493 Analele UniversităŃii din Oradea Fasci cula:Ecotoxicologie, Zootehnie și Tehnologii de Ind ustrie Alimentară, 2012 EFFECT OF PHYSIOLOGICAL… [601763]

493 Analele UniversităŃii din Oradea Fasci cula:Ecotoxicologie, Zootehnie și Tehnologii de Ind ustrie Alimentară, 2012

EFFECT OF PHYSIOLOGICAL FACTORS (AGE AND SEX) ON
THE FATTY ACID PROFILE OF MUSCLE TISSUE IN SHEEP

Mierlita Daniel, Stanciu Alina,

University of Oradea, Department of Animal Science, Oradea, Romania; [anonimizat]

Abstract
The aim of this study was to investigate the effect of age (young fattened intensively vs.
reformed adult sheep) and sex (male vs. females) on fatty acid profile (FA) of intramuscular fat
(Longissimus dorsi, LD), with particular reference the PUFA n-3 and CLA (mainly isomer, cis-9
trans-11 C18: 2). 8 heads were used intensively fat tened young sheep heads and 8 adult sheep breed
reformed pan (4 males and 4 females). The results r eveal a significant influence of age and sex on FA
profile or nutritional quality of fat in muscle tis sue. Highest proportion of n-3 FA and CLA (isomer
cis-9, trans-11 C18: 2) in LD muscle fat was found in intensively fattened males compared to females
and that fattened in the intensive sheep adult reco nditioned. Mainly young lambs fattened intensively
and male sex in intramuscular fat were significantl y higher proportions of PUFA n-3 (p <0.005),
especially C18: 3 n-3, whose weight was increased b y 52.0% and 30.8% in young males compared to
adult animals and that sex female lambs. The propor tion of cis-9, trans-11 CLA in intramuscular fat
was higher in young males gained intensive and comp ared with females and adult animals.

Key words : PUFA n-3, cis -9, trans -11 CLA, age and sex, meat, sheep

INTRODUCTION

Intramuscular fatty acid profile of fat storage and can be affected by
several factors, such as diet, breed, sex, age, and weight at slaughter
(Aharoni et al., 1995, Rule et al., 1995, Wood and Enser, 1997, Nürnberg et
al., 2005; Guler and Aktumsek, 2011). The most impo rtant factor role in
manipulating fatty acid profile of lambs, is the fo od.
Metabolic studies have shown that the total amount of fat in human
nutrition determine serum cholesterol levels, but i mportance is the type of
fat (Sanders, 2003). Additionally controlled trials have shown that replacing
saturated fat and high in trans fats with unsaturat ed fats and especially n-3
fatty acids, is more effective in preventing heart disease than reducing fat
(Renaud and Lanzmann-Petithory, 2002, Hu and Willet t, 2002; Sanders,
2003). Animal feeding strategies may lower proporti on of saturated fat and
increase weight polyunsaturated fatty acids n-3 and CLA (conjugated
linoleic acid) in intramuscular fat, which would im prove the quality of sheep
meat.
Lipids from ruminants are the richest sources of CL A, especially the
brown acid (C18: 2 cis -9, trans -11) which is the most important isomer of
CLA. This isomer, which represents over 80% of CLA in foods from
ruminant (Ha et al., 1990), proved very important f or human health because
it inhibits the proliferation of cancer cells (Schu ltz et al., 1992; Belury et al.,

494 1995), inhibits the accumulation of body fat (Park et al., 1997) and
antioxidant effect antidiabetogenic (Ip et al., 199 4).
Isomer cis-9, trans-11 CLA, are formed in the rumen by incomplete
biohidrogenation fatty acids in the diet, especiall y C18: 2 n-6, but a
substantial fraction of the amount of CLA in tissue s derived from
desaturation C18: 1 trans-11 ∆9-desaturase enzyme in action acting on
adipose tissue (Santora et al., 2000, Bauman et al. , 2001).
In terms of nutrition, fat from lambs fattened is m ore appropriate than
those from adult sheep, because it contains a highe r proportion of n-3
polyunsaturated fatty acids (PUFA n-3) and CLA and report n-6/n-3 lower
(Santos et al., 2002; Nuernberg et al., 2008; Guler and Aktumsek, 2011).
However there is little information on the influenc e of physiological
factors on nutritional quality of mutton fat. The a im of this study was to
investigate the effect of age and sex on the fatty acid profile of
intramuscular fat (LD), with particular reference t o PUFA n-3 and CLA
(mainly isomer cis -9, trans -11 C18: 2).

