI – selenate.
Data analysis
All values were reported as the means ± standard deviation (SD)
(Tables 1–3) or as the means (Table 4). The normality of distribution of all
analyzed traits was checked with the Shapiro–Wilk test before statistical
analyses. A one-way analysis of variance of the type of the diet as a fixed
effect was performed. The significance of differences between means (at the
level of Pα≤0.05 and Pα≤0.01) of different groups
was estimated by using Tukey's test. The simple Pearson linear
correlations between measured traits were calculated. The significance of
correlations was tested by means of an F test. Statistical analysis was conducted with
Statistica 10 software (StatSoft, Inc., 2011).
Results and discussion
Effect of lamb diets on meat quality characteristics
The analysis of the obtained data showed significant differences in pH value
of meat 48 h after slaughter. Samples of meat from group I (fed with 3 % rapeseed oil) were characterized by the lowest value of pH48, whereas
samples from group II (fed with 1 % addition of fish oil, FO) had the
highest pH48 (Table 1). No significant differences were observed
between other groups. It should be noted that pH values do not exactly reflect
the level of lactate and glycolytic potential at slaughter time (Table 1).
According to Van Laack et al. (2001), this inconsistency between ultimate pH
and lactate levels could be explained by the buffering capacity of muscles.
With respect to high ultimate pH observed in group II, similar findings were
reported by Najafi et al. (2012) for meat from goat kids fed with fish oil.
Nevertheless, pH48 values recorded in groups fed with CA and SE (groups III, IV, and V)
were intermediate between group RO (group I) and FO (group II). A study by Morán et al. (2012b) showed significant and lower
pH24 in meat of lambs fed CA (0.6 g kg-1) compared to the control group,
and no respective differences in the group with CA dose of 1.2 g kg-1. No
significant differences between ultimate pH of lambs fed CA in comparison to
the control group were found in studies by Bañón et al. (2012) and
Ortuño et al. (2014). Also, no effect of Se level or source on meat pH was stated
in the studies of Vignola et al. (2009) and Skřivanová et al. (2007).
Color parameters differed significantly between groups of animals. The
lightest meat was observed in groups III and IV (containing FO, CA, and SeY in
the diet). The higher lightness of meat was also stated in the group of lambs
fed the diet containing CA in a study conducted by Morán et al. (2012a, b)
and Ortuño et al. (2014), while no differences were reported by
Bañón et al. (2012) and Aouadi et al. (2014). Darker meat
(Pα≤0.05) was observed in the group with RO (group I), with
inorganic Se (group V) and with fish oil (group II). The results achieved by
Nute et al. (2007) also showed that FO in lamb diet reduced the intensity of
meat color. While Ponnampalam et al. (2012) stated that the content of
highly oxidizable PUFAs in muscle tissues has no major role in maintenance of
redness at days 3–4 of retail display, and that vitamin E and heme iron
contents are important. Considering a∗ and b∗ color
parameters, the meat from group I was more red and yellow. Results in Table 1
show also that the addition of FO and SeVI to the diet decreased redness and
yellowness of meat, while supplementation of lamb diet with CA and SeY
increased values of a∗ and b∗ coordinates of color
(Pα≤0.05) (Table 1). It is worth noting that studies of many authors
have shown no effects of diets containing CA (Bañón et al., 2012;
Morán et al., 2012b; Aouadi et al., 2014; Ortuño et al., 2014) or Se
on redness and yellowness of veal and lamb meat (Skřivanová et al.
2007; Vignolla et al., 2009) as well as on retarding color deterioration.
These differences in the presented results may be due to the combined effect
of FO and SeY added to diet. However, Taylor et al. (2008) found that meat
from cattle fed selenium-enriched diet tended to have a higher average values
of a∗ and b∗ during 12 days of exposition in comparison to
the control group. Also, Zhan et al. (2007) showed that diet supplementation
with Se increased the Hunter a∗ value, but more in the
selenomethionine-treated group than in the sodium selenite-treated group.
