Climatic environment is an external stress that represents a challenge for animal growth and energy use (Le Dividich et al., 1998). Depending on physiological stage, any variation of ambient temperature below the thermoneutral zone results in activation of thermoregulatory processes affecting energy metabolism. According to Le Dividich et al. (1998), a cold environment is defined as the 1 below the lower critical temperature estimated to be 26 to 28°C for weaned piglets and 18 to 20°C for 60 kg BW pigs.
Influence of a low rearing temperature on growth performance, body composition, muscle traits, and meat quality has recently been reported in different outdoor pig production systems (Lebret et al., 2002; Bee et al., 2004; Gentry et al., 2004). However, because of interactions with physical activity, housing type, or feeding, daily variations of rearing temperature in these studies were part of a multifactorial and complex pig adaptive response to rearing conditions. Long-term effects of environmental temperature exposure after postweaning (PW) and growing–finishing (GF) period were assessed by Lefaucheur et al. (1991) considering low (12°C) versus high (28°C) temperature in pigs between 8 and 92 kg BW. They reported growth performance responses and body composition modulation as well as muscle type-specific physiological adaptive changes, but these changes could not be ascribed to either the low or high rearing temperature. In young piglets (8 to 17 kg), a constant cold environment (8 vs. 23°C) increased oxidative capacity through greater mitochondrial respiration in both oxidative and glycolytic muscles (Herpin and Lefaucheur, 1992). Because cold stress induces dynamic balance between heat production and heat loss, muscle adaptation could be the result of an energetic stress involving energy and nutrient homeostasis regulation. The adenosine monophosphate (AMP)-activated protein kinase (AMPK) has been identified as a key sensor of cellular energetic status involved in energy balance in skeletal muscle (Winder, 2001; Hardie and Sakamoto, 2006). In response to an energetic depletion and once activated by phosphorylation, AMPK inhibits anabolic pathways and stimulates catabolic pathways to restore cellular energy level (Hardie and Sakamoto, 2006; Scheffler and Gerrard, 2007). In cold environment, AMPK activation would improve oxidative muscle capacity (Winder et al., 2000; Jäger et al., 2007). The present study was undertaken to compare the effects of both short- and long-term cold exposure on growth performance, carcass traits, and metabolic characteristics of oxidative and glycolytic striated pig muscles and to evaluate the reversibility of short-term cold effects. Special focus was given on the implication of AMPK in muscle energy metabolism adaptation to cold environment in both muscle metabolic types.
MATERIALS AND METHODS
The experiment was conducted using French guidelines for animal care and use. All people involved in the experiment have an individual agreement for conducting experimental procedures on animals, delivered by the Veterinary Services of French Ministry of Agriculture.
Animals and Experimental Design
The experiment included 84 castrated males and females Large White × (Large White × Landrace) pigs from 14 litters (produced from 3 fathers). In each litter, piglets were paired per sex and similar BW. After weaning, paired animals were randomly submitted to either Cold (from 23 ± 1 to 15 ± 3°C; n = 48) or thermoneutral (T; from 28 ± 1 to 23 ± 1°C; n = 36) environmental temperatures during PW period from 7.7 ± 0.6 to 24.7 ± 1.6 kg BW (25 BW).
After this 6-wk PW period, 12 Cold and 12 T piglets (6 castrated males and 6 females randomly selected in each treatment) were slaughtered the same day to evaluate the short-term effects of cold exposure. Eighteen remaining Cold piglets were randomly chosen and reared at 12 ± 2°C (CC) whereas 18 Cold and 24 T piglets were reared at 23 ± 4°C (CT and TT, respectively) up to 114.3 ± 5.9 kg BW (115 BW; i.e., during 98 ± 10 d).
During the experiment, room temperature was automatically set at selected temperatures using an air-conditioning system, and ambient temperature was registered at 0800 and 1700 h in addition to minimal and maximal daily values.
All pigs were free of the halothane-sensitive (n) and Rendement Napole alleles. Pigs were individually housed and fed ad libitum with a standard diet: first age diet during the first 2 wk of PW (10.2 MJ NE/kg, 19.2% CP, and 12.8 g/kg digestible lysine), second age diet during the next 4 wk of PW (9.9 MJ NE/kg, 18.4% CP, and 11.4 g/kg digestible lysine), then a growing diet (10.1 MJ NE/kg, 16.5% CP, and 10.5 g/kg digestible lysine) from 25 up to 70 kg, and a finishing diet thereafter (10.3 MJ NE/kg, 15.3% CP, and 10 g/kg digestible lysine). Animals had free access to water.
