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A dose-response study relating the concentration of carotenoid pigments in blood and reflectance spectrum characteristics of fat to carotenoid intake level in sheep¹

P. H. M. Dian^*,2, B. Chauveau-Duriot^*, I. N. Prado and S. Prache^*,3

^* INRA, UR1213 Herbivores, Site de Theix, 63122 St-Genès-Champanelle, France; and and Universidade Estadual de Maringa, Av. Colombo 5790, 87020-900, Bloco 32-Sala 3, Maringa-PR, Brazil

	Abstract

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study was conducted to describe the dose-response curve relating the concentration of carotenoid pigments in plasma and reflectance spectrum characteristics of fat to the carotenoid intake level in sheep, and to investigate the extent to which incorporation of dehydrated alfalfa in the diet affects the reliability of the discrimination between concentrate-fed and pasture-fed lambs based on these measurements. In Exp. 1, 6 treatments were compared in individually penned lambs: feeding 0, 250, 500, 750, 1,000, or 1,250 g/d of dehydrated alfalfa for 60 d before slaughter. Each treatment (T0 to T1,250) consisted of 8 male Romanov x Berrichon lambs with an initial average BW of 24.8 kg (SD 2.6). All lambs received straw for ad libitum intake and T0 to T1,000 lambs received a concentrate free of green vegetative matter in amounts to produce similar ADG in all treatments. In Exp. 2, 33 male Romanov x Berrichon lambs grazed a natural pasture maintained in a leafy green vegetative stage for at least 59 d before slaughter. Initial BW when turning out to pasture was 14.2 kg (SD 2.3). Plasma carotenoid concentration was measured at slaughter by spectrophotometry. Reflectance spectrum, lightness, redness, and yellowness were measured after 24 h of shrinkage in subcutaneous caudal and perirenal fat. The spectra were translated to 0 reflectance at 510 nm, and the integral of the translated spectrum was calculated between 450 and 510 nm (i.e., the range of light absorption by carotenoids). Reflectance measurement was replicated 5 times, from which we calculated the absolute value of the mean integral (AVMI). In Exp. 1, plasma carotenoid concentration at slaughter (PCCS) increased linearly with mean daily carotenoid intake (P < 0.01). Both subcutaneous caudal and perirenal fat AVMI increased linearly (P < 0.01) with mean daily carotenoid intake and PCCS, the slopes of the regressions being greater for perirenal than for subcutaneous caudal fat. The mean PCCS was greater for lambs of Exp. 2 than for lambs on any treatment of Exp. 1 (P < 0.01). We established the dose-response curves relating PCCS and AVMI of subcutaneous and perirenal fat to carotenoid intake level. The combined use of PCCS and of perirenal fat AVMI enabled discrimination of pasture-fed lambs of Exp. 2 from the lambs of Exp. 1 that received up to 500 g/d of dehydrated alfalfa.

Key Words: alfalfa • carcass • carotenoid • fat • reflectance • sheep

	INTRODUCTION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
One of the most important production factors affecting the composition of milk and meat from herbivores is the animal’s diet. Its effects are due to specific compounds that are transferred from the feed to the end product or transformed or produced by ruminal microorganisms or the animal’s metabolism in response to specific diets. In turn, some of these compounds can be used to authenticate the diet (Martin et al., 2005; Prache et al., 2005).

Carotenoid pigments are examples of such compounds. They are involved in the nutritional and sensory properties of herbivore products and have recently proved useful for diet authentication, particularly for distinguishing pasture-fed from grain-fed animals (Prache and Theriez, 1999; Nozière et al., 2006b; Serrano et al., 2006). On this point, Prache and Theriez (1999) proposed a mathematical analysis of the fat reflectance spectrum in the zone of light absorption by carotenoids as an indirect means of estimating the concentration of these pigments to discriminate pasture-fed and stall-fed lamb carcasses. A fat reflectance spectrum measurement is of practical interest because it can easily be implemented in the meat industry via a portable spectrophotometer. This method has been validated in sheep on a large cohort of animals (Dian et al., 2007), and it has been generalized to beef meat (Serrano et al., 2006) and milk (Nozière et al., 2006b). However, the carotenoid concentration in animal tissues can range widely according to supply in the diet.

