Fatty acid profiles and cholesterol of muscovy duck meats supplemented with cellulase, carnitine and fish oil

This present study was carried out to investigate the inclusion effect of carnitine, cellulase, and fish oil on fatty acids composition and cholesterol level of Muscovy duck eggs. A total of 120 Muscovy ducks in laying period were utilized and assigned into 4 dietary treatments, 5 replicates consisting of 6 ducks each, receiving treatments for 90 days. Experimental groups composed of T0 = basal ration, T1 = T0 supplemented with 0,1% cellulase, T2 = T1 supplemented with L-carnitine 40 ppm, and T3 = T2 supplemented with 4% tuna fish oil. This study revealed that the effects of fish oil and L-carnitine supplementation in rations containing cellulase enzyme significantly lower cholesterol level (P<0.01) of Muscovy duck eggs from 867.42 mg/dl to 712.33 mg/dl, Low Density Lipoprotein (LDL) from 28.33 to 22.12 mg/dl, saturated fatty acids from 30.88% to 25.02%. Meanwhile it increased the total concentration of High Density Lipoprotein (HDL) from 71.67 to 77.88 mg/dl, unsaturated fatty acid from 69.12% to 74.98%, linolenic acids from 5.71% to 8.40%, and linoleic acids from 28.74% to 37.30%. A diet rich in cellulase enzyme, carnitine, and fish oil notably increased HDL level, along with the reduction of LDL and total cholesterol.


