Sophie Allais
email: sophie.allais@jouy.inra.fr

Hubert H Levéziel

Nathalie Payet-Duprat

J.-F F Hocquette

Jacques Lepetit

Sylvie Rousset

Christophe Denoyelle

Carine Bernard-Capel

L Journaux

Aline Bonnot

# J Lepetit

‖ S Rousset

¶ C Denoyelle

G Renand

The two mutations, Q204X and nt821, of the myostatin gene affect carcass and meat quality in young heterozygous bulls of French beef breeds

Keywords: beef cattle, carcass trait, GDF8, meat quality, myostatin, polymorphism

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

ABSTRACT:

The availability of genetic tests to detect different mutations in the myostatin gene allows the identification of heterozygous animals and would warrant the superiority of these animals for slaughter performance if this superiority is confirmed. Thus, 2 mutations of this gene, Q204X and nt821, were studied in 3 French beef breeds in the program Qualvigène. This work was done with 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine young bulls from, respectively, 48, 36, and 30 sires and slaughtered from 2004 to 2006. In addition to the usual carcass traits recorded at slaughter (e.g., carcass yield, muscle score), carcass composition was estimated by weighing internal fat and dissecting the 6th rib. The muscle characteristic traits analyzed were lipid and collagen contents, muscle fiber section area, and pH. Regarding meat quality, sensory qualities of meat samples were evaluated by a taste panel, and Warner-Bratzler shear force was measured. Deoxyribonucleic acid was extracted from the blood samples of all calves, the blood samples of 78% of the dams, and the blood or semen samples of all the sires. Genotypes were determined for 2 disruptive mutations, Q204X and nt821. Analyses were conducted by breed. The superiority of carcass traits of calves carrying one copy of the mutated allele (Q204X or nt821) over noncarrier animals was approximately +1 SD in the Charolais and Limousin breeds but was not significant in the Blonde d'Aquitaine. In the Charolais breed, for which the frequency was the greatest (7%), young bulls carrying the Q204X mutation presented a carcass with less fat, less intramuscular fat and collagen contents, and a clearer and more tender meat than those of homozygous-normal cattle. The meat of these animals also had slightly less flavor. Also in the Charolais breed, 13 of 48 sires were heterozygous. For each sire, the substitution effect of the wild allele by the mutant allele was approximately +1 SD for carcass conformation and yield, showing that the estimate of the substitution effect was independent of family structure, as it ought to be for a causal mutation. These results illustrate the challenge of using genetic tests to detect animals with the genetic potential for greater grades of carcasses and meat quality.

INTRODUCTION

The GDF8 gene has been identified as the gene responsible for the muscle hypertrophy (mh) phenotype, or double-muscling, in cattle [START_REF] Grobet | A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[END_REF][START_REF] Mcpherron | Double muscling in cattle due to mutations in the myostatin gene[END_REF]. It has been mapped to the centrometric end of chromosome 2 in cattle [START_REF] Charlier | The mh gene causing double-muscling in cattle maps to bovine Chromosome 2[END_REF][START_REF] Dunner | Towards interbreed IBD fine mapping of the mh locus: Double-muscling in the Asturiana de los Valles breed involves the same locus as in the Belgium Blue cattle breed[END_REF][START_REF] Smith | Myostatin maps to the interval containing the bovine mh locus[END_REF]. The expression of the gene results in the production of myostatin, a protein that suppresses both the proliferation and differentiation of myogenic cells [START_REF] Grobet | A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[END_REF]. Several mutations in the gene (e.g., nt821, Q204X, C313Y) were shown to be involved in the production of a disrupted myostatin, which leads to an increase in muscle mass in the animals carrying 2 copies of inactive alleles [START_REF] Kambadur | Mutations in the myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle[END_REF][START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF].

In addition to adverse effects on fitness (dystocia, stress susceptibility, fertility), the beef value of homozygous double-muscled cattle is well documented: a dramatic increase in saleable meat yield as a consequence of improved dressing percentage (reduced digestive tract), reduced carcass fatness, and fineness of the limb bones (for reviews, see Ménissier, 1982b;[START_REF] Arthur | Double muscling in cattle: A review[END_REF]. The beef value of the heterozygous cattle was first estimated in experiments in which the homozygosity of the parent genotypes was inferred from visual phenotypes. However, it was often difficult to distinguish the heterozygote from the homozygote-normal cattle, leading to the hypothesis that the mh gene was partially recessive (Ménissier, 1982c). The availability of molecular tests then enabled researchers to distinguish genotypes unequivocally.

In breeds in which myostatin mutated alleles are segregating, there is a need to know the current frequency of mh genotypes and the actual beef value superiority of the heterozygous cattle over noncarrier cattle to help breeders make rational decisions about the mh breeding strategy. The objectives of this study were to take advantage of an informative French testing design to determine 1) the frequencies of the Q204X and nt821 disruptive mutations in the Charolais, Limousin, and Blonde d'Aquitaine breeds, and 2) the effect of 1 single copy of the inactive allele on carcass traits, muscle characteristics, and meat quality.

MATERIALS AND METHODS

Animals used in this study were slaughtered in accredited slaughterhouses according to the protection of animals rules defined in the French law (Code Rural, articles R214-64 to R214-71, http://www.legifrance. gouv.fr).

In a collaboration between AI enterprises, INRA, and Institut de l'Elevage, a research program named Qualvigène was implemented to study the genetic determinism of beef and meat quality traits. This study was fully integrated in the Qualvigène program.

Animals

The Qualvigène program was based on 3 successive years of progeny tests. Purebred young bulls, progeny of 48 Charolais, 36 Limousin, and 30 Blonde d'Aquitaine sires, were randomly procreated in a large number of herds from mostly unrelated dams. In each breed and each year, calves were born within a restricted period of 17 wk. After weaning on the farm of origin, bull calves entered the feedlots at 40, 37, or 24 wk of age on average (±3 wk) for the 3 respective breeds. The Charolais bull calves were fattened in 2 locations and fed ad libitum with whole-plant corn silage. The Limousin bull calves were fattened in a single feedlot and fed ad libitum with wet corn silage. The Blonde d'Aquitaine bull calves were fattened in a single feedlot with a dried corn diet. The young bulls were shipped to slaughterhouses without mixing animals from different fattening lots and in accordance with good animal transport practices. They were humanely slaughtered in commercial slaughterhouses (a single slaughterhouse for each feedlot) when they reached 730 (±15) kg of BW on average for the Charolais progeny and 479 (±3) or 417 (±4) d on average for the Limousin or Blonde d'Aquitaine progeny, respectively. After editing, the records of 10 animals were discarded from the database because of sanitary problems, penalized growth during fattening, or both, and 6 others were discarded for genotype incompatibility with the sire. A total of 1,114 Charolais, 1,254 Limousin, and 981 Blonde d'Aquitaine purebred young bulls were eventually used in this study.

Traits Analyzed

The traits usually recorded in the progeny tests were birth weight (kg), ADG during fattening (kg/d), age (d), and BW (kg) at the end of fattening. At slaughter, the HCW (kg), without trimming, was recorded and carcass yield (%) was calculated. The next day, the chilled carcass was graded by a technician from the Institut de l'Elevage in accordance with the European Union beef carcass classification system adapted from the European Association for Animal Production reference methods defined by De [START_REF] De Boer | Manual on E.A.A.P. reference methods for the assessment of carcass characteristics in cattle[END_REF]. Muscularity was assessed across 6 classes (SEUROP classification, where S is the highest score and P the least), and fatness across 5 classes (1 to 5). Each class was subdivided into minus (-), equals (=), and plus (+) subclasses. Scores were recoded to numeric variables: muscle score (scale of 1 to 18, where 1 is poorly muscled and 18 is heavily muscled); fatness score (scale of 1 to 15, where 1 is extremely lean and 15 is extremely fat). Eventually, the length of the leg (cm) and the maximum width of the thigh (cm) were measured according to the recommendations of De [START_REF] De Boer | Manual on E.A.A.P. reference methods for the assessment of carcass characteristics in cattle[END_REF].

In addition to these routinely recorded traits, other measurements were specifically recorded for this study. Internal (pelvic and kidney) fat was removed from the warm carcass and weighed (kg). The decrease in tem-perature during chilling was monitored with captors inserted in the carcass between the 10th and 11th rib of 2 or 3 one-half carcasses per slaughter batch (13 to 26 young bulls slaughtered per batch on average for the 3 breeds). The next day, a 4-rib section (approximately 20 cm from the 6th to 9th rib) was removed from the right half carcass. The 6th rib was separated and immediately frozen. The longissimus thoracis (LT) muscle was excised from the 7th to 9th ribs, sliced into 3 steaks. The first one was divided into small samples and immediately frozen for later analyses. The 2 others were vacuum-packaged and kept at 4°C for 14 d for aging before to be frozen.

After thawing, the pH and the rib eye area (cm 2 ) were measured on the 6th rib. The rib was then dissected into muscle, fat, and bone. The rib fat content (%) was calculated as the ratio of dissected fat (intermuscular + subcutaneous) to the weight of muscle + fat. The lightness (L*) was measured on the freshly cut LT muscle section using a Minolta spectrophotometer (CM 2002, Minolta France SA, Carrières sur Seine, France). Muscle characteristics were measured on the 7th-rib LT steak. Intramuscular lipid content was measured by the Soxhlet method using a Soxtherm apparatus (Gerhardt France SARL, Les Essarts Le Roi, France). Insoluble collagen content (%) was measured on a LT muscle sample after 2 h of heat treatment in a buffer solution at 90°C. This was estimated from the measurement of hydroxyproline content (collagen = 7.5 × hydroxyproline) according to the method of [START_REF] Bergman | Two improved and simplified methods for the spectrometric determination of hydroxyproline[END_REF] modified by [START_REF] Bonnet | Dosage du collagène dans les tissues conjonctifs, la viande et les produits carnés[END_REF]. Muscle fiber section mean area (µm 2 ) was determined on 10-µmthick sections cut perpendicularly to the muscle fibers with a cryotome at -25°C. Between 100 and 200 fibers of 2 samples per muscle were measured to calculate the mean fiber section area by computerized image analysis. The ratio of rib eye area to the mean fiber section area was calculated as a rough and approximate estimate of hyperplasia in LT muscle. The 8th-rib LT steaks were thawed and then cooked over an electric grill to an internal temperature of 55°C (rare cooking), using a standardized cooking temperature, cooking time, and cooking equipment. Cooked steaks were cooled to room temperature before 10 parallelepiped core samples were cut with the fiber orientation parallel to the long axis. The Warner-Bratzler shear force (N/cm 2 ) was averaged on the 10 core measures. The sensory quality attributes were evaluated by 3 test panels (1 for each breed) composed of 12 trained panelists. Within each panel, most of the panelists remained over the 3 yr. The same cooking procedure as above was applied on the 9th-rib LT steaks, and the cooked steaks were immediately served to panelists. The Limousin and Blonde d'Aquitaine test panels had to evaluate 12 samples in each session, and the Charolais test panel had to evaluate 15 samples. Panelists scored tenderness, juiciness, and flavor on nonstructured 100-point scales: from 1 (extremely tough, dry or weak) to 100 (extremely tender, juicy or intense). The scores were averaged over the scores of each panelist for each animal.

