Enhancement of Quails’ (Coturnix japonica domestica) Meat Quality: Effects of Dietary Supplementation of Dried Bergamot Pulp

1. Introduction

In the past few decades, meat consumption has changed significantly, and due to its indiscriminate consumption [1], there has been much debate about its potential negative effects for humans, affecting consumer attitudes over time. There are several epidemiological studies which demonstrate a positive association of high levels of consumption, especially in red and processed meat, with cancer incidence [2], leading to consumers being more mindful of their consumption and to increasingly foods that are seen as healthier and safer [3].

Following the COVID-19 pandemic, the human–nature relationship has come under increased scrutiny, with renewed emphasis on reducing wild meat consumption to protect both public health and biodiversity [4]. However, such reductions present challenges for the global food system, given the nutritional importance of wild meat for certain populations and its interconnectedness with other food production systems.

In Europe, consumption of the common quail (Coturnix coturnix L.) was particularly widespread during the last century, when quails were predominantly obtained through hunting [5]. It has been estimated that approximately 6.4 million hunters in Europe collectively harvest more than 52 million birds annually, including quail [6]. At present, however, the quail meat market is exclusively based on the Japanese quail (Coturnix japonica - Temminck & Schlegel, 1849), which has been progressively domesticated for egg and meat production (Coturnix japonica domestica) [7]. Due to their high seasonal reproductive rate, quails, along with other Galliformes, may represent a viable option for meeting the growing demand for poultry products, thereby supporting the diversification of poultry meat consumption.

The primary source of energy in quail diets is mainly represented by Maize (Zea mays L.) [8], which is also used extensively in the feeding of other livestock species as well as in beverages, biofuels, and human foods [9]. The continuous rise in the cost of raw materials and conventional feedstuffs has intensified the search for alternative dietary resources [3]. In this context, increasing attention has been directed toward the valorization of agro-industrial by-products.

The use of by-products (such as tomato pomace and fermented bean sprouts waste flour) in Japanese quail diets has been examined recently [10,11]. In the citrus industry, bergamot pulp—a by-product of the processing of Citrus bergamia Risso—appears to be a promising candidate for use in animal feed, with potential benefits in terms of waste reduction. Evidence from studies involving small ruminants and pigs indicates that the inclusion of bergamot pulp in the diet can positively influence meat quality and extend shelf life [12,13,14,15]. Bergamot is a citrus fruit cultivated in coastal areas, where it thrives on saline soils within a typical Mediterranean ecosystem. Particularly favorable ecological conditions are found in Calabria, in southern Italy (notably in the province of Reggio Calabria), as reflected by the high levels of fruit productivity recorded in this region [16]. Moreover, bergamot has been shown to possess bioactive properties, especially in terms of antioxidant activity [17]. However, to our knowledge, there are no studies evaluating the use of bergamot pulp in the feeding of small monogastric species such as quail. Accordingly, the present study aimed to evaluate the effects of dietary supplementation with bergamot pulp on growth performance and meat quality in Japanese quail.

2. Materials and Methods

The Animal Welfare Committee of the University of Reggio Calabria approved this study (protocol number 1214, 25-01-2023). The care and use of animals were performed in accordance with the EU Directive 2010/63.

In the present study, 140 Japanese quails with equal number of male-female (in mixed gender) aging 15 days (146,52 ± 9.15 g live-weight) were randomly assigned to 2 treatment groups of 70 quail each, and each group was divided into 7 replicates of 10 quails each, in an enclosed area (temperature 20–24 °C). The control group (C) received a basal diet composed of maize, soybean meal and faba bean, while the bergamot by-product group (BP10) fed the basal diet in which part of the maize was replaced with 10% (DM on the diet fed) of dried bergamot pulp (BP). A slightly greater quantity of faba beans was integrated into the BP10 diet compared to the control diet to avoid providing diets with different concentrations of crude protein (Table 1). The diets were fed ad libitum and the animals had continuous access to water. The quails were subjected to one week of adaptation to the experimental diet. Each quail was weighed at the beginning, in the middle and at the end of the experimental trial which lasted 3 weeks. Daily the animals’ feed consumption was calculated by removing the amount of feed remaining in the feeder from the previous day’s feeding, in order to evaluate the dry matter intake (DMI).