Introduction
In Indonesia, Muscovy ducks (MD) are commonly raised traditionally as either for eggs production or alternative food sources.Numerous species of local duck can also be found in different phenotypic characteristics, including Rambon, Pitalah, Alabio, Magelang, Pegagan, and Bayang ducks [1].Although some studies mentioned that diverse rearing system might be effective in modifying their performance and nutritional characteristics [2,3], they commonly come with a slow growth and reproductive performances, while possessing high content of saturated fat and cholesterol [4].With an increase of consumer consciousness toward healthy product that they consume, study to modify the nutritional content, especially that enriched with n-3 (n-3) and n-6 (n-6), while maintaining low level of cholesterol are great of interest [5].The effort to modify nutritional content of MD meat may through the inclusion of fish oil and l-carnitine within the mixed ration.They carry essential function during metabolism by converting fat into energy.
Up to date, rice bran and maize made up the highest portion for MD ration in Indonesian traditional raising system.Both maize and rice bran contain considerably high amount of fiber, causing it not easily to be digested by the animal owing to the limit enzymes capable of degrading fiber.Therefore, the addition of exogenous enzymes into feed rations are addressed in improving the nutritional content of the feed by providing higher availability and efficiencies of nutrient [6].Olgun et al [7] revealed that the addition of carbohydrase significantly upregulates the availability of energy within the diet.With this regard, cellulase as a part of carbohydrase enzymes have potential in increasing nutrient digestibility by providing potential available energy.Cellulase helps the catalysation process during the conversion of cellulose into glucose [6,7], provide more available energy source, thus reduce the waste from incomplete digestion process.Previous study mentioned that adding β-xylanase, cellulase, β-glucanase, and phytase containing enzymes into diets markedly improve the feed efficiency and consequently higher egg production [7].
L-carnitine is found naturally in plants, animals, and microorganisms.It is included as easily dissolve in water quaternary amines group [8].Carnitine involves essentially during lipid metabolism process by providing support to transport long-chain fatty acids (LCFAs) into the mitochondrial system, penetrating through membrane for oxidation [8,9].Therefore, deficit in LCFAs availability that can be processed and esterified into triacylglycerols and stored in adipose tissue will infer the metabolism process within the mitochondria, and less lipids available for peroxidation.Taking carnitine supplements increases fatty acid oxidation for energy production and decreases energy utilization [10].Carnitine levels are lower in plant-derived poultry feed ingredients like soybeans, maize, and other plant products than in animalderived feedstuffs.Therefore, a diet high in cereals for poultry may result in a lack of carnitine [9].Additionally, since carnitine has antioxidative properties, including it in a diet high in fat or oil may aid in the process of reducing or preventing rancidity [11].It has been demonstrated that adding carnitine to the diet of poultry can alter the chemical composition of poultry products and improve poultry performance [12,13].
An effective strategy to improve the percentage of unsaturated fatty acids (UFAs) within poultry products is by adding feed materials rich in UFAs, such as tuna fish, menhaden, and linseed oil.Marine fish and fish oil typically contain abundant UFAs, mainly n-3 and n-6 [14].Tuna fish oil as an example, is enriched with n-3 Eicosapentaenoic and docosahexaenoic acid, which accounts for 33.6 to 44.85% and 14.64%, respectively, while containing 5.8% fat, and 8260 kcal/kg total energy metabolism [14].The addition of UFAs sources to poultry products have been shown to have a positive effect in several studies [5,14,15].However, limited information is accessible with the topic of influence of supplementation with feed materials rich in UFAs on quality properties of Muscovy duck egg.Therefore, the objective of this study was to carefully investigate the addition effect of carnitine, cellulase, and tuna fish oil on cholesterol, lipid, and fatty acid composition of Muscovy duck eggs.All of the procedures of this experiment were approved by the committee of ethics for animal experiment of the Department of Animal Science, Universitas Sebelas Maret.A total of 120 muscovy ducks in layer phase were allotted into four treatment groups with 5 replications, in which every treatment was composed of six ducks.T0 was basal diet, consisted of rice bran, maize, and concentrate for layer phase, T1 was basal diet added with 0.1 percent of cellulase enzyme, T2 was basal diet added with 0.1 percent cellulase enzyme and 40 ppm of L-carnitine, while T3 was composed of basal diet added with 0.1 percent cellulase enzyme and 40 ppm of L-carnitine and 4% of tuna fish oil.Table 1 depicts the detail feed materials used and nutritional content of the tested feed ration.The ducks were raised according to the standard management procedures, kept in battery cages, and subjected to diverse dietary treatments (ad libitum) for 90 days.In addition, all Muscovy ducks were provided with ad-libitum water access throughout treatments, and eggs produced were collected daily.In order to analyse the cholesterol, lipid, and fatty acid composition, egg samples were randomly taken (10 replication each) and subsequently subjected to evaluation according to Association of Official Analytical Chemist's (AOAC) procedure [16].

Materials and methods
The obtained data were statistically calculated through the analysis of variance (ANOVA).The significant differences were set (p<0.05) and if statistical significance were present (α = 0.05), it was continuously subjected to an orthogonal contrast test.Set contrast applied were: T0 vs T1, T2, T3 (evaluate the effect of cellulase enzyme within the diet); T1 vs T2, T3 (evaluate the effect of L-carnitine within the diet); and T2 vs T3 (evaluate the effect of tuna fish oil within the diet).

Cholesterol contents
In this study, the cholesterol level of the tested eggs is shown by Table 2.The lowest cholesterol level on Muscovy duck eggs were detected under treatment with complete supplementation (T3), possessing 712,33 mg/dl.While as expected, the highest cholesterol level was detected on egg without any supplementation (867,42 mg/dl).In addition, this study revealed that treatment with the supplementation of cellulase enzyme or L-carnitine alone did not bring significant effect on both LDL, HDL, and cholesterol contents in eggs.a-b Means within the same row indicate significant differences from dietary treatments (P<0.01).
1 T0 was basal diet, consisted of rice bran, maize, and concentrate for layer phase T1 was basal diet added with 0.1 percent of cellulase enzyme, T2 was basal diet added with 0.1 percent cellulase enzyme and 40 ppm of Lcarnitine, while T3 was composed of basal diet added with 0.1 percent cellulase enzyme and 40 ppm of L-carnitine and 4% of tuna fish oil.
This study also inferred the essential role of fish oil and L-carnitin combination within the diet containing 0.1% cellulase enzyme, shown by a markedly notable effect (P<0.01) on cholesterol levels of Muscovy duck eggs.Further, following a thorough orthogonal contrast test, it was revealed that Mucovy duck receiving basal diet (T0) was not differ to that of T1, T2 (P>0.05).Similarly, insignificance was also observed between T1 with T2.Cellulase enzyme supplementation affected nothing (P>0.05) on cholesterol levels of Muscovy duck eggs, either did L-carnitine when only combined with cellulase.