MATERIAL AND METHODS

The study was conducted at the University of Oradea mainly aimed
profiling fatty acids (FA) of intramuscular fat ( longissimus dorsi ) in relation
to age (young fattened intensively vs. reformed adu lt sheep) and sex (males
vs. females).
For sampling analysis (muscle tissue) were used 8 y oung sheep
fattened heads and 8 heads intensively reformed adu lt sheep breed pans,
distributed as follows: 4 males and 4 females. Biol ogical material derived
from commercial farms in Bihor County.
With 60 days before sampling analysis, respectively slaughter for food
rations were used as fodder uniform structures are based on alfalfa hay,
green fodder and feed hay combined with specific nu tritional requirements.
Alfalfa hay and green fodder were provided ad libitum and premixtures
were administered twice a day for 200 g / head for adults and 300 g / head
youth. In this way food was removed influence the f atty acid profile of
housing. Mixed fodders used both youth nutrition an d adult sheep consisted
of: corn-45,5%; triticale-20,0%; meal sunflower-10, 0% and vitamin-mineral
premix-4,5%. Mixed fodder provided for 1 kg DM: 1.4 1 UNC (meat
nutrition units = 1481 kcal EN; 6.2 MJ), 127.3 g PD IN (protein digestible in
the intestine in the diet levels rumen degradable a zot) and 116.7 g PDIE
(protein digestible in the intestine in the diet le vels rumen degradable
energy).
Slaughtering control were preceded by a 12-hour die tary animal and
sampling LD muscle analysis was done in 24 hours po stmortem (after
drying carcass). Samples for analysis were collecte d from the lower back

495 after the 13 th thoracic vertebra to a length of 10 cm. Samples we re stored at
-20 0C until fat isolation and identification of their p rofile.
For extraction of total lipids from muscle tissue s amples was used
classical method described by Folch et al. (1987). Weighed sample (about 1
g) was triturated with an electric homogenizer and treated with 10 ml of
methanol and BHT. After mixing for 1-2 minutes, wer e added 20 ml of
chloroform and stirred again for 2 minutes. The mix ture was filtered and the
waste is treated again with a mixture of chloroform : methanol in the ratio of
2 : 1 (v / v). Total extract obtained was added to a volume of 88% KCl
solution so that the ratio of chloroform : methanol : potassium chloride to be
8 : 4 : 3 (v / v). After phase separation by centri fugation, recovered hipofaza
chloroform containing total lipids. Chloroform extr act was passed over
anhydrous sodium sulphate to remove traces of water , was evaporated to
dryness and restart in a known volume of chloroform (1 ml). Total lipid
extract was stored in glass bottles with ground gla ss stopper, in darkness and
temperatures of – 20 0C until use for further analysis.
For determination of fatty acids, total lipid extra ct was transesterificat
with methanol saturated with hydrochloric acid for 2 hours at 80 0C in Pyrex
tubes. Methyl esters were extracted in petroleum et her : benzene (8 : 1),
purified, separated and isothermal identified as me thyl esters by gas
chromatography with an HP 5890 gas chromatography c oupled with II/5972
GC-MSD mass spectrometer. Grout was used to identif y PUFA No. 2
(Animal Source). Fatty acids were expressed as a pe rcentage of total methyl
esters identified.
ANOVA was used to assess the effect of age and sex on FA profile of
intramuscular fat. Comparison of the means was perf ormed using Duncan
test. Differences between means were considered for p <0.05.

RESULTS AND DISCUSSION

The results presented in tables 1 and 2 reveals a s ignificant influence
of age and sex on nutritional quality of fat in mus cle tissue, that the fatty
acid profile. Highest proportion of n-3 FA and CLA (isomer cis -9, trans -11
C18: 2) in LD muscle fat was found in intensively f attened males compared
to females in system ingrasatate intensive (table 1 ), and reconditioned or
adult sheep (table 2). Share saturated FA (SFA) and monounsaturated FA
(MUFA) in intramuscular fat was higher by 10.0% and 18.2% in adult sheep
compared with youth reconditioned gained intensive, while the share of
polyunsaturated FA (PUFA) was lower by 32.5%. Simil ar issues were found
and about the influence of sex, meaning that the la rgest proportion of SFA
and MUFA in intramuscular fat and the lowest propor tion of PUFA were
found in females compared with males (Fig. 1). Thes e results are in

496 agreement with previous findings established in stu dies in fattening calves
(Steen et al., 2010).
Mainly young lambs fattened intensively and male se x in
intramuscular fat were significantly higher proport ions of PUFA n-3 (p
<0.005), especially C18: 3 n-3 (Fig. 2), whose weig ht was increased by
52.0% and 30.8% in young males compared to adult an imals and that sex
female lambs.

