Introduction consumer [1]. All these traits can be heavily affected by exercise immediately before slaughter. Two to three

1. Introduction consumer [1]. All these traits can be heavily affected by exercise immediately before slaughter. Two to three

Exercise changes muscle metabolism and related hours rest after stress before slaughtering seems to muscle composition, especially in untrained muscles. reverse the effect of exercise [2]. Pre-slaughter exercise Preslaughter pig transportation can be regarded as and time of rest between exercise and slaughter may exercise of untrained muscles. The resulting affect meat quality traits differently [3-8] because the biochemical changes affects proteome muscle underlying molecular mechanisms differ between traits. composition and biochemical processes related to Exercise is an energy consuming process [7, 9], and energy metabolism, which do also affect post mortem due to differences in the energy metabolism between processes affecting meat quality. different muscles the effect may vary among muscles Consumers’ perception of pork quality relies on the [7]. Pre-slaughter exercise e.g. during transportation stage in the entire consumption process. When buying may therefore affect post mortem metabolism and raw pork the appearance of the meat is consequently the meat quality [10]. The post mortem important-including technological meat quality traits proteome may be directly related to these processes and such as color, drip loss, inter and intra muscular fat, and changes in the proteome before or after slaughtering in texture. In the cooking process cooking loss is critical the search for biomarkers for post mortem protein and during consumption the taste and texture degradation [11-13]. Biomarkers known to be perceptions of the pork are most important for the associated with meat quality traits and effects of

exercise and rest could be used to optimize pig Corresponding author: Marinus F.W. te Pas, Ph.D., senior

treatment.

researcher, research fields: genomics, bioinformatics and systems biology. E-mail: marinus.tepas@wur.nl.

Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting 599

2.3 Statistical Analyses of Proteomics Data in Relation relations among the abundance of proteome profiles

The objective of this research was to investigate the

with Meat Quality Data and Stress and biochemical meat quality traits and exercise

The proteome profiles of all animals were analyzed followed by rest before slaughter, in samples removed for differences in abundance of individual peaks immediately after slaughter in two different pig between the treatment groups using the SELDI-TOF muscles. (BioRAD) software. Clustering was done using the

2. Materials and Methods

cluster algorithm that first ensures high signal to noise

2.1 Animal Treatment and Sampling peaks and than generates two-dimensional arrays of

peaks across multiple spectra. This cluster map was Animal management and experimental conditions

used for all downstream analyses. were as previously reported [14]. Briefly, the

Proteins were recognized by the M/Z values of their experiment was conducted with ten litters of four

peaks in the proteome profiles generated by the female pigs (Duroc × (Landrace and Yorkshire).

SELDI-TOF equipment. Statistical difference (P < Within litter, pigs were assigned to one of four

0.05) of the abundance of a protein between groups one treatments: control pigs without experimental exercise

and two was taken as an indication of immediate (group 1, control group), or pre-slaughter exercise (on

response to exercise, differences between groups two average 27 min running on a treadmill) followed by 0 h

and three, and between groups two and four were taken (group 2), or 1 h (group 3) or 3 h (group 4) of rest [14].

as an indication of a reaction to rest after exercise. Muscle biopsies were taken within 1 min after bleeding.

The proteome profiles were compared using The left longissimus dorsi (LD) was biopsy sampled 4

principal component analysis (PCA) using the SELDI cm cranially to the last rib and the biceps femoris (BF)

analysis software package. The procedure was done as

20 cm from the knee joint in the caudal direction indicated by the manufacturer and the four

towards the tail base. Samples were snap frozen in exercise-rest groups were visualized.

liquid nitrogen, transported on dry ice, and stored at Association between the abundance of a protein and

-80 ℃ until use for proteome isolation. meat quality or exercise-related biochemical traits [14]

2.2 Isolation of Proteome and Proteomics Data was studied using the SELDI-TOF software. The Generation

software calculates P-values using the Kruskal-Wallis non-parametric test. Afterwards these results were used

Isolation of the water soluble muscle proteome for comparison of the abundances of the proteins in the fraction, the sarcoplasmic proteome, was done as four treatment groups. The P-values were P < 0.05 for previously described [15, 16]. In Ref. [14] pH significant results and P < 0.1 for a tendency of effect. differences between the sample groups were described These final data indicate the relation of a protein and the isolation buffer has low buffering capacity. (represented by its M/Z value) to meat quality and/or However, the pH of the small sample size (30-50 mg) biochemical traits, as well as exercise/rest of the pigs. could be buffered by the large buffer volume (1.5 mL).

