Profile of fatty acids, muscle structure and shear force of musculus longissimus dorsi (MLD) in growing pigs as affected by energy and protein or protein restriction followed by realimentation

Meat Science 91 (2012) 339–346

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Profile of fatty acids, muscle structure and shear force of musculus longissimus dorsi (MLD) in growing pigs as affected by energy and protein or protein restriction followed by realimentation Grzegorz Skiba a,⁎, Stanisława Raj a, Ewa Poławska b, Barbara Pastuszewska a, Gabriela Elminowska-Wenda c, Joanna Bogucka c, Damian Knecht d a

The Kielanowski Institute of Animal Nutrition and Physiology, Polish Academy of Sciences, Jabłonna, Poland Polish Academy of Sciences, Institute of Genetics and Animal Breeding, Jastrzębiec, Wólka Kosowska, Poland c University of Technology and Life Sciences, Department of Histology, Bydgoszcz, Poland d University of Environmental and Life Sciences, The Faculty of Biology and Animal Science, Institute of Animal Breeding, Wrocław, Poland b

a r t i c l e

i n f o

Article history: Received 29 August 2011 Received in revised form 3 February 2012 Accepted 13 February 2012 Keywords: Pigs Compensatory growth Meat tenderness Muscle structure Fatty acids profile

a b s t r a c t Forty-eight gilts were submitted to a 30% restriction of feed (groups F and F1) or protein intake (group P) from 90 to 118 days of age, followed by realimentation from 119 to 168 days of age. Control pigs (C) were fed during the whole experiment according to a semi ad libitum scale. During realimentation all animals were fed according to semi ad libitum scale except pigs F1 which were fed ad libitum. Six pigs from each group were slaughtered at the end of restriction and realimentation. Restriction decreased the weight of musculus longissimus dorsi (MLD) and increased shear force. Restriction of feed intake depressed MUFA concentration and increased n-6/n-3 ratio while protein restriction decreased n-6/n-3 and PUFA:SFA ratios. Structure of fibers was not affected. After realimentation MLD mass was still lower in all previously restricted pigs, shear force was the lowest in F1 pigs. Only percentage of fast twich oxidative fibers was significantly greater in F1 pigs than in others. Significant correlations between parameters investigated during the study were found. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Nutrition is one of many factors which affect quality of pork. Many authors reported that level of nutrition can influence intramuscular fat content (Masson et al., 2005; Skiba, 2010; Więcek, Rekiel, & Skomiał, 2010), while recent studies of Stolzenbach et al. (2009) showed that feeding strategy including compensatory growth can improve also meat tenderness/shear force. A positive effect of compensatory growth on shear force of meat during few weeks of realimentation was also reported by Skiba (2010) but other authors did not find such effect (Heyer & Lebret, 2007). Feeding intensity and strategy of compensatory growth influence amount of fat deposited in the body (Skiba, Fandrejewski, Raj, & Weremko, 2005) and also its fatty acid profile (Więcek, Rekiel, Batorska, & Skomiał, 2011), mainly due to the changes of activity of lipogenic enzymes (Daza et al., 2007). The results are, however, often inconsistent because of differences in genetic origin of pigs and experimental design as well.

⁎ Corresponding author. Tel.: + 48 22 765 33 66. E-mail address: [email protected] (G. Skiba). 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2012.02.013

Some authors postulate that there are relationships between fatty acid profile and type of fibers in the muscle (Andres et al., 2001). It seems therefore, that modification of muscle structure can affect meat quality but in the studies on the morphology of the muscle the relationships between these traits and parameters of pork quality were not analyzed. Moreover, the muscle structure was usually determined only at one stage of growth, prevalently at slaughter weight. The objective of the present experiment was to determine the effect of different strategies of pig nutrition (restriction and realimentation) on intramuscular fat content and fatty acids profile, shear force and structure of the musculus longissimus dorsi, and to estimate possible interrelationships between these parameters. The pigs were subjected to the restriction of energy and protein or only protein intake in the early growth stage (91st to 118th day of life) followed by realimentation till slaughter weight at the age of 168 days. The compensatory growth was induced to diversify amount and composition of fat deposited in the muscle without supplementation of the diet with fat. It was assumed that different intake of energy and protein in early growth of pigs will change characteristic of the musculus longissimus dorsi and that these changes will be maintained till heavier body mass.

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2. Materials and methods

Table 2 Scheme of the experiment.

2.1. Animals, diets and experimental procedures

Growth stage

The experimental procedures were approved by the Local Ethical Commission. The experiment was performed on 54 gilts, progeny of a Danish Landrace boar and seven half-sisters Large White sows. At the age of 90 days the animals were allotted to four experimental groups of 12 gilts, and one additional “zero” group of 6 gilts, two or three pigs from each litter being assigned to the treatment to minimize genetic variability. Two diets were formulated: diet A and diet P containing about 70% less protein than diet A (Table 1). The experiment comprised a restriction period from 91st to 118th day of life and a realimentation period from 119th to 168th day of life. Control animals (group C) were fed during both periods on diet A according to a semi ad libitum scale (approximately 95% of ad libitum intake). During the restriction period two groups of animals (F and F1) were fed diet A at the feeding level of approximately 70% of feed intake in group C, and one group (P) was fed on a low-protein P diet to consume 70% less of protein only. During the realimentation period all groups were fed on diet A at the semi ad libitum scale (approximately 95% of ad libitum intake), except F1 group which was fed ad libitum. The scheme of the study is presented in Table 2. The aim of the application a semi ad libitum scale in feeding of pigs was to fully control a nutrient intake during restriction. Such kind of feeding during realimentation allowed pigs to exhibit a “clear” compensation (without influence of feed intake). According to results of earlier studies when pigs are re-alimented ad libitum (e.g. Bikker, 1994; De Greef,

Table 1 Composition and profile of fatty acids of experimental diets. Ingredients

