S Ghazal

† V Berthelot

N C Friggens

P Schmidely
email: philippe.schmidely@agroparistech.fr

Influence of a supplement containing conjugated linoleic acid on dairy performance, milk fatty acid composition, and adipose tissue reactivity to lipolytic challenge in mid-lactation goats

Keywords: conjugated linoleic acid, milk fatty acid composition, lipolytic challenge, goat

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

INTRODUCTION

Conjugated linoleic acids (CLA) are geometric and isomeric isomers of linoleic acid that are produced during ruminal biohydrogenation of linoleic acid [START_REF] Lourenço | The role of microbes in rumen lipolysis and biohydrogenation and their manipulation[END_REF]. Conjugated linoleic acids are mainly produced when high levels of PUFA and high percentages of rapidly fermentable carbohydrates are associated [START_REF] Bauman | The biology of conjugated linoleic acids in ruminants[END_REF]. Among the CLA isomers, the trans-10,cis-12 C18:2 isomer is known to be the most potent isomer for decreasing milk fat content (MFC) in dairy cows [START_REF] Baumgard | Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis[END_REF], even if cis-10,trans-12 and trans-9,cis-11 isomers induce similar responses [START_REF] Saebø | Milk fat synthesis is unaffected by abomasal infusion of the conjugated diene 18:3 isomers cis-6,trans-10,cis-12 and cis-6,trans-8,cis-12[END_REF][START_REF] Perfield | Trans-9, cis-11 conjugated linoleic acid reduces milk fat synthesis in lactating dairy cows[END_REF]. Molecular-based approaches indicated that trans-10,cis-12 C18:2 simultaneously downregulated the abundance of transcript of key genes involved in de novo synthesis of FA, uptake of long-chain FA, and desaturation of FA in the mammary gland of cows [START_REF] Shingfield | Role of trans fatty acids in the nutritional regulation of mammary lipogenesis in ruminants[END_REF][START_REF] Bauman | Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis[END_REF]). In dairy goats, feeding ruminally protected trans-10,cis-12 C18:2 resulted in a smaller decrease in MFC than in cows [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF][START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF], reflecting a lower sensitivity of the goat mammary gland to the antilipogenic effect of trans-10,cis-12 C18:2.

It has been shown that the stage of lactation, the duration of the trial, and the form of protection of dietary trans-10,cis-12 C18:2 affect the response of mammary gland to CLA supplementation [START_REF] Bauman | Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model[END_REF] but less attention has been paid to diet composition. In cows [START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF] and in ewes [START_REF] Weerasinghe | Effect of a supplement containing trans-10,cis-12 conjugated linoleic acid on the performance of dairy ewes fed 2 levels of metabolizable protein and at a restricted energy intake[END_REF], CLA may alter partitioning of nutrients to support an increase in milk and protein yield when dietary energy is limited. Moreover, feeding CLA to dairy cows fed a high-concentrate diet (high starch-low NDF) resulted in a smaller decrease in MFC than in cows fed a low-concentrate diet (low starch-high NDF), suggesting that dietary ingredients or diet composition (or both) may affect the response or sensitivity of the mammary gland to trans-10,cis-12 C18:2 [START_REF] Glasser | t10,c12-18:2-induced milk fat depression is less pronounced in cows fed high-concentrate diets[END_REF]. In dairy goats, reduced MFC was observed with a low-concentrate-low-starch diet based on alfalfa hay [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF], and with a high-concentrate diet based on hay [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF] during short-term trials. To date, no information is available on the response to trans-10,cis-12 C18:2 of dairy goats fed high-concentrate diets based on corn silage during a long-term trial.

In opposition to changes observed in the mammary gland, expression of lipogenic enzymes and of genes related to lipid synthesis in subcutaneous adipose tissue were increased during short-term trans-10,cis-12 C18:2 supplementation in cows [START_REF] Harvatine | Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows[END_REF], reflecting the partitioning of spared energy away from milk toward adipose tissue. Simultaneously, von Soosten et al. (2011) reported reduced mobilization of retroperitoneal adipose tissue in early lactating cows fed CLA, which may suggest reduced lipolysis in adipose tissue. Accordingly, changes in plasmatic NEFA after a β-adrenergic challenge (epinephrine) in cows fed trans-10,cis-12 C18:2 were small [START_REF] Baumgard | trans-10, cis-12 Conjugated Linoleic Acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows[END_REF][START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF]. However, trans-10,cis-12 C18:2 decreased adipocyte size in primiparous cows during early lactation, suggesting that CLA may stimulate adipose tissue lipolysis [START_REF] Akter | Physiological and conjugated linoleic acid-induced changes of adipocyte size in different fat depots of dairy cows during early lactation[END_REF]. Adipose tissue response to adrenergic challenge in cow is altered according to changes in body energy input and output [START_REF] Mcnamara | Regulation of bovine adipose tissue metabolism during lactation. 4. Dose-responsiveness to epinephrine as altered by stage of lactation[END_REF], which are affected by CLA. In dairy goats, no study has reported on the effect of trans-10,cis-12 C18:2 on reactivity of adipose tissue to lipolytic signals.

In consequence, this trial studied the long-term effect of trans-10,cis-12 C18:2 supplementation on milk performance, milk FA composition, energy metabolism, and reactivity of adipose tissue evaluated by a β-agonist challenge in early to mid-lactating goats fed a high-concentrate diet based on corn silage.

MATERIALS AND METHODS

Animals and Diets

Twenty-four multiparous dairy Saanen or Alpine goats (BW = 63.7 ± 8.0 kg, 30 ± 4 DIM; mean ± SD) were used for 10 wk, with the first 3 wk for adaptation to experimental diets. Twelve of the goats were fitted with a ruminal cannula and a T-type duodenal cannula for determination of the diet digestibility and measurement of duodenal flow (not described here) and they were housed in metabolism crates after the adaptation period. The 12 other goats were kept in individual pens. At the initiation of the trial, the average milk yield (mean ± SD) was 3.9 ± 1.3 kg/d, with a milk fat content (mean ± SD) of 35.0 ± 5 g/L and a milk protein content (mean ± SD) of 32 ± 2 g/L.

The goats were allocated to 2 groups according to their milk production and fat content measured 30 d after parturition. From parturition to the start of the trial (wk 0), all goats were fed ad libitum twice per day (0800 and 1700 h) a TMR consisting (on a DM basis) of a mixture of grass hay (20%), dehydrated alfalfa pellets (20%), beet pulp (30%), and a commercial concentrate (30%; Fluvia Selectunic, Sanders, France). The dietary transition was made in the first week of the adaptation period for corn silage, followed by the addition of lipid supplements in the second week. Throughout the experimental period of 7 wk (wk 3 to 9), the goats were fed ad libitum a TMR containing corn silage (35% on a DM basis), beet pulp (20%), barley (15%), and the same commercial concentrate (30%). The CP, OM, NDF, ADF, and starch contents (DM basis) of the basal diet without adding either Ca salts of palm oil (control, CTL) or CLA supplement were 118, 922, 381, 188, and 175 g/kg of DM, respectively. The NE L of the TMR was 1,500 kcal/kg of DM. The composition of FA of the basal diet is given in Table 1. Lipid supplements (CTL supplement vs. CLA, 45 g/d) were top-dressed on the TMR and thoroughly hand mixed. The CTL supplement consisted of Ca salts of palm oil (Saphir Intense; Eolia, Bruz, France). The CLA supplement (Lutrell; BASF Animal Nutrition, Château-Gontier, France) had a lipid content of 65% and a FA composition of 15% trans-10,cis-12 C18:2, 15% cis-9,trans-11 C18:2, 9% 16:0; 42% 18:0, 12% cis-9 C18:1, and 1% cis-9,cis-12 C18:2, corresponding to a daily distribution of 4.5 g of cis-9,trans-11 C18:2/d and 4.5 g of trans-10,cis-12 C18:2/d. The CTL supplement had a lipid content of 90% with 50% C16:0; 35% of C18:1 cis-9, and 10% C18:2 cis-9, cis-12. Lipid supplements distribution was 15 g and 30 g in the morning and afternoon feed allocations, respectively.

