Grape By-Products as Feedstuff for Pig and Poultry Production

Simple Summary Grape is one of the most produced fruits worldwide for juice and winery industries. Grape by-products include grape pomace, grape seed, and grape-seed oil, and are valuable, although underexplored, ingredients for pig and poultry feeding. Indeed, they are rich in fiber and bioactive phenolic compounds, which makes them promising sources to partially replace conventional and unsustainable feedstuffs. However, grape by-products are mostly discarded or misused (e.g., landfills) with a negative environmental impact. The present review focuses on the effects of grape by-products on poultry and pig production. Overall, these dietary sources could improve piglet growth when added up to 9% feed, conversely to poultry where this result was only obtained using by-products up to 3%. The beneficial effect on animal growth performance is caused by the presence of nutritional and bioactive compounds with consequent enhancement of intestinal health. The incorporation of high levels of grape by-products in poultry diets can impair growth performance due to the presence of anti-nutritional compounds. Therefore, the use of processes, such as enzymatic supplementation and pre-treatments, to degrade or inhibit these compounds, should be further explored to allow grape by-products to be used as feed ingredients for monogastric animals. Abstract Grape by-products are exceptional options for replacement of conventional and unsustainable feed sources, since large amounts are generated every year from the winery industry. However, the majority is wasted with severe environmental and economic consequences. The present review aimed to evaluate the effects of grape by-products on pig and poultry growth performance. The most recent literature was reviewed using ScienceDirect and PubMed databases and the results of a total of 16 and 38 papers for pigs and poultry, respectively, were assessed. Fewer studies are documented for pig, but the incorporation of grape by-products up to 9% feed led to an improvement in growth performance with an increase in average daily gain. Conversely, lower levels (<3% feed) are needed to achieve these results in poultry. The beneficial effects of grape by-products on animal performance are mainly due to their antioxidant, antimicrobial, and gut morphology modulator properties, but their high level of cell wall lignification and content of polyphenolic compounds (e.g., tannin) limits nutrient digestion and absorption by monogastric animals. The use of exogenous enzymes or mechanical/chemical processes can provide additional nutritional value to these products by improving nutrient bioavailability. Overall, the valorization of grape by-products is imperative to use them as feed alternatives and intestinal health promoters, thereby contributing to boost circular agricultural economy.


Introduction
The steady growth of the human population in the last decades has increased the demand for animal products worldwide, including the most consumed meats (i.e., poultry and pork) and eggs [1]. Feed supply contributes to the highest percentage of total livestock production costs [2]. Therefore, finding sustainable and economically viable alternatives to the presence of heavy metals in the whole grape pomace [27,28], with values (mg/kg DM) averaging 111 for Al [28], 16.3 for Pb, 3.5 for Ni, and 1.1 for Cd [27]. Indeed, most reports analyzed the accumulation of trace elements in grape stem [27,29], skin [30][31][32], or seed [26,32]. There is a great variability in heavy metal contents in grape by-products, since they depend on soil composition and contamination and on the grape genetic variety. For instance, 8.1 mg/kg DM of Al and 0.002 mg/kg DM of Pb was found in grape skin in the study from De Nisco et al. [30], but other grape varieties could accumulate 158-575 mg/kg DM of Pb in their skins [32]. However, in grape stem and seed, the predominant trace element was Al (average of 201 and 0.78 mg/kg DM, respectively) ( Table 2). Although high levels of these trace elements can be harmful to animals with potential carcinogenic effects [33] and kidney, nervous, and immune system toxicity [34], there is a lack of reference values for the evaluation of toxic effects of grape by-products used as feedstuffs [11]. Nevertheless, As, Cd, and Pb should not be higher than 0.5, 0.5, and 0.2 mg/kg in food ingredients [35], and, thus, Pb values, which can reach up to 26.2 mg/kg in grape stem, should be monitored. The minimization of anthropogenic sources of trace elements [25], selection of grape varieties [29], and application of bioremediation techniques to remove heavy metals from the soil [36], are possible solutions to face the concerning issues.
