L Liméa

G Alexandre
email: gisele.alexandre@antilles.inra.fr

V Berthelot
email: valerie.berthelot@agroparistech.fr

O Coppry

R Arquet

T Etienne

S Calif

G Saminadin

Fatty acid composition of muscle and adipose tissues of indigenous Caribbean goats under varying nutritional densities 1

Keywords: adipose tissue, concentrate, fatty acid, goat, muscle, tropical forage

The effects of a concentrate diet on growth, carcass fat, and fatty acid (FA) composition of muscle (supraspinatus), perirenal, and intermuscular adipose tissues of Creole goats (n = 32) were evaluated. Goats were fed a tropical green forage Digitaria decumbens ad libitum with no concentrate (G0) or 1 of 3 levels of concentrate: 140 (G100), 240 (G200), and 340 g•d -1 (G300), respectively. Goats were slaughtered according to the standard procedure at the commercial BW (22 to 24 kg of BW). Goats fed the concentrate diets (G100, G200, and G300) had greater ADG (P < 0.001), cold carcass weights (P < 0.001), and omental (P < 0.01), perirenal (P < 0.01), and intermuscular (P < 0.01) adipose tissues weights. Dietary intake of C18:0, C18:1n-9, C18:2n-6 increased as concen-trate supplementation increased (P < 0.001), whereas C18:3n-3 intake was not affected (P > 0.05). Increased concentrate supplementation did not affect (P > 0.05) the proportion of MUFA in all tissues and had very little effect on SFA in perirenal tissue, but increased the PUFA proportion in muscle (P < 0.05). The major effect of feeding increased concentrate was an increase in n-6 PUFA proportions in all tissues (P < 0.001) and, surprisingly, a decrease in n-3 PUFA (P < 0.001). Focusing on FA, which are supposed to have a beneficial or an adverse effect on human health, feeding increased concentrate did not increase the content of any cholesterol-increasing SFA in meat, but increased the n-6/n-3 ratio above 4 when more than 240 g of concentrate was fed per day.

INTRODUCTION

The consumption of fat in meat from ruminants has been associated with high-calorie intakes and fatty acid (FA) profiles that are associated with health problems. To overcome this problem, ruminant meat producers need to focus on producing a leaner meat with decreased proportions of SFA, especially C14:0 and C16:0, and increased proportions of cis-MUFA and PUFA, especially long-chain PUFA of the n-3 series. The potential health benefits would be improved if trans-MUFA were reduced and CLA were increased, especially the C18:2cis-9, trans-11 isomer [START_REF] Aurousseau | Effect of grass or concentrate feeding systems and rate of growth on triglyceride and phospholipid and their fatty acids in the m. Longissimus thoracis of lambs[END_REF]. In this context, goat meat has an advantage over beef and lamb due to its low fat content [START_REF] Webb | The animal fat paradox and meat quality[END_REF] and a favorable FA composition of fat [START_REF] Banskalieva | Fatty acid composition of goat muscles and fat depots: A review[END_REF].

Supplementing forage-feeding (FF) with concentrates (CF) improved ADG of kids but also increased carcass fat content because of greater energy intakes [START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF]. It has been speculated that concentrate feeding might be associated with changes in the FA profile of muscle and fat due to the differences in the FA content and profile of feed ingredients. Meat from forage-fed ruminants has a greater n-3 PUFA content than that from concentrate-fed-ruminants, despite increased ruminal PUFA biohydrogenation in these animals, due to the greater C18:3 content in forage compared with most concentrates [START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF][START_REF] Webb | The animal fat paradox and meat quality[END_REF]. However, the beneficial effect of a FF system on FA composition is not always clear; [START_REF] Rhee | Fatty acid composition of goat diets vs intramuscular fat[END_REF] observed that the intramuscular fat of range goats had more SFA than intramuscular fat from goats fed a grain diet.

Because local demand for Creole goat meat is high in the Caribbean area, different feeding systems were studied to improve its meat production. The growth performance, carcass quality, and noncarcass components of the indigenous Caribbean goats in these feeding systems have been reported in a companion paper (Liméa et al., 2009a). The present study examined the effect of these FF and CF systems on the FA composition of adipose tissues and muscle in Creole kids.

MATERIALS AND METHODS

The study was conducted on the Experimental Farm of the Institut National de Recherche Agronomique (INRA) Animal Production Research Unit in Guadeloupe and is fully described in Liméa et al. (2009a). All animal care, handling techniques, and slaughter procedures were approved by the INRA Animal Care and Use Committee before the research was initiated.

Experimental Diets and Animals

Thirty-two male indigenous Caribbean goats (9.0 ± 0.2 kg at 3 mo old) from a total of 40 (Liméa et al., 2009a) were reared in individual pens. Kids were blocked by BW and ADG between 30 and 70 d preweaning and were randomly assigned to 1 of the 4 groups. Each group (8 replicates per group) received a different diet with a different level of concentrate supplementation. The first group (G0) was fed ad libitum a basal diet exclusively made up of Digitaria decumbens, a green tropical forage, that was cut daily at 28 d of regrowth. The animals in the other 3 groups (G100, G200, and G300) received daily the same basal diet plus 140, 240, or 340 g (as fed) of a commercial concentrate, respectively. The concentrate was made up of maize (68%), soybean cake (15%), and wheat bran (11%). The chemical and FA composition of forage and concentrate are presented in Table 1.

Slaughtering Procedure

Animals were slaughtered at a fixed BW of 22 to 24 kg. They were weighed and fasted the day before slaughter. The slaughtering procedures followed the methods standardized by [START_REF] Colomer-Rocher | Standard methods and procedures for goat carcass evaluation, jointing and tissue separation[END_REF]. The omental fat (OF) was removed and weighed. Dressed carcasses were weighed within 1 h of slaughter (HCW) and after chilling for 24 h at 4°C (cold carcass weight). The perirenal fat (PR) was removed, weighed, sampled, and frozen at -30°C. The left shoulder was separated from the cold carcass for dissection and analysis (see Liméa et al., 2009a), and samples of muscle (supraspinatus, SS) and intermuscular adipose (IM) tissues were removed and frozen at -30°C until chemical analysis was done.

Chemical Analysis

Samples of forage (2 subsamples of 200 g) were taken each day from Monday to Friday. One of the subsamples was kept for daily DM determination. All the samples of the feed collected during 2 wk were mixed together for each kid, and a pooled subsample (200 g) was used for chemical analyses. Feed chemical analysis procedures were described in Liméa et al. (2009a). The DM content of PR, IM, and SS was determined after 48 h of freeze-drying. The lipid extract content of feed and tissues were determined by ether-petroleum extraction [START_REF] Aoac | Official Methods of Analysis[END_REF]. Feed and tissue FA extractions were carried out according to [START_REF] Bas | Fatty acid composition of mixed-rumen bacteria: Effect of concentration and type of forage[END_REF] and [START_REF] Rule | Direct transesterification of total fatty acids of adipose tissue, and of freeze-dried muscle and liver with borontrifluoride in methanol[END_REF] respectively, using tricosanoic acid as an internal standard in the extraction solvent mixture. Feed and Composition, per kilogram as-fed basis: 680 g of corn grain, 150 g of soybean cake, 110 g of wheat bran, 10 g of urea, and 50 g of vitamin and mineral supplement.

tissue methylations were described by [START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF]. Samples were then injected using an auto sampler CP-8410 into a Varian CP-3900 gas liquid chromatograph (GLC, Varian, Les Ulis, France) on a DB-wax fusedsilica capillary column (60 m length × 0.25 mm i.d. × 0.25 µm film thickness; JW, Folsom, CA). For the GLC procedure, the split/splitless injector, type 1177, and the flame-ionization detector were held at 250°C. The oven temperature was increased from 120 to 195°C at 4°C/min and then held for 60 min at constant temperature. The injector was in splitless mode for 1.0 min and then in split mode until the end of the run with a split ratio of 30:1. The column flow rate was 1.2 mL/min of He. Fatty acids were identified by comparison with retention time of reference standards (Sigma, St. Louis, MO; Interchim, Montlucon, France) analyzed under similar conditions and integrated using the Star Chromatography Workstation System (version 4.5, Varian Associates Inc., Walnut Creek, CA). The quantification of FA methyl esters was based on the internal standard technique.

Calculations and Statistical Analysis

The FA methyl esters were summed by families according to their structure: even straight-chain SFA (ESFA) = C10:0 + C12:0 + C14:0 + C16:0 + C18:0 + C20:0 + C22:0 + C24:0; odd-numbered straight-chain FA (oddFA) = C13:0 + C15:0 + C17:0 + C17:1n-8 + C19:0 + C21:0; methyl-branched chain FA of the iso and anteiso forms (iso and anteiso FA) = isoC14:0 + isoC15:0 + anteisoC15:0 + isoC16:0 + isoC17:0 + anteisoC17:0; straight-chain MUFA (MUFA) = C14:1 + ∑C16:1 (n-9 and n-7) +∑C18:1 (n-7, n-9, trans-10) + C17:1n-8 + C20:1 + C22:1 + C24:1; n-3 FA = C18:3n-3 + C20:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3; n-6 FA = C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:4n-6; PUFA = n-3 FA + n-6 FA.

Regressions of the different variables against the actual intake of concentrate were computed using the REG procedure (SAS Inst. Inc., Cary, NC) to test linear and quadratic effects. Significance was defined at P ≤ 0.05.

RESULTS

Growth Performance and Carcass Composition

The growth performance of the 32 kids used for FA composition analyses (Table 2) was not significantly different to the performance of the entire group of 40 kids (Liméa et al., 2009a). Total FA, C16:0, C18:0, and C18:2n-6 intakes (Table 2) increased with increased concentrate supplementation (linear, P < 0.001). The C18:1n-9 intake increased with increased concentrate supplementation (linear, P < 0.001; quadratic, P < 0.001), whereas C18:3n-3 intake was not affected by dietary group. It took animals on the G0 treatment 4 mo longer to reach slaughter weight compared with G300 (400 vs. 275 d, respectively). The ADG increased linearly with amount of concentrate in the diet. Compared with the FF kids, cold carcass weights of CF kids were greater when concentrate increased, with no difference between G100 and G200 groups. The weight of adipose tissue deposits (OF, PR, and IM) increased linearly with amount of concentrate in the diet; the quadratic term was significant only for PR weight. The SS weight was the least for the G0 group and the greatest for the G300 group.

