1. Introduction
Phytogenic feed additives have been recognized as antimicrobials, antioxidants, anti-toxigenic, anti-coccidiosis, and antiparasitic [
1,
2,
3]. In addition, phyto-feed additives improve the palatability and digestibility of feed, enhance the absorption of nutrients, as well as manipulate the microbial habitat and gut functions of domestic animals [
4,
5]. Moreover, they protect the feed lipids from oxidative damage and improve the antioxidant and immune status of the animal. Furthermore, phyto-feed supplementations are natural additives, less poisonous, residue-free, with more integrity and perfect as feed additives for poultry when compared to antibiotics [
1]. Consequently, they can be considered as an important tool in poultry nutrition for enhancing growth performance, feed efficiency and reproductive performance, and reducing the incidence of diseases and the house emissions of poultry [
1,
4]. The inclusion of phytogenic feed additives in the diet can improve the nutritional value of meat and tissue composition [
6].
As one of the phytogenic feed additives, vegetable oils, such as olive oil, rice bran oil, corn germ oil, and wheat germ oil are commonly used as food supplements in the human diet [
7]. Vegetable oils are natural, healthy, and nutritious due to their high content of unsaturated fatty acids and functional molecules, and their high energy value [
8].
Rocket (
Eruca sativa Mill.) belongs to the large family of Brassicaceae (also called Cruciferae or the mustard family). The rocket is an annual or biannual herb that originated in the Mediterranean region and has spread through the world [
9].
E. sativa species are widely used in folklore and traditional medicine for their therapeutic properties as digestive, astringent, laxative, emollient, depurative, diuretic, rubefacient, stimulant, and tonic [
10]. The composition of rocket seeds has shown the presence of many active compounds, such as glucosinolates (glucoerucin and glucoraphanin), flavonoids (quercetin, kaempherol, and isohamnetin), carotenoids, and vitamin C, which are ascribed to antioxidant activity [
10]. Rocket seeds contain up to 25–35% of oil [
11] and rocket seed oil (RSO) has about 18% of the total saturated fatty acids and 82% of the total unsaturated fatty acids. Rocket seed oil prompts the regeneration of hepatic tissue, decreases hepatic lipid levels, and possesses potent free radical scavenging [
12], as well as inhibits melanoma tumor growth in mice [
13].
Additionally, rocket seed oil inhibits the growth of some Gram-positive and Gram-negative bacteria and has approximately the same efficiency as the broad-spectrum antibiotic Gentamicine [
14]. Moreover, RSO ameliorated the harmful effect of aflatoxin on rabbit blood, semen, and pathological changes in the liver, kidney, and testes [
15]. Furthermore,
E. sativa improved significantly the final body weight, average daily gain, feed intake, and feed conversion ratio of rabbits [
16]. The dietary supplementation of 1 g RSO/kg diet alone or with 1 g onion seed oil/kg diet in the growing rabbit’s diet for 12 weeks under heat stress improved growth performance, carcass weight, and nutrient digestibility as well as enhanced immunity [
17]. In this vein, Alagawany et al. [
18] found that dietary supplementation with 0.5–2 g/kg diet of watercress oil alone or in combination with coconut oil for 8 weeks in intensive rabbit production improved growth performance, feed utilization, antioxidant status, and immunity, as well as reduced pathogenic cecal bacteria. On the other hand, the addition of high levels of RSO (1–3 mL/kg body weight) to the rabbits for 2 weeks resulted in a reduction of the body weight with an increasing RSO oil dose [
19].
Wheat (
Triticum aestivum L.) germ is produced during wheat milling and is used worldwide as a diet supplement in the feed formulation of farm animals [
20]. Wheat germ oil (WGO) represents about 10–15% of the whole wheat germ [
21]. In addition, it contains tocopherol derivatives and tocotrienols [
22], n − 3 fatty acids, especially alpha-linolenic acid [
23], fat-soluble carotenoids [
24], phytosterols, especially D5-avenasterol [
25] and phenolic compounds [
26]. Moreover, wheat germ oil has an anti-inflammatory effect and strong antioxidant effects [
21,
26]. Whereas, it reduces O
2-production and NADPH oxidase activity, and thereby, decreases oxidative stress [
23]. WGO manages the serum lipid profile and prevents hypercholesterolemia and atherosclerosis in male albino rabbits fed high cholesterol diet [
27]. Other benefits of wheat germ and its derivatives are lowering cholesterol absorption, retarding platelet aggregation, delaying ageing, improving physical endurance, enhancing fertility [
25], as well as preventing and curing carcinogenesis [
28]. Furthermore, dietary WGO supplementation increased the body weight of male broilers [
29].
Taking previous knowledge into account, the present study aimed to investigate the effects of RSO, WGO, and their mixture on growth performance, feed utilization, nutrient digestibility, carcass characteristics, meat fatty acid profile, and redox and immune status of growing rabbits.