Experimental Measurements and Sampling

From wk 2 onwards, feed intake was recorded daily. Three diet samples were collected at the end of the adaptation period (wk 2), in the middle (wk 6), and in wk 9 for feed composition analyses and total lipid determination. Goats were weighed once per week at the same time in the middle of the day. During the trial (from wk 0 to 9), 2 milk samples of 30 mL (one at the evening milking and one at the following morning milking) were individually collected each week with a preservative (Bronopol; Grosseron SA, Saint-Herblain, France) and stored at 4°C until milk composition analysis (protein, fat, and lactose content). During wk 1, 5, and 6, 2 additional milk samples of 30 mL (one at the evening milking and one at the following morning milking) were individually collected without preservative, and stored at -18°C for milk FA composition.

Isoproterenol Challenges

In the last week of the trial, an intravenous isoproterenol (ISOP) challenge was performed on the 12 cannulated goats fed the CTL or CLA diets. A solution of ISOP (Sigma-Aldrich, Saint-Quentin-Fallavier, France) of 40 μg/mL was prepared in NaCl (9‰) the day before the challenge as described by [START_REF] Gagliostro | Duodenal rapeseed oil infusion in early and midlactation cows. 4. In vivo and in vitro adipose tissue lipolytic responses[END_REF] and was kept at 4°C. The day of challenge, the solution was heated on a hot plate agitator (37°C), and a dose of 4 nM ISO/kg of BW (1 μg/kg of BW) was injected within 15 s into the left jugular vein of each goat immediately after morning milking. During the challenge, the 12 goats were not fed. Blood samples were collected from the jugular vein into heparin vials at -20, -5, +5, +10, +15, +20, +25, +30, +45, and +60 min relative to the injection. Blood samples were immediately centrifuged at 1,780 × g for 10 min at 4°C and plasma was stored at -18°C until analysis for glucose, NEFA, BHBA, and insulin.

Laboratory Analysis

For each time point, 1 feed sample was dried in a forced-air oven at 90°C for 24 h to determine DM content and then ground and stored at room temperature until determination of the OM, NDF, ADF [START_REF] Van Soest | Methods for dietary fiber, neutral detergent fiber, and non starch polysaccharides in relation to animal nutrition[END_REF], and starch (Gluco-sequant glucose/HK; Roche/Hitachi Diagnostics, Mannheim, Germany) content. Dry matter, ash, and enzymatic starch in feed were determined following International Organization for Standardization (ISO) standards: ISO (1999; 6496), ISO (2002;5984), and ISO (2004;15914), respectively. The 2 other feed samples were immediately stored at -20°C and lately lyophilized and ground for total lipid determination and FA analysis according to [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF].

Methyl esters from milk or feed samples were injected with an autosampler into a Varian CP 3800 gas chromatograph equipped with a flame ionization detector (Varian SA, Les Ulis, France) on a CP-Sil 88 (100-m × 0.25-mm i.d. fused silica capillary column; Interchim, Montluçon, France). The carrier gas was ultra-pure helium with a flow of 1 mL/min. Injector and detector temperatures were maintained at 250°C. The split ratio in the injector was 20:1. The initial oven temperature was 50°C (for 1 min) and was then increased at 10°C/ min to 170°C (held for 78 min) and then increased again at 5°C/min to 220°C (held for 15 min). Individual FA were identified by comparison of retention time to those of pure standards (Supelco Inc., Bellefonte, PA).

For plasma glucose, NEFA, and BHBA analyses, an autoanalyzer (Cobas-Mira Roche/Hitachi, Mannheim, Germany) was used with kits (Gluco-sequant glucose/ HK, Roche/Hitachi Diagnostics; NEFA-HR(2), Wako Chemicals GmbH, Neuss, Germany). β-Hydroxybutyrate was analyzed by the method of [START_REF] Barnouin | Automated micro-determination of bovine plasma 3-hydroxybutyrate without deproteinization[END_REF]. Insulin was analyzed with an ELISA kit (10-1202-01; Mercodia AB, Uppsala, Sweden).

Statistical Analysis

Milk yield; fat, protein, and lactose yields and contents; DMI; and BW measured during the trial were evaluated using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) for repeated measures according to the following statistical model:

Y ijkl = μ + A i + B j + AB ij + C k + D l + Cov l + ε ijkl ,
where Y ijkl is the response variable, μ is the overall mean, A i is the fixed effect of treatment (CTL vs. CLA), B j is the fixed effect of time (7 wk), AB ij is the interaction between the treatment and time, C k is the cannula effect (cannulated vs. intact), Cov l is a covariate measured at the end of wk 2, and ε ijkl is the residual error. Goat was considered as a random effect (D l ). A first-order autoregressive [AR (1)] covariance structure was used for repeated measures. The same model was used to evaluate the effect of CLA, time, and CLA × time interaction on each milk FA measured during wk 5 and 6, with wk 2 values used as a covariate. Preliminary statistical analysis showed that the cannula effect and CLA × cannula interaction effect were not significant; therefore, these effects were withdrawn from the model. Treatment effects on plasma concentrations of NEFA, glucose, insulin, and BHBA, after ISOP injection were tested for baseline concentrations (baseline values calculated as the average at -20 and -5 min before ISOP injection), maximal concentrations (at 5 or 10 min after injection), maximal increase above basal value, changes in the area under the curve (AUC; calculated as the positive incremental area that was above baseline value during the first 25 min relative to ISOP injection), and total AUC (total AUC, calculated as the area from -20 into 25 min after injection), as described by [START_REF] Cardoso | Technical note: Comparison of 3 methods for analyzing areas under the curve for glucose and nonesterified fatty acids concentrations following epinephrine challenge in dairy cows[END_REF]. These data were analyzed using the GLM procedure of SAS (SAS Institute Inc.) according to the following model:

Y ij = μ + A i + ε ij ,
where Y ij is the response variable, μ is the overall mean, A i is the fixed effect of treatment (CTL vs. CLA), and ε ij is the random residual error. Least squares means are reported and treatment effects are declared significant at P < 0.05.

RESULTS

Animal Performance

The BW, DMI, milk yield, and milk protein and lactose yields and contents were unaffected by CLA (Table 2). Compared with the CTL, the CLA treatment significantly decreased milk fat yield and content by 18 and 15%, respectively. The CLA treatment increased the net energy balance (NEB) by 260 Kcal/d. No interactions were observed between CLA treatment and time for BW, DMI, NEB, milk yield, or milk protein and lactose yields and contents, or milk fat yield and milk fat content.

Milk FA Profile

The trans-10,cis-12 C18:2 isomer was detected only in the milk of goats fed the CLA treatment, with no change in the proportion of the cis-9,trans-11 C18:2 isomer between the groups. Apparent efficiency of transfer of trans-10,cis-12 C18:2 from diet to milk {100 × [trans-10,cis-12 C18:2 output in milk (g/d)]/[trans-10,cis-12 C18:2 intake (g/d)]} was 1.9%. The interaction between the CLA treatment and time was not significant for most of milk FA. Compared with the CTL, the CLA treatment did not affect the proportions of the sums of SFA, PUFA, or FA with C <16 in milk fat. It increased the sum of ISOP FA (Table 3). When the FA were considered individually (Appendix Table A1), the CLA treatment decreased numerically or significantly the proportions of the linear FA from C8 to C13, and C16:0 in milk fat. It increased the proportions of C4:0, C14:0, C18:0, C19:0, C20:0, and C22:0. The CLA treatment did not affect the sum of trans C18:1 (Table 3), but it tended to increase the proportion of the trans-11, trans-13 + trans-14, and trans-16 isomers of C18:1. Compared with CTL, the CLA treatment reduced the sum of cis C18:1 isomers, cis-9 C18:1, and the sum of C16:0 + cis-7 C16:1. It also reduced the ratios of cis-5 C14:1/C14:0, cis-9 C18:1/C18:0, and cis-9,trans-11 3).

The CLA treatment significantly reduced milk secretion of <C16 FA synthesized de novo (353 vs. 290 mmol/d for CTL and CLA, respectively) and the sum of C16:0 + cis-7 C16:1 (194 vs. 154 mmol/d), and it tended to decrease (P = 0.07) the secretion of >C16 FA in milk (184 vs. 164 mmol/d; Figure 1).

Response of Plasma Concentrations to an Isoproterenol Challenge

Compared with CTL, the CLA treatment increased NEB by 77.5% for wk 8, which was before ISOP challenge. Baseline plasma concentrations (-20 and -5 min before the ISOP challenge) of glucose, NEFA, and BHBA were not affected by CLA treatment (Table 4). The baseline concentration of insulin was numerically higher in the CLA group than in the CTL group (P = 0.15). Compared with the CTL, the glucose-to-insulin ratio at baseline tended to be lower in the CLA treatment (P = 0.08). After ISOP challenge, concentrations of glucose, NEFA, and insulin increased for 5 or 10 min and decreased thereafter to return to baseline concentrations at 25 or 30 min (Figure 2). The maximal concentrations (peak) and the increases above basal concentrations of glucose, NEFA, and insulin were not affected by CLA treatment (Table 4). The concentration of BHBA decreased during the 60 min after ISOP injection.