Changes in Adipose Tissue and Muscle FA Proportions with Diet

With increased concentrate, the DM content increased (linear and quadratic, P < 0.05) in PR (Table Table 2. Fatty acid (FA) intakes, performance, and body adiposity of Creole goats fed diets supplemented with 0 (G0), 140 (G100), 240 (G200), or 340 (G300) g of concentrate (n = 8 per group) 3) and decreased in SS (linear, P < 0.05). The change in DM content of IM was curvilinear (quadratic, P < 0 0.05, Table 4). The lipid content of PR, IM, and SS was not significantly affected by the different levels of concentrate supplementation. Individual C14:0, C16:0, and C18:0 proportions were not affected by the different amounts of concentrate supplementation in PR, IM, and SS (Tables 3,4, and 5); neither were total ESFA proportions in IM and SS. However, ESFA proportion in PR was greater in the G100 group (linear, P < 0.05; quadratic, P < 0.01) than in the other groups. Total oddFA, C15:0, C17:0, and C19:0, as well as iso-and anteiso-FA proportions in PR, IM, and SS decreased (linear, P < 0.001; quadratic P < 0.001) with the greatest proportions being for the G0 group and the least for the G200 and G300 groups.

Total MUFA proportions were not affected by the different concentrate supplementation in PR, IM, and SS. Among MUFA, C16:1n-9 proportion was not affected by diets in PR and SS whereas it decreased linearly (P < 0.05) in IM with increased concentrate supplementation. The proportion of C16:1n-7 decreased (P < 0.02) linearly with increased concentrate supplementation in SS, whereas it was not modified in PR and IM. The proportion of C17:1n-8 decreased (linear, P < 0.0001; quadratic P < 0.01) with increased concentrate supplementation in PR and SS, whereas it was not affected by diet in IM. The proportion of C18:1n-9 increased linearly (P < 0.05) with increased concentrate supplementation in PR and IM, whereas it was not affected in SS. The proportion of C18:1n-7 decreased in PR, IM, and SS with increased concentrate supplementation. This effect was quadratic in PR (P < 0.05) and SS (P Table 3. Fatty acid (FA) composition of the perirenal adipose tissue of Creole goats fed diets supplemented with 0 (G0), 140 (G100), 240 (G200), or 340 (G300) g of concentrate (n = 8 per group) Quad = quadratic; ESFA = even straight-chain SFA; odd FA = C13:0 + C15:0 + C17:0 + C17:1n-8 + C19:0 + C21:0; iso and anteiso FA = isoC14:0 + isoC15:0 + anteisoC15:0 + isoC16:0 + isoC17:0 + anteisoC17:0; n-6 FA = C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:4n-6; n-3 FA = C18:3n-3 + C20:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3; ND = not detected; ND1 = not detected except 1 kid in group G0 value = 0.14 g/100 g of FA; ND2 = not detected except 1 kid in group G100 value = 0.30 g/100 g of FA.

< 0.01). The C18:1trans-10 was only detected in 1 SS sample, in 2 PR samples, and not at all in IM samples.

The proportions of C18:2n-6 and total n-6 FA increased in PR and IM (linear, P < 0.001) with increased concentrate supplementation, with the least proportion being for the G0 group and the greatest for the G300 group. In SS, C18:2n-6 and total n-6 FA proportions increased as concentrate supplementation increased from 0 to 140 g (G100) and did not change between G100 and G300 groups (linear, P < 0.001; quadratic, P < 0.05). The proportions of C18:3n-6 and C22:4n-6 decreased as concentrate supplementation increased from 0 to 240 g (G200), and remained the same between G200 and G300 groups in all tissues (linear, P < 0.001; quadratic, P < 0.05) except for C22:4n-6 in SS, which increased from 0 to 240 g (G200). The proportion of C20:4n-6 increased between G0 and G100 groups in IM and SS but not in PR. The proportions of C20:3n-6 increased in SS (linear, P < 0.001; quadratic, P < 0.05) but decreased (linear, P < 0.001; quadratic, P < 0.001) in IM and PR, as concentrate supplementation increased from 0 to 140 g (G100) and remained the same between G100 and G300 groups. The C20:2n-6 was not affected by diets in the 3 tissues studied. The proportion of C18:2cis-9, trans-11 decreased in PR and SS as concentrate supplementation increased from 0 to 140 g (G100; linear, P < 0.001; quadratic P < 0.05) but remained unchanged in IM.

Total n-3 FA, C18:3n-3, and C20:5n-3 proportions in PR, IM, and SS decreased with increased concentrate supplementation with the greatest proportions being for the G0 group and the least for the G200 and G300 groups (linear, P < 0.001; quadratic, P < 0.01). The proportion of C22:5n-3 decreased linearly with increased concentrate supplementation, whereas the C22:6n-3 proportion was not affected in PR, IM, and

DISCUSSION

Growth Performance and Carcass Composition

The aim of this study was to quantify the growth performance, body composition, and the changes in FA profiles of different tissues from goat carcasses fed increasing amounts of concentrate and slaughtered at a fixed commercial BW. Consequently, and by design, the goats fed increased concentrate had a younger slaughter age because they had a greater growth rate due to the greater daily energy intake. The daily NE g intake increased from 120 MJ•d -1 (group G0) to 250 MJ•d -1 (group G300), and the ADG ranged from 42 to 85 g•d -1 . Feeding greater amounts of concentrate increased the carcass weight and the dressing percentage in our study (see companion paper, Liméa et al., 2009a). This was in agreement with studies in local goats in which the animals were slaughtered at a fixed age [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF] or at a fixed BW [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF]. This could be related to a greater muscle development associated with greater energy and protein intakes, as indicated by the greater weight of SS in the carcass of goats fed increased concentrate in this study, and as observed in our previous study on Creole goats (Liméa et al., 2009b). In association with the greater growth rate and energy intake, Quad = quadratic; ESFA = even straight-chain SFA; odd FA = C13:0 + C15:0 + C17:0 + C17:1n-8 + C19:0 + C21:0; iso and anteiso FA = isoC14:0 + isoC15:0 + anteisoC15:0 + isoC16:0 + isoC17:0 + anteisoC17:0; n-6 FA = C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:4n-6; n-3 FA = C18:3n-3 + C20:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3; ND = not detected; ND1 = not detected except 1 kid in group G200 value = 0.17 g/100 g. OF, IM, and PR weights were at least doubled between G0 and G300 goats. A similar increase in body fat with increased concentrate supplementation was observed in goats slaughtered at fixed BW [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF] or at a fixed age [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF], indicating that after partition of energy toward carcass muscle deposition, excess dietary energy was channeled toward lipid deposition, at least in adipose tissues. This was confirmed in the present study (Liméa et al., 2009a). However, lipid proportion in muscle was not affected by level of concentrate because goats fed the all forage-diet had the greatest lipid content in SS, which confirms that lipid deposition in goats is mainly internal [START_REF] Sahlu | Nutrient requirements of goats: Developed equations, other considerations and future research to improve them[END_REF], as observed in the companion paper (Liméa et al., 2009a). This study demonstrated that, in the Creole goat breed, feeding increased concentrates increased growth rate and carcass fat content.

In the present paper, FA intake was reduced compared with the lipid intakes (based on ether extract analysis) presented in the companion paper (Liméa et al., 2009a). This was due to the decreased FA content measured in the forage (Table 1).

Overall FA Composition of Adipose Tissues and Muscle of Creole Kids

In the present study, DM of SS was in agreement with data reported on muscles of goats fed hay [START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF], hay and concentrate [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF], argan pulp [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF], or grazed grass supplemented with concentrate [START_REF] Werdi Pratiwi | Feral goats in Australia: A study on the quality and nutritional value of their meat[END_REF]. However, the Creole goat kids had greater SS lipid content (11 to 13% of DM) compared with most reported data: 5 to 6% of muscle DM reported by Werdi Pratiwi et al. ( 2007) and [START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF], and 7 to 9% reported by [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF] and [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF]. Only [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF] reported a greater lipid content (20% of DM in goats around 20 kg of BW) compared with our study. Such variations are related to differences in the age and breed of goats studied, as well as to feeding level and energy density of diets [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF]. Moreover, muscle differences (mostly LM vs. SS in the present trial) and differences in fat trimming procedures may also be responsible for these differences [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF]. In the present study, the DM and lipid content of PR were in the high range of reported measurements in goats [START_REF] Banskalieva | Fatty acid composition of goat muscles and fat depots: A review[END_REF][START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Werdi Pratiwi | Feral goats in Australia: A study on the quality and nutritional value of their meat[END_REF]. To our knowledge, few data are available on IM, for comparison.

The G0 goats had similar ESFA proportions in IM and PR adipose tissues (between 50 and 60% of total FA) but proportions below 40% in SS. These proportions are in agreement with data in goats fed argan pulp [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF], but slightly less than those in muscle and adipose tissues of goats fed concentrate and hay [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF], browse-based diet [START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF], or grazed grass supplemented with concentrate [START_REF] Werdi Pratiwi | Feral goats in Australia: A study on the quality and nutritional value of their meat[END_REF]. In this study, C18:0 was the predominant FA in PR, whereas, in SS and IM, C16:0 and C18:0 were in similar proportions. In goats fed concentrate and hay, C16:0 was greater than C18:0 in the muscle [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF][START_REF] Werdi Pratiwi | Feral goats in Australia: A study on the quality and nutritional value of their meat[END_REF] but in similar proportions in PR [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF]. However, goats fed argan pulp had similar proportions of C16:0 and C18:0 in both PR and muscle [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF]. In all studied tissues of the Creole goats and especially in SS, the C14:0 proportion was reduced, and less than most of the published values [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF][START_REF] Werdi Pratiwi | The fatty acid composition of muscle and adipose tissues from entire and castrated male Boer goats raised in Australia[END_REF], 2007). Because consumption of C14:0 has been implicated in increasing plasma cholesterol, meat from indigenous Caribbean goats with its reduced C14:0 may have positive health implications.