2. Materials and Methods
2.1. Animal Management and Feeding
Eighty-four V-line rabbits at 5 weeks of age (after weaning) with an initial BW of 535.60 ± 13.48 g were assigned randomly into four experimental groups (seven replicates in each group, three rabbits in each replicate). The first group served as the control and received 0.3 mL/kg BW of distilled water (CON), the second group received 0.3 mL/kg BW of rocket seed oil (RSO), the third group received 0.3 mL/kg BW of wheat germ oil (WGO), and the fourth group received a mixture of oils consisting of 0.15 mL of RSO and 0.15 mL of WGO/kg BW (MOs). The oils of wheat germ and rocket seeds were obtained from El Madina Factory for natural seed extract in Borg El Arab, Alexandria, Egypt.
Rabbits were given oils once daily via gavage (oral administration) for 7 weeks from 28 May to 15 July. The basal ration was formulated and pelleted to meet the nutrient requirements of rabbits, according to the NRC [
30]. The rations were offered to rabbits
ad libitum. The ingredients and chemical composition of the pelleted rations are shown in
Table 1. The rabbits were offered free access to freshwater.
All the rabbits were kept under similar management, as well as hygienic and environmental conditions. Freshwater was automatically available all the time through stainless steel nipples that were fixed in each cage. The rabbits were housed in galvanized wire cages (dimensions: 40 × 50 × 65 cm) located in a well-ventilated building. The daily photoperiod is a 16:8 h light-dark cycle. This study was conducted at the Rabbit Research Laboratory, Department of Animal and Fish Production, Faculty of Agriculture (Saba Basha), Alexandria University. All the protocols applied in the present experiment have been approved by the Alexandria University, Animal Care and Use Committee with approval no. AU: 19/21/03/25/3/16.
2.2. Body Weight and Feed Intake
The rations were removed at night before the days of rabbit weight. The growing rabbits were weighed weekly in the morning before being given a feed. The average daily gain (ADG) and weight gain percentage were calculated. The feed intake was recorded biweekly, then daily feed consumption was calculated by dividing the weekly feed intake by 14 days. The feed conversion ratio (FCR) was calculated by dividing the daily feed intake by the average daily gain.
2.3. Digestibility Trial
At 10 weeks of age, sixteen male rabbits were randomly taken to determine the nutrient digestion coefficients of the experimental diets. The rabbits were allocated to four different treatments (four rabbits in each group). The rabbits within each treatment were housed individually in metabolic cages that enabled the separation of urine and feces. The preliminary period was 2 days to adapt rabbits to the new cages and then followed by 5 days as a collection period for feces and urine. During the collection period, the total excreted feces and urine of each rabbit are collected daily in buckets before offering a morning meal and weighing them.
Representative samples (10%) of the total quantity of feces from each rabbit were oven-dried daily at 70 °C for 48 h to determine the total dry matter (DM) of the feces and to calculate the quantity of feces on a DM basis. At the end of the collection period, the faecal samples from each rabbit were mixed thoroughly, and representative samples (10%) of the mixtures were ground through a 1-mm screen on a Wiley mill grinder and then stored frozen at −20 °C prior to the chemical analysis.
Nutritive values in terms of total digestible nutrients (TDN) and digestible crude protein (DCP) were calculated according to the classic formula [
31] as follows:
where DCP is the digestible crude protein, DCF is the digestible crude fiber, DNFE is the digestible nitrogen-free extract, and DEE is the digestible ether extract.
Digestible energy (DE) was calculated using the equation according to [
32], as follows:
2.4. Lipid Content and Fatty Acid Profile of Rabbit Meat
The lipid content and fatty acid profile of rabbit meat were determined in the musculus semitendinosus of three slaughtered rabbits per group. Total lipids were extracted with chloroform:methanol (2:1
v/
v) from 0.8 g of meat, according to the procedure of Folch et al. [
33].
Lipid extraction from the meat samples was performed according to the procedure of Pearson [
34]. About 10 g of the sample was weighed in a 250 mL centrifuge bottle. The total volume was completed to 16 mL with distilled water, then 40 mL of methanol and 20 mL of chloroform were added and macerated for 2 min. After that, 20 mL of chloroform was added and macerated for 30 s, then 20 mL of water was added and macerated again for 30 s. The mixture was centrifuged for 10 min at 2000–2500 rpm. The bottom layer of chloroform was removed and filtered through a coarse filter paper into a dry-weight flask or beaker. Then, the chloroform was evaporated to dryness.