The purpose of this study was to establish the dose-response curve relating carotenoid concentration in plasma and fat to carotenoid intake level in sheep, and to investigate the extent to which incorporating dehydrated alfalfa in the diet of stall-fed lambs reduces the reliability of the discrimination between concentrate-fed and pasture-fed lambs based on carotenoid concentration in plasma and spectral characteristics of fat in the zone of light absorption by these pigments.

	MATERIALS AND METHODS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The experiments were conducted at the experimental farm of the INRA Center of Clermont-Ferrand Theix, France. The animals were handled by specialized personnel who cared for their welfare in accordance with the European Union Directive No. 609/1986.

Experimental Design, Animals, and Diets
Exp. 1.
Six levels of dehydrated alfalfa were compared: 0, 250, 500, 750, 1,000, and 1,250 g/d of dehydrated alfalfa. Each treatment (T0, T250, T500, T750, T1,000, and T1,250) comprised 8 male Romanov x Berrichon lambs, individually penned indoors and offered the feeding treatment for 60 d before slaughter. Forty-eight lambs were classified into 8 blocks according to birth weight and ADG between birth and the beginning of the experiment. They were then assigned at random from within blocks to 1 of the 6 treatments. Mean lamb birth weight and ADG between birth and the beginning of the experiment were 4.1 kg (SD 0.94) and 309 g/d (SD 51); lambs weighed 24.8 kg (SD 2.6) at the beginning of the experiment. The animals were housed in a sheepfold from birth to slaughter, and they were managed uniformly before the experiment. They were born on November 2, 2004, on average, and they received ad libitum access to commercial concentrate from 3 wk of age until the beginning of the experiment. The composition of the concentrate is listed in Table 1. The dams were also kept indoors, and they received a commercial concentrate containing no green vegetative matter and ad libitum access to hay. Lambs were progressively weaned from December 27 until the beginning of the experiment.

The concentrate offered in Exp. 1 was similar to that fed previously from 3 wk of age. During the first 2 wk of the experiment, the T1,250 lambs also received 100 g/d of the commercial concentrate (as-fed) to bring about a feeding transition and adaptation. Oat straw was supplied ad libitum to all the lambs. Dehydrated alfalfa and straw were offered in the morning to ensure the consumption of the assigned dehydrated alfalfa level, and the concentrate was offered in the afternoon. Feed tubs were emptied every morning, and feed refusals were weighed, recorded, and discarded daily. Samples of offered alfalfa, concentrate, and straw were collected twice weekly for DM and carotenoid concentration estimations.

Exp. 2.
Thirty-three male Romanov x Berrichon lambs born in March were pasture-fed. They grazed, from April 24 until slaughter, a natural pasture that was always maintained at a leafy green vegetative stage. The lambs received no supplementation. The botanical composition of the pasture was (DM basis) Lolium perenne (29.8%), Dactylis glomerata (28.6%), Festuca arundinacea (20.7%), Taraxacum officinale (10.0%), Bromus sterilis (9.9%), Trifolium repens (0.7%), and Ranunculus macrophyllus (0.3%). Lambs weighed 14.2 kg (SD 2.3) when they were turned out to pasture at a mean age of 38 d (SD 1.4). The duration of the grazing period ranged from 59 to 136 d. Most of the lambs were suckled as twins. Weaning took place on June 28. Lambs were slaughtered when they attained a satisfactory degree of fatness, which was manually assessed by skilled technicians according to the method of Russel et al. (1969), to obtain a subcutaneous fat thickness on the cold carcass ranging from 2 to 3 mm.

In both experiments, water and salt blocks were always available. The salt blocks contained (g/kg, as-fed) 60 Ca, 20 P, 10 Mg, 280 Na, 17.5 Zn, 1.5 Fe, 5.5 Mn, 0.03 Co, 0.03 I, and 0.01 Se.

Slaughter Procedures
All the lambs from both experiments were slaughtered. In Exp. 1, the lambs were fed their assigned experimental diet for 60 d, so there were 4 slaughter sessions, on March 7, 9, 14, and 16. Lambs were slaughtered in the morning and were not fed on the day of slaughter. In Exp. 2, lambs were slaughtered between June 22 and September 7. They were transported by truck to the slaughterhouse, which was located within 500 m of the stall and the pasture. Immediately after their arrival, the lambs were slaughtered by cutting their throat. The carcasses were placed in a refrigerated room at 4°C until 24 h postmortem and were always kept in the dark.