Pigs were slaughtered after a fasting period (lasting between 18 and 20 h at 25 BW and between 24 and 30h at 115 BW) by electrical stunning and exsanguination at the experimental slaughterhouse of INRA (PEGASE, St-Gilles, France) in compliance with the current national French regulations applied in slaughterhouses and with standardization of slaughter conditions. At 25 BW, 24 pigs (i.e., 6 pigs per sex and treatment) were slaughtered in 1 session. At 115 BW, pigs were slaughtered in 4 sessions, each including 2 or 3 pigs per sex and treatment. At each slaughter session, 1 pig from each treatment (alternatively) was slaughtered every 10 min, to ensure similar average fasting time between treatments. Growth performance and carcass traits were recorded on only 14 pigs (7 of each sex) out of the 18 from the CC group, either due to individual problems recorded at the end of the GF period or due to the removal by veterinary services.
Carcass Traits and Body Composition
Weights of hot carcass, internal fat, heart, and thyroid gland were recorded on the day of slaughter. At 115 BW, backfat (G2) and muscle (M2) depths were measured at 1 dorsal spot between the third and fourth last ribs using a Capteur Gras/Maigre-Sydel device (CGM) device as described by Daumas (2008).
After 24 h chilling at 4°C, carcass length was measured on half carcass from the atlas to the anterior edge of the pubic symphysis, and weights of fresh carcass and wholesale cuts (ham, loin, shoulder, belly, and backfat) of the left half carcass were recorded. Joint weights were expressed as percentage of the half-carcass weight. These data were used on 115 kg BW pigs to calculate lean meat content (LMC) from carcass cut percentages according to Daumas (2008). Carcass drip loss (difference between hot and cold carcass weights) and composition (proportion of wholesale cuts in the left side) were calculated on all pigs.
Because a major goal of the present study was to determine muscle metabolism adaptation to cold temperature, we analyzed 2 muscles differing in function and metabolism [i.e., the white LM and the red semispinalis (SS) muscle].
Samples of LM and SS (around 10 g) were taken just after exsanguination (T0) with a biopsy device at the last rib level and at the neck level, respectively, and were immediately frozen in liquid nitrogen and stored at –80°C to represent the “in vivo” physiological situation for the further determination of the AMPK status. Thirty minutes after slaughter (T30), other LM and SS samples were collected (close to the previous sampling site), frozen in liquid nitrogen, and stored at –80°C before determination of glycolytic potential (GP), as described previously by Lebret et al. (2006). Activities of lactate dehydrogenase (LDH), citrate synthase (CS), and β-hydroxy-acyl-CoA deshydrogenase (HAD) were also determined on these samples to assess the glycolytic, oxidative, and fatty acid β-oxidation capacities, respectively (Lefaucheur et al., 2011).
The day after slaughter (i.e., 24 h postmortem), LM and SS slices were trimmed of external fat, minced, and freeze-dried before determination of lipid content (Folch et al., 1957). Muscle water content was determined from the weight of minced muscle before and after freeze-drying and used for calculation of lipid content per gram of fresh muscle.
Determination of Adenosine Monophosphate-Activated Protein Kinase Phosphorylation by Immunoblotting
We have used AMPK phosphorylation as an indirect indicator of enzyme activation because several studies demonstrated positive relationships between the level of kinase phosphorylation and the activity of the enzyme (Yu et al., 2003). Muscle samples were analyzed by immunoblotting to quantify the level of AMPK phosphorylation as previously described by Faure et al. (2012).
Briefly, frozen muscle sample (approximately 300 mg) was crushed into powder in liquid nitrogen and homogenized on ice in 1.5 mL lysis buffer [150 mM NaCl, 10 mM Tris, 1 mM Ethylene glycol tetraacetic acid, 1 mM EDTA, pH 7.4, 100 mM sodium fluoride, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% Triton X-100, 1.5 mg/mL of complete protease inhibitor cocktail (Roche, Meylan, France), and 100 μL of each phosphatase inhibitor cocktail (cocktail 2 P0044 and cocktail 3 P5726; Sigma, St-Quentin Fallavier, France)]. The solution obtained was centrifuged (1,000 × g for 30 min at 4°C and 150,000 × g for 45 min at 4°C) before protein concentration determination. Protein concentration was determined using a bicinchroninic acid protein assay kit (Thermo Pierce Biotechnology, Rockford, IL) according to the protocol of the manufacturer with BSA as a standard. Proteins were then denatured in a 1x Laemmli solution and separated onto polyacrylamide gels [stacking gel: 4% acrylamide: N,N-methylene-bis-acrylamide (37:1); resolving gel: 10% acrylamide: N,N-methylene-bis-acrylamide (37.5:1)] by electrophoresis. Equal amounts of protein (30 μg per lane) were loaded onto gels set on a Mini-Protean tetra electrophoretic system (Bio-Rad, Hercules, CA) and run in a 1x buffer (Bio-Rad 10X, Tris/Glycine/SDS;ref 161-0772) for 1 h at 50 V at ambient temperature and 70 V until the blue color reached the bottom of the gel. After migration, separated proteins were transferred to polyvinylidene fluoride membranes. Membranes were incubated overnight at 4°C with primary antibodies specific for total AMPK α1+α2 isoforms (AMPKα; α1 + α2 isoforms; mouse monoclonal antibody, detecting total AMPKα protein, number 2793; Cell Signaling, Ozyme, St Quentin en Yvelines, France) or Threonine 172 phosphorylated form of AMPK α1+α2 isoforms (pAMPK; Thr 172; rabbit polyclonal, number 2531; Cell Signaling) diluted 1:1,000 and 1:500, respectively. Protein bands were visualized using enhanced chemiluminescence (ECL plus; GE Healthcare, Orsay, France), scanned with an ImageQuant LAS 4000 (GE Healthcare), and quantified using the Image Quant TL program for determination of AMPKα and pAMPK. Membrane normalization was based on a common reference LM sample previously selected as exhibiting average values of LM for lactate and ultimate pH. The reference sample was repeated on every gel in the first and last lanes and mean of both values were used for normalization of experimental samples.
