1. Introduction
Rabbit meat is traditionally part of a healthy Mediterranean diet and its nutritional characteristics, such as a low fat and cholesterol content and a favourable fatty acid profile, etc., [
1], are widely recognised. The high percentage of polyunsaturated fatty acids (PUFA), including long chain
n-3 fatty acids, means that this meat is valued in terms of human health, but at the same time this results in a greater proneness to lipid oxidation, reducing its shelf life [
2,
3]. Previous studies have shown that animal feeds can affect fatty acid composition, shelf life, and the amount of bioactive compounds in meat, and that by including certain ingredients, the potential of rabbit meat as a “functional food” can be capitalised [
1]. In this respect, incorporating in animal diets by-products of industrial fruit and vegetable processes [
4,
5,
6] is a well-accepted strategy due to its perceived naturalness and to the environmental benefits it affords by reducing waste.
During industrial winemaking processes large amounts of pomace are produced (mainly grape skins, pulp, seeds, and any unremoved stems), which represent almost 25% of the contents of grapes used for wine production [
7]. This by-product has phenolic compounds with antimicrobial and antioxidant properties that can be used in animal feed, with promising results in live animals (health, productivity) and in the shelf life of their meat [
8]. Additionally, grape pomace provides other secondary plant metabolites, such as tannins and lignin, and linoleic fatty acid, which could affect feed digestibility or change the fatty acid profile of the meat. In a previous study, grape pomace showed a slight reduction in protein and energy digestibility, as well as a lower feed efficiency, but it was concluded that this by-product could partially replace alfalfa hay in growing rabbits’ diets (from 100 to 300 g/kg total weight), depending on the availability and economic value of these two ingredients [
4]. In rabbit bucks, including up to 20% of grape pomace in their diet improved semen quantity and quality, and also increased their antioxidant capacity without affecting body weight gain [
9]. However, to the best of our knowledge, the effect of grape pomace in fattening rabbits’ diets on the quality of their meat has not been assessed hitherto. Based on the aforementioned previous studies, we expect that the inclusion of 20% of grape pomace in diets will not impair animals’ performance, but it could improve the shelf life of meat if the phenolic compounds from the by-product are transferred to the meat. Interesting findings have been observed in other species that could be applicable to rabbits, such as the inhibition of pathogenic bacteria in the gut in piglets [
10], methane reduction in dairy cows [
11], and an increase of polyunsaturated fatty acids and reduction in oxidation in the meat of chickens [
12,
13], pigs [
14], and lambs [
15].
Furthermore, in line with the European Union tendency, a specific Spanish national strategy has been developed to minimise the use of medical products in animal production. In this respect, the use of by-products rich in polyphenols can help produce food without using medicated feeds in rabbit diets, thus improving the image of the sector vis-a-vis consumers by presenting a more sustainable product.
The objective of this study was, therefore, to investigate the effect of including 20% of grape pomace by-product in non-medicated fattening rabbit diets on animal performance, and on the fatty acid composition, the phenolic profile, and shelf life of their meat, in comparison to a medicated commercial diet.