Furthermore, no protein concentration differences were

3. Results

noted. Proteomics profiles were generated using the

3.1 Proteomics Profiles of LD and BF Muscles SELDI-TOF PCS4000 Enterprise (BioRAD,

Veenendaal, The Netherlands) equipment and the array The proteome profiles of both muscles from all 40 types CM10, IMAC30, and Q10 each with different

animals were determined on three array types (Table 1). binding characteristics (BioRad).

The number of peaks in the profiles varies from 92 to

600 Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting

136 and the peaks vary from 1-278 kDa. The CM 10, a (control) and group 2 (exercised pigs) was not always more general protein binding array type showed the

statistically significant due to delayed reaction to stress. highest number of peaks. For a given array type the

Indeed, the abundance patterns in especially the BF samples of all animals were run on the equipment in a

muscle indicated that a delayed reaction to stress could single run making peak height as a measure for

be measured with this method in groups 3 and 4 (Figs. expression level better comparable and there is no

1C and 1F).

variation in number of peaks between the animals. In a In the cluster plot analysis shown for the LD muscle good replicate this variation is usually less than 5%.

seven proteins showed statistically different abundance Except for the Q10 array type the number of peaks

in the groups of exercise followed by rest for different within the profiles for the two muscles show good

time before slaughtering. Four proteins had increased similarity. When analyzing the peak identity based on

abundance after exercise and three peaks showed m/z values the similarity of the peaks between the two

reduced abundance after exercise. The BF muscle muscles is 70-86 percent.

showed that the abundance of 17 protein peaks differed between the four groups, nine of which showed

3.2 Relation between Proteome Peak Patterns and increased abundance and three of them showed Stress decreased abundance after exercise. The remaining five The effect of exercise on the abundance of muscle

proteins can be evaluated with different parameters. First, we measured the number of proteins with different abundance between the groups without exercise (control, group 1) and the group exercised on a treadmill without rest before slaughtering (group 2). In

the LD muscle six protein peaks showed significant differences with five proteins showing increased abundances and one protein with decreased abundance after exercise. In the BF muscle ten protein peaks were significantly different with five increased and five

decreased abundances. A small protein peak of 1.2 kDa was similar for LD and BF.

Using a cluster plot analysis method we evaluated the whole pattern of changed protein abundances from

Fig. 1 Examples of abundance profiles of proteins in LD or

control (group 1) to exercised pigs (group 2), and from

BF related to exercise and rest (groups 1-4). The

exercised pigs to those rested for 1 (group 3) and 3

SELDI-TOF equipment measured the amount of ions after

hours (group 4) before slaughter. It should be noted that

the separation on molecule mass during the time of flight,

thus the dimension of the Y-axis is μA. Measurements are absolute and normalized. A: reduced abundance after

the direct effect of exercise - i.e. comparison of group 1

Table 1 Proteome profile data of the LD and BF muscles. exercise followed by recovery of the situation before the

Peak numbers

Similar peaks

exercise during 3 h of rest. B: increased abundance after

Array type LD exercise followed by overcompensation during rest resulting BF LD - BF

in lower abundance after 3 h rest. C, D: delayed reaction to

CM10 136 133 95

exercise leading to reduced or increased abundance after 3 h

IMAC30 92 94 68

rest. E: delayed increased abundance of the protein followed Q10 95 111 82 by recovery. F: delayed increased abundance of the protein

Total 323 338 245

without recovery during the 3 h rest period.

Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting 601

showed a delayed response (i.e. after 1 or after 3 h rest). It is uncertain whether this response is due to the exercise or the rest. The protein peaks of LD and BF are different. Fig. 1 shows some examples of the diverse exercise-related protein abundance profiles. In response to exercise the abundance of a protein may be either decreased (Fig. 1A), increased (Fig. 1B) or expressed as a delayed reaction (Fig. 1C-F). During rest the situation that existed before exercise may be either restored (Fig. 1A, E), over compensated (Fig. 1B) or not restored (Fig. 1C, D, F).

Furthermore, the results indicate differences between animals for phenotypic resistance to exercise

e.g. the two high expressing animals in group 1 of Fig. 1A probably reacted more to exercise than the lower expressing animals for this protein. Such animal-specific differences have to be taken into account when developing a biomarker for exercise and rest.

Fig. 2 Principal component analysis of the exercise-rest

Because of the overlap in expression levels of the

groups using the whole proteome expression data. The LD

proteome between the four exercise-rest groups a PCA

data are shown on the left; the BF data are shown on the

analysis using the whole proteome dataset was

right, from top to bottom array types CM10, IMAC30, and

performed with the four exercise-rest groups as

Q10. The results of group 1 animals (i.e. control animals) are shown in red, group 2 animals (exercise, no rest) in blue,

determining factors. The analysis was performed for

group 3 (exercise followed by 1 h rest) in green, and group 4

each muscle and each array type individually. Only

(exercise followed by 3 h rest) in pink.

