1. Introduction
Proteins are essential for a balanced diet, and given the significant rise of the global population in the near future, much larger quantities of food-grade protein will be needed [
1]. Foods of animal origin are needed for global food security, since they comprise 25% of the protein intake worldwide [
1]. Protein feed stocks are expensive and limited in animals’ ratios. Currently, soybean meal, after soy oil extraction, is the main source of vegetable protein for animal feed [
2]. In particular, approximately 97% of the globally produced soybean meal is used as animal feed [
3]. However, feeding animals with soybean proteins and other grains increases the dependence of animal production on human-edible plants, negatively affecting human food security and sustainability [
4].
Therefore, there is an urgent need to use alternative, more sustainable protein feed sources to partly replace the current supply chains and reduce the environmental footprint of animal products [
2]. Moreover, such a necessity arises from the fact that traditional sources of protein for animal feed compete with human food for the use of high-quality land for agricultural uses. Thus, alternative animal feed proteins, such as protein-rich food industry by-products, macro-algae, insect meal, and single cell proteins (SCPs), have a high potential as sustainable protein sources, since they have a low footprint, and can be produced locally on low-quality agricultural land. [
5].
YLP is an oleaginous, non-pathogenic yeast, which can be utilized as a SCP in animal feed [
6]. Among others, this is due to its capability to produce considerable levels of proteins and lipids from low cost substrates [
7]. In addition, these strains can grow in biofuel-produced by-products, such as glycerol, to produce a yeast biomass rich in proteins and/or lipids [
8]. The annual production of SCPs reaches 1200 tons of dry mass, that contains approximately between 41% and 45%
w/
w of protein [
9]. Available research evidence shows that the YLP biomass has a high nutritional value. It is a source of high quality proteins (especially essential amino acids), minerals, vitamins and polyunsaturated fatty acids [
8,
10]. Currently, the American Food and Drug Administration, has examined the safety issues of YLP yeast, and has characterized both the yeast and its various fermentation products as being generally recognized as safe (GRAS) [
11]. Moreover, a YLP strain grown on raw glycerol has been considered safe for use as a high value foodstuff (EU 2017/1017) [
12]. Finally, the European Food and Safety Authority (EFSA) declared the YLP yeast biomass as a novel food (NF) safe for use, pursuant to Regulation (EU) 2015/2283 [
13] on dietary supplements intended for the general population over three years of age [
14].
Previous investigations using YLP as a feed supplement for turkeys [
15,
16,
17,
18,
19,
20], piglets [
8,
21,
22,
23], calves [
24], rats [
25], fish [
26,
27,
28,
29], crustaceans [
30,
31] and mollusks [
32,
33] have been reported, giving promising results, in terms of their health and performance. More specifically, the incorporation of YLP in the diet of productive animals has been shown to beneficially affect: (1) weight gain, (2) feed conversion ratio, (3) intestinal and ruminal microbiomes, (4) intestinal morphology, (5) antioxidant status and immune response, (6) erythropoietic processes and hematobiochemical profiles, (7) survival rate, (8) digestibility, (9) pathogen elimination (10) the fatty acid composition of fish fillets and (11) the metabolic status of the organism [
34]. However, to our knowledge, there are no indications in the available literature regarding the dietary effect of YLP yeast in broilers’ growth performance or meat quality. Moreover, no data are available on whether the use of feed with YLP yeast affects the welfare and behavior of productive animals, in general, and broiler chickens, in particular.
This study aims to assess the dietary effect of YLP yeast on broilers’ growth performance and meat quality, as well as certain welfare and quality behavior indicators of broilers. Moreover, different inclusion rates of YLP were tested to specify the optimal inclusion rate of YLP in chickens’ feed.
4. Discussion
This is the first report that investigates the potential use of YLP yeast in the diet of broilers. Relative previous studies in birds have been carried out only in turkey hens, aged 1–16 weeks old [
15,
16,
17,
18,
19,
20]. According to our results, the incorporation of YLP did not affect the overall growth performance of chicks, as indicated by the final BW at 41 days-old, and the BWG, FC and FCR during the whole experimental period (13–41 days of age). Only some differences in the BW, FC and FCR of broilers were recorded at weekly intervals among groups. The higher BW of the YLP3 chicks at 20 days of age, compared to the other two groups, could be attributed to the numerical increase of the FC and a net numerical increase of the BWG of birds in this group during the growing period. Moreover, the reduced FC recorded in the YLP groups, compared to the CON group, between 21 and 27 days of age, could be due to an adaption of the birds to the finisher 1 diet. Finally, the observed differences in the feed efficiency between the CON and YLP3 groups during the growing period is the net result of the numerical differences in the FC and BWG seen between the groups during this time period. Similar to our findings, previous reports demonstrated that the supplementation of 3% YLP in turkeys’ diet had no impact in the final BW, BWG, FC [
17,
19] and FCR [
17]. However, Czech et al. [
19] noted an amelioration of the FCR in YLP3-fed birds, compared to the controls. Moreover, Merska et al. [
17] found that increasing the incorporation rate of YLP to turkeys’ feed from 3% to 6%, resulted in a lower BW of birds, compared to the CON group and those fed with 3% YLP diets. The higher percentage of CON chicks that were recorded feeding at 20days of age, in comparison to that observed in the YLP groups is probably not related to the YLP supplementation. This is consistent with the non-significant difference in food consumption during the growing phase of birds of all groups. Thus, given the instant character of the quality behavior traits recording procedure, this finding could be considered random.
