A Szczepkowska

J Młynarczuk

A Grochowalski

L Dufourny

J.-C Thie ´ry

J Skipor
email: j.skipor@pan.olsztyn.pl

Effect of a two-week treatment with low dose of ortho-substituted polychlorinated biphenyls (PCB104 and PCB153) on VEGF-receptor system expression in the choroid plexus in adult ewes

Keywords: polychlorinated biphenyls: PCB104, PCB153, vascular endothelial growth factor, choroid plexus, ewes

Ortho-substituted polychlorinated biphenyl (PCB) congeners, which constitute a large part of PCB residues found in the environment and in animal tissues, are known to exert potent vascular effects and can activate endothelial cells in the periphery and in the brain. The choroid plexus (CP) is responsible for cerebrospinal fluid (CSF) production and its epithelial cell layer is responsible for structure and functions of the blood-CSF barrier. The aims of this study were: 1) to investigate if environmentally relevant doses of PCB153 and similar doses of PCB104 caused changes in the expression of vascular endothelial growth factor (VEGF) -receptor system, which maintains CP function, and 2) to determine the level of both congeners in blood plasma after their oral administration. Studies of both congeners were performed on ovariectomized ewes treated per os with low doses (0.1 mg/kg, three times a week for two weeks) of PCB153 (n=4) or PCB104 (n=4) and vehicle (control, n=4). The effects of PCB153 and PCB104 treatment on mRNA expression of two isoforms of VEGF (VEGF 120 and VEGF 164 ) and their receptors Flt-1 and KDR were determined using real-time PCR. Plasma concentration of PCBs was measured using high resolution chromatography/tandem mass spectrometry (HRGC/MS-MS). We observed that neither PCB153 nor PCB104 significantly altered the mRNA of the VEGF-receptor system in the CP. In PCB treated animals plasma concentration of PCB153 (1.425 ± 0.16 ng/g of dry mass, DM) was about 150 times higher than PCB104 (0.009 ± 0.007ng/g DM). In control animals the PCB153 level was 0.14 ± 0.031 ng/g DM, while the PCB104 level was below detection level. This indicates that increase in plasma PCB153 concentration to levels similar to those reported in humans and of PCB104 concentration to levels 100 times higher than those found in human plasma did not affect the VEGF-receptor system in the CP in adult ewes. The significantly lower increase of PCB104 than PCB153 concentration in blood after oral administration suggests different absorption of both congeners from the digestive tract.

Introduction

Polychlorinated biphenyls (PCBs) are chemicals that comprise a group of 209 congeners with varying degrees of chlorination, which determines their physical, chemical, and biological properties [START_REF] Safe | Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment[END_REF]. From a structural point of view, there are at least two distinct classes of PCBs: the non-ortho (coplanar) and ortho-substituted (planar) congeners. Ortho-substituted PCBs constitute a large part of the PCB residue found in the environment and animal tissues [START_REF] Kodavanti | Congener-specific distribution of polychlorinated biphenyls in brain regions, blood, liver, and fat of adult rats following repeated exposure to Aroclor 1254[END_REF][START_REF] Faroon | Effects of polychlorinated biphenyls on the nervous system[END_REF][START_REF] Costera | PCDD/F and PCB transfer to milk in goats exposed to a long-term intake of contaminated hay[END_REF]. The most environmentally relevant ortho-substituted PCB -2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) has been demonstrated to affect the reproductive function and puberty onset in young goats which were natally exposed to a low dose of PCB153 (Lyche et al. 2004[START_REF] Oskam | Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats[END_REF]. We recently demonstrated that a low dose of PCB153 affects gonadotropin secretion in adult female ewes through an action on both pituitary Luteinizing Hormone (LH) release and the hypothalamic Gonadotropin Releasing Hormone (GnRH) pulse generator [START_REF] Skipor | Photoperiod modulates access of 2,2',4,4', 5,5'-hexachlorobiphenyl (PCB153) to the brain and its effect on gonadotropin and thyroid hormones in adult ewes[END_REF]. Although the brain appears to be better protected against PCBs than other tissues because of the blood-brain and blood-cerebrospinal fluid (CSF) barriers [START_REF] Skipor | The choroid plexus-cerebrospinal fluid system: undervaluated pathway of neuroendocrine signaling into the brain[END_REF], ortho-substituted PCBs have been reported to accumulate preferentially in the brain and CSF compared to other PCBs [START_REF] Kodavanti | Congener-specific distribution of polychlorinated biphenyls in brain regions, blood, liver, and fat of adult rats following repeated exposure to Aroclor 1254[END_REF][START_REF] Takasuga | Ultratrace analysis of polychloronated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) in human serum and cerebrospinal fluid (CSF) samples[END_REF][START_REF] Montie | Organohalogen contaminants and metabolites in cerebrospinal fluid and cerebellum gray matter in short-beaked common dolphins and Atlantic white-sided dolphins from the western North Atlantic[END_REF]. Recently, measurement of PCB concentration in the ovine CSF showed that PCB153 was the most abundant congener in the CSF (Skipor et al. 2012).

There is evidence that PCB153 disrupts expression of tight junction proteins in the blood-brain barrier which restricts most paracellular movement of ions and solutes across the brain barriers [START_REF] Eum | PCB and tight junction expression[END_REF][START_REF] Seelbach | Polychlorinated biphenyls disrupt blood-brain barrier integrity and promote brain metastasis formation[END_REF]). Among ortho-substituted PCBs, 2,2',4,5,6'-pentachlorobiphenyl (PCB104) also affects tight junction proteins expression in brain endothelial cells, stimulates inflammatory mediators in vascular endothelial cells and induces prometastatic responses [START_REF] Choi | PCB 104-induced proinflammatory reactions in human vascular endothelial cells: relationship to cancer metastasis and atherogenesis[END_REF][START_REF] Sipka | Oral administration of PCBs induces proinflamatory and prometasstatic responses[END_REF]. It has been demonstrated that vascular endothelial growth factor (VEGF) is involved in PCB104 induced endothelial hyperpermeability [START_REF] Eum | VEGF regulates PCB 104-mediated stimulation of permeability and transmigration of breast cancer cells in human microvascular endothelial cells[END_REF]. VEGF is continuously and highly expressed in the choroid plexus (CP), in which the blood-CSF barrier is located [START_REF] Skipor | The choroid plexus-cerebrospinal fluid system: undervaluated pathway of neuroendocrine signaling into the brain[END_REF]. In the ovine CP two isoforms, VEGF 120 and VEGF 164 , are expressed [START_REF] Szczepkowska | Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods[END_REF]. VEGF plays an important role in regulation of the stability of the endothelial cells in the CP [START_REF] Maharaj | VEGF and TGF-β are required for the maintenance of choroid plexus and ependyma[END_REF] and is involved in maintaining endothelial cells' fenestrated phenotype in the CP capillaries [START_REF] Roberts | Increased microvascular permeability and endothelial fenestrations induced by vascular endothelial growth factor[END_REF]Palade 1995, Esser et al. 1998). The effects of VEGF are transduced mainly by two high-affinity receptors belonging to the tyrosine kinase-family: the fms-like tyrosine kinase (Flt-1) and the fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR). The CP has been shown to contain mRNA [START_REF] Nico | Vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2 and angiopoietin-2 expression in the mouse choroid plexuses[END_REF]) and protein [START_REF] Witmer | Expression of vascular endothelial growth factor receptors 1, 2, and 3 in quiescent endothelia[END_REF][START_REF] Maharaj | VEGF and TGF-β are required for the maintenance of choroid plexus and ependyma[END_REF][START_REF] Yang | VEGF/VEGFR-2 changes in frontal cortex, choroid plexus, and CSF after chronic obstructive hydrocephalus[END_REF] of VEGF receptors.

Studies on PCB toxicity are mainly based on in vitro models or in vitro experiments in which mice or rats are intraperitoneally injected with PCBs [START_REF] Sipka | Oral administration of PCBs induces proinflamatory and prometasstatic responses[END_REF]. The in vivo effects of PCB153 and PCB104 on expression of the VEGF system in the CP in domestic animals exposed to PCBs through the food chain, the most common route of PCB exposure, has not yet been reported. Therefore, the aim of this study was to evaluate the effect of chronic oral administration of low doses (0.1 mg/kg of body weight, bw) of PCB104 and PCB153 on expression of the VEGF system in the CP in ewes. Additionally, the concentration of both PCB congeners was measured in blood plasma.

Materials and Methods

Animal management and experimental design

The experiment was conducted on adult Polish Lowland ewes (n=16, 60-70 kg bw). To prevent variability linked with the estrous cycle or interaction with steroids the animals were ovariectomized (under xylazine anaesthesia (0.25 ml/kg, im; Rometar, Spofa, Prague, Czech Republic) about 20 days before PCB administration. All animals were maintained indoors under natural lighting conditions (October-November), each group being in separated pens in order to avoid contamination through the ingestion of PCB-contaminated faeces. The ewes were fed a diet of hay, straw, and commercial concentrates with water and mineral licks available ad libitum. Experimental procedures were conducted in accordance with the Polish Guide for the Care and Use of Animals (1997) and approved by the Local Ethics Committee (agreement no 4/2008).

After recovery, the animals were randomly allocated to three groups: group 1 -control (control), group 2 -PCB104 treated (PCB104) and group 3 -PCB153 treated (PCB153). Animals in the PCB104 and PCB153 groups were treated with appropriate PCB congeners by oral gavage three times weekly for 14 days, at a dose of 0.1 mg/kg of bw. Animals from the control group were treated with sunflower oil used as a vehicle for PCBs. Blood samples (20 ml) were collected from the jugular vein 15 days after beginning the PCB treatment (1 day after the end of PCB treatment) and then centrifuged and stored at -20 o C for further analysis of PCBs. The animals were then sacrificed and, immediately after decapitation, the brains were dissected out and the CP from the lateral ventricles were collected and stored at -80 o C until further analysis.

Calculation of doses and PCB administration

PCB104 (2,2',4,5,6'-pentachlorobiphenyl) and PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) from Ac-cuStandard (New Haven, CT, USA) were purchased locally at Tusnovics Instruments Polska and were 99.9% pure. Each PCB was dissolved in sunflower oil to a concentration of 1 mg/ml. Literature data (Lyche et al. 2004b) indicated that similar doses of PCB153 given orally in sheep for 30 days resulted in a plasma concentration of PCB153 of 30 ng/g (wet-weight). [START_REF] Costera | PCDD/F and PCB transfer to milk in goats exposed to a long-term intake of contaminated hay[END_REF] demonstrated that, in goats exposed to contaminated hay (concentration of PCB153 of about 460 ng/kg of dry mass (DM)), the time to reach a steady state concentration in milk was 15 days for most PCB congeners.