SNP Genotyping

Deoxyribonucleic acid was extracted from blood samples (1 mL) for all calves, blood samples (1 mL) for 78% of the dams, and blood (1 mL) or semen samples (0.22 to 0.25 mL) for all the sires. Genotypes were determined for 2 disruptive mutations (Q204X and nt821) of the GDF8 gene, which is located on the centrometric end of the bovine chromosome 2. The first mutation, Q204X, is a C→T substitution at nucleotide position 610 in the second exon, generating a premature stop codon in the N-terminal latency-associated peptide at AA position 204. The second mutation, nt821, is a deletion of 11 bp at nucleotide position 821 in the third exon, producing a premature stop codon [START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF]. The Q204X genotyping was performed using TaqMan SNP genotyping assays designed by Applied Biosystems (Courtaboeuf, France; forward primer: 5′-GGAATCCGATCTCTGAAACTTGACA-3′; reverse primer: 5′-GCTCTGCAACACTGTCTTCAC-3′; discriminantly labeled probes: CAATGCTCTGCCA-AATA and ATCAATGCTCTACCAAATA) with an ABI 7900HT Real-Time PCR System (Applied Biosystems). The nt821 deletion was genotyped by amplicon size determination on an ABI 3100 genetic analyzer (Applied Biosystems); PCR products were obtained using a labeled forward primer (5′-TCTTCTTTC-CTTTCCATACA-3′) and an unlabeled reverse primer (5′-ACATCTTTGTAGGAGTACAGC-3′).

Statistical Analysis

Analyses were conducted by breed. Data were analyzed by a mixed-model ANOVA with the MIXED procedure (SAS Inst., Inc., Cary, NC). The first analytical model was Y ijkl = µ + C i + G j + S k + e ijkl , where Y ijkl are phenotypic observations, µ is the overall mean, C i is the fixed effect of contemporary group (i.e., the cattle from the same fattening lot and slaughtered the same day) or the date of taste panel for the sensory quality traits, G j is the fixed effect of genotypes, S k is the random effect of sire, and e ijkl is random error. In this model, the substitution effect was estimated at the population level directly by the contrast between the effects of the genotypes (mh/+) and (+/+) and the associated probability value (DIFF option of the least squares means procedures of SAS).

To estimate the substitution effect between the mutated and normal allele within heterozygous sires, a second model was fitted: Y ijkl = µ + C i + S k + A(S) kj + e ijkl , where Y ijkl , µ, C i , S k , and e ijkl have the same definition as in the first model. The effect A(S) kj refers to the effect of the transmitted allele A j within sire S k . This model could be used only in the Charolais breed, for which the within-sire substitution effects were estimat-ed as the contrasts between performances of progeny that unequivocally received the mutated allele from the sire and performances of normal homozygous progeny from this sire.

RESULTS

Genotypic and Allelic Frequencies

The frequencies in each breed of heterozygous animals for the Q204X and nt821 mutations are reported in Table 1. No animal carrying 2 copies of inactive alleles of the myostatin gene could be found in any genotyped cattle in this study. Even in the Charolais breed, mutated homozygous animals were absent although the apparent allele frequency of the Q204X mutation would be compatible with a frequency 0.5% of mh homozygous animals (i.e., approximately 5 homozygous calves). The reasons for this absence were that 1) the AI enterprises never progeny test double-muscled sires for pure breeding; 2) the AI enterprises never use double-muscled dams as support for procreating test progeny; and 3) even if a double-muscled progeny were born by chance from 2 heterozygous parents, it would never enter the fattening station because it would be considered too extreme and too fragile. An unbiased estimate of the mutated allele frequency (p) in the 3 breeds could, however, be calculated from the observed frequency (h) of heterozygous dams, given that dams were a representative sample of each breed, except that there was no double-muscled female (homozygous mh/ mh) in this sample: p = h/(2 -h).

The Q204X mutation was present predominantly in the Charolais breed, with h = 13% of the dams being heterozygous. Less than h = 2% of the dams in the 2 other breeds were heterozygous. Consequently, the frequency of the Q204X allele could be estimated as p = 7% in the Charolais breed and p = less than 1% in the Limousin and Blonde d'Aquitaine breeds. The nt821 mutation was almost absent in the Charolais breed. In the Limousin breed, there were h = 4.4% heterozygous dams, corresponding to a frequency of the nt821 allele of p = 2%. In the Blonde d'Aquitaine breed, the fre-quency of the inactive nt821 allele was as small as the frequency of the inactive Q204X allele.

In each breed, sires are selected by AI enterprises according to their own selection objectives; therefore, they cannot be representative of the whole population. The proportion of heterozygous sires in the Charolais sample (27%) appeared to be greater than the proportion of heterozygous dams, leading to a marked frequency of heterozygous calves (17%) among the test progeny. Only 1 calf was observed carrying a copy of the nt821 allele in this breed. No heterozygous Blonde d'Aquitaine sire could be found, and a single heterozygous sire was found in the Limousin breed. The numbers of heterozygous progeny were very small in these 2 breeds, 1 and 3%, respectively.

Substitution Effect of Inactive Alleles of the Myostatin Gene on Carcass Yield and Muscle Score in the 3 Breeds

Given that the frequencies of the inactive alleles were very small in the Limousin and Blonde d'Aquitaine breeds, the 2 inactive Q204X and nt821 alleles were pooled in the same class, mh. The means and residual SD (RSD) of carcass yield and carcass muscle score are reported in Table 2 for the 3 breeds. The substitution effect of the normal allele by the mh allele at the population level was calculated as the contrast between heterozygous and normal homozygous cattle [a = (mh/+) -(+/+)] and the contrast divided by the RSD (Table 2) with the first model. In the Charolais and Limousin breeds, the young heterozygous bulls were 1 SD over homozygous animals for both carcass yield and conformation score (P < 0.001). In the Blonde d'Aquitaine breed, the superiority of the heterozygous over homozygous animals was only approximately 0.5 RSD and was not significant for both traits.

Effect of the Mutation Q204X in the Charolais Breed

The effect of a single copy of a mutated allele on all the recorded traits with the first model was estimated only in the Charolais breed, in which the increased frequency of heterozygous animals allowed very accurate estimations. In this Charolais population, only the Q204X mutation was studied because there was only 1 heterozygous animal for the nt821 mutation. This animal was discarded from analysis. The substitution effects at the population level of the normal allele by the inactive Q204X allele on all recorded traits are reported in Table 3. Heterozygous bull calves were slightly (+0.17 RSD), but significantly (P < 0.04), heavier at birth than homozygous-normal calves and they showed more difficulty at birth (+0.37 RSD, P < 0.001). Postweaning growth in the feedlot (daily BW gain, slaughter age and BW) was not affected by the presence of the inactive myostatin allele.

Similarly to carcass yield and muscle score, the carcasses of heterozygous animals were markedly heavier (+0.98 RSD), the thighs were thicker (+0.84 RSD) and the rib eye areas were larger (+0.65 RSD) than those of young homozygous bulls. On the contrary, their legs were significantly shorter (-0.32 RSD). The carcass composition was very different between heterozygous and homozygous-normal cattle. The heterozygous animals were markedly leaner, with less internal fat (-0.57 RSD) and less fat on the 6th rib (-0. 84 RSD). Significant differences in muscle characteristics were also 1 RSD = root mean square error of the basic model that includes only the fixed effect of the contemporary groups.

2 Scale of 0 to 4, where 1 is no difficulty, 2 is easy pull, 3 is hard pull, and 4 is caesarian section.

3 Scale of 1 to 15, where 1 is extremely lean and 15 is extremely fat. 4 Scale of 1 to 100, where 1 is extremely tough, dry, or weak and 100 is extremely tender, juicy, or intense.

observed. The LT muscle of young heterozygous bulls contained less intramuscular lipid (-0.56 RSD) and less insoluble collagen (-0.48 RSD). All these carcass and muscle characteristics were different (P < 0.001).

Significant differences in histology were also observed (P < 0.01). The heterozygotes had thinner muscle fibers (-0.24 RSD) and a greater ratio of rib eye area to fiber area (+0.62 RSD).

No meat quality information was discarded, first because no pH above 6.0 was found (a clue that animals were slaughtered without stress), in accordance with care transport and slaughter conditions [START_REF] Mounier | Variations in meat pH of beef bulls in relation to conditions of transfer to slaughter and previous history of the animals[END_REF], and second because the monitored decline in temperature always followed a normal curve compatible with the absence of cold shortening. The pH in the LT muscle of heterozygotes was not different from homozygous-normal animals. The LT muscle was markedly lighter (+0.57 RSD), however. Although no difference in meat texture was observed when studied via Warner-Bratzler shear force (-0.10 RSD, P = 0.25), moderate differences in sensory tenderness could be detected by the test panel in favor of heterozygotes (+0.27 RSD, P < 0.01). The panelists also detected a slightly lower flavor in heterozygotes (-0.21 RSD, P = 0.02), and no differences in juiciness.

Within-Sire Substitution Effects of the Inactive Q204X Allele on the Carcass Yield and Muscle Score of Charolais Young Bulls

In each of the 13 families of heterozygous Charolais sires, the parent origin of the Q204X allele could be determined unequivocally in 75% of the heterozygous calves because most of the dams were genotyped. For carcass yield and muscle score, the substitution effects within sire families (estimated with the second model) were significant (P < 0.001). The heterozygous calves that received the inactive allele from their sires had consistently greater carcass yields (+0.6 to +1.8 RSD) and muscle scores (+0.2 to +2.0 RSD) than their half sibs that received the normal allele (Table 4). The within-sire substitution effects averaged 1.17 RSD for both traits.

