The effect of fish oil, lycopene and organic selenium as feed additives on rabbit meat quality

ABSTRACT

The aim of the study was to compare the quality of rabbit meat depending on the type of feed used. The animals were divided into 5 groups of 10 individuals and were fed ad libitum with feed for 55 days. The control group was fed with the addition of sunflower oil (4%), the feed of group FO was enriched with 4% odourless fish oil (FO), the feed of group FOLyc was enriched with 4% FO and 15 ppm lycopene (Lyc), the feed of group ROSe was enriched with 4% FO and selenized yeast (SeY) and the feed of group FOLycSe contained 4% FO, 15 ppm Lyc and SeY. The obtained results showed that the enrichment of feed with fish oil resulted in an increase in the longissimus muscle mass by 7.3%. The feed supplementation with fish oil was beneficial in terms of nutrition due to the increase in the content of polyunsaturated fatty acids. Fat oxidation level, measured in the malondialdehyde test, indicated that the highest level was observed in muscles of rabbits fed diets enriched with only FO. The use of odourless FO in feed adversely affected the sensory quality of meat by increasing the intensity of negative notes like rancid.

KEYWORDS:

• Rabbit
• longissimus
• fish oil
• lycopene
• selenium

Introduction

Rabbit meat is of high biological value, appreciated for its favourable properties such as high protein level as well as meat leanness with less cholesterol, saturated fatty acids and high amount of unsaturated fatty acids; 60% of all fatty acids (FA) (Dalle Zotte 2002; Hermida et al. 2006; Hernández 2008; Nistor et al. 2013; Petrescu and Petrescu-Mag 2018). It has fewer calories and is healthier than beef, chicken, lamb or pork (Oliver et al. 1997; Escribá-Pérez et al. 2019). Due to nutritional value the World Health Organisation recommended rabbit meat for children over other types of meat (WHO) (Vergara et al. 2005). Among adult consumers rabbit meat is perceived as healthier and tastier than other meats and also more expensive (Petrescu and Petrescu-Mag 2018). Moreover, rabbit meat is also characterized by no religious taboo or social stigma regarding its consumption.

Assuming that health is recognized as a strong motivation for changing eating habits that can even lead to a change in the type of commonly consumed meat (Escribá-Pérez et al. 2017), it can be expected that the nutritional quality of rabbit meat will certainly meet health requirements of modern consumers (Cullere and Dalle Zotte 2018). The meat is perceived to be tender and juicy as well as delicately flavoured. Consumers believe that rabbit meat has good sensory properties, which are crucial for consumers’ choice.

The strategy of popularizing rabbit farms, enriching feed among breeders and increasing the consumption of rabbit meat could be a favourable direction as for improving the diet of the population. Feeding rabbits with diets, containing considerable amounts of linoleic acid, brings about an increase in the amount of this acid in the perirenal fat, though only up to a certain level. The possibility of impacting meat quality and manipulating the composition of the fatty acids through diet, means that rabbit meat could be more valuable in human nutrition (Hernández and Gondret 2006; Dalle Zotte and Szendrő 2011).

These characteristics, together with the possibility of manipulating the composition of the fatty acids through diet, mean that rabbit meat could be more valuable in human nutrition. The use of animal or vegetable fat, which reduces feeding costs, is of great interest, provided these do not impair the sensory or nutritional quality of the meat (Bhatt et al. 2017). It could be a good opportunity for the domestic market by reorienting the offer towards rabbit meat as ‘new products’ (Petracci et al. 2018).

Rabbit meat consumption could be a good way to provide bioactive compounds to consumers, since manipulation of rabbits’ diet is very effective in increasing the levels of n-3PUFA (especially n-3long-chain PUFA) (Hernández 2008). The high concentration of PUFA in rabbits’ tissues is associated with lipid oxidation, which reduces the quality of raw material by degrading physicochemical properties or sensory attributes – aroma and flavour. When the concentration of unsaturated FA (particularly LPUFA) in meat is increased by dietary means, the meat becomes more susceptible to lipoxidation and this is especially evident when no exogenous antioxidants are simultaneously fed. It is well established that when C18:3n-3 gets close to the 3% of meat FA, negative effects on the oxidative stability of meat due to lipid and myoglobin oxidation can occur (Wood et al. 2004). Similarly, Rozbicka-Wieczorek et al. (2012) suggest that lipid profiles of the meat of rabbits, whose diet was supplemented with fish oil (rich in pro-health n-3LPUFA), can be more susceptible to oxidation. The addition of substances with antioxidant properties may slow down or inhibit the unfavourable lipid oxidation process. Among them, the role of lycopene (Lyc) and selenium (Se) is often emphasized. In fact, Lyc belongs to the group of carotenoids which are known to be natural dyes. It occurs mainly in red fruit and vegetables. Tomatoes, and in particular their preserves, are characterized by high content of this component (Skiepko et al. 2015). Lyc is a hydrocarbon with many double bonds, thanks to which it has strong antioxidant properties (Sun et al. 2015). According to Przybylska (2020), it is twice more effective than β – carotene and 10 times as high as α – tocopherol, which is confirmed by in vitro studies of Martin et al. (2000). Lyc can be added in the production of meat and meat products among others as well as a supplement for animal feed. The improvement of meat quality and more beneficial production effect were observed in some research studies (Viuda-Martos et al. 2014).