Relative gene expression assays

CP tissue, frozen and cut into small pieces (20 mg), was homogenized in Fenozol in Lysing Matrix D (MP Biomedicals, Illkrich, France) with a Fas-tPrep-24 instrument (MP Biomedicals). RNA was extracted using a Total RNA Kit (A&A Biotechnology, Poland), and Amplification Grade DNase 1 (Sigma Aldrich, Germany) was used to eliminate possible genomic DNA contamination, according to the manufacturer's instructions. The concentration and quality of RNA isolated from the CP tissue were determined using a NanoDrop (Thermo Scientific, USA) and 2% agarose gel electrophoresis. One microgram of total RNA was retained for further use in RT (reverse transcription) reaction. The RT reaction was performed with Quanti Tect Reverse Transcription Kit according to the protocol supplied by the manufacturer (Qiagen, USA). The resulting cDNA was stored at -20 o C until further analysis.

To evaluate the effects of PCB treatment on mRNA expression of components of the VEGF-receptor system, real-time PCR was performed with the use of an ABI Prism 7900 sequence detection system and a Power SYBR green PCR master mix (Applied Biosystems by Life Technologies, Carlsbad, CA, USA). Specific primer pairs for the different genes were used according to the literature (Table 1). All primers were synthesized by IBB PAN (Poland).

PCR-derived DNA fragments (VEGF-A 120 , VEGF-A 164 , Flt-1, KDR, NRP-1, reference genes-RPL19 and PPIC) were separated by electrophoresis on 2% agarose gels supplemented with 0.01% ethidium bromide and examined under UV light (Gel Logic100, KODAK). After the cycles, a final melting curve analysis under continuous fluorescence measurement was performed to evaluate the specific amplification. The results were analyzed using Real-time PCR Miner (available on-line: http://www.miner.ewindup.info/ version2), based on the algorithm developed by [START_REF] Zhao | Comprehensive algorithm for quantitative real-time polymerase chain rReaction[END_REF].

PCB determination in blood plasma

Blood plasma samples (20 ml) were freeze dried and spiked with clean-up standards using 1 ml of 13 C-PCBs/g of the sample with standard mixture containing 10 ng/ml of 13 C-PCB104 and 20 ng/ml of 13 C-PCB153, and extracted with toluene for 16 h in 20 ml Soxhlet apparatus. The extract was cleaned up according to the previously published procedure (Surma-Zadora and Grochowalski 2008). The cleaned-up extract was evaporated to approximately 1 ml, transferred to 5 ml of dichloromethane and placed in a polyethylene semipermeable membrane. Dialysis was performed within 24 h with hexane as a recovery solvent. The hexane dialysate was then cleaned-up on a column which contained 10 g of 50% H 2 SO 4 on silicagel. The column was eluted with 100 ml of hexane, evaporated to approximately 1 ml and transferred to another column containing 5 g of acidic Alumina (Merck 101078, 0.063 -0.200 mm of activity I) and eluted firstly with 15 ml of hexane (fraction was discarded). The PCBs were then eluted in the second fraction of 50 ml of 2% dichloromethane in hexane. The eluate was evaporated just to dryness and reconstituted into 50 μl of nonane containing 100 pg/ml of 13 C-1,2,3,4-tetrachlorodiben- The method is of high specificity and gives non-interfered signals in the determination of PCB104 and 1CB153 congeners. Recoveries of PCB104 and PCB153 were in the range of 65 -90% and 85-120%, respectively.

Data analysis

PCB data are expressed as the mean ± SD. The real-time PCR results are presented as the relative gene expression (mean ± SEM) of the target gene vs. the reference gene (RPL19). The significance of differences were statistically analyzed using one-way ANOVA (PRISM 4, Graph Pad, USA).

Results

PCB determination in blood plasma

The mean level of PCB congeners in blood plasma of sheep after oral administration of a low dose (0.1 mg/kg/per day) of PCB153 and PCB104 is presented on Fig. 1. In control sheep concentration of PCB153 was 0.14 ± 0.031 ng/g of DM, while the PCB104 level was below the detection limit (0.0001 ng/g DM). After oral administration of PCB153 and PCB104, their concentrations in blood plasma were 1.425 ± 0.16 ng/g DM and 0.009 ± 0.007 ng/g DM, respectively.

Real Time PCR analysis

PCB104 and PCB153 treatment had no significant effect on the expression of all tested genes: VEGF-A 120 , VEGF-A 164 , Flt-1 and KDR (Fig. 2) in comparison with the control group.

Discussion

This is the first study investigating the effect of environmentally relevant PCB153 in vivo on the VEGF-receptor system in the CP. In this study PCB153 action was compared with that of PCB104, formerly documented as affecting the VEGF system in vitro [START_REF] Eum | VEGF regulates PCB 104-mediated stimulation of permeability and transmigration of breast cancer cells in human microvascular endothelial cells[END_REF]). Neither PCB153 nor PCB104 exposure significantly altered the mRNA of the VEGF-receptor system expression in the CP in ovariectomized adult sheep. The high variability of PCB concentration (78.5% for PCB104 and 21.8% for PCB153) observed in our study may account for large individual differences in PCB action on VEGF-receptor system mRNA expression. An earlier study demonstrated that maternal exposure to low doses (98 μg/kg bw) of PCB153 during gestation and lactation suppressed prepubertal plasma LH concentrations, delayed the onset of puberty of the female offspring, altered bone composition, and suppressed neonatal immunity in goats (Lyche et al. 2004a, b, Lyche et al. 2006[START_REF] Lundberg | Perinatal exposure to PCB 153, but not PCB 126, alters bone tissue composition in female goat offspring[END_REF]). Recently we observed symptoms of estrogenization (enlarged vulva and edematous uterus) in PCB153 treated ewes, in which the plasma concentration of PCB153 was 196 pg/ml, equivalent to 2.4 ng/g DM [START_REF] Skipor | Photoperiod modulates access of 2,2',4,4', 5,5'-hexachlorobiphenyl (PCB153) to the brain and its effect on gonadotropin and thyroid hormones in adult ewes[END_REF]. Moreover, in these sheep basal plasma LH and pulsatile LH release was also affected [START_REF] Skipor | Photoperiod modulates access of 2,2',4,4', 5,5'-hexachlorobiphenyl (PCB153) to the brain and its effect on gonadotropin and thyroid hormones in adult ewes[END_REF]). It should be noted that the mean plasma concentration of PCB153 found in our study (1.4 ng/g DM, corresponding to 0.116 ng/ml) as well as in our earlier study (0.196 ng/ml) is similar to the PCB153 concentration measured in the human population in the United States (http://www.cdc.gov/exposurereport). According to a national report on human exposure to environmental chemicals, the geometric mean of serum PCB153 concentration in humans was 0.167 ng/g (0.151-0.185 ng/g, 95% confidence interval, CI) in 2001-2002 and 0.121 ng/ml (0.114-0.128 ng/ml, 95% CI) in 2003-2004. In constrast to PCB153, PCB104 is not environmentally and tissue relevant (http://www.ewg.org/reports/bodyburden2, [START_REF] Fielden | Examination of the estrogenicity of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its hydroxylated metabolite 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and a further chlorinated derivative, 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155)[END_REF], mainly due to the low distribution in commercial PCB mixtures [START_REF] Frame | A collaborative study of 209 PCB congeners and 6 Aroclors on 20 different HRGC columns[END_REF]. In our studies, treatment of ewes with a low dose of PCB104 increased its concentration in blood plasma from an undetectable level to 0.009 ng/g DM (corresponding to 0.75 pg/ml). In humans, measurement of PCB104 in umbilical blood plasma gave 0.9 pg/g of lipid weight, corresponding approximately to 0.007 pg/ml (http://www.ewg.org/reports/ bodyburden2). Therefore, we demonstrated here that a 100 times higher concentration of PCB104 than those found in humans did not affect the VEGF-receptor system in the CP. The dose of PCB104 used in in vitro studies promoting an increase in the microvascular permeability and the transmigration of breast cancer cells following PCB104 treatment ranged from 2-15 μM, which corresponds to 650-4860 ng/ml. Such high concentrations of total PCBs (3-6 μM) were detected in human blood plasma in industrialized areas of the Netherlands and in the Aland/Turku Archipelago [START_REF] Koopman-Esseboom | Dioxin and PCB levels in blood and human milk in relation to living areas in The Netherlands[END_REF][START_REF] Hagmar | Consumption of fatty fish from the Baltic Sea and PCB in whole venous blood, plasma and cord blood from delivering women in the Aland/Turku Archipelago[END_REF].

In our studies, treatment of ewes with the same low doses (0.1 mg/kg bw) of PCB104 and PCB153 resulted in about 150 times higher concentration of PCB153 in blood plasma compared to PCB104. This could result from feeding ewes with hay containing PCB153 since the concentrations of PCB153 congener in hay is among the highest compared to other congeners [START_REF] Chauhan | Assessing the role of ortho-substitution on polychlorinated biphenyl binding to transthyretin, a thyroxine transport protein[END_REF]. [START_REF] Costera | PCDD/F and PCB transfer to milk in goats exposed to a long-term intake of contaminated hay[END_REF] showed that amounts of PCB153 in hay may reach 460 ng/kg/DM. It is also possible that our findings may result from the lower resistance to the biotransformation for PCB104 than for PCB153, which was demonstrated to be very resistant [START_REF] Thomas | PCBs in dairy cows -metabolism and body-burden[END_REF]. Indeed, about 22% of the total PCB residue in human tissue consists of PCB153 [START_REF] Jensen | Structures and levels of most chlorobiphenyls in two technical PCB products and in human adipose tissue[END_REF]. It has been suggested that binding of PCB with transthyretin in blood plasma protects against their rapid removal from circulation. PCB153 binds with transthyretin more effectively than PCB104 (IC50, 90 vs. >1000) [START_REF] Chauhan | Assessing the role of ortho-substitution on polychlorinated biphenyl binding to transthyretin, a thyroxine transport protein[END_REF]. In our study, the mean concentration of PCB153 in blood plasma was about 1.4 ng/ml. This is below the levels measured in the plasma of pregnant goats (30 ng/g wet weight) long-term treated with a similar dose of PCB153 (Lyche et al. 2004b). Such discrepancy between two closely related animal families could be due to species difference, as differences in reactivity to PCB treatment has also been reported between rats and mice [START_REF] Craft | Comparative responsiveness of hypothyroxinemia and hepatic enzyme induction in long-Evans rats versus C57BL/6J mice exposed to TCDD-like and phenobarbital-like polychlorinated biphenyl congeners[END_REF]. Alternatively, it could stem from the use of pregnant goats by Lyche et al. (2004b) and ovariectomized sheep in our study. Finally, the endocrine status of the female rather than a difference in regimen of the treatment may be responsible for such a discrepancy, since we demonstrated that in sheep two-week oral administration of low doses of PCB153 (0.3 mg/kg bw) induced higher levels of PCB153 in short days than long days [START_REF] Skipor | Photoperiod modulates access of 2,2',4,4', 5,5'-hexachlorobiphenyl (PCB153) to the brain and its effect on gonadotropin and thyroid hormones in adult ewes[END_REF].

In summary we demonstrated that increase of plasma PCB153 concentration to the level present in humans and PCB104 concentration to the level exceeding 100 fold that present in human plasma did not affect the VEGF-receptor system in the CP in adult ewes.