In our Creole goats, the proportions of n-3 FA were classically greater in the muscle than in the adipose tissues because muscle has a greater phospholipid content than adipose tissues, phospholipids that are characterized by greater 18:3n-3 and n-3 FA proportions than storage lipids [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF]. The most represented FA in all studied tissues was 18:3n-3, but the most biologically significant FA are C20:5n-3, C22:5n-3, and C22:6n-3 when dietary recommendations for human health are considered [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF]. From this point of view, total n-3 FA in muscle and PR were in the range observed in goats fed argan pulp [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF], but less than those observed in muscle of goats fed a browse diet [START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF]. Differences in n-3 FA proportions between these experiments may be related to differences in the C18:3 content of experimental diets, as well as in ruminal physico-chemical conditions that affect ruminal biohydrogenation of PUFA [START_REF] Glasser | Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors: 2. C18 fatty acids[END_REF]. Data on C20:5n-3, C22:5n-3, and C22:6n-3 are scarce for goat muscle and adipose tissues [START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF].

Proportions of n-6 FA in edible tissues are usually greater than those of n-3 FA, as dietary C18:2n-6 is less biohydrogenated than dietary C18:3n-3 [START_REF] Glasser | Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors: 2. C18 fatty acids[END_REF], and as C18:2n-6 and its long-chain products C20:4n-6 and C22:4n-6 are preferentially deposited in phospholipids compared with n-3 FA [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF]. Total n-6 FA, C20:4n-6, and C22:4n-6 proportions in muscle and PR of our Creole goats were less than those reported by [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF], but were in the range of those reported for C18:2n-6 in muscle and PR [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF]. With respect to n-3 FA, these results may be explained by differences in dietary C18:2 intake and conditions of ruminal biohydrogenation.

Trans-FA in goat muscle or in adipose tissues have been scarcely reported in the literature, especially for the profile of trans-C18:1 FA. In our study, only C18:1trans-10 can be quantified, C18:1cis-11 and C18:1trans-11 co-elute on the DB-Wax column used [START_REF] Kramer | Evaluation of two GC columns (60-m SUPELCOWAX 10 and 100-m CP Sil 88) for analysis of milkfat with emphasis on CLA, 18:1, 18:2 and 18:3 isomers, and short-and long-chain FA[END_REF][START_REF] Alves | Comparison of two gas-liquid chromatograph columns for the analysis of fatty acids in ruminant meat[END_REF] and cannot be quantified separately. Other positional isomers of C18:1trans FA in muscle or adipose tissues cannot be obtained because they are not well separated from each other. Under these conditions, C18:1trans-10 was never detected in SS or in IM from G0 goats, and only 1 goat fed this diet exhibited a value above the threshold of sensitivity of our method in the PR tissue. In goats fed the all-forage diet, these results are consistent with the fact that C18:1trans-10 mainly reflects changes in the ruminal biohydrogenation process of FA associated with changes in pH and microbial populations [START_REF] Harfoot | Lipid metabolism in the rumen[END_REF][START_REF] Jenkins | Board-invited review: Recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem[END_REF] induced by feeding high-grain diets, for example in lambs [START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF]. This is confirmed by the very low concentration of the trans-10, cis-12 isomer of the CLA (0.01 to 0.03 in the muscle or in the adipose tissues) that is generally produced when high-grain diets are fed. The proportions of C18:2cis-9, trans-11 were in the greater range of values reported in lambs [START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF].

The proportions of C18:1n-9 tended to be less than in other studies on growing goats [START_REF] Casey | Fatty acid composition of subcutaneous and kidney fat depots of Boer goats and response to varying levels of maize meal[END_REF][START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Dhanda | Part 2. Carcass composition and fatty acid profiles of adipose tissue of male goats: Effects of genotype and liveweight at slaughter[END_REF][START_REF] Werdi Pratiwi | The fatty acid composition of muscle and adipose tissues from entire and castrated male Boer goats raised in Australia[END_REF]. The total oddFA plus iso-and anteiso-FA proportions ranged from 4.4 to 8.4% of the total FA in all tissues with progressively increased proportions in SS, IM, and PR, respectively. These results are in the greater range of what is usually observed but agreed with results of [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF] in young Morocco goats in semi-intensive conditions.

Effect of Increased Concentrate Supplementation on FA Composition of Adipose Tissues and Muscle in Creole Goats

In this experiment, increased concentrate supplementation significantly increased the DM content of PR and tended to increase the DM content of IM, with only small changes in their lipid content. This could be due to a faster growth of adipocyte size for these younger goats at greater energy intakes. Conversely, increased concentrate supplementation slightly decreased the DM and lipid contents of SS. This is in contrast to several studies that have reported greater DM and muscle fat contents in goats [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF] or in lambs [START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF] fed increased concentrate. Nevertheless, some studies also reported no changes in DM or in lipid contents of muscle of goats fed different levels of concentrate [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF]. The discrepancy between this study and others could be explained by the type of muscle studied (LM in most experiments vs. SS in our study) but also by the younger age of the goats fed concentrate. For instance, [START_REF] Mahgoub | Fatty acid composition of muscle and fat tissues of Omani Jebel Akhdar goats of different sexes and weights[END_REF][START_REF] Werdi Pratiwi | Feral goats in Australia: A study on the quality and nutritional value of their meat[END_REF] reported that DM or lipid contents in adipose tissue or in muscle increased with increased age of growing goats (increased BW).

Increased concentrate supplementation did not increase the proportions of C14:0 and C16:0 in PR, IM, and SS, which is desirable for human nutrition (Micha and Mozaffarian, 2010). Moreover, it did not affect C18:0 and ESFA proportions in IM and SS, although in PR there was a small (maximal 2%) increase in the G100 group compared with the average proportion of ESFA (60%) observed in the other experimental groups. Increased concentrate supplementation has given rise to inconsistent results in the literature. Proportions of ESFA, C14:0, C16:0, and C18:0 were not affected by concentrate levels in IM lipids of goats [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF], whereas these FA were greatly increased in the muscle of goats fed increasing percentages of concentrate [START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF], or decreased in the muscle of grain-fed goats compared with range goats [START_REF] Rhee | Fatty acid composition of goat diets vs intramuscular fat[END_REF]. Moreover, these FA could be differently affected according to the tissue considered, as observed by [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF]. This author reported only small changes in C14:0 and C16:0 but a decrease of C18:0 and ESFA proportions in muscle, but not in PR tissue, of goats fed argan pulp with increased percentage of concentrate. Because part of the C16:0 and most of the C18:0 in adipose tissue and muscle arise from dietary FA, differences among experiments may reflect differences in FA intake, differences in PUFA intake, as well as differences in ruminal biohydrogenation of the PUFA and transfer into the tissues being considered [START_REF] Geay | Effect of nutritional factors on biochemical, structural and metabolic characteristics of muscles in ruminants, consequences on dietetic value and sensorial qualities of meat[END_REF]. Moreover, differences may also be due to the preweaning diet (milk FA composition), especially for C14:0, because FA of body fat stored before weaning influenced the FA composition of fat deposits and muscle several months after weaning [START_REF] Bas | Effect of nutritional factors on fatty acid composition of lamb fat deposits[END_REF]. However, all these data suggest that feeding concentrate has little, if any, effect on SFA proportions in edible parts of goat meat.

The C15:0, C17:0, and C19:0 proportions as well as iso-and anteiso-FA proportions decreased with increased concentrate level in all studied tissues, the PR being the most affected and SS being the least affected. These results are in agreement with a decrease in oddFA and methyl branched-chain FA proportions observed with increased concentrate level in most studies in goats [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF], but not in all studies [START_REF] Atti | The effect of spineless cactus (Opuntia ficus-indica f. inermis) supplementation on growth, carcass, meat quality and fatty acid composition of male goat kids[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF]. This probably reflects the decrease of microbial synthesis of FA in the rumen as these FA mainly originate from ruminal lipid microbial synthesis [START_REF] Vlaeminck | Effect of forage:concentrate ratio on fatty acid composition of rumen bacteria isolated from ruminal and duodenal digesta[END_REF], especially when diets have increased NDF contents [START_REF] Bas | Fatty acid composition of mixed-rumen bacteria: Effect of concentration and type of forage[END_REF]. With regard to health aspects, the decrease in oddFA can be considered as neutral because these FA are not known to have any effect on health.

In the present experiment, the commercial concentrate was rich in C18:2n-6 due to the components used (corn and soybean cake), and as stated above, greater intakes of C18:2n-6 were observed in goats fed increased concentrate supplementation. Despite extensive ruminal hydrogenation of C18:2n-6 in the rumen (on average 90%, [START_REF] Glasser | Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors: 2. C18 fatty acids[END_REF], the differences in the proportions of C18:2n-6 in the studied tissues reflected the changes in its dietary intake. Surprisingly, the C18:3n-3 intake was not different between groups.

This was due to the rather low C18:3 content of the forage (1.1 g•kg -1 of DM) and to the only slight decrease of forage intake when concentrate was fed (maximum -50 g•d -1 , which means -55 mg of C18:3•d -1 ). This slight decrease was compensated by the C18:3 from concentrate intake. Generally, goats fed grass pasture or forage had greater C18:3n-3 proportions in fat deposits and muscle than animals fed concentrate baseddiets when these concentrates are poor in C18:3n-3 [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF]. This was not the case when concentrates were rich in C18:3 [START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF], which reflected a positive relationship between intake of C18:3n-3 and its proportion in meat [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF]. In our study, the goats fed the forage diet had slightly greater C18:3n-3 proportions in all studied tissues, despite a similar intake when compared with the concentrate fed-groups. This may have been due to a reduction in C18:3n-3 ruminal biohydrogenation (95% on average, [START_REF] Glasser | Digestion of fatty acids in ruminants: A meta-analysis of flows and variation factors: 2. C18 fatty acids[END_REF] in the rumen of goats fed D. decumbens by a mechanism that remains to be elucidated or a dilution effect caused by the absorbed C18:3n-3 being distributed in a greater amount of lipid in the kids fed concentrates (this assumes that duodenal flows of C18:3n-3 remained the same between groups).