Similarly, Se has antioxidant and protective properties against oxidative stress as a cofactor of selenoenzymes (like glutathione peroxidase); therefore, it may reduce the risk of cancer. In addition, it participates in many biochemical pathways – lipid and amino acid metabolism, as well as stabilizing lipid membranes (Brigelius-Flohé and Maiorino 2013). Se is also an important mineral ingredient for the proper functioning of animals. Its deficiency may have a negative influence on development and growth, which contributes to a decrease in production efficiency. Due to the low content of Se in the soil in many European countries, which directly affects its low level in plant feeds, its deficiencies can occur quite often. Therefore, adequate Se-supplementation of animal feed is also important, for example in organic forms of Se, especially selenized yeast (SeY), which are usually more efficiently incorporated in tissues of animals and humans (Edens and Sefton 2017).

Considering the above, we hypothesized that the antioxidants (i.e. Lyc and SeY) added to the diet enriched in odourless fish oil (rich in LPUFA), would stimulate the bioaccumulation of UFA (especially pro-health n-LPUFA) and effectively reduce oxidative stress caused by adding the high concentration of fish oil (>3%) to the rabbits’ diet. However, diet manipulation could affect sensory quality of meat due to higher level of fatty acids in muscle. Decreasing the sensory quality of meat aroma and flavour could have a negative impact on consumers’ behaviour and their choices on the meat market since meat aroma is of crucial importance for the sensory quality perception.

Taking the above into consideration, the aim of our study was to compare the quality of the longissimus thoracis muscle of rabbits depending on the type of supplement(s) (i.e. odourless fish oil, Lyc and SeY) added to the diet. It can be hypothesized here that the addition of fish oil will enrich meat with omega 3 fatty acids and the addition of lycopene and selenium will improve the quality of meat and protect them against oxidation.

Material and methods

The studies were carried out on 50 New Zealand White rabbits, 25 males (♂) and 25 females (♀) with an average initial body weight (BW) of 0.858 ± 0.087 kg. The observations were made from weaning at 35 days to 90 days of age. All animals were housed individually in accordance with protocols approved by the Local Animal Care and Use Committee (the Third Local Commission of Animal Experiment Ethics at the Warsaw University of Life Sciences; Ciszewskiego 8, 02-786 Warsaw, Poland). All the rabbits were clinically healthy and of the same age group. The animals were divided into 5 groups, 10 individuals (5 ♂ and 5 ♀) in each and fed ad libitum with properly prepared feeds for 55 days of experiments on rabbits. The rabbits of the control group were fed with the basal diet (BD) with the addition of 4% sunflower oil (SO), group FO was fed with BD enriched with 4% odourless fish oil (FO), group FOLyc was fed with the BD enriched successively in 4% FO and 15 ppm Lyc, whereas group ROSe was fed with the BD with 4%: FO and 0.25 ppm Se as highly selenized yeast (as a source of organic selenium). In turn, the rabbits of group ROLycSe were fed with the BD enriched with 4% FO, 15 ppm Lyc and 0.25 ppm Se as highly selenized yeast (SeY) (Table 1). The rabbits had free access to water. The control diet and all experimental diets were formulated to be isoproteinous and isoenergetic. The chemical compositions of the BD, SeY, SO and odourless FO are presented in Table 1. The health status of all rabbits was monitored daily by professional veterinary staff. All rabbits’ welfare guidelines and handling procedures, recommended by the Third Local Commission of Animal Experiment Ethics at the University of Life Sciences, were strictly followed throughout the preliminary and experimental periods. Measurements of rabbits’ increments were carried out at the Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences (Jabłonna, Poland). The feed intake was controlled; during the course of the experiment, the animals were weighed several times: at 35th, 56th, 70th, 77th and 90th days of life. After the rearing period (90th day of life), all rabbits were slaughtered in an onsite slaughterhouse after 24-hour of starvation. Slaughter was carried out according to the currently binding methodology (Council Regulation (EC) No. 1099/2009), in iden­tical technological conditions for all groups.

Table 1. Division of experimental groups^a due to the way of feeding, chemical composition the basal diet (BD), highly selenized yeast (SeY), sunflower oil (SO) and odourless fish oil (OR).