Changes in the AUC for glucose, NEFA, BHBA, insulin, and total AUC of glucose and NEFA were not affected by CLA treatment. Total AUC of BHBA and insulin tended to be higher (P = 0.10 and 0.13, respectively) and the ratio of total AUC of glucose to total 2 Saturated FA including pair and odd FA.

3 Includes iso C14:0, iso C15:0, iso C16:0, and iso C17:0. 4 Monounsaturated FA = ∑ (cis-5 C14:1, cis-6 C15:1, cis-7 C16:1, total cis C18:1, total trans C18:1, and cis-11 C20:1).

5 Polyunsaturated FA = ∑ (all isomers of C18:2, cis-9,cis-12,cis-15 C18:3, cis-6,cis-9,cis-12 C18:3, and C20:3n-3). 6 Includes anteiso C17:0. AUC of insulin tended to be lower (P = 0.11) in the CLA group compared with the CTL (Table 4).

DISCUSSION

Animal Performance

Compared with cows (reviewed by [START_REF] Harvatine | Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows[END_REF][START_REF] Bauman | Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis[END_REF], dietary CLA have been less used in dairy goats [START_REF] Erasmus | Effect of level of rumen protected CLA supplementation on milk yield and composition in Saanen goats[END_REF][START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF][START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. Our data confirm previous conclusions demonstrating that milk fat yield is reduced after CLA supplementation in goats but that goats appear less sensitive to CLA than cows [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. Indeed, we obtained a mean proportion of 0.10% trans-10,cis-12 C18:2 in milk fat that decreased MFC by 15% and fat yield by 18%. In comparison, a similar proportion of trans-10,cis-12 C18:2 in milk induced a decrease in milk fat content and in milk yield between 25 and 30% in cows [START_REF] Perfield | Use of conjugated linoleic acid (CLA) enrichments to examine the effects of trans-8, cis-10 CLA, and cis-11, trans-13 CLA on milk-fat synthesis[END_REF][START_REF] Moore | Effect of supplemental conjugated linoleic acids on heat-stressed Brown Swiss and Holstein cows[END_REF]. Moreover, temporal patterns of decrease in MFC are different between goats and cows, as the response to CLA is obtained within 2 to 3 d in cows, whereas we found it takes at least 7 d in goats (see also [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF].

In our experiment, the decrease in milk fat content and fat yield were intermediary to those previously reported in goats [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF][START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. The main difference in response is largely related to the trans-10,cis-12 C18:2 intake and its bioavailability. Using a dose ranging from 7 to 22 g of trans-10,cis-12 C18:2/d [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF] and from 3 to 6 g/d [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF], respectively, the mean concentration of trans-10,cis-12 C18:2 in milk ranged from 0.2 to 0.8%, and from 0.1 to 0.2% respectively. In those studies, the calculated transfer efficiency of di- The maximal value was observed at 5 or 10 min after the ISOP injection.

3 Area under the curve (AUC) was calculated as the positive incremental area that was above baseline value during the first 25 min after injection.

etary trans-10,cis-12 C18:2 to milk was between 1.7 and 2.4% [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF] and between 1.8 and 1.9% [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF]. This was in line with the value in the present study (1.9%). The corresponding decreases in MFC were 25 to 36% and 5 to 18% for [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF] and [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF], respectively. The intake of 4.5 g of trans-10,cis-12 C18:2/d in our experiment produced a 15% decrease in MFC. However, closer comparison with the results of [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF] shows that at a similar trans-10,cis-12 proportion in milk, we obtained a greater decrease in MFC and in fat yield. This suggests that CLA-induced decrease in MFC is affected by the initial level of milk fat (31.2 g/L in Lock et al., 2008 vs. 45 g/L in our trial). In line with this, at similar levels of trans-10,cis-12 C18:2 intake, the decrease in milk fat was greater in goats fed supplements of FA than those fed no lipid supplement [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. Similarly, the response of MFC to CLA was less pronounced in cows exhibiting low initial MFC than those with high MFC (i.e., when cows were fed high-concentrate compared with low-concentrate diets; [START_REF] Glasser | t10,c12-18:2-induced milk fat depression is less pronounced in cows fed high-concentrate diets[END_REF]. In our conditions, a highconcentrate diet based on corn silage led to high MFC and it elicited a significant decrease in MFC in goats fed low doses of trans-10,cis-12 C18:2. Our results also show that supplementation with 4.5 g of trans-10,cis-12 C18:2/d in lactating goats fed a high-concentrate diet based on corn silage had no effect on DMI, BW, milk yield, or milk protein and lactose yields and contents throughout a 7-wk trial. This agrees with data in goats supplemented with a wide range of doses of trans-10,cis-12 C18:2 and fed alfalfa or corn silage diets with 35 to 60% concentrate [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF][START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF].

Because CLA treatment induced a decrease in the net energy needs due to a reduction in milk fat secre- tion (at constant DMI, BW, and milk yield relative to the CTL), CLA supplementation gradually increased net energy balance. We observed a mean increase in NEB of 260 Kcal/d between CLA and CTL treatments, larger than the calculated increase in NEB of 60 of [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF] and 90 Kcal/d of [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. These differences may be related to the fact that the above-mentioned studies in goats were conducted during late lactation (140 to 250 d postpartum), which can affect the CLA-induced changes in partitioning of energy nutrients away from mammary gland [START_REF] Gervais | Lactation response of cows to different levels of ruminally inert conjugated linoleic acids under commercial conditions[END_REF].

Response to an Isoproterenol Challenge

Before ISOP injection, plasma NEFA concentrations were not affected by CLA treatment, as reported in cows in positive energy balance or during established lactation [START_REF] Baumgard | trans-10, cis-12 Conjugated Linoleic Acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows[END_REF][START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF]. Plasma NEFA concentration is an index of basal fat mobilization that reflects the apparent balance between basal lipolysis of triglyceride and FA esterification rate or de novo lipogenesis in adipose tissue. These processes are known to be regulated by insulin that decreases lipolysis and increases reesterification and de novo lipogenesis [START_REF] Brockman | Hormonal regulation of metabolism in ruminants; a review[END_REF]. Insulin may control NEFA appearance through enhanced trapping into the adipose tissue of NEFA derived from intravascular triglyceride lipolysis [START_REF] Carpentier | On the suppression of plasma nonesterified fatty acids by insulin during enhanced intra-vascular lipolysis in humans[END_REF]. Conversely to data in dairy cows where no change in plasma insulin concentration were reported after CLA treatment [START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF][START_REF] Kay | Effects of dietary conjugated linoleic acid on production and metabolic parameters in transition dairy cows grazing fresh pasture[END_REF], we observed a trend for plasma insulin and insulin:glucose ratio to be higher in goats fed CLA, probably reflecting their higher NEB balance compared with CTL goats at this time. In dairy cows, the expression of lipogenic enzymes and of genes related to lipid synthesis in subcutaneous adipose tissue was increased during short-term trans-10,cis-12 C18:2 supplementation [START_REF] Harvatine | Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows[END_REF], reflecting the partitioning of spared energy away from milk toward adipose tissue. To date, no data are available reporting the effect of CLA on lipogenesis in adipose tissue of goats.

As no data are available on the measurement of lipolysis in adipose tissue of goats fed trans-10,cis-12 C18:2, we attempted to determine if β-agonist-stimulated lipolysis may have been enhanced by CLA supplementation. Isoproterenol is a synthetic catecholamine that is a lipolytic agent that stimulates both β1-and β2adrenergic receptors on adipocytes [START_REF] Lafontan | Advances in adipose tissue metabolism[END_REF]. Thus, the NEFA response to ISOP provides an efficient method of studying the adipose tissue lipolytic potential because it induces an increase in plasma NEFA concentration, which reflects the lipolytic properties of this β-agonist minus the fraction of released NEFA that is directly reesterified [START_REF] Chilliard | Plasma non-esterified fatty acid response to a beta-adrenergic challenge before or after feeding in energy underfed or overfed, dry or lactating cows[END_REF]. The response to a β-agonist injection (norepinephrine) is unchanged in dairy cows fed CLA and a low-ME diet during midlactation [START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF] or eventually reduced in dairy cows in positive net energy balance after CLA supplementation [START_REF] Baumgard | trans-10, cis-12 Conjugated Linoleic Acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows[END_REF]. In our study, no change in any of the parameters of NEFA response was observed after CLA treatment, as observed in cows [START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF].