At the end of the experiment trial, all the animals were slaughtered in a commercial abattoir, and the carcasses were subsequently weighed after the removal of feathers, legs and internal organs, and maintained at 4 °C. After 24h, the breast from each animal was removed for the evaluation of meat quality. In detail, a total of 70 breasts (n=5 breasts/replicant, n=35 breasts/treatment) were vacuum packed and stored at -30 °C for analyses of proximate composition. The left side of the other 70 breasts (n=5 breasts/replicant, n=35 breasts/treatment) was vacuum-packed and delivered to the laboratory, stored at 4 °C, for the determination of lipid oxidation and color stability, while the right side was vacuum-packed and stored at -30 °C and subsequently used to evaluate the fatty acid (the upper right breast) and α-tocopherol (the lower right breast) composition of the meat.

2.1. Feedstuff Analysis

The chemical composition (dry matter, crude ash, crude protein and ethereal extract) of the experimental diets was determined according to the AOAC methods [18], while the neutral detergent fibre (NDF) content was determined following the method of Van Soest et al. [19]. The Folin-Ciocalteu reagent [20] was used to determine the total phenols and tannins in feed, which were extracted in aqueous acetone. The α-tocopherol was evaluated by means of a methanol:acetone:petroleum ether (1:1:1, v:v:v) extraction process [21], as well as ultra-high performance liquid chromatography (UHPLC; Nexera, Shimadzu Corporation, Milan, Italy) equipped with a Zorbax ODS column (25 cm × 4.6 mm, 5 µm; Agilent Technologies, CA) and a Shimadzu spectrofluorometric detector (RF-20AXS) (excitation wavelength of 295 nm and emission wavelength of 330 nm). The analysis of retinol (absorbance at 325 nm) was conducted using a Shimadzu photodiode array detector (PDA; SPD-M40). The UHPLC system was controlled by LabSolutions software. The quantity of sample with methanol injected was 10 µL, and the analytes were identified by comparing the retention times with those of pure standards. The chromatographic conditions were described in detail in Natalello et al. [22]. The procedures used by Valenti et al. [23] were utilised to determine the fatty acid composition.

2.2. Fatty Acid Profile, α-Tocopherol and Proximate Composition

The fatty acid composition of the meat samples was analysed on the total lipids extracted following the procedures described by Folch et al. [24]. Five grams of minced meat samples were extracted with chloroform/methanol (2:1, v/v), then with 1 mL of hexane and 0.05 mL of 2 N methanolic KOH. A total of 100 mg of lipids were methylated [25] containing C9:0 as an internal standard. The evaluation of fatty acid methyl esters was conducted utilising a gas chromatography/flame ionization detector (CP 3900, Varian Inc., Netherlands), which was equipped with CP-Sil capillary column (100 m X 25 mm i.d. X 0.25 um film thickness; Agilent Technologies, CA, USA).

The gas chromatography conditions and the identification of fatty acid methyl esters (FAME) were performed in accordance with the methods described by Scerra et al. [14] The quantification of fatty acids (FA) was conducted in g/100 g of methyl esters.

The α-tocopherol content of the muscle samples was determined in accordance with the methodology outlined by Natalello et al. [22], employing the UHPLC configuration previously delineated. The chromatographic conditions employed for the analysis of the muscle samples were identical to those utilised for the feed samples.

The AOAC methods [18] were utilised to ascertain moisture, crude fat, ash, and crude protein in meat samples. The breast of each animal was ground and homogenized prior to analysis.

2.3. Lipid Oxidation and Color Measurement

To evaluate raw meat oxidative stability, three slices of meat (breast) from each sample were placed in a tray (covered with PVC film) and assessed through thiobarbituric acid reactive substances (TBARS) assay at 0, 3 and 7 days of storage at 4 °C [26]. In brief, 2.5 g of meat samples were homogenized (in a cold-water bath) with 12.5 mL of distilled water, mixed with 10% (w/v) trichloroacetic acid (12.5 mL) and filtered (Whatman No.1 filter paper). Subsequently, 4 mL of filtrate was mixed with 1 mL of 0.06 M aqueous thiobarbituric acid, following which the mixture was incubated in a water bath at 80 °C for a period of 90 min. The UV-1800 Shimadzu spectrophotometer (Shimadzu Corporation, Milan, Italy) was utilised to measure the samples’ absorbances at 532 nm. The assay was calibrated in accordance with the methodology outlined by Scerra et al. [14] The results were expressed as milligrams of malondialdehyde (MDA) per kilogram of meat.