The potential value of by-products in animal feeding depends essentially on their nutritive properties, such as fibrous, protein, and organic-matter digestibility and energy value. Although grape pomace, grape seeds, and grape-seed oil contain beneficial compounds for monogastric animals' metabolism and growth, such as essential fatty acids and antioxidant phytosterols and tocopherols (vitamin E) ( Table 2), these by-products are also composed of anti-nutritional compounds. Indeed, they contain a high amount of fiber and procyanidins (i.e., condensed tannins) and, in a lower quantity, phytic acid [2]. The highest proportions of dietary fiber (74% wt.), mostly composed of hemicelluloses and covered in a whitish bloom (a dusting of wild yeasts and bacteria) [37], and polyphenols (i.e., tannins) [38] are located in the grape skin. However, grape stem is woody and fully composed of tannins, containing more than 50% of total polysaccharides and, thus, representing an economically attractive source of fiber material [39]. However, depending on the dose and treatment of grape by-products included in animal feed, dietary fiber and polyphenolic compounds can maintain or improve pig [40][41][42] or poultry [2] growth performance and health. In fact, fiber increases intestinal peristalsis and acts as a buffer and a prebiotic that stimulates the development of beneficial bacteria in the digestive tract [2], whereas polyphenols can act as antioxidants, antimicrobials, and immunomodulators [20,[40][41][42][43][44][45].
Enzyme supplementation or pre-treatment methods, such as fermentation [2], polyethylene glycol treatment [46,47], steam explosion, and amination are possible solutions to release non-starch polysaccharides or linked tannins from grape by-product cell walls and biomass, thereby increasing their digestibility and bioactive properties [2]. Moreover, phytases might be used to hydrolyze phytate and, thus, release organically bound phosphorus that is unavailable for digestion and absorption by monogastric animals [48]. These treatments could increase the use of grape by-products in animal diets with concomitant benefits on animal production and health, reduction in the overexploitation of conventional feedstuffs, feed costs, and environmental impact derived from the discard or incineration of by-products.
The enzyme supplementation of grape by-products is scarcely studied and has controversial results, since the outcome depends on the by-product dose [2]. For instance, the ability of a carbohydrase complex, and especially tannase, to degrade polymeric procyanidin structures into monomeric and dimeric residues of catechin when acting on 5% and 10% of grape pomace was demonstrated, but the antimicrobial effect of these phenolic compounds against Clostridium perfringens in the intestine of broiler chickens was only present with 5% of substrate [49]. However, the activity of exogenous enzymes in the context of pre-treatments, such as fermentation, or in combination with polyethylene glycol, has been applied more and was shown to increase the antioxidant and antimicrobial bioactivities of grape compounds and hinder anti-nutritional effects [2]. Particularly, the fermentation Animals 2022, 12, 2239 5 of 18 process can increase the amount or effectiveness of bioactive polysaccharides, polyphenols, or mannoproteins [2]. Grape seed fermented with Aspergillus niger, which produces several enzymes such as amylase, protease, xylanase, cellulase, and lipase, was shown to modulate intestinal microbiota of broiler chickens causing an increase in beneficial Lactobacillus and a decrease in Staphylococcus aureus [20]. This process was also efficient in the bioconversion of grape pomace, stimulating both antioxidant (increase in serum catalase level) and microbial modulation (decrease in C. perfringens) effects of grape-derived compounds [44]. Moreover, the pre-treatment of 10% red grape pomace with polyethylene glycol, which is a strong tannin-binding agent [50], and a cellulolytic enzyme mixture was shown to ameliorate the anti-nutritional effects of condensed tannins [46]. In addition, Van Niekerk and Mnisi [47] reported a partial inactivation of grape pomace condensed tannins with polyethylene glycol, without affecting the health status of broiler chickens. Concerning the hydrolysis of phytate phosphorus, few studies evaluated the activity of phytase in grape by-products, whether using enzymes naturally present in the substrate [48] or biosynthesized by A. niger during a fermentation process [51]. This might be due to the residual amounts of phytic acid usually present in the by-products [48,52] and an inhibition of fermentation by phenolic compounds, such as resveratrol and anthocyanins, with anti-fungal activities [52].
