Effect of dietary inclusion of lampante olive oil on milk and cheese fatty acid profiles of ewes

El objetivo de este estudio fue evaluar el efecto de la inclusión de aceite de oliva lampante en la dieta de ovejas sobre el perfil de ácidos grasos de leche y queso. Nueve ovejas fueron utilizadas en un diseño de CuadradoLatino 3 × 3. Las dietas fueron suplementadas con 0 (control; T0), 36 (T1) y 88 (T2) g de aceite de oliva lampante por kg de materia seca de alimento. La ingesta, la producción de leche y la composición de leche (grasa y proteína) no se vieron afectados por los tratamientos. Los ácidos oleico y vaccénico incrementaron (P < 0.05) gradualmente mientras que los ácidos grasos saturados y el índice aterogénico disminuyeron (P < 0.05) en leche y queso a medida que la inclusión de aceite de oliva lampante se incrementó en la dieta. En conclusión, la inclusión de aceite de oliva lampante en dietas de ovejas lactantes aumenta la concentración de ácidos grasos monoinsaturados y disminuye la de los saturados en leche y queso con posibles efectos benéficos sobre la salud humana.


INTRODUCTION
In the Mediterranean-type region of Chile, unirrigated areas are characterized by harsh conditions and around 77% of these lands are poor quality grasslands that are usually designated for sheep production.In these areas, dual purpose (Merino Precoz) and meat (Suffolk and Hampshire) breeds are of particular economic interest; however, there have been efforts to improve prolificity, carcass quality and feed conversion efficiency through the crossbreeding of Merino Precoz ewes with Dorset and Sufflok rams and crosses between Merino Precoz and Finnish Landrace (Crempien, 1999).Usually, in traditional sheep production systems of the dry areas of Chile, lambs are left with their mothers and they become valuable on the market since their diet is based solely on milk (Aguilar et al., 2006).These production systems can attain high gross profit margins but low profitability per hectare and farmers need to increase their profits by adding value to their products (Vera et al., 2009).The sheep growing region of Chile is interspersed with a rapidly increasing area sown with olive trees dedicated to high quality olive oil production, and the oil extracting plants produce large amount of various by-products, including considerable amounts of discarded oil (lampant olive oil).
The impact of supplementation with vegetable oils on sheep's milk fatty acid (FA) profile has been studied previously (Bessa et al., 2005;Bouattour et al., 2008;Gómez-Cortes et al., 2008a, 2009;Manso et al., 2009Manso et al., , 2011)).Usually lipid-supplemented diets provoke decreases in dry matter intake mainly due to its palatability (Klevenhusen et al., 2011) and rumen effects.Gómez-Cortes et al. (2008b) reported that the supplementation of ewe diets with olive oil (60 g kg -1 of DM) does not have detrimental effects on animal performance but significantly modifies milk's FA profile.Also, Antongiovanni et al. (2002) and Martini et al. (2004) reported that feeding olive oil calcium soaps (50 g d -1 of DM and 7% as feed respectively) to dairy ewes can improve their milk FA characteristics (increased concentration of conjugated linoleic acid) towards a healthier pattern for human consumption.
E. VARGAS-BELLO-PÉREZ, R. R. VERA, C. AGUILAR, R. LIRA, I. PEÑA, A. VALENZUELA AND H. CERDA (to mimic real production conditions from the Mediterranean-type region of Chile) from 1300 to 1830 h and then housed in two 10 m 2 pens.During data collection periods, feed intake and milk yield were measured daily.Milk samples were obtained only from morning milking during the last 4 d of each experimental period, preserved with Bronopol TM (100mg sample -1 ) and pooled for each collection period; approximately, 80 ml/ewe/period of milk sample were stored at -20°C for later analysis.
The diets were sampled during data collection periods and stored at -20°C for later chemical analyses.Standard procedures described by AOAC (2006) were used to determine the DM, Kjeldahl N, and ether extract.Crude protein was calculated as N × 6.25.Neutral detergent fiber and ADF were determined according to the methods described by Goering and Van Soest (1970) and Van Soest et al. (1991).Milk samples were analyzed for pH, titratable acidity (Thörner degrees; °Th), fat content (Gerber method;British Standards Institution 696, 1969), crude protein (Kjeldahl N × 6.38), and total ash and solids according to the AOAC (1984) procedures.