2. Materials and Methods
2.1. Animals and Experimental Design
The study that was performed at the Animal Experimentation Service of the University of Zaragoza, Spain (latitude 41°41′ N). The care and use of animals were performed in accordance with the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. Thirty-six New Zealand white rabbits were weaned at 35 days of age and allotted to two treatments, with the animals in each group balanced according to initial live weight. Each treatment consisted of three cages housing six rabbits each, balanced by sex (three females and three males), making a total of 18 animals per treatment. Treatments consisted of two ad libitum diets during 30 days:
The commercial pellets contained alfalfa flour, vetch, fescue, ray-grass, barley, sunflower seed meal, corn gluten feed, soybean hulls, palm kernel pressure cake, sugar cane molasses, wheat bran, calcium carbonate, palm vegetable oil, sodium chloride, vitamins, and minerals. The CON diet had robenidine hydrochloride as the coccidiostat, unlike the withdrawal feed which was a non-medicated control diet without coccidiostat. Grape pomace from Granache grapes was supplied by a family winery from Cariñena (Aragón, Spain). The GPD was the result of mixing eight parts of ‘withdrawal feed’ and two parts of the grape pomace by-product. The withdrawal pellets and the by-product ground and new pellets were produced at the Animal Experimentation Service of the University of Zaragoza. Diet samples were taken during the experiment to analyse the chemical composition, total phenolic content, and antioxidant capacity (
Table 1). The housing conditions established were an ambient temperature of 20 °C, 16L:8D lighting schedule, and free access to drinking water. Animals were fattened in traditional, flat-deck type cages (73 cm long × 47 cm wide × 30 cm high; 572 cm
2/head). Feed consumption and rabbit weight were recorded weekly and at the end of the fattening period, as group data of the six animals per cage. The feed conversion ratio was calculated by dividing the feed consumed (kg) per kg of live weight gained.
2.2. Slaughtering and Meat Sampling
At 65 days of age, 18 rabbits per group were stunned and slaughtered without fasting, at a commercial slaughterhouse located at 28 km. After slaughter, carcasses (excluding all viscera and the distal parts of the tail and fore and hind legs, but including the head) were immediately transported to the Animal Production Unit Laboratory of the University of Zaragoza, where carcass weights were recorded. Carcass yield was obtained for the group of rabbits in each cage, calculated as the ratio between carcass weight and live weight at slaughter. Carcasses were refrigerated at 4 °C for 24 h and left hind leg samples were obtained, vacuum-packed, and stored at −20 °C until analysis. Before analysing, samples were thawed at 4 °C for 24 h, the meat was removed from the bone and minced to assess pH, thiobarbituric acid reactive substances (TBARs), and total volatile basic nitrogen (TVB-N) at 0, 4, and 6 days of storage at 4 °C, in overwrap. At day 0, samples of hind leg were freeze-dried to analyse the total phenolic content, antioxidant capacity, reducing power, and phenolic compounds. The fat percentage and fatty acid composition of the meat was analysed in a cranial section of the right Longissimus dorsi muscles, which were minced and freeze-dried.
2.3. Chemical Composition of the Diets, Grape Pomace, and Fat Percentage in Meat
Analyses of diet composition, grape pomace, and fat percentage of the meat were conducted at the Agrifood Research and Technology Centre of Aragón (CITA, Zaragoza, Spain) at the Physical-Chemical and Instrumental Analysis Laboratory. Feedstuff samples were oven-dried to determine the dry matter content. Crude protein and ether extract were determined according to the procedures of the Association of Official Analytical Chemists (AOAC) [
16]. Neutral detergent fibre, acid detergent fibre, and acid detergent lignin analyses were carried out using the sequential procedure of Van Soest et al. [
17], with the Ankom fibre analyser (Model 200/220, Ankom Technology, Gomensoro, Madrid, Spain). The fat percentage in freeze-dried, minced
Longissimus dorsi muscle was also analysed using the aforementioned methodology and was expressed as a percentage of fresh meat.
