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J. Anim. Sci. 2003. 81:360-367
© 2003 American Society of Animal Science

Persistence of carotenoid pigments in the blood of concentrate-finished grazing sheep: Its significance for the traceability of grass-feeding1

S. Prache*,2, A. Priolo*,3 and P. Grolier{dagger}

* Unité de Recherches sur les Herbivores and and {dagger} Unité Maladies Métaboliques et Micronutriments, INRA Clermont-Ferrand/Theix 63122 St. Genès Champanelle, France

2 Correspondence:
Phone: 33-4-73-62-40-63; fax: 33-4-73-62-41-18; E-mail:
prache{at}clermont.inra.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Carotenoid pigments are good biomarkers of grass feeding in sheep. However, grazing lambs are often stall-finished because of grass shortage. We investigated the nature of the carotenoids present in sheep blood and their persistence in this tissue. Four treatments were compared: 1) feeding a concentrate-based diet (n = 10 lambs), 2) grazing followed by a long stall-finishing period (n = 10), 3) grazing followed by a short stall-finishing period (n = 10), and 4) grazing to slaughter weight (n = 10). The concentrate supply was regulated to have similar average daily gain for all treatments. The 40 lambs were allocated to either the grazing or the stall treatments on the basis of their birth date, birth weight, and body weight. The 30 grazing lambs were further allocated to long-stall, short-stall, or grass treatment on the basis of their body weight and plasma carotenoid content. Plasma content of total carotenoids was measured by spectrophotometry during the grazing and the stall periods for all lambs and at slaughter weight for the eight heaviest lambs of each treatment. Analysis of the nature and the concentration of individual carotenoids was performed by HPLC on pasture and stall diets and on blood of grazing lambs. The carotenoid content of the stall diet was 2 to 3% that of the pasture diet. Lutein, zeaxanthin, and ß-carotene accounted for 43 to 58%, 3 to 17%, and 0 to 7% of total plasma carotenoids in grazing lambs, respectively. Two unknown polar carotenoids, expressed in lutein equivalent, accounted for 10 to 22% and 0 to 9% of total carotenoids. Plasma carotenoid content during the grazing and the finishing periods varied among animals (P < 0.001). At slaughter weight, plasma carotenoid content was higher for grass-fed than for stall-fed, long-stall finished, or short-stall finished lambs (P < 0.001), and reliably distinguished grass-fed lambs from all the others. Plasma carotenoid content decreased exponentially with the interval from starting on the stall diet (P < 0.005). The deceleration parameter of the model increased linearly with lamb average daily gain during the stall-finishing period, suggesting that the turnover of carotenoids in the blood may depend on the level of intake of the stall-finishing diet. After 4 to 13 d on the stall diet, depending on the initial plasma carotenoid concentration, plasma carotenoid concentration of previously grazed, stall-finished lambs fell to the values of lambs fed a concentrate diet without grazing. Such a low persistence is of interest for discriminating grazing lambs from stall-finished grazing lambs.

Key Words: Carotenoids • Grasses • Persistence • Sheep • Tracer Techniques


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Carotenoid pigments have been shown to be good biomarkers of grass feeding in herbivores (Prache and Theriez 1999; Prache et al., 2002; Priolo et al., 2002) and also to be of nutritional interest for human health (Mayne 1996; Bone et al., 1997; Borel et al., 1998). These pigments are widely distributed in plants and cannot be synthesized de novo by animals. Lutein is thought to be the only carotenoid in serum and adipose tissue of sheep, whereas cattle also store ß-carotene (Yang et al., 1992). Because of grass shortage, grazing animals are often stall-finished with low-carotenoid diets. Prache and Theriez (1999) observed that the plasma carotenoid content of stall-finished grazing lambs was lower at slaughter than at the end of the grazing period, but little information is available regarding the persistence pattern of these pigments in the animal tissues.