The decrease in 18:3n-3 proportions with increased level of concentrate supplementation was associated with decreased proportions of the different n-3 FA (C20:3n-3, C20:5n-3, and C22:5n-3, but not for the C22:6n-3) in all studied tissues. The increase in C18:2n-6 proportions with increased concentrate supplementation were also associated with an increase in C20:4n-6 in IM and SS but not in PR. All these results are in line with previous results on long-chain PUFA in tissues of goats or lambs fed diets with increased C18:2/C18:3 intake [START_REF] Aurousseau | Effect of grass or concentrate feeding systems and rate of growth on triglyceride and phospholipid and their fatty acids in the m. Longissimus thoracis of lambs[END_REF][START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF][START_REF] Lee | Chemical composition and quality characteristics of chevon from goats fed three different post-weaning diets[END_REF]. Therefore the n-6/n-3 ratio significantly increased in PR, IM, and SS from G0 to G300. With regard to the recommendation of a n-6/n-3 ratio below 4 [START_REF] Wood | Fat deposition, fatty acid composition and meat quality: A review[END_REF] to improve the nutritional value of meat, the most favorable meat based on the ratio in IM and SS was that produced from Creole goats fed 0 to 140 g of commercial concentrate.

The proportions of C18:2cis-9, trans-11 were greater in forage-fed goats than concentrate-fed goats in PR and SS. This agrees with results obtained on muscles of lambs fed grass or concentrate [START_REF] Nuernberg | Effect of grass vs. concentrate feeding on the fatty acid profile of different fat depots in lambs[END_REF][START_REF] Aurousseau | Indoor fattening of lambs raised on pasture: 2. Influence of stall finishing duration on triglyceride and phospholipid fatty acids in the Longissimus thoracis muscle[END_REF]. Similar results were reported in steers fed increased concentrate on grassbased diets [START_REF] French | Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grazed grass, grass silage or concentrate-based diets[END_REF]; they were related to a decrease in the C18:1trans-11-producing bacteria at a similar C18:2n-6 intake [START_REF] French | Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grazed grass, grass silage or concentrate-based diets[END_REF]. Factors that determine the amount of cis-9, trans-11 C18:2 in the tissues are the dietary intake of C18:2, the rumen conditions that may affect the growth and activity of C18:1trans-11-producing bacteria (Butyrivibrio fibrisolvens; [START_REF] Klieve | Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets[END_REF], and the Δ 9 -desaturase enzyme that desaturates C18:1trans-11 to C18:2cis-9, trans-11 C18:2 in the muscular and adipose tissues. Moreover, a C18:2n-6 increase in diet has also been shown to increase the C18:2cis-9, trans-11 proportion in tissues [START_REF] Beaulieu | Concentrations of conjugated linoleic acid (cis-9, trans-11-octadecadienoic acid) are not increased in tissue lipids of cattle fed a high-concentrate diet supplemented with soybean oil[END_REF][START_REF] Bas | Effect of level of linseed on fatty acid composition of muscles and adipose tissues of lambs with emphasis on trans fatty acids[END_REF]. Consequently, an increase in C18:2cis-9, trans-11 proportion in tissues of goats could also have been expected in our study. This was not the case, possibly because of the changes in fermentation patterns associated with high-concentrate diets or because of the dilution of this FA in the greater lipid content of PR and IM on these diets. It has been shown that high-concentrate diets promote the growth of the bacterial strain Megasphera elsdenii with cis-9, trans-10 isomerase activity resulting in C18:1trans-10 production, at the expense of B. fibrisolvens, which has a cis-9, trans-11 isomerase activity [START_REF] Klieve | Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets[END_REF] resulting in C18:1trans-11 production. However, we failed to detect the C18:1trans-10 in most tissues except in very few goats in our study, which is also in agreement with the absence of variation of C18:2trans-10, cis-12 in all tissues with concentrate supplementation. More probably, limited C18:1trans-11 availability from ruminal fermentation to muscle and adipose tissues may have resulted in a reduced proportion of C18:2cis-9, trans-11 in these tissues in our study. Indeed, we observed a decreased in C18:1n-7 proportions in all tissues. However, we were unable to distinguish between cis-11 and trans-11 C18:1. Because the C18:1n-7 corresponded to the sum of the C18:1cis-11 and C18:1trans-11, it is difficult to conclude which FA is involved in that variation. Even though [START_REF] Faucitano | Comparison of alternative beef production systems based on forage finishing or grain-forage diets with or without growth promotants: 2. Meat quality, fatty acid composition, and overall palatability[END_REF] showed that increased dietary concentrate increased the proportion of C18:1cis-11 in the muscle of steers, the greater variation of n-7 observed between groups might be due to the trans-11 isomer as in PR and muscle C18:1n-7 and C18:2cis-9, trans-11 were positively correlated (r = 0.545 in PR and 0.732 in SS; P < 0.001). Consequently, limited availability of C18:1trans-11 may account for the reduced C18:2cis-9, trans-11 proportions in all studied tissues.

The C18:1n-9 proportion in adipose tissues increased with increased concentrate supplementation. This result is in agreement with other studies [START_REF] Rhee | Composition and stability of goat meat patties as affected by breed type and feeding regimen[END_REF][START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF][START_REF] Ryan | Effects of concentrate level on carcass traits of Boer crossbred goats[END_REF][START_REF] Faucitano | Comparison of alternative beef production systems based on forage finishing or grain-forage diets with or without growth promotants: 2. Meat quality, fatty acid composition, and overall palatability[END_REF]. The C18:1n-9 in tissues originates partly from this FA intake not being completely biohydrogenated in the rumen, and partly from de novo conversion of C18:0 by the Δ 9 -desaturase enzyme in tissues. In the present study, a significant increase in C18:1n-9 intake was observed with increased concentrate supplementation. The best estimate of this enzyme activity is the Δ 9 -desaturase activity proxy, which is calculated as ID14 (ID14 = C14:1cis-9 × 100/C14:0 + C14:1cis-9). When calculating the Δ 9 -desaturase activity proxy, significant increases were observed with increasing intake of concentrate in PR (G0: 0.52; G100: 0.51; G200: 0.63; G300: 0.79) and in IM (G0: 1.72; G100: 1.68; G200: 2.50; G300: 2.44). This therefore suggests that desaturase enzyme activity could have been induced but not completely proportional to levels of concentrate, prob-ably in response to an increased desaturase gene expression induced by insulin [START_REF] Daniel | Differing effects of forage and concentrate diets on the oleic acid and conjugated linoleic acid content of sheep tissues: The role of stearoyl-CoA desaturase[END_REF] and downregulated by PUFA [START_REF] Bas | Effect of feeding on fatty acid composition of muscles and adipose tissues in young goats raised in the argan tree forest of Morocco[END_REF]. However, contrary to results from adipose tissues, we failed to show any differences in the C18:1n-9 proportion in SS with increased concentrate supplementation. However, a significant increase of the ID14 proxy with levels of concentrate was measured (G0: 29.5; G100: 27.5; G200: 40.9; G300: 40.6) in muscle.

In this study, the growth performance and FA composition of adipose tissues and muscle from male indigenous Caribbean goats were altered by feeding increased concentrate. This was due to the differences in energy and FA intake, and possibly also to the age of the animals slaughtered at a constant BW. From a nutritional point of view, meat from Creole goats fed the all-forage diet was similar to that of other goat breeds fed similar diets. Increased concentrate supplementation did not affect the proportion of MUFA, increased PUFA in muscle, and had very few effects on ESFA in tissues. One of the major modifications of FA composition was the decrease in n-3 PUFA and increase in n-6 PUFA in all tissues with increased concentrate supplementation. When evaluating the effect of changes in FA composition with regard to human health, concentrate supplementation did not increase any cholesterol-raising SFA in meat, but increased the n-6/n-3 ratio above 4 when more than 200 g of concentrate was added to the diet. Therefore, it is recommended to feed concentrates at levels less than 240 g/d to have FA compositions that are desired for current human nutrition guidelines.

Table 1 .

 1 Ingredients, chemical composition, and fatty acid (FA) content of the different components of the diets

	Item

Table 4 .

 4 Fatty acid (FA) composition of the intermuscular adipose tissue of Creole goats fed diets supplemented with 0 (G0), 140 (G100), 240 (G200), or 340 (G300) g of concentrate (n = 8 per group) 1

			Diet				P-value
	Item	G0	G100	G200	G300	SEM	Linear	Quad
	DM, %	58.2	65.4	67.7	66.7	2.323	0.483	0.036
	Lipid content, % of DM	75.9	70.4	89.1	80.1	3.14	0.864	0.824
	FA composition, g/100 g of FA
	C14:0	2.91	3.22	3.60	3.17	0.120	0.149	0.183
	C15:0	1.10	0.74	0.75	0.56	0.047	<0.0001	0.0005
	C16:0	22.61	22.76	22.28	23.04	0.199	0.464	0.420
	C17:0	2.02	1.61	1.46	1.39	0.048	<0.0001	<0.0001
	C18:0	27.31	27.05	21.63	24.35	0.766	0.347	0.669
	C19:0	0.26	0.18	0.16	0.14	0.010	<0.0001	0.003
	C14:1	0.050	0.056	0.077	0.146	0.015	0.217	0.476
	C16:1n-7	1.27	1.21	1.64	1.47	0.065	0.988	0.565
	C16:1n-9	0.54	0.51	0.44	0.45	0.011	0.035	0.361
	C17:1n-8	0.70	0.56	0.79	0.51	0.048	0.253	0.425
	C18:1n-7	5.74	4.85	4.32	4.74	0.132	0.0010	0.010
	C18:1n-9	24.51	27.05	30.08	32.16	0.759	0.025	0.169
	C18:1trans-10	ND	ND	ND	ND	-	-	-
	C18:2n-6	1.45	2.17	2.35	2.64	0.070	<0.0001	0.0002
	C18:2cis-9, trans-11	1.06	0.82	1.02	0.89	0.026	0.0798	0.152
	C18:2trans-10, cis-12	0.020	0.020	0.022	0.019	0.001	0.128	0.097
	C18:3n-6	0.59	0.26	0.20	0.16	0.031	<0.0001	<0.0001
	C18:3n-3	0.73	0.55	0.42	0.39	0.026	<0.0001	0.012
	C20:2n-6	0.035	0.038	0.038	0.039	0.001	0.392	0.456
	C20:3n-3	0.023	0.015	0.015	0.016	0.001	0.0021	0.086
	C20:3n-6	0.051	0.026	0.016	0.016	0.003	<0.0001	0.0005
	C20:4n-6	0.065	0.086	0.096	0.086	0.003	0.0008	0.003
	C20:5n-3	0.035	0.020	0.016	0.013	0.002	<0.0001	0.015
	C22:4n-6	0.092	0.065	0.047	0.046	0.004	<0.0001	0.0096
	C22:5n-3	0.17	0.15	0.10	0.09	0.008	0.057	0.883
	C22:6n-3	0.039	0.034	0.034	0.032	0.002	0.712	0.944
	Odd FA	4.11	3.12	3.09	2.62	0.125	<0.0001	<0.0001
	iso and anteiso FA	3.51	2.65	2.31	2.43	0.095	<0.0001	<0.0001
	ESFA	53.44	53.51	48.00	50.97	0.687	0.317	0.631
	MUFA	33.04	34.41	39.62	37.46	0.722	0.0919	0.367
	PUFA	4.38	4.27	4.46	4.50	0.059	0.944	0.838
	n-3 FA	0.99	0.76	0.58	0.54	0.035	<0.0001	0.021
	n-6 FA	2.30	2.68	2.82	3.05	0.067	0.0007	0.082
	n-6/n-3	2.32	3.51	4.76	5.77	0.250	0.0001	0.889

Table 5 .