Preparation of fatty acid methyl esters from the total lipids of the sample was performed according to the procedure of Radwan [
35]. A sample of total lipids (50 mg) was transferred into a Screw-Cap flask, then 2 mL of benzene and 5 mL of methanolic sulphuric acid (1 mL of conc sulphuric acid and 100 mL of methanol) were added. The vial was covered under a stream of nitrogen gas, then placed in a water bath at 90 °C for 90 min. The flask was cooled, then 10 mL of distilled water was added and the methyl esters in each flask were extracted with 5 mL of petroleum ether three times. The petroleum ether extracts were combined and concentrated to their minimum volume using a stream of nitrogen. The analysis of fatty acids was carried out by gas-liquid chromatography (HP, Hewlett Packard 6890 GC model) equipped with a flame ionization detector (FID). Separation was achieved in a column HP-INWAX (cross linked polyethylene glycol, 60 m, 0.25 mm ID, 0.25 μm film thickness) under the following conditions: Detector temperature, 250 °C; injector temperature, 220 °C; injection volume, 3 μL; split ratio, 50:1; carrier gas, nitrogen; gas flow, 1.5 mL/min. Before running the samples, a standard mixture of methyl esters was analyzed under identical conditions. The retention times of the unknown sample of methyl esters were compared with the standard. The proportions of methyl esters were calculated by the triangulation method.
2.5. Serum Biochemical Parameters
Before slaughter, 4 mL of blood sample was taken with a sterile syringe from the ear vein of five growing rabbits from each group. The blood sample was placed into a sterile vacutainer tube without an anticoagulant for the serum biochemical analysis.
The serum total protein, lipid profile, and urea were estimated colorimetrically using commercial kits produced by Bio Diagnostic Co., Giza, Egypt. The serum total protein and albumin were determined according to Doumas et al. [
36]. The serum globulin concentration was calculated by the difference between the total protein and albumin [
37].
Total lipids were estimated by the reaction with sulphuric and phosphoric acids and vanillin to form a pink chromophore [
38]. Triglycerides were measured colorimetrically using the quadruple enzymatic reaction [
39]. Cholesterol was determined after enzymatic hydrolysis and oxidation as described by Allain et al. [
40]. High-density lipoprotein-cholesterol (HDL-c) was determined according to the methods of Grove [
41]. Low-density lipoprotein-cholesterol (LDL-c) was determined using the following calculation according to Warnick et al. [
42] using the following equation:
The very low-density lipoprotein-cholesterol (vLDL-c) was calculated by dividing the value of TG by a factor of 5 according to the method of Warnick, Benderson, and Albers [
42]. Serum urea was assayed according to Chaney and Marbach [
43].
The triiodothyronine (T3) and thyroxine (T4) hormones were determined in the serum by a direct radioimmunoassay technique. Kits from the Diagnostic Products Corporation (Los Angles, CA, USA) with ready, antibody-coated tubes were used based on the manufacturer’s instructions, according to Kubasik et al. [
44].
2.6. Antioxidant Assays
Thiobarbituric acid reactive substances (TBARs) were measured colorimetrically according to Tappel and Zalkin [
45]. The catalase activity (U/mL serum; EC 1.11.1.6, CAT) was measured according to Luck [
46]. The superoxide dismutase activity (U/mL serum; EC 1.15.1.1, SOD) was evaluated according to Misra and Fridovich [
47]. The total antioxidant capacity (TAC) was estimated colorimetrically using commercial kits produced by Bio Diagnostic, Egypt according to the method of [
48].
2.7. Antibody Titers against SRBCs
The primary and secondary immune response was assayed by measuring antibody titer against sheep red blood cell counts (SRBCs), as the agglutination titer described by Wegmann and Smithies [
49]. The agglutination titer was calculated as the log of the reciprocal of the highest serum dilution for the whole agglutination [
50].
2.8. Chemical Analysis
Chemical analyses of the experimental rations and feces samples were carried out according to AOAC [
51] for crude protein (CP, method 968.06), ether extract (EE, method 920.39), crude fiber (CF, method 932.09), and ash (method 967.05). The nitrogen-free extract (NFE) was calculated according to the next equation:
Organic matter (OM) was calculated as the difference between 100% DM and ash. Gross energy (GE, kcal/kg) of the experimental diets was calculated based on 5.64, 4.11, and 9.44 kcal GE/g CP, NFE, and EE, respectively NRC [
52].
Digestible energy (DE) of the experimental diets was calculated according to the equation described by Cheeke [
53], as follows:
The concentration of hemicellulose was estimated as the difference between NDF and ADF. Nitrogen in urine was determined by the micro-Kjeldahl method [
51].
2.9. Statistical Analysis
Data of the experiment were analyzed statistically using the one-way analysis of variance (ANOVA) with the SPSS 11.0 statistical software [
54]. Differences among means were determined using the Duncan test [
55]. Data were analyzed using the following model:
where U is the overall mean, A
i is the effect of wheat germ oil, rocket seed oil, and their mixture treatments; and E
ij is the random error.