Diet A

P

Barley, g/kg Wheat, g/kg Triticale, g/kg Maize, g/kg Soybean meal, g/kg Rapeseed meal, g/kg Premixa, g/kg Cereal starch, g/kg Chemical composition Dry matter, g/kg Crude protein, g/kg Fat (extract ether), g/kg Ash, g/kg Nitrogen free extract, g/kg Crude fiber, g/kg ADF, g/kg NDF, g/kg ADL, g/kg Starch, g/kg

250 245 90 200 160 30 25 – C18:2n-6 874.4 167.5 26.4 43.0 602.1 35.4 52.5 135.8 10.9 461.0

310 200 90 – 100 – 25 275 25.25 888.7 112.3 16.5 43.5 685.8 30.6 42.9 107.8 8.6 572.8

Sugar, g/kg Organic matter, g/kg Lysineb, g/kg Methionineb, g/kg Threonineb, g/kg Tryptophanb, g/kg Metabolisable energy, MJ/kg

36.1 831.4 8.26 2.41 4.74 1.59 12.86

24.7 845.2 5.72 1.68 3.30 1.10 13.63

a

Profile of fatty acids, %

Diet A

P

C14:0 C16:0 C18:0 Σ SFA C16:1n-7 C18:1n-9 C18:1n-7 Σ MUFA 27.39 C18:3n-6 C18:3n-3 C20:3n-6 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 Σ PUFA PUFA/SFA C18:2n-6/ C18:3n-3 Σ n-6/Σn-3

0.21 24.77 3.84 32.78 0.3 34.08 2.09 37.92

0.33 28.94 4.25 37.27 0.3 26.20 1.71 30.05

0 1.36 0 0.52 0 0.07 0 27.41 0.84 18.57

0.04 1.65 0 0.68 0 0.06 0 30.07 0.8 16.60

17.65

15.88

Premix contained per kg (Diet A, values in parentheses refer to Diet P): 211.9 (232.9) g Ca; 34.52(46.46) g P; 45.46 (51.40) g Na; 2.46 (2.46) g Fe; 3.29 (3.29) g Zn; 1.03 (1.03) g Cu; 1.23 (1.23) g Mn; 12.3 (12.3) g I; 8.2 (8.2) g Se; 206,000 (206,000) IU vitamin A; 25,000 (25,000) IU vitamin D3; 1650 (1650) mg vitamin E; 2.46 (2.46) mg vitamin K; 82.3 (82.3) mg vitamin B1; 820 (820) mg vitamin B2; 0.82 (0.82) mg vitamin B12; 0.006 (0.006) mg biotin; 0.053 (0.053) mg folic acid; 620.7 (620.7) mg nicotinic acid; 410.9 (410.9) mg calcium pantothenate; 40.8 (40.8) mg choline chloride; 87.0 (69.0) g lysine; 7.84 (10.68) g methionine; 13.46 (17.23) g threonine; 5.54 (3.96) g tryptophan. b Apparent ileal digestible according to the CVB (1995).

Daily intake of energy/protein as a percent of intake of control group

Restriction (90–118 days of life) Realimentation (119–168 days of life) a

Ca

P

F

F1

100/100 100/100

100/70 100/100

70/70 100/100

70/70 Ad libitum

95% of ad libitum intake.

1992) these previously restricted with feed (protein and energy) intake show a higher appetite. Contrary, those previously restricted with protein intake only characterize feed intake similar to control animals. Thus, to measure an influence of voluntary feed intake on investigated parameters during realimentation period the ad libitum feeding of F1 pigs was used. Animal age plays a crucial role in the processes of both “normal” and compensatory growth (Hogg, 1991; Lawrence & Flower, 2002). For this reason scheme of the experiment assumed comparison of pigs at similar age (not body weight), thus body weight and other investigated features was only result of applied feeding procedure. The animals were maintained individually in pens equipped with automatic feeders and nipple drinkers, in thermally neutral environment. Feed allowances for pigs were changed every week adjusted to experimental protocol, except pigs of F1 group during realimentration when they were fed ad libitum. Semi ad libitum intake of control pigs during each week/period of the experiment was assumed based on ad libitum intake of this genotype of pigs recorded in our earlier studies (unpublished data). On the base of this intake a restriction level for F and F1 pigs were calculated to these animals consumed 70% of feed (lysine and ME) in group C. Due to a higher energy content in the diet P daily allowance during following week of the restriction for these animals was adjusted to they consumed daily the same amount of energy, but only 70% of protein as C pigs. The average quantities of feed and daily intake of apparent ileal digestible lysine and metabolisable energy, allowed per pig are presented in Table 3. At the age of 90 days six pigs from “zero” group were slaughtered by exsanguination after electrical stunning. Six gilts from each group were slaughtered at the end of restriction period and six at the end of realimentation, at the age of 118 and 168 days, respectively. Immediately after slaughter carcass was weighted afterwards halved, and samples of the musculus longissimus dorsi (MLD) from the last rib area of the right half of the carcass were taken for histological analysis and stored at — 20 °C. The carcasses were chilled at 4 °C for 24 h. Then, at the left half-carcass of each pigs a backfat thickness was measured at 5 points (above: neck, last rib, and musculus gluteus at three points — front, middle and hind). From these measurements an average value was calculated. Next, the entire MLD was dissected from the left half of the carcass and weighed. Meat content in the carcass of pigs at 118 and 168 days of life was calculated according to formula

Table 3 Diets fed during the experiment and average daily intake of feed, apparent ileal digestible lysine (Lys) and metabolisable energy (ME) in the experimental groups. Experimental period

Age (day)