Color-meter were assessed at 0 d (after 2 hours of blooming), 3 d, and 7 d of refrigerated storage, using the same slice for TBARS assay (color test before TBARS assay). The colour coordinates L* (lightness) a* (redness) and b* (yellowness) were measured using a Minolta CR300 colour-meter (illuminant A and 10° standard observer; Minolta Co. Ltd. Osaka, Japan). Hue angle (H*) was calculated as: H* = tan−1 (b*/a*) × (180/π), whereas Chroma (C*) was calculated as: C* = $\sqrt{{\left(a^{*}\right)}^2+({b^{*})}^2}$.

2.4. Statistical Analysis

Data on animal performance, chemical composition and intramuscular FA composition of meat were analyzed using a one-way ANOVA to evaluate the effect of the dietary treatment (C and BP10). Individual animal was considered as the experimental unit for body weight measurements, while pen was the experimental unit for other performance parameters. For meat quality parameters, sample quails (from which the breast was removed) were used as the experimental unit. Data of color and TBARS in meat were analyzed using a mixed model to evaluate the effect of experimental diets and of time of storage (0, 3 and 7 days), as well as of their interaction as the fixed factors, while individual animal sampled was included as a random factor.

Differences between means were assessed using Tukey’s multiple comparison test. Significance was declared when P ≤ 0.05, whereas trends were considered for 0.05 < P ≤ 0.10. Minitab software (version 19, Minitab Inc., State College, PA) was used for statistical analyses.

3. Results

The performance data of quails are shown in Table 2. The integration of dried bergamot pulp in quail diet reduced final body weight (FBW, 252 g and 234 g, in control and BP10 groups, respectively, P < 0.05), average daily gain (ADG, 5.38 and 4.60 g/d, in control and BP10 groups, respectively, P < 0.05) compared to control treatment. Furthermore, a reduction of daily intake (DMI, P < 0.05) was observed in BP10 group than in control group. Conversely, quails from BP10 group showed a higher feed conversion ratio (P = 0.05) than control quail. No statistical differences were observed for carcass weight (P = 0.154).

Regarding the chemical composition of meat (Table 2), no statistical differences were observed between the two experimental groups for moisture (P = 0.659), crude protein (P = 0.358), ethereal extract (P = 0.847) and ash (P = 0.524), while statistical difference was observed for α-tocopherol level (P < 0.05), showing higher values in meat from quails supplemented with bergamot pulp compared to meat from control quails.

Data on fatty acid composition of meat is shown in Table 3. Intramuscular fat (IMF) content was not affected (P = 0.349) by diet. As regards the individual fatty acids, the control group showed higher values of C20:2 ω-6 (P = 0.030) and C20:3 ω-6 (P = 0.025) compared to the BP10 group. Furthermore, meat from control group showed a tendentially higher values of arachidonic acid (C20:4 ω-6, P = 0.08) and of the ω-6/ω-3 ratio (P = 0.097) than meat from BP10 group. Conversely, the supplementation of bergamot pulp tended to increase the C20:5 ω-3 (EPA, P = 0.08) and the total of ω-3 fatty acids (P = 0.08) compared to the control treatment.

Finally, no statistical differences were found between experimental groups (P > 0.05) regarding the total saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids.

Data on oxidative stability (TBARS) of meat is shown in Table 4. The TBARS values increased over storage time in raw meat (P < 0.001), while experimental diets did not affect the lipid oxidation (P > 0.05).

Regarding color descriptor in meat (Table 4), the lightness (L*) and redness (a*) descriptor were significantly affected by the diet (P = 0.001), with values of 45.8 vs 51.2 for L* and 14.6 vs 12 for a* in control and BP10 groups respectively, while storage had no significant effect. In contrast, the yellowness index (b*) was influenced by both diet and time (P = 0.001), with values that increase constantly over time and higher in BP10 group than in control one. Finally, meat saturation (C*) was affected only by the time of storage (P < 0.001), with values increasing from 0 to 3 days and stabilizing thereafter, whereas hue angle (H*) increased along the storage time (P < 0.01), accompanied by the influence of dietary treatment (P < 0.01).