The incorporation of YLP in broilers’ diet at both studied levels (3% and 5%), had a positive effect on FPD, as evaluated at 41 days of age. This finding is considered of critical importance for chickens’ health and welfare, but also for the farmers’ financial income. FPD is a multifactorial problem with litter quality, nutrition and gut health as some of the factors implicated in its incidence [
57,
58,
59]. However, litter condition is considered the most important risk factor for the development of FPD [
60]. The litter moisture and ammonia concentration from accumulated fecal material can burn and weaken the dermis of the footpad [
61], with an increased severity of FPD resulting from the prolonged exposure of feet to wet litter. Moisture causes the outer layer of the dermis to soften, posing a risk of microbial contamination, leading to necrosis [
62]. FPD is a very common and well recognized problem in the broiler industry [
58] that negatively affects birds’ productivity and welfare [
57] and it has been associated with a reduced mobility, lameness and consequently with behavioral restrictions of birds [
59,
63]. FPD has also been shown to be highly associated with systemic bacterial infections, as pathogens can enter chickens’ bodies through the damaged epithelium in the foot pads [
64]. Furthermore, financial losses due to FPD are basically attributed to the abattoir condemnation of carcasses with contact dermatitis lesions [
58]. Chicken legs is a highly profitable industrial by-product, and poor footpad conditions due to FPD degrade the product quality, leading to rejections and loss of income [
59].
The exact mechanism involved in the positive nutritional effect of YLP on FPD observed in this study is presently unknown and requires further investigation as there are no similar studies in the available literature. Two possible explanations could be given for the interpretation of this result. According to the first one, it is possible that YLP ameliorated the gut health of YLP birds, resulting in a reduction of their fecal moisture and consequently reduced litter moisture, which is one of the major causative factors of FPD. The beneficial effect of YLP in intestinal function and gut microbiota has been previously demonstrated in turkey hens [
19,
20]. The addition of 3% YLP in turkeys’ diet increased the intestinal villus length, as well as the ratio of the villus length to the crypt depth and the intestinal muscular layer thickness, thus improving the birds’ intestinal health [
19]. In another study it was proved that feeding turkey hens with 3% YLP favorably influenced the intestinal microbiota, since it reduced the number of fungi and coliforms, including
Escherichia coli [
20]. In similar studies performed on growing piglets, the incorporation of YLP in the piglets’ diet decreased the total number of coliform bacteria and
Escherichia coli in their enteric contents [
22]. The beneficial dietary effect of YLP on gut health has been linked to the presence of β-glucans in the yeast cell wall which protects the gastrointestinal tract against the colonization by dangerous pathogens, such us
Salmonella enterica [
20]. In support of this, previous studies have demonstrated that β-glucans and mannans, the two major components of the yeast cell wall, are bioactive components with potential benefits for the development of the intestinal immune system in animals [
23]. They mitigate the release of pro-inflammatory cytokines and prevent the colonization of pathogens in intestinal mucosa.
According to a second explanation, the higher percentage of birds with no evidence of FPD recorded in the YLP groups, compared to the CON group, could also be due to the beneficial properties of the bioactive compounds of YLP for healthy skin, such as polyunsaturated fatty acids (PUFAs). YLP used in the present study consisted of approximately 39.7% PUFA, mainly linoleic acid (35.2%). It has been previously demonstrated that oils containing high levels of essential fatty acids improve skin hydration, regenerate the damaged epidermal lipid barrier and regulate skin metabolism [
65]. Moreover, omega-3 and omega-6 FAs are significant components of the cell membrane, necessary for the function of the epidermal barrier; they display anti-inflammatory and anti-allergic effects by enhancing the repair processes and soothing irritation [
65].