Data were analyzed using the GLM procedure (SAS Inst. Inc., Cary, NC). The model (Model 1; Tables 1 and 2) used for analyses of growth performance and carcass composition included the rearing temperature (RT), sex, father, and RT × sex interaction as fixed effects. Hot carcass weight was added as covariable in the model of carcass composition (Model 2). For each muscle, the model used for analyses of muscle metabolism (Model 3; Tables 3 and 4) included the same RT, sex, father, and RT × sex effects as well as slaughter date and included HCW for energy metabolic traits or western blot series for AMPKα quantification. Results are expressed as least square means and model residual SD. Comparison of AMPKα quantification (Model 4; Fig. 1) between muscles (LM and SS) was evaluated using an ANOVA analysis (GLM procedure) at each physiological stage (25 BW and 115 BW) including animal, muscle, RT, and muscle × RT effects in the model. The Tukey post hoc test (P < 0.05) was performed to compare least square means between TT, CT, and CC groups.
|Growth performance 8 to 25 kg|
|No. of pigs||36||48|
|Initial BW, kg||7.7||7.7||0.6||0.78||0.60|
|Final BW, kg||25.2||24.3||1.6||0.008||0.93|
|Feed conversion ratio, kg/kg||1.46||1.67||0.10||<0.001||0.34|
|Body composition at 25 kg|
|No. of pigs||12||12|
|Slaughter BW, kg||24.6||23.8||1.4||0.18||0.59|
|Internal fat, kg||0.91||0.72||0.08||<0.001||0.20|
|Carcass length, cm||64.3||62.1||1.3||0.001||0.86|
|Carcass drip loss, %||3.27||3.05||0.55||0.39||0.52|
|Carcass composition, %|
|Growth performance 25 to 115 kg|
|No. of pigs||24||18||14|
|Initial BW, kg||25.3||24.0||24.0||2.0||0.08||0.85|
|Final BW, kg||116.0||113.8||112.9||5.9||0.27||0.002|
|Final age, d||161||162||161||10||0.98||0.199|
|Feed conversion ratio, kg/kg||2.72b||2.69b||3.28a||0.22||<0.001||0.09|
|Body composition at 115 kg BW|
|Slaughter BW, kg||113.6||111.4||110.5||5.8||0.25||<0.001|
|Mean back fat depth, mm||16.7||16.7||17.5||4.1||0.35||0.89|
|Muscle depth, mm||51.5||52.1||50.0||2.6||0.66||0.001|
|Internal fat, kg||1.59||1.48||1.50||0.30||0.48||0.003|
|Carcass length, cm||102.0||102.7||100.6||2.4||0.06||0.11|
|Carcass drip loss, %||2.40||2.84||2.76||0.78||0.21||0.25|
|Carcass composition, %|
|Free glucose + G6P,6 µmol/g||10.5||11.5||2.0||0.27||0.56||9.1||7.4||9.0||2.8||0.19||0.76|
|Glucose (glycogen),7 µmol/g||72.6||61.0||11.1||0.04||0.32||34.0b||30.0b||50.1a||11.4||<0.001||0.64|
|Intramuscular fat content, %||1.11||1.32||0.31||0.17||0.10||1.54b||1.59b||2.12a||0.43||0.001||0.008|
|Free glucose + G6P,6 µmol/g||6.8||4.0||1.47||<0.001||0.08||3.1||2.7||3.9||1.54||0.13||0.007|
|Glucose (glycogen),7 µmol/g||15.4||6.8||7.1||0.02||0.03||5.4||4.4||6.7||3.9||0.31||0.003|
|Intramuscular fat content, %||5.45||6.24||0.99||0.099||0.18||8.67||8.87||9.32||1.85||0.61||0.005|
Pearson’s correlation coefficients were calculated between muscle traits and AMPKα status (CORR procedure of SAS). Comparison of pAMPK:AMPKα ratio and GP (Model 5; Fig. 2 and 3) between stages (25 BW and 115 BW) was evaluated in each muscle using an ANOVA analysis (GLM procedure) including animal, stage, RT, and stage × RT effects in the model. Relationships between GP and pAMPK:AMPKα ratio and between intramuscular fat (IMF) and pAMPK:AMPKα ratio used individual values. Plots were built with R software R Development Core Team (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URLwww.R-project.org.; version 2.11.1) using Rcmdr package (Fox, 2005). Ellipse confidence limit for each group was fixed at 90%. Inertia value of each point cloud, calculated as variance of GP (or IMF) + variance of pAMPK:AMPKα ratio for each group (Cold, CC, T, and TT) and muscle (LM and SS), characterized the variability of each point cloud and the scattering around a hypothetical individual mean.