Table 1
Influence of age on fatty acid profile of muscle ti ssue (% of FAME)
Age
Youth
intensive
fattening Mature
reconditioned SEM 1 p
Total lipids 6,25 8,45 0,54 ***
C 12:0 0,02 0,03 0,01 *
C 14:0 4,60 5,97 0,10 **
C 16:0 18,87 21,48 0,41 ***
C 18:0 12,29 11,94 0,37 NS
C 20:0 0,12 0,13 0,10 NS
C 18:1 28,30 33,46 0,71 ***
C 18:2 n-6 9,74 6,45 0,69 **
C 18:3 n-3 3,71 2,44 0,15 ***
CLA (cis -9, trans -11 C18:2) 2,64 1,31 0,20 ***
C 20:2 n-6 0,75 0,82 0,07 NS
C 20:4 n-6 3,80 3,48 0,23 NS
C 20:5 n-3, EPA 2,69 1,45 0,18 ***
C 22:3 n-3 0,74 0,32 0,07 ***
C 22:5 n-3 DPA 0,58 0,41 0,02 **
C 22:6 n-3 DHA 2,64 1,73 0,11 ***
SFA 35,95 39,55 0,65 **
MUFA 28,30 33,46 0,71 ***
PUFA 27,29 18,41 1,16 ***
FUFA n-3 10,36 6,35 0,29 ***
PUFA n-6 14,29 10,75 0,97 *
n-6/ n-3 1,38 1,69 0,21 *
SFA/MUFA 1,27 1,18 0,10 *
MUFA/PUFA 1,03 1,82 0,12 **
PUFA/SFA 0,76 0,46 0,08 ***
FAME = methyl esters of fatty acids; n=4; SEM = sta ndard error of least sqare means;
SFA = fatty acids saturated, MUFA = fatty acids mon ounsaturated, PUFA = fatty acids polyunsaturated
PUFA n-6 = C 18:2 n-6 + C 20:2 n-6 + C 20:4 n-6
PUFA n-3 = C 18:3 n-3 + EPA + C 22:3 n-3 + DPA + DHA
CLA = conjugated linoleic acid, isomer c9, t11 C18:2 (aci d rumenic)
DPA = acid docosapentaenoic, EPA = acid eicosapenta enoic, DHA = acid docosahexaenoic,
NS = insignificant, * = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001

497 Table 2
The influence of sex on fatty acid profile of muscl e tissue (% of FAME)
Sex
Males Females SEM p
Total lipids 5,41 7,39 0,35 **
C 12:0 0,02 0,02 0,01 NS
C 14:0 3,72 5,02 0,12 **
C 16:0 18,25 19,30 0,51 **
C 18:0 12,49 13,74 0,45 NS
C 20:0 0,10 0,14 0,01 *
C 18:1 26,53 29,19 0,86 **
C 18:2 n-6 9,50 7,87 0,85 **
C 18:3 n-3 4,08 3,12 0,18 **
CLA (cis -9, trans -11 C18:2) 2,97 2,18 0,13 **
C 20:2 n-6 1,28 0,85 0,08 *
C 20:4 n-6 4,17 3,43 0,19 **
C 20:5 n-3, EPA 2,58 1,74 0,08 ***
C 22:3 n-3 0,95 0,67 0,03 *
C 22:5 n-3 DPA 0,50 0,37 0,01 *
C 22:6 n-3 DHA 2,18 1,95 0,05 NS
SFA 34,58 38,22 0,80 **
MUFA 26,53 29,19 0,86 **
PUFA 28,31 20,72 1,12 ***
FUFA n-3 10,29 7,85 1,19 **
PUFA n-6 15,05 10,69 0,69 **
n-6/ n-3 1,46 1,36 0,30 NS
SFA/MUFA 1,30 1,31 0,24 NS
MUFA/PUFA 0,94 1,41 0,10 **
PUFA/SFA 0,81 0,54 0.09 ***
FAME = methyl esters of fatty acids; n=4; SEM = sta ndard error of least sqare means;
SFA = fatty acids saturated, MUFA = fatty acids mon ounsaturated, PUFA = fatty acids polyunsaturated
PUFA n-6 = C 18:2 n-6 + C 20:2 n-6 + C 20:4 n-6
PUFA n-3 = C 18:3 n-3 + EPA + C 22:3 n-3 + DPA + DHA
CLA = conjugated linoleic acid, isomer c9, t11 C18:2 (aci d rumenic)
DPA = acid docosapentaenoic, EPA = acid eicosapenta enoic, DHA = acid docosahexaenoic,
NS = insignificant, * = p ≤ 0,05; ** = p ≤ 0,01; *** = p ≤ 0,001