  Each real-time PCR reaction well (20 μl) contained 2 μl of diluted RT product, 0.2 μM forward and reverse primers each and 10 μl of Power SYBR green PCR master mix. The following protocol was used: 95 o C for 15 min for Hot Start AmpliTaq Gold DNA polymerase and 40 cycles of 95 o C for 10 s (denaturation), 55 o C for 20 s (annealing), and 72 o C for 20 s (extension).

Fig. 1 .

 1 Fig. 1. Mean levels (±SD) of PCB104 (A) and PCB153 (B) in blood plasma of ovariectomized ewes receiving vehicle (C-PCB104 and C-PCB153), PCB104 (T-PCB104 -A) or PCB153 (T-PCB153 -B) per os (0.1 mg/kg of body weight; 6 doses over 14 days). Note that PCB104 in control group was below detection limit.

  zodioxin ( 13 C-1,2,3,4-TCDD) as a precision and recovery standard. The determination of PCBs was performed in an accredited laboratory (Laboratory for Trace Organic Analyses, Krakow University of Technology, Poland) using the isotope dilution method, high resolution chromatography/tandem mass spectrometry (HRGC/MS-MS) on a Thermo Scientific GCQ-1100/Trace2000 system adjusted to double fragmentation mode equipped with Xcalibur data acquisition and analysis software. Separation of PCB congeners was performed on a 30 m × 0.25 mm i.d. DB5MS capillary column of 25 μm film and DB17 30 m × 0.25 mm i.d. A sample of 2 μl volume was injected into a SSL injector at 260 o C. The GC oven temperature was held at 100 o C for 1 minute, and then raised to 180 o C at 20 o C/min, then 2 o C/min to 260 o C, and then 20 o C/min to a final temperature of 300 o C. The temperature was held at 300 o C for 5 minutes.

Fig. 2 .

 2 Fig. 2. Effect of PCB104 and PCB153 on mRNA expression of VEGF120 (A), VEGF164 (B), and its receptors Flt-1 (C) and KDR (D) in the choroid plexuses of adult ovariectomized sheep. Results were normalized to control group receiving vehicle (control) which was considered as 100%.

Table 1 .

 1 Sequences of oligonucleotide primers used for Real Time -PCR analyses.

	Gene	Primers (5'→3')	Product size	Literature source
	VEGF-A 120	Forward: AAGGCCAGCACATAGGAGAG	101bp	Kaczmarek et al. (2008)
		Reverse: CCTCGGCTTGTCACATTTTT
	VEGF-A 164	Forward: GAGGCAAGAAAATCCCTGTG	150bp	Kaczmarek et al. (2008)
		Reverse: TCACATCTGCAAGTACGTTCG
	Flt-1	Forward: TGGATTTCAGGTGAGCTTGGA	68bp	Redmer et al. (2005)
	VEGF receptor 1	Reverse: TCACCGTGCAAGACAGCTTC
	KDR	Forward: CTTCCAGTGGGCTGATGACC	67bp	Redmer et al. (2005)
	VEGF receptor 2	Reverse: GCAACAAACGGCTTTTCATGT
	NRP-1	Forward: GATTGCGGTGGACGATATTAGC	60bp	Vonnahme et al. (2006)
	neuropilin	Reverse: GGTTTTGCGCAGTCCTCTTG
	RPL19	Forward: AATCGCCAATGCCAACTC	156bp	Drouilhet et al. (2010)
	ribosomal protein L19	Reverse: CCCTTTCGCTTACCTATACC
	PPIC	Forward: TGGCACTGGTGGTATAAGCA	145bp	Herman et al. (2010)
	peptidyl-prolyl cis-	Reverse: GGGCTTGGTCAAGGTGATAA
	trans isomerase C

Unauthenticated | 194.167.77.1 Download Date | 3/19/13 2:15 PM

Acknowledgement

This work was supported by the Polish Ministry of Science and Higher Education and Grant 258/N-INRA/2008/0 and DWM/N19/INRA/2008.


Louis L Lefaucheur

Bénédicte Lebret

Patrick P Ecolan

Isabelle Louveau

Marie M Damon

Armelle Prunier

Yvon Billon

Pierre P Sellier

Hélène Gilbert

Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs

Keywords: composition, muscle fiber type, residual feed intake, selection line, swine

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT: Residual feed intake (RFI) is defined as the difference between the observed feed intake and that expected based on requirements for maintenance and production. A divergent selection was conducted during 4 generations in Large White male pigs to produce low and high RFI lines. The present study aims at determining the influence of this selection on biochemical and histological traits of skeletal muscle, and relating these changes to correlated effects on growth, carcass composition, and meat quality traits. At 8 d preslaughter, biopsies from the LM were taken in the fed state on 14 females from each RFI line fed ad libitum. Animals were slaughtered at 107.8 ± 8.0 kg of BW without any previous fasting. Samples of LM, semimembranosus (SM), biceps femoris (BFM), and rhomboideus muscles were taken at both 30 min and 24 h postmortem. Myofiber typing was only assessed in LM. Low RFI pigs ("efficient") had leaner carcasses with greater muscle content (P < 0.001), less backfat thickness (P < 0.001), and less intramuscular fat content in all 4 muscles (P < 0.01 to P = 0.04). Their greater muscle content was associated with hypertrophy of all fast-twitch fibers. Glycogen content in all glycolytic muscles (i.e., LM, SM and BFM), was greater in low than high RFI pigs.

The greater accumulation of glycogen in LM of low RFI pigs was specifically located in the fast-twitch glycolytic IIBW fibers, which correspond to fibers containing IIb, IIb + IIx, or IIx myosin heavy chains. The difference in muscle glycogen content between RFI line pigs was more significant in the living animals (P = 0.0003) than at 30 min postmortem (P = 0.08). This was associated with a decreased ultimate pH (P = 0.001), and greater lightness of color (P = 0.002) and drip loss (P = 0.04) in LM of low than high RFI line pigs, suggesting that selection for reduced RFI may impair some meat quality traits, such as water-holding capacity. Pigs from the low RFI line exhibited a greater (P = 0.02) percentage of IIBW fibers in LM and tended (P < 0.10) to have less lipid β-oxidative capacity in LM, SM, and BFM. In contrast, no difference (P > 0.10) between lines was found for citrate synthase and lactate dehydrogenase activities, mitochondrial activity, and expression of genes coding for uncoupling proteins 2 and 3. Differences between RFI pigs in plasma leptin, cortisol, and thyroid hormone concentrations are presented and discussed. In conclusion, selection for low RFI influenced muscle properties in a way favoring muscle mass, but likely impairing meat quality.

INTRODUCTION

Improving feed efficiency is a relevant way to reduce both the cost of pork production and the environmental impact of pig production. Residual feed intake (RFI) is defined as the difference between the observed feed intake and that expected on the basis of requirements for maintenance and growth [START_REF] Kennedy | Genetic and statistical properties of residual feed intake[END_REF]. At INRA, a divergent selection experiment for RFI has been undertaken under ad libitum feeding conditions in Large White (LW) male pigs to produce a low ("efficient") and a high ("luxurious") RFI line [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]. After 4 generations of selection, reducing RFI was genetically correlated with decreased carcass backfat thickness (r a = 0.44 ± 0.16) and increased lean meat content (LMC; r a = -0.55 ± 0.14). Interestingly, a correlated genetic response to selection was a decreased ultimate pH and an increased lightness in glycolytic Muscle characteristics and meat quality traits are affected by divergent selection on residual feed intake in pigs 1muscles of pigs from the low vs. high RFI line, suggesting an impairment of some pork quality traits in the low RFI line. A recent review shows that multiple physiological processes are involved in interanimal variations in RFI, such as feed intake and digestion processes, metabolic utilization of feed, thermogenesis, physical activity, stress susceptibility, and health status [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. Because skeletal muscle accounts for approximately 50% of body mass and about 25% of basal metabolic rate [START_REF] Henriksson | The possible role of skeletal muscle in the adaptation to periods of energy deficiency[END_REF], changes in contractile and metabolic properties of skeletal muscle could explain some variation in RFI and correlated effects on muscle development and meat quality. The major objective of the current study was to compare biochemical and histological traits of skeletal muscle between low and high RFI pigs after 4 generations of divergent selection, and relate these changes to correlated effects on muscle growth, meat quality traits, and hormonal status of the animals. Different muscle sampling and myofiber typing technologies were evaluated to compare the 2 RFI lines.

MATERIALS AND METHODS

During the whole experiment, animals were raised in accordance with French legislation on animal experimentation. Pigs were slaughtered by electrical stunning and exsanguination in compliance with the current national French regulations applied in slaughterhouses.

Animals and Experimental Design

The selection experiment was conducted at INRA within the French LW breed as described previously in detail by [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]. All pigs were free of the RyR1 halothane-sensitive (n) and AMP-activated protein kinase (AMPK) γ3 R225Q mutations [START_REF] Fujii | Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia[END_REF][START_REF] Milan | A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle[END_REF]. Briefly, the phenotype for RFI was predicted from a selection index derived as a multiple regression of ADFI, ADG, backfat thickness, and metabolic BW at the phenotypic level. Divergent selection for RFI was conducted during 4 generations in noncastrated males recorded between 35 and 95 kg of BW, whereas random selection was performed on females, each dam being replaced randomly by 1 daughter. Eight to twelve animals per pen were offered ad libitum a pelleted diet based on cereals and soybean meal (10 MJ of NE/kg, 160 g of CP/kg, with at least 0.80 g of digestible Lys/MJ of NE). Feed intake of each candidate and some sibs was recorded in the INRA facilities in Rouillé (Poitou-Charentes, France) using a single-place electronic feeder ACEMA 64 (Pontivy, France) as described previously [START_REF] Labroue | Feeding behavior of group-housed Large White and Landrace pigs in French central test stations[END_REF]. The estimated difference in RFI between the high and low lines after 4 generations was 0.72 phenotypic SD (i.e., 79 g/d). The present study involved 14 females from each line (from 5 different litters and 3 to 4 sires within lines) at similar final slaughter BW (107.8 ± 8.0 kg). Two series of animals from 2 pens (1 per line, 6 to 8 pigs) equipped with collective feeders and tested in 2 successive batches were transported from Rouillé to INRA experimental facilities in Saint-Gilles (4 h transport) at about 100 kg of BW, without any mixing of pigs between pens. Genetic analyses using the REML methodology [START_REF] Patterson | Recovery of inter-block information when block sizes are unequal[END_REF] applied to a multi-trait individual animal model [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF] were carried out including the pedigree of generations 0 to 4. In generation 4, differences of genetic values between the low and high RFI lines for the 27 animals involved in the present study were similar to those estimated between their contemporary groups. Thus, differences were 10.5 points for the selection index (compared with 12.8 points for the contemporary group), corresponding to 76.7 g/d (compared with 93.0 g/d). Differences were 108.6 g/d for daily feed intake (compared with 141.8 g/d), 0.19 kg/kg for feed conversion ratio (compared with 0.18 kg/kg), -4.1 g/d for ADG (compared with 7.0 g/d), -1.44% for LMC (compared with -1.05%), and 3.66 points for the meat quality index (compared with 3.19). Tested on the contemporary groups, all traits but ADG were significantly different between the lines.