Measurements
Carotenoid Concentration in the Feed.
Carotenoids of alfalfa, concentrate, and straw were extracted using the procedure described by Cardinault et al. (2006). Lipophilic components of 50 mg of lyophilized and ground food were first extracted with acetone and then purified with diethyl ether containing echinenone kindly donated by Hoffman La Roche (Basel, Switzerland) as internal standard. After saponification and cleaning with water, carotenoids were then analyzed by HPLC using the method described by Lyan et al. (2001). The HPLC apparatus consisted of a Waters Alliance 2996 HPLC system (Waters S.A., Saint Quentin en Yvelins, France) with photodiode array detector monitoring between 280 and 600 nm. Carotenoids were separated on a 150 x 4.6 mm, RP C18, 3 µm, Nucleosil column coupled with a 250 x 4.6 mm, RP C18, 5 µm, Vydac TP 54 column (Interchim, Montluc on, France). Millennium 32 software from Waters SA (Saint Quentin en Yvelines, France) was used for instrument control, data acquisition, and data processing. Wavelength detection for carotenoids was at 450 nm, and the compounds were identified by comparing retention times and spectral analyses with those of pure standard (>95% of zeaxanthin gas and 13-as ß-carotene kindly donated by Hoffman La Roche and of all-trans ß-carotene and lutein (Sigma Chemical Co., St. Louis, MO). Concentrations of each compound were calculated using external standard curves and were then adjusted by percent recovery of the added internal standard.

Plasma Carotenoid Concentration.
Plasma carotenoid concentration was measured for all lambs at slaughter. Blood samples were taken from the jugular vein of each lamb at 0800 (lithium heparin purchased from Consortium du Material pour Laboratoires, Nemours, France). Plasma was stored at –20°C until required for assay. Extraction of carotenoids from plasma was performed within 3 mo after sampling.

Crude estimation of total carotenoids was obtained by a spectrophotometric procedure using the following method. Protein from 3 mL of plasma diluted with 2 mL of distilled water was precipitated with 4 mL of ethanol. Carotenoids were then extracted with 4 mL of hexane. Absorption of the upper layer obtained after centrifugation at 5,000 x g for 5 min was measured between 600 and 400 nm using a Kontron Uvikon 860 recording spectrophotometer (Kontron Instruments S.A., Montigny-le-Bretonneux, France). The concentration of total carotenoids was calculated from absorption maxima (Karijord, 1978), assuming a value of 2,500 for the E1% extinction coefficient (Patterson, 1965; Karijord, 1978) and allowing for the dilution of the original sample. Care was taken throughout the experimental and analytical procedure to protect samples from natural light (i.e., samples and test tubes were wrapped in aluminum foil to keep light out and extraction under dim artificial light).

Animal Characteristics at Slaughter.
Lambs were weighed just before slaughter. Carcass weight, perirenal fat weight, and subcutaneous fat thickness were measured after 24 h of shrinkage. Perirenal fat together with kidneys was removed from both carcass halves. The fat was separated from the kidneys using a knife and then weighed. The cold carcass was weighed before removal of these items. Subcutaneous fat thickness was measured by making 2 incisions through the fat along lines extending 4 cm ventrolaterally from the dorsal midline at the last rib and, at the limit of that cut, extending 4 cm cranially. A flap of fat was raised, and subcutaneous fat thickness was measured at the intersection of the incisions (Fisher and de Boer, 1994).

Reflectance Spectrum of Perirenal and Caudal Fat.
We measured the reflectance spectra of subcutaneous caudal and perirenal fat at wavelengths between 700 and 400 nm and color coordinates expressed as lightness (L*), redness (a*), and yellowness (b*) in the CIELAB uniform color space (CIE, 1986), using a Minolta CM- 2002 spectrophotometer (illuminant D₆₅, observer angle 10°, Minolta France S.A., Carrieres sur Seine, France). The instrument was fitted with protective glass to shield the eye of the apparatus from the fat sample. This apparatus measures the proportion of light reflected every 10 nm. Measurements were made after a 24-h shrinkage. For perirenal fat, a plane surface was obtained with a knife to allow perfect adherence of the fat to the eye of the apparatus. For each site of fat deposition, 5 measurements were made.