Fatty acid analysis
Lipids from the milk, cheese, lampante olive oil and diets were extracted with chloroform/methanol (2:1, v/v) according to the Folch et al., (1957) method and methylated with the modifications of Sukhija and Palmquist (1988).Analyses of the fatty acid methyl esters (FAME) of the experimental diets, olive oil and milk samples were performed using a gas chromatograph (GC; Shimadzu Scientific Instruments AOC-20s, Columbia, MD, USA) equipped with a 30-m fused-silica capillary column (Rtx 2330, 30m × 0.32mm i.d., 0.20 µm film thickness; Restek Corporation, Bellefonte, PA) but cheese fatty acids were determined using an Rtx column 100 m × 0.32 mm × 0.20 um column.The GC conditions were as follows: initial temperature of 40 °C, 2.5 °C min -1 to 226 °C, injector and detector temperatures were 260 °C, the column flow was 3.0 ml min -1 , the split ratio was 1:10 and 1 µL injection volume was used.The hydrogen carrier gas flow to the detector was 40.0 mL min -1 , the airflow was 400.0 mL min -1 and the flow of nitrogen makeup gas was 30.0 mL min -1 .Fatty acid (FA) peaks were identified by using two FAME standards (GLC 60; Nu-Check-Prep, Elysan, MN, USA), and the Food Industry 37 FAME mix, 35077 Restek Co, Bellefonte, PA, USA).

Cheese-making process
Milk from the same treatment and period across Latin squares was pooled and made into cheese.The cheeses were made in a pilot plant as follows: 5 L of milk were heated to 60 °C for 15 min, and commercial rennet was added to curdle the milk at 38 °C.No starter culture was added for cheese making.After the milk had clotted (30 min), the curd Sheep's milk and cheese are gourmet products in Chile and there is an increasing demand from a growing and diversified market that includes an incipient sector of functional and nutraceutical foods.Despite the fact that there are previous reports on the use of olive cake silage (Sadeghi et al., 2009), olive oil (Gómez-Cortes et al., 2008b) and Ca salts of olive oil (Antongiovanni et al., 2002, Martini et al., 2004) in dairy ewe diets, there are no studies on the use of lampante olive oil in ewe (with dual purpose animals) diets under the conditions of the Mediterranean-type region of Chile.Therefore, the objective of this experiment was to evaluate the effect of the dietary supplementation of lampante olive oil on the fatty aid profiles of the milk and cheese of ewes.

MATERIALS AND METHODS
All animals were handled following the guidelines of the Animal Care Committee of the Pontificia Universidad Católica de Chile.

Animals and diets
Nine lactating ewes (crossbreed between Finnish Landrace, Border Leicester, Poll Dorset and Merino Precoz) (BW 56.5 ± 5.2 kg) with 45 d of lactation at the beginning of the experiment were assigned in a replicated (n = 3) 3 × 3 Latin Square design, that included three experimental periods of 10 d each (6 d of diet adaptation and 4 d of data collection) and three dietary treatments.The animals were housed in individual pens (2 m 2 ) with free access to water.
Their diets were elaborated according to the lactating ewe requirements of the National Research Council (2007).Three iso-caloric, isonitrogenous and iso-fibrous diets were formulated.The diets consisted of a total mixed ration (TMR), including molasses to avoid selection of dietary ingredients, based on alfalfa hay, ground corn grain and soybean expeller meal supplemented with 0 (control; T0), 36 (T1) and 88 g of lampante olive oil/ kg of DM (T2).These amounts of lampante olive oil were chosen to mimic those supplied by olive oil cake in a parallel experiment (unpublished).Lampante olive oil was added daily to the TMR to avoid rancidity and allow for homogenous inclusion in the diet.The rations were supplied twice daily (1030 and 1830 h).Orts were measured daily during each collection period to determine feed intake for each ewe.Lampante olive oil was obtained from olive oil that is not suitable for human consumption due to its high acidity and defective aroma, color and taste (donated by Bethania, Chile) (EC regulation No. 2568, 1991).