Restriction

90–118

Realimentation

119–168

Group

Diet kg/day Lys, g/day ME, MJ/day Diet kg/day Lys, g/day ME, MJ/day

C

P

F

F1

A 2.13 17.6 27.3 A 2.70 22.3 34.7

P 2.00 12.3 27.3 A 2.70 22.3 34.7

A 1.50 12.3 19.1 A 2..70 22.3 34.7

A 1.50 12.3 19.1 A Ad libitum (2.85) 23.5 36.6

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used in Poland (Różycki, 1996). The sections from the 8th to 12th thoracic vertebra were taken for determination of chemical composition, fatty acids concentration and the Warner–Bratzler shear force. Measurement of shear force was recorded only 24 h after slaughter because in this study an influence of temporary restriction feeding and following realimentation on among other things was investigated. According to previous studies such feeding scheme influence on rate of protein turn-over, especially activity of proteolytic enzymes (mainly from calpain family) which largely influence shear force/ tenderness of meat (e.g. Skiba, Raj, Weremko, & Fandrejewski, 2009; Therkildsen et al., 2002). Thus, it was assumed that compensating animals will differ from controls in rate of muscle proteolysis (due to different enzymes activity) and consequently value of shear force just at slaughter time.

2.2. Analysis and measurements Chemical composition of diets and fat content in MLD were determined according to Association of Analytical Chemists methods (AOAC, 1994). Fatty acids were analyzed in homogenized samples using methyl esters method (Folch, Lees, & Stanley, 1957). Methyl esters of fatty acids were separated by gas chromatography on GC-2010 AF Schimadzu chromatograph equipped with a BPX70 capillary column (60 m × 0.25 mm × 0.25 μm) with helium as a carrier gas. The Warner–Bratzler shear force (WBSF) was determined in samples (approximately 400 g) thawed at 4 °C for 24 h next heat-treated in circulating water at 70 °C for 90 min. Heated samples were cut parallel to muscle fibers into cubes of 3 × 1 × 1 cm (length × height × width, respectively), eight cubes per sample. WBSF measurements were performed on a TA-HDi Texture Analyzer (UK) mounted with a 50 kg load cell and rectangular blade, using a crosshead speed of 50 mm/min, the maximum force (kg/cm 2) being recorded. Characteristics of muscle fibers were determined in the cross sections of MLD (10 μm thick) cut with cryostat microtome (Leica, Bensheim, Germany) at −20 °C. The histochemical reaction of NADH dehydrogenase (Fiedler & Weber, 1981) was used to differentiate the type of fibers for STO-slow twich oxidative — red, FTO — fast twitch oxidative/intermediate, and FTG — fast twich glycolytic/white, and to measure their diameter. Microstructural traits were estimated by counting the number of fibers per type (for the calculation of fiber type semi quantitative image system AMBA (IBSB, Berlin, Germany)). Because of the special structure of the MLD and clustered slow fibers, only fibers of the primary bundles, which were completely visible, were analyzed. Bundles were randomly selected from the slices. The total number of fibers was calculated by multiplying the number of fibers per cm 2 by the area of the muscle, determined by planimetry.

2.3. Calculations and statistics Results were analyzed separately for the period of restrictive feeding and realimentation. They comprised final body mass, daily body gain, MLD mass, content of intramuscular fat in MLD, proportions of SFA, MUFA, PUFA (including 18:2n-6 and 18:3n-3) in the sum of fatty acids, ratios of 18:2n-6/18:3n-3 and PUFA/SFA, proportions (%) of STO, FTO and FTG fiber types and their diameter (μm) and shear force (WBSF) in kg/cm 2. The results were analyzed using one-way ANOVA. Differences between groups were tested using the Tukey test. Due to the close relation between animals (litters) and the identical ages of pigs at slaughter, influence of these treatments was omitted in statistical analyses. Pearson's correlation was used to estimate the relationships between WBSF and the analyzed features. Statistical analysis was performed using Statgraphics Centurion (2005) software.

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3. Results 3.1. Growth performance and carcass characteristics of pigs during restriction and realimentation periods Carcass characteristic at the beginning of the study are presented in Table 4. Restriction of protein intake caused considerable decrease of daily body weight gain but the negative effect of feed restriction (energy and protein) was greater (Table 5). Consequently, at the end of the restriction period mean body weight and mass of the warm carcass of pigs from P group was lower respectively by 4.6 and 3.4 kg than of controls while weight of the F and F1 pigs was about 11.6 and 8.5 kg lower. Backfat thickness of P did not differ from C pigs, however in F and F1 animals was lower (P b 0.01). Carcass of F and F1 pigs characterized over 4% higher meat content (P b 0.01) compared to P and C animals. Compensatory growth rate was much smaller in protein than in feed restricted pigs and was the greatest in F1 pigs which during realimentation were fed ad libitum. Also the final body weight was the greatest in gilts from F1 group, followed by P and C group, and was the smallest in F pigs. Mass of the warm carcass in F1 and C animals was similar (on average 96.1 kg) and the heaviest (P b 0.05) compared to P and F pigs (91.2 and 89.3 kg). Backfat thickness of F pigs was the lowest, however F1 animals the highest compared to P and C gilts (21.6 and 26.8 vs. 25.2 and 24.3 mm, respectively, P b 0.01). Pigs of the F and C group had higher meat content in the carcass compared to P and F1 animals (on average 53.2 vs. 51.8%, P b 0.05). 3.2. Muscle characteristics after restriction period The mass of MLD and its characteristic at the beginning of the study are presented in Table 4. After restriction mass of the MLD was negatively affected by both types of restriction but the effect of lower feed intake was significantly greater than of protein intake since the MLD mass was depressed from 1863 to 1566 g (mean of F and F1 values) by feed, and to 1677 g by protein restriction (Table 6). Intramuscular fat concentration tended (Pb 0.09) to be lower in pigs submitted to feed than to protein

Table 4 Carcass characteristics, mass of the longissimus dorsi muscle, intramuscular fat concentration, profile of fatty acids, structure and diameter of muscle fibers and meat shear force at the beginning of the study (90 days of life). Item

Value

S.D.