Since all animals were fed the same diet, we assume that differences
in the concentration of C18: 3 n-3 in intramuscular fat is mainly due to the
peculiarities of rumen fermentation processes which alter the rate of linoleic
acid in the rumen biohidrogenare food reducing its level in intramuscular fat
(Chilliard et al., 2001; Nuernberg et al., 2005). I ncrease of C18: 3 n-3 in
intramuscular fat is considered beneficial to consu mer health (Gill et al.,
1995; Warnants et al., 1996).
High content of intramuscular fat in C20: 5 n-3 (EP A), C22: 5 n-3
(DPA) and C22: 6 n-3 (DHA) in young intensive fatte ning versus adultele
and males versus females that suggests higher bioav ailability of C18: 3

498 which favored the synthesis of these long chain pol yunsaturated fatty acids
of carbon atoms.

38.22
20.72 34.58 39.55
35.95
27.29
18.41 28.31
15 20 25 30 35 40 45 50
Youth Mature Males Females SFA MUFA PUFA

Fig. 1. Influence of age and sex on FA profile of m uscle tissue

7.85
2.18 3.12
1.36 10.29
6.35 10.36
1.31 2.97
2.64 3.71
2.44 4.08
1.38 1.69 1.46
12345678910 11 12
Youth Mature Males Females FA n-3 c9,t11 CLA C18:3 n-3 n-6/2-3

Fig. 2. Influence of age and sex on the content of FA n-3 and CLA muscle tissue

The proportion of cis -9, trans -11 CLA in intramuscular fat was higher
in young males gained intensive and compared with f emales. The content of
cis-9, trans-11 CLA may be due to biohidrogenarea l inoleic acid (C18: 2 n-
6) in the rumen, and increased production of C18: 1 trans -11, of which C18:

499 2 c9, t11 can be produced in tissues by enzymatic d esaturation processes
(Griinari et al., 2000; Santora et al., 2000, Coope r et al., 2004).
Report PUFA / SFA in intramuscular fat ranged betwe en 0.81 and
0.46 which are much higher than those mentioned in similar studies aimed
at enabling improved fatty acid profile of intramus cular fat. The best PUFA
/ SFA, in terms of impact on human health, intramus cular fat was obtained
coming from intensively fattened rams.

CONCLUSION

The results demonstrate the opportunity of using su stainable local
animal genetic resources in order to improve the fa tty acid profile of muscle
tissue, analyzed in terms of impact on consumer hea lth. Young male breed
fattened intensively pans recorded in intramuscular fat increased levels of n-
3 polyunsaturated FA (especially C18: 3 n-3), cis -9, trans -11 CLA and best
relationships PUFA / SFA. High content of intramusc ular fat in C20: 5 n-3
(EPA), C22: 5 n-3 (DPA) and C22:6 n-3 (DHA) and int ensively fattened
young, especially in males suggests a higher bioava ilability of C18: 3 out of
food that favored the synthesis of these FA.

Acknowledgements: This work was supported by CNCSIS – UEFISCSU,
project number PN II – IDEI 679/2008.