The amination nd steam explosion treatments have not been exploited for grape byproducts, although they are known to increase the digestibility of fibrous cell walls in highfiber feedstuffs [2]. Amination breaks down hemicellulose and lignocellulose and enhances available nitrogen and soluble sugar contents by using chemical compounds (e.g., ammonia), whereas steam explosion is also effective as a lignocellulose material treatment through the application of high temperature and pressure, but it does not increase nitrogen content [53,54]. Even though steam explosion has more advantages than amination, since it is cost-effective, does not use chemicals, and has low energy expenditure [2], both methods could be used for delignification of cell walls in grape pomace, solving the problem of the high level of lignification of this by-product that compromises monogastric animals' digestibility.  [75]. 3 Includes minor amounts of asparagine. 4 Includes minor amounts of glutamine. 5 n.a., not available.   [82]. 4 Values were detected in grape stem. 5 Values were detected in grape skin. 6 More than 99.5% of the total phenols are flavonoids. 7 Values are expressed as mg GAE/g. 8 Values are expressed as mg epicatechin equivalents/g. 9 Values are expressed as mg TAE/g.

Effect of Dietary Grape By-Products on Production Performance of Monogastric Animals
The literature review on the influence of dietary grape by-products on growth performance parameters of monogastric species is presented in Tables 3 and 4. The studies herein presented used grape by-products at up to 10% feed with variable effects for pigs and poultry. In general, although different concentrations and experimental periods were reported, dietary grape by-products did not impair or even improved growth performance with an increase in average daily gain (ADG) in pigs, with positive results for poultry when added in low amounts (<3% feed) in the diet (Figure 1). The effects on animal growth performance were mostly attributed to the bioactive properties of grape by-products compounds (e.g., polyphenols), which include prevention of oxidative stress, as well as immune, microbiota, and gut morphology modulation, in pigs [40][41][42]83] and poultry [20,[43][44][45]. For instance, Hao et al. [40] and Fang et al. [41] showed that procyanidins added at up to 1.5 and 1% in piglet diets increased glutathione peroxidase and superoxide dismutase activities in the serum and liver, respectively. Fang et al. [41] reported an increase in serum immunoglobulins, interleukins, and complements caused by these compounds. In addition, polyphenols could improve the disease resistance in piglets by enhancing the proportion of beneficial intestinal bacteria (e.g., Lactobacillus sp., Olsenella sp., Selenomonas sp.) [42] and decreasing the incidence of diarrhea [40,41]. The bioactive compounds were also responsible for an increase in intestinal villus height/crypt depth ratio when grape pomace was fed at 5% to piglets [42]. This bioactivity might explain the increase in average daily gain (ADG) and decrease in feed conversion ratio (FCR) [41] or the ameliorated effects [40,42] on growth performance in piglets fed grape by-products. ratio when grape pomace was fed at 5% to piglets [42]. This bioactivity might explain the increase in average daily gain (ADG) and decrease in feed conversion ratio (FCR) [41] or the ameliorated effects [40,42] on growth performance in piglets fed grape by-products.
Similar bioactivities of grape compounds were detected in poultry, but the outcomes were shown to be dependent on by-product dose, treatment, and type. Indeed, 1.5% of grape pomace fed to broiler chickens increased serum glutathione peroxidase and superoxide dismutase levels, had no effect on intestinal bacteria count, and enhanced ileum lamina thickness. On the other hand, 1.5% of fermented grape pomace increased serum catalase, decreased cecal C. perfringens, and did not change ileal morphology [44]. Furthermore, Viveros et al. [43] reported an increase in ileal Lactobacillus with 0.72% of grape-seed extract, and an increment in Enterococcus and decrease in Clostridium with grape-seed extract and 6% of grape pomace fed to broilers. Grape pomace increased intestinal villus height/crypt depth ratio, but an opposite effect occurred when feeding grape-seed extract. Additionally, Erinle et al. [45] found an increase in beneficial Lactobacillus and improvement in gut morphology in broilers fed 2.5% of grape pomace. Overall, the bioactivity of grape compounds was variably associated with an increase in final body weight [44] and ADG [20] and decrease in FCR [43].