In the INRA experimental facilities at Saint-Gilles, animals were kept for 12 d before slaughter in pens equipped with collective feeders, and feed composition was similar to that delivered in Rouillé. Because muscle glycogen is a labile compound that can be quickly metabolized during preslaughter stress and postmortem processing, biopsies from the LM were taken 8 d before slaughter without any fasting or stress to assess in vivo muscle glycogen content and its distribution among myofibers. The LM biopsies, approximately 500 mg, were taken at the level of the last rib curvature, 8 cm from the midline. The entire biopsy procedure was carried out within a few minutes using a shot-biopsy device (Biotech, Nitra, Slovakia) as described previously [START_REF] Geverink | The effect of shot biopsy on behavior, salivary cortisol, and heart rate in slaughter pigs[END_REF][START_REF] Hamilton | The impact of longissimus glycolytic potential and short-term feeding of magnesium sulfate heptahydrate prior to slaughter on carcass characteristics and pork quality[END_REF][START_REF] Damon | Number of intramuscular adipocytes and fatty acid binding protein 4 content are significant indicators of intramuscular fat level in crossbred Large White × Duroc pigs[END_REF], and approved by the French Veterinary Services [START_REF] Damon | Number of intramuscular adipocytes and fatty acid binding protein 4 content are significant indicators of intramuscular fat level in crossbred Large White × Duroc pigs[END_REF]. Immediately after sampling, the collected biopsies were trimmed of fat and skin, oriented according to muscle fiber longitudinal axis, embedded in Tissue-Tek OCT compound (Sakura, Tokyo, Japan) on a piece of cork, frozen in 2-methylbutane (isopentane) prechilled with liquid nitrogen, and stored at -80°C until further processing. Pigs were slaughtered 8 d after the biopsy in our INRA experimental slaughterhouse (Saint-Gilles, France) after a very short individual transportation (about 300 m) and without any previous fasting or stress (no mixing of animals) to limit preslaughter glycogen breakdown, and thus be as close as possible to the in vivo physiological situation. Our experimental facilities allowed increased standardization of preslaughter and slaughter conditions.

Carcass Traits and Organ Weights

Weights of hot carcass, rhomboideus muscle (RM), heart, liver, and thyroid gland were recorded at slaugh-ter. After 24 h at 4°C, wholesale cuts of the left halfcarcass (ham, shoulder, belly, loin, and backfat) were weighed. Lean meat content was estimated from a linear combination of the weights of the ham, loin, and backfat, and expressed as a percentage of the half-carcass weight using the equation LMC = 5.684 + 1.197% (ham) + 1.076% (loin) -1.059% (backfat) [START_REF] Métayer | Estimation, par découpe, de la teneur en viande maigre des carcasses de porcs[END_REF]. The whole LM was dissected from the left loin and weighed.

Meat Quality Traits

Thirty minutes after slaughter, samples of LM (3rd/4th lumbar vertebra level), semimembranosus (SM), biceps femoris (BFM), and RM were taken, mounted on tongue depressors, frozen in isopentane prechilled with liquid nitrogen, and stored at -80°C until further processing. The pH at 30 min postmortem was determined on LM, SM, and BFM muscles after homogenization of 2 g of muscle in 18 mL of 5 mM iodoacetate (Ingold Xerolyte electrode, Knick pH-meter, Berlin, Germany). Muscle glycolytic potential (GP), a trait directly related to in vivo muscle glycogen content at the time of slaughter, defined as GP = 2([glycogen] + [glucose] + [glucose 6P]) + [lactate], was measured on LM, SM, BFM, and RM and expressed as micromoles of lactate equivalents per gram of fresh tissue [START_REF] Lebret | Influence of rearing conditions on performance, behavioral, and physiological responses of pigs to preslaughter handling, carcass traits, and meat quality[END_REF]. The next day, ultimate pH (pH 24 h postmortem) was measured directly in LM, SM, and BFM muscles using the same pH meter as described above. The CIE L* (lightness), a* (redness), and b* (yellowness) parameters were determined in triplicate in the central part of LM, SM, and BFM muscles at 24 h postmortem using a Minolta chromameter CR-300 (Osaka, Japan), with a D65 illuminant and a 1-cmdiameter aperture. A fresh section of each muscle was allowed to bloom for 1 h at 4°C under artificial light before L*, a*, and b* measurements. The same day, approximately 100 g of LM (cranial to the last rib cut) was collected, trimmed of external fat and perimysium, weighed, and kept at 4°C in a plastic bag for a subsequent 24 h for determination of drip loss after 2 d postmortem [START_REF] Honikel | Reference methods for the assessment of physical characteristics of meat[END_REF]. Muscle slices were also taken on all 4 muscles, trimmed of external fat, minced, and freeze-dried before determination of intramuscular fat content after chloroform-methanol extraction, as described previously [START_REF] Lebret | Influence of rearing conditions on performance, behavioral, and physiological responses of pigs to preslaughter handling, carcass traits, and meat quality[END_REF]. Lipid content of fresh tissue (g/100 g) was calculated by taking into account the DM content determined from the weight of minced tissue before and after freeze-drying.

Skeletal Muscle Biological Traits

Histological Analyses. Histological measurements were performed on 10-µm-thick serial transverse sections cut on a cryostat (2800 frigocut N, Reichert-Jung, Heidelberg, Germany) at -20°C. Histoenzymology was undertaken on both biopsies and 30-min postmortem samples of LM. One muscle serial section was stained using the conventional acto-myosin-ATPase histochemistry after preincubation at pH 4.35 to identify types I, IIA, and IIB fibers, and another one was processed for succino-dehydrogenase to identify red oxidative (R) and white glycolytic (W) fibers as described previously [START_REF] Lefaucheur | Myosin heavy chain composition of different skeletal muscles in Large White and Meishan pigs[END_REF]. Both I and IIA fibers are oxidative (type R), whereas IIB fibers can be either moderately oxidative (type R) or not oxidative (type W). Myofibers were classified as types I, IIA, IIBR, and IIBW as a result of both stains [START_REF] Larzul | Phenotypic and genetic parameters for longissimus muscle fiber characteristics in relation to growth, carcass, and meat quality traits in Large White pigs[END_REF]. For an accurate estimation of the proportions of types I, IIA, IIBR, and IIBW fibers, several entire bundles of myofibers from 30-min postmortem samples were selected in 3 random fields to obtain a total of at least 1,000 fibers that were counted using a projection microscope (Visopan, Reichert, Heidelberg, Germany). Immunocytochemistry was undertaken on LM biopsies using NLC-MHCs, 6B8, 8F4, and BFF3 monoclonal antibodies, specific for pig type I, IIa, IIx, and IIb myosin heavy chains (MyHC), respectively, as outlined in a previous study [START_REF] Lefaucheur | Myosin heavy chain composition of different skeletal muscles in Large White and Meishan pigs[END_REF]. Thus, myofibers were classified as types s, a, x, and b based on the presence of types I, IIa, IIx, and IIb MyHC, respectively. Hybrid sa, ax, and xb fibers could also be observed. On both LM biopsies and samples taken at 30 min postmortem, glycogen concentration at the myofiber level was determined on serial sections stained with the periodic acid Schiff method [START_REF] Pearse | Histochemistry: Theoretical and Applied[END_REF] using luminance (256 gray levels scale) determined by computerized image processing [START_REF] Fernandez | Comparative study of two classifications of muscle fibres: Consequences for the photometric determination of glycogen according to fiber type in red and white muscle of the pig[END_REF]. Indeed, these previous reports showed that luminance of the periodic acid Schiff staining was closely related to glycogen content. An additional serial section was stained with azorubine to uniformly stain the myofibers in red, but keeping the interfiber network unstained, which allowed the extraction of the interfiber network by computerized image analysis. A total of 500 fibers from 3 random fields were used to evaluate glycogen content and cross-sectional area (CSA) of the different fiber types classified by histoenzymology or immunocytochemistry or both on biopsies and 30-minpostmortem samples of LM.

Biochemical Analyses. Activities of lactate dehydrogenase (LDH), citrate synthase (CS), and β-hydroxy-acyl-CoA dehydrogenase (HAD) were determined on LM, SM, BFM, and RM samples taken at 30 min postmortem as described previously [START_REF] Lebret | Influence of the three RN genotypes on chemical composition, enzyme activities and myofiber characteristics of porcine skeletal muscle[END_REF]. These enzymes were chosen to assess the glycolytic pathway, Krebs cycle activity, and fatty acid β-oxidation potential, respectively. Activities were measured at 30°C and expressed as micromoles of substrate degraded per minute per gram of fresh tissue. Expression of genes coding for uncoupling proteins 2 and 3 (UCP2 and 3, respectively) was investigated on a subset of 6 animals from each line by real-time reversetranscription PCR [START_REF] Damon | Number of intramuscular adipocytes and fatty acid binding protein 4 content are significant indicators of intramuscular fat level in crossbred Large White × Duroc pigs[END_REF]. The potential of mitochondrial respiration was measured on fresh thin bundles of 20 to 30 permeabilized myofibers from LM taken immediately after slaughter to determine the maximal rate of oxygen consumption and respiratory control ratio (RCR; i.e., the ADP-activated state III respiration/state IV respiration), using a 2-chamber high resolution respirometer (Oroboros oxygraph-2k, Innsbruck, Austria; [START_REF] Gueguen | Ca 2+ -activated myosin-ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbit[END_REF]. Because respiration measurements had to be done on fresh myofibers, subsets of 4 animals from each RFI line were analyzed.

Hormone and Metabolite Concentrations

Ten milliliters of blood was collected on all pigs in the home pen 2 wk before slaughter via jugular venipuncture into heparinized (20 IU of heparin/mL of blood) in the morning after an overnight fasting (fasted animals), and on all pigs at exsanguination at the slaughterhouse without any previous fasting (fed animals). Blood was centrifuged at 2,500 × g for 10 min at 4°C and plasma was stored at -20°C until further analysis. All samples were analyzed within a single assay. Except for glucose, plasma determinations were undertaken on both fasted and fed pigs. Concentrations of plasma IGF-I were determined using a validated RIA [START_REF] Louveau | Effect of a growth hormone infusion on plasma insulin-like growth factor-I in Meishan and Large White pigs[END_REF] that used recombinant human IGF-I (GroPep, Adelaïde, Australia). The quantification limit of the assay was 0.08 ng/mL, and the intra-and interassay CV were 7 and 15%, respectively, at 302 ng/mL. Plasma leptin was measured using the multi-species RIA kit (Linco Research, St. Charles, MO) previously validated for use in porcine plasma [START_REF] Qian | Leptin mRNA expression and serum leptin concentrations as influenced by age, weight, and estradiol in pigs[END_REF]. The quantification limit of the assay was 1 ng/mL, and the intra-and interassay CV were 5 and 14%, respectively, at 1.82 ng/mL. The plasma concentrations of total (T) thyroxine (T 4 ), T triiodothyronine (T 3 ), and free T 3 (FT 3 ) were determined using RIA kits (M.P. Biomedicals, Illkirch, France). The quantification limits of the assays were 7.6 ng/mL, 0.07 ng/mL, and 0.09 fmol/mL for TT 4 , TT 3 , and FT 3 , respectively. The intra-and interassay CV were 8. 1, 5.6, 8.4 and 11.3, 7.5, 8.9% at 7.6 ng/mL, 1 ng/mL, and 2.3 fmol/mL, respectively. Plasma cortisol was determined using a competitive 125 I RIA kit (Immunotech, Prague, Czech Republic). The quantification limit of the assay was 5 ng/mL, and the intra-and interassay CV were 4.2 and 10.0% at 71 ng/mL, respectively. Plasma glucose concentration was measured only on fasted pigs using a commercial kit (Glucose HK, Thermo Electron, Cergy Pontoise, France). Saliva was collected on all pigs in the home pen, in the morning after an overnight fasting. Saliva cortisol was measured with a luminescence immunoassay kit (IBL, Hamburg, Germany). The quantification limit of the assay was 0.16 ng/mL. The intraand interassay CV were 6.1 and 7.8% at 2.1 ng/mL, respectively.