REFERENCES

1. AOAC, 1996, Official Methods of Analysis. Vol I 16t h ed. Association of Official
Analytical Chemists, Arlington, VA.
2. Ashes, J.R., Thompson, R.H., Gulati, S.K., Brown, G .H., Scott, T.W., Rich, J.C.,
1993, A comparison of fatty acid profiles and carca ss characteristics of feedlot
steers fed canola seed and sunflower seed mal suppl ements protected from
metabolism in the rumen. Aust. J. Agr. Research, 44 , p. 1103-1112.
3. Bolte M.R., Hess B.W., Means W.J., Moss G.E., Rule D.C., 2002, Feeding lambs
high-oleate safflower seed differentially influence s carcass fatty acid composition.
J. Anim. Sci. 80: 609-616.
4. Chilliard, Y., Ferlay, A., Rouel, J., Lambere, G., 2003, A review of nutritional and
physiological factors affecting goat milk synthesis and lipolysis. J. Dairy Sci.
86:1751-1770.
5. Cividini A., Levart A., Zgur S., 2008, Fatty acid c omposition of lamb meat as
affected by production system, weaning and sex. Act a agriculturae Slovenica,
suplement 2, 47–52. http://aas.bf.uni-lj.si
6. Cooper S.L., Sinclair L.A., Wilkinson R.G., Hallett K.G., Enser M., Wood J.D.,
2004, Manipulation of the n-3 polyunsaturated fatty acid content of muscle and
adipose tissue in lambs. J. Anim. Sci., 82: 1461-14 70.
7. Folch, A.J., M. Lees, and G.H. Stanley., 1957, A si mple method for the isolation
and purification of total lipids from animal tissue s. J. Biol. Chem. 226: 497-509.

500 8. Ghoorchi, T., Gharabash, A.M., Torbatinejad, N.M., 2006, Effects of calcium salt
of long chain fatty acid on performance and blood m etabolites of atabay lambs.
Asian Journal of Animal and Veterinary Advances 1(1 ), p. 70-75.
9. Griinari, J.M., Bauman, D.E., 1999, Biosynthesis of conjugated linoleic acid its
incorporation into meat and milk in ruminants, in: Yurawecz, M.P., Mossoba,
M.M., Kramer, J.K.G., Pariza, M.W., Nelson, G.J. (E ds.), Advances in Conjugated
linoleic acid research, vol.1, AOCS Press, Champaig n, IL, pp. 180-200.
10. Guler G. O., Aktumsek A., 2011, Effect of feeding r egime on fatty acid
composition and conjugated linoleic acid content of perirenal, omental and tail fat
in Akkaraman lambs. African Journal of Biotechnolog y vol. 10(36): 7099-7108,
http://www.academicjournals.org/AJB
11. Jagusch, K.J., Rattray, P.V., Oliver, T.W. & Cox, N .R., 1979, The effect of
herbage yield and allowance of growth and carcass c haracteristics of weaned
lambs. Proc. N.Z. Soc. Anim. Prod. Vol. 39, p. 254- 259.
12. Kaczor U., Borys B., Pustkowiak H., 2010, Effect of intensive fattening of lambs
with forages on the fatty acid profile of intramusc ular and subcutaneous fat. Czech
J. Anim. Sci., 55(10): 408–419.
13. Lourenco, M., Van Rans, G., De Smet, S., Raes, K., Fievez, V., 2007, Effect of
grazing pastures with different botanical compositi on by lambs on rumen fatty
acid metabolism and fatty acid pattern of longissim us muscle and subcutaneous
fat. Animal, 1- The Animal Consortium, p. 537–545.
14. Mierlita, D., Pascal, C., Daraban, St., Lup, F.G., Maerescu, C., 2011, The
influence of forage/concentrate ratio and full fat soya by-pass supplementation on
the fatty acids profile from the carcass of fatting lambs. Journal of Agricultural
Science and Technology, 5(1), p. 67-76.
15. Piasentier, E., 2003, The effect of grazing on the quality of lamb meat.
Internationale Fachtagung fur Schafhaltung, Innsbru ck.
16. Potkanski A., Cermak B., Szumacher-Strabel M., Kowa lczyk J., Cieslak A., 2002,
Effects of different amounts and types of fat on fa tty acid composition of fat
deposit in lambs. Czech J. Anim. Sci., 47(2): 72-75 .
17. Steen R.W.J., Lavery N.P., Kilpatrick D.J., Porter M.G., 2003, Effects of pasture
and high concentrate diets on the performance of be ef cattle, carcass composition
at equal growth rates, and the fatty acid compositi on of beef, New Zealand. J. of
Agric. Research, 46:2, 69-81. http://dx.doi.org/10.1080/