Control group		Experimental groups
Control	Group FO	Group FOLyc	Group FOSe	Group FOLycSe
BD	BD	BD	BD	BD
4% SO	4% FO	4% FO	4% FO	4% FO
–	–	15 ppm Lyc^b	–	15 ppm Lyc^b
–	–	–	0.25 ppm Se as SeY	0.25 ppm Se as SeY
Ingredients (g/kg BD)		The basal diet (BD)
Post-extraction soya middings		100
Wheat bran		186
Barley middings		250
Dried alfalfa		230
PreLak milk replacer		20
NaCl		4
Feed phosphate		10
Vitamins and mineral premix^b		10
Composition of highly selenized yeast (SeY)
Total Se, mg Se/g dry mass SeY		1.8
Identified Se species, % of total Se		88.3
Se-methionine, % of total Se		83.0
Se-cysteine, % of total Se		5.0
Sodium selenite, % of total Se		0.3
		Sunflower oil (SO):
The energy content: 37.0 MJ kg^−1 The iodine value: 119–135 g/100 g SO The acid value: 1.3 mg KOH/g SO		The fatty acid composition (%): C12:0 ∼0.05, C14:0 ∼1.9, C16:0 5.4, C16:1 0.3, C17:0 0.2, C17:1 ∼0.03, C18:0 2.7, cis9C18:1 (c9C18:1) 39.1, c9c12C18:2 48.3, c9c12c15C18:3 0.3, C20:0 0.25, C20:1 0.14, C22:0 0.9, C22:1 0.15, C22:2 ∼0.05, C24:0 0.25 and C24:1 ∼0.27.
		Odourless fish oil (FO):
The energy content: 36.8 MJ kg^−1 The iodine value: 50–65 g/100 g FO The acid value: 20 mg KOH/g FO		The fatty acid composition (%): C12:0 0.01, C14:0 1.23, c9 C14:1 0.02, C15:0 0.047, C16:0 5.69, c7C16:1 0.03, c9C16:1 0.04, ΣC16:2 1.55, C17:0 0.04, c9C17:1 0.02, C18:0 0.9452, c6C18:1 0.02, c7C18:1 0.08, c9C18:1 29.06, c12C18:1 1.58, c14C18:1 0.02, c9c12C18:2 11.45, c9c12c15C18:3 2.10, c11C20:1 2.42, c7c9c12c15C18:4 0.04, c11c14C20:2 0.24, c8c11c14C20:3 0.03, c5c8c11c14C20:4 0.03, c8c11c14c17C20:4 0.06, C22:0 0.014, c13C22:1 1.11, c11C22:1 0.17, c5c8c11c14c17C20:5 0.69, c13c16C22:2 0.01, c7c10c13c16C22:4 0.11, c15C24:1 0.04, c7c10c13c16c19C22:5 0.20 and c7c10c13c16c19C22:6 2.67.

^a Experiments were carried out on 50 New Zealand White rabbits (25 males (♂) and 25 females (♀)) with an average initial body weight of 0.858 ± 0.087 kg at 35 days of age.

^b Lycopene.

^c 1 kg of vitamins and mineral premix (provided by LNB Sp. z.o.o. Kiszowo, Poland) comprised: vitamins A as E672-retinol (10,00,000 IU), D₃ as E671-cholecalciferol (1,50,000 IU), E as Dl-α- tocopherol (2 727 mg), K₃ as menadione (52 mg), B₁ (50 mg), B₂ (400 mg), B₃ (2000mg), B₅ (786 mg), B₆ as pyridoxine hydrochloride (50 mg), B₁₂ (1500 mcg), biotin (10,000 mcg), choline chloride (12,500 mg), folic acid (57 mg), trace elements (Fe – 5000 mg, Mn – 7500 mg, Cu – 750 mg, Zn – 5000 mg, I – 100 mg, Co – 100 mg, Se – 20 mg); vitamin-mineral premix carrier: calcium carbonate containing 22.2% Ca. The BD without vitamins and mineral premix contains 0.05 ppm Se.

The average final BW of rabbits at 90th days of life was 2.277 ± 0.257. The averages of hot carcass weight (g), giblets weight (g), inedible parts weight (g), inguinal fat weight (g), shoulder fat weight (g), kidney and abdominal fat weight (g), carcass muscle weight (g) and dressing percentage (%) were 1358 ± 16, 112 ± 2.7, 1064 ± 19, 6.01 ± 0.9, 12.1 ± 1.2, 32.2 ± 2.1, 1067 ± 19 and 52.8 ± 1.1, respectively. The carcasses of rabbits were divided into three parts: the anterior part, with the cut made at the height of the last rib; the saddle, cut at the height of the last lumbar vertebra; and the posterior part, including the hind legs. A scalpel was used to separate the subcutaneous fat from the shoulders in the anterior part, organ fat from around the kidneys and stomach in the middle part (saddle) and subcutaneous inguinal fat in the posterior part. The longissimus thoracis muscle dissection was carried out according to the method described by Bieniek (1997). The impact of the control and experimental diets on all internal organs, muscles and adipose tissues of slaughtered rabbits were visually evaluated by professional veterinary staff. Removed longissimus thoracis muscles were frozen; muscle samples were stored in sealed tubes at −32°C until analyses.