Statistical Analyses

The SAS software (SAS Inst. Inc., Cary, NC) was used in all statistical analyses of the data. For growth performance, muscle chemical composition, meat quality traits, and metabolic enzyme activities, data were first analyzed by a 2-way ANOVA using the GLM procedure of SAS, with the RFI group (pigs from the low vs. high RFI line) and slaughter day as main effects. The effect of the slaughter day was removed from the final model because it did not significantly influence any of the characteristics studied, likely due to the increased standardization of our preslaughter and slaughter conditions. The model used to study the effects of RFI line on raw muscle and organ weight data included RFI line as fixed effect, carcass weight as a covariate, and their interaction. For histological characteristics of LM, data were submitted to a 2-way ANOVA, with the RFI group, fiber type, and their interaction as main effects. For hormone concentrations, the effects of the RFI group were analyzed by a 1-way ANOVA within each nutritional status (fed or overnight fasted). Differences in hormone concentrations between the fed and overnight fasted states were analyzed using the paired Student's t-test, each animal being its own control. To assess the validity of the muscle sampling technique to predict in vivo characteristics of the muscle, the differences for myofiber CSA and glycogen content between muscle biopsies taken at 8 d before slaughter and muscle samples at 30 min after slaughter on the same animals were analyzed using the paired Student's t-test. In addition, different traits were sometimes included as covariates in the statistical models to refine the interpretation of the overall differences between RFI groups. Significant effects were those with P < 0.05, and marginally significant effects were those with P < 0.1.

RESULTS

Growth Performance, Carcass Traits, and Organ Weights

Animals from both lines were slaughtered at similar BW and age (Table 1), and ADG did not differ significantly between RFI groups. Pigs from the low RFI line exhibited leaner carcasses, as reflected by a greater LMC (P < 0.0001) and loin:backfat ratio (P < 0.001). The proportion of backfat, expressed as a percentage of carcass weight, decreased by 24%, whereas weight of LM increased by 13% (P < 0.001) in pigs from the low compared with the high RFI line. In contrast, RM, heart and liver weights were not affected by the RFI line. The weight of thyroid gland was 19.4% lighter (P = 0.01) in animals from the low compared with the high RFI line.

Meat Quality Traits

Low RFI line pigs exhibited about 30% less intramuscular fat content in all 4 muscles, LM, SM, BFM, and RM, than high RFI line pigs (Table 2). The opposite was observed for GP, which was 17% greater in LM, SM, and BFM of pigs from the low RFI line (P < 0.04), with no significant difference in the RM between lines. The pH 30 min and 24 h postmortem were only measured in LM, SM, and BFM. No difference between RFI groups was observed for pH 30 min, whereas pH 24 h postmortem was significantly less in LM (-0.16 pH unit) and BFM (-0.12 pH unit) in low compared with high RFI line pigs. The same trend occurred in SM (-0.09 pH units) but did not reach statistical significance (P = 0.19). The low RFI line animals had significantly greater L* values in LM, SM, and BFM, which was associated with a greater LM drip loss assessed at 2 d postmortem (2.13 vs. 1.15%; P = 0.04). The redness parameter (a*) did not differ between RFI groups in any muscle, whereas yellowness (b*) tended to be greater only in LM of pigs from the low vs. high RFI line.

Metabolic Enzyme Activities

The LM, SM, and BFM are mostly glycolytic muscles (greater LDH activity), with an increasing oxidative capacity in the rank order LM < SM < BFM, whereas RM muscle is mostly oxidative (greater CS and HAD activities) and poorly glycolytic. The activities of LDH and CS were not significantly different between RFI groups in any muscle (Table 3). Only HAD activity, an indicator of fatty acid β-oxidation potential, tended (P < 0.1) to be greater in LM, SM, and BFM of high than low RFI line pigs, whereas no difference between lines was observed in RM. Data from a subset of animals from each line did not show any significant difference between lines for the expression of genes coding for UCP2 and 3 (n = 6), or the potential and regulation of mitochondrial respiration (maximal rate, state III, state IV, RCR; n = 4; data not shown).

Myofiber Characteristics in LM

Myofiber Typing Technology. Four fiber types (i.e., I, IIA, IIBR, and IIBW) were identified by conventional histoenzymology combining actomyosin-ATPase and succino-dehydrogenase staining on serial sections (Figure 1A,B). On average, LM contained 12.4, 6.1, 7.8, and 73.7% of I, IIA, IIBR, and IIBW fibers, respectively. Using monoclonal antibodies specific of each MyHC, fibers could be classified as types s, a, x, and b based on the presence of MyHC I, IIa, IIx, and IIb, respectively (Figure 1C to 1F). Moreover, some hybrid sa, ax, and xb fibers could be observed. Thus, on average, LM contained 12.9% s, 0.9% sa, 5.9% a, 5.2% ax, 9.0% x, 13.5% xb, and 52.6% pure b fibers. The correspondence between both myofiber classifications (i.e., histoenzymology vs. immunocytochemistry) is shown on Figure 2. Most I and IIA fibers contained type s (97%) or type a (87%) MyHC, respectively, which suggested a relatively good correspondence between both classifications for these fibers. In contrast, type IIBW fibers were a mixture of b (71%), xb (18%), and x (11%) fibers and about 65% of IIBR fibers corresponded to hybrid ax fibers, and 23% to x fibers. Thus, 85% of type x, 97% of type xb, and 100% of type b fibers were IIBW fibers, showing that conventional fiber typing using histoenzymology (I, IIA, IIBR, IIBW) could not make any distinction between types x, xb, and b fibers under our experimental conditions. Residual SD (root mean square error of the full ANOVA model).

3

Probability of a type I error.

Muscle Sampling Technology.

In the present study, one of the main objectives was to quantify glycogen content within myofibers according to their contractile and metabolic types to explain the genetic correlation between RFI and ultimate pH. The comparisons between muscle biopsies taken at 8 d before slaughter and muscle samples at 30 min after slaughter for myofiber CSA and intrafiber glycogen content according to I, IIA, IIBR, and IIBW fiber types are shown in Figure 3. On average, intrafiber glycogen content was 1.26% in muscle biopsies and 0.82% in 30-minpostmortem samples, denoting a 35% decrease in glycogen content within 30 min postmortem. No significant difference in the decrease of glycogen content was observed between the different fiber types. Therefore, to be close to the in vivo situation, priority was given to results obtained on muscle biopsies to assess the influence of divergent selection for RFI on myofiber glycogen content with respect to both fiber type classifications [i.e., histoenzymology (I, IIA, IIBR, IIBW) and immunocytochemistry based on MyHC composition (s, sa, a, ax, x, xb, and b)]. Because myofiber typing on serial sections is tedious and time consuming, myofibers were only classified as types I, IIA, IIBR, and IIBW in 30-min-postmortem samples to make comparisons with biopsies taken at 8 d before slaughter. Regarding CSA of myofibers, we noticed an artifactual 52% increase in CSA of all fiber types (Figure 3A) in muscle biopsies compared with 30-min postmortem samples, likely due to the fact that myofibers contracted during the quick conditioning and freezing of muscle biopsies, whereas such contraction was not present at 30 min postmortem. Therefore, the influence of divergent selection for RFI on myofiber CSA was evaluated only on muscle samples taken at 30 min postmortem.

Influence of Divergent Selection for RFI on Histological Traits. Compared with high RFI line pigs, animals from the low RFI line exhibited greater proportions of fast-twitch glycolytic type IIBW (75.5 vs. 71.9%; P = 0.016) and pure type b MyHC fibers (54.1 vs. 51.2; P = 0.06; Table 4). Animals from the low RFI line also tended to have less IIBR fibers (P = 0.08), suggesting a shift to a more glycolytic phenotype. Selection for low RFI induced a 23% general increase in myofiber CSA, in particular of type II fibers. As a combination of changes in fiber type proportions and myofiber CSA, the relative area of IIBW fibers was slightly greater in pigs from the low than high RFI line (P = 0.06). On average, within myofiber glycogen content assessed by image analysis in muscle biopsy samples was 1.38% in low RFI line pigs, and 1.11% in the high RFI line pigs (Figure 4), corresponding to 25% more glycogen in the former group (P < 0.001). As shown by a highly significant RFI group × fiber type interaction (P < 0.001), the influence of RFI group on myofiber glycogen content differed according to fiber types (Figure 4). Thus, glycogen content increased by 30% in glycolytic fibers (IIBW, x, xb, and b), 23 to 26% in IIBR and ax fibers, and only 16 to 18% in IIA and a fibers, whereas no significant changes were observed between RFI groups in types I, s, and sa fibers. This led to a greater heterogeneity of glycogen content between fiber types in low RFI (IIBW > IIBR = IIA > I) than high RFI line pigs where weaker differences between fiber types could be observed (Figure 4A). When differences between RFI groups were assessed on muscle samples taken 30 min postmortem, only IIBW fibers tended to contain more glycogen in low than high RFI line pigs (0.95 vs. 0.75%, P = 0.08; data not shown). Thus, the line difference in myofiber glycogen content was stronger when evaluated on LM biopsies taken at 8 d preslaughter than on muscle samples taken at 30 min after slaughter.

Hormone Concentrations

Plasma concentrations in IGF-I were not affected by either the nutritional status (fed vs. overnight fasted) or the RFI group (Table 5). Plasma leptin was not influenced by the nutritional status, but was significantly increased (P = 0.04) in pigs from the high vs. low RFI line in the fed state, whereas differences between lines were not significant in the fasted state. Total T 4 , TT 3 , and FT 3 were all significantly increased (P < 0.001) in the fed vs. fasted state. Pigs from the low RFI line tended to exhibit greater TT 4 and FT 3 concentrations (P < 0.10) than high RFI line pigs in the fed state, whereas no line difference were observed in the fasted state. Plasma cortisol was unaffected by the nutritional status and tended to be greater in pigs from the low RFI line (P < 0.10), whatever the nutritional state. This difference was not observed in the saliva from fasted animals. Finally, fasted plasma glucose did not differ between RFI groups.