Furthermore, as it was stated by Caputi-Jambrenghi et al. (2005), α-tocopherol acetate stabilized meat color more efficiently than rosemary
extract did. The same result was observed in gluteus medius muscle
upon diet supplementation with low doses of CA and vitamin E
(0.6 g kg-1). Only the highest dose of carnosic acid
(1.2 g kg-1) tended to show a similar effect to vitamin E. But
Ortuño et al. (2014) found that a rosemary diterpenes extract
significantly delayed lamb meat discoloration at 11 and/or 14 days of
storage. In turn, the effect of tannins on improving meat color stability of
fresh lamb during extended refrigerated storage was shown by Luciano et
al. (2009a). They demonstrated that tannin supplementation of lamb diet
increased a∗ values and reduced b∗ values as well as resulted
in a higher heme pigment content in the SM during refrigerated storage.
No significant differences were observed in glucose, lactate, and glycolytic
potential contents and drip loss among dietary groups (Table 1). In relation
to drip loss, in the study of Morán et al. (2012b), no significant
differences were observed in water-holding capacity between control and
groups supplemented with CA and vitamin E. Also, Skřivanová et al. (2007) and Vignola et al. (2009) demonstrated no effect of Se on natural
drip loss in meat. In turn, Li et al. (2011) showed that increased levels of
SeY decreased the drip loss in the muscle. Finally, Zhan et al. (2007)
showed significantly reduced drip loss in Se-methionine-treated group in
comparison to the control and the Na-selenite-treated group. These authors
concluded that dietary Se-methionine contributed to a significant decrease
in drip loss of the muscle by protecting membrane integrity.
The results showed that meat from lambs fed a diet with FO addition was
characterized by higher pH48 and lesser lightness, redness, and yellowness.
Similar results (regarding the indicated color parameters) were observed in
the group fed with addition of FO, CA, and inorganic Se (the V diet, Table 1). Zhan et al. (2007) reported that organic Se did not seem to affect meat
quality, but inorganic Se might have a detrimental effect on it.
Effect of different diets on protein profile of the longissimus muscle of lambs after 7 days of ageing (%).
Protein
Group
I
II
III
IV
V
Diet
Control
+1 % FO
+1 % FO +0.1 % CA
+1 % FO +0.1 % CA +0.35 ppm Se as SeY
+1 % FO +0.1 % CA +0.35 ppm Se as SeVI
Myosin HC
22.33ab ± 1.69
21.60ab ± 2.32
23.10b ± 2.83
20.37ac ± 2.26
18.36c ± 1.38
150 kDa
10.64a ± 1.45
10.85a ± 1.52
9.75ac ± 1.33
7.49b ± 0.94
8.42bc ± 1.15
α-actinin
7.20a ± 0.68
6.46b ± 0.26
6.79ab ± 0.59
7.03a ± 0.39
7.26a ± 0.27
μ-calpain
2.09 ± 0.30
2.16 ± 0.49
2.25 ± 0.24
2.38 ± 0.36
2.48 ± 0.23
80 kDa
2.29 ± 0.41
2.23 ± 0.88
2.13 ± 0.52
2.65 ± 1.27
2.68 ± 0.91
75 kDa
3.59 ± 0.41
4.34 ± 0.55
4.01 ± 0.54
4.12 ± 0.43
4.25 ± 0.37
60 kDa
4.32ab ± 0.71
4.06a ± 0.68
3.94a ± 0.52
4.91bc ± 0.53
5.30c ± 0.77
Actinin
23.96 ± 1.39
24.62 ± 1.81
25.83 ± 1.35
25.64 ± 1.93
24.63 ± 1.07
TnT
6.38ab ± 0.71
6.11a ± 0.38
4.99c ± 0.32
6.72b ± 0.41
6.24ab ± 0.42
Tropomyosin
6.82 ± 0.75
6.45 ± 0.54
5.68 ± 0.82
6.64 ± 1.43
6.42 ± 0.62
33 kDa
4.56 ± 1.73
4.23 ± 0.37
3.84 ± 0.56
4.34 ± 0.64
3.84 ± 0.51
30 kDa
1.78a ± 1.21
1.04a ± 0.62
1.93a ± 0.65
2.05ab ± 0.94
3.03b ± 0.76
Myosin LC1
2.67b ± 1.24
4.09a ± 0.42
4.22a ± 0.31
3.77ab ± 1.42
4.62a ± 0.70
TnI
1.35 ± 0.96
1.75 ± 1.19
1.49 ± 1.11
1.88 ± 1.34
2.44 ± 0.84
a,b,c – means with different superscripts in the same row differ
significantly at Pα≤0.05.