 5 Fatty acid (FA) composition of the supraspinatus muscle of Creole goats fed diets supplemented with 0 (G0), 140 (G100), 240 (G200), or 340 (G300) g of concentrate (n = 8 per group) 1

			Diet				P-value
	Item	G0	G100	G200	G300	SEM	Linear	Quad
	DM, %	25.1	23.4	23.5	23.6	0.157	<0.0001	0.234
	Lipid content, % of DM	12.6	11.4	10.7	10.7	0.333	0.112	0.348
	FA composition, g/100 g of FA
	C14:0	0.22	0.35	0.23	0.27	0.019	0.142	0.124
	C15:0	0.60	0.40	0.35	0.31	0.024	<0.0001	0.004
	C16:0	19.63	18.72	18.87	18.62	0.266	0.265	0.336
	C17:0	1.34	1.05	1.00	0.90	0.034	<0.0001	0.007
	C18:0	17.01	17.15	16.23	15.98	0.281	0.937	0.651
	C19:0	0.22	0.15	0.12	0.11	0.008	<0.0001	<0.0001
	C20:0	0.14	0.12	0.08	0.09	0.006	0.025	0.2851
	C14:1	0.09	0.16	0.16	0.19	0.016	0.124	0.307
	C16:1n-7	1.61	1.34	1.45	1.58	0.038	0.013	0.010
	C16:1n-9	0.53	0.51	0.48	0.48	0.007	0.106	0.471
	C17:1n-8	0.92	0.69	0.72	0.65	0.023	<0.0001	0.008
	C18:1n-7	4.04	3.41	3.21	3.21	0.061	<0.0001	0.008
	C18:1n-9	33.08	33.28	35.54	35.47	0.465	0.335	0.838
	C18:1trans-10	ND	ND	ND1	ND	-	-	-
	C18:2n-6	4.19	7.00	6.89	7.71	0.309	0.0004	0.013
	C18:2cis-9, trans-11	1.22	0.85	0.89	0.87	0.033	<0.0001	0.0002
	C18:2trans-10, cis-12	0.031	0.025	0.029	0.031	0.001	0.012	0.006
	C18:3n-6	0.45	0.22	0.17	0.17	0.022	<0.0001	<0.0001
	C18:3n-3	1.50	1.08	0.72	0.67	0.063	<0.0001	<0.0001
	C20:2n-6	0.055	0.063	0.060	0.067	0.002	0.298	0.578
	C20:3n-3	0.040	0.069	0.029	0.026	0.008	0.503	0.299
	C20:3n-6	0.022	0.227	0.190	0.215	0.015	<0.0001	<0.0001
	C20:4n-6	1.04	1.72	1.56	1.77	0.069	0.001	0.021
	C20:5n-3	0.46	0.31	0.19	0.17	0.022	<0.0001	0.002
	C22:4n-6	0.07	0.13	0.14	0.17	0.007	0.0004	0.069
	C22:5n-3	0.74	0.67	0.45	0.45	0.028	0.0051	0.301
	C22:6n-3	0.18	0.16	0.15	0.15	0.006	0.094	0.302
	ESFA	37.38	36.70	35.92	35.41	0.407	0.464	0.788
	MUFA	40.42	39.55	41.00	41.64	0.433	0.999	0.652
	PUFA	10.01	12.56	11.52	12.23	0.329	0.049	0.141
	iso and anteiso FA	2.86	2.59	2.40	2.42	0.050	0.003	0.046
	Odd FA	3.12	2.34	2.21	2.00	0.081	<0.0001	<0.0001
	n-3 FA	2.91	2.29	1.53	1.46	0.117	<0.0001	0.008
	n-6 FA	5.86	9.39	9.06	10.16	0.381	0.0006	0.016
	n-6/n-3	2.01	4.12	5.93	6.93	0.349	<0.0001	0.007
	1

at INRA Institut National de la Recherche Agronomique on August 13, 2013 www.journalofanimalscience.org Downloaded from

Nutrient composition based on laboratory analyses.

Quad = quadratic; LWS = BW at slaughter.

Fatty acid composition of Creole goat meat at INRA Institut National de la Recherche Agronomique on August 13, 2013 www.journalofanimalscience.org Downloaded from

Quad = quadratic; ESFA = even straight-chain SFA; odd FA = C13:0 + C15:0 + C17:0 + C17:1n-8 + C19:0 + C21:0; iso and anteiso FA = isoC14:0 + isoC15:0 + anteisoC15:0 + isoC16:0 + isoC17:0 + anteisoC17:0; n-6 FA = C18:2n-6 + C18:3n-6 + C20:2n-6 + C20:3n-6 + C20:4n-6 + C22:4n-6; n-3 FA = C18:3n-3 + C20:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3; ND = not detected.

This study was supported by the Region Guadeloupe and the European Community (Fonds Européen d'Orientation et de Garantie Agricole).

2 The first author is the PhD student who was in charge of all field and laboratory measures, the constitution of the experimental database, and the literature review.


Etienne E Couturier

Nicole N Brunel

Stéphane S Douady

Naomi N Nakayama
email: naomi.nakayama@ips.unibe.ch

Abaxial growth and steric constraints guide leaf folding and shape Acer Pseudoplatanus

Keywords: abaxial growth, Acer pseudoplatanus, Aceraceae, biomechanics, leaf folding, leaf shape determination, maple leaf development, palmate leaf, space-dependent growth, steric constraints

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Leaf morphology is a classic botanical interest. The leaf has become a model system to dissect lateral organ development in plants, and leaf morphogenesis has become a model system to study regulation of plant form (e.g., [START_REF] Sinha | Leaf development in angiosperms[END_REF][START_REF] Fleming | The control of leaf development[END_REF][START_REF] Braybrook | How a plant builds leaves[END_REF][START_REF] Efroni | Morphogenesis of simple and compound leaves: A critical review[END_REF][START_REF] Hasson | Leaving the meristem behind: The genetic and molecular control of leaf patterning and morphogenesis[END_REF][START_REF] Moon | How a leaf gets its shape[END_REF]. While genetic and physiological studies continue to identify molecular regulators of leaf shapes, contributions of nonmolecular factors, such as the physical constraints acting upon the developing primordia, have recently started to be addressed [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF][START_REF] Prusinkiewicz | Constraints of space in plant development[END_REF]. In particular, transient folding of leaf primordia inside the bud can explain the palmate leaf shapes of a diverse array of dicotyledon species. This observation led to a theoretical characterization of the lobed leaf shape formation and formulation of the kirigami theory [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF].

The theory proposes that this type of leaf morphogenesis works like kirigami, the Japanese craft in which a piece of paper is fi rst folded and then cut. At the shoot apex under the many layers of older primordia, a young primordium grows via cell division and expansion. As the lamina grows, it folds inward along the veins and again outward between two veins, forming folds and antifolds . This process describes the paper folding part of kirigami. The primordium grows until it fi lls the available space, halting growth when it meets the other primordia within the bud. Such space-fi lling growth of leaf primordia has been found in all dicotyledon species examined to date and thus seems to be a widely relevant phenomenon [START_REF] Couturier | The fi lling law: A general framework for leaf shape diversity and its consequences on folded leaves[END_REF]. Depending on the species, the geometry of the space available for a primordium delimits the lamina growth on a nearly fl at plane: this comprises the cutting part of kirigami. For example, in some plants with decussate phyllotaxy, two opposite leaves grow to meet each other in the middle, mutually inhibiting further growth of the facing primordium ( Fig. 1A ).

Kirigami is a method to create a repetitive and symmetrical outline on a piece of paper. Similarly, the kirigami regulation acts on the outlines of the leaf lamina. A sinus is the indentation between two lobes ( Fig. 1B ). Each fold and antifold of a young primordium corresponds to the lobe and sinus of the mature leaf, respectively ( Fig. 1B,C ). The number and position of the lobes coincide with vein pattern, and the sizes of the lobes are determined by the timing of vein formation. We should note

• Premise of the study: How leaf shape is regulated is a long-standing question in botany. For diverse groups of dicotyledon species, lamina folding along the veins and geometry of the space available for the primordia can explain the palmate leaf morphology. Dubbed the kirigami theory, this hypothesis of fold-dependent leaf shape regulation has remained largely theoretical. Using Acer pseudoplatanus , we investigated the mechanisms behind the two key processes of kirigami leaf development.

• Methods: Cytological examination and quantitative analyses were used to examine the course of the vein-dependent lamina folding. Surgical ablation and tissue culturing were employed to test the effects of physical constraints on primordia growth. The fi nal morphology of leaves growing without steric constraints were predicted mathematically. • Key results: The cytological examination showed that the lamina's abaxial side along the veins grows substantially more than the adaxial side. The abaxial hypergrowth along the veins and the lamina extension correlated with the lamina folding. When a primordium was released from the physical constraints imposed by the other primordia, it rapidly grew into the newly available space, while maintaining the curvature inward. The morphology of such a leaf was predicted to lack symmetry in the lobe shapes. • Conclusions: The enhanced growth on the abaxial side of the lamina along the veins is likely to drive lamina folding. The surgical ablation provided clear support for the space-fi lling nature of leaf growth; thus, steric constraints play a role in determination of the shapes of folded leaves and probably also of the fi nal leaf morphology. [Vol. 99 dicotyledon leaves or contact-dependent growth inhibition are as yet unknown. To gain a greater biological basis for the theory, we have conducted cytological and surgical examinations of kirigami leaf development, using a maple, Acer pseudoplatanus , as an example. The results suggest that a common mechanismasymmetric growth across the abaxial-adaxial axis along the veins-drives the lamina folding and guides the physical constraints acting in the space-dependent growth, emerging as the key process in the kirigami regulation of lobed leaf shape.