DISCUSSION

Muscle Sampling and Myofiber Typing Technologies

Complementary analyses were used to evaluate the effects of divergent selection on muscle properties. Myofiber typing by histoenzymology (types I, IIA, IIBR, and IIBW) and immunocytochemistry (types s, sa, a, ax, x, xb, and b) clearly show that types I, IIA, IIBR, and IIBW do not fully correspond to type s, a, x, and b fibers, respectively, and that IIBW fibers are a mixture of b, xb, and x MyHC-containing fibers. This confirms our previous findings obtained on skeletal muscle in LW and Meishan pigs [START_REF] Lefaucheur | Myosin heavy chain composition of different skeletal muscles in Large White and Meishan pigs[END_REF], showing that these classifications are not interchangeable. From a practical point of view, both classifications are relevant and the choice between them will depend on the objectives of each study and the availability of MyHC monoclonal antibodies. Indeed, some antibodies are unfortunately not commercially available, in particular the IIa (6B8) and IIx (8F4) MyHC monoclonal antibodies. In the present study, because biopsies were physiologically more relevant to study in vivo muscle glycogen content, both classifications were used to study the influence of divergent selection on glyco-gen distribution among fiber types on muscle biopsies taken at 8 d before slaughter. Differences in muscle glycogen content between RFI groups were indeed much more significant when evaluated on muscle biopsies (P = 0.0003) compared with 30-min-postmortem samples (P = 0.08). The weaker difference between RFI groups at 30 min postmortem is likely related to an increased uncontrolled variability of muscle glycogen content due to preslaughter and slaughter conditions, as well as to variable early postmortem glycogen breakdown. In contrast, muscle samples taken at 30 min postmortem were more relevant than biopsies to determine myofiber CSA, due to an artifactual myofiber contraction when conditioning muscle biopsies.

Carcass LMC and Muscle Properties

Because RFI is, by definition, phenotypically independent of BW and level of production, the present divergent selection on RFI was initially expected to have no effect on ADG and carcass composition. Thus, pigs from both RFI lines exhibited actually similar ADG, but contrary to what was initially expected, low RFI pigs also had leaner carcasses, in accordance with the genetic correlations previously reported on pigs from the same selection experiment [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]. For different traits not measured on present animals, it could be confirmed from estimated genetic values that our low RFI pigs exhibited less feed intake and feed conversion ratio, leaner carcasses, and unaffected ADG at the genetic level. This is in agreement with other data in growing pigs indicating that selection for low RFI decreased backfat and increased LMC without TT 4 = total thyroxine; TT 3 = total triiodothyronine; FT 3 = free triiodothyronine.

2

Residual SD (root mean square error of the full ANOVA model).

3 Probability of a type I error.

affecting ADG [START_REF] Mrode | Genetic variation in measures of food efficiency in pigs and their genetic relationships with growth rate and backfat[END_REF][START_REF] Johnson | Genetic parameters for production traits and measures of residual feed intake in Large White swine[END_REF][START_REF] Nguyen | Responses in residual feed intake in lines of Large White pigs selected for growth rate on restricted feeding (measured on ad libitum individual feeding)[END_REF][START_REF] Hoque | Genetic parameters for feed efficiency traits and their relationships with growth and carcass traits in Duroc pigs[END_REF]Cai et al., 2008). Present data show that the greater LMC and LM weight of the low vs. high RFI pigs was at least partly explained by a general 23% increase in myofiber CSA, in particular of fast-twitch type II fibers. Whether an increase in the total number of fibers, a characteristic definitely fixed before birth in pigs [START_REF] Wigmore | Muscle development in large and small pig fetuses[END_REF], also contributed to explain the greater LMC and LM weight of the low RFI line pigs remains to be studied. The finding of a greater proportion of glycolytic IIBW fibers in the LM of low RFI pigs is consistent with the generally accepted idea that faster lean growth is genetically associated with a greater proportion of IIBW fibers [START_REF] Lefaucheur | A second look into fibre typing-Relation to meat quality[END_REF]. The contribution of these differences in LMC on RFI variability remains to be asserted, but available data in cattle suggest that body composition is likely not a major contributor to variation in RFI because energy retained in the body explained only 5% of the total variation of RFI [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. In this model, most variation in RFI is ascribed to differences in the efficiency of metabolic processes and heat production. Supporting this, other high RFI pigs from our selection experiment were shown to exhibit a 10% greater basal metabolism than low RFI pigs (Barea et al., 2010), in accordance with previous results reported in laying hens (Bordas et al., 1992). Beyond the decreased adiposity, we also found that pigs from the low RFI line had less intramuscular fat content in all muscles studied, in agreement with results previously reported in pigs (Cai et al., 2008) and cattle [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. Based on the 0.30 positive genetic correlation reported between fatness and intramuscular fat content in pigs [START_REF] De Vries | Genetic parameters of pork quality and production traits in Yorkshire populations[END_REF][START_REF] Sellier | Genetics of meat and carcass traits[END_REF], the decreased intramuscular fat content in low RFI pigs was expected. However, differences in intramuscular fat content between RFI groups are likely not related to changes in fiber type composition because no consistent relationship between intramuscular fat content and fiber type composition has been documented [START_REF] Lefaucheur | A second look into fibre typing-Relation to meat quality[END_REF].

Hormonal Status

To explain differences in RFI and other production traits between lines, the concentrations of several hormones were assessed. Only plasma leptin concentration and thyroid gland mass differed significantly between the 2 RFI lines. Leptin is primarily produced by adipose tissue and is involved in the regulation of feed intake [START_REF] Houseknecht | The biology of leptin: A review[END_REF]. It has been associated with decreased feed efficiency and increased fatness [START_REF] Suzuki | Genetic correlations among carcass cross-sectional fat area ratios, production traits, intramuscular fat, and serum leptin concentration in Duroc pigs[END_REF]. This is in agreement with the current data showing a greater leptin concentration in (fatter) high RFI pigs in the fed state, and a positive phenotypic correlation between plasma leptin concentration in the fed state and backfat proportion on all 27 pigs under study (r = 0.64). Serum leptin concentrations or leptin mRNA abundance in adipose tissue were also positively correlated with RFI in other studies using pigs [START_REF] Hoque | Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs[END_REF][START_REF] Lkhagvadorj | Gene expression profiling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed efficiency[END_REF] and cattle [START_REF] Richardson | Metabolic differences in Angus steers divergently selected for residual feed intake[END_REF][START_REF] Nkrumah | Genetic and phenotypic relationships of serum leptin concentration with performance, efficiency of gain, and carcass merit of feedlot cattle[END_REF][START_REF] Hoque | Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs[END_REF]. However, the line difference in plasma leptin disappeared when LMC was included as a covariate in the ANOVA (data not shown), suggesting that this line difference may mostly be due to differences in body composition. Our data about plasma IGF-I are more difficult to interpret. Indeed, although IGF-I is a key player in cell proliferation and protein deposition [START_REF] Florini | Growth hormone and the insulin-like growth factor system in myogenesis[END_REF], plasma concentrations in IGF-I did not differ between RFI groups, whatever the nutritional state of the animal, suggesting that plasma IGF-I measured at the end of the growing period is likely not a good predictor of RFI. In contrast, other studies did show a high positive genetic correlation (0.63) between plasma IGF-I at 35 d of age and RFI in a population of Yorkshire pigs (Bunter et al., 2010). Similarly, juvenile IGF-I has been reported to be genetically positively related to backfat thickness and intramuscular fat content, and negatively to loin muscle area at 110 kg of BW [START_REF] Suzuki | Genetic correlation between serum insulin-like growth factor-1 concentration and performance and meat quality traits in Duroc pigs[END_REF]Bunter et al., 2010). Thus, contrary to IGF-I at slaughter, juvenile IGF-I seems to be a good physiological indicator of genetic merit for RFI. The increased concentrations of plasma thyroid hormones in the fed vs. fasted state is consistent with previous data [START_REF] Dauncey | Thyroid hormones and thermogenesis[END_REF]. Because of the stimulating effects of thyroid hormones on basal energy metabolism, a less active thyroidal function can be expected in the low vs. high RFI line, as shown in cockerels [START_REF] Gabarrou | Glucose-insulin relationships and thyroid status of cockerels selected for high or low residual food consumption[END_REF]. Present data showed differences in the thyroidal function between RFI lines based on the fact that low RFI pigs had smaller thyroid glands. However, contrary to expected, low RFI pigs tended to have greater plasma FT 3 and TT 4 than high RFI pigs in the fed state. Another study carried out in pigs also reported a greater serum TT 3 concentration in low than high RFI pigs [START_REF] Lkhagvadorj | Gene expression profiling of the short-term adaptive response to acute caloric restriction in liver and adipose tissues of pigs differing in feed efficiency[END_REF]. Further studies including more animals as well as the determination of thyroid-hormone-stimulating hormone are still needed to get a clear picture of changes in the thyroidal function between RFI lines. Finally, plasma cortisol concentrations tended (P = 0.08) to be greater in low than high RFI pigs, both in the fed and fasted states. An opposite trend has been reported in cattle, sheep and chicken in which low RFI animals were hypothesized to be less susceptible to stress than high RFI animals [START_REF] Herd | Physiological basis for residual feed intake[END_REF]. Our results rather suggest that low RFI pigs would be more susceptible to stress but, in view of these highly controversial data, additional studies are warranted to investigate the relationships between stress susceptibility and RFI.

Muscle Glycogen Content and Distribution

According to our data, the absolute decrease in glycogen content during the first 30 min postmortem did not differ between RFI lines, in accordance with the absence of any line effect on pH at 30 min postmortem.

In contrast, pigs from the low RFI line exhibited decreased ultimate pH and greater lightness in LM, SM, and BFM compared with pigs from the high RFI line, in agreement with previously reported genetic correlations from the same selection experiment [START_REF] Gilbert | Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits[END_REF]. This is in accordance with data obtained in a large scale study involving Dutch Yorkshire pigs [START_REF] De Vries | Genetic parameters of pork quality and production traits in Yorkshire populations[END_REF] where reduced RFI was associated with decreased meat ultimate pH and increased lightness and drip loss. However, such effects were not found in a selection experiment built to reduce RFI in Yorkshire pigs (Cai et al., 2008;[START_REF] Fan | Identification of genetic markers associated with residual feed intake and meat quality traits in the pig[END_REF] or in another study where reduction in RFI was achieved through selection for high growth rate under restricted feeding [START_REF] Nguyen | Genetic selection strategies for efficient lean growth in pigs[END_REF]. The discrepancy between current results and those of Cai et al. (2008) might be due to differences in the equations used to calculate RFI, the genetic background of the pigs, preslaughter and slaughter conditions, and the fact that they only compared a low vs. a randomly selected RFI line. In the present study, in vivo LM glycogen content was greater in low RFI pigs, which likely explains the decreased LM ultimate pH of these pigs. Interestingly, we also observed a decreased ultimate pH in BFM, another glycolytic muscle, in the low RFI pigs. The greater LMC and in vivo muscle glycogen content in the low RFI line pigs are in agreement with the positive genetic correlation generally reported between lean-to-fat ratio and in vivo muscle glycogen content [START_REF] Sellier | Genetics of meat and carcass traits[END_REF][START_REF] Larzul | Selection for reduced muscle glycolytic potential in Large White pigs. II. Correlated responses in meat quality and muscle compositional traits[END_REF].