The purpose of this study was to determine: 1) the nature of the carotenoid pigments present in lamb plasma, and 2) their persistence pattern in the plasma of lambs stall-fed a low-carotenoid diet after pasture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
This experiment was 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 according to the European Union Directive No. 609/1986.

Experimental Design
Four feeding treatments were compared: 1) grass-feeding, 2) stall-feeding, 3) grass-feeding followed by a short stall-finishing period, and 4) grass-feeding followed by a long stall-finishing period. Grass-fed lambs were allowed continuous access to pastures predominated by cool-season grasses, whereas stall-fed lambs were fed concentrate and hay. The feeding level of stall-fed lambs was adjusted every week to achieve similar growth rates in the four treatments.

Animals and Diets
Forty male Ile-de-France lambs born in spring (April 3 ± 7 d) and suckled by their dams were used. Each of the four treatments included 10 lambs. First, 10 and 30 lambs were allocated at random to the stall and grass treatments on the basis of their BW 2 d before grass-fed lambs were turned out to grass (April 18). Second, 10 grazing lambs were allocated at random to the long stall-finishing treatment on the basis of their BW and plasma carotenoid concentration on June 17. Finally, 10 grazing lambs were allocated at random to the short stall-finishing treatment on the basis of their BW on July 8 and plasma carotenoid concentration on June 28. The 40 lambs were thus allocated to 10 blocks of four lambs. The long and short stall-finishing periods began on June 18 and July 9, respectively. Grass-fed animals rotationally grazed a natural pasture divided into five paddocks. During the first grazing cycle, the lambs grazed on uninterrupted spring growths, and thereafter on regrowths. After grazing, residual herbage was topped with a forage harvester, and the trimmings were removed. The botanical composition of the pasture was (DM basis) Lolium perenne (29.9%), Dactylis glomerata (28.7%), Festuca arundinacea (20.8%), Taraxacum officinale (10.1%), Bromus sterilis (9.9%), Trifolium repens (0.7%), and Ranunculus macrophyllus (0.3%). Hand-plucked samples of herbage were taken monthly using scissors to simulate herbage grazed by the animals. About 50% of the sample was dried at 65°C for 48 h for CP and NDF estimation. The remaining 50% was used for determination of the carotenoid content of the pasture. It was stored at -20°C until required for assay. The 10 stall lambs were penned indoors as one group in a 5 x 12-m pen without any artificial lighting. They were fed commercial concentrate and hay. The concentrate included 200 g/kg of barley, 118 g/kg of wheat, 50 g/kg of wheat red shorts, 50 g/kg of legume seeds hulls, 150 g/kg of wheat bran, 88 g/kg of soybean meal, 21 g/kg of malt sprouts, 150 g/kg of dried sugar beet pulp, 44 g/kg of cocoa beet shells, 40 g/kg of sugar beet molasses, 3.5 g/kg of formalin, 83.5 g/kg of calcium carbonate, sodium chloride, ammonium chloride, minerals (14 g/kg of Ca, 4.5 g/kg of P, 150 mg/kg of Zn, 108 mg/kg of Fe, 50 mg/kg of Mn, 2 mg/kg of Co, 6.5 mg/kg of I, 0.182 mg/kg of Se), and vitamins (6,000 IU/kg of vitamin A, 1,200 IU/kg of vitamin D3, 20 IU/kg of vitamin E), and 2 g/kg of water. The concentrate always constituted 85% of the diet and the hay constituted the remaining 15% on an as-fed basis. Samples of hay and concentrate offered to the animals were taken weekly for CP, NDF, and carotenoid content estimations. Lambs were weaned at 77 d on average.

Grazing lambs and stall-finished grazing lambs received an anthelminthic drench on July 5 (febantel). All lambs were treated against coccidiosis on July 9 (using sulfadimethoxine). Water and salt blocks were always available. The salt blocks included 60 g/kg of Ca, 20 g/kg of P, 10 g/kg of Mg, 280 g/kg of Na, 17,500 mg/kg of Zn, 1,500 mg/kg of Fe, 5,500 mg/kg of Mn, 30 mg/kg of Co, 30 mg/kg of I, and 10 mg/kg of Se.