Growth Performance and Body Composition
During PW, cold exposure induced strong responses for growth performance (Table 1). Despite a greater ADFI (+58 g/d; P < 0.001), Cold piglets exhibited a less ADG than T piglets. Consequently, Cold piglets presented greater feed conversion ratio (FCR) than T piglets (+0.21 kg/kg; P < 0.001) and a growth retardation at the end of this period (–0.9 kg BW; P = 0.008). Short-term cold exposure induced also morphological adaptation with changes in body and carcass composition. After 6 wk PW, Cold piglets exhibited lesser carcass length (–2.2 cm; P = 0.001) and adiposity but greater shoulder percentage and loin:backfat ratio (+18%; P = 0.03) than T piglets. Greater thyroid gland (P = 0.06) and heart (P = 0.03) weights were also recorded, as a result of an early and acute physiological adaptation to the cold condition.
During GF period, CC pigs still exhibited a greater ADFI (+394 g/d; P < 0.001) and FCR (+0.56 kg/kg; P < 0.001) than TT pigs, but ADG did not differ between CC and TT pigs (Table 2). Interestingly, during the growing period (25 to 65 kg), CC pigs showed a compensatory feed intake (+662 g/d ADFI; P < 0.001; data not shown) and therefore exhibited a compensatory growth (+131 g/d ADG; P < 0.001; data not shown) compared with TT group. From 25 to 115 kg, growth performances were similar for CT and TT pigs. Thus, effects of low temperature applied during an early stage of growth were reversible for ADFI, ADG, and FCR during the GF period. Body and carcass composition of CC pigs was not different from that of CT and TT pigs (P > 0.14), except for greater heart weight (P < 0.001) and shoulder proportion (P = 0.02). The CC pigs tended to have greater thyroid weight (P = 0.07) than TT and CT and shorter carcasses (P = 0.06) than CT.
Therefore, a long-term cold adaptation drastically increased feed intake but had only slight effects on body composition and did not influence carcass lean meat content and global amount of muscle deposition. Although gender effects on growth performances and body composition were weak at 25 BW, castrated males exhibited greater ADFI (+398 g/d; P < 0.001) and carcass adiposity (–2.0 points of LMC and –1.19 points of loin:backfat ratio; P < 0.001) than females after the GF period, without any interaction between sex and RT.
Muscle Energy Substrates and Metabolic Enzyme Activities
As expected, LM muscle is mostly glycolytic (high LDH:CS ratio and GP; Table 3) whereas SS muscle is mostly oxidative (high CS and HAD activities and low LDH:CS ratio and GP; Table 4). Cold rearing conditions induced muscle metabolism adaptations depending on pig physiological stages and muscle metabolic type. In LM (Table 3), a cold PW period induced a lesser GP (P = 0.07), due to lesser glycogen content (P = 0.04), but had no effect on LDH, HAD, and CS enzymes activities. By contrast, a long-term exposure to cold temperature resulted into greater GP (P < 0.001), glycogen (P < 0.001), and IMF (P < 0.001) contents, enhanced LDH, HAD, and CS enzymes activities, and decreased both LDH:CS and HAD:CS ratios in CC pigs compared with TT pigs. Pigs from CT group had slightly less GP (P < 0.05) and similar activities of LDH, HAD, and CS to TT pigs. In SS muscle (Table 4), GP was drastically reduced in Cold compared with T piglets (P = 0.005) due to both lesser glycogen (P = 0.02) and glucose + glucose-6-phosphate (G6P; P < 0.001) contents. The HAD and CS activities as well as HAD:CS ratio were increased in Cold piglets, without any difference in LDH activity, whereas LDH:CS ratio was slightly decreased in Cold piglets (P = 0.07). After the GF period, energy substrates as well as LDH activity were not different between CC, CT, and TT pigs. In contrast, oxidative metabolism was still enhanced after long exposure to cold temperature with greater HAD and CS activities in CC pigs compared with TT and CT pigs. By contrast, CT and TT pigs exhibited similar HAD and CS activities denoting a reversibility of short-term effects of cold exposure on HAD and CS activities in SS. Remarkably, cold temperature stimulated first oxidative metabolism in the SS during a short cold exposure during the PW period (that continued afterward during the GF period) and then stimulated glycolytic and oxidative pathways in the LM during the GF period, that is, after longer exposure to cold environment (CC pigs). Although gender effects were weak on LM muscle, in SS muscle castrated males exhibited lesser GP (–17.95 μmol/g; P = 0.02) due to lesser glycogen content (–7.04 μmol/g; P = 0.03) at 25 BW and greater GP (+13.92 μmol/g; P = 0.004) and IMF (+2.45 point; P = 0.005) at 115 BW.