TnT – troponin T; TnI – troponin I; LC1 – light chain of myosin; FO – fish
oil; CA – carnosic acid; SeY – the organic chemical form of selenium; SeVI
– selenite.
Regarding CA addition to diets for lambs, it may be concluded that it
changed the ultimate pH and color parameters of meat. According to Morán
et al. (2012a, c) CA shows a lower antioxidant activity when compared to vitamin E but it
seems to be useful in protecting meat from discoloration. Bañon
et al. (2012) stated that dietary inclusion of rosemary clearly seems to be
promising as a nutritional strategy for improving meat quality. In a study
by Ortuño et al. (2014), dietary rosemary extract delayed lean and fat
discoloration, odor deterioration and microbial spoilage. However, in the
research by Aouadi et al. (2014), essential oils from a rosemary extract
were ineffective in preventing color deterioration.
Effect of lamb diets on the proteolysis and profile of muscle
proteins
Table 2 shows the protein profile of muscle tissue in five lamb groups.
Average values for all groups regarding contents of myosin, α-actinin, μ-calpain, actin, troponin T (TnT), tropomyosin, myosin LC1,
troponin I (TnI), represented 21.2, 6.9, 2.3, 24.9, 6.1, 6.4, 3.9, and
1.8 %, respectively. Kołczak et al. (2003) stated that the differences
of the composition of meat protein depended on the age of the animal, type of
muscle, and duration of chilled storage. The residual products of the
proteolytic changes during meat maturation were divided into polypeptides
with molecular weights between 60 and 78 kDa and polypeptides with an
approximate molecular weight of 30 kDa. The bands 76 and 78 kDa are
probably products of μ-calpain autolysis (Bee et al., 2007;
Huff-Lonergan and Lonergan, 2005, 2007). The 30 kDa polypeptides are the
products of post mortem proteolysis of TnT (Huff-Lonergan and Lonergan, 2005;
Huff-Lonergan et al., 2010) and are used as indicators of proteolysis (Hopkins and Thompson,
2002; Santé-Lhoutellier et al., 2008). It should be mentioned that
group II, fed FO, was characterized by higher ultimate pH (Table 1). The
results for the TnT also showed lower proteolysis of meat from groups I, II,
IV, and V because – as mentioned by Hopkins and Geesink (2009) – its
degradation can be interpreted as an indicator of post mortem proteolysis.
Electrophoretic profiles of muscle protein were affected by animal diet
(Table 2). Meat from the lambs fed the diet containing fish oil (FO) (group II) was characterized by higher abundance of myosin LC1 and a lower content
of α-actinin compared to control meat (group I). LC1 myosin content
was similar in group with Ca and inorganic Se (group III and V) as in
FO + CA group (II) (Table 2). The addition of CA caused troponin T (TnT)
to be less abundant in comparison to the samples from the control group (group I) and group II. However, the Se addition eliminated the influence
of CA on TnT content. The samples from groups IV and V contained more TnT
than these from group III (Table 2).
Effect of different diets on protein profile in drip loss of the
longissimus muscle of lambs after 7 days of ageing (%).
