Biotechnology in Animal Husbandry 25 (5-6), p 945-952, 2009 Publisher: Institute for Animal Husbandry, Belgrade-Zemun

ISSN 1450-9156 UDC 637.12.39

VARIATION IN FATTY ACID COMPOSITION OF MILK FROM TSIGAI SHEEP IN THE REGION OF CENTRAL BALKAN MOUNTAINS
G. Gerchev1, G. Mihaylova2
1 2

Institute of Mountain Stockbreeding and Agriculture, Troyan, Bulgaria Agricultural Faculty of Thracian University, Stara Zagora, Bulgaria Corresponding author: gercho_g@abv.bg Original scientific paper

Abstract: The study was conducted with individual sheep milk samples taken every month during the milking period from 7 Tsigai sheep raised in the conditions of Central Balkan Mountains. Extraction of fat from the milk samples was performed by the method of Rose-Gottlieb and the methyl esters of fatty acids were analyzed through a system of two gas chromatographs allowing separation of isomers of the unsaturated fatty acids. It was found that the milk fatty acid content in Tsigai sheep showed tendencies to variation in various directions in the months of milking period. It was characterized by relatively high total content of saturated fatty acids in the grazing period - from 57.20% in April to 65.98% in May. The breed and month of lactation period had a greater effect on their concentration in the milk than the pasture type. The content of poly saturated fatty acids was the highest at the beginning of grazing period April and May, 7.66% and 9.10% respectively and decreased in July 6.22%. The sum of short-chain fatty acids was the greatest in the milk obtained in June, that of medium-chain ones in July and that of long-chain ones in April. The breed, lactation period and sward stage had an effect on the content of vaccenic and conjugated linolic acid. Key words: sheep milk, milk fat, fatty acids

Introduction
Milk fat is a milk component, in which saturated fatty acids prevail. Its composition includes also specific functional substances: poly saturated fatty acids, including also conjugated linolic acid (CLA), having a number of favourable curative effects (Jarheis et al., 2000), as well as anticancerogene properties. Prodi (1999) showed that similar properties were also found in butyric and vaccenic acid (VA), natural components of milk fat. Therefore, the studies are directed towards the influence of fatty acids and mostly that of trans-fatty acids and conjugated

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linolic acid on human health, on the one hand and towards the possibilities to increase the quantity of these acids in milk and milk products, on the other hand. According to Nudda et al. (2005), seasonal variation in CLA and VA content in milk fat is related to a change in pasture composition and their concentration in milk products depends on their content in raw sheep milk. Addis et al. (2005) found that the feeding with appropriate fresh forage rich in linolic acid could change the composition of milk fat in the obtained sheep milk, including CLA content. Trials of phenotypic analysis were carried out (Carda et al., 2004) with the purpose of identifying a possible gene in crossbred sheep (Sarda X Lacaune), responsible for the fatty acid synthesis in mammary gland and determining a coefficient between CLA/VA. The objective of this study was to follow up the variation during the grazing period in the content of fatty acid composition of fat of milk yielded during the milking period of Tsigai sheep raised in the conditions of Central Balkan Mountains.

Materials and Methods

The study was conducted with individual milk samples taken from sheep of Tsigai breed. The milk samples were taken from 7 animals every month during milk tests. The milk was analyzed during the period from April to July when the animals were raised in confinement and by grazing in mountain pastures. From mid-June the sheep were put to alpine grazing on high-mountain matgrass pastures. The samples for analysis were taken from the milk quantity obtained from each animal according to the rules for milk sampling. Extraction of fat from the milk samples was performed in the laboratory of Dairying Department of Agricultural Faculty of Tracian University, town of Stara Zagora by the method of Rose-Gottlieb and the obtained fat was sent for study to University Friedrich Schiller, Jena, Germany. Methyl esters of fatty acids were separated through a system of two gas chromatographs (GC) and the CLA results were controlled through a liquid chromatograph (HPLC). Variance-statistical processing of data was performed through Statistica for Windows (Release, 4.3, Stat. Soft. Inc., 1994) and the average values were compared according to the tables of t-test of Student-Fisher.