MATERIALS AND METHODS

Plastic sections -Apical buds of Acer peudoplatanus were collected from the campus of les Cezeaux of the University Blaise Pascal in Clermont-Ferrand, France. After dissection to 2-3 mm in length, they were fi xed in ice-cold FAA that we call all tips on the leaf margin lobes , regardless of the more precise terminologies applicable ( Fig. 1B ). The lobes in a more traditional sense, which we call large lobes, form along the primary veins. The smaller lobes, which are often referred to as serrations or crenations, develop along the secondary and tertiary veins. This generalization stems from the observation that all tips and indentations are indistinguishable in how they form according to the kirigami model. It also is supported by the recent fi ndings that the different degrees of lamina indentations, from leafl et formation to serration, result from a common molecular mechanism [START_REF] Nikovics | The balance between the miR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]Barkoulas et al., 2008 ;[START_REF] Blein | A conserved molecular framework for compound leaf development[END_REF][START_REF] Koenig | Auxin patterns Solanum lycopersicum leaf morphogenesis[END_REF][START_REF] Kawamura | Mechanisms of leaf tooth formation in Arabidopsis[END_REF].

The kirigami theory is contingent on two processes of lamina development: folding along the veins and space fi lling growth. However, the mechanisms underlying transient folding of in long-day condition (16 h day/8 h night). Many did not grow well in culture and died within 5 d, while others survived longer than 20 d. Those lasting samples were analyzed ( N = 25). Growth was recorded daily using Nikon stereoscope SMZ 1500 and Nikon Digital Sight DS-Fi1 camera with the software NIS-Elements (Nikon Instruments Europe, Amsterdam, Netherlands).

Image analysis -Images were processed and assembled using Adobe Illustrator (San Jose, California, USA) and Microsoft Powerpoint (Redmond, Washington, USA). Noises, such as original scale bars and uneven background signals, were fi xed, and the image was processed for similar orientations and the best brightness and contrast.

Data were quantitatively analyzed with the programs Image J (http://rsbweb. nih.gov/ij/) and Matlab 7.7 (MathWorks, Natick, Massachusetts, USA). For Figs. 2 and3 , we used a complete set of transverse plastic sections of two opposite maple leaves (Appendices S1 and S2, see Supplemental Data with online version of article). We analyzed 3-19 successive sections from the mid-height region of the lamina. For Fig. 2 , the fold opening angle α , the lamina thickness L , and the lamina thickness at the vein V were measured for each vein, and the mean was calculated and graphed. The variations in the measurements are shown for a representative vein, as crossed standard deviation lines. For Fig. 3 , solution (3.7% formaldehyde, 50% ethanol, 5% acetic acid, all v/v), vacuum infi ltrated for 1 h and incubated at 4 ° C for 3-5 h. Tissues were then dehydrated through ethanol series and then gradually embedded into LR White Resin (Sigma Aldrich, St. Louis, Missouri, USA) as reported earlier ( Azri et al., 2009 ). Infi ltration was completed in 100% LR-White resin overnight at 4 ° C. The samples were then placed in gelatin embedding capsules, fi lled with fresh resin, and heat polymerized at 55 ° C for at least 15 h before semithin (3-4 µm) sectioning with a OMU2 microtome (Reichert Technologies, Depew, New York, USA). Semithin sections were stained with Astra blue and safranin. Observations were performed with a light microscope Zeiss Axioplan 2 (Zeiss, Jena, Germany), and data were recorded using a digital camera AxioCam HR with AxioVision digital imaging software (Zeiss).

Surgical ablation experiments -Actively growing axial branches were collected from the Aare river bank in Bern, Switzerland. They were fi rst dissected down to the shoot apex (the meristem and 3-4 pairs of young leaves smaller than 2 mm), and some were further dissected for ablation. After dissection, plants were placed onto the growth medium containing 1/2 × Murashige-Skoog medium, 1% (w/v) sucrose, and 0.5-0.6% (w/v) agarose. To prevent infection, Plant Preservative Mixture (Plant Cell Technology, Washington, D.C., USA) was also added to 0.1% (v/v). Plants were cultured at 21 ° C and 80% humidity, The ratio L / V of the folds normal to the plane of the section was measured from one leaf (•) and its pair leaf ( ). Standard deviations are shown as crossed bars. We collected measurements from two opposite leaves within a bud, for which we could obtain every section along the entire leaf. The data collapse on the same curve. (C) Dashed boxes highlight epidermal cells on the adaxial and abaxial sides of the folds and antifolds, respectively. Epidermal cells appear compressed on the adaxial side of the fold and the abaxial side of the antifold, while they appear extensively expanded on the opposite side. Vasculature is marked with an orange line (A, C). remaining leaf was measured, whereas for the control samples both intact leaves were measured. The leaves were monitored every 2 to 3 d over the 18 d after the surgery.

RESULTS

Lamina folding along the vein -At the shoot apex of A. pseudoplatanus , two primordia emerge in decussate phyllotaxy

the distance A between the vein and the antifold (the most curved place) and the lamina thickness at the vein V were measured for each antifold, as well as the antifold angle β and the sum of the lamina thickness of the two adjacent veins V 1 + V 2 . To determine the antifold angle β , we chose the approximate mid-point of the curved region farthest from the two adjacent veins as the central vertex and where the adaxial side of the lamina meets the two adjacent veins as the lateral vertices. For Fig. 5 , the central vein contour was plotted with the Segmented line tool of ImageJ at different time points of growth, and the total turning angle of the vein (the sum of the successive angles between the segments) and the length of the leaf were calculated. For the ablated samples, the mordia margin in the middle of two future lobes ( Fig. 1D,E ). First, the three folds appear along the primary veins, and later more folds form along the secondary veins. Furthermore, folding spreads in the proximal to distal direction. Therefore, a primordium contains folds and antifolds at varying stages of fold formation. and grow at the same rate opposite to one another ( Fig. 1A ; [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF]. The lamina of mature leaf typically consists of three large lobes and numerous smaller lobes within a large lobe ( Fig. 1B ). The apex of each lobe corresponds to the tip of a vein. Lamina folding occurs along the veins ( Fig. 1C ), initiating around the time when small nicks are visible at the pri- adjacent veins V 1 + V 2 ( Fig. 3B,D ). There was a negative correlation between the two parameters ( r = -0.6862, P = 0.0096, N = 21), indicating that the larger the abaxial side of the veins grows, the more folded the lamina becomes, leading to the antifolds.

Space-dependent growth of the primordia -

We next examined the effects of physical constraints on kirigami leaf morphogenesis. The space available for a primordium's growth is determined by other primordia in the vicinity, which we removed by surgical ablation. Maple leaves emerge in decussate phyllotaxy inside the older leaves or bud scales ( Fig. 4A ). Young primordia are curved slightly inward, so that the lamina of the two opposite leaves meet each other in the middle. The curvature indicates that the abaxial side grows more than the adaxial side. Longitudinal sections show that the cells on the abaxial side begin to expand early in development ( Fig. 4B ). We dissected actively growing buds down to the youngest 3-4 pairs of primordia, cultured them on a solid medium, and observed the growth of the outermost primordia. We fi rst verifi ed that they could survive and grow in the cultured condition. Although fungal and bacterial infection consumed the plants in long-term incubations, we could observe leaf growth over several days to a few weeks.

One of the two pairing leaves was removed by surgical ablation, and the development of the remaining leaf was monitored. Of 25 samples total, two clearly unfolded within a day, indicating that physical constraint keeps folds in their place and is important for proper folding. The remaining 23 showed a common behavior in which the intact leaf grew extensively, especially on the abaxial side of the primordia, and curved into the space where the ablation took place ( Fig. 4D,5A,5B ). The increase in the total turning angle measured along the abaxial side of the central lobe in side view, is signifi cantly correlated with the ratio between the fi nal to initial length of the leaf ( r = 0.51, P = 0.01, N = 23). There was no signifi cant change in the control without the ablation ( r = -0.28, P = 0.27, N = 17) ( Fig. 4C,5A,5B ). There was no correlation between the mean turning angle variation (mean taken along the vein) and the increase in leaf To gain more insights into what drives lamina folding, transverse sections of two primordia in the process of folding were examined. The areas of the lamina near the veins were observed at different stages of fold formation ( Fig. 2A ). The lamina on the abaxial side of the veins grows much more than the adaxial side. This hypergrowth seems to occur through more cell division and expansion. The differential growth may exert a compressive force on the adaxial side, since the epidermal cells on the adaxial side of the folds (the upper panel) and the abaxial side of the antifolds (the lower panel) are narrower toward the surface ( Fig. 2C ). Enhanced growth on the outer side of the leaf could impose folding on the lamina.

To quantify the correlation between the abaxial growth and the lamina folding, we measured the lamina thickness away from the veins L , the lamina thickness at the veins V , and the fold angle α ( Fig. 2B ). The fold angle was plotted as a function of the ratio L / V . When L / V is close to 1, which is when the abaxial hypergrowth has not happened yet, α is close to 180 ° , or the lamina is fl at. When the ratio is small, α approaches 0 ° (i.e., the fold is closed). Around the L / V ratio of 0.4, the fold does not close further but the abaxial side of the lamina along the veins continues to grow and form the rib. Overall, the bigger the rib becomes, the more folded the lamina ( r = -0.96, P < 10 -4 , N = 39). Fold formation coincides with the corresponding vein's differential growth across the abaxial-adaxial axis. Interestingly, the rib cell differentiation that leads to the abaxial hypergrowth initiates before the onset of folding ( Fig. 2Aa ).