Data Analysis
The reflectance spectrum between 510 and 450 nm was translated to make the reflectance value at 510 nm equal to zero (TR). On the translated spectrum, the integral value (I_450–510) was calculated as follows:

The integral value was averaged over the 5 measurements. The mean integral values were all negative; hereafter we use the absolute value of the mean integral (AVMI). The variance of plasma carotenoid concentration at slaughter (PCCS, µg/L) was stabilized using the natural logarithmic transformation.

The data were subjected to ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC) to examine the effect of the feeding treatment and animal block. We used the Newman-Keuls multiple range test for pairwise comparisons. Regression analyses were carried out using the GLM procedure of SAS to examine whether the responses of PCCS, of AVMI, and of b* value of the fat to the feeding treatment had a linear or a curvilinear relation (quadratic effect) to the mean daily carotenoid intake level (MDCI, mg). Because lutein is the only carotenoid stored in the fat of sheep (Yang et al., 1992; Prache et al., 2003a), we also examined whether the response of AVMI and of b* value of the fat to the feeding treatment had a linear or a curvilinear relation (quadratic effect) to the mean daily lutein intake level (MDLI, mg). Regression analysis of AVMI and b* value of the fat on PCCS was also carried out.

	RESULTS

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animal characteristics in Exp. 1 are summarized in Table 2. Average daily gain from birth to slaughter, age at slaughter, and perirenal fat weight did not differ among treatments (P = 0.25, 0.08, and 0.08, respectively). Slaughter weight differed among treatments (P< 0.01), but carcass weight did not (P = 0.53). Subcutaneous fat thickness was lower in T1,000 than in T0 (2.1 vs. 3.5 mm, P < 0.05).

Carotenoid Intake Levels
In Exp. 1, dehydrated alfalfa, concentrate, and straw contained 883, 871, and 872 g of DM/kg and 280.6, 2.3, and 2.0 µg of carotenoid/g of DM, respectively (Table 3). Lutein was the predominant carotenoid (71.3, 91.3, and 100% of total carotenoid pigments in dehydrated alfalfa, concentrate, and straw, respectively). Mean daily intake of dehydrated alfalfa was 99.2, 99.0, 99.9, 98.1, and 95.8% of the assigned levels for T250, T500, T750, T1,000, and T1,250, respectively (Table 4). The differences between actual and assigned levels occurred mainly at the beginning of the experiment. The MDCI and MDLI differed among feeding treatments (P < 0.01) and increased as expected with the mean daily dehydrated alfalfa intake level.

[1]

where r² = 0.98, residual SD (RSD) = 3.4, and n = 6. The intercept was not different from 0 (P = 0.88).

In Exp. 2, PCCS averaged 112 µg/L (SD 52), ranging from 43 to 258 µg/L. The mean natural logarithm of PCCS was greater in Exp. 2 than for any treatment of Exp. 1 (P < 0.01). There was no overlapping in the frequency distribution of the PCCS of pasture-fed lambs in Exp. 2 and lambs receiving up to 500 g/d of dehydrated alfalfa in Exp. 1. Half of the T750 lambs had values greater than 43 µg/L. The majority of T1,000 and T1,250 lambs (13 of 16) had PCCS greater than 43 µg/L, the lowest value observed in pasture-fed lambs.

Reflectance Spectrum Between 450 and 510 nm and Color of Fat
In Exp. 1, the AVMI of subcutaneous caudal fat ranged from 114 to 236, 132 to 269, 160 to 286, 134 to 325, 218 to 381 and 218 to 391 units for T0, T250, T500, T750, T1,000, and T1,250, respectively. The AVMI of perirenal fat ranged from 79 to 214, 111 to 269, 163 to 266, 232 to 354, 264 to 373, and 183 to 411 units for T0, T250, T500, T750, T1,000, and T1,250, respectively.

For both subcutaneous caudal and perirenal fat, AVMI were affected by treatment (P < 0.01 for both sites; Table 4). The AVMI of subcutaneous caudal fat was greater for T1,000 and T1,250 than for T0 and T250; it was greater for T1,000 than for T0, T250, and T500. The AVMI of perirenal fat was greater for T750, T1,000, and T1,250 than for T0, T250, and T500. For subcutaneous caudal and perirenal fat, AVMI did not differ significantly among T750, T1,000, and T1,250. The mean AVMI had linear relationships to MDCI (P < 0.01 for both fat sites; Table 4), without significant quadratic effects (P = 0.83 for subcutaneous fat and P = 0.35 for perirenal fat). Similarly, mean AVMI had linear relationships to MDLI (P < 0.01 for both fat sites; Table 4), without significant quadratic effects (P = 0.83 for subcutaneous fat and P = 0.35 for perirenal fat). The regression equation varied with the site of measurement, with a common intercept, but a greater slope for perirenal than for subcutaneous caudal fat (P < 0.05). The regression equation was

[2]

where r² = 0.91, RSD = 17, n = 12, and a = 0.378 for subcutaneous caudal fat and 0.532 for perirenal fat.