4. Discussion

The aim of the present trial was to evaluate the effects of dietary inclusion of dried bergamot pulp on growth performance and meat quality in Japanese quail. The incorporation of bergamot pulp into the diet resulted in significant differences in performance parameters among the experimental groups. In particular, supplementation with 10% dried bergamot pulp over a period of approximately three weeks led to a reduction in ADG. This outcome may be attributed to the lower DMI observed in the BP10 group, likely associated with reduced diet palatability. Bergamot pulp is characterized by a high concentration of flavonoids [27], compounds commonly associated with a bitter taste [28], and birds are known to be highly sensitive to bitter substances [29]. This may have negatively influenced feed intake. Comparable results were reported by Goliomytis et al. [30], who found that dietary supplementation with orange pulp in laying hens increased feed conversion ratio (FCR) and decreased DMI compared to control. Similarly, Ojabo et al. [31] observed reduced DMI in pullet chicks fed sun-dried sweet orange peel, whereas Florou-Paneri et al. [32] reported no significant effect on body weight in quails during egg production when diets were supplemented with 6% citrus pulp.

In addition to flavonoids, bergamot pulp contains other compounds such as oxalates, saponins, tannins, and phytates, also present in other citrus fruits including mandarin, lemon, and orange, which may further impair palatability and consequently reduce DMI [33]. Conversely, supplementation with citrus essential oils has been associated with increased body weight and DMI in Japanese quail [34], highlighting differences in the effects of citrus-derived products depending on their chemical composition.

Other studies investigating the use of citrus pulp in chick diets [35] reported an increase in dietary fiber content, particularly non-starch polysaccharides (NSP). These compounds promote morphological adaptations of the gastrointestinal tract, such as an increased length of the small intestine. The NSP are poorly degraded in the small intestine, which may prolong digesta retention time and reduce overall feed intake [36]. Gastrointestinal tract expansion, including increased intestinal length, represents an adaptive response to the higher fiber content of the diet, but may also affect nutrient absorption efficiency [37]. Furthermore, bergamot peel is known to be rich in pectins, compounds with well-documented negative effects on poultry growth performance [38,40]. Patel et al. [38] and Bishawi and McGinnis [39] suggested that these effects are related to the viscous properties of pectins, which can exert antinutritional effects by impairing nutrient utilization.

Partial replacement of maize with dried bergamot pulp (BP) did not induce marked changes in the fatty acid composition of quail meat. Although the diet of the BP10 group contained higher levels of α-linolenic acid, no significant differences were detected for this fatty acid between the two experimental groups, whereas the total ω-3 fatty acid (FA) was only tendentially slightly higher in the BP10 group compared with the control group. Different results were reported by Ciftci [41] when quail diets were supplemented with orange peel extracts. In that study, a significant increase in total ω-3 fatty acid and a significant reduction in the ω-6/ω-3 ratio was observed in the meat of quails fed diets containing the extracts. Significantly lower ω-6/ω-3 ratios were reported also in lambs fed fresh bergamot pulp [12], with values closer to the Department of Health recommendation of ≤4 [42]. However, the results obtained by Scerra et al. [12] are not fully comparable to those observed in the present study, both in terms of inclusion levels (35% vs. 10%) and, most importantly, because the studies involve different animal species. Among the long chain ω-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (C20:5 ω-3) and docosahexaenoic acid (C22:6 ω-3) are the most relevant due to its recognized health benefits [43,44]. In the present study, no differences were observed for docosahexaenoic acid (C22:6 ω-3), whereas for eicosapentaenoic acid (C20:5 ω-3) only a slight increasing trend was observed in the meat from BP10 group compared to the control group. Higher levels of these fatty acids have been reported by Çiftci et al. [41] who, in addition to observing increased ω-3 FA deposition in breast muscle lipids and a reduced ω-6/ω-3 ratio, found higher levels of desaturation and elongation products of α-linolenic acid (e.g., C20:5 ω-3) in quails supplemented with orange peel extract. Çiftci et al. [45] also demonstrated that supplementation with a mixture of essential oils (thyme, orange peel, bay leaf, and eucalyptus) improved ω-3 FA levels and reduced the ω-6/ω-3 ratio in quails. In layer hens, dietary inclusion of bergamot oil was likewise shown to significantly increase C20:5 ω-3, docosahexaenoic acid (C22:6 ω-3), and total ω-3 FA concentrations, while reducing the ω-6/ω-3 ratio in egg yolk [46]. These differences could be attributed to the longer duration of the experimental trial conducted by Ciftci et al. [41,45] (7 days more), as well as to substantial differences between the citrus-derived products used in the experiments, orange peel extracts in Ciftci et al. [41], bergamot oil [46], versus dried bergamot pulp in the present study. As regards the ω-6 FA fatty acids, the higher values of C20:2 ω-6, C20:3 ω-6 and tendentially higher value of C20:4 ω-6 observed in control group compared to the BP10 group should be related to the higher level of C18:2 ω-6 in control diet than in BP10 diet.