Warm carcass weight, kg Backfat thickness, mma Meat content in the carcass, % Mass of the longissimus muscle, g Intramuscular fat content, % SFA, % MUFA, % PUFA, % C18:3n-3, % C18:2n-6, % C18:2n-6:C18:3n-3 PUFA/SFA STO, % FTO, % FTG, % STO, μm FTO, μm FTG, μm Warner–Bratzler shear force, kg/cm2

30.7 11.8 49.9 932 1.01 34.5 38.5 20.6 0.58 14.8 25.6 0.60 14.7 20.3 64.9 42.2 44.2 52.4 2.42

1.51 0.99 1.01 35.0 0.27 1.46 1.85 1.70 0.05 1.57 3.02 0.07 2.98 6.12 8.19 7.06 6.81 9.98 0.97

SFA—saturated fatty acids; PUFA—polyunsaturated fatty acids; MUFA— monounsaturated fatty acids; STO—slow twitch oxidative (red); FTO—fast twitch oxidative (mediate); FTG—fast twitch glycolytic (white), WBSF—Warner–Bratzler Shear Force. a Average from five points measured above: neck, last rib, front-, middle and hind musculus gluteus.

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Table 5 Growth performance and carcass characteristic of pigs during restriction and realimentation periods. Group C

Final meat content in the carcass, % Realimentation period (119–168 days of life) Daily gain, g Final body weight, kg Final warm carcass weight, kg Backfat thickness, mm⁎ Final meat content in the carcass, %

F

P value

F1

41.0

41.1

41.2

40.9

1.15

b 0.8101

972C 68.5C 54.6c 15.0B 55.8a

815B 63.9B 51.2b 16.5B 56.0a

581A 57.3A 46.1a 12.5A 60.5b

575A 56.5A 46.4a 12.7A 60.0b

21.5 1.52 1.10 0.66 0.96

b 0.0099 b 0.0103 b 0.0292 b 0.0010 b 0.0365

1063B 110.7a 89.3a 21.6A 53.5b

1261C 118.4c 96.2b 26.8C 52.1a

32.5 3.2 1.53 1.45 0.81

b 0.0105 b 0.0412 b 0.0385 b 0.0096 b 0.0251

Initial body weight, kg Restriction period (90–118 days of life) Daily gain, g Final body weight, kg Final warm carcass weight, kg Backfat thickness, mm⁎

SEM P

991A 113.8ab 96.0b 24.3B 53.0b

1022AB 116.5bc 91.2a 25.2B 51.5a

C — control (fed diet A during both periods at a semi ad lib scale); P — consumed by 30% less protein, diet P, during restriction and during realimentation, diet A, at the same scale as the C; F — consumed 30% less feed, diet A, during restriction and during realimentation, diet A, at the same scale as the C F1 — consumed 30% less feed, diet A, during restriction and during realimentation, diet A, ad libitum; a, b, c — value in row marked a different letters differ significantly; A, B, C — value in row marked a different letters differ significantly. ⁎ Average from five points measured above: neck, last rib, front-, middle and hind part of musculus gluteus.

restriction (1.40 and 1.41 vs. 1.95% in F1, F and P groups, respectively). Proportion of saturated fatty acids (SFA), expressed as the percentage of total fatty acids, was not affected by restriction while the proportion of monounsaturated acids (MUFA) was lower (Pb 0.01) in pigs submitted to feed restriction than in protein restricted and control pigs (mean 39.7 vs. 44.6 and 44.4%, respectively). Inversely, proportion of polyunsaturated acids (PUFA) and, consequently, PUFA/SFA ratio, were significantly greater (Pb 0.01) in feed restricted than in protein restricted animals. Concentration of alpha-linolenic acid (C18:3n-3) did not differ among groups while proportion of linoleic acid (C18:2n-6) was the greatest in pigs from both feed restricted groups and the smallest in protein restricted animals (12.1 and 12.2 vs. 7.8%, respectively, P b 0.01), with control group as intermediate (10.3%). The n-6/n-3 ratio followed similar pattern and was the greatest in F and F1 and the smallest in P group (23.4, 22.5 and 14.4, respectively, P b 0.01). Restriction had not significant influence on proportions and diameter of muscle fibers except a tendency (P b 0.01) to greater proportion of slow twitch oxidative (STO) fibers in protein and feed restricted pigs than in controls (12.9, 10.9, 10.8 and 8.6%, respectively).

Warner–Bratzler shear force (WBSF) was significantly greater (P b 0.01) in all restricted pigs than in controls, which means that restriction affected meat tenderness negatively. 3.3. Muscle characteristics after realimentation period In all previously restricted pigs the MLD weight was lower (P b 0.05) than in controls. It did not differ significantly among protein and energy restricted animals but was slightly greater in F1 pigs fed during realimentation ad libitum than in their F analogs fed at the semi ad libitum scale (Table 7). Intramuscular fat concentration was the smallest (P b 0.05) in the F group and did not differ among the others. Proportion of MUFA was not affected, while those of SFA was increased (P b 0.05) only by intensity of realimentation being greater in F1 than in F group. Proportions of PUFA were influenced significantly (P b 0.01) both by type of restriction and intensity of realimentation. Among pigs fed at the same level during realimentation, PUFA proportion was the greatest in previously feed restricted pigs and the smallest in protein restricted animals, values of control pigs being intermediate (13.4,

Table 6 Mass of the longissimus dorsi muscle, intramuscular fat concentration, profile of fatty acids, structure and diameter of muscle fibers and meat shear force at the end of restriction period (118 days of life). Group

Mass of the longissimus muscle, g Intramuscular fat content, % SFA, % MUFA, % PUFA, % C18:3n-3, % C18:2n-6, % C18:2n-6/C18:3n-3 PUFA/SFA STO, % FTO, % FTG, % STO, μm FTO, μm FTG, μm Warner–Bratzler shear force, kg/cm2