Ragents and analytical methods

All chemicals were of analytical grade and organic solvents were of HPLC grade. Dichloromethane (DCM), KOH, NaOH, Na₂SO₄ and conc. HCl were purchased from POCH (Gliwice, Poland). Acetonitrile, methanol and n-heptane (99%, GC) were supplied by Lab-Scan (Ireland), while the conjugated linoleic acid (CLA) isomer mixture (2.1% trans,transCLA, 7.1% cis11trans13CLA, 40.8% cis9trans11CLA, 41.3% trans10cis12CLA, 6.7% cis8trans10CLA and 2.0% cis,cisCLA) by Industrial Chemistry Research Institute (Warsaw, Poland). Fatty acid methyl ester (FAME) standards and 25% BF₃ in methanol were purchased from Supelco and Sigma (USA). Lyc 10% in SO (LycoVit Dispersion 10%; product-No. 30264388) was obtained from BASF The Chemical Company (Germany). SO was donated by Company AGSOL (Pacanów, Poland). Odourless FO was donated by Meals Manufacturing Company ZPM (Bokiny-Łapy, Poland). Vitamins and mineral premix were provided by LNB Sp. z.o.o. Kiszowo, Poland. The highly selenized yeast (SeY) was purchased from Sel-Plex (Alltech Inc., Nicholasville, KY, USA). The total Se-amount of SeY is incorporated into the proteins of Saccharomyces cerevisiae.

Water used for the preparation of mobile phases and chemical reagents was prepared using an Elix^TM water purification system (Millipore). The mobile phases were filtered through a 0.45 μm membrane filter (Millipore).

Determination of fatty acid concentrations in the longissimus thoracis muscle

Saponification and fatty acid extraction

The homogenized muscle samples (∼50 mg) were placed in vials and treated with a mixture of 2 ml of 2 M KOH in water and 2 ml of 1 M KOH in methanol. Next, 50 μl of the internal standard (IS) solution (17 mg ml⁻¹ nonadecanoic acid in chloroform) was added to the obtained mixture. The resulting mixture was flushed with argon (Ar) for ∼4 min. The vial was then sealed and the mixture vortexed and heated under Ar at 95°C for 10 min, cooled for 10 min at room temperature and sonicated for 10 min. The resulting mixture was protected from the light and stored in the sealed vial under Ar at ∼22°C overnight. Next, 3 ml of water was added to the hydrolysate and the solution was again vortexed. The obtained solution was acidified with 4 M HCl to ∼pH 2 and free fatty acids were extracted four times with 3 ml of DCM. Extraction was repeated 4 times using 3 ml of n-hexane. The upper n-hexane layer was combined with the DCM layer, and next the resulting organic phase was dried with ∼0.1 g of Na₂SO₄. The organic solvents were removed under a stream of Ar at room temperature. The obtained residue was stored at −20°C until base – and acid-catalyzed methylation.

Preparation of fatty acid methyl esters (FAME)

Two ml of 2 M NaOH in methanol was added to the residue while mixing, then flushed with Ar and reacted for 1 h at 40°C. After cooling the reaction mixture to ∼4°C, 2 ml of 25% BF₃ in methanol was added, flushed with Ar and heated for 1 h at 40°C. To the cooled reaction mixture 5 ml of water was added and then FAME were extracted with 5 ml of n-hexane. The supernatant was transferred to a GC vial.

Analytical equipment

The analyses of all FAME were performed on a SHIMADZU GC-MS-QP2010 Plus EI equipped with a BPX70 fused silica capillary column (120 m × 0.25 mm i.d. × 0.25 μ m film thickness); SHIM-POL, quadrupole mass selective (MS) detector (Model 5973N) and injection port. Helium, as the carrier gas, operated at a constant pressure (223.4 kPa) and a flow rate of 1 ml/min. Injector and MS detector temperatures were maintained at 200 and 240°C, respectively. The total FAME profile in a one μl sample at a split ratio of 10:1 was determined using the column temperature gradient programme. The column was operated at 70°C for 4 min, then the temperature programmed at 12°C/min to 150°C, held for 6 min, programmed at 8°C/min to 168°C, held for 27 min, programmed at 0.75°C/min to 190°C, held for 10 min, programmed at 1.8°C/min to 210°C, held for 15 min, programmed at 6°C/min to 234°C, held for 4 min, programmed at 6°C/min to 236°C, held for 20 min. FAME identification was validated based on electron impact ionization spectra of FAME and compared with authentic FAME standards and NIST 2007 reference mass spectra library. Each FAME assay was done in duplicate.

Based on the obtained FA-peak areas, the percentage of individual fatty acids and the sum of saturated FA (SFA), polyunsaturated FA (PUFA), FA from the n-3 and n-6 family, as well as the ratios of PUFA/SFA and n-6PUFA/n-3PUFA in the longissimus thoracis muscles were calculated.

Fat oxidation stability (malondialdehyde; MDA) in the longissimus thoracis muscle

The method determines the content of MDA in muscle, which is the final product of fat oxidation processes – decomposition of hydroperoxides. MDA reacts with 2-thiobarbituric acid and stains. The extent of muscle lipid oxidation was evaluated with a spectrophotometer (Hitachi U-2000; Hitachi, Mannheim, Germany) set at 532 nm that measured the absorbance of TBARs and a 1,1,3,3-tetraethoxypropane calibration curve (Konieczka et al. 2014). Oxidation products were quantified as MDA equivalents (mg MDA/kg muscle).

