INTRODUCTION

Animal product usually contains high lipid; therefore nowadays people are more selective to choose their diet. This condition surely affects people preference on animal product. Now, is the right time for an innovation to produce high quality meats that have low content of cholesterol and fat. Herbal materials use as a mean to decreasing cholesterol and fat on blood and meat, have been published so many times, one of the herbal material is sweet orange (Citrus sinensis) waste. It has so much potential to decrease cholesterol and fat level on animal meat (Hernawan E & Adriani L 2014).

One option of fitofarmaka is sweet orange (Citrus sinensis) waste , because Indonesia produces 2,355,550 tons Citrus sinensis each year, (Ministry of Agriculture 2010), while only about 35-40 % which can be used by consumers, for food and beverage processing industry, whereas the remaining 65% is wasted  that can not be utilized. This is a good opportunity in livestock feed because it has a superior active compound (Mirzae & Naser 2008 ).

Efforts to reduce cholesterol and triglycerides using fitofarmaka for example adding bay leaf flour (Syzygium polyanthum Wight) have done by group of Animal Husbandry student, Universitas Padjadjaran. The result showed that blood cholesterol levels decreased from 297.75±51.73 to 139±12.03 mg/dl, with use 4% bay leaf in ration of Broiler. Other Research using Citrus sinensis waste in quail, showed a good result and adding up to 6% can reduce the cholesterol content of the meat from 60.11 to 29.82 mg/dL, (Fadilah 2011) and the optimum dose for decreasing fat and cholesterol in poultry  not more than 6 % in ration.

The content of the active compound in citrus waste including volatile oil 0.91%, tannins 0.95%, flavonoids 0.46%, and 0.84% saponin (LKO Unpad 2013).

The active compound in sweet oranges such as tannins, saponins, flavonoids and essential oil is able to inhibit the absorption of cholesterol in the intestine. The presence of tannins will stick or lining the intestine membrane thus inhibiting the absorption of cholesterol (Oluremi et al. 2007).

Essential oils are believed have the function of lowering the activity of Glycerol-3-Phosphate Dehydrogenase (GPHD), an enzyme that plays role in triglycerides synthesis. Essensial oil could inhibit triglyceride synthesis in the liver and small intestine may result to decrease the triglycerides (He et al. 2009). The dominant component compound of Citrus sinensis is hesperidin and narirutin classified as flavonoids that act as antioxidant (Peterson et al. 2006)

Hesperidin worked through the mechanism of inhibition the activities of HMG-CoA reductase, so the enzyme activity involved in the biosynthesis of cholesterol is inhibited.

Padjadjaran sheep, is a local breed that has related with Garut sheep from Wanaraja area still in purification for meat products, has white feathers and width ear characteristics (Bandiati et al. 2012). They have high potential as meat source and are highly adaptive.

Attempt to decrease cholesterol and fat levels on Padjadjaran sheep’s blood by using waste citrus sinensis was feared to harm the sheep’s biological function especially rumen’s ecology and the value of  hematology would improved.There is a trend to modify the animal cholesterol and fat content in order to produce high quality products. Citrus sinensis waste has been reported to reduce blood cholesterol and triglyceride level, therefore this study was conducted with the objectives to study the effect of dietary inclusion of Citrus sinensis waste on meat cholesterol and fat of Padjadjaran sheep.

MATERIALS AND METHODS

Animal Experimental

The sheep were procured from Animal Breeding Station, Animal Husbandry Faculty Universitas Padjadjaran. The research trial was conducted at Faculty of Animal Husbandry, Universitas Padjadjaran, Indonesia from April 2015 to August 2015. Twenty sheep were used for the study. The sheep were reared in cages having age group between 24-30 months with average body weight 29.66±2.74 kg and coefficient of variation <10 %. The experimental period lasted after five (5) weeks. The concentrate ration was used andgrass was used asfed. The consentrate  are rice bran, cassava flour, tofu waste, coconut cake, sweet orange (Citrus sinensis) waste, molasses, pollard and cassava peal.The sheep were randomly allocated to four treatment groups as T1, T2, T3 and T4, in a Completely Randomized Design (CRD). The sheep in the first group (T1) were givenrasion without the addition of Citrus sinensis while in other groups Citrus sinensis was supplemented at the rate of 7, 12, and 17% in T2, T3, T4 respectively.