Protein
Group
I
II
III
IV
V
Diet
Control
+1 % FO
+1 % FO +0.1 % CA
+1 % FO +0.1 % CA +0.35 ppm Se as SeY
+1 % FO +0.1 % CA +0.35 ppm Se as SeVI
200 kDa
0.36a ± 0.29
0.50a ± 0.39
0.40a ± 0.18
0.99b ± 0.54
1.56c ± 0.48
150 kDa
0.90 ± .74
0.82 ± 0.43
0.47 ± 0.14
0.87 ± 0.59
1.00 ± 0.24
PHb/PHbK
3.87 ± 1.01
4.23 ± 0.80
3.33 ± 0.35
3.88 ± 0.87
4.73 ± 0.71
AMPDA
1.81a ± 0.45
1.87a ± 0.25
1.39a ± 0.37
1.80a ± 0.34
3.16b ± 0.26
PGM
10.58a ± 0.61
11.31b ± 0.55
10.52a ± 0.36
12.06c ± 0.90
10.51a ± 0.49
65 kDa
2.46a ± 0.11
2.35a ± 0.13
2.44a ± 0.19
2.96b ± 0.37
2.90b ± 0.19
PK/PGI
8.05ab ± 0.18
8.57c ± 0.35
8.07ab ± 0.56
8.49bc ± 0.38
7.80a ± 0.35
55 kDa
1.79a ± 0.30
1.10b ± 0.63
2.20a ± 0.17
2.16a ± 0.65
1.95a ± 0.33
53 kDa
1.13 ± 0.40
1.53 ± 0.79
0.58 ± 0.04
0.70 ± 0.49
0.94 ± 1.11
EN
7.86 ± 0.85
8.40 ± 0.43
8.01 ± 0.16
8.20 ± 0.56
8.09 ± 0.26
45 kDa
3.15 ± 1.06
2.96 ± 0.35
3.37 ± 0.19
3.35 ± 0.35
3.60 ± 0.54
CK/PGAK
15.62a ± 0.78
15.85a ± 0.38
16.32a ± 0.96
14.25b ± 0.56
14.12b ± 0.57
ALD
12.54c ± 0.70
12.42c ± 0.63
12.52c ± 0.15
11.14b ± 0.41
10.43a ± 0.32
GAPDH
11.54 ± 0.75
11.07 ± 0.69
11.49 ± 0.53
11.44 ± 0.86
11.74 ± 1.11
LDH
6.52a ± 0.48
6.62a ± 0.42
6.80a ± 0.26
5.84b ± 0.28
5.57b ± 0.21
PGAM
6.56a ± 0.46
5.70b ± 1.06
6.80a ± 0.33
6.21ab ± 0.28
6.19ab ± 0.48
TPI
3.66ac ± 0.21
3.29c ± 0.87
3.82ab ± 0.08
4.23b ± 0.16
4.01ab ± 0.22
23 kDa
1.57 ± 0.08
1.40 ± 0.13
1.45 ± 0.20
1.40 ± 0.32
1.67 ± 0.23
a,b,c – means with different superscripts in the same row differ
significantly at Pα≤0.05.
FO – fish oil; CA – carnosic acid; SeY – the organic chemical form of
selenium; SeVI – selenate; PHb/PHbK – phosphorylase b/phosphorylase b
kinase; PFK – phosphofructokinase; AMPDA – AMP deaminase; PGM –
phosphoglucomutase; PK/PGI – pyruvate kinase/phosphoglucose isomerase; EN –
enolase; CK/PGAK – creatine kinase/phosphoglycerate kinase; ALD – aldolase;
GAPDH – glyceraldehyde-3-phosphate dehydrogenase; LDH – lactate
dehydrogenase; PGAM – phosphoglycerate mutase; TPI – triosephosphate
isomerase.
Se supplementation decreased the content of myosin HC and 150 kDa
polypeptides in meat samples in comparison to group III. Simultaneously, Se
and CA addition caused the longissimus muscle to contain more α-actinin when
compared to group II. The addition of Se and CA eliminated the influence of
FO on α-actinin content. Furthermore, a significant increase in the
60 kDa bands was observed in meat samples from groups IV and V in comparison
to those from group III. Similarly, the 30 kDa peptides were significantly
more abundant in the samples from group V than from group III (Table 2). The observed results may also be due to the effect of combination of CA,
FO and Se added to the diet.
The significant effects of feeding on myofibrillar protein proteolysis,
sarcoplasmic protein profile and proteolytic enzymes activity have been
studied by several authors (McDonagh et al., 1999; Santé-Lhoutellier et
al., 2008; Costa-Lima et al., 2015; Maqsood et al., 2015). The results
presented in Table 2 indicate a relation between antioxidant compounds of
the diet and post mortem degradation of muscle proteins. Nevertheless, the results are
difficult to make some generalization. It may be, however, concluded that CA
promotes degradation of TnT, whereas the addition of SeVI to the feed
increases degradation of myosin HC and content of 30 kDa proteins. These
results in relation to TnT are in accordance with works of Rowe et al. (2004), Santé-Lhoutellier et al. (2008) and Maqsood et al. (2015) and
also confirm the protective and antioxidant effects of CA and Se in relation
to proteolysis of myofibrillar proteins. Berardo et al. (2015) reported that
protein oxidation reduces its proteolysis. Additionally, Shibata et al. (2009) demonstrated a significant effect of cattle feeding on troponin T,
troponin I and myosin HC content of semitendinosus muscle. Another study showed that
feeding of animals could influence on the content of amino acids such as
glutamine, aspartic acid, and taurine in muscle (Cornet and Bousset, 1999).