Adenosine Monophosphate-Activated Protein Kinase Phosphorylation Status
The AMPKα status was diversely influenced by muscle type (LM or SS), physiological stage (25 BW or 115 BW), and ambient temperature (Cold or T; CC, CT, or TT; Fig. 1). Levels of total AMPKα were not different between muscles whatever the physiological stage. In contrast, the levels of pAMPK and the pAMPK:AMPKα ratio were drastically greater in the red SS than white LM at both 25 BW and 115 BW (Fig. 1B and 1C). In addition, a significant interaction between muscle and physiological stage was found. Total AMPKα and pAMPK abundance was not changed during the GF period in LM whereas they increased in SS between 25 BW and 115 BW (P < 0.001).
In response to cold temperature, Cold piglets exhibited less total AMPKα in LM than T piglets at 25 BW. On the opposite, total AMPKα in LM was greater in CC pigs compared with TT pigs at 115 BW. No difference in total AMPKα was noticed between treatments in SS muscle at both stages. In LM, pAMPK and pAMPK:AMPKα ratio did not differ between treatments at both 25 BW and 115 BW. In SS, a long-term exposure to cold temperature influenced AMPKα phosphorylation at 115 BW by decreasing pAMPK and pAMPK:AMPKα ratio in CC pigs compared with CT and TT pigs. The AMPK status was not influenced by sex (data not shown).
Adenosine Monophosphate-Activated Protein Kinase and Adaptation of Energy Metabolism
Considering GP and LDH activity on the 1 hand and IMF, HAD, and CS activities on the other hand as indicators of glycolytic and oxidative metabolisms, respectively, Pearson’s correlation coefficients were calculated to describe relationships between AMPKα phosphorylation status and energy metabolism in both contrasted LM and SS muscles (Table 5). In LM, pAMPK:AMPKα ratio was negatively correlated with HAD and CS activities at 25 BW (r = –0.48, P = 0.02 and r = –0.47, P = 0.02, respectively) and negatively correlated with GP at 115 BW (r = –0.46, P = 0.007). In SS muscle, pAMPK:AMPKα ratio was negatively correlated with GP at 25 BW and 115 BW (r = –0.57, P = 0.004 and r = –0.54, P < 0.001, respectively) whereas it was not correlated with HAD and CS activities at 25 BW or 115 BW. Whatever the muscle, within physiological stage (25 BW or 115 BW), the AMPK status was not correlated with LDH or IMF content. However, pooled data from the 2 muscles and physiological stages underlined highly significant correlations between pAMPK:AMPKα ratio and GP (r = –0.84, P < 0.0001), IMF content (r = 0.77, P < 0.0001), and LDH, HAD, and CS activities, suggesting a potential general role of AMPKα activity in changes of muscle metabolism between muscles.
|Intramuscular fat content||NS||NS||NS||NS||0.76***|
For a better understanding of the correlations between pAMPK:AMPKα ratio and energy use in regard to environmental temperature, individual projections of GP (Fig. 2) and IMF (Fig. 3) were performed in both thermoneutral and cold exposure conditions. Comparison between panels A and B in Fig. 2 and 3 underlined a specific muscle adaptation to cold rearing conditions. In LM, GP decreased between 25 BW (LM T) and 115 BW (LM TT) more strongly in thermoneutral (–32%; P < 0.01) than in cold rearing conditions (–10%; P = 0.004) without any significant variations of pAMPK:AMPKα ratio between stages. In SS, changes from 25 BW (SS T) to 115 BW (SS TT) denoted an increase of pAMPK/AMPKα (+30%; P < 0.01) and a decrease of GP (–37%; P < 0.01) under thermoneutral conditions. No difference was found between stages (Cold vs. CC groups), mostly due to reduced GP and increased variability of pAMPK/AMPKα at 25 BW in Cold group. Overall, our results indicated an earlier decrease of GP in SS of Cold than T group and a weaker long-term decrease of GP in LM of CC than TT group, which drastically changed the curvilinear shape of the curve observed at thermoneutrality (Fig. 2A).
Under thermoneutral conditions the positive correlation found between pAMPK:AMPKα ratio and IMF content (r = 0.85; P < 0.0001; Fig. 3A) was likely due to the effect of age on IMF in both muscles and on pAMPK in the SS. The greater scatter of individual projections along the pAMPK/AMPKα axis in SS of Cold group (Fig. 3B) attested of a precocious adaptation to cold conditions in SS and broke the relationship between pAMPK/AMPKα and IMF content observed at thermoneutrality. Altogether, cold conditions changed the evolution of muscle metabolism with age and affected the relationships between AMPK activity and glycolytic or lipid muscular energy stores.