Measurements
Crude Estimation of Total Plasma Carotenoids.
We measured plasma carotenoid concentration on July 19 for the stall-fed lambs; on the starting day of the stall-finishing period and 10 d afterwards for the long stall-finishing treatment; on June 17, June 28, the starting day of the stall-finishing period, then daily for four consecutive days, and then three times weekly for the short stall-finishing treatment; on June 17 and 28 and July 9 and 19 for the grass-feeding treatment; and when the lambs reached slaughter weight (about 35 kg) for the eight heaviest lambs of each treatment. Blood samples were taken from the jugular vein of each lamb. 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 (samplings and tests tubes wrapped in aluminium foil to keep light out and extraction under dim artificial light).

High-Performance Liquid Chromatography Analysis.
Analysis was performed by HPLC on pasture and stall diets and on blood samples taken from the jugular vein of each lamb on June 6. Echinenone, zeaxanthin, and 9-cis and 13-cis ß-carotene were generously donated by Hoffman La Roche (Basel, Switzerland). Lutein and all-trans ß-carotene were purchased from Sigma Chemical Co. (St. Louis, MO). Ammonium acetate (7.5 M) was supplied by Sigma Chemical Co. (l’Isle d’Abeau Chesnes, France). High-performance liquid chromatography-grade acetonitrile, methanol, isopropanol, tetrahydrofuran, and hexane were obtained from Carlo Erba (Chaussée du Vexin, France). Dichloromethane, stabilized with amylene (25 mg/mL), was purchased from Mallinckrodt (Deventer, Holland). High-performance liquid chromatography water was obtained using a MilliQ Plus water purification system (Millipore SA, Saint Quentin en Yvelines, France).

Stock solutions of carotenoid standards (1.8 to 2.1 mM) were stored at -20°C in tetrahydrofuran. These solutions were then diluted in isopropanol followed by mobile phase (2.5 µM) (working solution). Using the respective extinction coefficient (E1%) (Britton 1995), their concentrations were determined spectrophotometrically after dilution in ethanol (xantophylls) or hexane (carotenes). Echinenone was used as internal standard. The HPLC apparatus consisted of a Waters system (Waters SA, Saint Quentin en Yvelines, France) equipped with a pump (Waters 610 fluid unit), a regulator (Waters 600 controller), a cooled autosampler (Waters 717 plus), and a UV-visible photodiode-array detector (Waters 996). Millennium 32 software (version 3.05.01) from Waters was used for instrument control, data acquisition, and data processing.

Analyses were performed as described by Lyan et al. (2001) on a 150 x 4.6 mm, RP C18, 3-µm Nucleosil column (Interchim, Montluçon, France) coupled with a 250 x 4.6 mm RP C18, 5-µm Vydac TP54 column (Hesperia, CA), and a 20 x 4.6 mm C18, 5-µm Hypersil-guard column.

The mobile phase consisted of acetonitrile/methanol containing 50 mM ammonium acetate/water/dichloromethane (70/15/5/10; vol/vol/vol/vol). The methanol fraction was first prepared by dilution of ammonium acetate (7.5 M) in methanol. This mixture was then added to the other solvents of the mobile phase.

The flow rate was 2 mL/min. The run time was 50 min. The detection wavelength was set up at 450 nm. Absorption spectra of each standard were recorded between 300 and 600 nm in the mobile phase and stored in a spectra library to support the identification of plasma carotenoids.