Fatty acid contents
Table 3 displays dietary supplementation effects of L-carnitine, cellulase, and fish oil alone or in combination on the profile of Muscovy duck eggs' FAs.As shown, Muscovy ducks fed with ration enriched with a complete combination of cellulase, L-carnitine, and fish oil resulted in significant improvement of n-3 fatty acids, mainly Linoleic acid (8.4%) when compared to remaining treatments.Meanwhile, treatment with only a basal diet resulted in an egg with the lowest content of n-3 (5.71%).This study also revealed that supplementation of cellulase alone, or in combination with L-carnitine did not show significant influence on the content of n-3, n-6, saturated, and UFAs in eggs.Eggs' levels of n-3, n-6, and UFAs significantly increased when cellulase enzyme, L-carnitine, and fish oil were included together within the animal diet, while they maintained the lowest saturated fatty acids (P<0.01). 1 T0 was basal diet, consisted of rice bran, maize, and concentrate for layer phase T1 was basal diet added with 0.1 percent of cellulase enzyme, T2 was basal diet added with 0.1 percent cellulase enzyme and 40 ppm of Lcarnitine, while T3 was composed of basal diet added with 0.1 percent cellulase enzyme and 40 ppm of L-carnitine and 4% of tuna fish oil.

Cholesterol contents
Exogenous enzymes like cellulase did not lower cholesterol levels because this enzyme is mostly used to make nutrients easier to digest, especially when a diet is high in fiber [17,18].Previous research [19] demonstrated that the addition of cellulase enzymes to chicken eggs had no effect on HDL or LDL levels, supporting this finding.
Carnitine holds an essential role during energy production and lipid metabolism within eukaryotic mitochondria.They transport LCFAs and regulate the conversion ratio of free coenzyme A to acyl coenzyme A (Acyl-CoA) [9].The addition of essential substances were reported to modify the animal performance and meat quality indexes [10,20].In this present study, adding carnitine to a diet that included cellulase enzyme significantly reduced the amount of cholesterol in the eggs (p<0.05).Similar effect from dietary carnitine supplementation on reduction of lipid and cholesterol accumulation were demonstrated by previous studies [8,13,14].However, similar supplementation effect did not observed with regard to the LDL, HDL, and lipid concentration (P>0.05).The significant modification on cholesterol level is assumed due to the major function of carnitine during molecular lipid metabolism, wherein they facilitate the transfer of LCFAs across the membrane of mitochondria for energy generating, create reduction on cholesterol and lipid deposition [21].The result of this study in Muscovy duck eggs presented slightly different result from that of hens.Wherein, the dietary supplementation of carnitine within the diets create significant reduction in the concentration of blood cholesterol, with the exception of the eggs [21].Besides, carnitine addition to quail diets did not greatly affect to the profile of very low-density lipoprotein (VLDL), LDL and HDL of quail blood [22].
Fish oil from tuna are enriched with abundant amount of UFAs, mainly n-3 fatty acids.High concentration of n-3 within the diet are reported to modify the ratio of n3 to n6, creating a healthier animal product for consumption, due to an increased in lipids and HDL levels, along with the reduction of LDL and cholesterol [9,23].Similar observation were revealed by this study, in which a combination of fish oil, carnitine and cellulase enzyme within the Muscovy ducks' diet modified the FAs profile of eggs.Tuna oil addition into animal diet are purposed as the energy and UFAs source [24].In accordance, its addition to poultry products, mainly egg were proven to modify its lipid profiles [18].Moreover, adding oils rich in the concentration of UFAs, such as hemp seed oil to the diet of laying hens was proven to stimulate the more deposition of eggs lipid [11], which also observed in this present study.In a similar fashion, the lipid content on rats' body tissue increased when PUFAs were included in their diet [9].
Apart of lipid and lipoprotein profiles, this present study also observed the effect of carnitine, fish oil, and cellulase enzyme on the modification of serum and egg yolks [25].This is in line with those of Woods & Fearon [25] who described the significant effect of supplementary FAs on the profile of egg HDL.The high HDL content then makes it easier for cholesterol and lipids to travel to the bladder for elimination.In addition, the body tissue's HDL content increased when PUFAs supplements were included in the diet [15,19].UFAs supplementation has also been previously shown to increase HDL content in chicken eggs [25].The mechanism by which n-3 FAs contribute for the reduction of LDL and cholesterol is by upregulating the transport of cholesterol by the help of HDL to the liver, which in turn followed by continuous delivery to the intestine for excretion [21].Therefore, it is revealed that the administration of n-3 FAs contributed for the reduction of dairy goat and cow cholesterol level [24,27].Similarly, the chicken's body tissue's LDL content decreased when PUFAs supplementation were included in the diet [25].