Because glycolytic IIBW fibers generally contain more glycogen than other fiber types at slaughter (Lefaucheur, 2010), the greater muscle glycogen content in LM of low RFI pigs may be related to their greater percentage of IIBW fibers. However, neither the percentage of IIBW fibers used as a covariate nor the (RFI line × covariate) interaction were significant (data not shown), suggesting that differences in the percentage of IIBW fibers between RFI lines did not explain differences in LM glycogen content. Moreover, in vivo glycogen content was not different between fiber types in the high RFI pigs in our study, likely because muscle biopsies were taken without any previous physical exercise, stress, or fasting. The differences in glycogen content usually reported between fiber types at slaughter have indeed been ascribed to be due to different preslaughter physical activity or stress or both, leading to a greater muscle glycogen decrease in oxidative compared with glycolytic fibers [START_REF] Karlsson | Muscle glycogen depletion pattern in halothane-gene-free pigs at slaughter and its relation to meat quality[END_REF]. Because pigs from both RFI lines were biopsied in similar conditions in the present experiment, most of the line divergence in glycogen content can be attributed to the divergent selection for RFI, and not to environmental factors, with a specific accumulation of glycogen in glycolytic IIBW fibers in low RFI pigs. Interestingly, a specific accumulation of glycogen in IIBW fibers has also been reported in pigs carrying the dominant AMPK γ3 R225Q mutation [START_REF] Marinova | Relationship between metabolism and glycogen content in skeletal muscle fibers of Large White and Hampshire crossbred pigs[END_REF]Lebret et al., 1999). However, in contrast to low RFI animals, AMPK γ3 R225Q-mutated pigs exhibited decreased percentage and relative area of IIBW fibers in LM, less LDH activity, and greater CS and HAD activities, suggesting a greater reliance of glycolytic muscles on fatty acid oxidation. Therefore, muscle metabolic changes in AMPK γ3 R225Q-mutated pigs resemble those seen when muscles adapt to an increased physical activity and are drastically different from those observed in low RFI pigs (i.e., an increased proportion of IIBW fibers in LM and a decreased HAD activity in LM, SM, and BFM), denoting a decreased reliance of glycolytic muscles on fatty acid oxidation. Beside the greater glycolytic potential in LM, SM, and BFM, low RFI pigs also exhibited reduced intramuscular fat content, suggesting a negative relationship between both traits. However, a direct functional relationship between intramuscular fat content and muscle glycogen stores or fiber type composition is unlikely and still highly controversial [START_REF] Lefaucheur | A second look into fibre typing-Relation to meat quality[END_REF]. Thus, the mechanisms underlying the increased glycogen content in glycolytic muscles of low RFI pigs and its specific accumulation in the IIBW fibers are unknown. Because of its strong implication in the regulation of glycogen and fat metabolism in skeletal muscle [START_REF] Kahn | AMPactivated protein kinase: Ancient energy gauge provides clues to modern understanding of metabolism[END_REF], the AMPK pathway is an interesting candidate to better understand differences in muscle properties, and more generally in energy metabolism efficiency, between RFI lines.

Muscle Metabolic and Contractile Properties

Based on the HAD activity, our data suggest that high RFI pigs could potentially oxidize more fatty acids in mitochondria in glycolytic muscles, and that differences in mitochondrial activity could be involved to explain differences in energy metabolism efficiency between RFI lines. Mitochondria isolated from LM of low RFI steers have indeed been reported to have a greater degree of coupling, as assessed by RCR determination (Kolath et al., 2006a). Moreover, breast muscle mitochondria from broilers with high feed efficiency have been shown to exhibit a more tightly coupled respiratory chain along with decreased electron leak and reactive oxygen species production compared with broilers with low feed efficiency (Bottje et al., 2004). Improved efficiency of feed utilization could thus be related to a greater degree of mitochondrial coupling. However, our results obtained from a subset of animals from both lines did not show any significant line difference for the maximal rate of oxygen consumption and RCR. This discrepancy could be due to the limited sampling used in the present experiment but could also be ascribed to the biological material used to study mitochondrial respiration (i.e., permeabilized fibers in the present work vs. isolated mitochondria in other studies). Indeed, the cytoskeleton present in permeabilized fibers has been shown to play a key role in the regulation of mitochondrial respiration (Appaix et al., 2003). Consistently with the lack of line difference in coupling between electron transport chain and oxidative phosphorylation, we did not find any line difference in the amount of UCP2 and UCP3 mRNA in LM, in accordance with data reported for mRNA and protein UCP abundance in cattle LM (Kolath et al., 2006b). Finally, because protein turnover is also an energetically expensive process, a greater protein turnover in high RFI pigs could contribute to their decreased feed efficiency. Data supporting this hypothesis are the decreased myofibril fragmentation index and greater calpastatin content found in low vs. high RFI steers at slaughter, suggesting a decreased fractional protein degradation in the low RFI animals [START_REF] Mcdonagh | Meat quality and the calpain of feedlot steers following a single generation of divergent selection for residual feed intake[END_REF][START_REF] Castro Bulle | Growth, carcass quality, and protein and energy metabolism in beef cattle with different growth potentials and residual feed intakes[END_REF]. This would be consistent with the greater percentage of IIBW fibers in our low RFI pigs because glycolytic fibers have been reported to exhibit a decreased protein turnover than oxidative fibers [START_REF] Lewis | Pre-and postnatal growth and protein turnover in smooth muscle, heart and slow-and fast-twitch skeletal muscles of the rat[END_REF].

Among the multiple sources of variation in RFI, physical activity is potentially an important one because of its large effect on muscle energy expenditure. Indeed, divergent selection for RFI in chicken and cattle, or for heat loss or food intake corrected for BW in mice, all show that less efficient lines are physically more active than more efficient ones [START_REF] Luiting | Metabolic differences between white Leghorns selected for high and low residual feed consumption[END_REF][START_REF] Mousel | Locomotor activity, core body temperature, and circadian rhythms in mice selected for high or low heat loss[END_REF][START_REF] Herd | Physiological basis for residual feed intake[END_REF][START_REF] Lancaster | Characterization of feed efficiency traits and relationships with feeding behavior and ultrasound carcass traits in growing bulls[END_REF][START_REF] Kelly | Effect of divergence in residual feed intake on feeding behavior, blood metabolic variables, and body composition traits in growing beef heifers[END_REF]. In pigs, high RFI animals have been shown to exhibit a greater total daily feeding time, a decreased rate of feed consumption, and a greater number of visits to a feeding station, denoting a greater feeding related physical activity [START_REF] De Haer | Relations among individual (residual) feed intake, growth performance and feed intake pattern of growing pigs in group housing[END_REF][START_REF] Gilbert | Sélection divergente sur la consommation alimentaire résiduelle du porc en croissance: Effets corrélatifs sur le comportement alimentaire[END_REF][START_REF] Young | Effect of selection for residual feed intake on feeding behavior in pigs[END_REF]. Therefore, high RFI pigs could be less efficient because of a greater physical activity. Due to the known positive effects of physical exercise on oxidative metabolism in skeletal muscle [START_REF] Pette | Mammalian skeletal muscle fiber type transitions[END_REF], high RFI pigs were expected to exhibit a greater muscle oxidative metabolism. This was confirmed by the decreased percentage and relative area of glycolytic IIBW fibers in LM, and the tendency for a greater HAD activity in LM, SM, and BFM of high RFI pigs, but this was not supported by the lack of line differences in CS activity. The relationships between physical activity and RFI, and underlying mechanisms, will have to be further studied on next generations of selection.

Meat Quality Traits

In pigs, increasing the proportion of IIBW fibers and myofiber CSA, or decreasing intramuscular fat content are associated with an impairment of some meat quality traits such as water-holding capacity, flavor, juiciness, and tenderness [START_REF] Devol | Variation in composition and palatability traits and relationships between muscle characteristics and palatability in a random sample of pork carcasses[END_REF][START_REF] Larzul | Phenotypic and genetic parameters for longissimus muscle fiber characteristics in relation to growth, carcass, and meat quality traits in Large White pigs[END_REF][START_REF] Fernandez | Influence of intramuscular fat content on the quality of pig meat-1. Composition of the lipid fraction and sensory characteristics of M. longissimus lumborum[END_REF][START_REF] Rehfeldt | Myogenesis and postnatal skeletal muscle cell growth as influenced by selection[END_REF]. Moreover, increasing muscle glycolytic potential at slaughter can lead to a low ultimate pH and a decrease in water-holding capacity of fresh and cooked pork [START_REF] Monin | Evolution post-mortem du tissu musculaire et conséquences sur les qualités de la viande de porc[END_REF]. Therefore, the greater proportion of IIBW fibers, myofiber CSA, and glycolytic potential, along with the decreased intramuscular fat content in LM of efficient low RFI pigs are likely to impair their meat quality, as suggested by their decreased ultimate pH and greater lightness and drip loss compared with the high RFI pigs. This is consistent with the unfavorable genetic correlations often reported between pork quality and feed conversion efficiency [START_REF] Cameron | Meat quality of Large White pig genotypes selected for components of efficient lean growth rate[END_REF][START_REF] Hermesch | Genetic parameters for lean meat yield, meat quality, reproduction and feed efficiency traits for Australian pigs. 2. Genetic relationships between production, carcase and meat quality traits[END_REF][START_REF] Lonergan | Selection for lean growth efficiency in Duroc pigs influences pork quality[END_REF][START_REF] Hoque | Genetic associations of residual feed intake with serum insulin-like growth factor-I and leptin concentrations, meat quality, and carcass cross sectional fat area ratios in Duroc pigs[END_REF]. Interestingly, de [START_REF] De Vries | Genetic parameters of pork quality and production traits in Yorkshire populations[END_REF] evoked a greater risk of DFD meat in high RFI pigs, in connection with their reduced muscle glycogen content at slaughter and greater ultimate pH of meat. Depending on preslaughter conditions, various meat quality defaults can arise and further work is needed to establish the relationships between RFI and pork quality under different preslaughter conditions, in particular regarding technological and sensory traits.