2.4. Fatty Acid Composition of Diets and Rabbit Meat
Fatty acid profiling of freeze-dried diets and
Longissimus dorsi muscle samples were conducted according to the adapted methodology of Lee et al. [
18] at the Institute of Food Science, Technology and Nutrition (ICTAN) in Madrid. Samples of 0.1 g were weighed and placed in an ultrasound cleaned (USC) culture tube. Two mL of 0.5 M sodium methoxide in methanol and 1 mL hexane containing 1 mg/mL C13:0, as an internal standard, were added and then heated for 15 min at 50 °C. Acetyl chloride in methanol (1:10;
v/
v; 2 mL) was added before mixing thoroughly and heating for 1 h at 60 °C. Three mL of hexane, 1 mL of deionised water, and 0.2 g of anhydrous sodium sulphate were added, mixed, and centrifuged at 4 °C for 5 min at 1500 rpm. The organic solvent top layer was pipetted into a vial to be used for gas chromatography (GC) analysis. Fatty acid methyl esters (FAME) were assayed by gas chromatography with a flame ionization detector (Agilent 7820A), using a column (60 m × 0.25 mm × 0.25 μm, Agilent HP-23) with split injection, 1 μL (40:1), and helium at a constant flow of 1 mL/min, as the carrier gas. The detector temperature was set at 260 °C and the injector oven temperature at 250 °C. The initial temperature of the oven was 100 °C (held for 2 min) and then increased by 8 °C/min to 145 °C. This temperature was held for 20 min and then increased by 5 °C/min to 195 °C and held for 5 min. Afterwards, the temperature was increased by 5 °C/min to 215 °C and held for 5 min, and again increased by 5 °C/min to 230 °C and held for 5 min. The total analysis time was 59.6 min. Identification of fatty acids and their response factors was aided by the use of a reference standard (FAME 37 Supelco Ref CRM47885 + PUFA No. 2 Animal Source Ref 47015-U Sigma + PUFA No. 3 Menhaden oil Ref 47085-U) and quantified using the internal standard (C13:0).
Atherogenicity (AI) and thrombogenicity (TI) indices were calculated [
19]: AI = (C12:0 + 4 × C14:0 + C16:0)/((MUFA +
n-6) +
n-3)); TI = (C14:0 + C16:0 + C18:0)/((0.5 × MUFA + 0.5 ×
n-6 + 3 ×
n-3) + (
n-3/
n-6)), together with the peroxidability index (PI), according to Arakawa and Sagai [
20]: PI = (% monoenoic × 0.025) + (% dienoic × 1) + (% trienoic × 2) + (% tetraenoic × 4) + (% pentaenoic × 6) + (% hexaenoic × 8).
2.5. HPLC Analysis of Polyphenols
The analysis was carried out on freeze-dried diets and twelve meat samples (hind legs, 6 per group), with a Waters H-Class high-performance liquid chromatography (HPLC) system coupled to a quadrupole time-of-flight mass spectrometry (QTOF) equipped with an electrospray ionization (ESI) source (microTOF-Q, Bruker Daltonik), according to the modified method of Mena et al. [
21]. The column used was an analytical HPLC column (ZORBAX Eclipse Plus C18, 50 mm × 2.1 mm i.d., 1.8 μm spherical particle size, Agilent). Autosampler and column temperatures were 10 and 30 °C, respectively. The injection volume was 5 μL and the flow rate was 0.4 mL/min. The mobile phase was built using two solvents: 0.1% formic acid in water (A), and 0.1% formic acid in acetonitrile (B). Initial conditions were 5% B for 1 min, then a 11-min linear gradient to 30% B followed by 1-min linear gradient to 80% B and maintaining this composition for 4 min. In order to regenerate the column a 5 min pre-run with 5% B was achieved before the next injection. Electrospray ionization collision-induced dissociation mass spectrometry ESI-CID-MS (QTOF) analysis was carried out in positive and negative ion mode, with capillary and endplate offset voltages of 4500 and −500 V in positive mode, and 4000 and −500 V in negative mode, using nitrogen as the collision gas. For MS/MS measurements, collision cell energies of 25 eV were used for positive and negative mode, with an isolation width for the precursor ion of 4 m/z units. To allow coupling with the HPLC system, the nebulizer gas (N
2) pressure and the drying gas (N
2) flow rate were 1.6 bar and 8.0 L min
−1, respectively. The mass axis was calibrated externally and internally using Na-formate adducts. Bruker Daltonik o-TOF Control v.3.4, HyStar v.3.2 and Data Analysis v.4.2 software packages were used to control the MS (QTOF) apparatus, for the interface of the HPLC with the MS system and to process data, respectively. The two experimental diets, the wine industry grape pomace by-product and the meat sample extracts were ionized using the negative mode to detect phenolic acids, favan-3-ols, flavonols, and stilbenes, and the positive mode to detect anthocyanins. The resulting pseudomolecular ions (M − H)
− or (M + H)
+ after fragmentation by collision-induced disassociation (CID) were separated by mass to charge ratio (
m/
z). Then, when enough collision energy was supplied, the precursor ions with a particular m/z were fragmented again and separated in a second stage of mass spectrometry (MS/MS). Phenolic compounds were tentatively identified based on the calculated mass of pseudomolecular ions, m/z of fragmented product ions, and retention times previously reported in the specific literature [
22,
23,
24,
25,
26,
27,
28,
29,
30,
31].