PCA components 1, 2, and 3 were used as they

3.3 Relation between Protein Abundance Patterns and explained 45-69 percent of the variation. The results Biochemical Metabolism and Meat Quality Traits indicate that the variation within the groups is such that

the four exercise-rest groups show overlap, but can also The number of peaks relating to metabolic traits and

be grouped as distinct groups (Fig. 2). Fig. 2 shows the meat quality traits are shown for each muscle in Table PCA analysis with ovals indicating the suggested

2. The number of associating proteins was higher for boundaries of the groups. However, in three of the

the Biceps femoris (BF) muscle than for the LD muscle analyses the results of group 1 covered the whole area

especially for the ADP, ATP and Pro glycogen. For the and drawing this oval was omitted. Fig. 2 shows that

other investigated meat quality traits comparable either two PCA components or the three-dimensional

numbers of proteins between the two muscles (LD and representation with PCA components 1, 2, and 3 draw

BF) with association to meat quality traits were found. the distinction between the exercise-rest groups best.

Possible interactions were studied further using the Fig. 2 shows that the general expression level of the

Biomarkers Pattern software (BPS) where more muscle proteome in both muscles seem to change due

proteins are added to a protein pattern thereby to the respective treatments, but also show a large

improving the association between protein abundance amount of overlap.

patterns and the measured traits. While the BPS mainly

602 Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting

Table 2 Number of proteins associated significantly with

exercise/rest groups 2 and 4 (3 h rested pigs) (e.g. Figs.

biochemical and meat quality exercise-related traits in two muscles.

1A, 1B, 1D, 1F, but not 1E). Thus, it can be concluded Trait LD BF

that rest following exercise affected the expression of CP 18 14

the exercise-induced alterations in the protein IMP 2 22

abundances. Furthermore, most of these effects are ADP 4 24

maintained after one hour of rest (group 3), and even ATP 3 31

Pro glycogen

22 41 after three hours rest following exercise (group 4) the Total Glycogen

26 27 effect of exercise on the protein abundance may not be pH 27 17

reversed (e.g. Fig. 1D).

Drip loss

Minolta L

2 2 Moreover, 22 of 49 proteins did not show significant Minolta b

15 14 differences in these two rest groups (groups 3 and 4)

found combinations of peaks with individual indicating that the exercise-associated difference was association to the traits in the LD muscle it included

not followed by an effect of rest to reverse the effect of new peaks lacking individual association to the groups

exercise. Alternatively, there may be a sequence of in the BF muscle. However, despite this the best

steps for recovery from exercise, and these proteins specificity and sensitivity of these trees never exceeded

could be related to a late effect. This hypothesis was 70-75 percent (data not shown). Thus, explaining

supported by the observation that five peaks showed no exercise and predicting the effect of exercise and rest

significant differences between groups 2 and 3, while remained difficult.

differences were observed between groups 2 and 4 indicating a late reaction to exercise. Furthermore, we

3.4 Relation of Protein Abundances to Meat Quality observed seven proteins with significant differences

Traits and Stress/Rest Effects between groups 2 (exercised, no rest) and 3 (1 h rest)

Using the peaks with association with meat quality and groups 2 and 4 (3 h rest), but without significant data (Table 2) we investigated association to exercise

difference between groups 1 and 2 (e.g. Fig. 1E, 1F). and rest of these peaks (Table 3). Using the difference

This may also indicate a late reaction to exercise, between group 1 (control) and group 2 (exercised pigs)

and/or rest.

indicating the direct exercise-associated effect as a

A number of associations were found between selection criteria we obtained 22 proteins (18 for the

proteins and the metabolic traits ADP, ATP, creatine BF muscle, 4 for the LD muscle) that associated both to

phosphate, pro-glycogen and total glycogen; the the effect of exercise and rest time, and to meat quality

number of associations was 1, 11, 5, 11 and 8 proteins traits. To get a better picture for the LD muscle we

respectively. Eight associations were observed between increased the P-value to P < 0.1. This increased P-value

protein peaks, exercise-rest, and the Inositol mono increased the number of associations to 49 (12 LD, 37

phosphate measurements. Two proteins were associated BF muscle associations). The P-values for the

with the pH 24 hour trait and four proteins were differences between groups 2 (exercised pigs) and 3 (1

associated with drip loss. Some protein-associations

h rest), and groups 2 and 4 (3 h rest) indicate the with the meat color-determinants b* and L* were reaction of the proteome to rest after exercise. Of these

observed in both muscles. However, these color

49 proteins 21 also showed significant difference determinants of the BF muscle were not significantly between exercise/rest groups 2 (exercised) and 3 (1 h

affected by exercise and rest like in the LD muscle. rested) (e.g. Figs. 1A, 1B, 1E, 1F), and 17 of these 21

Finally, in searching for protein biomarkers that could also showed significant difference between

be both indicators for meat quality and exercise-related

Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting 603

Table 3 Proteins (indicated by their M/Z values) associated with meat quality/biochemical stress-related traits, and exercise-rest treatment groups. A: LD, B: BF muscle.