Conclusions

The current study indicates that pigs from the low RFI line when fed ad libitum exhibited similar growth rate but leaner carcasses and decreased intramuscular fat content compared with pigs from the high RFI line. The greater LMC of low RFI pigs was associated with hypertrophy of fast-twitch type II myofibers, a shift toward more glycolytic IIBW myofibers, those fibers containing b, xb, or x MyHC, and greater muscle glycogen content specifically in IIBW fibers. This was associated with a decreased ultimate pH, and greater lightness and drip loss in LM of low RFI pigs, suggesting that selection for reduced RFI may impair some meat quality traits. Because consumers and processing industry put increasing emphasis on the quality of pork, further work is needed to better evaluate the influence of RFI on technological and sensory quality of pig meat. Regarding muscle metabolic characteristics, pigs from the low RFI line tended to exhibit a decreased reliance of muscle energy metabolism on fatty acid β-oxidation in glycolytic muscles. However, a clear picture of the differences in muscle glycogen and lipid metabolism between RFI lines still deserves further investigation.

LITERATURE CITED

Figure 1 .

 1 Figure 1. Identification of fiber types by enzyme histochemistry and immunocytochemistry on serial transverse frozen sections of LM sampled at 30 min after slaughter. Histochemical determination of actomyosin ATPase after preincubation at pH 4.35 (A) and succino-dehydrogenase (SDH; B). Immunocytochemical staining with type I (C), IIa (D), IIx (E), IIb (F), and I + IIx + IIb (G) monoclonal antibodies. Glycogen staining with periodic acid Shiff (H). Myofibers are classified as types I, IIA, and IIB (A); red oxidative (R) and white glycolytic (W) fibers (B); or s, a, x, and b based on the presence of myosin heavy chain (MyHC) I, IIa, IIx, and IIb, respectively (C to F). The star symbol denotes a corresponding fiber on the serial sections. Bar = 100 µm.

Figure 2 .

 2 Figure 2. Histochemical fiber type classification as types I, IIA, IIBR, and IIBW plotted vs. presence of the myosin heavy chains s, a, x, and b revealed by immunocytochemistry on serial transverse sections of LM.

Figure 3 .

 3 Figure 3. Cross-sectional area (CSA; A) and glycogen content (B) of types I, IIA, IIBR, and IIBW myofibers in LM sampled at 8 d before slaughter (biopsy) or 30 min postmortem. Level of significance: ***P < 0.001.

3I

  , IIA, IIBR, and IIBW fibers: histoenzymatic classification on muscle samples taken 30 min postmortem. Types s, sa, a, ax, x, xb, and b: immunocytochemical classification based on myosin heavy chain composition on muscle biopsies taken 1 wk before slaughter. 4 Cross-sectional area of muscle fibers determined on muscle samples taken at 30 min postmortem.

Figure 4 .

 4 Figure 4. Glycogen content of myofibers in LM biopsies taken at 8 d before slaughter in pigs with low and high residual feed intake (RFI). Myofibers were classified as type I, IIA, IIBR, and IIBW (A) or based on the presence of the myosin heavy chains s, a, x, and b (B). Levels of significance: ***P < 0.001; **P < 0.01; *P < 0.05; †P < 0.10.

  Appaix, F., A. V. Kuznetsov, Y. Usson, L. Kay, T. Andrienko, J.Olivares, T. Kaambre, P. Sikk, R. Margreiter, and V. Saks. 2003. Possible role of cytoskeleton in intracellular arrangement and regulation of mitochondria. Exp. Physiol. 88:175-190. Barea, R., S. Dubois, H. Gilbert, P. Sellier, J. van Milgen, and J. Noblet. 2010. Energy utilization in pigs selected for high and low residual feed intake. J. Anim. Sci. 88:2062-2072. Bordas, A., M. Tixier-Boichard, and P. Mérat. 1992. Direct and correlated responses to divergent selection for residual feed intake in Rhode Island Red laying hens. Br. Poult. Sci. 33:741-754. Bottje, W. G., M. Iqbal, N. R. Pumford, C. Ojano-Dirain, and K. Lassiter. 2004. Role of mitochondria in the phenotypic expression of feed efficiency. J. Appl. Poult. Res. 13:94-105. Bunter, K. L., W. Cai, D. J. Johnston, and J. C. M. Dekkers. 2010. Selection to reduce residual feed intake in pigs produces a correlated response in juvenile insulin-like growth factor-I concentration. J. Anim. Sci. 88:1973-1981. Cai, W., D. S. Casey, and J. C. Dekkers. 2008. Selection response and genetic parameters for residual feed intake in Yorkshire swine. J. Anim. Sci. 86:287-298.

Table 1 .

 1 Growth performance of pigs from low and high residual feed intake (RFI) lines

		Low RFI line	High RFI line
	Item	(n = 14)	(n = 13)	RSD 1	P-value 2
	RFI, g/d (genetic values)	-54.2	22.5	13.8	<0.001
	Growth traits
	Initial BW, kg	27.7	27.6	3.9	0.948
	Final BW, kg	109.5	106	7.9	0.252
	Final age, d	179.1	179.6	1.8	0.437
	ADG, 3 g/d	795	762	69	0.24
	Carcass trait
	HCW, kg	91.9	87	7	0.08
	LMC, 4 %	58.6	54	2.6	<0.001
	Loin, %	29.2	27.2	1.1	<0.001
	Backfat, %	5.94	7.8	1.24	<0.001
	Loin:backfat	4.98	3.67	0.82	<0.001
	Muscle and organ weight, g
	LM	2,879	2,541	212	<0.001
	Rhomboideus	43.5	46.1	7.3	0.398
	Heart	377	372	44	0.772
	Liver	2,067	2,064	187	0.964
	Thyroid gland	6.98	8.66	1.46	0.01

Table 2 .

 2 Muscle chemical composition and meat quality traits of LM, semimembranosus (SM), biceps femoris (BFM), and rhomboideus (RM) muscles in pigs from low and high residual feed intake (RFI) lines

		Low RFI line	High RFI line
	Item	(n = 14)	(n = 13)	RSD 1	P-value 2
	Intramuscular fat, %
	LM	1.21	1.79	0.37	<0.001
	SM	1.66	2.43	0.91	0.039
	BFM	1.43	2.11	0.43	<0.001
	RM	1.88	2.37	0.55	0.032
	Glycolytic potential 3
	LM	162	138	21	0.007
	SM	144	123	19	0.009
	BFM	149	127	26	0.04
	RM	91	83	21	0.365
	pH 30 min postmortem
	LM	6.35	6.45	0.15	0.092
	SM	6.44	6.49	0.19	0.423
	BFM	6.42	6.43	0.15	0.823
	pH 24 h postmortem
	LM	5.42	5.58	0.1	0.001
	SM	5.5	5.59	0.18	0.187
	BFM	5.51	5.63	0.14	0.052
	L*
	LM	54.1	50.7	2.5	0.002
	SM	53.3	50.4	3.8	0.057
	BFM	50.4	47.7	3.1	0.036
	a*
	LM	7.78	7.63	1.2	0.749
	SM	8.93	8.89	1.94	0.951
	BFM	10.14	10.62	1.69	0.466
	b*
	LM	5.79	4.94	1.17	0.068
	SM	7.92	6.38	3.17	0.218
	BFM	7.23	6.6	1.31	0.23
	Drip loss 2 d postmortem, %
	LM	2.13	1.15	1.18	0.041

1

Residual SD (root mean square error of the full ANOVA model).

2 Probability of a type I error.

3 Expressed as micromoles of lactate equivalents per gram of fresh muscle.

Table 3 .

 3 Metabolic

	re-

enzyme activities of lactate dehydrogenase (LDH), citrate synthase (CS), and β-hydroxy-acyl-CoA dehydrogenase (HAD) in LM, semimembranosus (SM), biceps femoris (BFM), and rhomboideus (RM) muscles of pigs from low and high 1 Activities expressed as micromoles of substrate per minute per gram of fresh muscle.

2

Table 4 .

 4 Histochemical characteristics of LM of pigs from low and high residual feed intake (RFI) lines

		Low RFI line	High RFI line
	Item	(n = 14)	(n = 13)	RSD 1	P-value 2
	Proportion of fiber types, 3 %
	I	11.7	13	4.4	0.452
	IIA	5.78	6.53	2.66	0.468
	IIBR	7.04	8.51	2.13	0.085
	IIBW	75.5	71.9	3.6	0.017
	s	12.1	13.9	3.7	0.23
	sa	0.95	0.77	0.66	0.478
	a	5.77	6.06	2.73	0.792
	ax	4.77	5.7	2.46	0.346
	x	8.16	9.7	3.33	0.252
	xb	14.2	12.7	3.2	0.255
	b	54.1	51.2	3.7	0.059
	Cross-sectional area, 4 µm 2
	I	2,457	2,142	450	0.081
	IIA	2,175	1,545	336	<0.001
	IIBR	2,832	2,134	757	0.025
	IIBW	4,718	3,937	781	0.016
	Mean	4,163	3,385	683	0.006
	Relative area, %
	I	6.82	8.16	2.72	0.215
	IIA	2.99	2.91	1.21	0.87
	IIBR	4.69	5.4	1.64	0.271
	IIBW	85.5	83.5	2.6	0.062

1

Residual SD (root mean square error of the full ANOVA model).

2 Probability of a type I error.

Table 5 .

 5 Concentrations of growth factors, hormones, and metabolites in fed and overnight-fasted pigs from low and high residual feed intake (RFI) lines

		Low RFI line	High RFI line
	Item 1	(n = 14)	(n = 13)	RSD 2	P-value 3
	Fed animals
	Blood plasma
	IGF-I, ng/mL	145	147	50	0.916
	Leptin, ng/mL	2.40	2.96	0.66	0.038
	TT 4 , ng/mL	32.5	28.1	6.4	0.088
	TT 3 , ng/mL	1.16	1.04	0.27	0.271
	FT 3 , fmol/mL	0.69	0.42	0.40	0.092
	Cortisol, ng/mL	64.9	51.2	22.6	0.129
	Fasted animals
	Blood plasma
	IGF-I, ng/mL	142	126	55	0.480
	Leptin, ng/mL	2.57	2.40	0.53	0.418
	TT 4 , ng/mL	25.9	27.3	4.96	0.473
	TT 3 , ng/mL	0.85	0.80	0.26	0.595
	FT 3 , fmol/mL	0.28	0.22	0.21	0.480
	Cortisol, ng/mL	65.1	52.4	18.1	0.081
	Glucose, g/L	0.75	0.76	0.04	0.460
	Saliva
	Cortisol, ng/mL	1.70	1.93	0.67	0.387
	1

at INRA Institut National de la Recherche Agronomique on September 18, 2013 www.journalofanimalscience.org Downloaded from

The study was supported by INRA (Institut National de la Recherche Agronomique, France). The authors gratefully acknowledge the staff of the INRA experimental farm, slaughterhouse, and laboratories involved in this

study.2 Corresponding author: Louis.Lefaucheur@rennes.inra.fr Received September 6, 2010. Accepted December 2, 2010.

Residual SD (root mean square error of the full ANOVA model).

Probability of a type I error.

ADG during the growing-finishing period (i.e., from 27.7 kg to slaughter).

Lean meat content.

This article has been cited by 8 HighWire-hosted articles: at INRA Institut National de la Recherche Agronomique on September 18, 2013 www.journalofanimalscience.org Downloaded from