Lipid oxidation during storage is influenced by several factors, including dietary treatment. It is well established that meat enriched in PUFAs is more susceptible to oxidative deterioration [47,48], a susceptibility that increases with the number of double bonds, whereas the presence of antioxidants such as vitamin E and polyphenols exerts protective effects [49]. In the present trial, no significant differences in TBARS values were observed between treatments. This finding could be attributed to the comparable PUFA levels between the experimental groups, and thus a similar level of highly peroxidizable compounds. It is noteworthy that vitamin E levels in the meat were significantly higher in the BP10 group compared to the control group. Dried bergamot pulp supplementation increased α-tocopherol levels in the diet, which was reflected in the meat. Additionally, the elevated polyphenolic content of the BP10 diet may have indirectly preserved vitamin E during digestion due to its antioxidant activity, thereby enhancing the oxidative stability of the meat [50]. Citrus flavonoids are polyphenolic secondary metabolites produced by plants and are recognized for a range of biological activities, most notably their antioxidant properties [51]. Bergamot fruit are particularly rich in flavonoids, with naringin and neohesperidin being the most abundant [52]. While also considering the potential antioxidant effect exerted by vitamin E, several authors [53] have reported that even low concentrations of α-tocopherol in meat can exert protective effects against lipid oxidation, at levels lower than those provided by control diet in the present trial. Although malondialdehyde (MDA) concentrations were increased significantly after 7 days of storage in both experiment group, the values remained below the recommended threshold of 2 mg MDA/kg of meat [54]. It has been suggested that the influence of dietary treatments on oxidative stability becomes more pronounced when samples are subjected to stronger oxidative stress [55]. In this regard, TBARS analysis of cooked meat might have revealed clearer differences, although such evaluations could not be performed under the present experimental conditions. Comparable observations were reported by Scerra et al. [14] in pork meat from animals supplemented with ensiled bergamot pulp.

Colorimetric analysis revealed that dietary supplementation with bergamot pulp increased both lightness (L*) and yellowness (b*) values compared with the control group, likely due to the deposition of pigments such as carotenoids in muscle tissue. The yellow hue, in particular, may be attributed to xanthophyll accumulation [56,57]. Previous studies have similarly indicated that increases in these parameters, especially lightness, can be linked to higher polyphenol intake, as demonstrated in trials employing polyphenol-rich by-products in lambs [50,58]. With regard to redness (a*), in agreement with earlier findings [35], values were significantly lower in the BP10 group than in the control group.

This study reports the first data on the use of dried bergamot pulp in quail nutrition. However, the results are limited to a 10% inclusion rate over a 3-week feeding period, showing a reduction in growth performance. In order to determine the more appropriate levels of inclusion for improved growth performance and to assess the effects of longer feeding periods, it will be necessary to conduct further studies.

5. Conclusions

Partial replacement of maize with dried bergamot pulp in the quail diet resulted in a tendency toward higher ω-3 fatty acid deposition in meat, leading to an approximate 35% reduction in the ω-6/ω-3 ratio. Colorimetric analysis showed increased lightness (L*) and yellowness (b*) values in the BP10 group compared with controls, likely due to the deposition of carotenoid pigments in muscle tissue. In the present trial, no significant differences in TBARS values were observed between treatments. This finding could be attributed to the comparable PUFA levels between the experimental groups, and thus a similar level of highly peroxidizable compounds. However, dietary supplementation with 10% dried bergamot pulp reduced dry matter intake (DMI) and consequently average daily gain (ADG), likely as a result of lower diet palatability. The present findings are limited to a single inclusion level (10%) evaluated over a 3-week feeding period, during which a reduction in growth performance was observed. To identify the most appropriate inclusion rates capable of supporting improved growth performance, and to elucidate the effects of extended feeding durations, further investigations are warranted.