*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:

$$\text{TDN\%} = \text{DCP\%} + \text{DCF\%} + \text{DNNFE\%} + \text{(DEE\%} \times \text{2.25)} \tag{1}$$

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.

$$\text{NR: Multiitive value} = \text{(TDN (\%)/DCP (\%))} - 1 \tag{2}$$

Digestible energy (DE) was calculated using the equation according to [32], as follows:

$$\text{DE (kcal/kg diet)} = 44.3 \times \text{TDN}\% \tag{3}$$

#### *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 lipoproteincholesterol (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:

$$\text{LDL-c} = \text{cholesky-t} \text{-} (\text{FDL-c} + \text{vLDL-c}).\tag{4}$$

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].