[3]

where r² = 0.91, RSD = 17, n = 12, and b = 0.532 for subcutaneous caudal fat and 0.747 for perirenal fat.

In Exp. 1, mean subcutaneous caudal and perirenal fat AVMI increased linearly (P < 0.01 for both sites) with mean PCCS, without significant quadratic effects (P = 0.10 for subcutaneous fat and P = 0.25 for perirenal fat; Figure 1). The regression equation varied with the site of measurement, with a common intercept, but a greater slope for perirenal than for subcutaneous caudal fat (P< 0.01), the equation of regression for the mean data of 8 animals being

View larger version (8K): [in this window] [in a new window]   Figure 1. Response curve relating mean absolute value of the mean integral between 450 and 510 nm (AVMI) for subcutaneous caudal (open symbols) and perirenal (solid symbols) fat to mean plasma carotenoid concentration at slaughter. Triangles and squares refer to Exp. 1 and 2, respectively. Bars represent SEM.

[4]

where r² = 0.95, RSD = 13, n = 12, and c = 1.87 for subcutaneous caudal fat and 2.62 for perirenal fat.

All lambs receiving up to 500 g/d of dehydrated alfalfa in Exp. 1 had perirenal fat AVMI less than 270 units, whereas 28 of 33 pasture-fed lambs in Exp. 2 had perirenal fat AVMI greater than 270 units. For T750, T1,000, and T1,250, 19 of 24 lambs had perirenal fat AVMI greater than 270 units.

In Exp. 1, there was no significant effect of treatment on L*, a*, and b* values of subcutaneous caudal fat nor on L* and a* values of perirenal fat (Table 5). The b* value of perirenal fat was affected by treatment (P < 0.01). It was greater for T1,000 and T750 than for T500. The response of the mean b* value of perirenal fat to the treatment had a linear relation to MDCI and MDLI (P < 0.01 in both cases), without any quadratic effect (P = 0.97 in both cases).

	DISCUSSION

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In Exp. 1, the feeding management led to 1) mean daily intake of dehydrated alfalfa reaching 95.8 to 99.9% of the assigned levels, and 2) a similar ADG and perirenal fat deposition for all treatments, therefore avoiding confounding effect of fatness level on carotenoid concentration in the fat (Prache et al., 2003a).

The carotenoid concentration in dehydrated alfalfa was 280.6 µg/g of DM [i.e., about 40 to 65% of the concentrations observed by Prache et al. (2003b) in pasture herbage (433 to 697 µg/g of DM)]. This lower level compared with pasture herbage is probably due to the dehydration process (Nozière et al., 2006a). However, it is worth noting that the carotenoid profile was different between dehydrated alfalfa and pasture herbage, particularly in that lutein accounted for 71% of total carotenoid pigments in dehydrated alfalfa but only 54 to 60% in pasture herbage (Prache et al., 2003b). This is important because lutein is the only carotenoid stored in the fat of sheep (Yang et al., 1992; Prache et al., 2003a). Dehydrated alfalfa was therefore a good model to study the dose-response curve relating the concentration of lutein in the fat to the daily lutein intake in sheep. Although the carotenoid intake level was probably lower than in pasture-fed lambs, the proportion of lutein was greater in dehydrated alfalfa than in the pasture herbage of Prache et al. (2003b), and both the lutein concentration in the diet and the MDLI could be easily controlled in individually penned lambs. The ß-carotene accounted for 14% of total carotenoid pigments in dehydrated alfalfa but 22 to 26% in the pasture herbage of Prache et al. (2003b). The contribution of zeaxanthin was almost similar in both types of forages [i.e., 8% in dehydrated alfalfa and 6 to 9% in the pasture herbage of Prache et al. (2003b)]. If we assume that grazing lambs are eating about 1 kg of DM herbage daily at the time of slaughter (Delagarde et al., 2001) and that the lutein concentration of pasture herbage is 252 mg/kg of DM (Prache et al., 2003b), then, at the time of slaughter, the MDLI in this study for T1,250 lambs was about 16% lower than for pasture-fed lambs.