Sensory quality of the longissimus thoracis muscles

Due to the limited amount of material it was decided to conduct only the odour quality notes valuation. The sensory evaluation was carried out after heat treatment. The tested material (leg muscle) was placed in a special baking bag and directed to the heat-treated process in an oven at 180°C. This process was carried out until the temperature, inside each of the samples, reached 75°C. After cooling the samples a discussion and a preliminary evaluation were carried out and the attributes of the meat odours quality such as meat, sharp, fatty, sour, rancid and ‘other’ as well as an overall odour quality were chosen and defined. The intensity of each distinguishing attribute was indicated on an unstructured graphical scale (0–10 c.u. – convenient units). The marks of anchors of the tested attributes were for most of them as follows: no intensity – high intensity and overall quality (very low to very high). The evaluation panel consisted of 10 assessors all of whom were familiarized with the scaling methods and trained in accordance with ISO 8586-2: 2008. The assessors were characterized by many years of experience in meat and meat products assessments. The testing was conducted in rooms free from off odours, with daily light at room temperature (22 ± 1°C). The conditions and the assessment mode were established in accordance with Lawless and Heymann (2009) and Meilgaard et al. (1999).

Statistical methods

Results were elaborated with the use of STATISTICA software, version 10. The normality of distribution of all analysed traits was checked using the Shapiro–Wilk test. Dietary treatment effects on growth performance, chemical composition and fatty acid profile of muscle were tested according to ANOVA, while the effects of the diet, session and panellist for studied sensory traits were analysed using multifactor analysis (ANOVA). The significance of differences between means was calculated on the basis of the least significant differences test (LSD) at the level of P ≤ 0.05. Correlation coefficients were calculated based on the correlation matrix (P ≤ 0.05).

Results and discussion

The results of our current studies documented that 4% FO supplemented to the diets, irrespective of the presence of Lyc and/or SeY, do not cause harmful symptoms (e.g. vomiting and diarrhoea) to rabbits. Moreover, no visual pathological changes, acute Se toxicity and toxic changes of the experimental diets enriched in SeY were observed in the liver, heart, spleen, pancreas, kidneys, muscles and adipose tissues, blood and bones of rabbits.

The obtained results (Table 2) indicated that the supplemented feed was consumed by the animals in greater quantity compared to the control group. Higher average feed intake per day was observed in the group of the rabbits (for both sexes) fed the experimental diet containing FO with lycopene compared to the control diet (P < 0.05). When analysing the food intake of rabbits with regard to body weight gain (BWG), the higher BWG was found in the groups of the rabbits (for both sexes) fed the diet containing FO and the diets including FO and lycopene, irrespective of the presence of SeY, compared to the experimental diet with FO and SeY. The lowest BWG was observed in the rabbits (for both sexes) fed the experimental diet enriched with FO and SeY. The higher feed consumption per 1 kg of body weight gain was in the group of the rabbits fed the experimental diets containing FO and Lyc or with SeY in comparison with the control diet. The addition of fish oil to animal feed had no significant effect on the muscling of animals (Table 2). The obtained results also documented that the enrichment of feed in fish oil (FO) resulted in an increase in animal longissimus thoracis muscle mass by 7.3% on average compared to the control group. Research by Kowalska and Bielański (2009) confirmed these results. The above-mentioned differences in body weight of the rabbits can be explained by the effect on carcass fatness. Fat deposition in the body is affected by the degree of saturation of dietary fatty acids (Kowalska and Bielański 2009). However, these authors stated that low saturated fats can have an effect on lower fatness. This hypothesis is partly in opposition to own research results.

Table 2. Comparison of average feed intake, feed conversion ratio (FCR), body weight gain (BWG) of rabbits and longissimus thoracis muscle mass of the studied groups of rabbits (♀ = 5; ♂ = 5; Σ(♀ and ♂) = 10) at 90 days of life (i.e. after 55 days of experiments on rabbits).

		Control group	Experimental groups				SEM	P
		Control	FO	FOLyc	FOSe	FOLycSe
Average feed intake by the rabbit per day [g/day]	♀	70.1 a	90.8 b	92.8 b	78.7 ab	85.4 ab	4.16	0.05
	♂	74.1 a	75.8 a	91.7 b	74.2 a	84.5 ab	3.49	0.05
	Σ(♀ and ♂)	72.1 a	83.3 ab	92.2 b	76.4 a	84.9 ab	3.50	0.05
FCR (feed intake [kg] per kg of BWG of rabbits) FCR	♀	2.77 a	3.31 b	3.46 b	3.77 b	3.35 b	0.16	0.05
	♂	2.98 a	2.85 a	3.27 b	3.38 b	3.05 ab	0.10	0.05
	Σ(♀ and ♂)	2.88 a	3.08 ab	3.37 b	3.58 b	3.20 ab	0.12	0.05
	♀	1.378 b	1.534 b	1.501 b	1.166 a	1.423 b	0.06	0.05
Average BWG, [kg]^a	♂	1.356 ab	1.490 b	1.569 b	1.226	1.549 b	0.06	0.05
	Σ(♀ and ♂)	1.367 ab	1.512 b	1.535 b	a1.196 a	1.486 b	0.06	0.05
Average longissimus thoracis	♀	119.6	138.2	127.0	125.0	119.8	3.39	NS
muscle mass [g]	♂	127.0	141.5	136.3	127.2	140.3	3.12	NS
	Σ(♀ and ♂)	123.3	139.9	131.7	126.1	130.1	2.83	NS

^a The average body mass gain = (BW_{90 days} – BW_{35 days}); BW_{35 days} – the body weight of rabbits at 35 d of age; BW_{90 days} – the body weight of rabbits at 90 days of age; NS – non-significant.

