3. Results and Discussion
Whey is a highly nutritious byproduct of the cheese industry, which may substantially affect performance, bone morphology, microbiota and physico-chemical parameters in the intestinal tract of poultry. Lactose, the main ingredient of whey, acts as a prebiotic since it cannot be digested by the birds, but it is fermented by the intestinal microbiota [
2,
11,
12,
13,
19].
The feed conversion ratio and average daily feed consumption data are presented in
Table 3. They were collected and calculated on a pen basis. It can be concluded that whey inclusion levels in the diet higher than 2% may negatively affect birds’ performance. Therefore, the effect of whey at various concentrations in poultry diets should be further evaluated regarding lactose, sodium, chloride and protein contents.
The body weight in all experimental groups was not significantly different (
p > 0.05) at 1st, 13th and 23rd day of age (
Table 3). However, the addition of 1–2% of whey in poultry diets increased significantly (
p ≤ 0.05) the final body weight on day 37, whereas the addition of 5% whey decreased significantly (
p ≤ 0.05) the final body weight, compared to control group. Moreover, the addition of 1–2% of whey in poultry diets reduced the feed conversion ratio for the periods of a 23rd–37th day of age and 1st–37th day compared to the control diet, whereas addition of 5% of whey in poultry diets significantly (
p ≤ 0.05) deteriorated the cumulative feed conversion ratio. The addition of whey did not affect the mortality rate of birds in any group since no dead birds were recorded at any group.
The positive effect of low quantities of whey up to 4% in poultry performance has been reported in many studies [
12,
19,
20,
21]. In another recent study, it was shown that growth of chickens was improved by dietary incorporation of either 6% dry whey powder or 8% dry whey concentrate because of a higher mineral availability, increased feed intake and modulation of caecal microbiota communities [
11]. Whey is an excellent source of nutrients, providing high quality protein, peptides, amino acids, lactose, minerals, vitamins and varying quantities of lipids [
3,
4].
In addition, whey contains a wide range of bioactive components, such as β-lactoglobulin, α-lactalbumin, immunoglobulins and peptides that pose a range of biological and physiological effects, such the regulation of skeletal muscle protein synthesis, reduction of oxidative stress and regulation of cellular processes [
1,
3]. In particular, whey proteins contain a high level of branched-chain amino acids (leucine, isoleucine, and valine), essential amino acids (cysteine) and peptides, which are considered an excellent amino acid source for birds [
3,
4]. These proteins have a higher biological value compared to the main protein source in poultry feed, soybean meal, which could further promote the performance of broiler chicks [
11].
According to the Gülşen et al. [
21] and Ocejo et al. [
22], the improvement in the performance after whey dietary supplementation could be attributed to its effect on the intestinal integrity and morphology. In particular, the addition of whey resulted in a significantly (
p ≤ 0.05) greater villus height. The larger villus area means that the absorptive surface of the intestinal tract is increased, leading to a more efficient nutrient utilization and, subsequently to enhanced performance of broiler chicks [
23].
On the other hand, the addition of high concentrations of whey reduced the bodyweight of birds, which could be attributed to the lactose intolerance and/or increased osmolarity, due to its sugar and mineral content. In the current study, the maximum concentration of whey was 5% of a whey product containing 73% of lactose. This is equivalent to a dietary inclusion level of 3.65% of lactose, just above the bird’s lactose tolerance level of 3.5% [
2,
7].
The effect of whey, at various concentrations in the poultry diets, on the pH and viscosity values of the digesta of broiler chicks on the intestinal parts is displayed in
Table 4. The addition of 5% whey increased significantly (
p ≤ 0.05) the pH of duodenum digesta compared to other experimental groups, while the addition of 1, 2 and 5% whey increased significantly (
p ≤ 0.05) the pH of jejunum digesta and reduced significantly (
p ≤ 0.05) the pH of caecum digesta compared to the control group A. Ileal viscosity values were reduced significantly (
p ≤ 0.05) by whey addition at all inclusion levels, however, this was noticed in jejunum at the very low whey inclusion level of 1%. Viscosity is related with main absorption sites, as low viscosity values allow higher absorption rates [
8]. For that reason measurement of viscosity is less meaningful at caecal digesta or the very first part of duodenum.
The pH of the intestinal digesta depends on the feed composition and subsequently on the fermentation activity of intestinal microbiota [
24,
25]. The effect of whey on the pH of intestinal digesta has been observed in many studies [
13,
26]. The most plausible explanation for the effect of whey on the pH of the intestinal digesta is the lactose fermentation by the intestinal microbiota of the lacto- and bifido- bacteria. Lactose fermentation leads to the production of lactic acid and subsequently to the reduction of the pH value of intestinal digesta [
13].