The dynamics of the antifold formation were also quantitatively examined from the same transverse sections. We measured the lamina thickness at vein V , which mostly refl ects the abaxial growth, and the distance A between the vein/fold and the nearest antifold ( Fig. 3A,C ). For each antifold, we plotted A as a function of V. They were strongly linearly correlated ( r = 0.6846, P < e -4 , N = 42). The distance between the vein and the antifold is directly proportional to the lamina thickness at the vein; in other words, the lamina extends proportionally to the rib enlargement. Next the relationship between the antifold formation and the vein thickness was examined. The antifold angle β was plotted against the sum of the lamina thickness at two Fig. 5. Quantitative assessment of ablation induced growth and curving of the leaf. The total turning angle Γ of the abaxial side of the midvein from the intact leaf along the central lobe (radian) as a function of (A) days after surgery T or (B) length of the leaf L (mm). Two biological replicates are indicated in red and green (for ablated samples) or black and blue (for nonablated controls). ○ , ◊, *, × , and + denote value on 1, 3, 6, 9, 18 (red) and 1, 3, 8, 12, 16 (green) days after surgery.

A leaf grown without steric constraints would be different; the sizes of the lobes and sinuses could be more random ( Fig. 6B ). Moreover, morphology could vary leaf to leaf within an individual plant.

To gain more detailed information on the ablation effects, we examined longitudinal sections of the apices that were cultured for 3 d after the surgery ( Fig. 7 ). In the control samples, in which the primordia were left inside the bud, one can draw an almost straight line between the two pairing primordia, between the ordered rows of lamina folds ( Fig. 7A ). When the leaves were cultured without the bud, the lamina folds lost some of the even and uniform organization ( Fig. 7B ). In ablated samples, the remaining leaf of the pair, as well as the younger primordia inside, grew into the newly available space ( Fig. 7C-7E ). The lamina of the remaining leaf lost its ordered pattern of folding and was unfolded in some places, being twisted almost 90 ° and resembling transverse sections. Therefore, the physical constraints from the older primordia and the opposite primordium enforce the proper lamina folding. Interestingly, the excess growth still conformed to the space-fi lling rules [START_REF] Couturier | The fi lling law: A general framework for leaf shape diversity and its consequences on folded leaves[END_REF] and stopped when the lamina came close to the new boundary of physical constraints even at the wounded sites ( Fig. 7D,7E ). length (r = -0.01, p = 0.94, N = 23), indicating that the curvature was constant and that only the growing tip curves during the growth. The petiole of the leaf was already curved at the time of ablation and did not curve farther ( Fig. 5B ). When the leaf survived beyond 3 d, all the lobes curved independently from one another ( Fig. 4D ).

Since technical diffi culties prevented full maturation of the leaves in the cultured condition, we theoretically deduced the fi nal morphology of the maple leaves growing with or without the physical constraints from the opposite leaf ( Fig. 6 ). The number of lobes and sinuses and the angles in between them are fi xed due to vein pattern. Each lobe could grow indefi nitely and independently from the others. It has been observed for natural maple leaves, however, that the fi nal sizes of the lobes and sinuses are not random, but follow numerical relations that make them foldable to align their margins on one plane [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF]. If we remove the constraint of the opposite leaf, the lamina does not grow on a plane, as observed in the ablation assay. The resulting mature leaf will no longer hold these relationships among the shapes of the lobes and sinuses. The kirigami effects separate the successive lobes and sinuses at the same angle, making them also nearly equal in size ( Fig. 6A ). Fig. 6. Mathematical prediction of leaf morphology. Shapes of a hypothetical fi ve-lobed leaf unfolded (left) and folded (right). The lobes/folds are marked green, the sinuses/antifolds red. (A) A leaf that respects the kirigami constraints and can be folded to align the lamina margin on a plane, as in naturally growing maple leaves. The most lateral lobes L 1 and L 5 have the shape of lamina characterized by α > β . The heights of the lobes have the relationships: L 1 < L 2 , and L 5 < L 4 . The three central lobes ( L 2 , L 3 , L 4 ) have the same angle α with respect to the sinuses/antifolds S 1 , S 2 , S 3 , S 4 ; so L 2 = L 3 = L 4 , and S 1 = S 2 = S 3 = S 4 . (B) A leaf that does not respect the kirigami constraints and cannot be folded to align the lamina margin on a plane, as observed in the ablation experiment. The most lateral lobes L 1 and L 5 have the shape of lamina characterized by α > β , but the heights of the lobes have the relationships: L 1 = L 2 , and L 5 > L 4 . The three central lobes ( L 2 , L 3 , L 4 ) hold the same angle α with respect to sinuses/antifolds S 1 , S 2 , S 3 , S 4 ; however, it is not coherent with their respective lengths. For example, in this case, L 2 < L 3 , L 4 < L 3 , S 2 < S 1 , S 2 < S 3 , and S 4 < S 3 . the two phenomena. The rib cell differentiation that will drive the enhanced growth on the abaxial side of the vein begins prior to lamina folding ( Fig. 2A ). Therefore, the abaxial hypergrowth along the vein can be the driving force for fold formation and probably imposes folding on the lamina. As folds tighten, the lamina grows and creates the antifolds. The positions of the folds are specifi ed by the pre-existing venation, whereas the positions of antifolds may be decided by the spontaneous folding of the extending lamina in the midline between the two folds as a topological consequence. The physical constraints from the other primordia restrict the space in which the lamina can extend and would facilitate antifold formation. Small nicks were observed on the lamina margin that mark the midpoint between two adjacent veins prior to folding ( Fig. 1D,1E ); such incisions could demarcate antifold formation to start at that specifi c location.

Surgical ablation also indicated the importance of the enhanced growth on the abaxial side of the lamina in regulation of the space-dependent growth. Because of the excess abaxial growth in the proximo-distal direction, a primordium curves toward and meets the opposite leaf on the middle plane. Once free from the physical constraints imposed by the other leaves, a primordium quickly grew into the newly available space while maintaining its curvature ( Figs. 4,5 ), suggesting that physical constraints limit the growth at the both adaxial and abaxial sides of the lamina. The geometrical calculation predicts that without the growth inhibition from the opposite primordium, the lobes and sinuses would become less even in shape and size ( Fig. 6 ).

The newly gained mechanistic information supplemented the original kirigami model [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF], which is now revised as follows ( Fig. 9A ). The enhanced growth on the abaxial side of the lamina along the veins causes lamina folding. The same enhanced growth on the abaxial side curves the primordia slightly inward so that the two opposing primordia meet each other at the plane in the middle. Proper folding also depends on the geometry of the space available for the new primordia to grow, which are defi ned by the older primordia in the vicinity A priori this phenomenon could be a wound effect due to the surgery. To address this concern, we conducted a partial ablation experiment by removing only the central lobe from one of two pairing leaves, leaving all the lateral lobes ( Fig. 8A ). In this case, the central lobe of the intact leaf curved toward where ablation took place. The lateral lobes, which were still constrained by the lateral lobes of the opposite leaf, grew normally and did not curve ( N = 4) ( Fig. 8B ). Even when the leaf did not grow much over several days, a clear curving occurred within the fi rst day without any growth ( N = 8) ( Fig. 8C ), which may indicate the presence of residual stresses (i.e., the inhomogeneous growth of the different layers of the lamina creates stress inside, which was released upon the ablation). If curving were a wound effect, the whole leaf would have been affected, as well as the half-ablated leaf itself. Moreover, similar curving growth is found naturally, when the leaves grow outside of the bud and without physical constraints during their development ( Fig. 8D ). Therefore, ablation-induced leaf curving is a physiological response.

DISCUSSION

To uncover the biological mechanisms behind the kirigami regulation of palmate leaf shape, we observed how maple leaves fold and grow during their development. A fl at tissue that extends more on one side than on the other is geometricaly equivalent to curving or folding the tissue toward the side of less expansion. We observed in cytological sections that this abaxial growth is concentrated behind the veins and that the asymmetric growth of the lamina along the veins and the fold formation are correlated. These correlations point to the possibility of coregulation; in other words, abaxial growth along the vein, lamina extension, and folding are all controlled by a common regulator. Alternatively, lamina growth may regulate folding or vice versa. The fact that folds are not positioned randomly but are always localized on the veins suggests dependency between ( Fig. 7 ). In normal apices, the division plane between two pairing primordia appears almost fl at, but slightly fl uctuates on close inspection ( Fig. 1A ), indicating reciprocal growth limitation all along the margins of the primodia. Two opposing leaves inhibit growth of each other and stabilize at the central plane by mutual contact, exerting the cutting effect of kirigami.

A common mechanism-abaxial hypergrowth along the vein-mediates the ways by which the leaves fold their lamina and determine the geometry of the space inside the bud and thus surfaced as the critical developmental process for the kirigami model of leaf morphogenesis. The fact that a common process underlies both folding and space-fi lling growth of the primordia could explain why folding and the kirigami property coincide in evolution [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF]. Lamina folding along the veins in the primordial stages occurred many times independently during the angiosperm evolution [START_REF] Couturier | A global regulation inducing the shape of growing folded leaves[END_REF], indicating that this evolutionary transition probably does not require complicated genetic changes. The observations in the maple could apply generally to the other dicotyledonous species with kirigami leaves.

Surgical ablation also revealed some mechanistic clues on how the steric constraints inhibit growth of the primordia. It requires fi ne regulation that probably involves perception and response to physical contacts [START_REF] Williams | The shoot apex and leaf growth[END_REF][START_REF] Couturier | The fi lling law: A general framework for leaf shape diversity and its consequences on folded leaves[END_REF]. The fact that partial ablation could induce local growth indicates that spatial exploration takes place locally in each lobe and sinus, if not all over the lamina or primordia. Perhaps the simplest explanation would be to draw an analogy between growth of the lamina in the bud and expansion of a piece of gel confi ned inside a box. The gel absorbs water and increases its volume until it reaches the surface of the box. The pressure exerted by the wall of the box stops the fl ux of water into the gel. It is a passive, spontaneous phenomenon and can occur when the force exerted by growth is smaller than that from the physical barrier.