Analyses of concentrations of fatty acids in the muscles showed that the sex of rabbits did not have a statistically significant effect on the concentrations of SFA, MUFA and PUFA in muscles. Therefore, the results summarized in Table 3 are the average of the examined rabbits of both sexes (♀ and ♂). The concentration sum of all assayed fatty acids (ΣFA) (Table 3) in the longissimus thoracis muscle indicated that there was a significant increase in (P ≤ 0.05) the concentration of ΣFA in the group of the rabbits fed the diet with only FO in comparison to the control group as well as other experimental groups. Research by Kowalska (2011) on rabbits fed with fodder with the 3% fish oil addition confirmed such a tendency, whereas rabbits fed the experimental diets containing FO, irrespective of the presence of Lyc or/and SeY, were characterized at the same time by the lowest content of ΣFA and did not significantly differ from each other (P > 0.05). The addition of FO to the diet increased the concentration of PUFA in muscle, which was coherent with expectations, as fish oil is characterized by a high content of these components. Moreover, the experimental diets containing FO (rich in n-3PUFA), irrespective of the addition of Lyc or/and SeY, decreased the ratio of Σn-6PUFAs (Σn-6) to Σn-3PUFAs (Σn-3) in muscle compared to the control diet; the higher concentration sum of n-3PUFA was found in muscles of the rabbits fed the diet with FO and SeY compared to the control diet. Comparing the concentrations of PUFA and n-3PUFA in muscles of the rabbits fed the experimental diets, regardless of the presence of Lyc or/and SeY, no statistical differences were noticed. Moreover, Kowalska and Bielański (2009) in their study of the impact of rabbits’ feed enrichment with fish oil on the FA profile of meat lipids noticed that the meat of animals supplemented with feed also contained significantly more PUFA than the control group meat. Analysis of the impact of the addition of fish oil to rabbit feed on the fatty acid profile of their meat quality allowed us to conclude on the appropriateness of this nutritional supplementation. Dal Bosco et al. (2004) studied the effect of linseed oil added to rabbit feed on the FA profile. Meat of the rabbits from the above mentioned group was characterized by a significantly higher content of PUFA and lower level of saturated fatty acids (SFA). In addition, the content of n-3PUFA was twice higher in the experimental group in comparison to the control group. Supplementation of fodder with vegetable oils in order to improve FA profile and meat quality is often used in studies on various animals. Kawęcka et al. (2016) assessed the quality of lamb meat fed with flax seed. Based on the obtained results, it was found that the meat of the experimental group was characterized by a higher content of α-linolenic acid (c9c12c15C18:3). A higher share of FA from the n-3 group in the total share was found, as well as a lower (more favourable) ratio of Σn-6/Σn-3 acids compared to the control group. Moreover, there was also an increase in SFA content and a decrease in PUFA, which may be an adverse effect of supplementation in other studies observed (Lauridsen et al. 2005; Intarapichet et al. 2008; Cordero et al. 2010). In the literature the calculated ratio of n-6 and n-3 is different, from very low such as 2.95 in Longissimus dorsi of rabbits fed with the diet containing α-linolenic acid (Dal Bosco et al. 2004; Szabó et al. 2004; Ramírez et al. 2005), to high such as 11.6 for hind legs of rabbits (Dalle Zotte 2002; Ramírez et al. 2005). The calculated ratios of 1.82–3.54 obtained in the presented study (Table 3) are similar to the results reported by Dal Bosco et al. (2004). Our studies documented that the experimental diets containing Lyc or/and SeY decreased the concentration sum of atherogenic SFA (A^SFA) in muscle in comparison with the control diet and the experimental diet with only FO. Moreover, the experimental diets including Lyc or SeY also reduced the concentration sum of thrombogenic SFA (T^SFA) in muscle compared to the control and other experimental diets.

Table 3. The concentration sums (mg/g) of saturated fatty acids (ΣSFAs)^a, atherogenic^b (A^SFA) and thrombogenic^c (T^SFA) saturated fatty acids, monounsaturated fatty acids (ΣMUFAs)^d, polyunsaturated fatty acids (ΣPUFAs)^e, all assayed fatty acids (ΣFAs), Σn-3PUFAs (Σn-3), Σn-6PUFAs (Σn-6) and the ratio of Σn-6 to Σn-3 (Σn-6/Σn-3) in the longissimus thoracis muscle of rabbits.