2.6. Total Phenolic Content, Antioxidant Capacity, and Reducing Power
The phenolic extract preparation was conducted in two steps. Samples of hexane-defatted and freeze-dried meat and animal diets (1.5 g) were homogenized in 10 mL of ethanol:water (80:20) using an Ultra-Turrax T25 (Janke & Kunkel, Staufen, Germany) for 60 s. The homogenate was then centrifuged at 4 °C for 15 min at 4000 rpm and the supernatant was collected. The extracted pellets were homogenised in 10 mL of acidified methanol 10% with HCl 1N using a vortex shaker for 60 s before being incubated for 60 min at 85 °C to extract hydrolysable polyphenolics following the procedure described by Hartzfeld et al. [
32] and Lei et al. [
33] with slight modifications. They were then centrifuged at 4000 rpm for 15 min at 4 °C and evaporated to dryness in a rotary evaporator at 40 °C before being reconstituted with the supernatant obtained in the previous step, resulting in a crude extract containing both free and hydrolysable phenolic fractions. Crude extracts were subjected to the solid phase extraction (SPE) treatment with the Oasis HLB cartridge (200 mg, 3 cc, 30 μm particle size) filtered through a 0.45 μm membrane filter and kept at −18 °C until analysis. The final supernatant obtained was used for the estimation of total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and for polyphenolic compound profiling by HPLC-ESI-CID-MS (QTOF).
The total phenolic content (TPC) in meat and animal diets was determined using the Folin–Ciocalteu reagent following the modified method of Singleton et al. [
34]. Folin–Ciocalteu was added (0.5 mL) to 0.5 mL final extract, followed by the addition of 0.5 mL sodium carbonate solution (7.5%) and 7 mL of distilled water. The reaction mixture was vortexed before being incubated for 1 h in the dark. The absorbance of phenolic content was measured spectrophotometrically with a UV-20 Onda Spectrophotometer at 760 nm. The quantification of phenolics was based on the standard curve generated with the use of gallic acid and expressed as mg of gallic acid equivalent/100 g dry weight.
The DPPH radical scavenging activity was estimated according to Llorach et al. [
35]. Results were expressed as μmol of Trolox equivalents/100 g dry weight after measuring the absorbance of the DPPH radical-extract complex and the Trolox standard curve (0–60 μM) at 515 nm.
The FRAP assay was performed according to Thaipong et al. [
36] with some modifications. The stock solutions included a 23 mM acetate buffer (3.13 g C
2H
3NaO
2·3H
2O and 16 mL acetic acid), pH 3.6, 10 mM TPTZ (2, 4, 6 tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM ferric chloride hexahydrate solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL ferric chloride hexahydrate solution and then warmed at 37 °C before using. Samples of the meat extract (120 μL) were allowed to react with 900 μL of the FRAP solution for 20 min in the dark condition. Readings of the coloured product [ferrous tripyridyltriazine complex] were then taken at 595 nm. The standard curve was linear between 50 and 1000 μM Trolox. Results were expressed in μmol of Trolox equivalents/100 g dry weight. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve.
2.7. Evaluation of pH
The pH was determined in minced left hind leg meat at 0, 4, and 6 days of refrigerated storage (4 °C) in overwrap using an XSPH7 + DHS portable pH-meter with a penetration probe.