Before ISOP injection, plasma glucose concentration was not affected by CLA treatment, as observed in cows [START_REF] Baumgard | trans-10, cis-12 Conjugated Linoleic Acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows[END_REF][START_REF] De Veth | Response to conjugated linoleic acid in dairy cows differing in energy and protein status[END_REF]. In the present study, glucose concentration increased rapidly after ISOP injection, probably because of its effects on liver glycogenolysis [START_REF] Drackley | Adaptations of glucose and long-chain fatty acid metabolism in liver of dairy cows during the periparturient period[END_REF] and possibly on liver gluconeogenesis [START_REF] Faulkner | Effects of glucagon and alphaagonists and beta-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes[END_REF]. This increase in glucose was also associated with an increase in insulin, with a maximal response at 10 min relative to ISOP injection, which is probably due to pancreas stimulation of insulin synthesis by glucose [START_REF] Brockman | Hormonal regulation of metabolism in ruminants; a review[END_REF][START_REF] Oda | Adrenergic modulation of pancreatic glucagon and insulin secretion in goats[END_REF]. Nevertheless, this increase in glucose and insulin concentration after the ISOP challenge was not affected by CLA treatment in our study. The changes in AUC above baseline value and total AUC of glucose remained unaffected by CLA treatment. Nevertheless, total AUC of insulin tended to be higher and the total AUC of glucose-to-total AUC of insulin ratio tended to be lower in the CLA treatment, which may suggest increased pancreas sensitivity to hyperglycemia induced by ISOP. It is plausible that energy spared through the effects of CLA on milk energy output could affect the pancreas sensitivity to glucose to favor an increase in body fat lipogenesis [START_REF] Harvatine | Expression of enzymes and key regulators of lipid synthesis is upregulated in adipose tissue during CLA-induced milk fat depression in dairy cows[END_REF].

Milk FA Secretion and Profile

The reduction in fat yield in our experiment was due to the simultaneous decrease in the mammary secretion of <C16 FA (-18%), C16 FA (-20%), and to a lesser extent, a decrease in >C16 FA secretion (-11%). This reflects a CLA-coordinated downregulation of mRNA expression of lipogenic enzymes and enzymes of lipid metabolism in the mammary gland, implicating a role for sterol response element-binding protein 1 and Spot 14 at the cellular signaling level [START_REF] Bauman | Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis[END_REF]. Generally, the decrease in FA secretion is greater for short-chain and medium-chain FA than for long-chain FA, leading to a higher proportion of long-chain FA in the milk fat of cows [START_REF] Maxin | Combined effects of trans-10,cis-12 conjugated linoleic acid, propionate, and acetate on milk fat yield and composition in dairy cows[END_REF] and of sheep [START_REF] Sinclair | A supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat yield but does not alter organ weight or body fat deposition in lactating ewes[END_REF]. Our results are in agree-ment with these data, as CLA decreased the secretion of all FA, whatever their chain-length, but long-chain FA secretion was less reduced, which resulted in higher proportion of >C16 FA in milk fat.

However, some differences existed between our results and those previously reported in goats. With a similar concentration of trans-10,cis-12 C18:2 in goat milk, [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF] reported a smaller reduction in the secretion of short-chain FA and total C16 FA, and similar reductions as in our experiment were reported only with higher trans-10,cis-12 C18:2 proportion (0.3% of total FA; [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF]. Moreover, >C16 FA secretion was not reduced, whatever the dose of trans-10,cis-12 C18:2 in diet in the study of [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF], and it was significantly reduced only at a high trans-10,cis-12 C18:2 concentration in milk in the study of [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF]. Differences in lactation stage and in duration of supplementation could explain these between-experiment differences [START_REF] Bauman | Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model[END_REF]. The difference in >C16 FA secretion may reflect changes in lipoprotein lipase (LPL) activity or synthesis that are differentially affected by stage of lactation, energy balance, and dietary factors [START_REF] Bernard | Expression and nutritional regulation of lipogenic genes in the ruminant lactating mammary gland[END_REF], as mammary LPL activity is high during midlactation and it decreased during late lactation in goats [START_REF] Chilliard | A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis[END_REF]. This suggests that the ability of CLA to decrease the expression of genes involved in mammary uptake (LPL) is more effective during established lactation than during late lactation in dairy goats.

Among long-chain FA, our results are in agreement with those of [START_REF] Shingfield | Effect of calcium salts of a mixture of conjugated linoleic acids containing trans-10, cis-12 in the diet on milk fat synthesis in goats[END_REF] and [START_REF] Lock | A conjugated linoleic acid supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat synthesis in lactating goats[END_REF] who reported that the reduction in milk fat synthesis in goats was associated with a decrease in the proportions of total cis C18:1 and an increase in those of C18:0 and total trans C18:1. However, in our study, the increase in total trans C18:1 was not significant, reflecting only small changes in individual isomers of trans C18:1, except for an increase in trans-11 C18:1 proportion. This differs from most reports in dairy cows and goats where trans C18:1 were generally increased. Among cis C18:1 FA, only the proportion of the cis-9 isomer was reduced, which indicates a decrease in the activity of the mammary stearoyl-CoA desaturase as reflected first by the increase in C18:0 and second by the decrease in the calculated desaturase indices ratios that are a proxy of the stearoyl-CoA desaturase activity. Reductions in mammary desaturase indices have been reported with high doses of CLA in lactating cows [START_REF] Peterson | The inhibitory effect of trans-10, cis-12 CLA on lipid synthesis in bovine mammary epithelial cells involves reduced proteolytic activation of the transcription factor SREBP-1[END_REF] and sheep [START_REF] Sinclair | A supplement containing trans-10, cis-12 conjugated linoleic acid reduces milk fat yield but does not alter organ weight or body fat deposition in lactating ewes[END_REF], but not at low dosage [START_REF] Maxin | Combined effects of trans-10,cis-12 conjugated linoleic acid, propionate, and acetate on milk fat yield and composition in dairy cows[END_REF], although this is not a prerequisite for the decrease in milk fat yield [START_REF] Peterson | The inhibitory effect of trans-10, cis-12 CLA on lipid synthesis in bovine mammary epithelial cells involves reduced proteolytic activation of the transcription factor SREBP-1[END_REF][START_REF] De Andrade | Effect of duodenal infusion of trans10,cis12-CLA on milk performance and milk fatty acid profile in dairy goats fed high or low concentrate diet in combination with rolled canola seed[END_REF].

CONCLUSIONS

Conjugated linoleic acid supplementation (45 g/d) in goats fed a high-concentrate diet based on corn silage had no effect on DMI, BW, milk yield, or milk protein and lactose yields and contents, but it increased NEB. Conjugated linoleic acid supplementation significantly decreased milk fat content and yield, the secretion of short-chain and medium-chain FA, and to a lesser extent, the secretion of long-chain FA. In goats that had adequate energy intake in midlactation, the response of adipose tissue to an ISOP challenge was unaltered by CLA.

  Influence of a supplement containing conjugated linoleic acid on dairy performance, milk fatty acid composition, and adipose tissue reactivity to lipolytic challenge in mid-lactation goats Souzan Ghazal, Valérie Berthelot, Nicolas N.C. Friggens, Philippe Schmidely To cite this version: Souzan Ghazal, Valérie Berthelot, Nicolas N.C. Friggens, Philippe Schmidely. Influence of a supplement containing conjugated linoleic acid on dairy performance, milk fatty acid composition, and adipose tissue reactivity to lipolytic challenge in mid-lactation goats. Journal of Dairy Science, 2012, 95 (12), pp.7308. 10.3168/jds.2012-5808. hal-01521001

Figure 1 .

 1 Figure 1. Milk FA secretion (mmol/d) in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C18:2). Fatty acids are categorized according to metabolic origin: <C16 synthesized de novo, C16:0 + C16:1, and >C16 taken up from circulating plasma lipids. Values presented are least squares means, with data measured during wk 2 as covariates. ***P < 0.001; × indicates P = 0.07. Error bars indicate standard deviations.