Short- and Long-Term Body Adaptations as Influenced by Feed Intake
As anticipated, short- or long-term exposure to cold induced an increase in ADFI (+7.7 and +17.2% between Cold and T or CC and TT pigs, respectively) attesting a greater increase of energy requirements at both PW and GF stages when temperature is below the lower critical value (Le Dividich et al., 1998). Nevertheless, the increased feed intake of Cold piglets during PW was not sufficient to meet the increased energy requirements and energy expenditure in cold conditions (Bee et al., 2004) because they exhibited a slower growth rate than T piglets. This also explains the reduced body fat and energy reserves in Cold compared with T piglets (Rinaldo and Le Dividich, 1991). During the GF period, responses to cold environment resulted in greater ADFI (presented above) and FCR (+20.6% between TT and CC pigs vs. +14.4% between T and Cold pigs) indicating a greater ability to increase voluntary feed intake and a greater impact of cold exposure on FCR during GF than PW period. This did not significantly influence ADG during the GF period but led to a compensatory growth response during the early stage of GF. These growth adaptations involved in optimization of energy retention to meet energy requirements could likely be explained by a balance between a greater ADFI and a decreased heat production per unit of metabolic BW along the GF period in cold environment compared with thermoneutrality (Demo et al., 1995).
Depending on growth stage, environmental temperature influences the physical appearance of pigs, corresponding to an adaptive response aiming at reducing heat loss in the cold environment (Lefaucheur et al., 1991; Demo et al., 1995). Accordingly, Cold pigs exhibited shorter carcass length at 25 BW, and the same trend was observed on CC pigs at 115 BW. By contrast to feeding conditions allowing an equal rate of BW gain but fatter carcasses (Verstegen et al., 1985; Lefaucheur et al., 1991), pigs placed in the cold environment and fed ad libitum were not fatter at 115 kg BW. The less adiposity of Cold compared with T pigs at 25 BW indicates that CC pigs deposited fat at a greater rate than CT or TT pigs afterward, as observed in pigs exhibiting compensatory growth response (Lebret et al., 2007). In compliance with other studies, 25 BW pigs were leaner, less insulated, and finally more sensitive to low temperature than older pigs (Lefaucheur et al., 1991; Rinaldo and Le Dividich, 1991). Only a few changes were reported on carcass traits of 115 BW pigs reared at low temperature and fed ad libitum (Rinaldo and Le Dividich, 1991). Interestingly, short cold exposure applied during PW had reversible effects on pig performance insofar as CT pigs exhibited growth performance and carcass traits similar to TT pigs, thus confirming the lack of interaction between early and long-term effects of cold rearing environment (Gentry et al., 2004). Our experiment suggests also that protein and body fat depositions were dynamic processes during growth but finally exhibited the same ratio after a long-term adaptation (8 to 115 kg BW) to cold conditions because CC, CT, and TT pigs had similar carcass composition except a slightly greater shoulder percentage in CC carcasses. By progressively reducing heat production during growth in cold environment (Demo et al., 1995), acclimation of pigs is supported by thermoregulatory mechanisms affecting energy metabolism and regulation. Indeed, heavier weights of heart and thyroid gland in pigs maintained in the cold environment reflect a more active global energy metabolism. Trends to larger thyroid gland of Cold and CC pigs suggest an altered thyroidal function, in agreement with elevated concentrations of thyroid hormones reported in response to cold stress (Silva, 2006). Effects of thyroid hormones on energy homoeostasis and balance regulation involving AMPK pathway have been identified in hypothalamus and peripheral tissues (Herwig et al., 2008; López et al., 2010) and could be involved in metabolic adaptation of skeletal muscle to environmental temperature.
Short- and Long-Term Muscle Metabolic Adaptations Supported by Different Strategies in Glycolytic and Oxidative Muscles
Physiological muscle adaptations to ambient temperature were evaluated at both 25 and 115 kg BW in 2 contrasted muscles, the white glycolytic LM and the red oxidative SS. As previously reported, glycogen store and LDH activity were greater in LM in agreement with its greater content of fast-twitch type IIB glycolytic fibers (about 75%) than in SS (Fernandez et al., 1995; Larzul et al., 1997; Lebret et al., 1999). Conversely, SS muscle exhibited greater IMF content and CS and HAD enzymes activities than LM in accordance with a greater percentage of slow-twitch type I fibers (around 50%; Lefaucheur et al., 1991) and a greater mitochondrial fatty acid oxidation in these fibers (Schiaffino and Reggiani, 2011). Moreover, muscle glycolytic and oxidative capacities evolve during postnatal pig growth (Lefaucheur and Vigneron, 1986). Thus, our experiment provides additional data on age-related changes in pig muscle metabolism, including the AMPK pathway, between 2 different physiological stages potentially important for pig production and meat quality management.