Data Analyses
Variance of the plasma carotenoid concentration was stabilized using the logarithmic transformation. Plasma carotenoid concentration at slaughter weight was subjected to an ANOVA using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) to examine the effect of feeding treatment, and by using the Duncan’s contrast procedure for pairwise comparisons. The plasma carotenoid concentration of stall-finished lambs was subjected to an analysis of variance using the GLM procedure of SAS to examine the effect of animal and time elapsed since the end of the grass-feeding period. We used a two-factor model with repeated measures on one factor (time) as described by Cody and Smith (1991). The plasma carotenoid concentration of grass-fed lambs was subjected to an ANOVA using the same analysis (Cody and Smith, 1991) to examine the effect of animal and variation with time during the grass-feeding period. Single animals were considered experimental units. When significant differences among animals were observed, the animal factor was replaced by animal characteristics (BW, ADG) to evaluate the effect of these characteristics in regression models.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Mean ADG of the slaughtered animals was not different (P = 0.27) among feeding treatments (204, 229, 245, and 234 ± SE 7.56 g/d for grass-fed, short-stall-finished, long-stall-finished, and stall-fed lambs, respectively). The stall period of the slaughtered animals lasted 30 d on average (minimum and maximum of 15 and 43 d), and 42 d (minimum and maximum of 21 and 58 d) for the short- and the long-finishing treatments, respectively. During the stall-finishing period, lambs deposited on average 6.01 kg of live weight (minimum and maximum of 2.8 and 12.1 kg) and 7.22 kg of live weight (minimum and maximum of 1.6 and 10.8 kg) for the short- and the long-stall finishing treatments, respectively. Mean ADG during the stall-finishing period was not different between the short- and long-stall-finished lambs (239 and 193 ± SE 21.1 g/d for the eight lambs that were fattened until slaughter weight, P = 0.32). Average daily gain during the stall-finishing period ranged from 126 to 373 g/d and from 89 to 337 g/d for the short- and the long-stall-finished grazing lambs. Mean ADG during the first 9 d of the short-stall-finishing period was 268 g/d and varied in the range 46 to 523 g/d.

The sward offered was green vegetative herbage and had an average of 16.3% CP and 51.2% NDF, 19.1% CP and 50.6% NDF, 16.6% CP and 56.9% NDF, and 14.0% CP and 51.9% NDF (DM basis), in May, June, July, and August, respectively. The carotenoid content of the pasture (Table 1Go) remained steady from the beginning of May until the end of June (696, 616, and 684 µg/g of DM on May 9, June 6, and June 26, respectively), but decreased sharply at the beginning of August (433 µg/g of DM on August 2).


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  		Table 1. Carotenoid content of the pasture, of the hay and of the concentrate
 
The concentrate contained 18.6% CP and 27.2% NDF (DM basis), and the hay had 12.8% CP and 60.5% NDF (DM basis). The carotenoid content of the hay and the concentrate was 86 and 0 µg/g of DM, respectively (Table 1Go). Thus, the stall diet contained very low levels of carotenoid pigments compared with the pasture diet. If we assume similar DM intake levels for both diets, the carotenoid content of the stall diet was about 1.9 to 3.1% of the carotenoid content of the pasture diet. In both diets, lutein was the predominant carotenoid (54 to 60% and 70% of total carotenoid pigments for the pasture and stall diets, respectively).

High-performance liquid chromatography analyses showed that lutein was the main carotenoid present in the plasma of grazing lambs (43 to 58% of total carotenoids). Zeaxanthin (3 to 17%) and ß-carotene (0 to 7%) were also detected (Figure 1Go). In addition, two compounds were eluted before the lutein peak and exhibited typical absorption spectra. We have not yet identified these components, but when quantified using the lutein calibration curve, they contributed to 10 to 22% (Unknown 1) and 0 to 9% (Unknown 2) of total carotenoids.



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  		Figure 1. Chromatogram and absorption spectra of carotenoids extracted from sheep plasma. High-performance liquid chromotography conditions: columns, 150 x 4.6 mm Nucleosil C18, 3-µm particle size in series with a 250 x 4.6 Vydac TP 254 C18, 5-µm particle size equipped with a 20 x 4.6 mm Hypersil column, 5-µm particle size; detection 450 nm; flow rate, 2 mL/min; eluent, acetonitrile-methanol containing 50 mM acetate ammonium:dichloromethane:water (70:15:10:5, vol/vol/vol/vol). Carotenoids were identified by comparison with pure standards.