In Exp. 1, mean PCCS increased linearly with MDCI, and our Eq. 1 enables prediction. The mean PCCS was much less in T1,250 than in pasture-fed lambs (59 vs. 112 µg/L, P < 0.01) presumably owing to differences in carotenoid intake level.

In Exp. 1, the AVMI in subcutaneous caudal and perirenal fat increased with MDCI and MDLI. Our linear Eq. 2 and 3 enable prediction of AVMI from MDCI or MDLI. The intercept of the regression equation was similar for both measurement sites, but the slope was greater for perirenal than for subcutaneous caudal fat. This result agrees with previous studies reporting that the carotenoid concentration (Kirton et al., 1975) or the AVMI (Priolo et al., 2002b) was greater in perirenal than in subcutaneous caudal fat.

The subcutaneous and perirenal fat AVMI were not significantly different among T750, T1,000, and T1,250 lambs. This raised the question of whether a plateau was reached from T750 onward or whether this result was due to variability between replicates in lutein absorption. We did not detect any significant quadratic effect in the response curve. Moreover, combining the results of Exp. 1 and 2 provides evidence in support of a linear relationship. The subcutaneous and perirenal fat AVMI of the pasture-fed lambs in Exp. 2 averaged 293 and 374, respectively. Extrapolating Eq. [3] beyond intakes observed in Exp. 1, the corresponding daily lutein intake level would be predicted to be 230 to 270 mg, in line with the expected herbage intake level in pasture-fed lambs and the lutein concentration in pasture herbage observed by Prache et al. (2003b). Finally, the relationship between mean AVMI and mean PCCS remains quite linear when pooling data from the 2 experiments [i.e., when adding data from pasture-fed lambs in which mean PCCS was much greater than for lambs in Exp. 1 (Figure 1)]. The regression equation for the combined data was

[5]

where r² = 0.88, RSD = 23, n = 14, and d = 1.12 for subcutaneous caudal fat and 1.86 for perirenal fat.

It should be noted that the carotenoid intake in Exp. 2 was more subject to variations than in Exp. 1 because of likely variations in carotenoid concentration in pasture herbage and in forage intake.

The data of the current study enables us to propose equations to predict mean PCCS and mean fat AVMI from MDCI. This dose-response study may therefore shed some light on questions regarding the effects of sward availability, sward carotenoid content, and concentrate supplementation at pasture on the carotenoid concentration in plasma and fat in pasture-fed lambs, which are all factors that can affect the animal’s carotenoid intake level. Further work is needed to study the interaction of the dose-response curve with factors that may affect the animal’s response, such as growth pattern and breed.

In previous studies, the fat was slightly more yellow in pasture-fed than in concentrate-fed lambs (Priolo et al., 2002a) because of carotenoid pigments. In this study, we showed that the mean b* value of perirenal fat increased linearly with MDCI and MDLI despite a large interindividual variability.

The data in the current study clearly show the level of the interindividual variability in carotenoid absorption and storage. Although carotenoid intake level was similar for all lambs within a treatment in Exp. 1, inter-individual variability in plasma and fat carotenoid concentrations was observed. In T1,250 lambs, for example, PCCS ranged from 28 to 95 µg/L (i.e., a 3.4-fold variation), and AVMI for perirenal fat ranged from 183 to 411 units (i.e., a 2.3-fold variation).