Pigs
The effect of dietary incorporation of grape by-products on pig growth performance is mostly dependent on animal growth stage and by-product dose on a dry basis. In general, lower concentrations of grape by-products had fewer effects on growth performance, although depending on the concentrations applied and on the age of the animal. For instance, considering animals with similar weights (4.8-19.3 kg), pigs fed 9% of grape pomace had an increase in ADG during all experiments, while those fed 5% suffered no change in ADG (Table 3). The piglets fed 3% of the by-product improved their growth performance only during the growing stage (36-70 days) possibly due to increased digestibility [84]. Beside the differences in trial conditions, the pre-treatment of grape pomace should be considered, since the by-product used in the study by Yan and Kim [84] was submitted to a fermentation process, and, thus, contained a high phenolic content (62.1 g/mg). Moreover, Kafantaris et al. [85] observed that 9% of grape pomace fed to piglets for 30 days increased ADG, without affecting average daily feed intake (ADFI) and FCR. In growingfinishing pigs, Trombetta et al. [86] reported no effects on ADG and carcass traits with 3.5 Similar bioactivities of grape compounds were detected in poultry, but the outcomes were shown to be dependent on by-product dose, treatment, and type. Indeed, 1.5% of grape pomace fed to broiler chickens increased serum glutathione peroxidase and superoxide dismutase levels, had no effect on intestinal bacteria count, and enhanced ileum lamina thickness. On the other hand, 1.5% of fermented grape pomace increased serum catalase, decreased cecal C. perfringens, and did not change ileal morphology [44]. Furthermore, Viveros et al. [43] reported an increase in ileal Lactobacillus with 0.72% of grape-seed extract, and an increment in Enterococcus and decrease in Clostridium with grapeseed extract and 6% of grape pomace fed to broilers. Grape pomace increased intestinal villus height/crypt depth ratio, but an opposite effect occurred when feeding grape-seed extract. Additionally, Erinle et al. [45] found an increase in beneficial Lactobacillus and improvement in gut morphology in broilers fed 2.5% of grape pomace. Overall, the bioactivity of grape compounds was variably associated with an increase in final body weight [44] and ADG [20] and decrease in FCR [43].

Pigs
The effect of dietary incorporation of grape by-products on pig growth performance is mostly dependent on animal growth stage and by-product dose on a dry basis. In general, lower concentrations of grape by-products had fewer effects on growth performance, although depending on the concentrations applied and on the age of the animal. For instance, considering animals with similar weights (4.8-19.3 kg), pigs fed 9% of grape pomace had an increase in ADG during all experiments, while those fed 5% suffered no change in ADG ( Table 3). The piglets fed 3% of the by-product improved their growth performance only during the growing stage (36-70 days) possibly due to increased digestibility [84]. Beside the differences in trial conditions, the pre-treatment of grape pomace should be considered, since the by-product used in the study by Yan and Kim [84] was submitted to a fermentation process, and, thus, contained a high phenolic content (62.1 g/mg). Moreover, Kafantaris et al. [85] observed that 9% of grape pomace fed to piglets for 30 days increased ADG, without affecting average daily feed intake (ADFI) and FCR. In growing-finishing pigs, Trombetta et al. [86] reported no effects on ADG and carcass traits with 3.5 and 7% of grape pomace. Additionally, the supplementation of 5% of flax meal and 1% of grape seeds did not affect ADG and FCR, although increased ADFI [66]. A lack of a significant effect of grape-seed extract on these parameters was also observed when piglets were fed Animals 2022, 12, 2239 9 of 18 0.015% of the by-product [83]. However, a high dose of grape by-product might not always influence growth performance in pigs, since piglets born from sows fed with grape pomace (25% feed) and fed with the same ration before weaning, showed no difference in ADG and FCR [87]. Even though the results obtained across studies are variable, the increase in ADG demonstrated in some reports should be underlined, since it is a promising effect for animal production. ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

Poultry
Poultry feeding with grape by-products showed both dosage-and form-dependent effects, and, thus, they are normally incorporated at up to 6-10% feed (dry basis) in the diet [2]. Indeed, the inclusion of high levels of grape by-products is normally associated with a considerable amount of anti-nutritional compounds, such as fiber and polymeric polyphenols (e.g., proanthocyanidins), which can reduce the digestion and absorption of nutrients, and ultimately impact negatively on body weight gain. On the other hand, low levels of by-products (<6% feed) have been related to beneficial bioactive effects, such as modulation of gut morphology and microbiota and antioxidant activity, mostly due to the presence of polyphenols [2]. For instance, feeding broilers with 2.5% of grape pomace increased the relative abundance of beneficial Bacteroides and Lactobacillus genera and reduced the Firmicutes to Bacteroidetes ratio in the cecum [45]. Erinle et al. [45] also demonstrated an increase in intestinal villus height: crypt depth ratio caused by the grape by-product. In addition, Abu Hafsa and Ibrahim [96] reported that 1-4% of grape seeds increased Lactobacillus and decreased detrimental Streptococcus spp. and Escherichia coli in the ileum of broilers. Similarly, Viveros et al. [43] found an increase in Lactobacillus sp. in the ileal contents of broiler chicks fed 0.72% of grape-seed extract. Dietary grape seeds at up to 4% can help prevent oxidative stress by increasing the activity of several enzymes with antioxidant activity in the plasma, such as superoxide dismutase and glutathione peroxidase, and, at the same time, reduce thiobarbituric-acid-reactive substances [96]. Overall, the bioactivity of grape by-products contributes to the integrity of intestinal barrier function and prevention of diseases in the gut, thereby enhancing poultry growth [2].