Figure 2 .

 2 Figure 2. Mean plasma concentrations (a: glucose, b: NEFA, c: BHBA, and d: insulin) in response to isoproterenol challenge (4 nmol/kg of BW) in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C18:2). Standard errors of the means for glucose, NEFA, BHBA, and insulin were 0.014 g/L, 16.82 μmol/L, 2.65 mg/L, and 0.086 μg/L, respectively.

Table 1 .

 1 Fatty acid profile and contents of the basal diet

	Item	Basal diet
	FA profile, % of total FA
	C14:0	2.23
	C16:0	23.34
	C18:0	3.79
	cis-9 C16:1	0.49
	cis-9 C18:1	27.41
	cis-9,cis-12 C18:2	32.48
	cis-9,cis-12,cis-15 C18:3	3.33
	FA, mg/g of DM
	C14:0	0.031
	C16:0	0.324
	C18:0	0.053
	cis-9 C16:1	0.007
	cis-9 C18:1	0.381
	cis-9,cis-12 C18:2	0.451
	cis-9,cis-12,cis-15 C18:3	0.046
	Total FA	1.29

Table 2 .

 2 Live BW, DMI, net energy balance, and milk production and composition in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C18:2) 1

		Treatment			P-value
	Item	CTL (n = 12)	CLA (n = 12)	SEM	CLA	Time (T)	CLA × T
	Live BW, kg	60.2	61.8	1.714	0.51	0.0004	0.89
	DMI, kg/d	3.04	3.01	0.096	0.81	<0.0001	0.71
	Milk production, kg/d	3.82	3.74	0.14	0.18	<0.0001	0.91
	Milk yield, g/d
	Fat	175.8	144.7	7.5	<0.0001	<0.0001	0.80
	Protein	122.3	117.1	4.11	0.14	<0.0001	0.95
	Lactose	190.6	188.2	6.93	0.52	0.76	0.76
	Milk composition, g/L
	Fat	45.8	38.9	1.37	<0.0001	<0.0001	0.82
	Protein	32.1	31.6	0.53	0.16	0.28	0.60
	Lactose Energy balance, 2 kcal/d	48.6 -82	48.8 177	0.34 97.89	0.44 0.02	0.80 0.14	0.91 0.88

C18:2/trans-11 C18:1 by 33.3% 27.6, and 21.1% respectively. The CLA treatment increased the sum of >C16 FA by 7.2% (Table

Table 3 .

 3 Summation of some groups of milk FA (g/100 g of milk fat) in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C18:2) 1 Values presented are least squares means, with data measured during wk 2 as covariates. Full details of milk FA composition are given in the Appendix.

		Treatment			P-value
	Item	CTL (n = 12)	CLA (n = 12)	SEM	CLA	Time (T)	CLA × T
	∑SFA 2 ∑iso 3	72.49 0.97	72.56 1.06	0.41 0.04	0.41 0.01	0.82 <0.0001	0.04 0.17
	∑cis C18:1	13.44	11.79	0.51	0.001	0.55	0.61
	∑trans C18:1 ∑MUFA 4 ∑PUFA 5	5.62 19.3 4.32	6.23 20.1 4.32	0.46 0.61 0.12	0.32 0.23 0.98	0.31 0.38 0.78	0.08 0.06 0.06
	∑ C <16 ∑C16:0 + C16:1 6	38.13 28.99	37.30 27.56	0.54 0.57	0.29 0.05	0.37 0.0002	0.08 0.45
	∑ C >16	31.48	33.74	0.59	0.02	0.46	0.22
	Desaturation ratio
	cis-5 C14:1/C14:0	0.012	0.008	0.008	0.0001	0.99	0.98
	cis-9 C18:1/C18:0	1.96	1.42	0.51	<0.0001	0.45	0.57
	cis-9,trans-11 C18:2/trans-11 C18:1	0.38	0.30	0.02	0.02	0.60	0.22
	1

Table 4 .

 4 Changes in plasma concentrations of glucose, NEFA, BHBA, and insulin to isoproterenol (ISOP) challenge (4 nmol of ISOP/kg of BW) in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C18:2) 1

		Treatment		P-value
	Item	CTL (n = 6)	CLA (n = 6)	SEM	CLA
	Net energy balance, kcal/d	373	662	148	0.03
	BW, kg	61.63	62.82	2.93	0.85
	Baseline concentration
	Glucose, g/L	0.586	0.601	0.019	0.72
	NEFA, μmol/L	178.3	157.6	14.24	0.49
	BHBA, mg/L	42.3	51.1	3.39	0.21
	Insulin, μg/L	0.236	0.415	0.06	0.15
	Glucose:insulin ratio Maximal concentration 2	3.15	1.77	0.395	0.08
	Glucose, g/L	0.729	0.738	0.019	0.83
	NEFA, μmol/L	311.3	322.3	21.56	0.81
	BHBA, mg/L	28.3	36.7	2.42	0.08
	Insulin, μg/L	0.662	0.818	0.072	0.30
	Increase above basal value
	Glucose, g/L	0.128	0.151	0.020	0.58
	NEFA, μmol/L	133.1	164.8	13.5	0.26
	BHBA, mg/L	-14.1	-14.4	1.69	0.91
	Insulin, μg/L Changes in AUC 3	0.426	0.403	0.034	0.76
	Glucose, mg/L × 25 min	1.76	2.65	0.42	0.31
	NEFA, μmol/L × 25 min	1,019.4	1,416.5	174.3	0.27
	BHBA, mg/L × 25 min	-162.5	-121.2	31.07	0.53
	Insulin, μg/L ×25 min Total AUC 4	5.02	4.74	0.87	0.88
	Glucose, mg/L × 25 min	29.1	29.4	0.02	0.86
	NEFA, μmol/L × 25 min	9,435	8,948	684	0.74
	BHBA, mg/L × 25 min	1,723	2,160	133	0.10
	Insulin, μg/L × 25 min Glucose:insulin ratio, 5 × 25 min	17 1.88	24 1.38	2.4 0.16	0.13 0.11

1

Values represent mean response to ISOP (4 nmol of ISOP/kg of BW) challenge. 2

Journal of Dairy Science Vol. 95 No. 12, 2012

Values represent least squares means, with data obtained during wk

as covariates. 2 Net energy balance during the trial = (net energy intake + net energy value of the CLA or Ca salt of palm oil) -(NE M + NE L ).

Total AUC was calculated as the area from -20 into 25 min relative to ISOP injection.

Ratio of total AUC of glucose:total AUC of insulin (between -20 and +25 min relative to ISOP injection).

ACKNOWLEDGMENTS

We are grateful to J. Teissier (INRA, UMR791 Modélisation Systémique Appliquée aux Ruminants, Paris, France) and his team for obtaining research data on goats. We thank O. Dhumez, F. Ternois, and L. Péricard (all of INRA, UMR791) for the analysis of the samples.

APPENDIX

Table A1. Milk FA composition (g/100 g of milk fat) in lactating goats fed 45 g/d of Ca salts of palm oil (control, CTL) or 45 g/d of conjugated linoleic acid (CLA; mixture of 2 isomers: trans-11,cis-9 C18:2 and trans-10,cis-12 C8:2)


DIGESTIVE EFFICIENCY IN RABBIT DOES ACCORDING TO ENVIRONMENT AND GENETIC TYPE

Keywords: rabbit does, heat stress, dietary energy, genetic selection, digestibility

Ninety lactating rabbit does of 3 different genetic types [2 from a line differentiating 20 generations by selection for litter size at weaning (V16 and V36) and 1 from a line founded under reproductive longevity criteria and then selected by litter size at weaning (LP)] were subjected to 3 environmental conditions: NC, females housed under normal conditions (14 to 20°C) and fed with a control diet [333 g neutral detergent fibre (NDF)/kg dry matter (DM)]; HC, females housed under heat conditions (25 to 35°C) and fed with the control diet; or NF, females housed under normal conditions and fed with a fibrous diet (443 g NDF/kg DM). The apparent digestible coefficients of dry matter (DMd), organic matter, crude protein (CPd), gross energy, NDF (NDFd) and acid detergent fibre, as well as the daily intake of DM, digestible protein and digestible energy (DE), were determined (14 to 18 d post-partum). The environment affected all variables analysed. In general, heat conditions reduced the daily DM intake (around -30%; P<0.05) and increased main apparent digestible coefficients (+4.5 percentage points for DMd). In contrast, the use of a fibrous diet led to reduce DE intake (-217 kJ/d; P<0.05) and main apparent digestibility coefficients (-13.5 percentage points for DMd). Females from line V, regardless of generation, showed lower daily DM intake (-19.2 g/d; P<0.05) and NDFd (-1.5 percentage points; P<0.05) than line LP. Interactions between genetic type and environment were found for daily DM intake, NDFd and CPd. When receiving fibrous diet, LP females showed a higher increment in daily DM intake (+65.6 g/d; P<0.05) than V36 females, compared to control. Under heat conditions, NDFd obtained for LP females were higher to those in normal conditions (+3.14 percentage points), while V females showed similar NDFd. In addition, the increase in CPd observed under heat conditions was higher for LP (+9.87 percentage points) and V36 (+8.74 percentage points) than V16 females (+3.84 percentage points). In conclusion, rabbit females from a line founded for reproductive longevity seem to show a higher flexibility in their digestive capacity under constrained conditions.