DISCUSSION

The study of allelic frequencies showed that Q204X and nt821 occurred at a very small frequency in the Limousin and Blonde d'Aquitaine breeds. Previous studies of the myostatin polymorphism in the Limousin breed showed that this breed is characterized by an increased frequency of the F94L allele [START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF][START_REF] Dunner | Haplotype diversity of the myostatin gene among beef cattle breeds[END_REF]. This F94L mutation was not investigated in the present study, first because this mutation was thought to be almost fixed in the Limousin breed and absent in the 2 other breeds, and second because [START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF] predicted that the F94L mutation did not interfere drastically with the activity of the myostatin protein. However, this latter assumption was negated by the results of [START_REF] Esmailizadeh | Effects of the myostatin F94L substitution on beef traits[END_REF], [START_REF] Lines | Limousin myostatin F94L variant affects semitendinosus tenderness[END_REF][START_REF] Alexander | A Limousin specific myostatin allele affects longissimus muscle area and fatty acid profiles in a Wagyu-Limousin F 2 population[END_REF], who showed a significant effect of the F94L mutation. In the present study, even if the association between the beef value of the Limousin cattle and the F94L genotypes could not be studied, the presence of 1 copy of a disruptive mysotatin allele (Q204X or nt821) was shown to increase the beef value of these animals drastically, despite the small frequency of the allele. The 2 mutations, Q204X and nt821, certainly appeared in this breed as a consequence of the extension of the breed by absorbing local populations. Because of the adverse effects on fitness and maternal traits, the French Lim- The situation in the Blonde d'Aquitaine breed is different because [START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF] and [START_REF] Dunner | Haplotype diversity of the myostatin gene among beef cattle breeds[END_REF] found that the majority of animals had no mutation in the myostatin gene. In the present study, the effect of a single copy of the Q204X and nt821 disruptive mutations appeared to be less in this breed as compared with the Charolais and Limousin breeds. The power to detect effects in Blonde d'Aquitaine was small because of the low mutated allele frequency, so the number of animals may be too small to accurately estimate an effect as small as 0.5 RSD. To detect a difference of 0.5 phenotypic SD between normal homozygous and young heterozygous bulls with f (frequency of the mutated allele) = 1%, a power of 90%, and a P-value of 5%, we should have more than fourfold the number of Blonde d'Aquitaine animals genotyped in our program [START_REF] Whitley | Statistics review 4: Sample size calculations[END_REF]; hence, we cannot draw conclusions on the actual effect of the mutated allele in the Blonde d'Aquitaine breed.

Because of the increased frequency of the mutation of the myostatin gene in the Charolais breed, we studied the effect of the inactive allele, Q204X, more thoroughly in this breed. Our results showed that young heterozygous bulls had more muscled (+1.17 RSD for carcass yield, +1.01 RSD for muscle score, and +0.65 RSD for rib eye area) and leaner (-0.57 RSD for internal fat and -0.84 RSD for rib fat) carcasses than homozygous animals. The same results were obtained within sire families for carcass yield and muscle score (1.1 RSD on average), although the limited sample size led to less precise estimates (from 0.27 to 2.01 RSD). These within-family results showed that the effect of the myostatin mutated allele did not depend on the family structure, as it ought to for a causal mutation. These results follow the pattern that was reported previously [START_REF] Casas | Association of the muscle hypertrophy locus with carcass traits in beef cattle[END_REF] with the Belgian Blue and Piedmontese inactive alleles in heterozygous animals. In that study, the mutated allele inherited from presumed heterozygous sires was followed by a set of 5 microsatellites. The authors showed that a single copy of the mh allele (mh = mutated myostatin allele) increased the retail product yield and the rib eye area (+1.60 and +1.35 RSD, respectively) and decreased the yield grade, fat thickness, and estimated percentage of KPH respectively). [START_REF] Casas | Association of myostatin on early calf mortality, growth, and carcass composition traits in crossbred cattle[END_REF] studied the effect of the inactive nt821 allele in a crossed Belgian Blue-British breed (Angus and Hereford) population. They reported that the effects of 2 copies of the mutated allele were generally threefold the effects of a single copy. The individuals inheriting 1 copy of the inactive allele had a greater HCW, retail product yield, and LM area (between 1/3 and 2 RSD) and had decreased fat yield and fat weight (approximately -1 RSD) compared with the homozygous-normal animals. [START_REF] Wiener | Muscle-related traits in cattle: The role of the myostatin gene in the South Devon breed[END_REF] reported significant effects of a single nt821 allele on the muscle score and fat depth of heterozygous animals in the South Devon breed. These effects were one-third of the effect of 2 copies. They were less but were consistent with most of the previous studies (between 1/7 and 1/3 RSD). In the same way, the effects of nt821 on carcass conformation, found by [START_REF] Gill | Associations between the 11-pb deletion in the myostatin gene and carcass quality in Angus-sired cattle[END_REF] in a Aberdeen Anguscross population, were small (approximately +1/2 RSD of the conformation class grade, LM area, and carcass weight). Moreover, they found no difference between heterozygous and homozygous-normal animals for fat traits, but the small frequency of the nt821 allele clearly suggests that the effects were difficult to estimate in this population.

Concerning muscle composition, we estimated a large difference in intramuscular fat content between heterozygous and normal calves (-1 RSD), similarly to other studies for the marbling score [START_REF] Casas | Association of the muscle hypertrophy locus with carcass traits in beef cattle[END_REF][START_REF] Casas | Association of myostatin on early calf mortality, growth, and carcass composition traits in crossbred cattle[END_REF]. Heterozygous animals had less collagen content than normal homozygous animals (-0.48 RSD). This finding is consistent with the results of [START_REF] Uytterhaegen | Effects of double-muscling on carcass quality, beef tenderness and myofibrillar protein degradation in Belgian Blue White bulls[END_REF]. They showed that intramuscular collagen content of LT muscle was significantly reduced by 40% in double-muscled Belgian Blue-White bulls compared with normal-muscled animals. [START_REF] Ngapo | Perimysial collagen crosslinking and meat tenderness in Belgian Blue double-muscled cattle[END_REF] had the same result in the semitendinosus and the gluteobiceps muscles, but they found no significant difference in intramuscular collagen between normal and heterozygous Belgian Blue bulls. We also showed a greater number of LT muscle fibers (+0.62 RSD) in heterozygous animals. In the study by [START_REF] Wegner | Growth-and breed-related changes of muscle fiber characteristics in cattle[END_REF], the authors reported that the muscle fiber number of semitendinosus muscle from doubled-muscled young bulls was on average approximately twice that of semitendinosus muscle from German Angus, Galloway, and Holstein-Friesian bulls.

Regarding meat quality, this study indicated that the LT of young heterozygous bulls was more tender than that of homozygous animals according to the trained panelists (+0.27 RSD). This better tenderness can be a consequence of a reduced collagen content and a smaller mean area of the muscle fiber section (-0.24 RSD) because both characteristics have been shown to be related to muscle tenderness [START_REF] Renand | Relationships between muscle characteristics and meat quality traits of young Charolais bulls[END_REF]. The mh allele had no significant effect on Warner-Bratzler shear force but the effect was negative (-0.10 RSD), which is consistent with a more tender meat. The results for tenderness in other studies are divergent. [START_REF] Bailey | Muscle and adipose tissue from normal and double muscled cattle: Collagen types, muscle fibre diameter, fat cell size and fatty acid composition and organoleptic properties[END_REF] reported a more tender meat (LT) in double-muscled Charolais young bulls compared with non-double-muscled animals, as determined by a taste panel with confirmation by a measure of compression force. Wheeler et al. (2001b) measured the tenderness of 4 muscles as determined by a sensory panel and a measure of the ease of myofibrillar fragmentation in a population of Piedmontese crossbred steers and heifers. For the 2 traits, they found that all muscles were more tender in mh/+ than +/+ (between 1/5 and 1 RSD), and they estimated no difference between the tenderness of mh/+ and mh/mh except in the biceps femoris.

Several studies reported no effect of the myostatin gene on meat tenderness. Ménissier (1982a) did not find a difference in Warner-Bratzler shear force of LT between progeny of double-muscled Charolais sires and progeny of non-doubled-muscled Charolais sires. Similarly, [START_REF] Casas | Association of the muscle hypertrophy locus with carcass traits in beef cattle[END_REF] concluded that a single copy of the mh allele had no effect on LM Warner-Bratzler shear force in crossbred animals. In the same way, the results of [START_REF] Gill | Associations between the 11-pb deletion in the myostatin gene and carcass quality in Angus-sired cattle[END_REF] did not support a difference in meat tenderness (measured by a tenderometer) between normal homozygous and heterozygous Angus-cross animals for the nt821 mutation. [START_REF] Short | Pleiotropic effects in Hereford, Limousin and Piedmontese F 2 crossbred calves of genes controlling muscularity including the Piedmontese myostatin allele[END_REF] had the same conclusion after comparing Warner-Bratzler shear force of meat from +/+, mh/+, and mh/mh Piedmontese calves. [START_REF] Uytterhaegen | Effects of double-muscling on carcass quality, beef tenderness and myofibrillar protein degradation in Belgian Blue White bulls[END_REF] found the opposite result; they showed that the meat (LT muscle) of normal-muscled Belgian Blue-White bulls was more tender than that of doubled-muscled Belgian Blue-White bulls by a measure of Warner-Bratzler shear force.

Regarding meat flavor and juiciness, we reported in this study that the meat of heterozygous animals had less flavor and the same juiciness compared with the meat of young homozygous-normal bulls. In light of the previous results, this decreased meat flavor could be a consequence of the reduced intramuscular lipid content. The results of Wheeler et al. (2001b) showed less flavor intensity for the mh/mh genotype than for the +/+ genotype (-1/4 RSD), but they did not report a difference between the mh/+ and +/+ genotypes in the Piedmontese breed. According to the same reference, juiciness ratings were less for the mh/+ genotype than for the +/+ genotype (except in biceps femoris) and were less for the mh/mh genotype than for the mh/+ or +/+ genotype. However, [START_REF] Wheeler | Characterization of biological types of cattle (Cycle IV): Carcass traits and longissimus palatability[END_REF] found that the LM of F 1 mh/+ Piedmontese had less flavor intensity than that of Hereford × Angus. In addition, Wheeler et al. (2001a) reported that the LM of F 1 mh/+ Belgian Blue and F 1 mh/+ Piedmontese had less juiciness than that of Hereford × Angus, but there was no difference in flavor intensity ratings.