Results presented in Table 4 also showed that the addition of FO, CA, and Se
to animal diet influenced amino acid content in the longissimus muscle in the studied
groups of lambs.
Effect of different diets on the content (g/100 g fresh muscle
tissues) of amino acids (AAs) in the longissimus muscle of lambs.
Amino acids
Group
I
II
III
IV
V
Diet
Control
+1 % FO
+1 % FO +0.1 %CA
+1 % FO +0.1 % CA +0.35 ppm Se as SeY
+1 % FO +0.1 % CA +0.35 ppm Se as SeVI
Aspartic acid
1.74b
1.54a
1.56a
1.62ab
1.69ab
Glutamic acid
2.92b
2.62a
2.64a
2.62a
2.75ab
Asparagine
0.15
0.14
0.12
0.13
0.14
Histidine
1.54b
1.41a
1.33a
1.29a
1.34a
Serine
0.77b
0.68a
0.66a
0.71ab
0.67a
Arginine
2.02c
1.85c
1.72cb
1.58ab
1.63ab
Glycine
0.46b
0.41a
0.40a
0.42ab
0.44ab
Threonine
0.46b
0.40a
0.41a
0.43ab
0.45ab
Tyrosine
2.11ab
1.98ab
1.83a
2.07ab
2.23b
Alanine
0.06a
0.04a
0.04a
0.07a
0.12b
Methionine
0.19a
0.20a
0.22a
0.24ab
0.28b
Valine
0.46b
0.46b
0.47b
0.42ab
0.36a
Phenylalanine
0.55bc
0.48a
0.52ab
0.59bc
0.60c
iso-Leucine
0.63b
0.55a
0.56ab
0.61b
0.62b
Leucine
1.50b
1.36a
1.32a
1.32a
1.36a
Cysteine
0.26b
0.25b
0.23ab
0.19a
0.19a
homo-Cystine
0.006
0.005
0.006
0.006
0.006
Lysine
1.48c
1.34b
1.27ab
1.11a
1.21ab
∑AAs
17.32b
15.72a
15.31a
15.43a
16.08ab
∑IAAs
5.27b
4.80a
4.76a
4.72a
4.87ab
∑DAAs
12.04b
10.92a
10.54a
10.70a
11.20ab
∑IAAs / ∑DAAs
0.438a
0.440ab
0.451b
0.441ab
0.435a
∑IAAs / ∑AAs
0.305a
0.305a
0.311b
0.306a
0.303a
Met / ∑AAs
0.0108a
0.0130a
0.0143a
0.0158b
0.0171b
a,b,c – means with different superscripts in the same row differ
significantly at Pα≤0.05.
FO – fish oil; CA – carnosic acid; SeY – the organic chemical form of
selenium; SeVI – selenite; ∑AAs – the sum of amino acids content;
∑IAAs – the sum of indispensable amino acids content; ∑DAAs
– the sum of dispensable amino acids content; Met / ∑AAs – the ratio
of methionine (Met) to ∑AAs content.
Effect of lamb diets on drip loss protein profile
Table 3 presents the protein profile of the drip loss from meat samples of
the five analyzed groups. Among the identified proteins, the following ones
were the most abundant: creatine kinase/phosphoglycerate kinase (CK/PGAK),
15.2 %; aldolase (ALD), 11.8 %; glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), 11.5 %; phosphoglucomutase (PGM), 11.0 %;
pyruvate kinase/phosphoglucose isomerase (PK/PGI), 8.2 %; enolase (EN),
8.1 %; lactate dehydrogenase (LDH) 6.3 %; and phosphoglycerate mutase
(PGAM), 6.3 %. The study of the available literature shows only results in
relation to drip loss protein profile in other species than lambs.