Fatty acid contents
This present study demonstrated the positive effect of tuna fish oil administration into Muscovy diets, particularly an increased concentration of n-3 and n-6 profiles in eggs.Prior study revealed the efficacy of n-3 supplementation to modify the UFAs profile, mainly the increased of n-3 and n-6 in the body [25].Accordingly, previous research in dairy cow's milk [15], dairy goat's milk [24,27], and chicken eggs [18] all showed an increase in n-3 and -6 content when the diet were supplemented with tuna fish oil.
Fish oil functions as energy and UFAs source, wherein its administration into animal diet are proven to modify the animal products' FAs [28].Fish oil will easily be absorbed within the intestine, providing additional source of UFAs which further converted into energy and lipid deposits.An increased of n-3 was observed in laying hens' eggs [19].Accordingly, tuna fish oil displayed similar effect when compared to that of soybean oil fed animal.The egg profiles in fish oil fed chicken generated notably higher n-3 contents.Additionally, LCFAs content was increased when fish oil and peanut oil were combined together within the diet [10].
Abundant UFAs in fish oil is linked to a reduction of egg SFAs, and more optimum when cellulase enzyme and carnitine were also included in the diet.The concentration of eggs' SFAs was notably decreased under treatment with tuna fish oil, which confirmed previous study [19].In addition, the level of body tissue's SFAs were also decreased when UFAs were introduced in the diet [15,24,27].The profile of SFAs in dairy goat and cow milk's were also reduced under treatment using fish oil rich diet [24,27].

Conclusion
This study investigated the single or combined effect of carnitine, cellulase enzyme and fish oil into the fatty acid profiles of Muscovy duck eggs.A diet rich in cellulase enzyme, carnitine, and fish oil notably increased HDL level, along with the reduction of LDL and total cholesterol.In addition, treatment with this supplementation resulted in a markedly higher levels of n-3 and n-6 FAs of Muscovy duck eggs.Meanwhile, supplementation with the mixture composed of fish oil, cellulase enzyme, and L-carnitine produced Muscovy duck eggs with lower levels of SFAs.

Table 1 .
Detail composition and nutritional content of tested feed ration

Tabel 2 .
The level of Cholesterol, LDL and HDL from Muscovy duck eggs

Table 3 .
Fatty acid composition of Muscovy duck eggs Means within the same row indicate significant differences from dietary treatments (P<0.01).