The increased LDH and decreased CS activities in both LM and SS between 25 BW and 115 BW confirm previous results (Lefaucheur and Vigneron, 1986) and denotes a shift to a more glycolytic metabolism with increasing age and maturation of muscles. Besides, the greater decrease of HAD activity in LM than SS (–27 vs. –12%) indicates a decreased potential to oxidize fatty acids with increasing age in the LM. Independently of muscle type, GP, an indicator of in vivo glycogen concentration, decreased by 30% between 25 BW and 115 BW in thermoneutral conditions. A drastic decrease of glycogen content was also reported in LM and oxidative muscles of pigs during the first days after birth (Okai et al., 1978; Herpin et al., 2002). However, kinetics of muscle glycogen store at older stages is not well documented because most studies measured this trait at commercial slaughter weight in relation to body composition and postmortem metabolism determining meat quality, instead of considering changes in level of energy stores during growth (Monin and Sellier, 1985; Fernandez and Tornberg, 1991). The significant increase in IMF content between 25 BW and 115 BW in LM and SS is consistent with the literature (Herpin et al., 2002; Lebret et al., 2007; Bosch et al., 2012). Increasing IMF during growth provides local energy reserve relevant for muscle adaptation. Nevertheless, in thermoneutral conditions, the slight decrease in HAD activity between 25 BW and 115 BW indicates that increased IMF is not associated with greater fatty acid oxidation for muscle energy supply.
Our results underline the increased metabolic differences between SS and LM muscles during growth but also highlight muscle plasticity to cold exposure. Cold exposure induced a progressive increase of muscle oxidative capacity, evaluated first by greater HAD and CS activities in SS in 25 BW and then in SS and LM in 115 BW pigs, indicating an earlier and more pronounced metabolic adaptation of the oxidative muscle. At 25 kg BW, cold exposure drastically reduced GP in SS but keeping LDH unchanged and increasing lipid oxidation as attested by the greater HAD:CS ratio. Thus, proportionally more energy was derived from IMF in cold than neutral environment to satisfy energy supply. These changes are consistent with the early increase in percentage of slow-twitch type I fibers in oxidative muscles of cold exposed piglets (Herpin and Lefaucheur, 1992; Harrison et al., 1996). Conversely, in LM of CC pigs at 115 BW, the increased GP and LDH, CS, and HAD activities highlight the ability of this muscle to increase both its glycolytic and oxidative metabolisms and diversify its energy substrates, at the advantage of carbohydrate oxidation as attested by the decreased HAD:CS ratio. Besides, it is worth noting that increased IMF content in LM of CC compared with CT and TT pigs occurred independently of carcass fatness. Thus, in cold conditions, the potential to oxidize both carbohydrates and fatty acids increased in LM, which does not fit with the conventional Randle cycle theory suggesting that increasing fatty acid oxidation decreases carbohydrate oxidation and vice versa (Hue and Taegtmeyer, 2009). Similar responses on LM energy stores and metabolism to cold outdoor rearing conditions have been found in free-range pigs produced in winter (Bee et al., 2004) whereas muscle responses are weaker for pigs reared outdoors in intermediate climatic conditions (Lebret et al., 2002; Lebret, 2008).
Finally, the controlled cold environment in our experiment influenced the dynamic of muscle metabolism during growth in a muscle type dependent manner corresponding to an earlier and acute adaptation in the SS muscle from 8 kg onward and a belated adaptation in the LM from 25 to 115 kg BW. The faster response of the SS suggests an earlier and greater plasticity of oxidative muscles to low ambient temperature. To better understand the underlying mechanisms, AMPK status as a key sensor of cellular energy level was characterized in both SS and LM of our animals.
Adenosine Monophosphate-Activated Protein Kinase Involved in Muscle Energy Homeostasis in a Muscle Type Dependent Manner
As reported before, cold environment exposure represents a thermic and metabolic stress involving muscle homeostasis regulation and metabolic processes to maintain cellular ATP concentration. In case of cellular energy deficiency, AMPK activation occurs to adapt substrate supply and degradation to energy requirement (Hardie and Sakamoto, 2006; Scheffler and Gerrard, 2007). By switching off ATP-consuming processes (lipogenesis, protein synthesis, and glycogenesis) and switching on catabolic processes that produce ATP (fatty acid oxidation, glycogenolysis, and glycolysis; Hardie et al., 2006), the role of AMPK in physiological regulation of glucose and lipid metabolism is well recognized (Witczak et al., 2008; Carling et al., 2011). Thus, the more AMPK is phosphorylated, the faster cell homeostasis can be restored by a cascade of metabolic reactions (Witczak et al., 2008). Our study aimed at better understanding the basal modulation of the AMPK pathway in contrasted muscles under cold environment. Interestingly, our results show a much greater pAMPK:AMPKα ratio in the oxidative SS than the glycolytic LM as reported by Derave et al. (2000) and Durante et al. (2002). This could be explained by the fact that SS is a postural muscle exhibiting long lasting contractions of low intensity, which create a chronic energy deficit, as illustrated by its low GP. Indeed, physical muscle contractions represent a high consuming energetic process requiring ATP, which is notably provided by glycogen breakdown (Derave et al., 2000). Because AMPKα activity partly depends on muscle glycogen content (Derave et al., 2000; Sibut et al., 2008), we can hypothesize that the greater AMPKα activity in the SS muscle could result from its lesser glycogen concentration due to permanent contraction activity of this muscle involved in animal posture. In addition, preslaughter fasting could have also decreased muscle glycogen content, in particular in the red SS muscle (Fernandez et al., 1995). Altogether, this could explain the lesser GP at slaughter and greater AMPKα activation status of the SS compared with the LM. By contrast, less basal AMPKα activation has been found in resting red compared with resting white muscles in rats (Winder et al., 2000; Ljubicic and Hood, 2008), even though some of these results were not always confirmed (Ljubicic and Hood, 2009). Discrepancies between studies could be due either to the species, muscle types and physiological function, or experimental conditions and make uneasy to make overall general conclusions about the level of response on AMPKα activation in case of muscle energy adaptation.