Proceesing of Citrus sinensis

The Citrus sinensis was obtained from the sweet orange processing units located around Bandung, was sun dried (<50ºC) until the moisture content reached 10% and followed with milled with hammer mill at Faculty of Animal Husbandry, Universitas Padjadjaran.

Samples Collection

Meat samples were collected at the end of experimental period, randomly selected from three sheeps from each treatment group, so the total is 12 healthy individuals (60% of total sheep) was slaughtered with a cutting technique streak (Butterfield 1988).

Halal slaughter method is done after fasting for 22 hours to reduce the slaughter weight variation. Meat samples analyzed were taken from the right side of carcass longisimus dorsi – (LD) muscle and the biceps femoris (BF) muscle, were taken at the thigh. The meat was taken randomly as much as 10 grams each, at multiple locations on longisimus dorsi and biceps femoris.

Purbowati & Suryanto (2000) research showed that LD muscle meat fat content (3.10%) higher than the BF (2.16%) because the thigh muscles more used for moving. There is no significantly different (P>0.05) because the sheep were kept in cages so the motion

Table 1 . Ration composition, nutrient content and energy

Source: Caltucated aplication from Winfeed

activities in the thigh muscle is limited, as a result fat tends to stockpiled (Lambuth et al. 1970).

Meat cholestrol was estimated using Cholesterol kit (Biolab), using CHOD-PAP (Cholesterol Oxidase Phenylperoxidase Amino Phenozonphenol) method (Richmond 1973 in Kasturi & Singhania 2014). Meat fat was estimated using Soxhlet method (AOAC 2006).

Estimation of rumen pH (ruminal fluid from slaughtered sheep) was collected and pH was measured using pH-meter.

Statistical analysis

Data collected were subjected to analysis of variance (ANOVA) (Gaspersz 2006) was used to test the significance of difference between means considered significant at P<0.05.

RESULT AND DISCUSSION

It can be seen from Table 2, that the feed intake of all treatments are relatively equal, concentrate between 375.19±27.89 to 390.32±8.72 and grass consumption 2438.86±154.74 to 2538.21±210.65. Average daily gain ranged from 8.73-64.28 g. The feed intake and body weigh for all treatments was not significantly different (P>0.05).

Forage dry matter intake can be seen in Table 2. Consumption of dry matter forage and concentrates are relatively the same in all treatment, 400-440 g/head/day and exhausted. Average consumption of feed dry matter is 3.3% of body weight that is in the normal range.

The highest daily weight gain achieved by treatment T3 with body weight gain 64.28 g for 5 weeks (Table 2), with adaptations for 1 week. This illustrates that the provision of Citrus sinensis up to 12% shows the highest body weight gain compared to other treatments, is closely related to the active compounds including flavonoids and essential oils to improve metebolisme, and its impact on body weight gain (Hernawan & Adriani 2014).

Triglycerides were decreased in all treatment, its serves an energy reserve used (Malinow et al. 1987; Oluremi et al. 2007), so the blood levels to be reduced. The presence of flavonoids also believed to play a role in suppressing the concentration of triglycerides. Flavonoids are thought to activate cAMP synthesis resulting in increased protein kinase resulting in increased triglyceride hydrolysis so the triglycerides in the blood and liver will reduce (Olivera et al. 2007;

Table 2 . Feed consumption and body weight

Table 3 . Blood Cholestrol and Triglyceride Contain

Rusell 2009). Protein kinase activates triglyceride lipase through forforilasi here in after described triglycerides into free fatty acids and glycerol by triglyceride lipase (Rusell 2009).