Milk yield, milk composition and DMI
There was statistical evidence of treatment differences in dry matter intake (Table 2, P = 0.057) which tended to decrease with the highest inclusion of lampante olive oil (T2).Dietary supplementation with vegetable oils rich in unsaturated FA often results in a reduction in DMI (Shingfield et al., 2006), which is related to potentially detrimental effects on ruminal fermentation such as reduced digestibility of non-lipid energy sources that is often accompanied by a reduced production of methane, hydrogen, and volatile FA (Jenkins, 1993).
In the Mediterranean-type region of Chile farmers often leave the ewes with their lambs after milking for several weeks to ensure adequate lamb growth while grazing low quality pastures.Milk consumed by suckling lambs was not estimated and that explains the low daily milk yield reported in this experiment.
was cut to the size of a hazelnut and then the vat temperature was gradually increased to 37 °C at a rate of 1°C 3 min -1 and maintained for 2 h.
At the same time, the curd was stirred to remove the whey and favor grain aggregation.The curds were placed into 350-g molds and pressed in a horizontal mechanic press until the pH was about 5.5.The cheese were salted in brine at 10 °C for 12 h and then transferred to a ripening room where they remained at a temperature of 9-10 °C and ~90% RH for 60 d.Three cheeses per treatment were allowed to mature for 60 d, at which time two cores from each cheese were removed for FA analysis.

Statistical analyses
The data were analyzed as a replicated (n = 3) 3 × 3 Latin Square using the General Lineal Models procedure of SAS Statistical Package (SAS Institute Inc., 2004).The fixed effects were experimental periods and treatment; the random effect was the ewe.A probability of P < 0.05 was used to determine significant differences among means.
The statistical model was as follows: where Y ijkl is the dependent variable, µ is the general mean, θ i is the effect of the Latin square, D (i)j is the effect of animal within each Latin square, ρ (i)k is the effect of period in each Latin square, τ l is the treatment effect, θ • τ il is the corresponding interaction, and E (ijkl) is the experimental error.
In an effort to overcome the inevitable correlation among fatty acids, treatment profiles were initially analyzed with SAS multivariate analysis of variance, and since significant differences (not shown) were found among the respective vectors, univariate analyses were performed for individual fatty acids.