INTRODUCTION

Selection for litter size at weaning (LSW) in reproductive rabbit has not only succeeded in improving the target trait, but also changes important physiological aspects in the animals. In this sense, [START_REF] Quevedo | Effect of selection for litter size and feeding programme on the performance of young rabbit females during rearing and first pregnancy[END_REF] described a possible better effi cacy in the digestible energy use for foetal growth and even an improvement in feed intake capacity at early lactation [START_REF] Quevedo | Long-term effect of selection for litter size and feeding programme on the performance of reproductive rabbit does. 2. Lactation and growing period[END_REF], both changes directly related to the selection objective.

On the other hand, the foundation of a rabbit line by screening for females with very long reproductive career [START_REF] Sánchez | Constitution and evaluation of a long-lived productive rabbit line[END_REF]) resulted in animals with different capacity to manage the resources along their reproductive life. In fact, this strategy resulted in animals well adapted to manage the resources under adverse environmental conditions (i.e. high temperatures and feed restriction). They delayed reproductive senescence [START_REF] Theilgaard | Late reproductive senescence in a rabbit line hyper selected for reproductive longevity, and its association with body reserves[END_REF] and improved their lifespan [START_REF] Theilgaard | Effect of body fatness and selection for prolificacy on survival of rabbit does assessed using a cryopreserved control population[END_REF]Sanchez et al., 2008), resulting in more robust animals [START_REF] Theilgaard | Differences in productive robustness in rabbits selected for reproductive longevity or litter size[END_REF]. All of these findings could be related to a better use of the energy resources available to cope with the reproduction effort, as hypothesised by [START_REF] Pascual | Genetic selection of maternal lines and digestive efficiency in rabbits: long term selection for litter size at weaning versus hyper selection for reproductive longevity[END_REF].

The current knowledge shows that maternal rabbit lines founded under different criteria but which had been selected for the same trait (i.e. LSW) are different for the selected trait [START_REF] Ragab | A comparison of reproductive traits of four maternal lines of rabbits selected for litter size at weaning and founded on different criteria[END_REF], and previous works demonstrated that they adopted different fitness strategies when the environmental conditions were not at all suitable.

Therefore, the aim of the present work was to evaluate how selection for LSW (comparing animals separated by 20 generations of selection) or the foundation for hyper-reproductive longevity criteria could have affected female digestive efficiency under normal and constrained environmental conditions (heat or nutritional challenges).

MATERIALS AND METHODS

The experiment was designed and carried out according to the European Union recommendations on the protection of animals used for scientific purposes (European Union, 2010) and followed the guidelines for applied nutrition experiments in rabbits [START_REF] Fernández-Carmona | Recomendations and guidelines for applied nutrition experiments in rabbits[END_REF].

Animals

Ninety lactating rabbit females in first or second lactation were used to evaluate the apparent digestibility of animals belonging to 3 genetic types and kept under 3 different environmental conditions. The genetic types consisted of animals from a line founded under hyper-reproductive longevity criteria (see foundations details at [START_REF] Sánchez | Constitution and evaluation of a long-lived productive rabbit line[END_REF] and then selected for LSW over 6 generations (line LP), and animals from line V, selected for LSW [START_REF] Estany | Mixed model methodology for estimation of genetic response to selection in litter size of rabbits[END_REF] and belonging to generations 16 and 36 (V16 and V36, respectively).

Diets

Two diets differing in energy, mainly modifying the forage content, were formulated and pelleted (Table 1). The control diet (C) was formulated following the recommendations of de [START_REF] Blas | Feed formulation[END_REF] for lactating rabbit does [having on average 11.7 MJ of digestible energy (DE), 120 g digestible protein (DP), 168 g acid detergent fibre (ADF) and 333 g neutral detergent fibre (NDF) per kg dry matter (DM)]. A second fibrous low energy diet (F) was designed to induce a nutritional challenge (to have on average 9 MJ DE, 105 g DP, 266 g ADF and 443 NDF per kg DM).

Experimental procedure

At parturition, the rabbit does were housed in individual metabolic cages and randomly allocated to 1 of the 3 different environments resulting from the combination of environmental temperature and the diet received. Two room temperatures were used: a climatic chamber set up to obtain heat conditions (H) by means of a daily sigmoid temperature curve with a range from 25 to 35°C (see description by [START_REF] García-Diego | Technical note: design of a large variable temperature chamber for heat stress studies in rabbits[END_REF] and a normal conditions room (N) with a temperature range between 14 and 20°C. All animals housed at H received diet C while half of the animals placed at N were fed on diet C and half on diet F. As a result, the tested environments were HC (heat conditions with diet C), NC (normal conditions with diet C) or NF (normal conditions with diet F). Table 2 summarises the experimental groups in terms of genetic type, environmental temperature and parturition order. Diets and water were provided ad libitum throughout the experiment, the daylight scheme was a 16 h light and 8 h dark period and the animals were artificially inseminated at day 11 post-partum.

Litters were standardised to 9 and 10 kits at first and second parturition, respectively, to compare the digestive efficiency at similar productive effort. Rabbit does were transferred daily to the production cage to suckle their respective litters. At 14 d of lactation, similar live weight were observed for the different animal types (on average 3 837±51 g), although females housed under heat conditions were slightly lighter (-197±73 g; P<0.05). Feed intake and faecal collections were recorded daily just after the milk production measurement (using the does double weight method) from the 14 th to 18 th day of lactation. This period was selected to perform the digestibility trial, as it corresponds to the period of maximum milk yield in rabbit does [START_REF] Casado | Modelling the lactation curve of rabbit does: Towards a model including fit suitability and biological interpretation[END_REF] and is characterised by a regular females' feed intake. The chemical analyses of diets and faeces for DM, crude protein (CP), ADF, acid detergent lignin (ADL) and ash followed the AOAC methods (934.01, 976.05, 973.18 and 942.05, respectively;[START_REF] Aoac | Official methods of analysis of the Association of Official Analytical Chemists[END_REF]. NDF was analysed by the method described by [START_REF] Mertens | Gravimetric determination of amylasetreated neutral detergent fibre in feeds with refluxing beakers or crucibles: collaborative study[END_REF] and the gross energy (GE) was determined in adiabatic bomb as recommended by [START_REF] Egran | Technical note: attempts to harmonize chemical analyses of feeds and faeces, for rabbit feed evaluation[END_REF].

Statistical analysis

Data on the apparent digestibility coefficients (d) of DM, organic matter (OM), CP, GE, NDF, ADF and insoluble hemicelluloses (HEM=NDF-ADF), as well as the daily intake of DM (DMI), DP (DPI) and DE (DEI), were analysed using the GLM procedure of SAS software (SAS Institute, 2009). The model included as fixed effects the genetic type (G i , 3 levels), the environment (E j , 3 levels) and their interactions. Although the trial was not designed to study the parturition order effect (PO k , 2 levels), it was treated as a fixed effect, increasing the hypothesis test exigency. No interactions and no inference were performed with respect to PO k . The model used was: Finally, for variables where G i ×E j interaction was declared significant, the means comparisons were performed using a t-test.