A Array type M/Z value

P-value 1 P1-2 2 P2-3 2 P2-4 2 CM10

Trait

0.08 0.03 2.30E-04 IMAC30 6700

Inositol mono phosphate

0.01 IMAC30 8492

Minolta l

creatine phosphate

0.06 IMAC30 72648

Minolta b 0.010885

0.06 Q10 5015

pH24h

0.007281 0.06 Q10 5015

Pro glycogen

9.48E-04 0.06 Q10 12458 Pro glycogen

Total glycogen

0.019172 0.06 Q10 12458 Total glycogen

0.005583 0.06 Q10 33617 Total glycogen

0.017758 0.06 Q10 36324 Total glycogen

0.035421 0.06 0.08 B

Array type M/Z value

P-value 1 P1-2 2 P2-3 2 P2-4 2 CM10 5468

Creatine phosphate

0.05 0.002 CM10

Drip loss

Inositol mono phosphate

Inositol mono phosphate

Creatine phosphate

0.004 0.003 CM10

Drip loss

Inositol mono phosphate

Pro glycogen

Minolta b 0.039833 0.05

Pro glycogen

Minolta b 0.03617 0.08

0.03 0.04 CM10 17368

pH24h

Inositol mono phosphate

Minolta b 0.048107 0.1

CM10 58061

0.03 0.001 CM10 58061

Drip loss

Pro glycogen

0.025282 0.06 (to be continued)

604 Proteome Profiles of Longissimus and Biceps Femoris Porcine Muscles Related to Exercise and Resting

0.029985 0.06 IMAC30 7681

IMAC30 7634

Pro glycogen

0.014793 0.06 IMAC30 8485

Pro glycogen

0.005924 0.1 IMAC30 8485

ATP

9.48E-04 0.1 IMAC30 8485

Pro glycogen

0.006127 0.1 IMAC30 15053

Total glycogen

0.017758 0.05 IMAC30 15053

Pro glycogen

0.036085 0.05 IMAC30 15252

Total glycogen

0.010142 0.03 IMAC30 15252

Pro glycogen

0.025472 0.03 IMAC30 15346

Total glycogen

Pro glycogen

0.040373 0.04 0.1 Q10 20167 Drip loss

Total glycogen

0.1 Q10 20167 IMP

0.1 Q10 20167 Minolta b 0.039833 0.03 0.1

Q10 20164 Minolta l 0.025347 0.03 0.1 Q10 58571 Creatine phosphate

0.019915 0.08 1: P-value of the association with the biochemical/meat quality traits (trait column). 2: P-values of the association with the exercise/rest traits.

effects it was noted that several proteins associated

a cause of severely reduced meat quality. Rest for a with more than one trait (Table 3). The protein with

period of time before slaughter reverses the effect of M/Z of 8112 associated with ADP, ATP, creatine

exercise and restores biochemical parameters and meat phosphate, inositol mono phosphate and drip loss in the

quality. For that reason pigs are rested at the BF muscle. No protein peaks with such associations

slaughterhouse facility after transportation, which can could be shown in the LD muscle. Interestingly, the

be considered as an exercise event. One could imagine pro- and macro-glycogen associated with several peaks

that the nature and the amount of exercise affect the (M/Z: 5015 (in LD), 8485, 12458, 15053, 15252,

required length of the rest period. In the present study 15346). In only three of these associations also ATP

we focused on an exercise model that can be used as a concentration associated with the two glycogen traits,

model for transport exercise stress: exercise for 27 but none of the other traits associated with these traits

minutes on a treadmill. Compared to conventional within a single protein peak. These results may indicate

transport the length of the exercise is limited, but the different independent mechanisms taking place during

intensity of the exercise is high [3, 17, 18]. A high the biochemical reaction to the exercise and rest

intensity of the exercise may enable to study all effects periods, with ATP as the central energy carrying

generated by the exercise, including effects that molecule connecting the mechanisms. Furthermore,

otherwise may not have become significant. On the several proteins associated with more than one

other hand, it should be realized that transport exercise biochemical/meat quality trait and also with stress is a combination of exercise stress and noise, exercise/rest. Such protein peaks could potentially

housing, and different handling related to driving the become biomarkers to monitor exercise-rest effects,

animals into a desired direction, i.e. the stressful and meat quality development during rest.

unknown housing of a truck. So, this model of exercise and transport generates also a valuable tool to study