Nevertheless, the levels of AMP deaminase, CK/PGAK, PK/PGI, LDH,
triosephosphate isomerase (TPI), GAPDH, and PGAM were similar to the ones
obtained by Pérez et al. (2003) for pork. While the PGM and ALD were
more abundant, EN was a little less abundant compared to the results reported
by Pérez et al. (2003). In comparison to the study of Marino et al. (2014), the profile of TPI and LDH in our results was similar. The presence
of other proteins proved to be similar in our study. For example, there was
more PGM, ALD, and GAPDH, whereas there was less EN than in the work of Marino
et al. (2014). However, a different content of PGAM was shown by these authors
as affected by cattle breed. The study by these authors demonstrated that
the percentages of the major sarcoplasmic protein bands extracted from the longissimus muscle were influenced by breed and ageing time. As was noted by
Marino et al. (2014), these changes could be related to the chemical
modification or other different mechanisms such as isoelectric precipitation
due to pH decline or post mortem degradation.
The dietary inclusion of FO was observed to influence the content of PGM,
PK/PGI, 55 kDa peptides, PGAM, and TPI in drip loss. Meat of lambs from
group II was characterized by a higher content of PGM and PK/PGI in drip
loss compared to meat of control lambs. The opposite dependency was observed
for 55 kDa peptides, PGAM, and TPI. The addition of CA eliminated such an
effect as samples from group III had a similar content of these proteins and
peptides as the control ones (Table 3).
Drip loss from meat samples from groups IV and V contained significantly
more 200 and 65 kDa polypeptides and less CK/PGAK, ALD, and LDH when
compared to group III. Moreover, drip loss from meat of animals fed with
SeVI contained more 200 kDa polyleptides and less ALD in comparison to
animals fed with SeY. The influence of SeVI on the content of AMPDA has also
been observed – this protein was more abundant in the samples from group V
than in group IV. Additionally, SeVI supplementation caused a decrease
in PK/PGI content in drip loss. However, a significantly higher
content of PGM was measured in drip loss from meat of lambs fed with SeY
(Table 3). Many studies have shown that sarcoplasmic proteins appeared as
indicators of meat ageing and as biomarkers of some quality attributes,
tenderness in particular (Di Luca et al., 2011; Sierra et al., 2012; Ouali
et al., 2013; Bowker et al., 2014; Marino et al., 2014; Picard et al.,
2015). These proteins include GAPDH, EN, LDH, PGM, TPI, and
phosphorylase b/phosphorylase b kinase (PHb/PHbK). Di Luca et al. (2013b)
stated that muscle exudate could be a good source of these proteins because
it provides valuable information about the pathways and processes underlying
the post mortem ageing period as well as highlighting its post mortem modifications. Some studies
have also shown that sarcoplasmic protein profile in drip loss is also
determined by the feeding of animals (Shibata et al., 2009; Costa-Lima et
al., 2015). These results demonstrated effects of feeding factors on the
abundance of glycolytic enzymes and indicated that they could potentially
affect changes occurring in muscle tissue after slaughter and, consequently,
the quality of meat. In our study, we also observed a feeding effect on
contents of the following sarcoplasmic proteins: AMPDA, PGM, PK/PGI,
CK/PGAK, ALD, LDH, PGAM, and TPI (Table 3).
According to the results presented in Table 3, the groups fed diets with Se
(IV and V) were characterized by lower contents of CK/PGAK, ALD, and LDH in
comparison to group III. These results are in part consistent with the study
of El-Neweehy et al. (2000), who showed that selenium contributed to a
significant decrease in serum muscle-specific enzymes such as LDH. Bostedt (1976) reported on a significant increase in muscle CPK (creatine
phosphokinase) in lambs of an endemic flock although they were clinically
healthy and returned to normal levels within 1 week after single injection
of selenium. Additionally, a research by Morán et al. (2012b) showed no
effect of CA (0.6 and 1.2 g kg-1) in the diet of lambs on the serum levels
of glucose, CPK, and LDH. However, it demonstrated a lower content of
creatine phosphokinase in the antioxidant groups (fed with carnosic acid and
vitamin E).