2.8. Thiobarbituric Acid Reactive Substances Assay
Lipid oxidation was quantified using the thiobarbituric acid reactive substances (TBARs) assay according to Pfalzgraf et al. [
37]. Meat samples weighing 10 g ± 0.02 were homogenised with 20 mL of trichloroacetic acid (10%) using an Ultra-Turrax T25 (Janke & Kunkel, Staufen, Germany). Samples were centrifuged (Gyrozen 1248R, Daejeon, Korea) at 4000 rpm for 30 min and the supernatants filtered through qualitative paper (F1093 grade; Chmlab, Barcelona, Spain). Two milliliters of the filtrates were taken in duplicates and mixed with 2 mL of thiobarbituric acid, homogenized, and incubated for 20 min in a water bath at 97 °C. Absorbance was measured at 532 nm. The results were expressed as mg of malondialdehyde (MDA)/kg of the sample, using a standard curve that covered the concentration range of 1 to 10 mM 1,1,3,3-tetramethoxypropane. Lipid oxidation assays were performed at 0, 4, and 6 days of storage at 4 °C, in overwrap samples.
2.9. Total Volatile Basic Nitrogen Assay
The total volatile basic nitrogen (TVB-N) assay was carried out according to the protocol described in the European Union Commission Regulation 2074/2005, Chapter III, “
Determination of the concentration of TVB-N in fish and fish products” [
38] with some modifications. A 2 g sample of homogenised minced rabbit meat was blended with 90 mL of 6% perchloric acid solution using an Ultraturrax (IKA, Staufen, Germany). The extract obtained was filtered, and 50 mL aliquots were dispensed into an apparatus for steam distillation. Analyses were run in triplicate. The 50 mL of filtrate was rendered alkaline with 20% hydroxide and distilled in a Udk 129 Kjeltec Distillation Unit (VELP Scientifica Srl, Usmate (MB)—Italy. The titration was carried out with 0.01 N HCl, and the results were reported as mg N
2 non-protein/100 g fresh rabbit meat.
2.10. Statistical Analyses
Statistical analyses were performed using the SPSS software package (version 26.0). Analysis of variance was used to evaluate productive performance traits, fat percentage, fatty acid profile, total phenolic content, and DPPH and FRAP of rabbit meat, according to the dietary treatment (CON vs. GPD) as the source of variation. TBARs, TVB-N, and pH data on meat were processed using a general linear model, with diet, days of storage and their interaction as fixed factors. Marginal mean values and standard error were reported. A probability value of p ≤ 0.05 was considered statistically significant. Tukey’s multiple range test was used to determine whether there were significant differences between the mean values of storage days.
4. Conclusions
Adding 20% grape pomace to the diet did not affect the live weights of rabbits during the fattening period, however it reduced the feed conversion rate and carcass weight and yield. The resultant meat had a higher intramuscular fat percentage and a beneficial fatty acid composition of the Longissimus dorsi with respect to the polyunsaturated/saturated fatty acid ratio and the lower atherogenicity and thrombogenicity risks. However, the n-6/n-3 ratio was found to be unfavourable. Several phenolic compounds, such as quercetin, kaempferol, myricetin, and resveratrol derivatives, were highlighted in the diet with added grape pomace, likewise, the total phenolic content, antioxidant capacity, and reducing power were higher compared to the control diet. However, these phenolic compounds were not detected in the meat, and the two treatments, showed similar levels of antioxidant capacity and reducing power. Regarding meat shelf life, dietary inclusion of grape pomace apparently decreased protein spoilage of rabbit meat, but no effect on lipid oxidation was found in minced hind leg meat stored up to 6 days.
Finally, the present study shows that grape pomace is a suitable ingredient for use in fattening rabbit diets and could help reduce the use of medical products in animal nutrition and further the sustainability of farming systems, however there are other aspects such as feed costs that must be taken into consideration.