The inclusion of grape pomace at 6% in broiler chicken diets showed no effects [97] or a slight improvement [43] on growth performance. However, a negative impact on performance was observed when the same dose of grape-seed extract [43] or unfermented grape skin [38] was used, which was due to the presence of polyphenols, mostly purified in the case of grape seed. Feeding broilers with a lower dose grape seed (4%), which corresponds to 11.14 g polyphenols/kg feed [96], could still impair growth performance by decreasing final body weight and ADG and increasing FCR. Similarly, a linear increase in FCR over the growing and finishing periods of ducklings was observed with 0.01 and 0.02% of grape-seed extract providing 0.005-0.015 g polyphenols/kg feed, although an increase in final body weight and ADG was reported [98].
For a level of by-products higher than 6%, only Kumanda et al. [99] reported beneficial effects on broiler growth performance with a decrease in FCR when incorporating 7.5% of grape pomace in the diet. Other studies showed either no effect [59,100] or an impairment (decrease in final body weight, ADG, and hot carcass weight) [46] of animal performance with 10% grape pomace.
On the other hand, studies showed that the inclusion of grape seed or grape pomace at up to 3% feed (dry basis) had a positive impact on animal growth performance. Indeed, 0.5% of fermented or unfermented grape seed fed to broilers increased their final body weight and ADG [20] and even grape-seed extract at 2% feed led to similar results with also a reduction in FCR [96]. In addition, 1.5% of fermented grape pomace could increase the final body weight of broilers [44], whereas 2.5% of grape pomace raised ADG in the first two weeks of the trial, even though no effect was observed in the overall period [45].
The fermentation of grape by-products was reported to hinder the negative effects on growth performance caused by the presence of anti-nutritional compounds [2]. However, this occurrence was mostly shown for low levels of by-products in broiler diets. For instance, Nardoia et al. [38] observed that the pre-treatment reverted the increase in FCR found with unfermented grape skin at 3%. Moreover, Gungor et al. [44] reported a beneficial effect on animal growth with the fermentation of 1.5% grape pomace, while the same was not found with the unfermented source.   ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

Conclusions and Future Perspectives
Grape by-products have several industrial applications, including feed, biofuel, bioenergy, and fertilization/They are valuable feedstuffs due to their richness in nutritional and bioactive compounds, such as dietary fiber and polyphenols, which make them suitable for maintaining or improving animal performance. Indeed, conversely to the anti-nutritional properties found with high amounts of these compounds, low amounts were shown to modulate intestinal morphology and microbiota and stimulate antioxidant capacity, thereby maintaining intestinal health and preventing the occurrence of diseases in monogastric animals. Grape pomace can improve growth performance in pigs, with an increase in ADG, particularly when fed at higher levels (up to 9%). However, in poultry, the effect of grape by-products is more variable, and these sources should not be incorporated in broiler diets at more than 6-10% feed to prevent an impairment of animal growth performance. Forthcoming studies should concentrate their efforts on the optimization of dosages and digestibility of grape by-products for pig and poultry feeding.