We also showed in this paper that heterozygous animals were slightly heavier at birth than homozygous-normal calves, which is consistent with the results reported in other studies [START_REF] Casas | Association of the muscle hypertrophy locus with carcass traits in beef cattle[END_REF][START_REF] Casas | Quantitative analysis of birth, weaning, and yearling weights and calving difficulty in Piedmontese crossbreds segregating an inactive myostatin allele[END_REF][START_REF] Casas | Association of myostatin on early calf mortality, growth, and carcass composition traits in crossbred cattle[END_REF][START_REF] Short | Pleiotropic effects in Hereford, Limousin and Piedmontese F 2 crossbred calves of genes controlling muscularity including the Piedmontese myostatin allele[END_REF][START_REF] Wiener | The effects of a mutation in the myostatin gene on meat and carcass quality[END_REF] in which heterozygous calves were heavier at birth than homozygous-normal calves (+1/4 RSD on average). As a consequence of the increase in birth weight, calving difficulties also increased significantly, as observed by [START_REF] Casas | Quantitative analysis of birth, weaning, and yearling weights and calving difficulty in Piedmontese crossbreds segregating an inactive myostatin allele[END_REF], [START_REF] Short | Pleiotropic effects in Hereford, Limousin and Piedmontese F 2 crossbred calves of genes controlling muscularity including the Piedmontese myostatin allele[END_REF][START_REF] Wiener | The effects of a mutation in the myostatin gene on meat and carcass quality[END_REF]. However, [START_REF] Short | Pleiotropic effects in Hereford, Limousin and Piedmontese F 2 crossbred calves of genes controlling muscularity including the Piedmontese myostatin allele[END_REF] pointed out that calving difficulty was increased in Piedmontese heifers as 1 and 2 copies of the inactive allele were added in the calf, but they found no effect with cows. Therefore, knowledge of the mh genotype of the mating bulls is required to avoid procreating heterozygous calves when mating heifers.

In conclusion, because of adverse effects on dystocia and fitness, double-muscled animals are not desired by producers despite their greater beef value. On the ba-sis of our findings, producing young bulls with 1 copy of the inactive myostatin allele can improve carcass yield and muscle score in the Charolais and Limousin breeds. Moreover, in the Charolais breed, we showed that heterozygous animals had better carcass and meat qualities without a marked increase in birth weight. This slight increase in the birth weight of the calf could have a prejudicial effect on calving ease if the dam is a heifer. Consequently, controlling the inheritance of the myostatin allele by genetic tests could be a solution to have better beef performance if the appropriate mating strategy is applied to adult cows.

Table 1 .

 1 Number of sire, dam, and progeny genotypes in the 3 breeds and the corresponding frequency within each category of animals and breed

	Genotype	Charolais	Limousin	Blonde d'Aquitaine
	Sire
	+/+	35 (73%)	35 (97%)	30 (100%)
	+/Q204X	13 (27%)	0 (0%)	0 (0%)
	+/nt821	0 (0%)	1 (2.8%)	0 (0%)
	Dam
	+/+	748 (87%)	919 (94%)	616 (97%)
	+/Q204X	115 (13%)	14 (1.4%)	8 (1.3%)
	+/nt821	1 (0.1%)	43 (4.4%)	8 (1.3%)
	Progeny
	+/+	901 (82%)	1,208 (97%)	949 (99%)
	+/Q204X	191 (17%)	8 (0.6%)	5 (0.5%)
	+/nt821	1 (0.1%)	34 (2.7%)	5 (0.5%)

Table 2 .

 2 Number of young bulls, means, and muscle hypertrophy (mh) allele effects [a = (mh/+) -(+/+)], with SE, and divided by the phenotypic SD (residual SD 1 ) for carcass yield and carcass muscle score in the 3 breeds, with the corresponding P-value

	Breed and trait	No.	Mean	a ± SE	a/RSD	P-value
	Charolais
	Carcass yield, %	1,113	57.70	1.93 ± 0.13	1.17	<0.001
	Carcass conformation score 2	1,114	9.96	1.47 ± 0.12	1.01	<0.001
	Limousin
	Carcass yield, %	1,254	61.96	1.68 ± 0.20	1.30	<0.001
	Carcass conformation score 2	1,254	10.94	1.38 ± 0.24	0.90	<0.001
	Blonde d'Aquitaine
	Carcass yield, %	981	64.98	0.72 ± 0.46	0.51	0.11
	Carcass conformation score 2	981	10.90	0.83 ± 0.43	0.60	0.06
	1 RSD = root mean square error of the basic model that includes only the fixed effect of the contemporary
	groups.

Table 3 .

 3 Number of young bulls, means, and muscle hypertrophy (mh) allele effects [a = (mh/+) -(+/+)], with SE, and divided by the phenotypic SD (residual SD 1 ) for growth, carcass, muscle, and meat quality traits in the Charolais breed, with the corresponding P-value

	Trait	No.	Mean	a ± SE	a/RSD	P-value
	Birth weight, kg	1,111	48.73	0.98 ± 0.48	0.17	0.04
	Calving difficulty score 2	1,108	1.43	0.24 ± 0.05	0.37	<0.001
	ADG, kg/d	1,113	1.65	12.18 ± 14.85	0.07	0.41
	Slaughter age, d	1,114	500.50	2.45 ± 2.05	0.10	0.23
	Slaughter weight, kg	1,113	729.99	-0.77 ± 1.06	-0.06	0.46
	HCW, kg	1,114	421.47	13.17 ± 1.12	0.98	<0.001
	Thigh width, cm	1,114	32.86	8.17 ± 0.83	0.84	<0.001
	LM area, cm 2	1,114	53.38	548.98 ± 72.55	0.65	<0.001
	Leg length, cm	1,114	87.31	-5.95 ± 1.63	-0.32	<0.001
	Fatness score 3	1,114	8.65	-0.20 ± 0.12	-0.14	0.11
	Internal fat, kg	1,107	8.74	-1.21 ± 0.18	-0.57	<0.001
	Rib fat, %	1,113	20.41	-3.06 ± 0.30	-0.84	<0.001
	Intramuscular lipids, %	1,114	1.53	-0.47 ± 0.07	-0.56	<0.001
	Insoluble collagen, %	1,114	0.30	-0.02 ± 0.004	-0.48	<0.001
	Muscle fiber section mean area, µm 2	1,101	2,920.51	-183.93 ± 67.46	-0.24	<0.01
	LM area/muscle fiber area, ×10 3	1,101	1,958.74	357.60 ± 49.79	0.62	<0.001
	pH	1,114	5.61	-0.004 ± 0.006	-0.05	0.52
	Muscle lightness, L*	1,114	34.75	2.05 ± 0.30	0.57	<0.001
	Shear force, N/cm 2	1,114	38.09	-0.73 ± 0.64	-0.10	0.25
	Tenderness score 4	1,113	62.42	2.13 ± 0.69	0.27	<0.01
	Juiciness score 4	1,113	59.96	0.49 ± 0.45	0.09	0.28
	Flavor score 4	1,113	55.33	-0.96 ± 0.21	-0.21	0.02

Table 4 .

 4 Substitution effect of the normal allele by the Q204X myostatin allele in each of the 13 families of heterozygous Charolais sires for carcass yield and muscle score, with SE, divided by the phenotypic SD (residual SD 1 ) and the corresponding P-value

			Carcass yield			Muscle score
	Sire	a ± SE	a/RSD	P-value	a ± SE	a/RSD	P-value
	1	1.04 ± 0.86	0.63	0.23	1.63 ± 0.81	1.13	0.04
	2	1.21 ± 1.00	0.73	0.23	2.85 ± 0.94	1.97	<0.01
	3	1.23 ± 0.79	0.75	0.12	2.59 ± 0.74	1.79	<0.001
	4	1.26 ± 0.75	0.77	0.09	0.38 ± 0.70	0.27	0.58
	5	1.43 ± 0.64	0.87	0.03	1.91 ± 0.60	1.31	<0.01
	6	1.83 ± 0.76	1.11	0.02	0.91 ± 0.71	0.63	0.20
	7	1.93 ± 0.69	1.17	<0.01	0.75 ± 0.65	0.52	0.24
	8	1.95 ± 0.79	1.18	0.01	2.30 ± 0.74	1.59	<0.01
	9	2.51 ± 0.77	1.52	<0.01	2.92 ± 0.71	2.01	<0.01
	10	2.56 ± 0.79	1.55	<0.01	1.66 ± 0.74	1.14	0.03
	11	2.62 ± 0.75	1.59	<0.001	0.99 ± 0.70	0.68	0.16
	12	2.76 ± 0.64	1.68	<0.001	1.89 ± 0.60	1.30	<0.001
	13	2.92 ± 0.77	1.77	<0.001	1.19 ± 0.72	0.82	0.10

at INRA Institut National de la Recherche Agronomique on September 2, 2013 www.journalofanimalscience.org Downloaded from

Scale of 1 to 18, where 1 is poorly muscled and 18 is heavily muscled.

RSD = root mean square error of the basic model that includes only the fixed effect of the contemporary groups.

This article has been cited by 3 HighWire-hosted articles: at INRA Institut National de la Recherche Agronomique on September 2, 2013 www.journalofanimalscience.org Downloaded from

1 This work was supported by the Agence Nationale de la Recherche (contract numbers 05-GANI-017-04, ANR-GANI-017, ANR-06-GANI-001), Apis-Gène (Paris, France), the Office de l'Elevage (Montreuil sous Bois, France), and the FNE (Fonds National de l'Elevage, Paris, France). The authors thank the AI enterprise partners of the Qualvigène project: Gene Diffusion (Douai, France), Midatest (Soual, France),


B A N Silva

Jean Noblet

J L Donzele

R F M Oliveira

Yvan Primot

Jean-Luc L Gourdine

David Renaudeau

† J Noblet

Effects of dietary protein level and amino acid supplementation on performance of mixed

Keywords: feed intake, lactation, lysine, protein, sow, tropical climate

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

INTRODUCTION

According to Food and Agriculture Organization predictions, pig production in tropical and subtropical countries will rapidly increase as a result of increasing human population. Although many factors are obviously involved, the combination of high temperatures and high relative humidity (RH) resulting in heat stress remains one of the major problems that affects the production efficiency of pigs in these regions. According to their high nutrient requirements, lactating sows are particularly sensitive to high ambient temperatures.