Correlation between sarcoplasmic proteins and some meat quality
traits
The statistical analysis showed intermediate and significant negative
correlations between pH48 and TPI and PGAM (r=-0.40;Pα≤0.05 for both enzymes). The correlation between sarcoplasmic proteins and
some meat quality traits as color stability or water-holding capacity has
been studied by many authors (Sayd et al., 2006; Di Luca et al., 2013b; Canto
et al., 2015; Gao et al., 2016; Nair et al., 2016). Di Luca et al. (2013b)
demonstrated lower abundance of TPI in the samples with high drip loss. This
enzyme plays an important role in glycolysis and catalyzes the
interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate
(Di Luca et al., 2013a). However, PGAM catalyzes the internal transfer of a
phosphate group, which results in the conversion of 3-phosphoglycerate to
2-phosphoglycerate (Scheffler and Gerrard, 2007). The lower level of these
two enzymes could be related to insufficient glycolysis and higher ultimate
pH in group II (Table 1), in other groups, characterized by higher
concentrations of these enzymes (especially PGAM), the pH48 was
significantly lower. Similarly, pH48 was related to EN, but positively;
the calculated correlation was r=0.40(Pα≤0.05). EN catalyzes
the penultimate reaction of the glycolysis pathway: dehydration of
2-phosphoglycerate to phosphoenolpyruvate. Sayd et al. (2006), Nair et
al. (2016) and Gao et al. (2016) indicated that EN was related rather to
color parameters of meat.
The obtained results showed also a significant correlation between color
parameter a∗ and ALD (r=0.50;Pα≤0.05) and LDH
(r= 0.40; Pα≤0.05). ALD is involved in the aldol cleavage
reaction. The substrate for ALD is fructose-1,6-bis-phosphate, which is
cleaved to the glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.
This is the final reaction of the first stage of glycogenolysis. Whereas LDH
catalyzes the last step of the glycolytic pathway, i.e., the conversion of
pyruvate to lactate. In a study conducted by Przybylski et al. (2016), a
significant correlation (r= 0.58) was observed between ALD and color
parameters. However, Gao et al. (2016) demonstrated a significant
correlation between redness (a∗ value) of sheep cardiac muscle and
CK. Żelechowska et al. (2012) and Ramirez et al. (2004) also reported on
significant correlations between ALD and the b∗ color parameter in pork
and rabbit meat, respectively.
Effect of lamb diets on amino acid profile in Longissimus muscle
The results summarized in Table 4 show that the diets fed to groups II to V
showed at least a tendency to decrease the sum of all amino acids (∑AAs) as well as of indispensable amino acids (∑IAAs) and
dispensable amino acids (∑DAAs) in the muscle as compared to the
control diet. In fact, the lower ∑AAs, including ∑IAAs and
∑DAAs, in the muscle of lambs fed the diets containing FO,
irrespective of the addition of CA, SeY or SeVI (the II–V diets), can be
attributed to the diminished ruminal microbial protein synthesis (i.e.,
bacterial growth in the rumen) or to stimulated ruminal bacterial
degradation of a larger portion of amino acids originating from dietary
protein in the rumen, especially when the diet was supplemented with FO and
CA (the III diet). Really, Maia et al. (2007) showed that the fish oil
long-chain polyunsaturated fatty acids (like eicosapentaenoic acid (EPA;
C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3)) inhibited the
growth of ruminal bacteria; toxicity to growth was ranked EPA > DHA > C18:3n-3
(αLNA) > C18:2n-6 (LA).
Similarly, Buccioni et al. (2012) showed that dietary FO (especially
long-chain polyunsaturated fatty acids) revealed a toxic effect on ruminal
microorganisms. Moreover, according to Dimov et al. (2012), the diet
composition may affect the capacity of the bacterial degradation of amino
acids originating from dietary proteins as well as bacterial protein
synthesis in the rumen.