After a long-term exposure to a cold environment compared with TT, a lesser level of AMPKα phosphorylation was found in the red SS muscle whereas total AMPKα was increased in the white LM. By contrast, activation of AMPKα has been observed in gastrocnemius muscle of rats kept at 4°C for 4 d (Oliveira et al., 2004), but this has been associated with short-term metabolic changes induced by contractile activity during shivering. In our study considering more than 4 mo cold exposure, shivering was no more observable in CC pigs. In another experiment performed with mice after 4 to 5 wk of cold exposure, Bruton et al. (2010) did not observe any difference in the extent of AMPKα phosphorylation in the flexor digitorum brevis, a 70% fast-twitch type II muscle. Thus, differences between studies could be likely due to different species, muscle types, physiological stage, or experimental conditions such as type, duration, or intensity of treatments. The lesser level of AMPKα phosphorylation in SS muscle of our long-term cold acclimated pigs can be compared with effects of exercise training. Indeed, there is a general consensus showing that previous training decreases muscle AMPKα activation after acute exercise, probably because of reduced metabolic stress in trained muscles (Durante et al., 2002; Ljubicic and Hood, 2008, 2009). Thus, the reduced AMPKα phosphorylation in SS of CC pigs could denote a better adaptability and reduced metabolic stress of this muscle after a long-term adaptation to cold environment.
Relationships between GP and level of AMPKα phosphorylation were reinforced in our study. In thermoneutral conditions, the hyperbolic shape of this relationship indicates that AMPKα activity decreases when GP increases, confirming negative correlations previously reported between these 2 traits (Sibut et al., 2008; Faure et al., 2011, 2012). However, cold conditions modulated this general trend because the relationship within muscle was weaker, even though muscles were still clearly discriminated according to GP or AMPK status. In accordance with the greater increase of AMPK phosphorylation due to contraction in muscles exhibiting decreased glycogen concentration (Derave et al., 2000), the cold conditions maintained a greater level of AMPKα phosphorylation and a reduced glycogen content in the SS. The greater GP of LM at both 25 and 115 kg BW in particular in CC pigs was probably sufficient to provide energy without further AMPKα activation and disturbance of cell homeostasis in this phasic muscle, likely poorly involved in physical exercise and thermogenesis. On the opposite, the very low GP of SS suggests a chronic energy deficiency, explaining its greater level and variability of pAMPK/AMPKα.
Taken together, our results suggest that AMPKα has a major role in long-term adaptation of energy metabolism to a cold environment in the oxidative SS muscle. Interactions between cold environment and muscle energy stores as affected by feeding level of animals (fasted/fed before sampling) remain further studies.
Cold ambient temperature influenced growth performance and muscle metabolism of pigs through increased feed intake for growth and homeostasis regulation requirements. Muscle-specific strategies were found in energy metabolism adaptation. The oxidative SS muscle exhibited an earlier and more intense response toward a more oxidative metabolism, without any early modulation of the AMPKα status. The long-term adaptation of SS to cold environment involved less AMPKα phosphorylation than in thermoneutral environment, which could denote a reduced metabolic cold stress and improved adaptability of red muscles to overcome any other environmental stress. By contrast, the belated response of LM metabolism suggests its lesser sensitivity to cold environment. Long-term adaptation of LM attested a greater potential for use of both carbohydrates and lipidsas energy substrates, thereby preventing this muscle from any chronic energy deficit and explaining its lesser activation of AMPK pathway. Thus, LM may display a greater resistance than SS to protect its energy metabolism regulation to another external stress during growth or postmortem hypoxia. Finally, muscle metabolic responses to constant low ambient temperature could likely influence postmortem muscle metabolism with muscle-type dependent consequences on technological and sensory meat quality.