Group	A^SFA	TSFA	ΣSFAs	ΣFAs	ΣMUFAs	ΣPUFAs	Σn-3	Σn-6	Σn-6/Σn-3
Control	5.55b	6.72b	6.96b	27.19a	3.49b	16.70 a	3.48a	12.31a	3.54b
FO	6.73b	8.95b	9.15bc	31.88b	3.63b	19.13 b	5.24ab	13.86a	2.64a
FOLyc	3.10a	3.93a	4.04ab	26.39a	1.87a	20.51 b	6.29ab	13.99a	2.22a
FOSe	2.09a	2.15a	2.53a	25.67a	1.63a	21.52 b	7.49b	13.58a	1.82a
FOLycSe	2.03a	4.96ab	6.01b	26.99a	1.47a	19.53 b	5.51ab	13.88a	2.52a
SEM	0.95	1.17	1.14	1.09	0.47	0.81	0.66	0.31	0.28
P	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05

Note: Mean values in columns having the different superscripts are significantly different at a, b P < 0.05.

^a The sum of C8:0, C10:0, C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 and C24:0.

^b A^SFA: C12:0 + C14:0 + C16:0.

^c T^SFA: C14:0 + C16:0 + C18:0.

^d The sum of c7C14:1, c9C14:1, c7C15:1, c9C16:1, c10C16:1, c9C17:1, c10C17:1, c6C18:1, c9C18:1, t11C18:1, c12C18:1, c11C20:1 and c13C22:1.

^e The sum of c9c12C18:2, c9t11CLA, t10c12CLA, c6c9c12C18:3, c9c12c15C18:3, c6c9c12c15C18:4, c11c14C20:2, c8c11c14C20:3, c11c14c17C20:3, c5c8c11c14C20:4, c8c11c14c17C20:4, c5c8c11c14c17C20:5, c7c10c13c16C22:4, c7c10c13c16c19C22:5, c4c7c10c13c16c19 C22:6 and c13c16C22:2.

Our recent studies have shown that dietary Lyc increased the concentration of unsaturated fatty acids (UFA) in chickens’ thigh muscles (Rozbicka-Wieczorek et al. 2012) as well as decreased oxidative stress and peroxidative damage of UFA and affected the biosynthesis yield of FA in chickens’ tissues (Rozbicka-Wieczorek et al. 2012). Lyc elevated chick birth body weight, increased liver total antioxidant capacity, glutathione to oxidized glutathione ratio, glutathione peroxidase activity and reduced liver malondialdehyde (MDA) concentration (Sun et al. 2015).

The organic chemical form of Se is Se-Met, Se-amino acid which is the most abundant in SeY (Rozbicka-Wieczorek et al. 2012). The availability of selenium from the organic form (Se-Met) is higher than that of sodium salts (Stewart et al. 2012). This correlation is also confirmed by Calamari et al. (2010), stating that selenium in the form of yeast passes into milk in 16.3% and as selenite in only 3.2%. Increased selenium concentration in meat during feed supplementation was also demonstrated in rabbits (Dokoupilová et al. 2007).

Due to its high content of PUFA, rabbit meat is susceptible to lipid oxidation processes, which may cause deterioration of the quality of the raw material. Gašperlin et al. (2006) state that the factors that determine the speed and direction of fat oxidation are the chemical composition of meat, the water content in meat, the presence of pro- and antioxidants in meat, technological processes and storage conditions such as time, temperature and oxygen availability. Evaluation of fat oxidation level (Figure 1) indicated that the highest level was observed in the samples with fish oil addition (FO) and in the sample with fish oil with lycopene (FPLyc) (P < 0.05). The use of antioxidants was justified, particularly in the case of lycopene and selenium used simultaneously; it caused a decrease in oxidation processes, which confirms a similar value of TBARS indicator to the control sample. The group with the fish oil only was characterized by the significantly highest level of MDA. Lycopene supplementation reduced the content of the lipid oxidation product. It is worth noting that group FOLycSe, whose feed was enriched with two antioxidants – Lyc and SeY, was characterized by the lowest level of MDA compared to the groups with one antioxidant alone. This shows a better antioxidant effect due to a possible synergistic effect of these components. The content of MDA in group FOLycSe was comparable with the control group. The obtained results indicate that in order to obtain meat enriched with omega-3 acids of unprocessed oxidation processes, it would be necessary to supplement feed for animals in SeY and Lyc. It could be pointed that such a meat is not characterized by good storage ability. A study by Chwastowska Siwiecka et al. (2016) showed that the content of MDA in rabbit meat increased along with the length of refrigeration storage time. Oxidative changes in fats can worsen the sensory quality of meat as many compounds responsible for the formation of rancid odour and flavour are formed. Consumers perceive it as very unfavourable, which may disqualify the raw material from consumptions. In the studies of Chwastowska Siwiecka et al. (2016) it was found that longer storage time of meat influenced the reduction of the value of sensory features such as aroma, juiciness and palatability. Moreover, Kowalska (2011) studied the influence of the rabbit meat storage length in freezing conditions on the TBARS level. After 90 days of storage at −10°C, the TBARS index was about 1 mg malondialdehyde/kg. Similarly, Kowalska (2011) investigated rabbit meat with 2% fish oil addition and formulated similar conclusions.