C

P

F

F1

1863c 1.71 37.3 44.4B 14.8b 0.59 10.3b 17.7b 0.40B 8.6 28.5 62.9 48.5 40.9 60.4 2.72A

1677b 1.95 38.4 44.6B 11.2a 0.54 7.8a 14.4a 0.29A 12.9 24.9 62.2 44.4 51.2 60.3 3.26B

1583a 1.41 37.7 39.6A 16.7bc 0.52 12.1c 23.4c 0.44B 10.9 26.3 62.8 50.1 53.1 63.4 3.32B

1550a 1.40 37.6 39.8A 16.8bc 0.54 12.2c 22.5c 0.45B 10.8 27.6 61.6 51.8 55.7 66.6 3.24B

SEM

P value

45 1.30 0.65 0.92 0.70 0.03 0.62 1.73 0.03 1.10 1.75 2.40 4.14 3.75 3.88 0.15

b 0.0315 b 0.5625 b 0.3960 b 0.0110 b 0.0225 b 0.7519 b 0.0257 b 0.0336 b 0.0103 b 0.2145 b 0.3019 b 0.6120 b 0.3165 b 0.3312 b 0.2960 b 0.0099

C (control) — fed diet A at the semi ad libitum scale; P — consumed by 30% less protein, diet P; F and F1 — consumed 30% less feed, diet A; SFA — saturated fatty acids; PUFA — polyunsaturated fatty acids; MUFA — monounsaturated fatty acids; STO — slow twitch oxidative (red); FTO — fast twitch oxidative (intermediate); FTG — fast twitch glycolytic (white); a, b, c — value in row marked a different letters differ significantly; A, B — value in row marked a different letters differ significantly.

G. Skiba et al. / Meat Science 91 (2012) 339–346

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Table 7 Mass of the longissimus dorsi muscle, intramuscular fat concentration, profile of fatty acids, structure and diameter of muscle fibers and meat shear force at the end of realimentation period (168 days of life). Group

Mass of the longissimus muscle, g Intramuscular fat content, % SFA, % MUFA, % PUFA, % C18:3n-3, % C18:2n-6, % C18:2n-6/18:3n-3 PUFA/SFA STO, % FTO, % FTG, % STO, μm FTO, μm FTG, μm Warner–Bratzler shear force, kg/cm2

C

P

F

F1

3135cb 2.25b 41.9b 44.8 12.5B 0.73b 9.42B 12.9b 0.30b 8.8 22.1A 69.1 58.6 59.2 74.8 2.70B

2920a 2.58b 42.2b 46.8 10.1A 0.65a 7.37A 11.3a 0.24a 11.5 20.1A 68.4 54.7 56.6 70.3 2.76B

2888a 1.74a 40.6a 44.2 13.4C 0.71b 9.56B 13.5b 0.33b 9.0 23.5A 67.5 57.8 54.5 65.9 2.72B

2980ab 2.43b 42.6b 46.4 10.0A 0.67a 7.43A 11.1a 0.23a 12.8 31.6B 55.6 57.7 55.6 68.7 2.37A

SEM

P value

95 0.62 0.50 0.61 0.39 0.02 0.30 0.85 0.01 1.41 1.21 1.80 2.52 2.89 2.52 0.08

b 0.0318 b 0.0391 b 0.0413 b 0.5025 b 0.0104 b 0.0265 b 0.0111 b 0.0356 b 0.0264 b 0.5731 b 0.0099 b 0.3113 b 0.4151 b 0.5210 b 0.3096 b 0.0103

C — control (fed diet A during both periods at a semi ad lib scale); P — consumed by 30% less protein, diet P, during restriction and during realimentation, diet A, at the same scale as the C; F — consumed 30% less feed, diet A, during restriction and during realimentation, diet A, at the same scale as the C; F1 — consumed 30% less feed, diet A, during restriction and during realimentation, diet A, ad libitum; SFA — saturated fatty acids; PUFA — polyunsaturated fatty acids; MUFA — monounsaturated fatty acids; STO — slow twitch oxidative (red); FTO — fast twitch oxidative (intermediate); FTG — fast twitch glycolytic (white); a, b, c — value in row marked a different letters differ significantly; A, B, C — value in row marked a different letters differ significantly.

10.1 vs. 12.5 in group F, P and C, respectively). In animals submitted previously to feed restriction PUFA proportion was significantly higher in the group fed during realimentation less intensively than ad libitum (13.4 vs. 10.0% in F and F1, respectively). PUFA/SFA ratio was higher (P b 0.05) and did not differ between C and F group, and was lower and did not differ between P and F1 group. Concentrations and ratio of alpha-linolenic and linoleic acids depended in similar way both on type of restriction and realimentation and were greater in the C and F than in the P and F1 groups. Among muscle fibers, only the proportion of fast twich oxidative (FTO) fibers was significantly (P b 0.01) affected by intensity of realimentation being greater in the F1 than in the F and in all other groups. On the contrary, the proportion of fast twitch glycolytic fibers (FTG) in the F1 pigs tended to be the smallest, while it did not differ among other groups. On the contrary slow twitch oxidative — STO fiber in pigs of the group F1 and P tended (P b 0.10) to be higher than in the C and F animals. The diameter of the muscle fibers did not differ between the groups. Shear force was affected only by intensity of realimentation and was smaller in feed restricted pigs realimented ad libitum than according to semi ad libitum scale (2.37 vs. 2.72 kg/cm 2 in F1 and F, respectively), and then in all other groups. 4. Discussion As it was expected feed (simultaneously energy and protein) restriction reduced growth rate to greater extent than protein restriction only. Both types of restriction resulted in smaller mass of the body, warm carcass and musculus longissimus dorsi (MLD) as well. However feed restriction resulted in lower backfat thickness and consequently increased percentage content of meat in the carcass comparing to protein restriction and “normal” feeding. During realimentation pigs previously restricted with protein compensated mass of the body, but body mass of pigs previously restricted with feed and then fed ad libitum even exceeded body mass of C pigs. As far as mass of the carcass, the only animals fed ad libitum during realimentation were able to fully compensate its mass. Despite of this mass of MLD in these pigs remained lower similarly like in other experimental group still remained lower than in animals fed adequately through the study. Ad libitum feeding of pigs previously restricted with feed increased fatness of their carcass and consequently decreased