Results and Discussion

The content of saturated fatty acids (4-18) in milk fat by months is presented in Table 1. Quantity of 4:0 in the studied sheep milk increased in May as compared to that obtained in April and then decreased until the end of the

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studied period (July) at > 0.01. The variation in 6:0, 8:0, 10:0 and 12:0 levels marked an increase from April to June (>0.001) and a decrease in July end of lactation period. Concentration of C13:0, 14:0 and 15:0 showed a tendency to continuous increase from beginning to end of the studied period at high significance of differences (>0.001). Medium-chain C16:0 decreased in May as against April, but at the end of milking period again marked a tendency to gradual increase in June and July (<0.01).
Table 1. Saturated fatty acids in milk from the studied Tsigai sheep (n=7) Fatty acids C4:0 C6:0 C8:0 C10:0 C12:0 C13aiso C13:0 C14iso C14:0 C15iso C15aiso C15:0 C16iso C16:0 C17iso C17aiso C17:0 C18:0 April x Sx 3.68 0.214 1.75 0.224 1.12 0.175 2.57 0.416 1.69 0.172 0.03 0.007 0.05 0.005 0.16 0.012 5.79 0.435 0.26 0.016 0.47 0.033 0.89 0.065 0.32 0.016 20.74 0.508 0.48 0.029 0.46 0.022 0.90 0.040 14.97 0.878 May x Sx 4.02 0.158 2.81 0.191 2.11 0.152 4.97 0.366 2.53 0.138 0.04 0.009 0.07 0.002 0.14 0.008 6.88 0.243 0.35 0.016 0.65 0.029 1.15 0.031 0.29 0.014 19.49 0.447 0.46 0.033 0.50 0.016 0.74 0.018 11.41 0.454 June x Sx 3.44 0.161 2.87 0.121 2.40 0.109 6.46 0.378 3.29 0.156 0.04 0.006 0.09 0.004 0.17 0.013 8.56 0.276 0.42 0.025 0.73 0.052 1.38 0.071 0.30 0.017 21.02 0.500 0.53 0.020 0.56 0.018 0.90 0.020 10.81 0.565 July x 3.28 2.45 1.94 5.28 3.14 0.06 0.09 0.22 9.87 0.63 0.96 1.44 0.41 22.08 0.64 0.64 0.81 11.15 Sx 0.143 0.143 0.147 0.421 0.190 0.004 0.004 0.013 0.371 0.036 0.046 0.048 0.029 0.619 0.026 0.033 0.026 0.417

Similar variation in the content was also recorded in C17:0. Quantity of 18:0 decreased from April to June and increased in July up to lower levels than those found in May. The other found saturated fatty acids had small quantities and showed fluctuations in various directions. In April, traces of 11:0 were found only in one sample from the studied total of seven individual samples of sheep milk and in June, traces of 13:0 iso. The variation in the concentration of saturated fatty acids during the milking period resulted from the changes having occurred in consequence of the stage of vegetation of the sward. After the move of the sheep into the matgrass pasture with fresh sward (July) there was a tendency to decrease of the quantity of short-chain saturated fatty acids (from 4:0 t 12:0) and increase of concentration of the medium-chain ones (from 14:0 t 16:0), the differences as against June being mathematically

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proven for myristic (<0.01), isomyristic (<0.001) and isopentadecanoic (<0.001) acids. These results were close to the dynamics found for fatty acids 14:0, 15:0 and 16:0 and contrary for 18:0 in milk from sheep of Karakachan breed raised in the pastures during the same period (Mihaylova et al., 2008), which shows that there are also breed differences in milk fat synthesis. In addition, the changes in some of the saturated fatty acids (myristic, etc.) corresponded to the increase of milk fat at the end of lactation period associated with a change in milk palatability. Concentrations of the found monounsaturated fatty acids (Table 2) were lower than 1%, except for 18:1 cis9, 18:1 trans11 and 16:1 in July. Oleic acid 18:1 cis9 showed the greatest quantity in April and the smallest one in June, whereas vaccenic acid 18:1 trans11 had the highest value in May and the lowest one in April (>0.001). The other trans-isomers of C18:1 decreased considerably (at p>0.001) at the end of lactation (July) as against April and May. The same tendency was also observed for the cis-forms of oleic acid, except for 18:1cis9.
Table 2. Monounsaturated fatty acids in milk from sheep of Tsigai breed (n=7) April x Sx C10:1 0.09 0.015 C14:1 0.09 0.010 C16:1 0.99 0.047 C17:1 0.32 0.064 C18:1tr6/7 0.32 0.012 C18:1tr9 0.34 0.009 C18:1tr10 0.34 0.022 C18:1tr11 1.72 0.139 C18:1tr12 0.43 0.037 C18:1tr13 0.92 0.156 C18:1tr15 0.37 0.043 C18:1tr16 0.51 0.063 C18:1cis9 27.36 1.714 C18:1cis11 0.56 0.019 C18:1cis12 0.29 0.023 C18:1cis15 0.26 0.028 C20:1n9 0.21 0.023 C18:2tr9,12 0.73 0.073 C18:2cis9,12 3.12 0.135 C18:3gcis6,9,12 0.19 0.012 CLA 9c,11t 1.35 0.102 C20:4n6 0.16 0.009 C20:5n3 0.10 0.011 C22:5n3 0.13 0.010 Fatty acids May x Sx 0.16 0.013 0.12 0.012 0.98 0.049 0.30 0.059 0.30 0.021 0.31 0.015 0.37 0.027 3.28 0.342 0.56 0.035 1.49 0.103 0.51 0.032 0.75 0.039 21.24 0.937 0.44 0.013 0.21 0.017 0.35 0.019 0.09 0.008 1.06 0.055 2.40 0.084 0.25 0.014 2.30 0.201 0.15 0.006 0.13 0.005 0.15 0.008 June x Sx 0.25 0.021 --0.19 0.023 1.00 0.071 0.44 0.164 0.21 0.012 0.25 0.008 0.22 0.010 2.66 0.265 0.28 0.017 0.63 0.060 0.26 0.020 0.42 0.023 20.15 0.884 0.40 0.011 0.12 0.012 0.24 0.020 0.07 0.002 0.61 0.032 2.00 0.133 0.16 0.009 2.28 0.184 0.15 0.011 0.14 0.009 0.19 0.010 July x 0.26 0.26 1.11 0.17 0.23 0.18 2.30 0.19 0.40 0.17 0.32 21.41 0.38 0.07 0.16 0.04 0.49 1.21 0.11 2.29 0.12 0.10 0.16 Sx 0.018 0.018 0.077 0.010 0.008 0.011 0.185 0.009 0.016 0.007 0.008 0.787 0.012 0.004 0.011 0.002 0.024 0.058 0.004 0.128 0.005 0.008 0.008