Prache and Theriez (1999) were the first to find that carotenoids in plasma and fat could be used as markers of pasture-feeding in sheep, and this result was confirmed in subsequent experiments comparing pasture-fed to concentrate-fed sheep and cattle (Prache et al., 2002, 2003a,Prache et al., b; Priolo et al., 2002b; Serrano et al., 2006; Dian et al., 2007). However, it was uncertain whether and to what extent the inclusion of dehydrated alfalfa (and more generally of green vegetative matter) in concentrates could affect the reliability of the discrimination between pasture-fed and concentrate-fed lambs. Actually, feeding alfalfa indoors does not have the same "natural" connotation in the popular sense as pasture-feeding. This study helps answer this question. The combined use of all the plasma and perirenal fat data for individual animals in both experiments (Figure 2) enabled discrimination of all pasture-fed lambs from all those receiving up to 500 g/d of dehydrated alfalfa in this study. All 24 lambs receiving up to 500 g/d of dehydrated alfalfa presented values for PCCS and AVMI of perirenal fat less than 40 µg/L and 270 units, respectively, values that were never observed in the 33 pasture-fed lambs of Exp. 2. Lambs presenting values for PCCS greater than 95 µg/L or values for AVMI of perirenal fat greater than 411 units were all pasture-fed (15 of 33 pasture-fed lambs). There was some overlapping in the distribution of pasture-fed lambs and lambs fed more than 500 g/d of dehydrated alfalfa indoors. However, the MDCI were probably very high in the pasture-fed lambs, given the quality and the availability of the sward offered together with the observed PCCS and ADG. Further evaluations are therefore required to gauge the extent to which the results from this study regarding diet authentication could be generalized to less favorable pasture-feeding conditions.

View larger version (11K): [in this window] [in a new window]   Figure 2. Distribution of lambs according to plasma carotenoid concentration at slaughter and absolute value of the mean integral between 450 and 510 nm (AVMI) for perirenal fat. For Exp. 1, the treatments are represented as T0 (), T250 (), T500 (), T750 (x), T1,000 (), and T1,250 (•), where T = treatment and the number indicates the g/d of dehydrated alfalfa that was fed; and for Exp. 2 the treatment is shown as (pasture-fed lambs).

In conclusion, we demonstrated that the concentrations of carotenoid pigments in plasma and fat increase linearly with mean daily carotenoid intake. This study confirms that carotenoid fixation is lower in subcutaneous caudal fat than in perirenal fat. Although the carotenoid intake level was similar for all the lambs within treatment levels, some interindividual variability in plasma and fat carotenoid concentration was still observed. The inclusion of high levels of dehydrated alfalfa in the diet of stall-fed lambs may affect the reliability of the method based on carotenoid pigments in plasma and fat reflectance spectra used to authenticate pasture-feeding in sheep. Future research should therefore be directed toward combining other molecular and atomic compounds with carotenoid pigments.

	Footnotes

 
¹ We thank M. Bernard, R. Jailler, J. Pourrat, and all the staff of the experimental farm and abattoir of INRA Theix for their collaboration. We also thank P. Gasqui for valuable advice on data analysis.

² P. H. M. Dian was financed by the Brazilian Ministry of the Education ’Coordenação de Aperfeic oamento de Pessoal de Nivel Superior’ (CAPES-PDEE). Present address: University of Maringá, UEM, Av. Colombo 5790, 87020-900, Bloco 32-Sala 3, Maringá-PR, Brazil.

³ Corresponding author: prache{at}clermont.inra.fr

Received for publication July 18, 2006. Accepted for publication June 29, 2007.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 


Cardinault, N., M. Doreau, C. Poncet, and P. Nozière. 2006. Digestion and absorption of carotenoids in sheep fed fresh red clover. Anim. Sci. 82:49–55.[CrossRef]

CIE. 1986. Colorimetry. 2nd ed. CIE Publ. No. 15.2. Commission International de l’Eclairage, Vienna, Austria.

Delagarde, R., S. Prache, M. Petit, and P. D’Hour. 2001. Ingestion de l’herbe par les ruminants au pâturage. Fourrages 166:189–212.

Dian, P. H. M., D. Andueza, C. M. P. Barbosa, S. Amoureux, M. Jestin, P. C. F. Carvalho, I. N. Prado, and S. Prache. 2007. Methodological developments in the use of visible reflectance spectroscopy for discriminating pasture-fed from concentrate-fed lamb carcasses. Br. Soc. Anim. Sci. 1:1198–1208.

Fisher, A. V., and H. de Boer. 1994. The EAAP standard method of sheep carcass assessment. Carcass measurements and dissection procedures. Report of the EAAP Working Group on Carcass Evaluation, in cooperation with the CIHEAM Instituto Agronomico Mediterraneo of Zaragoza and the CEC Directorate General for Agriculture in Brussels. Livest. Prod. Sci. 38:149–156.[CrossRef]

Karijord, O. 1978. Correlation between the content of carotenoids in depot fat and in plasma of sheep. Acta Agric. Scand. 28:355–359.