In the present study, the broilers of all experimental groups were evaluated with very good scores for hock burn, indicating no evidence of such a welfare issue. Regarding the feather cleanliness results, even though a higher percentage of birds with moderate feather soiling (Score 2) was recorded in the YLP5 group, compared to that observed in the CON and YLP3 groups, this finding was not reflected in the FPD or hock burn outcomes. In a number of previous studies however, dirty feathers and FPD were highly correlated [
66,
67,
68].
In current investigation, a significant antioxidant effect was detected in both thigh and breast meat of the broilers that were fed YLP diets, compared to the CON-fed birds, as indicated by the MDA results. This finding is very important because oxidation of the lipid components in muscle tissues is a major cause of quality deterioration and short shelf life after slaughter. The high concentration (42.5%) of oleic acid in YLP used in this study might have played a key role in this result. It has been previously demonstrated that oleic acid reduces oxidative stress and inflammatory response by activating the peroxisome proliferator-activated receptors in animals [
69]. Complementarily, the antioxidant effect of YLP in broiler meat could also be attributed to the bioactive components of the yeast cell wall, such as β-glucans and mannans. Previous research evidence indicated that adding mannan oligosaccharides and β-glucans in poultry feed decreases the accumulation of the lipid oxidation end product (MDA) in the tissues of broiler chickens [
70]. The beneficial dietary impact of YLP on the antioxidant status of turkey hens has been demonstrated in previous reports [
16,
18]. According to Merska et al. [
16], supplementing turkeys’ feed with 3% and 6% YLP yeast, increases the catalase activity (CAT) and decreases the plasma concentrations of lipid peroxidation products, such as hydroperoxide (LOOH) and MDA. Those results were confirmed in a later study by Czech et al. [
18] who indicated that the role of YLP in the activation of antioxidant enzymes and the reduction of peroxidation products, when incorporated in turkeys’ diet at 3% inclusion rate.
The supplementation of broilers’ diet with 3% YLP yeast ameliorated the lipid profile of breast meat, as indicated by the increased concentration of MUFAs and PUFAs and the decreased levels of SFAs observed in this group. The reduced SFA concentration recorded in the breast meat of YLP3 birds could be primarily attributed to a reduction of palmitic acid, which was the most abundant among the SFAs recorded in the breast meat of all dietary treatments and, to a lesser degree, to a similar decrease of the stearic acid concentrations. The bioactive components of the yeast cell wall, such as β-glucans and mannans, might have played a key role to the reduction of the SFA levels in the breast meat of YLP3 birds, since they act as antioxidants and could suppress meat lipid oxidation. The increased level of PUFAs in the breast meat of the YLP3 group was mainly linked to an elevation of linoleic acid, the most abundant fatty acid among PUFAs found in the breast meat of all dietary treatments. Taking into consideration the high level of linoleic acid (35.2%) in the composition of the YLP used in the present study, it could be supported that the addition of YLP at a 3% incorporation rate in broilers’ diet resulted in a concomitant increase of linoleic acid concentration in the breast meat of birds of this group. However, this beneficial dietary effect of YLP was not proportional to the incorporation rate of YLP in the birds’ diet, as it was revealed by the breast lipid profile results of the YLP5 birds, which were similar with the CON group. It has been previously documented that the FA profile of chicken meat is affected by the birds’ diet [
71] and genetic factors [
72]. Moreover, a FA diet composition, fat metabolism and fat deposition in edible tissues are often correlated in monogastric animals. Dietary supplementation with polyunsaturated FAs, such as linoleic, α-linolenic and arachidonic acids is often associated with increased levels of these acids in the muscle and adipose tissues, both through direct incorporation and modification of the unsaturated FA synthesis in these tissues [
73,
74]. However, the mechanisms involved are complicated and affect the lipogenic genes’ expression [
75,
76].
The supplementation of broilers’ diet with 3% YLP, positively affected the nutritional quality of their breast meat, as indicated by the improved PUFA/SFA ratio and the better health lipid indices recorded in the breast meat of YLP3 birds. The PUFA/SFA ratio is the most-commonly used index for estimating the effect of a certain food on cardiovascular health, considering that all PUFAs are capable to decrease low-density lipoprotein cholesterol and serum cholesterol, whereas all SFAs could contribute to the elevation of serum cholesterol [
77]. Thus, higher values indicate a better (positive) effect given by a particular meat or meat product intake. The dietary ratios of PUFA to SFA, that are higher than 0.45, are regarded as safe for human consumption [
78], and suitable to protect against the development of ischemic heart disease [
79]. The optimal PUFA/SFA ratio from a nutritive point of view was achieved with the 3% incorporation rate of YLP, compared to the CON and YLP5 groups.