Figure 1. Average concentration of malondialdehyde (MDA) in meat samples [mg/kg product] in the test groups (n = 10). a, b – means marked with different letters differ significantly at P < 0.05.

Lipid oxidation processes are advanced and the content of reaction products is also higher, which have an effect on the deterioration of the sensory quality of meat, whereas in the studies of Tedesco et al. (2005) and Peiretti et al. (2013) in the case of rabbit meat there was no positive effect of Lyc and vitamin E addition to feed on oxidative stability observed.

The results of the sensory evaluation and their analysis indicated a significant effect of the use of feed additives on the intensity of the odours’ attributes (Figure 2). The samples of the meat from the animals fed with fish oil enrichment, Lyc and selenium were characterized by a lower intensity of the meat odour. In terms of the overall sensory quality, the control sample was of the highest odour quality, whereas the lowest notes were observed in the case of the samples enriched with fish oil (FO) and fish oil with selenium (FOSe) at the same time. Lowering the perceptibility of meat notes was associated with a higher intensity of notes such as sharp, rancid and ‘other’. Regarding the rancid note, the lowest level of detection was observed in a control sample and with the addition of selenium and lycopene simultaneously. The addition of fish oil to feed increased the perceptibility of fatty and rancid note in meat, which may indicate the presence of unfavourable lipid autoxidation products in meat, which is confirmed in MDA level (Figure 1). The group supplemented with Lyc and selenium simultaneously, which were expected to significantly inhibit the unfavourable oxidation processes, was rated quite low in terms of overall odour quality. It could be the result of the low perceptibility of meat notes and the relatively high intensity of notes described by the ‘other’. Also Olivier et al. (1997) shows that diet with higher level of fat also affected the organoleptic quality of the rabbit meat. However, Colin et al. (2005) and Bianchi et al. (2006) did not observe any alteration of hedonic characteristics of fresh rabbit meat with a raised content of n-3 PUFA obtained by dietary use of extruded linseed. Nevertheless, at 6 months of storage, differences were shown. These results indicate that some variation in the sensory properties of the meat (off flavours, rancidity, etc.) occurred during frozen storage, and suggest that rabbit meat products with raised PUFA should not be stored for long periods.

Figure 2. Comparison of sensory quality of odour notes of rabbit meat in the experimental groups (n = 50).

Analysing the correlations between variables one can notice a set of relationships, which are presented in Table 4. The overall quality of aroma was positively correlated to the greatest extent with the meat aroma. In turn, the occurrence in high intensity of sharp, rancid and ‘other’ notes lowered the overall sensory quality of aroma to the highest extent. Research by Chwastowska Siwiecka et al. (2016) confirms this relationship as the authors stated that the storage time of rabbit meat influenced the value of sensory attributes such as aroma, juiciness and palatability reduction. This study showed that attributes with a negative sensory influence (rancid odour, sharp odour) determine the overall sensory quality of examined products and are of crucial importance for the overall sensory quality. It is known (Lawless and Heymann 2009) that after exceeding a critical value those negative attributes strongly decrease overall sensory quality of tested products.

Table 4. Values of correlation coefficients (r) between the analyzed variables (n = 50).

Attribute	meat o.	sharp.o	fat o.	sour o	rancid o	other o.	overall quality
meat o.	1	−0.53*	−0.47	0.34	−0.62	−0.65	0.82
sharp.o		1	0.12	0.27	0.45	0.59	−0.75
fat o.			1	0	0.45	0.17	−0.39
sour o				1	0.37	0.28	−0.27
rancid o					1	0.56	−0.75
other o.						1	−0.74
overall quality							1

Note: o- odour.

*Correlation coefficients marked in bold are significant at the level of significance P < 0.05.

As rabbit meat has unique nutritional and dietary properties, not only for children but also for adults, the sensory quality of meat should be not overlooked in studies. Similar observation was shown by Escribá-Pérez et al. (2019) who claimed that rabbit industry should think about the child segment. Promoting rabbit meat consumption among young consumers could have several consequences, as in future they will be in charge of household purchases or share this responsibility.

Conclusion

Despite the importance of the nutritional properties of essential fatty acid, care must be taken to avoid lipid oxidation and detrimental effects on the sensory qualities of meat and meat products during processing and subsequent storage. Feed supplementation with fish oil was only beneficial in terms of nutrition due to the decrease of the ratio of Σn-6/Σn-3. The use of selenium and lycopene simultaneously showed antioxidant activity which slows down the fat oxidation process. Despite the increase in muscle mass of animals and high content of FA from the family n-3 in meat, its low sensory quality and far-reaching oxidative changes in muscle fat indicate the lack of practicality of enriching rabbit fodder with fish oil.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the statutory funds from The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences (Jabłonna, Poland) and by Polish Ministry of Science and Higher Education within funds of Faculty of Human Nutrition and Consumer Sciences, Warsaw University of Life Sciences (WULS), for scientific research.