Fatty acid composition of dietary treatments, milk and cheese
The fatty acid composition of lampante olive oil and dietary treatments are shown in Table 3.As expected, about 70% of the FA identified in the lampante olive oil used in this experiment was 18:1 cis-9.
Except for 4:0, 6:0, 8:0 and 17:0, the milk saturated FA (10:0 to 18:0) was affected by dietary treatments (Table 4).Compared with T0 and T1, T2 was higher (P < 0.05) in 18:1 cis-9, 18:1 trans-11, and 20:1 cis-11 and lower (P < 0.05) in 14:1 cis-9 and 17:1 cis-10.The supplementation of olive oil (TI and T2) reduced (P < 0.05) 18:2n6 cis-9, cis-12 and 18:3n3 cis-9, cis-12, cis-15 compared with T0.Myristic (14:0), palmitic (16:0) and oleic (18:1 cis-9) acids were the major FA present in milk samples.Oleic and stearic acids gradually rose (P < 0.05) in milk as the concentration of lampante olive oil increased in dietary rations.Following dietary supplementation with oils rich in long-chain FA , a reduction in short-and medium-chain FA contents in milk has been reported in studies on sheep (Zhang et al., 2006;Chiofalo et al., 2004;Gómez-Cortés et al., 2008b) and goats (Schmidely and Sauvant, 2001).The decrease in medium-chain FA may represent an improvement in the profile of milk fatty acids because medium-chain FA have been reported to constitute the hypercholesterolemic portion of milk fat (Ney, 1991); however, a reduction in 6:0 to 10:0 may be undesirable because of their potential hypocholesterolemic effects.Unfortunately, the transfer of mediumchain FA from diet to milk is very inefficient; hence dietary supplementation is not useful for increasing these FA in milk (Grummer, 1991).Also, when the bioavailability of C18 FA increases as a result of increased dietary intake, 10:0 to 16:0 de novo Milk yield and milk composition were not affected by dietary treatments (36 and 88 g d -1 of lampante olive oil) which might be related to no negative effects of lampante olive oil on ruminal processes.This is in agreement with a Gómez-Cortés et al. (2008a) study in which no differences in the DMI, milk yield or milk composition in lactating ewes supplemented with soybean oil (60 g kg -1 DM) were reported.However, Gómez-Cortés et al. (2008b) observed that olive oil supplementation (60 g kg -1 DM) in dairy ewes did not affect DMI but increased milk yield.Hervás et al. (2008) reported no effect on ewes supplemented with sunflower oil (60 g kg -1 DM); however, they found an increase in milk fat content and decrease in milk protein content, which agrees in part with the tendency to increase milk fat content (Table 2; P = 0.063) as lampant olive oil was increased in the diet in this experiment.
Although it has been suggested that milk yield increases when sheep diets are supplemented with oils, these changes might be due to the greater energy contents of supplemented diets vs. non-supplemented standard diets (Chilliard et al., 2003).The effects of oil supplementation on milk production, fat and protein yield depend on the genetic potential of the ewe to increase milk production as it consumes more dietary nutrients (Appeddu et al., 2004).Manso et al. (2011) supplemented sheep diets with the same amount (3% DM) of hydrogenated palm, olive, soybean and linseed oils and fed animals with iso-nitrogenous and iso-energetic diets.They found no difference in milk yield or milk composition.Similarly, in the current experiment, animals were offered equal amounts of feed with the same ingredients.Hence, it is unlikely that milk composition would be affected by dietary energy and protein content differences, because all dietary treatments used were balanced to be iso-nitrogenous, iso-fibrous and iso-energetic.et al., 2002;Martini et al., 2004).Compared with T0; SFA, PUFA, PUFA n-3, and PUFA n-6 in the milk were lower (P < 0.05) in T1 and T2.Monounsaturated (MUFA), and MUFA/ SFA and PUFA n-6/PUFA n-3 ratios were higher (P < 0.05) in T2 than in T0 and T1 (Table 6).The reduction in PUFA content in the milk from ewes fed T1 and T2 was due to reduced contents of 18:2n6 cis-9, cis-12 and 18:3n3 cis-9, cis-12, cis-15. Conversely, Antongiovanni et al. (2002), reported no differences in 18:2n6 cis-9, cis-12 and 18:3n3 cis-9, cis-12, cis-15 contents in the milk from ewes fed olive oil calcium soap.Martini et al. (2004) reported a decrease in 18:2n6 cis-9, cis-12 and an increase in 18:3n3 cis-9, cis-12, cis-15 contents in milk from ewes fed olive oil calcium soap.On the other hand, Gómez-Cortés et al. (2008b) reported a decrease in both FA; they attributed those results to the reduced dietary contents of those FA.
The MUFA/SFA ratio in milk and cheese was higher in T2 than in T0 and T1, which may be explained by a combined effect between the increases in unsaturated FA (mainly MUFA) and decreases in SFA.Similar results were reported by Antongiovanni et al. (2002), Martini et al. (2004), andHervás et al. (2008).Despite the fact that PUFA n-6 and PUFA n-3 were reduced (12.3 and 47.5% respectively) from T0 to T2, the PUFA n-6/ PUFA n-3 ratio in milk was increased in T2.One of the main considerations when trying to meet synthesis decreases as does their (10:0 to 16:0) concentration in milk (Gómez-Cortés et al., 2008b).Our findings agree with Chiofalo et al. (2004) who reported more 18:1 and 18:0 and less mediumchain saturated FA in milk of ewes supplemented with olive cake.
Compared with other studies (Antongiovanni et al., 2002;Martini et al., 2004;Gómez-Cortes et al., 2008b;Sadeghi et al., 2009), the concentrations of short-chain FA found in milk appeared to be low, however, the nature (FA composition), the type (oilseeds, protected or unprotected oils) and the amount of dietary fat, and their interactions with the basal diet ingredients have been found to affect the ruminal biohydrogenation process (Chilliard et al., 2000).
In the current experiment, the contents of 10:0-17:0, 18:2n6 cis-9, cis-12 and 18:3n3 cis-9, cis-12, cis-15 were diminished and 18:0, 18:1 trans-11 and 18:1 cis-9 were increased in cheese as lampante olive oil supplementation increased (Table 5).The higher levels of 18:1 cis-9 observed in the milk and cheese of ewes fed T2 diet compared with those in ewes fed T0 and T1, may be partly due to the action of the Δ 9 -desaturase enzyme in the mammary gland (Griinari et al., 2000) over a portion of 18:0 produced by ruminal biohydrogenation, and partly to the fact that T2 contains higher concentrations of oleic acid than T0 and T1.In ruminants, roughly 40% of 18:0 absorbed by the mammary gland is desaturated to 18:1 cis-9 (Lock and Garnsworthy, 2003).The increase in monounsaturated FA milk content found in this experiment also agrees with Similarly, Castro et al. (2009) reported a reduction in this index when lactating ewes were supplemented with 12 g kg -1 of sunflower oil.In the human diet, lipids (particularly SFA) are known to contribute to coronary heart disease (CHD; Williams, 2000).Conversely, some milk unsaturated FA can diminish the risk of cardiovascular disease, including 18:2 cis-9, trans-11, MUFA (in particular 18:1 cis-9), and PUFA.In the present experiment, the addition of lampante olive oil in lactating ewes' diets decreased most SFA.The recognized atherogenic SFA are 12:0, 14:0, and 16:0 (Keys et al., 1965) with 14:0 being the most atherogenic, with about four times more cholesterol-raising potential than 16:0 (Ulbricht and Southgate, 1991).The present experiment showed that atherogenic FA in milk were reduced with the inclusion of lampante olive current recommendations for PUFA n-3 is that the current intake of PUFA consists primarily of PUFA n-6.The competition for desaturases and elongases in PUFA n-6 and PUFA n-3 metabolism results in inverse affects on tissue concentrations of these FA (Gebauer et al., 2006).The results obtained from PUFA n-6 and PUFA n-3 content in milk and cheese from ewes fed T2, are important because they show that milk from ewes fed with lampante olive oil could be used to produce cheese with enhanced FA quality (Manso et al., 2011).The atherogenicity index in milk was reduced (P < 0.05) as dietary lampante olive oil supplementation increased (Table 6).The atherogenicity index was lower in milk and cheese obtained from ewes fed with the highest level of lampante olive oil inclusion in the diet (T2).