In our study, it was noticed that dietary increase of whey was accompanied with a slight increase in pH values in proximal parts of the intestine, especially in duodenum and jejunum in a dose-dependent manner, whereas pH values in the ileal digesta was remained unaffected. However, the dietary inclusion of whey affected caecal content that remains longer time under stable fermentation process, by presenting considerably lower pH values. Caecum is the main site for bacterial fermentation in chickens due to its special habitat [
27]. Bacteria metabolize soluble nondigestible carbohydrates (sNDCs) into short chain fatty acids (SCFAs) and lactate which consequently lowers the pH [
28]. Reduced pH may inhibit the growth of acid sensitive bacteria such as members of the family Enterobacteriaceae [
13,
26,
28,
29].
Viscosity, a physico-chemical property of the intestinal digesta, is associated with the feed materials, mainly the undigested part, and the digestive chyme [
30,
31]. It has been calculated that birds’ lactose tolerance is about 3.5%. If higher concentrations of lactose are used in the birds’ feed, then the excess of lactose remains undigested and osmotic diarrhoea may be observed leading to growth retardation [
2,
23]. The results of the present study confirmed the results of the previously published studies since the addition of 5% of whey increased significantly (
p ≤ 0.05) the viscosity in ileum digesta and reduced the bodyweight of birds. On the other hand, the addition of low quantities of whey (1–2%) reduced significantly (
p ≤ 0.05) the viscosity in ileum digesta and increased the bodyweight of birds. Low quantities of whey, although not absorbed by the digestive system of the birds, may act as a prebiotic and are used as a substrate by the intestinal microbiota, promoting gut health and performance [
13]. In this study, evaluation of different carbohydrate substrates such as starch and pentozans of different grains or lactose at levels 30 g/kg or inulin supplemented at a concentration of 20 g/kg of feed, were tested.
Increased viscosity in the gastrointestinal tract of poultry, through addition of cereals containing high amounts of soluble non-starch polysaccharides (NSPs), has been shown to negatively interfere with digestion and absorption of nutrients and consequently reduce their performance [
32]. Although many studies have investigated the anti-nutritive effect of increased gastrointestinal contents’ viscosity [
28].
Poultry diets with high soluble NSP increase intestinal viscosity that hampers the nutrient digestibility and have deleterious effects on the bird’s health and performance. New generation carbohydrases may help in digestion of a broad range of dietary fibers with reduction of intestinal viscosity and competition between host and microbiota for SCFA in the small intestine and improve digestibility of nutrients. This enzyme activity may also reduce the loads of pathogenic microbes and improve intestinal health [
33].
According to the data presented in
Table 5, dietary whey had a significant effect (
p ≤ 0.05) on the caecal microbiota of broiler chicks. In particular, the addition of 1, 2 and 5% of whey increased significantly (
p ≤ 0.05) the caecal counts of
Lactobacillus spp. compared to the control group, which confirms its effect on the pH of intestinal digesta. Furthermore, the addition of 5% whey increased significantly (
p ≤ 0.05) the total anaerobic counts in the caeca compared to control group possibly due to the available quantity of lactose or lactic acid on sight. As mentioned above, lactose in chicks’ acts as a prebiotic, since it cannot be hydrolyzed or absorbed in the intestinal tract of chicks and is fermented by the intestinal microbiota stimulating the growth of beneficial caecal bacteria [
11,
13].
It is generally acknowledged that normal intestinal microflora is associated with increased numbers of
Lactobacilli and decreased numbers of
E. coli that can partly explain improvement in healthy status especially of young animals. In opposite cases, increased numbers of
E. coli are associated with high incidence of diarrhoea in young animals causing mortality and growth retardation [
23]. In our study we found that counts of
Lactococcus lactis were increased. Recently, an excellent review of Markowiak and Śliżewska [
34] explained in detail that lactic acid that is produced after bacterial fermentation of sugars, is partly dissociated, while the undissociated form passes through lipid cell membranes and by dissociating within the cell, it acidifies cell contents and inhibits the growth of pathogenic microorganisms, including putrefactive bacteria, Gram-negative bacteria, and also some moulds. Also, it was reported that lactic acid can be further fermented into acetic acid, which neutralizes electrochemical cell potential and can decrease the growth of putrefactive bacteria, including those of the genera
Clostridium spp. and
Salmonella spp. Bacteria of the genera
Lactobacillus spp.,
Enterococcus faecium, Lactococcus lactis and
Streptococcus thernophilus are the main producers of bacteriocins [
18,
34]. However, in the current study, the bacteriocin content in the gut was not evaluated.