Cholesterol comes from food into the digestive tract and in the small intestine to be absorbed by enterocytes of the small intestine mucosa, the next will be esterification into cholesterol esters. After that, lipoprotein cholesterol esters will formed chylomicrons, then get into the flow of lymph and end up in the bloodstream (Shepherd 2001; Linder 2006).

The active compounds contained in sweet oranges such as tannins, saponins, flavonoids and essential oil is able to inhibit the absorption of cholesterol in the intestine (Malinow et al. 1987; Francis et al. 2002; Oluremi et al. 2007). The presence of tannin can stick or coat the intestinal membrane thereby inhibiting the absorption of cholesterol (Oluremi et al. 2007). In addition, the saponin in the gastrointestinal tract to form a bond with cholesterol complexes that are difficult absorbed by the intestine so that most cholesterol will come out with feces (Malinow et al. 1987). The presence of flavonoids are also believed to inhibit the absorption of cholesterol by inhibiting the formation of micelles ration so that the absorption of cholesterol settles and can be pressed (Olivera et al. 2007; Gropper et al. 2009).

Table 4 is the major research,the results revealed that dietary inclusion sweet orange waste in all levels decreasing the meat cholesterol and fat content of sheep meat, when compared to the control group (T1). Highest reduction of thigh and breast meat cholesterol level 9.43±0.04 and 9.71±0.04 μg/mg respectively, was significantly different (P<0.05) recorded in all treatments, fed 7% citrus waste (T2) in the diet when compared to control groups. Further, the highest decrease in fat content of thigh and breast were 9.70±3.98 and 10.48±1.85 μg/mg respectively, was observed in the group fed 17% citrus waste (T4) in the diet compared to other treatments including control.

Flavonoids also produce multi-enzyme systems such as cytochrome P-450; affecting on lipid metabolism and bile acids. The enzyme cytochrome P-450 has the ability to mediate the formation of bile acids so the amount of bile acids was decreased.

Bile acid decreasing is the main route to eliminate the cholesterolThe presence of flavonoids is also believed to play a role in suppressing the concentration of triglycerides (Table 2). Flavonoids which can activated cAMP synthesis, will increase the kinase protein and also increased the triglyceride hydrolysis, which decreased the blood triglycerides (Olivera et al. 2007).

The decreased lipid profile related with Galleano et al. (2012) study, that flavonoids may have beneficial role in regulating the fatty oxidation and improve adipocyte function, also flavonoids act as antioxidants, thus decreased the cholesterol levels in the blood (Oluremi et al. 2007), by release one hydrogen atom from the group and reducing the formation of free radicals, resulting in the synthesis of 3-hydroxy-3-metilglutaril-CoA (HMG CoA) which serves as a precursor in obstruction of cholesterol formation (Reynertson 2007). Naringenin flavonoid in citrus has been reported prevented the accumulation of adipose, adipocyte hypertrophy and dyslipidemia (Mulvihill et al. 2010).

The cholesterol from food, was entered into the digestive tract and small intestine then absorbed by enterocytes of the small intestine mucosa and will esterified into cholesterol esters. After that, with phospholipids and apolipoprotein, lipoprotein cholesterol esters will forming chylomicrons then get into the flow of lymph and the ending in the bloodstream (Linder 2006).

In addition, the saponin in the gastrointestinal tract forming a complex bond with difficult cholesterol absorbed blood cholesterol, but has no significant difference to blood cholesterol and triglycerides, even there are tendency to decrease from 69.6 mg/dL to 64.00 mg/dL (Hernawan E & Adriani L 2014) by the intestine so most of cholesterol was release with feces). The presence of flavonoids are also believed to inhibit the absorption of cholesterol by inhibiting the formation of micelles

Table 4. Effect of Citrus sinensis on meat cholesterol, meat fat and rumen pH

Means within the row with different superscripts are significantly different (P<0.05)