The most frequently used index for estimating the nutritional value of dietary foods is the PUFA/SFA ratio. However, it is regarded too general and inappropriate for evaluating the atherogenicity of foods [
80] as specific SFAs and PUFAs have different metabolic effects [
79]. Thus, AI was established [
54] in order to estimate the atherogenic potential of the FAs in food. A lower value indicates better nutritional characteristics of the food [
77]. Another important and commonly used index to further characterize the thrombogenic potential of FAs is TI [
80]. This index points out the trend to form clots in blood vessels and indicates the contribution of different FAs, showing the relationship between the pro-thrombogenic FAs (C12:0, C14:0 and C16:0) and the anti-thrombogenic FAs (MUFAs and the n-3 and n-6 families) [
54]. It has been documented that animal products with a low index of thrombogenicity reduce the threat of atrial fibrillation [
81]. Summarizing, AI and TI can be employed for the estimation of the potential impact of the FA composition on cardiovascular health (CVH). An FA composition with a lower AI and TI, has a better nutritional quality, thus its consumption may decrease the risk of coronary heart disease (CHD), but the recommended values for the AI and TI have not been provided yet [
80]. The results of this study indicated that the addition of 3% YLP in broilers’ diet decreased significantly both the AI and TI, compared to the CON and YLP5 groups. At the same time, this incorporation rate of YLP yeast significantly reduced the h/H ratio of the produced breast meat. Santos-Silva et al. [
82] supported that the higher the ratio between the hypocholesterolemic and the hypercholesterolemic fatty acids, the more the oil or fat is suitable to human nutrition. These findings indicate the health benefit potential related to the fat intake from breast meat produced from YLP3-fed broilers, since they presented a higher nutritional quality than the CON and YLP5 breast meat.
The FA profile analysis of the thigh meat revealed a significant elevation of n-3 PUFAs in the YLP-fed groups, compared with the CON group. This elevation is attributed to the higher concentration of α-linolenic acid observed in the thigh meat of the YLP groups, compared to the CON group, possibly related to the α-linolenic acid concentration of YLP used in the study. This finding is very important from the consumers’ point of view. Intake of the recommended amounts of PUFAs, and particularly n-3 acids, is absolutely necessary for ensuring the correct functioning of the human body and essential for the prevention and mitigation of several diseases, such as cardiovascular diseases, skin diseases, autoimmune conditions and certain forms of cancer—breast, colon and prostate cancer [
83,
84,
85,
86]. Differences in the breast and thigh meat FA profile observed in the current study could be attributed to the different roles of FAs in these tissues or to their different phospholipid contents. The PUFAs are preferentially incorporated into phospholipids [
87], and more phospholipids are in found in breast than in thigh muscles [
88]. Since there is lack of research evidence regarding the effect of YLP in the lipid profile of breast and thigh meat, as well as on the relative health lipid indices when supplementing broilers’ feed, the comparison of our results with those in literature is not possible. However, similar studies curried out on Atlantic salmon, have shown a beneficial effect of YLP on the FA composition in fish fillets [
27].
In the present study, the response of the panelists for the breast and thigh meat of broilers fed diets supplemented with YLP yeast did not influence the sensory attributes of the broiler meat. However, according to the SET results, the thigh meat of YLP5 chicks scored the highest values for all sensory characteristics, leading to significantly higher overall acceptance scores, compared to the other dietary treatments. These findings are very important from the consumers’ point of view, since they imply that YLP does not compromise the organoleptic characteristics of broiler meat; on the contrary, it increases the overall likeability of thigh meat. One of the main concerns when using foods rich in polyunsaturated lipids is the deterioration of the sensory quality of the poultry products. The results of several studies have demonstrated that the meat of animals containing more PUFAs is more susceptible to oxidative processes [
89], which has a negative impact on its organoleptic characteristics and shelf life [
90]. In the present study however, despite the high concentrations of PUFAs in breast meat of the YLP3 group, as well as the higher concentration of n-3 PUFAs in the thigh meat of the YLP-fed broilers, compared to the CON group, the tenderness, juiciness, flavor, color and overall acceptance of YLP meat were not reduced. Instead, a positive effect on the overall acceptance of thigh meat from the YLP groups was detected with significant differences observed in the YLP5 group. This positive effect could be attributed to the antioxidant activity of oleic acid (present in high concentration 42.5%) in YLP used in this study, as well as to the bioactive components of the yeast cell wall, such as β-glucans and mannans, that have antioxidant properties protecting meat from lipid peroxidation. This is also supported by the lower values of MDA recorded in the breast and thigh meat of YLP birds, compared to the CON birds.