Moreover, SeVI (the inorganic form of Se) added to the diet (the V diet)
tended to increase (P=0.07) the total content of amino acids (∑AAs) in the muscle in comparison with SeY (the organic form of Se) added to
the diet (the IV diet). In fact, the higher content of amino acids in the
longissimus muscle results in a lower content of fatty acids in the longissimus muscle (Jaworska
et al., 2016). In fact, numerous investigations support the concept that the
Se pro-oxidative effect of diets containing extra inorganic Se compounds
(like SeVI or selenite) is due to the catalysis of hydrosulfide oxidation
that results in decreasing the biosynthesis yield of lipogenic enzymes
(e.g., acetyl-CoA carboxylase and fatty acid synthase) in tissues of animals
(Navarro-Alarcon and Cabrera-Vique, 2008). In this study the lower content
of ∑AAs in the muscle of lambs fed the III diet compared with the
control group (the I diet) was observed and also lower content of FAs,
including saturated FAs, in the longissimus muscle was observed in work of Jaworska et al. (2016). Interestingly, this diet most efficiently increased the ratio of
indispensable amino acids (∑IAAs) to ∑AAs (∑IAAs / ∑AAs) content in the muscle. Indeed, the nutritive quality of the
longissimus muscle of lambs is related primarily to such indispensable amino acids like
histidine, methionine, lysine, and phenylalanine (Brzostowski et al.,
2008). Moreover, the current studies and our recent research (Jaworska et
al., 2016) showed that CA added to the diet enriched in FO (the III
diet) most efficiently increased the accumulation of unsaturated fatty acids
as well as improved the n-3PUFA to n-6PUFA (n-3PUFA / n-6PUFA) and ∑IAA
to ∑DAA (∑IAA / ∑DAA) ratios in the longissimus muscle of lambs.
Moreover, our recent research showed that dietary RO, FO, and CA, regardless
of the presence of SeVI, improved sensory texture quality of the
longissimus muscle of lambs (Jaworska et al., 2016). In fact, the inability of many
farm animals and humans to synthesize certain AAs has long triggered
tremendous interest in increasing contributions of IAAs (especially
methionine, lysine, and tryptophan) in diets (Ufaz and Galili, 2008).
Interestingly, the simultaneous addition of FO, CA, and SeVI to diet with RO
(the V diet) stimulated the incorporation of methionine in the muscle as
compared with the control, II, and III diets. Moreover, SeVI added to the
diet with FO, RO, and CA (group V) more efficiently increased the ∑AAs,
including tyrosine, alanine, methionine, and phenylalanine, in the muscle
than the III diet. Considering the above results, we argued that especially
the inorganic chemical form of dietary Se (i.e., SeVI) and particularly SeVI
metabolites (like metabolites of selenides or seleno-cysteine) reduced
bacterial degradation of these amino acids in the rumen and/or stimulated
the biosynthesis of proteins rich in these amino acids in the longissimus muscle of
lambs. Indeed, numerous studies have shown that dietary Se compounds
affected ruminal microorganism profile and activity of microbiota; hence, it
might be concluded that dietary Se compounds affect volatile fatty acid
production and yield of microbial fermentation in the rumen (Kim et al.,
1997; Del Razo-Rodrigues et al., 2013).
Considering the above results, we argued that dietary SeY and particularly
SeVI metabolites (like metabolites of selenides or seleno-cysteine) reduced
bacterial degradation of these amino acids in the rumen and/or stimulated
the biosynthesis of proteins rich in these amino acids in the longissimus muscle of
lambs.
Interestingly, the addition of Se (as SeY or SeVI) to the diet with FO, RO,
and CA most effectively increased the content of methionine, as well as the
ratio of methionine content to ∑AAs (Met / ∑AAs) in this muscle
(Table 4). Contrary to the V diet, the IV diet less efficiently (P=0.08)
stimulated the accumulation of methionine in the muscle because only a part
of dietary SeY (rich in Se-Met) is metabolized into metabolites of selenides
or seleno-cysteine in the body system of animals fed a diet with the adequate
concentration of Se (Navarro-Alarcon et al., 2008). These findings are consistent
with the effects of diets enriched in SeY or SeVI (the IV and V diets) on
the content of cysteine in the muscle of the examined lambs; the conversion
of methionine to cysteine is an irreversible process in the animal body
(Navarro-Alarcon et al., 2008). Consequently, the IV and V diets more effectively
decreased the content of cysteine in the muscle than the III diet (P < 0.1; the tendency) and especially the control and II diets (P≤0.05).
Interestingly, all supplemented diets (the II–V diets), especially the
diet with SeY (the IV diet), decreased (P≤0.05) the content of lysine in
the muscle compared with the control diets. Furthermore, the IV diet reduced
the content of lysine in the muscle as compared with the muscle of lambs fed
the II diet. Considering the above, we argued that the II–V diets
including FO (rich in long-chain PUFA), irrespective of the presence of
CA, SeVI, and especially SeY, decreased the biosynthesis yield of proteins
rich in lysine in the lambs' muscle.