 
Mean plasma carotenoid concentration on June 17 ranged between 43 and 280 µg/L (i.e., a sixfold interindividual variation in the plasma carotenoid concentration of the 30 lambs still at pasture at that date. It was not related to lamb live weight or to lamb ADG. It did not differ (P = 0.67) between grass-feeding treatments (153, 137, and 150 ± SE 10.9 µg/L for grass-fed, short-stall-finished, and long-stall-finished lambs, respectively). Mean plasma carotenoid on June 28 and July 9 ranged from 31 to 208 µg/L and from 55 to 202 µg/L, respectively. It did not differ (P = 0.77, P = 0.72) between grass-feeding treatments (101 and 106 ± SE 11.5 µg/L for grass-fed and short-stall-finished lambs, respectively, on June 28 and 101 and 110 ± SE 9.6 µg/L for grass-fed and short-stall-finished lambs, respectively, on July 9).

Mean plasma carotenoid concentration of the 10 grass-fed lambs varied with time (P < 0.01) (Figure 2Go) and animal (P < 0.001). It was higher on June 17 than on the other sampling dates. Values on June 28, July 9, and July 19 did not differ (P > 0.05). Values at slaughter weight, which was reached between July 24 and August 21, were lower than on the preceding dates (P < 0.01).



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  		Figure 2. Means of the plasma carotenoid concentration of grass-fed lambs according to sampling date (slaughter occured between July 24 and August 21). Bars represent standard errors. Values on June 17 differed (P < 0.01) from those on all other dates. Similar values (P > 0.05) were recorded on June 28, July 9, and July 19. Plasma carotenoid concentrations recorded at slaughter differed (P < 0.01) from concentrations measured at all other sampling dates.

 
There was an effect of feeding treatment (P < 0.001, Figure 3Go) on plasma carotenoid concentration at slaughter weight. It was higher (P < 0.001) in the grass treatment than in the other feeding treatments (63, 9, 7, and 12 µg/L for grass-fed, short-stall-finished, long-stall-finished, and stall-fed lambs, respectively). It was lower (P < 0.05) for the long-stall-finished vs stall-fed lambs. Short-stall-finished lambs did not differ from long-stall-finished or stall-fed lambs (P > 0.05). Plasma carotenoid concentration at slaughter weight varied in the range of 37 to 105, 3 to 17, 4 to 11, and 6 to 21 µg/L for grass-fed, short-stall-finished, long-stall-finished, and stall-fed lambs, respectively. At slaughter, all the lambs with a plasma carotenoid concentration below 21 µg/L were stall-fed or stall-finished lambs, whereas all the lambs with a plasma carotenoid concentration above 37 µg/L were grass-fed. There was no overlap in the frequency distribution of the plasma carotenoid concentration at slaughter weight between grass-fed and stall-fed or stall-finished grazing lambs.



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  		Figure 3. Means of the plasma carotenoid concentration at slaughter weight (35 kg). Bars represent standard errors. Means with unlike superscripts differ (A, b: P < 0.001, b, c: P < 0.05).

 
In the long-stall-finished treatment, the frequency of blood sampling during the stall-finishing period was insufficient to describe the persistence profile correctly. The frequency of blood sampling was therefore increased for the short-stall-finished treatment. Plasma carotenoid concentration (PCC, µg/L) of short-stall-finished grazing lambs varied with the animal (P < 0.001) and with the interval from starting on the stall diet (P < 0.005, Figure 4Go). It decreased curvilinearly with the interval from starting on the stall diet after pasture, according to a decreasing exponential model, PCC = a x e(-b x d), the key equation being:



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  		Figure 4. Means of the plasma carotenoid concentration of short-stall-finished grazing lambs according to the interval from starting on the stall diet. Bars represent standard errors. Pairwise comparisons of plasma carotenoid concentrations among dates yielded the following results: d 0 = d 1 = d 2 (P > 0.05); d 2 = d 3 (P > 0.05), d 3 = d 4 (P > 0.05); d 3 < d 0 and 1 (P < 0.05); d 4 < d 0, 1, and 2 (P < 0.05); d 7 < d 0 to 4 (P < 0.005); d 10 < d 0 to 7 (P < 0.05); d 14 < d 0 through 10 (P < 0.01). A semineperianlogarithmic representation of the same data is presented in the inset.