CONCLUSIONS
Overall, the supplementation of lampante olive oil (36 or 88 g d -1 ) to lactating ewes' diets can increase the contents of 18:1 cis-9 and 18:1 trans-11, and reduce the atherogenicity index in their milk and cheese .The results from the present experiment support the possibility of implementing oil in ewe diets suggesting a positive health effect for human consumption.Despite the fact that 14:0 and 16:0 and PUFA were diminished and 18:0 and 18:1 cis-9 were increased in the milk from ewes fed T2, the thrombogenic index was not different between dietary treatments suggesting that there was a biological distinction among different FA that are accounted for in this index.2005.Effect of lipid supplementation on growth performance, carcass and meat quality and fatty acid composition of intramuscular lipids of lambs fed a management system to produce sheep milk with a naturally enhanced FA composition that has a positive impact on cheese fatty acid quality.

Table 1 Ingredients and chemical composition of dietary treatments (DM basis)
EFFECT OF DIETARY INCLUSION OF LAMPANTE OLIVE OIL ON MILK AND CHEESE FATTY ACID PROFILES OF EWES E. VARGAS-BELLO-PÉREZ, R. R. VERA, C. AGUILAR, R. LIRA, I. PEÑA, A. VALENZUELA AND H. CERDA

Table 2 Milk yield, milk composition and dry matter intake from ewes supplemented with lampante olive oil
3 P-value represents the probability of a treatment effect; a,b,c Means in the same row with different superscripts are different (P < 0.05).EFFECT OF DIETARY INCLUSION OF LAMPANTE OLIVE OIL ON MILK AND CHEESE FATTY ACID PROFILES OF EWES other studies which used unprotected olive oil (Gómez-Cortés et al., 2008b) and olive oil calcium soap (Antongiovanni

Table 5 Effect of treatment diets on the fatty acid composition of the cheese from ewes' milk supplemented with lampante olive oil
Official Methods of Analysis of the Association of Official Agricultural Chemists, 18th ed.AOAC Int., Gaithersburg, MD., USA (1st revision).Appeddu LA, Ely DG, Aaron DK, Deweese WP, Fink E. 2004.Effects of supplementing with calcium salts of palm oil fatty acids or hydrogenated tallow on ewe milk production and twin lamb growth.J. Anim.Sci.82, 2780-2789.Bessa RJB, Portugal PV, Mendes IA, Santos-Silva J.