percentage content of meat. Similar effect gives temporary protein restriction followed by controlled feeding. However, pigs previously temporary restricted with feed next fed similar to control animals maintained lower backfat thickness without deterioration of percentage content of meat in the carcass. During the realimentation period all previously restricted groups of animals showed the compensatory growth since their growth rate was 7 to 27% faster that pigs fed adequately throughout the experiment. However, this compensation was not sufficient to support recover of the musculus longissimus dorsi mass to value found in the control pigs. It was probably due to allocation of enhanced growth in parts of the body other than muscle tissue, probably representing offal (e.g. Skiba et al., 2005), which is significantly depressed during restriction and firstly recovered during the realimentation (especially when feed restriction was previously applied). The increase shear force in all restricted animals shows that both types of restriction influence this parameter of meat negatively. Similar effects of alimentary restriction on shear force were found by Kristensen et al. (2002) and Więcek et al. (2011). After realimentation shear force of meat was improved in our study only in animals fed ad libitum and growing faster than others. This points toward other factors influencing shear force, such as the rate of muscle metabolism, and specially activity of proteolytic enzymes (Kristensen et al., 2002; Skiba et al., 2009). Our results are not in accordance with those of Więcek et al. (2011), who did not find differences in shear force between compensating and normally growing pigs. The intramuscular fat concentration is an important parameter contributing to meat quality, and its optimal concentration in musculus longissimus dorsi is in the range of 1.5 to 2.0% (Flachowsky, Schulz1, Kratz, & Glodek, 2008). It is affected by genetic and nutritional strategy, comprising feeding level and composition of the diets. It was found that dietary restriction strongly influences intramuscular fat concentration, but results are ambiguous (e.g. Teye et al., 2006; Wood et al., 2004). In our study intramuscular fat concentration was decreased in pigs restricted with feed (protein and energy), but it was increased in animals restricted only in protein. Our findings on the depressive effect of feed restriction on intramuscular fat concentration in musculus longissimus dorsi are in agreement with those reported by Lebret, Juin, Noiblet, and Bonneau (2001), Heyer and Lebret (2007), Więcek et al. (2010) but not by Lebret, Heyer, Gondret, and Louveau (2007). The effect of protein restriction on

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intramuscular fat concentration found in this study agreed with the results reported by Lebret et al. (2001); Teye et al. (2006) and Alonso, del Mar Campo, Provincial, Roncales, and Beltran (2010). In some studies the response to alimentary restriction differed between muscles (Heyer & Lebret, 2007; Lebret et al., 2001; Lebret et al., 2007). The intramuscular fat concentration in musculus longissimus dorsi of control animals slaughtered at the end of this experiment was greater over 30% compared to pigs of 118 days of age over 120% when compared to pigs of 90 days of age. It final concentration final even slightly exceeded optimal concentration reported by Flachowsky et al. (2008). After realimentation intramuscular fat concentration was the smallest in animals previously restricted with feed and fed during recovery at the lower level of feed intake. Obviously, pigs restricted similarly but fed ad libitum during realimentation, increased fatness of the muscle as compared to with those realimented at lower feeding level. The greater intramuscular fat concentration found in protein restricted animals before realimentation was maintained till the end of the experiment. According to results reported by Więcek et al. (2011) fatness of the longissimus muscle, in animals realimented ad libitum during 63 days, initially increased, next reached level similar to controls and finally decreased compared to normally growing pigs. Studies performed by many authors led to a widely accepted presumption that meat tenderness/shear force is positively (Channon, Kerr, & Walker, 2004; Stolzenbach et al., 2009; Teye et al., 2006) related to its fat concentration. Our results concerning the effect of the restriction and partly realimentation do not confirm this relationship. After the restriction period, the smallest shear force had the nonrestricted (controls) pigs with the intermediary intramuscular fat concentration, while the shear force of meat of pigs having the lowest (both groups restricted with feed) and the highest (animals restricted with protein) fatness of muscle did not differ and was lower (higher shear force) than in control pigs. However at the end of realimentation meat of pigs realimented ad libitum contained more fat and had also lower shear force than meat of animals realimented at the lower feeding level. On the other hand, shear force of pigs differing significantly in intramuscular fat content (group F, C and P) was uniform. In conclusion we did not find significant relationship between the Warner–Bratzler shear force and intramuscular fat concentration expressed as Pearson correlation. This finding is in line with results reported by Rincker, Killefer, Ellis, Brewer, and McKeith (2008). However other authors found a negative but low relation between intramuscular fat concentration and shear force (e.g. Fortin, Robertson, & Tong, 2005). On the other hand, Fernandez, Monin, Talmant, Mourot, and Lebret (1999) also found a low or any relation between intramuscular fat concentration and meat toughness depending on crossbreed pigs used in the experiment (Duroc × Landrace vs. Tia Meslan × Landrace, respectively) even so range of intramuscular fat concentration was comparable in both genotype. Thus, our results and those of cited authors indicated that relation between intramuscular fat concentration and quality of pork is not systematic and not completely recognized. Many, other factors, e.g. rate protein turnover, activity of the proteolytic enzymes, which are varied in pigs differ in growth rate also influenced meat shear force, what is confirmed by other authors (Skiba et al., 2009; Therkildsen et al., 2002). Moreover, other meat quality traits like ultimate pH (Lonergan et al., 2007), time of meat ageing (van Laack, Stevens, & Stadler, 2001). Thus, in the study on relation of intramuscular fat concentration and meat tenderness/shear force also above mentioned factors should be also simultaneously considered, however it could be difficult due to exist a possible interaction between these traits. The result of our study showed that changes of feeding patterns may modify fatty acids profile of intramuscular fat to some extent even when diet is not supplemented with fat sources. In pigs grown according to “normal” pattern we found an increased concentration of C18:3n-3 (especially during finishing phase), however content of C18:2n-6, and ratio between these fatty acids was decreased.