 

[1]

where r2 = 0.997, residual standard deviation = 4.0, n = 8, and d = day.

Plasma carotenoid persistence of short-stall-finished grazing lambs also varied with the animal (P < 0.001), as illustrated in Figure 5Go. The percentage of variance explained by a decreasing exponential model ranged between 95.7 and 99.6 depending the animal. The deceleration parameter of Eq. [1] (i.e., parameter b) increased linearly (P < 0.05) with ADG (g/d) during the first 9 d of the finishing period after pasture, according to the model:



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  		Figure 5. Plasma carotenoid concentration (PCC, µg/L) of stall-finished grazing lambs according to the interval from starting on the stall diet (D, days). Solid and open symbols represent data from the lamb having the highest and the lowest plasma carotenoid concentration at the end of the grass-feeding period, respectively. A semineperianlogarithmic representation of the same data is presented in the inset. The curvilinear equations: i) solid symbol: PCC = 197 (SE 5.4) x e[-0.1781 (SE 0.01085) x d] where r2 = 0.996, residual standard deviation = 7.0, n = 10, and d = day. ii) open symbol: PCC = 55 (SE 3.3) x e[-0.2917 (SE 0.03579) x d] where r2 = 0.984, residual standard deviation = 3.8, n = 9, and d = day.

 

[2]

where r2 = 0.55, residual standard deviation = 0.03301, and n = 10. In Eq. [1]Go, the deceleration parameter (i.e., parameter b) tended to be inversely correlated with the intercept (parameter a; P = 0.09).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
On any given date, there was a marked interindividual variability in the plasma carotenoid concentration of grazing lambs; it ranged, for example from 43 to 280 µg/L on June 17. This range is of interest for the generalization of the results of the present study. It is of similar amplitude to that observed by Karijord (1978; 25 to 250 µg/L), but higher than that observed by Yang et al. (1992; 6 µg/L on average). The differences among authors may be related to the analysis methods. When carotenoid concentrations were measured by spectrophotometry (i.e., the present study and that of Karijord [1978]), total carotenoids were quantified. Using HPLC, Yang et al. (1992) detected only lutein in sheep plasma. In the present experiment, we detected mainly lutein, but for the first time, zeaxanthin, ß-carotene, and two minor unknown carotenoids were also detected. In the present study, the sum of individual carotenoid levels was very close to the total carotenoid concentration measured by spectrophotometry. Thus, the crude estimation of carotenoids by the spectrophotometry method, which is rapid and inexpensive, can provide a good estimation of the plasma carotenoid content. The interindividual variability in plasma carotenoid content is most likely due to variations in carotenoid absorption and metabolism (Rock, 1997) rather than to variations in grass intake level since plasma carotenoid concentration was not related to animal BW or to ADG. Its magnitude highlights the need to take plasma carotenoid concentration into account in allocating animals to different treatments to avoid bias, as anticipated in the present study. This variability, however, did not impair the quality of the discrimination between grass-fed and stall-fed lambs based on plasma carotenoid concentration, demonstrating its soundness. Mean plasma carotenoid concentration at slaughter weight was higher (P < 0.001) for grass-fed than for stall-fed lambs and no overlapping occurred in the frequency distribution of the plasma carotenoid concentration at slaughter of grass-fed lambs and stall-fed lambs. The stall-fed lambs had values between 6 and 21 µg/L, whereas the grass-fed lambs had values between 37 and 105 µg/L. These results confirm those of Prache and Theriez (1999), who were the first to demonstrate that carotenoid pigments can act as biomarkers of grass-feeding.