Y ijk =G i +E j +PO k +G i ×E j +e ijk

RESULTS AND DISCUSSION

The environment affected all the variables studied (Table 3). Animals submitted to the heat challenge (HC) showed a lower DMI, DPI and DEI (34.3,24.3 and 29.6%, respectively; P<0.05), while their main apparent digestibility coefficients (DMd, OMd, CPd, GEd and ADFd) increased (from 4 to 7 percentage points; P<0.05) in comparison to animals kept at NC. As recently reviewed by [START_REF] Cervera | Nutrition and the climatic environment[END_REF], high temperatures usually depress feed intake of rabbits and consequently increase the apparent digestibility coefficients of main nutrients. A decrease in the amount of feed eaten normally leads to a lower passage rate, hence the ingested feed is exposed to the action of digestive enzymes for a longer period, thereby increasing digestibility of nutrients [START_REF] Carabaño | The digestive system of the rabbit[END_REF].

The only nutrient whose apparent digestibility coefficient not increased at high environmental temperature was insoluble hemicellulose (HEM 40.6 and 42.4% for HC and NC groups,respectively;P<0.05). This discordant response is difficult to explain from our results. In any case, [START_REF] Gidenne | Microbial activity in the caecum of the rabbit around weaning: impact of a dietary fibre deficiency and of intake level[END_REF], and [START_REF] Gidenne | Feed restriction strategy in growing rabbit. 1. Impact on digestion, rate of passage and microbial activity[END_REF] described similar bacterial fibrolytic activity independently of the feed intake level, suggesting that digestibility of some cell-wall constituents is not substrate-dependent. Moreover, [START_REF] Hannah | Effects of increasing dietary levels of endophyteinfected tall fescue seed on diet digestibility and ruminal kinetics in sheep[END_REF] observed that NDF digestibility was also similar in sheep housed at 27 to 34°C.

On the other hand, animals at NF showed higher DMI and DPI (17.3 and 6.6%, respectively; P<0.05) and a reduction in the digestibility (DMd, OMd, CPd, GEd and NDFd; P<0.05) except for ADFd, which was higher (3.6 percentage points; P<0.05) compared to those at NC. DMI, clearly increased by the use of a high fibre low energy diet during lactation, agrees with the literature [START_REF] Fernández-Carmona | Effect of diet composition on the production of rabbit breeding does housed in a traditional building and at 30°C[END_REF][START_REF] Fernández-Carmona | Effect of diet composition on the production of rabbit breeding does housed in a traditional building and at 30°C[END_REF][START_REF] Quevedo | Long-term effect of selection for litter size and feeding programme on the performance of reproductive rabbit does. 2. Lactation and growing period[END_REF][START_REF] Nicodemus | Effect of level of fibre and type of grinding on the performance of rabbit does and their litters during the first three lactations[END_REF], where the response on DMI was related to the dietary DE content. On average, DMI of lactating rabbit does increased about 8% per each MJ of DE decreased in the diet. In spite of this, the DE content of the diet F (about 9.0 MJ/kg DM) did not allow females to compensate the DE intake completely (5.9% lower compared to control; P<0.05).

The reduction of DMd, OMd and GEd (13-14%) in diet F mainly reflects the change in carbohydrate composition, higher fibre and lower starch than diet C, as well as the effect of increased DMI. However, NDFd and ADFd were less affected by the dietary change. The design of a high fibre diet by changing the forage content also involved a change in the fibre sources (Table 1) and, consequently, in the nature of fibre constituents, and this also affected the digestibility of fibre fractions [START_REF] Gidenne | The nutrition of the rabbit[END_REF]. Moreover, the higher addition of unsaturated oil in diet C could have affected its fibre digestibility [START_REF] Fernández | The effect of fat inclusion on diet digestibility in growing rabbits[END_REF].

Regarding the genetic type, a higher DMI was observed for LP in comparison to V16 animals (+26.2 g DM/d; P<0.05) whereas compared to V36 animals it was similar (+3.9%). Similarly, DEI was higher for LP than for V16 animals (3 345.8±77.9 and 3 096.2±78.1 kJ/d, respectively; P<0.05). Moreover, the NDFd was higher (1.4%; P<0.05) for LP than for V36 animals (+1.8%, P<0.05). As observed by [START_REF] Pascual | Genetic selection of maternal lines and digestive efficiency in rabbits: long term selection for litter size at weaning versus hyper selection for reproductive longevity[END_REF] and [START_REF] Theilgaard | Differences in productive robustness in rabbits selected for reproductive longevity or litter size[END_REF], females from LP line showed a higher feed intake (19.21 g DM/d) during lactation period than the V line. However, the difference between V16 and V36 (-14.0 g DM/d) was not statistically significant.

Interactions between genetic type and environment were observed for DMI, NDFd and CPd. DMI of LP animals was significantly higher compared to V16 and V36 animals, but only when fed with diet F (+47.9±16.6 and +57.4±17.1 g DM/d, respectively; P<0.05) (Figure 1a). Furthermore, the higher NDFd observed for LP animals was mainly due to the increase observed under heat challenge (HC) relative to normal room temperature (NC) (+3.1±1.3 percentage points; P<0.05), which was not observed for V animals (Figure 1b).

In the current study, only the NDFd was higher for the LP line, relative to line V. However, [START_REF] Pascual | Genetic selection of maternal lines and digestive efficiency in rabbits: long term selection for litter size at weaning versus hyper selection for reproductive longevity[END_REF] observed better digestibility of DM, OM and GE in favour of line V. This difference in the NDFd in the present study did not seem to be a direct response to differences in feed intake, because the highest NDFd occurred in animals submitted to a heat challenge (Table 3), where feed intake was similar among genetic types (208.0±12.7, 194.2±11.5 and 217.6±11.5 g DM/d for LP, V16 and V36, respectively). In fact, the reduction in DMI of line LP at HC to similar levels of line V could explain these results, because the passage rate would also decrease, promoting an increase in fibre digestibility. Generations 16 and 36 of line V presented similar raw results for this variable, but a possible different pattern in restricted environments (especially at high temperatures) should be confirmed.

Finally, Figure 1c shows the genetic-type×environment interaction for CPd as changes due to heat and nutritional challenges (HC and NF, respectively) compared to NC. Heat challenge led LP animals to an increase of CPd significantly higher than that observed for V16 animals (9.9±1.6 vs. The genetic type fixed effect per se did not affect the CPd but the environment did (+7.5 % under heat challenge and -4.0% for a high fibre-low energy diet). However, there was a significant genetic type×environmental interaction. Generation 16 of line V presented a high CPd at NC compared to generation 36 and line LP (+6.6%; P<0.05). However, under the constrained environment, this difference disappeared, which suggests that line LP and generation 36 of line V adjusted the CPd better in unfavourable conditions. [START_REF] Theilgaard | Late reproductive senescence in a rabbit line hyper selected for reproductive longevity, and its association with body reserves[END_REF]2009) described the flexibility of LP line, especially in constrained environments and later in productive life. In summary, the LP line seems to have a greater ability to obtain resources when the environment is not suitable, which is manifested in its higher DMI, when fed a high fibre low-energy diet (NF), in the better digestibility of fibre under heat stress (HC), and the advantage in CPd (both under HC and NF environment). All of this suggests that the foundational criteria applied to line LP also selected animals with a greater digestive flexibility.

On the other hand, digestive efficiency of females did not seem to be affected after 20 generations of selection for litter size. Although females more selected for this criteria could have increased their ability to obtain resources at the beginning of lactation, as observed by [START_REF] Quevedo | Long-term effect of selection for litter size and feeding programme on the performance of reproductive rabbit does. 2. Lactation and growing period[END_REF], and which could be also guessed from our results, digestive efficiency for main nutrients was not changed to any great extent. However, the higher DEI reduction of more selected animals under a nutritionally constrained environment (NF) could be related to a higher sensitivity to that environment, which must be studied in further works.

CONCLUSIONS

Both heat and the nutritional challenges affected the feed intake (reduction and increasing, respectively) and consequently the apparent digestible coefficients (improved and decreased, respectively). The selection over 20 generations for litter size at weaning was not a factor improving the feed intake capacity or the digestibility of the animals, but did improve the flexibility under constrained conditions. The foundation criteria for long-productive animals followed by a selection for litter size at weaning was effective, improving the feed intake capacity without reducing the digestive capacity and flexibility under constrained conditions. These patterns seem to contribute to a longer productive lifespan.

  3.8±1.6 percentage points, respectively; P<0.05), with V36 animals showing an intermediate increment (8.7±1.6). However, in NF environment, V16 showed a greater reduction on CPd (-6.1±1.7 percentage points; P<0.05), while V36 females did not seem to be affected when fed with diet F (-1.7±1.7 percentage points) compared to NC environment. Females from line LP showed an intermediate reduction in CPd with diet F (-4.2±1.6%) compared to diet C.

