Effect of forage / concentrate ratio and oil supplementation on C 18 : 1 and CLA isomers in milk fat from Sarda ewes

Sixteen lactating Sarda ewes were fed 4 diets differing in the forage/concentrate ratio (2 diets 75/25, 2 diets 60/40, DM) and in soyabean oil supplementation (2 diets with 100 g/head/d oil, 2 diets with no oil), in a 4x4 Latin square design. The inclusion of soyabean oil in the diet resulted in a signifi cant increase of both rumenic acid (cis 9 trans, 11 CLA) and vaccenic acid (trans 11 C18:1) in milk fat, with higher increases with the 2 high forage diets. The 2 low forage diets allowed increases of trans 10 C18:1 and of trans 10, cis 12 CLA, probably due to a shift of rumen biohydrogenation of linoleic acid.


INTRODUCTION
The forage/concentrate (F/C) ratio and the quality of lipids of the diet are the major factors affecting the rate and extent of rumen biohydrogenation of unsaturated fatty acids (UFA), included those involved in the production of conjugated linoleic acid (CLA) and of vaccenic acid (trans 11C 18:1 , VA) (Chilliard et al., 2002).Kukuk et al. (2001) demonstrated that interactions between F/C ratio and dietary UFA may occur when oils are added to the diet of lactating ewes, but no information on the milk fat composition were reported in that paper.
Aim of the present work was to verify whether and how the supplementation with unprotected soyabean oil (SO) to diets with different F/C ratio may affect the C 18:1 and CLA isomers content in milk fat of Sarda ewes.

MATERIAL AND METHODS
The experimental design was 4×4 Latin square with 4 replicates per diet.The animals were 16 Sarda ewes in mid lactation, fed 4 different diets based on lucerne hay and a concentrate mixture of barley meal, soyabean meal, minerals and vitamins.The diets were isonitrogenous (15% CP on DM) at 2 F/C ratios (75/25 or 60/40 DM), supplemented or not with rumen unprotected SO (100 g/head/d).
Milk fat was analysed for cis and trans C 18:1 isomers by gas chromatography (Christie, 2001) and for CLA isomers by Ag + ion HPLC (Sehat et al., 1998).
Statistical analysis was performed using a linear model including the fi xed effects of: 1. diet; 2. replicate; 3. period within replicate and 4. ewe within replicate.Contrasts for F/C ratio and SO treatment were tested for signifi cance.

RESULTS AND DISCUSSION
All cis and trans C 18:1 isomers resulted enhanced in the milk of ewes fed the diets with SO added, but trans ones were increased at a higher extent (Table 1).In particular, trans 11 was increased more than 10 times with diet HF/O as compared with HF/NO and almost 8 times with LF/O against LF/NO.The contrast HF vs LF was statistically signifi cant as well.The same effect of the SO supplementation was observed for trans 10, but with smaller differences.
Also CLA isomers were affected by the diet composition (Table 2).Trans 10, cis 12 reached the highest level with diet LF/O, while diet HF/O accounted for the highest concentration of cis 9, trans11 (rumenic acid, RA).A similar pattern was reported by Kukuk et al. (2001) who monitored the duodenal fl ow of fatty acids in sheep fed SO supplemented diets with different F/C ratios; low forage diets resulted in a greater duodenal fl ow of UFA, including all trans C 18:1 isomers and trans 10, cis 12 CLA; the highest fl ow of RA resulted from high forage diets.LF/O -low forage with oil 2 contrasts: F/C -forage/concentrate ratio; SO -soyabean oil * (P<0.05);** (P<0.01)Piperova et al. (2000) reported that both RA and VA were increased only slightly in milk of dairy cows fed high concentrate diets supplemented with SO.In the present experiment, even though the F/C ratios didn't reach the extreme levels of the Piperova's experiment, a similar behaviour was observed: diet HF/O induced the highest amount of RA, probably due to the accumulation of a higher amount of VA, the mammary precursor of RA in the desaturation pathway biocatalysed by stearoyl-CoA desaturase (Griinari et al., 2000).
The shift toward trans 10 isomers most probably occurred in the rumen of the ewes fed the low F/C ratio diets: actually, higher amounts of trans 10 C 18:1 , cis 12 C 18:1 and of trans 10, cis 12 CLA could be detected in milk fat.This is in accordance with what reported by Chilliard et al. (2002).
The levels of RA in milk from ewes fed diets with no SO supplementation resulted very low and comparable with the levels found in the milk of cows fed traditional diets in Northern Italy, based on dry forages (Secchiari et al., 2003).These data seem to indicate that when dairy ewes are fed similarly to dairy cows, the differences between the two species, as far as CLA in milk is concerned, are quite small.

CONCLUSIONS
The supplementation with SO of both HF and LF diets resulted in an important increase of RA and VA levels in milk fat, but the HF diet induced higher levels of both these acids.On the contrary, the LF diets allowed an increase of trans 10 C 18:1 and of trans 10, cis 12 CLA, probably due to a shift of the rumen biohydrogenation pathway of linoleic acid.
In conclusion, the interaction between the F/C ratio and the lipid fraction of the diet appeared to affect the trans C 18:1 and CLA isomers profi le of milk fat of dairy ewes, as it does in cows.

Table 1 .
Effect of F/G ratio and SO supplementation on C 18:1 isomers in milk, g/100 g total lipids

Table 2 .
Effect of F/C ratio and SO supplementation on CLA isomers in milk, mg/g total lipids HF/NO -high forage no oil; HF/O -high forage with oil; LF/NO -low forage no oil;