Mean plasma carotenoid concentration of grass-fed lambs varied with time (P < 0.01), being higher on June 17 than on June 28, July 9, or July 19, and twice the mean slaughter value. This variation is most likely due to variations in the carotenoid content of herbage, which was much lower in August than in the preceding sampling dates. It may explain why Prache and Theriez (1999) observed that plasma carotenoid concentration was below the detection limit for three out of 100 of their grazing blood samples. This may limit the method for detecting lambs grazing herbage containing very low carotenoid concentrations, but this was not the case in the present study.

There was an effect of feeding treatment (P < 0.001) on plasma carotenoid concentration at slaughter weight, values for stall-finished lambs being lower than for grass-fed lambs and close to those for stall-fed lambs. No overlapping occurred in the frequency distribution of the plasma carotenoid concentration at slaughter of grass-fed lambs and stall-fed or stall-finished grazing lambs. The stall-fed or stall-finished grazing lambs had values between 3 and 21 µg/L, whereas the grass-fed lambs had values between 37 and 105 µg/L.

Persistence of carotenoid pigments in the blood was actually low. Their concentration decreased curvilinearly with the interval from starting on the stall diet after pasture (d), according to a decreasing exponential model = a x e(-b x d). Our model predicts that mean plasma carotenoid concentration decreases to a level similar to that of the stall-fed lamb with the highest carotenoid level (21 µg/L) after 8.1 d on the stall diet, and to a level comparable to the mean plasma carotenoid concentration of the stall-fed lambs (12 µg/L) after 10.5 d on the stall diet. Plasma carotenoid persistence of stall-finished grazing lambs also varied with the animal. The decreasing exponential model fit all individual data, the percentage of variance explained by the model ranging between 95.7 and 99.6. Our model predicts that the plasma carotenoid concentration of the stall-finished lamb with the highest persistence decreases to a value similar to that of the stall-fed lamb with the highest carotenoid level after 12.7 d on the stall diet, and to a level comparable to the mean plasma carotenoid concentration of the stall-fed lambs after 15.6 d on the stall diet. Corresponding values for the stall-finished lamb with the lowest persistence are 3.4 and 5.2 d, respectively.

The deceleration parameter of the decreasing exponential model increased linearly with ADG during the first 9 d of the finishing period after pasture (P < 0.05). Turnover of carotenoid pigments in blood has been shown to increase with fat mobilization in sheep stall-fed after pasture (Patterson, 1965). Results of the present study suggest that the turnover of carotenoid pigments in the blood may also depend of the level of intake of the stall-finishing diet.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
We have confirmed that carotenoids are good traceability biomarkers of grass feeding in lambs, although there is considerable interindividual variability. We have demonstrated that the persistence of carotenoids in the blood is low: after 4 to 13 d on the stall diet, plasma carotenoid content of stall-finished lambs decreased to the values of the stall class. This result is robust considering the range of variability of plasma carotenoid content at the end of the grazing period. Using this traceability method means that stall-finished grazing lambs will be considered grass-fed during the first 4 to 13 d of the stall-finishing period, and as stall-fed thereafter. We propose a decreasing exponential model to predict plasma carotenoid content during the stall-finishing period. The deceleration parameter of the model increases linearly with lamb daily gain, suggesting that the turnover of carotenoids in the blood may depend on the level of intake of the stall-finishing diet.


   Footnotes
 
1 We thank L. Lavelle, J. B. Teuma, H. Tournadre, J. Atzeni, M. Krogmann, and all the staff of the experimental farm for their collaboration. Back

3 Present address: University of Catania, DACPA Sezione di Scienze delle Produzioni Animali, Via Valdisavoia 5, 95123 Catania, Italy. Back

Received for publication June 24, 2002. Accepted for publication October 17, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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