Somewhat different result presented Kouba, Enser, Whittington, Nute, and Wood (2003), who found decreased concentration of both C18:3n-3 and C18:2n-6 acids but unchanged ratio between these acids in growing pigs fed a diet not supplemented with. While in our pigs slaughtered at the end of restriction period, the proportion of C18:2n-6, was increased in feed restricted pigs having the lowest fatness of the muscle, and decreased in protein restricted pigs with the highest intramuscular fat concentration. Similar changes were found by Alonso et al. (2010) in pigs fed on diets with different protein level and having different fatness of longissimus dorsi muscle. The proportion of C18:3n-3 was not influenced by type of feed restriction, but due to differences in concentration of C18:2n-6, the ratio between these fatty acids was considerably greater in feed than in protein restricted animals. Also Więcek et al. (2011) found a decrease in intramuscular fat concentration in feed restricted pigs was accompanied by the increase proportion of PUFA n-6, but contrary to our results these authors reported that also proportion of PUFA n-3 was increased. The difference between our and Więcek et al. (2011) results cannot be ascribed to different age of animals as it was similar in both experiments. The possible reason is a difference of genetic background since our animals represented a leaner type of pigs than the Duroc crossbreed used in study by Więcek et al. (2011). It is possible that fatty acids composition of the muscle may be more easily changed by dietary treatment/feeding strategy in fatter than in lean pigs. Pigs previously restricted with feed (energy and protein) than realimented at lower feeding level had the lower proportion of SFA than those realimented ad libitum and previously restricted with protein as well. The difference in PUFA proportion between protein restricted and control group was maintained during realimentation, while in feed restricted pigs realimented at the lower feeding level proportion of these acids was greater than in pigs realimented ad libitum. In consequence, the difference of PUFA/SFA ratio between protein restricted and control pigs was maintained during realimentation while in pigs previously restricted with feed the lower feeding level during realimentation increased PUFA/SFA ratio as compared to animals fed ad libitum. Our data show that the linoleic and alpha-linolenic acids concentration in the MLD fat were smaller in the pigs previously protein restricted and realimented ad libitum than of controls and realimented at lower feeding level. A similar relation was found for the PUFA n6:n-3 ratio. Thus, it may be concluded that type of previous restriction has considerable but dissimilar effects on the important components of dietary value of intramuscular fat such as fatty acid profile and n-6/ n-3 ratios. In the study by Więcek et al. (2011) changes provoked during restriction were hold only during 3 first weeks of realimentation and disappeared later. When consider above mentioned findings it seems that in order to improve fatty acids composition of meat of realimented pigs they should be slaughtered after few weeks of realimentation, or restriction and realimentation should be evoked in slightly older animals. However this improvement regarding ratio of n-6:n-3 acids is very far from those prescribed from human health point of view. It could be also noted that amount of intramuscular fat strongly influences a composition/profile of fatty (e.g. Wood et al., 2008). Changes in fatness of muscle and consequently composition/profile of fatty acids resulted from different amount of daily deposited muscle fat between investigated group of pigs during restriction and particular phase of the realimentation what was presented in earlier studies on this issues (e.g. Lebret et al., 2007; Skiba, 2010). Our results concerning histological structure of longissimus dorsi muscle show that the proportion of fast-twitch glycolytic (white) fibers was slightly greater in older than in younger pigs, which is in agreement with other studies (Bee et al., 2007; Brocks et al., 2000; Henckel, Oksbjerg, Erlandsen, Barton-gade, & Bejerholm, 1997;

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Kłosowska et al., 1985; Larzul et al., 1997; Russenen & Puolanne, 1996). The increase of fast twitch glycolytic (white) fiber proportion was a consequence of their earlier development as compared with slow twitch oxidative (red) fibers (Bee et al., 2007; Ono, Solomon, Evock-Clover, Steele, & Maruyama, 1995). The results of present study indicate that nutritional manipulation applied during restriction period does not affect significantly either proportions or diameter of the fibers in the musculus longissimus dorsi. A tendency to a greater proportion of slow twitch oxidative (red) in both protein and feed restricted pigs than in controls was observed. Curiously enough, against all the odds restriction has not influenced diameter of particular type of fiber. The lack of significant effects may be probably caused by the small number of animals per treatment, short duration and small intensity of restriction or high individual variation of myofiber typing. After realimentation the only significant difference in fibers proportion was found in the contribution of fast twitch oxidative (mediate), which was considerably greater in pigs fed ad libitum than in pigs fed on the lower level of feed intake, and other groups. The increase of the fast twitch oxidative (mediate) proportion was accompanied by tendency for decrease of fast twitch glycolytic (white) and increase of slow twitch oxidative (red) fibers. These results indicate that the increase of feeding intensity provokes the increase of contribution of fast twitch oxidative (intermediate) and slow twitch oxidative (red) at the expense of fast twitch glycolytic (white) fibers in the muscle. The literature data concerning the effects of feeding intensity on the proportions of particular types of muscle fibers, are controversial. Solomon, Campbell, Steele, Caperna, and McCurty (1988) reported the increased proportion of slow twitch oxidative (red) fibers in MLD of pigs fed restrictively while Bee et al. (2007) found the decreased proportion of slow twitch oxidative (red) and increased of fast twitch glycolytic (white) fibers but only in animals compared at similar body weight, not age. Other authors did not find such relationship (Candek-Potokar, Lefaucheur, Zlender, & Bonneau, 1999; Harrison, Rowlerson, & Dauncey, 1996). Contrary to expectation in our study, fiber diameter was affected neither by type of restriction nor intensity of realimentation. However, the diameter of fast twitch oxidative (intermediate) fibers was nonsignificantly but considerably greater in protein and feed restricted pigs than in controls, the difference disappearing in the realimented pigs. Harrison et al. (1996) and Bee et al. (2007) reported a reduction of fiber hypertrophy in restrictively fed pigs compared with animals fed ad libitum at similar age but at lower body mass. On the other hand, when restricted animals were compared with non-restricted at similar body weight but older age, some researchers found that size of muscle was greater (Lefaucheur, 1983), smaller (Solomon et al., 1988) or unaffected (Bee et al., 2007). Moreover, the impact of feed restriction on histological traits differed among the muscles (Bee et al., 2007). Design of our experiment assumed comparison of animals at similar age but different body weight thus the results differ from those reported above since the size of