Alternatively, growing primordia may actively recognize physical contact with chemical or physical signals. A primordium could sense physical constraints when it is exposed to a chemical signal present on the other primordia. Interestingly, the maple leaf primordia do not seem to require mutual communication, since primordia showed contact-inhibited growth even at the wound sites that lack a normal surface ( Figs. 7D,7E,9B ). The contact regulation could also be mediated by mechanical stimuli. Touch stimuli and physical stresses are known to infl uence plant growth via modulation of gene activities [START_REF] Braam | In touch: Plant responses to mechanical stimuli[END_REF][START_REF] Coutand | Biomechanical study of the effect of a controlled bending on tomato stem elongation: Local strain sensing and spatial integration of the signal[END_REF]. Repetitive touch application inhibits growth throughout the plant body [START_REF] Braam | In touch: Plant responses to mechanical stimuli[END_REF], while a site-specifi c application of loads induced a more local response [START_REF] Coutand | Biomechanical study of the effect of a controlled bending on tomato stem elongation: Local strain sensing and spatial integration of the signal[END_REF]. What we observed in the maple leaf primordia resembles the latter, since growth could be inhibited even at the suborgan levels ( Fig. 8 ).

The kirigami model of palmate leaf development entails both molecular and nonmolecular regulation of morphogenesis. While steric constraints oversee the size and the shapes, the numbers and the positions of the lobes are determined by venation, which is patterned by the dynamic fl ow of the phytohormone auxin [START_REF] Nelson | Leaf vascular pattern formation[END_REF][START_REF] Sieburth | Auxin is required for leaf vein pattern in Arabidopsis[END_REF][START_REF] Scarpella | Control of leaf vascular patterning by polar auxin transport[END_REF]. The initiation of a lobe or leafl et on a primordium starts with local accumulation of auxin, just like leaf initiation [START_REF] Reinhardt | Auxin regulates the initiation and radial position of plant lateral organs[END_REF][START_REF] Nikovics | The balance between the miR164A and CUC2 genes controls leaf margin serration in Arabidopsis[END_REF]Barkoulas et al., 2008 ;[START_REF] Blein | A conserved molecular framework for compound leaf development[END_REF][START_REF] Koenig | Auxin patterns Solanum lycopersicum leaf morphogenesis[END_REF][START_REF] Kawamura | Mechanisms of leaf tooth formation in Arabidopsis[END_REF]. It promotes outgrowth, and CUC genes are expressed on the surrounding area to mark the border of the outgrowth. Since the kirigami leaves develop through repetitive reiteration of outgrowth (lobe) and border (sinus), it would be interesting to see if folds and antifolds can induce or maintain auxin accumulation and CUC gene expression, respectively. Physical forces also infl uence the direction of cell growth and division, probably through regulation of the microtubule organization [START_REF] Lintilhac | Stress-induced alignment of division plane in plant tissues grown in vitro[END_REF][START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. We anticipate that future investigations of kirigami leaf morphogenesis will address the integration of chemical and physical regulation of biological form. (A, B) Summary of growth regulation by available space in normal conditions (A) or when one leaf was ablated from a pair (B). The envelope (designated E) made of the bud scales or older primordia, as well as the opposite leaf, set the physical constraint for lamina folding and growth. The lamina folds along primary and secondary veins inside the envelope, representing the paper-folding part of kirigami. The lamina grows until it fi lls the space, i.e., until they reach surrounding primordia. Because growth (double headed arrow) is enhanced on the abaxial side, the pairing leaves grow curving slightly inward, meeting one another in the middle (A). Facing leaves inhibit each other's growth, acting as the paper-cutting part of kirigami. When one leaf is removed, the remaining leaf grows excessively, while maintaining the curvature inward (B). The contact inhibition does not require normal surfaces on both ends, since it was also observed at the wound sites. P, primordia.

Fig. 1 .

 1 Fig. 1. Morphology and folding of Acer pseudoplatanus leaf. (A) A pair of young leaves. The lamina of the opposite leaves fold and form a near mirror-image set of lobes that press against one another. The lamina margins are positioned along a plane in the middle with fl uctuations, indicating reciprocal limitation of growth between the opposite leaves. The lamina folding and mutual inhibition between facing leaves together comprise kirigami leaf morphogenesis and produce the lobed shape of mature, opened leaves. (B) Typical lamina morphology of lobed mature leaf, which correlates with the organization of the developmentally transient folds. The abaxial side of the leaf is shown. (C) Folds and antifolds in an opening leaf, pictured from the adaxial side. (D) Fold formation in a young leaf visualized by scanning electron microscopy. The lamina has already folded along the primary veins, but not yet along the secondary veins. Nicks delimit future small lobes (marked with asterisks) on the lamina margin prior to folding along the secondary veins. Close-up of the lamina before folding (E). Scale bars = 100 µm.

Fig. 2 .

 2 Fig. 2. Correlation between abaxial growth along the vein and fold formation. Because the different orders of veins mature at different times during leaf development, and the size of the veins varies along the proximo-distal axis, we can fi nd a collection of different stages of folding dynamics in one leaf. Transverse sections along the proximo-distal axis were examined. (A) Successive stages (a-d) of vein growth. The cells in the region of the lamina abaxial to the veins grow much more than the adaxial cells. (B) The fold angle follows the vein development.The ratio L / V of the folds normal to the plane of the section was measured from one leaf (•) and its pair leaf ( ). Standard deviations are shown as crossed bars. We collected measurements from two opposite leaves within a bud, for which we could obtain every section along the entire leaf. The data collapse on the same curve. (C) Dashed boxes highlight epidermal cells on the adaxial and abaxial sides of the folds and antifolds, respectively. Epidermal cells appear compressed on the adaxial side of the fold and the abaxial side of the antifold, while they appear extensively expanded on the opposite side. Vasculature is marked with an orange line (A, C).

Fig. 3 .

 3 Fig. 3. Antifold formation correlates with lamina growth. The same set of transverse sections of folding leaves as in Fig. 2 were used for examination of antifold formation. (A, C) The lamina thickness along a vein V is plotted as a function of the lamina length A between the vein and the closest antifold. The lamina extension (medio-lateral growth) is proportional to the lamina thickening (abaxial-adaxial growth). Note that most of the lamina thickening is due to abaxial growth. (B, D) The antifold angle β is plotted against the sum of the lamina thickness at the two closest veins V 1 + V 2 . Antifold formation correlates with the abaxial growth along the vein. Each dot represents the mean of 3-19 measurements made for each vein at different proximo-distal positions. : data from one leaf; : data from the pairing leaf. Standard deviation is shown by the crossed error bars for two representative veins.

Fig. 4 .

 4 Fig. 4. Ablation of one leaf induces growth and curving of the opposite leaf. (A) Maple leaves form in pairs with a 90 ° lateral shift in position. A pair of primordia (a's) develop inside the previous pair (b's). One leaf from the pair (b) was removed to reveal the younger pair (a) growing inside. (B) Longitudinal sections of young primordia. The cells in the abaxial side are much larger than ones on the adaxial side. (C, D) Shoot apices were dissected and then tissue cultured. Both leaves in a pair were left intact for the control samples (C), or one leaf was removed in the ablated samples (D). In the ablated samples, the remaining leaf grew into the space previously occupied by the ablated leaf. The leaf grew curving inward; within a leaf, each lobe curved independently. Numbers indicate days after surgery. Scale bars = 500 µm.

Fig. 7 .

 7 Fig. 7. Histological examination of the ablation effects. Longitudinal sections of tissue-cultured maple shoot apices. The apices were cultured for 3 days after surgery. (A) Representative of controls, in which leaves were left inside the bud. The apices were dissected down to 3-4 pairs of primordia before fi xation. Opposite leaves meet each other in the middle, and the lamina ( L ) is folded in a uniform and ordered manner. (B) Representative of another kind of controls in which the apices were dissected down to 3-4 pairs of primordia before culturing. The opposite leaves still meet each other in the middle, but the order of the folds is disturbed. (C) Representative of ablated samples in which one leaf was removed from a pair. Ablation resulted in induction of growth toward the ablated area. The lamina showed clearly abnormal folding. (D, E) Close-ups of the remaining leaf near the ablation wound. Space-dependent growth regulation is apparent even at the wound sites (marked W ).

Fig. 8 .

 8 Fig. 8. Ablation affects growth locally. (A) Schematics of total and partial ablation. Either the entire leaf or only the central lobe was removed from a pair of facing leaves and tissue cultured for a day. Lobes curve into the newly available space independently. (B, C) The central lobe was removed from a leaf at two different heights, at lower (B) and higher (C) positions. The central lobe of the intact leaf grew and curved into the newly available space. The remaining parts prevented the curving of the corresponding lateral lobes of the intact leaf. Arrows point at the ablation sites. Numbers indicate day(s) after dissection. Scale bars = 500 µm. (D) Naturally occurring neo-morphed leaf of Acer pseudoplatanus , which grew outside the bud. Naturally free from surrounding physical constraints, lobes were loose, and the leaf curved without any ablation. Scale bar = 1 mm.

  LITERATURE CITED AZRI , W. , C. CHAMBON , S. HERBETTE , N. BRUNEL , C. COUTAND , J.-C. LEPLÉ , I. BEN REJEB , ET AL . 2009 . Proteome analysis of apical and basal regions of poplar stems under gravitropic stimulation. Physiologia Plantarum 136 : 193 -208 . BARKOULAS , M. , A. HAY , E. KOUGIOUMOUTZI , AND M. TSIANTIS . 2008 . A developmental framework for dissected leaf formation in the Arabidospis relative Cardamine hirsuta. Nature Genetics 40 : 1136 -1141 .

Fig. 9 .

 9 Fig.9. Mechanism of kirigami leaf shape regulation via folding and physical constraints. (A, B) Summary of growth regulation by available space in normal conditions (A) or when one leaf was ablated from a pair (B). The envelope (designated E) made of the bud scales or older primordia, as well as the opposite leaf, set the physical constraint for lamina folding and growth. The lamina folds along primary and secondary veins inside the envelope, representing the paper-folding part of kirigami. The lamina grows until it fi lls the space, i.e., until they reach surrounding primordia. Because growth (double headed arrow) is enhanced on the abaxial side, the pairing leaves grow curving slightly inward, meeting one another in the middle (A). Facing leaves inhibit each other's growth, acting as the paper-cutting part of kirigami. When one leaf is removed, the remaining leaf grows excessively, while maintaining the curvature inward (B). The contact inhibition does not require normal surfaces on both ends, since it was also observed at the wound sites. P, primordia.