When ambient temperature increases above the evaporative critical temperature (i.e., 22°C;[START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF], the sow reduces voluntary feed intake to reduce heat production due to the thermal effect of feed (TEF). This reduced voluntary feed intake has negative consequences on body reserves mobilization, milk production, and future reproductive and productive career of the sow [START_REF] Dourmad | Effect of protein and lysine supply on performance, nitrogen balance, and body composition changes of sows during lactation[END_REF]. Alternatives to reduce heat stress have been developed to maintain sows performance under high temperature conditions by increasing heat loss using a neck drip cooling system [START_REF] Mcglone | Management of lactating sows during heat stress: Effects of water drip, snout coolers, floor type and a high energy-density diet[END_REF], chilled drinking water [START_REF] Jeon | Effects of chilled drinking water on the performance of lactating sows and their litters during high ambient temperatures under farm conditions[END_REF], or a floor cooling system [START_REF] Silva | Effect of floor cooling on performance of lactating sows during summer[END_REF]. An alternative approach consists of attenuating the change of feed intake in hot conditions. The TEF of digestible fat, starch, and protein averages 10, 18, and 42% of the ME content [START_REF] Noblet | Prediction of net energy value of feeds for growing pigs[END_REF]. Therefore, diets with reduced protein content decrease heat production and may attenuate the effect of elevated temperature on feed consumption. These nutritional solutions were successfully tested in temperate conditions (Renaudeau et al., 2001;[START_REF] Bellego | Effect of high temperature and low protein diets on performance of growingfinishing pigs[END_REF]. In contrast, very little has been published on the effects of diet manipulation on performance of sows raised in a tropical, humid climate.

The first objective of our study was to evaluate the effect of diets with low TEF on feed consumption and performance of lactating sows under tropical, humid conditions. The second objective of the experiment was to determine if adding AA to basal diet can attenuate the effect of heat stress on maternal body reserve mobilization in heat-stressed lactating sows.

MATERIALS AND METHODS

Care and use of animals were performed according to the certificate of authorization to experiment on living animals issued by the French Ministry of Agriculture.

Experimental Design

Ten successive replicates of 8 to 10 mixed-parity Large White sows (n = 89 in total) were used in an experiment conducted at the INRA experimental facilities in Guadeloupe, French West Indies (latitude 16°N, longitude 61°W) considered a tropical humid climate area [START_REF] Berbigier | Description des climats équatoriaux et tropicaux[END_REF]. This study covered the period between February 2007 and January 2008. Two seasons were distinguished a posteriori from climatic measurement variables continuously recorded in the farrowing room.

Within each replicate, sows were distributed in a completely randomized experimental design among 3 dietary treatments according to backfat thickness, parity order, and BW after farrowing. The dietary experimental treatments were a normal protein diet (NP), a low protein diet (LP), and a NP diet supplemented with an AA complement (NP+). The experimental diets (Table 1) were formulated using corn, wheat middlings, and soybean meal, which met or exceeded AA requirements of lactating sows (NRC, 1998). The NP and LP diets contained the same levels of standardized digestible Lys (i.e., 0.80 g/MJ of NE), and the NP+ diet contained 0.95 g/MJ of NE. For the calculation of the AA complement composition in the NP+ treatment, an ADFI of 5 kg was assumed, and the Lys level was increased until the first essential AA became limiting (i.e., Phe + Tyr), after available synthetic AA (l-Lys, dl-Met, l-Thr, l-Trp, l-Val, l-Ile) were added to maintain a constant ratio between the essential AA and Lys. Each morning, 53 g of the calculated AA complement was incorporated manually into the NP diet before distribution to the sows. The levels of digestible essential AA relative to digestible Lys were similar for the 3 diets. The ratio between digestible essential AA and digestible Lys in the experimental diets was calculated to ensure that they were not below that of the ideal protein recommended for this animal category [START_REF] Renaudeau | Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate[END_REF]. Chemical composition and nutritional value of diets are presented in Table 2. Diets were offered as pellets. Feeds were prepared for 1 or 2 successive replicates and stored in a temperaturecontrolled room (24°C, 50 to 60% RH).

Animal Management

During the gestation period, sows were housed in open-fronted gestating pens in groups of 5 sows each The AA complement calculation: lysine content was increased in the NP diet until the other essential AA (Phe + Tyr, His, Arg, Leu) become limiting following the NRC recommendations for AA/Lys: 111, 39, 55, 110, for Phe + Tyr, His, Arg, Leu, respectively. For the calculations of the AA complement, an estimated ADFI of 5,000 g/d was used. Fifty-three grams of the AA complement was offered daily to each NP+ sow.

3

Mineral and vitamin mixture supplied (g/kg of diet): 10 of Cu (as CuSO 4 ); 80 of Fe (as FeSO 4 •7H 2 O); 40 of Mn (as MnO); 100 of Zn (as ZnO); 0.6 of I (as Ca(IO 3 ) 2 ); 0.10 of CO (as CoSO 4 •7H 2 O); 0.15 of Se (as Na 2 SeO 3 ); 5,000 IU of vitamin A; 1,000 IU of vitamin D 3 ; 15 IU of vitamin E; 2 mg of vitamin K 3 ; 2 mg of thiamine; 4 mg of riboflavin; 20 mg of nicotinic acid; 10 mg of d-pantothenic acid; 3 mg of pyroxidine; 0.02 mg of vitamin B 12 ; 1.0 mg of folic acid; and 0.2 mg of biotin.

and restrictively fed a conventional diet containing 13 MJ of DE/kg, 140 g of CP/kg, based on maize, wheat middling, and soybean meal. Feed allowance during the first 30 d after mating was calculated to standardize body condition at farrowing, according to the model proposed by [START_REF] Dourmad | Prédiction de la de la composition chimique des truies reproductrices à partir du poids vif et de l'épaisseur de lard dorsal[END_REF]. The feeding level was fixed at 2.5 kg/d from d 30 to 114 of gestation. Ten days before parturition, sows were moved to openfronted farrowing pens (2.1 × 2.2 m) on a slatted metal floor. Variations in ambient temperature, RH, and photoperiod closely followed outdoor conditions. On d 1 postpartum, sows received 1 kg of the standard gestation diet and the allowance increased by 1 kg each day until d 4 of lactation to avoid overconsumption at the beginning of lactation and agalactia problems. The proportion of gestation diet decreased progressively over the 4-d postpartum (100, 75, 50, and 25 on d 1, 2, 3, and 4, respectively), and sows were fed only the lactation diet on d 5. From d 6 to 26 postpartum, sows were allowed to consume feed ad libitum. The day before weaning (i.e., d 27), sows were allowed only 3 kg of feed (i.e., at least 1.5 kg less than their usual feed intake) to standardize consumption for all sows for determination of sow BW at weaning.

After birth, piglets were handled for tooth cutting, umbilical cord treatment, and ear tagged for labeling. On d 3, they received an intramuscular injection of 200 mg of iron dextran. As necessary, cross-fostering was conducted within the first 48 h after birth to standardize litter size at 11 piglets. On d 14, male piglets were castrated. After 21 d of lactation, piglets were offered creep feed containing 15.3 MJ of DE/kg, 20% CP, and 1.47% total Lys. Infrared lights provided supplemental heat for the piglets during the first 21 d of the lactation period. At weaning, sows were moved to a breeding facility and were presented to a mature boar twice daily to detect onset of standing estrus. From 28 d after mating, all sows were checked for pregnancy diagnosis using ultrasonography (Agroscan, ECM, Angoulême, France).

Measurements and Chemical Analyses

Sows were weighed after farrowing and at weaning. Backfat thickness measurements were taken ultrasonically (Agroscan, ECM) at 65 mm from the midline at the point beside the shoulder and at the last rib on each flank 2 d before farrowing and at weaning. The total number of piglets born, born alive, stillborn, and piglet deaths during lactation were recorded for each litter. Piglets were individually weighed at birth, at d 14 and 21 of lactation, and at weaning. Every morning, feed refusals were collected, and fresh feed was immediately distributed once per day between 0700 and 0900 h. Feed consumption was determined as the difference between feed allowance and the refusals collected on the next morning. Every day, 1 sample of feed and feed refusals were collected daily for DM content measurement, and successive samples were pooled and stored at 4°C for further analyses. Before sampling the NP+ refusal, these were homogenized manually to guarantee that during the sampling, residues of the AA complement were also incorporated to the sample. Rectal temperatures and respiratory rate of each sow were measured on Monday and Thursday at 0700 and 1200 h from Monday before farrowing to the Monday after weaning. Ambient temperature and RH were continuously recorded (1 measurement every 30 s) in the farrowing room, using a probe (Campbell Scientific Ltd., Shepshed, UK) placed 1 m above the floor.

At d 14, piglets were separated from the sows after suckling, and 50 min later (i.e., equivalent to average suckling interval; Renaudeau and Noblet, 2001), the sow was injected with 10 IU of oxytocin (Intervet, Angers, France) in an ear vein, and all functional mammary glands were hand milked. Samples (approximately 100 mL) were immediately stored at -20°C for further analyses.

Feed (2 samples per diet and per replicate) and milk (3 samples per sow) samples were analyzed for DM, ash, fat content (AOAC, 1990), and CP (N × 6.25 for feed and N × 6.38 for milk) according to the Dumas method (AOAC, 1990). Feed was analyzed for crude fiber and for cell wall components (NDF, ADF, and ADL) according to [START_REF] Van Soest | Use of detergents in the analysis of fibrous feeds. IV. Determination of plant cell-wall constituents[END_REF]. Lactose content of milk was determined using an enzymatic method (Boehringer Mannheim, reference No. 176303). Net energy values were estimated from the chemical composition of the diet using the equation of [START_REF] Noblet | Prediction of net energy value of feeds for growing pigs[END_REF]. Standardized digestible AA contents were calculated from the analyzed AA content and estimated standardized digestibility coefficients from INRA Tables [START_REF] Sauvant | Tables de composition et de valeur nutritive des matières premières destinées aux animaux d'élevage[END_REF].