Kirton, A. H., B. Crane, D. J. Paterson, and N. T. Clare. 1975. Yellow fat in lambs caused by carotenoids pigmentation. N. Z. J. Agric. Res. 18:267–272.

Lyan, B., V. Azais-Braesco, N. Cardinault, V. Tyssandier, P. Borel, M. C. Alexandre-Gouabau, and P. Grolier. 2001. Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method. J. Chromatogr. Biomed. Sci. Appl. 751:297–303.[CrossRef][Medline]

Martin, B., A. Cornu, N. Kondjoyan, A. Ferlay, I. Verdier-Metz, P. Pradel, E. Rock, Y. Chilliard, J. B. Coulon, and J. L. Berdagué. 2005. Milk indicators for recognizing the types of forages eaten by dairy cows. Pages 127–136 in Indicators of Milk and Beef Quality. J. Hocquette and S. Gigli, ed. Proc. Eur. Assoc. Anim. Prod. No. 112, Bled, Slovenia.

Nozière, P., B. Graulet, A. Lucas, B. Martin, P. Grolier, and M. Doreau. 2006a. Carotenoids in ruminants: From forages to dairy products. Anim. Feed Sci. Technol. 131:418–450.[CrossRef]

Nozière, P., P. Grolier, D. Durand, A. Ferlay, P. Pradel, and B. Martin. 2006b. Variation of carotenoids, fat-soluble micronutrients and color in cow’s plasma and milk following changes in forage and feeding level. J. Dairy Sci. 89:2634–2648.[Abstract/Free Full Text]

Patterson, D. S. P. 1965. The association between depot fat mobilization and the presence of xanthophyll in the plasma of normal sheep. J. Agric. Sci. 64:273–278.

Prache, S., A. Cornu, J. L. Berdagué, and A. Priolo. 2005. Traceability of animal feeding diet in the meat and milk of small ruminants. Small Rumin. Res. 59:157–168.[CrossRef]

Prache, S., A. Priolo, and P. Grolier. 2003a. Effect of concentrate finishing on the carotenoid content of perirenal fat in grazing sheep: Its significance for discriminating grass-fed, concentrate-fed and concentrate-finished grazing lambs. Anim. Sci. 77:225–233.

Prache, S., A. Priolo, and P. Grolier. 2003b. Persistence of carotenoid pigments in the blood of concentrate-finished grazing sheep: Its significance for the traceability of grass-feeding. J. Anim. Sci. 81:360–367.[Abstract/Free Full Text]

Prache, S., A. Priolo, H. Tournadre, R. Jailler, H. Dubroeucq, D. Micol, and B. Martin. 2002. Traceability of grass feeding by quantifying the signature of carotenoid pigments in herbivores meat, milk and cheese. Pages 592–593 in Proc. 19th Gen. Meet. Eur. Grassl. Fed., La Rochelle, France.

Prache, S., and M. Theriez. 1999. Traceability of lamb production systems: Carotenoids in plasma and adipose tissue. Anim. Sci. 69:29–36.

Priolo, A., D. Micol, J. Agabriel, S. Prache, and E. Dransfield. 2002a. Effect of grass or concentrate feeding systems on lamb carcass and meat quality. Meat Sci. 62:179–185.[CrossRef]

Priolo, A., S. Prache, D. Micol, and J. Agabriel. 2002b. Reflectance spectrum to trace grass feeding in sheep: Influence of measurement site and shrinkage time after slaughter. J. Anim. Sci. 80:886–891.[Abstract/Free Full Text]

Russel, A. J. F., J. M. Doney, and R. G. Gunn. 1969. Subjective assessment of body fat in live sheep. J. Agric. Sci. Camb. 72:451–454.

Serrano, E., S. Prache, B. Chauveau, P. Pradel, J. Agabriel, and D. Micol. 2006. Traceability of grass-feeding in young beef using carotenoid pigments in plasma and adipose tissue. Anim. Sci. 82:909–918.[CrossRef]

Yang, A., T. W. Larsen, and R. K. Tume. 1992. Carotenoid and retinol concentrations in serum, adipose tissue and liver and carotenoid transport in sheep, goats and cattle. Aust. J. Agric. Res. 43:1809–1817.[CrossRef]

Young, O. A., G. A. Lane, A. Priolo, and K. Fraser. 2003. Pastoral and species flavour in lambs raised on pasture, lucerne or maize. J. Sci. Food Agric. 83:93–104.

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