Table 8 Pearson's correlation between meat shear force (WBSF) and intramuscular fat concentration (%), fatty acids profile (%), muscle structure (%) and diameter of muscle fiber (μm). Item

r2

IMF SFA PUFA MUFA PUFA/SFA

0.04 0.07 − 0.16 0.16 − 0.16

(P b 0.3836) (P b 0.1838) (P b 0.0017) (P b 0.020) (P b 0.0023)

Item

r2

STO FTO FTG STO FTO FTG,

− 0.24 0.16 − 0.01 0.02 0.09 0.07

(P b 0.0001) (P b 0.0019) (P b 0.9155) (P b 0.7381) (P b 0.0877) (P b 0.1961)

IMF — intramuscular fat content; SFA — saturated fatty acids; PUFA — polyunsaturated fatty acids; MUFA — monounsaturated fatty acids; STO — slow twitch oxidative (red); FTO — fast twitch oxidative (intermediate); FTG — fast twitch glycolytic (white).

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particular muscle fibers did not differ between control and restrictively fed animals, in spite of their lower body weight. Diversity of the reported results shows that the comparison of the results of our experiment with other data is difficult because of variable experimental schemes and the interactive effects of age and body weight of animals. Also our observation on the influence of intensity of realimentation on the proportions of particular types of muscle fibers is difficult to interpret since there is no available data on influence of compensatory growth no histological parameters of the muscle. The measurements of the content and composition of intramuscular fat, and muscle histology, taken during the experiment, were employed in an attempt to establish relationship between Warner– Bratzler shear force of the musculus longissimus dorsi and selected parameters under study. The results presented in Table 8 show that shear force was not related to the intramuscular fat content and proportion of saturated fatty acids but is negatively correlated with proportion and ratio of polyunsaturated to saturated fatty acids. Admittedly these correlation are low but they could suggest that increase of the proportion of PUFA in the intramuscular fat can positively affects, in some extent, shear force of the MLD. On the contrary, the proportion of MUFA was correlated with shear force positively indicating for their possibility negative effect on this trait. Shear force was also related to the structure of the muscle since it was negatively correlated with proportion of slow twitch oxidative (red) fibers and positively correlated with fast twitch oxidative (intermediate) fibers. On the contrary, there was no significant relationship between shear force and diameter of particular types of fibers. 5. Conclusions Based on the results of this study it could be concluded that nutritional manipulation (including both restriction and realimentation) could be a way to change meat shear force (toughness), the concentration of intramuscular fat in longissimus dorsi muscle and, to some extent its proportion of fatty acids, and contribution but not size of particular muscle fiber type. However, the estimated relationships allow for the following conclusions. Firstly, contrary to a widely accepted assumption, shear force is not necessarily related to the intramuscular fat content, due to the possible effects of other factors which influence these parameters. Secondly, shear force of meat could be affected by the fatty acid profile and seems that it is decreased by the PUFA acids. Thirdly, the proportion between slow twitch oxidative and fast twitch oxidative fibers may be an important factor influencing meat shear force. References Alonso, V., del Mar Campo, M., Provincial, L., Roncales, P., & Beltran, J. A. (2010). Effect of protein level in commercial diets on pork meat quality. Meat Science, 85(1), 7–14. Andres, A. I., Cava, R., Mayoral, A. I., Tejeda, J. F., Morcuende, D., & Ruiz, J. (2001). Oxidative stability and fatty acids composition of pigs muscles as affected by rearing system, crossbreeding and metabolic type of muscle fibre. Meat Science, 59, 39–47. AOAC (Association of Official Analytical Chemists) (1994). Official methods of analysis (16th ed). Washington (DC).. Bee, G., Calderini, M., Biolley, C., Guex, G., Herzog, W., & Lindemann, M. D. (2007). Changes in the histochemical properties and meat quality traits of porcine muscle during the growing — Finishing period as affected by feed restriction, slaughter age, or slaughter weight. Journal of Animal Science, 85, 1030–1045. Bikker, P. (1994). Protein and lipid accretion in body components of growing pigs: Effects of body weight and nutrient intake. PhD Thesis. Department of Animal Nutrition, Wageningen Agricultural University, The Netherlands, pp 1–203. Brocks, L., Klont, R. E., Buist, W., de Greef, K., Tieman, M., & Engel, B. (2000). The effect of selection of pigs on growth rate vs. leanness on histochemical characteristic of different muscle. Journal of Animal Science, 78, 1247–1254. Candek-Potokar, M., Lefaucheur, L., Zlender, B., & Bonneau, M. (1999). Effect of slaughter weight and/or age on hiostological characteristic of pig longissimus dorsi muscle as related to meat quality. Meat Science, 52, 195–203. Channon, H. A., Kerr, M. G., & Walker, P. J. (2004). Effect of Duroc content, sex and ageing period on meat and eating quality attributes of pork loin. Meat Science, 66, 881–888.

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