Figure 1 :

 1 Figure 1: Change on (a) dry matter intake, (b) apparent digestibility coefficients of neutral detergent fibre and (c) crude protein caused by the heat (HC-NC) or nutritional (NF-NC) challenges respect to the control conditions for the different genetic types (£ LP, ¢ V16 and ¢ V36). LP: line constituted for longevity-productive criteria; V16 and V36: line V selected for litter size at weaning during 16 and 36 generations. HC: heat conditions (25-36ºC) and diet C (11.7 MJ digestible energy/kg DM); NC: normal conditions (14-20ºC) and diet C; and NF: normal conditions and diet F (9 MJ digestible energy/kg DM)]. Error bars represent the standard errors. a, b: means for the different genetic types on same environment not sharing letters were significant different (P<0.05).

Table 1 :

 1 Ingredients and chemical composition of the experimental diets.

	Ingredients (g/kg)	Control diet (C)	Fibre diet (F)
	Barley grain	315	0
	Wheat bran	50	100
	Sunflower meal (30% CP)	100	52
	Soybean meal (44% CP)	101	0
	Alfalfa hay	370	660
	Sugar beet pulp	0	138
	Soybean oil	40	10
	L-Lysine HCl	0.45	2.8
	DL-Methionine	0.95	2.6
	L-Threonine	0.6	1.6
	Arginine	0	0.8
	Cycostat 66G ®1	1	1
	Bacipremix 50 ®2	2	2
	Calcium carbonate	1	0
	Dicalcium phosphate	10	0
	Monosodium phosphate	0	21.2
	Sodium chloride	4	4
	Vitamin/mineral mixture 3	4	4
	Chemical composition (g/kg DM)
	Dry matter (DM; g/kg)	951	938
	Organic matter	917	881
	Neutral detergent fibre	333	443
	Acid detergent fibre	168	266
	Acid detergent lignin	25	44
	Crude protein (CP)	175	162
	Gross energy (MJ/kg DM)	18.69	18.31

1 Alpharma, Antwerp (Belgium), provides 66 ppm of robenidine. 2 Andreés Pintaluba SA, Reus (Spain), provides 100 ppm of zinc bacitracin. 3 Contains (g/kg): thiamine, 0.25; riboflavin, 1.5; calcium pantothenate, 5; pyridoxine, 0.1; nicotinic acid, 12.5; retinol, 2; cholecalciferol, 0.1; α-tocopherol, 15; phytylmenaquinone, 0.5; cyanobalamin 0.0006; choline chloride, 100; MgSO 4 H 2 O, 7.5; ZnO, 30; FeSO 4 7H 2 O, 20; Cu SO 4 5 H 2 O, 3; KI, 0.5; CoCl 2 6 H 2 O, 0.2; Na 2 SeO 3 , 0.03.

Table 2 :

 2 Number of animals (n) according to environment condition and genetic type by parity order. Environment: HC, heat conditions (25-35°C) and diet C (on av. 11.7 MJ digestible energy/kg dry matter); NC, normal conditions (14-20ºC) and diet C; and NF, normal conditions and diet F (on av. 9 MJ digestible energy/kg dry matter). 2 Genetic type: LP, line constituted for longevity-productive criteria; V16 and V36, line V selected by litter size at weaning during 16 or 36 generations.

	Environment 1		HC			NC			NF
	Genetic type 2	LP	V16	V36	LP	V16	V36	LP	V16	V36
	Parity order
	First	5	6	6	5	6	6	6	5	5
	Second	4	5	5	5	3	4	5	5	4
	Total	9	11	11	10	9	10	11	10	9

1

Table 3 :

 3 Apparent digestible coefficients [dry matter(DMd), organic matter (OMd), crude protein (CPd), gross energy (GEd), neutral detergent fibre (NDFd), acid detergent fibre (ADFd) and insoluble hemicellulose (HEMd)] and daily intake [dry matter (DM), digestible protein (DP) and digestible energy (DE)] according to environment and genetic type of females on lactation (from 14 to 18 d). Genetic type: LP, line constituted for longevity-productive criteria; V16 and V36, line V selected by litter size at weaning during 16 or 36 generations. 3 Contrast within genetic type or environment were calculated on complete data; e.g. LP vs.V=(LPHC+LPNC+LPNF)/3-{[(V16HC+V16NC+V16NF)/3 +(V36HC+V36NC+V36NF)/3]/2}. a, b, c, d Means not sharing superscripts, in rows, are different at P<0.05. Contrasts (standard error) followed by * are significant at P<0.05.

	Contrast 3	LPvs.V LPvs.V16 LPvs.V36 V16vs.V36 HCvs.NC NFvs.NC		0.27 0.10 0.44 0.33 4.51* -13.51*	(0.45) (0.51) (0.51) (0.51) (0.51) (0.52)	0.17 -0.03 0.30 0.27 4.07* -13.96*	(0.43) (0.50) (0.50) (0.50) (0.50) (0.50)	-0.28 -0.78 0.21 0.99 7.48* -3.99*	(0.81) (0.93) (0.94) (0.94) (0.93) (0.95)	0.14 -0.04 0.32 0.36 4.32* -12.93*	(0.49) (0.56) (0.56) (0.56) (0.56) (0.57)	1.47* 1.15 1.79* 0.64 1.15 -3.17*	(0.65) (0.75) (0.75) (0.75) (0.76) (0.76)	0.78 0.40 1.15 0.75 5.08* 3.61*	(0.77) (0.88) (0.90) (0.89) (0.90) (0.90)	1.94* 2.01* 1.86* -0.15 -2.21* -7.26*	(0.73) (0.84) (0.85) (0.84) (0.85) (0.85)		19.2* 26.2* 12.2 -14.0 -107.8* 65.9*	(8.5) (9.9) (9.9) (9.9) (9.9) (9.9)	1.46 2.25 0.67 -1.58 -10.11* 2.51*	(1.1) (1.2) (1.2) (1.2) (1.2) (1.2)	172.6 249.6* 95.6 -154.0 -1084.7* -217.4*	(95) (110) (110) (110) (110) (111)
	NF	V16 V36		49.02 a 48.67 a	49.58 a 49.40 a	64.83 ab 65.10 ab	49.84 a 49.83 a	24.40 a 25.03 ab	17.50 c 17.72 c	34.72 a 36.00 ab		367.5 d 358.0 dc	38.65 bc 38.84 bc	3 355 bc 3 267 b
		LP		48.25 a	48.90 a	63.29 a	48.92 a	24.56 a	17.33 c	35.40 a		415.4 e	42.56 c	3 720 d
	NC	V16 V36		63.03 b 61.52 b	64.08 b 62.74 b	70.89 c 66.83 b	63.29 b 61.74 b	28.55 c 26.59 abc	14.34 ab 13.89 ab	43.11 d 41.24 cd		299.5 b 327.5 bc	37.13 b 38.27 b	3 541 bcd 3 778 d
	Environment 1 HC	Genetic type 2 LP V16 V36 LP	Apparent faecal digestibility coefficients (%)	DMd 67.85 d 65.64 c 66.50 cd 61.90 b	OMd 68.37 d 66.47 c 67.16 cd 62.95 b	CPd 4 77.35 d 74.73 d 75.57 d 67.48 b	GEd 67.80 c 66.03 c 66.52 c 62.33 b	NDFd 4 31.50 d 28.01 c 27.43 bc 28.36 c	ADFd 21.15 d 18.90 cd 16.89 bc 13.48 a	HEMd 43.36 d 38.44 bc 39.47 c 43.55 d	Daily intake	DM (g /d) 4 208.1 a 194.2 a 217.6 a 316.3 b	DP (g /d) 28.16 a 25.30 a 28.73 a 37.11 b	DE (kJ /d) 2 642 a 2 393 a 2 706 a 3 675 cd

1 Environment: HC, heat conditions (25-35°C) and diet C (11.7 MJ digestible energy/kg DM); NC, normal conditions (14-20°C) and diet C; and NF, normal conditions and diet F (9.0 MJ digestible energy/kg DM). 2 4 Interaction environment×genetic type significant (P<0.05).

Acknowledgements:

The authors would like to thanks Luis Ródenas, Eugenio Martínez-Paredes and Juan Carlos Moreno for their technical support and to the Spanish Ministry of Education and Science (Project AGL2011-30170-C02-01) for the economic support to conduct this study.


teaching and research institutions in France or abroad, or from public or private research centers.