Feed and milk AA contents were analyzed by Ajinomoto Eurolysine (Amiens, France) using ion-exchange chromatography, except for Trp, which was analyzed using HPLC and fluorimetric detection (Waters 600E, St. Quentin en Yvelines, France).

Calculations and Statistical Analyses

Daily maximum, minimum, mean, and variance of daily ambient temperature and RH were averaged for each replicate. These data were used to split the total experiment period between 2 seasons through a principal component analysis (PRINCOMP procedure, SAS Inst. Inc., Cary, NC). The effects of season, diet composition, replicate, parity number, and their interactions on performance of sows and litters were tested the GLM procedure of SAS. The effect of group of sows was tested within the effect of season. The average daily rectal temperature and respiratory rate (defined as the mean of values measurements at 0700 and 1200 h) measurements were pooled per sow over the lactation period. These data were analyzed using the same previous model. The effect of lactation stage on daily feed intake was tested with a mixed linear model (MIXED procedure of SAS) for repeated measurements with diet composition, season, and replicate as main effects. The least squares means procedure (PDIFF option of SAS) was used to compare means when a significant F-value was obtained. The number of sows returning into estrus before and after 5 d postweaning were compared using a χ 2 test (FREQ procedure of SAS).

RESULTS

Climatic Measurements

The warm season was determined to be between February and April 2007 and between November 2007 and January 2008, whereas the hot season corresponded to May to October 2007 period. The average minimum and maximum ambient temperatures and average RH for the warm season were 20.5 and 28.2°C, and 93.8%, respectively. The corresponding values for the hot season were 22.7 and 29.4°C, and 93.7%, respectively. The mean temperature values for warm and hot season were 23.7 and 26.1, respectively (Table 3).

Sow and Litter Performance

Three sows were removed from the study due to low litter size (<6 piglets). No interaction (P > 0.10) between season and diet composition was found for all criteria studied. According to the experimental design, parity number averaged 3.2 and was not affected by season or by diet composition. Lactation length was greater in the hot season than in the warm season (29.2 vs. 28.1 d; Table 4), but no difference in lactation length was observed between dietary treatments (28.6 d on average).

As presented in Table 4, ADFI was affected (P < 0.001) by parity and season with reduced feed consumption in the hot season during the entire lactation period and during the ad libitum period (4.72 vs. 3.69 kg; and 5.36 vs. 4.13 kg, respectively). After farrowing, sows were restrictively fed for 5 d according to the same feeding plan, and the increase of ADFI was similar for both seasons until d 4 (Figure 1). After d 4, ADFI tended to be greater for LP and NP+ diets as compared with the NP diet during the entire lactation period and during the ad libitum period (i.e., 4.35 vs. 3.89 and 4.91 vs. 4.39 kg, respectively; P = 0.08). The ADFI of sows fed LP and NP+ diets increased with the advancement of lactation after d 4 similarly to NP sows (P < 0.05; Figure 2). Lactation BW and backfat losses were not affected (P > 0. 10) by season (Table 4). Lactation BW loss was numerically less for the NP+ than the NP or LP sows (21.8 vs. 26.2 kg; P > 0.10). The LP and NP+ sows showed numerically less backfat loss than NP sows (3.5 vs. 5.4 mm; P = 0.10). Body protein, lipid, and energy losses were not affected by the treatments (P > 0.10). Rectal temperature was greater (P < 0.001) in the hot season than in the warm season (38.8 vs. 38.4°C).

Litter size and the average piglet BW at birth were not affected (P > 0.10) by season. Litter BW gain between birth and weaning and mean BW of piglets at weaning were greater (P < 0.05) during the warm season than during the hot season (2.3 vs. 1.8 kg/d and 7.5 vs. 7.1 kg). Litter BW gain during the lactation period was not affected by dietary treatment (P > 0.10). Daily consumption of solid creep feed (Table 5) during wk 4 of lactation was not affected by diet composition or by season (P > 0.10).

Milk production between d 1 and 21 was greater (P < 0.01) in the warm season than in the hot season (8.1 vs. 6.8 kg/d, respectively; Table 5). Milk lipid content was greater in the hot season than in the warm season (38.7 vs. 33.1%; P < 0.05), and protein content tended to be less in the warm season (27.1 vs. 25.1%; P = 0.05; Table 6). Milk production and composition were not affected by diet.

Reproductive performance was measured for a total of 86 sows, but 5 sows (3, 1, and 1 sows fed NP, LP, and NP+, respectively) did not show estrus until 15 d after weaning and the weaning-to-estrus interval (WEI) was calculated on a total of 81 sows. The WEI averaged 3.7 and 4.5 d (P < 0.05) for the warm and hot seasons, respectively (Table 7). The percentage of sows with a delayed estrus was greater in the hot than in the warm season (6.2 vs. 0%; P < 0.05).

DISCUSSION

Effect of Season on Sow and Litter Performance

The effect of high ambient temperatures on the performance of lactating sows and their litters is well established [START_REF] Black | Lactation in the sow during heat stress[END_REF][START_REF] Renaudeau | Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate[END_REF]. In tropical, humid conditions, the average temperatures observed during the warm and the hot seasons are above the upper limit of the thermoneutral zone of the sows (i.e., 22°C; [START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF]. Under our tropical humid conditions, the mean temperatures observed during the warm and hot season (23.7 and 26.1°C, respectively) exceeded 22°C. In addition, the 22°C value was determined at low RH, and it probably overestimates the upper limit of the zone of thermoneutrality when the RH is close to 100%. Therefore, lactat-ing sows suffered from heat stress most of the time in our experimental conditions. This effect was accentuated during the hot season due to the greater average daily temperature (i.e., +2.5°C). This observation is consistent with the increase of rectal temperature (i.e., +0.4°C), increase of the respiratory rate (i.e., +9 breaths/min), and the sharp reduction of ADFI during the hot season. However, the average rectal temperature recorded during the warm and the hot seasons in this study was less than values measured by [START_REF] Lorschy | Food intake, heat production and milk yield of lactating sows exposed to high temperature[END_REF] and [START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF] at 38.2 vs. 39.3°C and 38.6 vs. 39.5°C, respectively. Moreover, the increase in respiratory rate observed from warm to hot season (+4.0 breaths•min -1 •°C -1 ) is less than values reported in other studies using high temperatures (+7.1, +8.9, and +8.0 breaths•min -1 •°C -1 , according to [START_REF] Lorschy | Food intake, heat production and milk yield of lactating sows exposed to high temperature[END_REF][START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF]Renaudeau and Noblet, 2001, respectively). The smaller increase observed in the respiratory rate between seasons in our study may be related to the fact that sows in the warm season were already heat stressed, leading to a relatively smaller increase in respiratory rate when exposed to hot season temperature. Taking into account the low cutaneous evaporative capacities in pigs, a significant rise in respiratory rate is required to cause an effective increase of evaporative heat losses. All these From a GLM analysis including the effects of season (S), diet composition (D), the effect of parity (P), and the effect of batch of sows (G), and their interactions as fixed effects. The interaction between diet and season was not significant (P > 0.10).

4 Estimated from equations published by [START_REF] Dourmad | Prédiction de la de la composition chimique des truies reproductrices à partir du poids vif et de l'épaisseur de lard dorsal[END_REF]. Protein (kg) = 2.28 (2.22) + 0.178 (0.017) × empty BW -0.333 (0.067) × P2 (RSD = 1.9); lipids (kg) = -26.4 (4.5) + 0.221 (0.030) × empty BW + 1.331 (0.140) × P2 (RSD = 6.1); energy (MJ) = -1.075 (159) + 13.67 (1.12) × empty BW + 45.98 (4.93) × P2 (RSD = 208). Empty BW (kg) = a × BW 1.013 (kg), with a = 0.912 at farrowing and a = 0.905 at weaning. P2 = P2 backfat thickness (mm).

processes produce to a short-term adaptation to hot climatic conditions via increased heat dissipation.

The negative effect of high temperatures on feed intake has been extensively described [START_REF] Christon | Dietary fat and climate alter food intake, performance of lactating sows and their litters and fatty acid composition of milk[END_REF][START_REF] Johnston | Effect of room temperature and dietary amino acid concentration on performance of lactating sows[END_REF][START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF][START_REF] Renaudeau | Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate[END_REF][START_REF] Gourdine | Effects of season and breed on performance of lactating sows in a tropical humid climate[END_REF]. [START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF] showed that the negative effect of temperature on ADFI is accentuated as the ambient temperature increases. In their study, they indicated that each degree increase in ambient temperature between 25 and 27°C at a RH fluctuating between 50 and 60% resulted in a reduction of feed intake equivalent to 214 g/d. In our study, over a similar temperature range between the 2 seasons and at approximately 94% RH, the corresponding value was more than twice as large (492 g•d -1 •°C -1 ). [START_REF] Renaudeau | Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate[END_REF], evaluating the effects of dietary fiber content in the same tropical conditions as ours (i.e., a similar range of temperature and ambient humidity), observed a reduction of 584 g•°C -1 in ADFI between both seasons. These results suggest that the reduction in feed intake of lactating sows kept in tropical climates is related to the combined effects of high temperatures and high humidity. This emphasizes that the high humidity accentuates the effect of high temperatures by limiting the capacity of evaporative heat loss from the lungs through increased respiratory rate [START_REF] Renaudeau | Effects of short-term exposure to high ambient temperature and relative humidity on thermoregulatory responses of European (Large White) and Caribbean (Creole) restrictively fed growing pigs[END_REF].