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Quantity of linolic acid was the greatest in April and that of linolenic acid in May. The values of conjugated linolic acid were almost equal in May, June and July. The low values of conjugated linolic acid in April corresponded with the low levels of vaccenic acid in the same month. In our opinion, that was due to the fact that the sward was at the initial stage of development, i.e. it had lower content of some nutrients. During the intensive sward growth in May the concentration of the above fatty acids in the milk reached its maximum and during the next months (June and July) their content decreased gradually (at >0.001). CLA cis9 trans11 was an exception: its concentration in the milk fat was maintained during the whole period, as well as in July during alpine grazing in fresh matgrass sward of the highmountain pasture. For the same period of study and in the same pastures it was found that in the milk fat of Karakachan breed sheep (Mihaylova et al., 2008) the values of milk fatty acids C18:1 trans11 and CLA were considerably higher, whereas the values of oleic and linolic acid were similar in concentration for the two breeds, which was probably rather an influence of the breed than an influence of other environmental factors. The total quantity of saturated fatty acids in the milk from the studied Tsigai sheep increased continuously from April (57.20%) to July (65.98%) (Table 3). The content of monounsaturated fatty acids was the highest in April and that of the polyunsaturated ones in May. The total sum of the isomers of conjugated linolic acid was almost equal in May, June and July and their values in Tsigai breed were unidirectional, but considerably lower for the whole period than those of Karakachan sheep raised in the same pastures (Mihaylova et al., 2008). Unidirectional variation was observed during the milking period for the total quantity of trans-isomers of oleic acid and that of polyunsaturated fatty acids, the results obtained by us being considerably higher than those found by Mihaylova et al. (2006) for Rhodope Tsigai. Similarly to this study, Federica et al. (2008) found differences in the concentration of the different fatty acids in the milk from some local Italian breeds, but not for poly saturated fatty acids, including CLA.
Table 3. Groups of fatty acids in milk from sheep of Tsigai breed (n=7) Fatty acids CLA C18:1 trans SFA MUSFA PUSFA C4:0-C11:0 C12:0-C16:1 C17iso-C25:0 April x Sx 1.62 0.119 5.00 0.440 57.20 1.632 35.14 1.613 7.66 0.383 9.21 0.919 31.46 1.150 59.31 1.987 May x Sx 2.83 0.219 7.60 0.576 59.36 0.950 31.54 0.865 9.10 0.264 14.15 0.807 32.67 0.620 53.18 1.069 June x Sx 2.77 0.206 4.96 0.309 64.83 1.014 27.58 0.854 7.58 0.303 15.48 0.617 37.20 0.776 47.32 1.091 July x Sx 2.75 0.162 3.99 0.244 65.98 0.952 27.79 0.846 6.22 0.230 13.23 0.777 40.34 0.798 46.40 1.086

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The content of trans-fatty acids (sum of 18:1 trans 18:2 tr9,12) in the studied milk from Tsigai sheep raised in the region of Central Balkan Mountains reached 8.66% in May and gradually decreased to 4.48% in July. Lower concentrations of mono- and polyunsaturated and higher ones of saturated fatty acids corresponded to those low levels of trans-fatty acids. Mihaylova et al. (2008) found in milk from Karakachan sheep that the content of these acids was considerably higher with the respective higher values of mono- and polyunsaturated fatty acids and lower values of saturated ones. The ratio of omega6/omega-3 was within the limits of the optimum values, higher values being maintained in April and May, 2.38 and 2.56% respectively and then decreasing to about 2.0 in June and July. The biologically important ratio of PUSFA/SFA or the so called P/S ratio, which is relatively low in sheep milk, was not an exception in the Tsigai breed: 0.13-0.15 at the beginning of the grazing period to 0.09 at the end.