Elevated intestinal coliform and
E. coli counts are generally associated with adverse health effects. These bacteria are often contrasted with
Lactobacillus spp. [
35]. On the contrary, the outcomes in some novel studies hinted a relation between higher intestinal
E. coli/Enterobacteriaceae load and improved performance [
36], meaning that the interrelationships of intestinal microbiota need further elucidation. It is generally accepted that indigenous
Lactobacillus spp. are considered beneficial bacteria as they positively contribute to microbial balance and gut health through competitive exclusion and through the production of lactic acid [
37]. In the study of Molnar et al. [
30] diets with high sNDC content did not increase caecal
Lactobacillus spp. numbers. Instead, sNDC diets resulted in a microbial shift towards a higher caecal coliform load relative to the control group. This notice of higher caecal coliform numbers was associated with enhanced intestinal functions such as lower caecal pH or higher butyrate concentration; pointing out an augmented bacterial fermentation in the tested sNDC groups due to higher substrate availability.
Another similar study showed that high viscosity is also associated with higher incidence of necrotic enteritis (NE) [
38]. Mortality due to NE and intestinal lesions of randomly selected birds were associated with substantially higher median
Clostridium perfringens (CP) counts than those found in bird groups without such lesions. Those findings suggested that median counts above one million per gram predicted a high probability of concurrent NE-specific gut lesions in at least 10% of randomly selected, killed birds [
38]. It is obvious that intestinal microbiota can play a major role mainly in the escalation of intestinal diseases. Very recently, it has been shown that coccidian diseases can modify the homeostasis of this dynamic ecosystem, negatively affect gut health and growth performance [
39]. Although, the gastrointestinal ecosystem is well organized and very complicated that can be regarded resistant to changes, even in presence of a coccidian infection [
39] the severity of clinical coccidiosis in individual chickens was quantified by microbial changes associated with different lesion scores. The coccidian infection, despite the fact that diversity of taxa within the caecal microbiome remained largely stable, it paused major changes in the abundance of microbial taxa. The major changes were detected in birds displaying severe caecal pathology; taxa belonging to the order Enterobacteriaceae were increased, while taxa from Bacillales and Lactobacillales were decreased. Notable were the profiles in birds that remained asymptomatic (lesion score 0), with taxa belonging to the genera Bacteroides to be decreased, whereas Lactobacillus be increased. Many differential taxa from the order Clostridiales were identified supporting the view that caecal microbiome dysbiosis was associated with Eimeria infection [
39].
In
Table 6, the effect of diet supplementation with whey is presented on bone morphology and breaking strength of tibiotarsus.
The weight and diameter of the tibiotarsus, as well as the ratio breaking strength to bone weight, were not significantly different (
p > 0.05) among experimental groups. But the addition of 5% of whey reduced significantly (
p ≤ 0.05) the tibiotarsus length compared to the control group. The results of our study are in accordance with the results of the study of Scott [
40] who observed that the addition of 5% whey resulted in an increase of the occurrence of enlarged hocks in young turkeys, while the addition of delactosed whey, resulted in the absence of enlarged hocks. Although the underlying mechanism has not been elucidated, it can be hypothesised that increased viscosity reduces the absorption of minerals and fat-soluble vitamins having a deleterious effect on the occurrence of enlarged hocks [
41]. It was an interesting finding that dietary addition of whey at levels of 1 and 2% did not adversely affect tibiotarsus bone weight and breaking strength. The importance of retaining bone health is clear, as intensive genetic selection for performance in modern broilers has increased growth rate but at the same time has compromised skeletal development and integrity [
42]. Skeletal problems, particularly those affecting leg bones are associated with chronic pain in broilers [
43] and high prevalence of lameness causing substantial financial losses due limitations in access to feed [
42,
44]. Several factors such as genetics, gender, endocrinal hormones, growth and aging, nutrition and health status, toxins and antinutrient compounds and physical loading can either directly or interactively affect bone strength in poultry [
45]. Therefore, healthy bone development should be at first sight of examination on research trials with new dietary components [
46].
The current manuscript addresses a value-added potential for an agricultural waste such as cheese whey that offers potential poultry feed additive benefits, while reducing the negative effects of such a food waste and its accompanied environmental hazards. In addition, it has been successfully used to control intestinal pathogens, such as Salmonella spp., Campylobacter spp. and Clostridium perfringens in broiler chicks. The novelty of the current research is that whey may be used in low inclusion levels with positive effects on the growth performance, physico-chemical properties of intestinal digesta and the intestinal microbiota, while the high inclusion levels of whey may have deteriorated effects on the performance and bone characteristics. Therefore, the poultry industry could be an alternative reservoir absorbing large quantities of whey and minimizing environmental pollution. However, further studies are needed in order to investigate possible whey interaction or synergy with other feed additives, such as probiotics, prebiotics and organic acids, as well as its effect on the gut immunity.