In the warm season, piglet growth rate over the first 3 wk of lactation (194 g/d) was less than results reported in temperate conditions (Auldist and King, 1995: 265 g/d; Hulten et al., 2002: 290 g/d;[START_REF] Quiniou | Influence de la quantité d'aliment alloué à la truie en fin de gestation sur le déroulement de la mise bas, la vitalité des porcelets et les performances de lactation[END_REF] 253 g/d). As observed for ADFI, this suggests that the sows in our study were heat stressed, and their milk production was depressed, even in the warm season. According to [START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF] and [START_REF] Gourdine | Effects of season and breed on performance of lactating sows in a tropical humid climate[END_REF], milk yield is reduced at elevated temperatures. In the current study, the effect of season on milk production and on litter BW gain was significant, with decreased values observed during the hot season. Moreover, when milk production was expressed on a per piglet basis, the amount of milk available for each piglet decreased during the hot season. This indicates that the negative effect of heat stress on sow milk production was emphasized during the hot season. Independent of the season, piglet BW gain between d 21 and weaning was greater than piglet BW gain from wk 1 to wk 3 of lactation, likely due to creep feed consumption during this period. In the present study, piglets consumed similar amounts of creep feed during the warm and hot seasons (14 vs. 13 g•d -1 •piglet -1 , respectively). In contrast to our findings, Renaudeau et al. (2001) reported a greater creep feed intake in piglets from sows maintained under heat stress conditions (23 vs. 38 g•d -1 •piglet -1 , respectively, for 20 and 29°C; P < 0.01). According to these authors, piglets compensated for the decreased milk production by increasing their creep feed consumption, with a subsequent attenuated effect of heat stress on performance of the litter. The difference observed between our findings and the latter authors can be related to the observation that the Effect of season on daily feed intake during the lactation period. Feed intake was not different between seasons from d 1 to 5 (P > 0.10), whereas it differed from d 6 to 25 (×; P < 0.05). A total of 33 and 53 sows were used for the warm and hot seasons, respectively. range of temperatures in our experiment was too small to affect creep feed intake (2.4 vs. 9.0°C, respectively, for our study and Renaudeau et al., 2001).

In agreement with Renaudeau and Noblet (2001), our results show an increased lipid content of milk in sows exposed to hot climatic conditions. Similar results were obtained at thermoneutrality when dietary energy supply was reduced and mobilization of body fat reserves was accentuated [START_REF] Noblet | Effect of energy level in lactating sows on yield and composition of milk and nutrient balance of piglets[END_REF]. Collectively, these studies suggest that elevated milk lipid concentrations that result when sows are exposed to high ambient temperatures are related to an increased mobilization of body fat reserves.

Sow BW, protein, lipid, energy, and backfat thickness losses were not affected by season. In contrast to our results, most studies investigating this area demonstrated that BW loss increases in heat-stressed sows in connection with a reduced ADFI [START_REF] Christon | Dietary fat and climate alter food intake, performance of lactating sows and their litters and fatty acid composition of milk[END_REF][START_REF] Johnston | Effect of room temperature and dietary amino acid concentration on performance of lactating sows[END_REF][START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF][START_REF] Renaudeau | Effects of dietary fiber on performance of multiparous lactating sows in a tropical climate[END_REF] and a smaller relative reduction of performance. A possible explanation for our findings is that the sows were more efficient to produce milk from feed energy than from energy mobilized from body stores in the hot season, resulting in no effects on chemical composition of BW loss [START_REF] Noblet | Energy utilization in pregnant and lactating sows: Modelling of energy requirements[END_REF]. Our study showed a significant effect of season on weaning to es-trus interval, whereby the sows showed a greater WEI in the hot season than in the warm season (i.e., 4.5 vs. 3.7 d). [START_REF] Shaw | Relationships between LH, FSH and prolactin secretion and reproductive activity in the weaned sow[END_REF], [START_REF] Barb | Endocrine changes in sows exposed to elevated ambient temperature during lactation[END_REF], [START_REF] Koketsu | Influence of feed intake during individual weeks of lactation on reproductive performance of sows on commercial farms[END_REF][START_REF] Van Den Brand | Dietary energy source at two feeding levels during lactation of primiparous sows: I. Effects on glucose, insulin, and luteinizing hormone and on follicle development, weaning-toestrus interval, and ovulation rate[END_REF] also observed a negative effect of high temperature on the return to estrus of sows after weaning. According to these authors, the delay in the return to estrus after weaning was related to decreased secretion of LH in sows maintained in a hot environment.

Effect of Dietary Protein Content on Sow and Litter Performance

In the present study, LP sows tended to have increased ADFI (+0.540 kg) when compared with the NP diet sows. Our results show that the increase of ADFI with LP diet was quite similar in multiparous and in primiparous sows (+11%, respectively). Renaudeau and Noblet (2001) evaluating the effect of protein reduction (14.2 vs. 17.6%) also reported a numerical increase of ADFI at 29°C for sows fed LP diet (+0.639 kg). [START_REF] Lynch | Voluntary food intake of sows and gilts[END_REF] also observed an increased feed consumption (+0.700 kg/d) in multiparous lactating sows fed a low CP diet (14 vs. 20%) at 28°C. In contrast, [START_REF] Quiniou | Influence of high ambient temperatures on performance of multiparous lactating sows[END_REF] did not report any effect of diet on performance of lactating sows kept at 29°C when dietary protein content was reduced from 17 to 14%. In that study, the authors attributed the lack of interaction between temperature and diet to the few number of observations, the reduced supplies of sulfur AA and Trp expressed as a percentage of Lys in the 14% CP diet, or a combination of both. In addition, the reduction of dietary protein content with a supplementation of synthetic AA leads to increase the ratio between Trp and branched chain AA [long neutral AA (LNAA): Leu,Ile,Val,Phe,Tyr;i.e.,4.52 vs. 5.37% in NP and LP diet,respectively]. According to [START_REF] Tackman | Dietary disproportions of amino acids in the rats: Effects on food intake, plasma and brain amino acids and brain serotonin[END_REF], Trp and LNAA share the same neutral carrier system to cross the blood-brain barrier, and they compete for uptake by the brain. Serotonin and its precursor, Trp, are known to be involved in the control of feed intake; an increased ratio of Trp:LNAA is reported to increase appetite linearly [START_REF] Henry | Feed intake and dietary amino acid balance in growing pigs with special reference to lysine, tryptophan and threonine[END_REF]. [START_REF] Trottier | Dietary and plasma branched-chain amino acids in relation to tryptophan: Effect on voluntary feed intake and lactation metabolism in the primiparous sow[END_REF] reported that a reduction in the Trp:LNAA ratio through dietary addition of LNAA decreased feed intake of primiparous lactating sows. Thus, the increased ADFI in LP treatment may also be related to a reduced Trp:LNAA ratio.

Litter BW gain, milk production, and composition were not affected by dietary CP content. Similarly, [START_REF] Johnston | Effect of room temperature and dietary amino acid concentration on performance of lactating sows[END_REF] and Renaudeau et al. (2001) showed that lactating sows kept at 29°C exhibited no From a GLM analysis including the effects of season (S), diet composition (D), the effect of parity (P), and the effect of batch of sows (G) and their interactions as fixed effects. The interaction between diet and season was not significant (P > 0.10). 4 After cross fostering.

5

Daily milk production over the first 21 d of lactation was calculated from litter growth rate, litter size between d 1 and 21, and milk DM using the equation from [START_REF] Noblet | Estimation of sow milk nutrient output[END_REF].

***P < 0.001, **P < 0.01, *P < 0.05, †P < 0.10. From a GLM analysis including the effects of season (S), diet composition (D), and the effect of batch of sows (G) and their interactions as fixed effects. The interaction between diet and season was not significant (P > 0.10).

***P < 0.001, **P < 0.01, *P < 0.05, †P < 0.10.

change in litter BW gain when dietary CP level was decreased (from 16.7 to 13.3%; and from 17.6 to 14.2%, respectively).

Evaluating the effect of dietary CP content on the performance of lactating sows under heat stress, Renaudeau et al. (2001) observed that the LP sows showed a greater daily feed intake and lost less BW in comparison with NP sows. In the present study, the reduction of BW mobilization in sows fed LP diet was very small (-1.6 kg). The estimated chemical composition of BW loss was not affected by the diet composition. On average, each 100 g of BW loss contained 14 g of protein and 47 g of lipid. Evaluating diets comparable with ours, Renaudeau et al. (2001) reported similar values of body nutrient change (12 and 46%, respectively, for protein and lipid content in BW loss) for multiparous sows over a 27-d lactation. In our study, the lack of a significant impact of diet on reproductive performance was attributed to the observation that dietary treatment did not affect mobilization of body reserves.

Effect of Dietary AA Content on Sow and Litter Performance

In the present study, sows fed NP+ diet showed, on average, +10.6% greater ADFI when compared with sows fed the NP diet. Because of the small number of primiparous sows, we did not evaluate their performance independently. However, greater feed intake observed for the NP+ sows can be partly attributed to a greater effect on primiparous sows, which had greater feed intakes when fed an AA complement. In contrast to our results, [START_REF] Tokach | Characterization of luteinizing hormone secretion in the primiparous sows: Relationship to blood metabolites and return-to-estrus[END_REF] and [START_REF] Paiva | Lisina em raçoes para femeas suinas primiparas em lactataçao[END_REF], evaluating the effects of dietary Lys levels in lactating primiparous sows, via an addition of synthetic AA, did not observe a significant effect of the increase of AA content on ADFI.

Piglet and litter performance and sows milk production were not affected by the dietary AA complement. Similarly, [START_REF] Trottier | Dietary and plasma branched-chain amino acids in relation to tryptophan: Effect on voluntary feed intake and lactation metabolism in the primiparous sow[END_REF], [START_REF] Dourmad | Effect of protein and lysine supply on performance, nitrogen balance, and body composition changes of sows during lactation[END_REF][START_REF] Paiva | Lisina em raçoes para femeas suinas primiparas em lactataçao[END_REF] also did not observe any effect of increasing dietary AA content on piglet and litter performance and on the milk yield of sows. Milk composition was not affected by dietary AA content. Similarly, [START_REF] Trottier | Dietary and plasma branched-chain amino acids in relation to tryptophan: Effect on voluntary feed intake and lactation metabolism in the primiparous sow[END_REF] also did not observe any effect of increasing dietary AA content on milk composition of sows.

The results of BW, protein, lipid losses, as well as changes in backfat thickness obtained in our study, were not affected by the dietary AA supplementation. Nevertheless, when compared between NP+ and NP, a numerical less BW mobilization was observed for the sows fed the NP+ diet, but these results are related to the performance of the primiparous sows, which were more affected by AA supplementation. This result can be associated to the effect of the greater Lys intake observed in these primiparous sows (i.e., +11 g/d), which can also be related to the numerical greater ADFI observed for the sows (i.e., +460 g). [START_REF] Stahly | Milk yield responses of sows nursing large litters to dietary amino acid levels[END_REF], [START_REF] Johnston | Response of maternal-line sows to dietary protein concentration during lactation[END_REF][START_REF] Dourmad | Effect of protein and lysine supply on performance, nitrogen balance, and body composition changes of sows during lactation[END_REF] also reported that the loss of BW was affected by Lys supply, whereas increasing Lys levels in the diets reduced BW loss.