Conclusion
The milk from the studied Tsigai sheep was characterized by relatively high total content of saturated fatty acids (57.2-66%). Breed and month of lactation period had a greater effect on their concentration in the milk than the pasture type. The content of poly saturated fatty acids was the highest at the beginning of grazing period April and May, 7.66% and 9.10% respectively and decreased in July 6.22%. The sum of short-chain fatty acids was the greatest in the milk obtained in June, that of medium-chain ones in July and that of long-chain ones in April. The breed, lactation period and sward stage had an effect on the content of vaccenic and conjugated linolic acid in the studied sheep milk.

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Varijacije u sastavu masnih kiselina u mleku cigaja ovaca u regionu centralnih Balkanskih planina
G. Gerchev, G. Mihaylova

Rezime
Istraivnje je uraeno na pojedinanim uzorcima mleka koji su sakupljani svakog meseca tokom perioda mue 7 cigaja ovaca koje su drane I gajene u uslovima centralnih Balkanskih planina. Izdvajanje masti iz uzoraka mleka je uraeno metodom po Rose-Gottlieb i metal estri masnih kiselina analizirani kroz system dva gasna hromatografa koji su omoguili separaciju izomera nezasienih masnih kiselina. Utvreno je da sadraj masnih kiselina u mleku cigaja ovaca pokazuje tendencije vriranja u razliitim pravcima u mesecima perioda mue. Karakterie ga relativno visok sadraj zasienih masnih kiselina u periodu ispae od 57.20% u aprilu do 65.98% u maju.Rasa i mesec perioda laktacije su imali vei efekat na njihovu koncentraciju nego tip ispae. Sadraj poli zasienih masnih kiselina je bio najvii na poetku perioda ispae - april i maj, 7.66% i 9.10% respektivno i smanjio se u julu 6.22%. Zbir masnih kiselina kratkog lanca je bio najvei u mleku dobijenom u junu, kiselina srednjeg lanca u julu i dugakog lanca u aprilu. Rasa, period laktacije i stadijum/faza panjaka su imali uticaj na sadraj n11-oktadecenska kiselina (C18H34O2) i konjugovane linolenske kiseline.

References
MIHAYLOVA G., ODZHAKOVA T.S., KAFEDZHIEV V. (2006): Variation in milk fatty acids in Tsigai sheep raised in conditions of the Rhodopes. Agricultural Science, 3, 37-42. MIHAYLOVA G., GERCHEV G., NAYDENOVA N. (2008): Variation in milk fat composition in Karakachan sheep from the region of Central Balkan Mountains. Book of Papers 80 Years Agrarian Science in the Rhodopes, 110-113. ADDIS M., CABIDDU A., PINNA G., DECANIA M., PIREDDA G., PIRISI A., MOLLE G. (2005): Milkand cheese fatty acids composition in sheep Fed Mediterranean forages with reference to conjugated linolic acid cis-9, trans-11. J. Diry Sci., 88, 3443-3454.

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FEDERICA S., CONTARINI G., ANNICCHIARICO G., NAPOLITANO F., ORRU L., CATILLO G. (2008): Breed differences in sheep milk fatty acid profiles, Opportunitie for sustinablesuse of animal genetic resources. J. Small Ruminant Research, V., 78, 24-31. JAHREIS G. (2000): Functional foots. Fettsuren und fetthnliche inhalsstoffe der milch mit functional-food-charakter. Deutche Molkerei Zeittung, 7, 320-324. CARTA A., PIREDDA G., ADDIS M., CABIDDU A., FIORI M., LEROOX C., BARILLET F. (2004): Fatti acid composition of sheep milk from a backcross Sarda x lacauneresource population, Preliminary QTL detection for CLA content. IURA SAGA, BP 27, 31326, Castanet, Tolosan, FranceCiheam - Options Mediteraneennes, 107-113. NUDDA A.M., GUIRE M.C., BATTACONE C., POLINA G. (2005): Seasonal variation in conjugated Linolic acid and vaccenic acid in milk fat sheep and its transfer to cheese ricotta. J. Dairy Sci., 88, 4, 1311-1319. PARODI P.W. (1999): Conjugated linolic acid and other anticarcinogenic agents of bovine milk fat. J. Dairy Sci., 82, 1339-1349.
Received 31 May 2009; accepted for publication 15 August 2009