In conclusion, the present study demonstrates that high ambient temperature had an important negative effect on feed intake and more generally on performance of lactating sows, and these negative impacts are exacerbated when the RH is high. Regardless of season, the use of diets with reduced TEF or supplemented with an AA complement in lactation may allow an increase in ADFI and partially attenuate the negative effects of tropical climate on performance of sows. The addition of AA supplementation in diets for primiparous sows may attenuate the effects of heat stress on the performance of these animals, but this assumption needs to be more thoroughly investigated with a greater number of sows. AOAC. 1990 From a GLM analysis including the effects of season (S), the effect of batch of sows (G), and their interactions as fixed effects. The interaction between diet and season was not significant (P > 0.10).

LITERATURE CITED

*P < 0.05.

2[

  Percentage of Trp/(% Ile + % Leu + % Val + % Phe + % Tyr)] × 100. LNAA = long neutral AA.

  3

Figure 1 .

 1 Figure1. Effect of season on daily feed intake during the lactation period. Feed intake was not different between seasons from d 1 to 5 (P > 0.10), whereas it differed from d 6 to 25 (×; P < 0.05). A total of 33 and 53 sows were used for the warm and hot seasons, respectively.

Figure 2 .

 2 Figure 2. Effect of diet composition on daily feed intake during the lactation period. Feed intake was not different from d 1 to 3 and on d 5, 11, 13, and 19 (P > 0.10), whereas it differed between diets on d 4, from d 6 to 10, d 12, d 14 to 18, and from d 20 to 25 (×; P < 0.05). A total of 30, 30, and 26 sows were used for NP, LP, and NP+, respectively. NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement.

Table 1 .

 1 Composition of the lactation diets, as-fed basis 1

	Item	NP	LP	NP+ 2
	Ingredient, %
	Corn	59.5	67.4	59.5
	Soybean meal	24.4	10.6	24.4
	Wheat middlings	8.4	14.3	8.4
	Soybean oil	3.4	2.4	3.4
	l-Lysine•HCl	0.020	0.415	0.020
	dl-Methionine		0.109
	l-Threonine		0.175
	l-Tryptophan		0.064
	l-Isoleucine		0.127
	l-Valine		0.140
	Monocalcium phosphate	1.0	1.0	1.0
	Calcium carbonate	2.1	2.1	2.1
	Salt	0.1	0.1	0.1
	Minerals and vitamins 3	1.1	1.1	1.1
	AA complement, g			53
	1 NP = normal protein diet; LP = low protein diet; NP+ = NP diet
	supplemented with an AA complement.

2

Table 2 .

 2 Analyzed chemical composition of the lactation diets 1

	Analyzed composition	NP	LP	NP+
	Ash	5.5	5.3	5.6
	CP	17.3	14.1	17.6
	Starch	39.0	45.2	39.0
	Ether extract	4.3	5.6	4.3
	NDF	10.0	10.8	10.0
	ADF	2.5	2.7	2.5
	Standardized digestible AA
	Lys	0.80	0.80	0.97
	Met + Cys	0.49	0.48	0.68
	Thr	0.54	0.54	0.66
	Trp	0.18	0.17	0.21
	Ile	0.63	0.54	0.77
	Leu	1.36	1.07	1.36
	Val	0.71	0.65	0.86
	Phe	0.82	0.56	0.82
	Tyr	0.59	0.41	0.59
	Trp:LNAA, 2 %	4.52	5.37	4.83
	Calculated nutritional value 3
	NE, MJ/kg	10.2	10.1	10.2
	Digestible Lys, g/MJ of NE	0.80	0.80	0.95
	NE/ME, %	71.6	73.5	71.6

1

Measured or calculated values adjusted for 88% DM. NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement.

Table 3 .

 3 Main characteristics of climatic variables 1 Seasons correspond to the means of daily values of ambient temperature and relative humidity. Warm season: February to April 2007 and November 2007 to January 2008. Hot season: May to October 2007.

		Season
	Item	Warm	Hot
	Temperature, °C
	Minimal	20.7	23.3
	Maximal	28.0	29.4
	Mean	23.7	26.1
	Relative humidity, %
	Minimal	82.3	87.0
	Maximal	98.4	97.7
	Mean	93.5	93.7
	1

Table 4 .

 4 Effects of season and diet composition on performance of lactating sows over a 28-d lactation (least squares means)

			Diet 1		Season
	Variable	NP	LP	NP+	Warm	Hot	RSD 2	Statistical analysis 3
	No. of lactations	30	30	26	33	53
	Average parity	3.3	3.2	3.3	3.3	3.2	1.2	P***
	Lactation length, d	28.4	29.3	28.2	28.1	29.2	2.7
	ADFI, as fed
	From d 1 to weaning, kg	3.89	4.36	4.35	4.72	3.69	0.65	D †, S***, G*, P***
	From d 6 to 26, kg	4.39	4.93	4.91	5.36	4.13	0.81	D †, S***, G*, P***
	BW, kg
	After farrowing	224.6	226.9	229.6	226.9	227.2	29.1	P***
	At weaning	197.6	201.5	207.8	203.5	201.0	30.9	P***
	Loss during lactation	27.0	25.4	21.8	25.8	23.6	12.7
	Backfat thickness, mm
	After farrowing	17.7	16.9	18.8	18.4	17.3	3.8
	At weaning	12.3	13.6	14.9	14.7	12.5	2.8	D †, S*, G †, P*
	Loss during lactation	5.4	3.3	3.8	3.6	4.7	2.5	D †, P †
	Chemical composition of BW loss 4
	Protein, kg	3.2	3.6	2.8	3.6	2.8	2.0
	Lipid, kg	13.4	10.3	10.2	10.8	11.8	5.2
	Energy, MJ	635	517	493	538	558	245
	Rectal temperature, 5 °C	38.6	38.5	38.6	38.4	38.8	0.2	S***
	Respiratory rate, 5 breaths/min	61	59	58	55	64	9

1 NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement. 2 RSD = residual SD.

3

Table 5 .

 5 Effect of season and diet composition on performance of litters over a 28-d lactation (least squares means)

			Diet 1		Season
								Statistical
	Variable	NP	LP	NP+	Warm	Hot	RSD 2	analysis 3
	No. of lactations	30	30	26	33	53
	Parity	3.3	3.2	3.3	3.3	3.2	1.2	P***
	Lactation length, d	28.4	29.8	28.5	28.1	29.2	2.7
	Litter size
	At d 1 4	12.2	12.4	11.4	12.2	11.8	1.9	G**
	At weaning	10.1	10.1	10.7	10.7	9.8	1.3	S*, G**
	Piglet BW, kg
	At d 1	1.4	1.4	1.4	1.4	1.4	0.2
	At d 14	4.2	4.2	4.2	4.3	4.2	0.7	G*
	At d 21	5.7	5.7	5.6	5.8	5.5	0.7
	At weaning	7.5	7.4	7.1	7.5	7.1	0.8	S †
	Litter BW gain, kg/d
	Wk 1 to 3	2.1	2.0	2.0	2.2	1.8	0.4	S**, G*
	Wk 4	2.5	2.4	2.2	2.6	2.1	0.6	S*
	Wk 1 to 4	2.2	2.1	2.1	2.3	1.8	0.4	S***
	Total creep-feed consumption, piglet/wk 4	99	108	82	102	91	131	P †
	Total creep-feed consumption, litter/wk 4	981	1,100	883	1,064	912	1,335	P †
	Sow milk production, 5 kg/d	7.6	7.3	7.5	8.1	6.8	1.4	S**, G*
	1 NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement.

2

RSD = residual SD.

3

Table 6 .

 6 Effect of season and diet composition on milk composition 1

			Diet		Season
	Item	NP	LP	NP+	Warm	Hot	RSD 2	Statistical analysis 3
	No. of lactations	22	21	21	13	51
	Milk composition
	DM, %	19.7	20.3	19.4	19.8	19.8	1.4	G***
	Protein (N × 6.38), %	26.7	25.9	26.3	27.1	25.1	1.6	S †, G**
	Ash, %	4.3	4.2	4.1	4.3	4.1	0.3	G*
	Lipid, %	34.1	38.4	35.2	33.1	38.7	5.5	S*, G**

1 Data represent least squares means of milk samples collected at d 14. NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement. 2 RSD = residual SD.

3

  . Official Methods of Analysis. 15th ed. Assoc. Off. Anal. Chem., Washington, DC. Auldist, D. E., and R. H. King. 1995. Piglets' role in determining milk production in the sow. Pages 114-118 in Manipulating

Table 7 .

 7 Effect of season and diet composition on the weaning-to-estrus interval (least squares means)

			Diet 1		Season
	Item	NP	LP	NP+	Warm	Hot	RSD 2	Statistical analysis 3
	No. of lactations	27	29	25	32	49
	Weaning to estrus interval, n
	1 to 3 d	6	6	6	12	6
	3 to 5 d	19	22	17	21	40
	>5 d	2	1	2	20	5
	Weaning to estrus interval, d	4.2	4.2	4.0	3.7	4.5	1.0	S*, G*
			χ 2 = 0.74, P = 0.94	χ 2 = 9.50, P = 0.009
	1 NP = normal protein diet; LP = low protein diet; NP+ = NP diet supplemented with an AA complement.

2

RSD = residual SD.

3

The authors gratefully acknowledge Ajinomoto Eurolysine (Paris, France) for their financial support and for measurement of AA content in feeds; and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brasilia, Brazil) for the grant to B. A. N. Silva during his PhD research at INRA. The authors also gratefully acknowledge C. Anäis, M. Bructer, K. Benoni, T. Etienne, and G. Saminadin for their technical assistance.

2 Corresponding author: David.Renaudeau@antilles.inra.fr ReceivedMay 13, 2008. Accepted August 6, 2009.

at INRA Institut National de la Recherche Agronomique on August 14, 2013 www.journalofanimalscience.org Downloaded from

Measured at 0700 and 1200 h every Monday and Thursday during lactation. ***P < 0.001, *P < 0.05, †P < 0.10.