This study showed that birds treated to SC at a concentration of 10⁷ cfu/g and GS at 10% (T5) presented significantly higher levels of creatine kinase (CK) as compared to the control groups (T1, T2, and T3). CK concentration in plasma is known to increase when muscle and liver damage occurs, mainly in striated skeletal muscles, in situations such as necrosis or reversible cell injury (Latimer, 2011). In addition, the SC-supplemented specimens exhibited higher weight as compared to those fed the control diet. Probiotics stimulate appetite and improve the intestinal environment, thus improving production parameters such as higher breast weight and better body-to-legs ratio in broilers (Barros Barrios et al., 2021). These changes, which imply an increase in CK activity, could be related to the effect of the yeast on the metabolism of the animals with regard to acceleration of morphological development and growth stimulation (Çalışlar & Kanat, 2021). On the other hand, GS supplementation is likely to have contributed a higher percentage of crude protein to the basal diet fed to the broilers in this experiment. In animals, increased bioavailability of proteins promotes muscle growth, which increases serum CK levels (Ogungbesan, et al., 2013).
This study found that T1 (control; commercial concentrate feed, no SC, no GS) exhibited significant and positive correlations of glucose with triglycerides and cholesterol (total, VLDL, and LDL) (r = 0.99). In animals, carbohydrates are stored in the form of large polymers called glycogens. Carbohydrates constitute an excellent source of instant energy and stored energy (as polysaccharides). Carbohydrates conjugated to glycoproteins or glycolipids are part of a variety of structural components of cells and organs (Hernández & Puig, 2010). In the digestive tract, hydrolysis of carbohydrates to monosaccharides allows their intestinal absorption and subsequent transport through the portal blood to the liver, where they are metabolized for energy, stored as glycogen or converted to amino acids or fats (Latimer, 2011). It is a well-known fact in poultry farming that lipid absorption is altered when changes in diet composition affect the intestinal microflora in chickens, disturbing the conversion of primary bile salts (chenodeoxycholic and cholic acids) to secondary ones (lithocholic and deoxycholic acids) needed to digest fat (Osorio & Flórez, 2011)
Broilers from the control group (T1 exhibited significant and negative correlations (r = -0.98) of glucose with protein and globulin. Gluconeogenesis employs amino acids or fatty acids as raw material (Arneson & Brickell, 2007), which can explain this negative correlation by indicating that amino acids — fundamental components of proteogenesis — may have been used for glucose synthesis. Glucose binds strongly and steadily to proteins throughout the lifespan of a particular protein. Therefore, levels of glucose-bound proteins reflect average blood glucose concentrations during the half-life of the protein. When the protein in the complex is albumin or total serum protein, the product is called fructosamine (FrAm) (Kaneko et al., 1997).
In the control groups (T1 and T3), cholesterols exhibited a significant correlation (r = 0.99) with total cholesterol and VLDL (r = 1). Lipids are transported by lipoproteins in the bloodstream. There are four classes of lipoproteins in the blood: chylomicrons, VLDL, LDL, and HDL. Chylomicrons transport triglycerides (TAG) to vital tissues (heart, skeletal muscles, and adipose tissues). Synthesized by the liver, VLDL redistributes TAG to adipose tissues, heart, and skeletal muscles. LDL transports cholesterol into cells whereas HDL transports cholesterol from cells back to the liver. TAG-rich lipoproteins and their remnants are atherogenic and attributed to other lipid risk factors (Carvajal, 2014).
There is vast evidence of metabolic differences between birds and mammals, including lipid metabolism (Osorio & Flórez, 2011). In birds, lipoproteins called portomicrons (PMs), which are equivalent to chylomicrons in mammals, are transported to the liver via the bloodstream instead of the lymphatic route, through the endothelial intracytoplasmic vesicles, reaching the liver directly through the portal vein. However, due to their large size, PMs are not metabolized in said organ (Fraser et al., 1986 cited by Osorio et al., 2016). After overnight fasting, endogenous triglycerides are synthesized in the liver, together with apolipoprotein B and cholesterol, and released into the bloodstream as very low-density lipoprotein particles (VLDL). Upon contact with fat cells and muscle capillary beds, VLDL triglycerides are hydrolyzed by LP-lipases. Hence, VLDL particles are catabolized into intermediate forms within hours, which are in turn further catabolized into low-density lipoproteins (LDL) (Wahl et al., 1984).
In T2 (control; no SC + 10% GS), the presence of 10% GS is associated to a significant correlation between VLDL cholesterol and triglycerides (r = 1). In VLDL lipoproteins, the lipid component consists of triglycerides, cholesterol, and phospholipids at an approximate ratio of 4:1:1, which are mainly synthesized in the liver. VLDLs export triglycerides and cholesterol from the liver and distribute triglycerides to adipose tissues and skeletal muscles (Latimer, 2011).
Lipogenesis in adipose tissues of birds is limited, which implies that fat deposition depends on lipids present in the diet and hepatic lipogenesis (Griffin et al., 1989). Therefore, accumulation of triglycerides in adipocytes is related to the metabolism of VLDL and other substances. However, it can equally be affected by substances that do not directly alter VLDL (Xu et al., 2003).
T3 broilers (control; no SC + 15% GS) exhibited a close and significant correlation of total proteins and albumin with all cholesterols (total, LDL, HDL, VLDL) and triglycerides. A typical broiler starter feed contains 21–24% protein, with grains and milling by-products meeting about half of the protein needs in most poultry feeds. Additional protein is provided by protein-rich concentrates, which can be of animal or vegetable origin (Saavedra, 2020).
In this study, GS contributed 22% more crude protein to the control diet, indicating that raising the amount of GS in the diet increases protein availability. This, in turn, increases the bioavailability of proteins and amino acids in the bloodstream for later use in the formation of apoproteins needed to transport lipids. Likewise, an increase in apoproteins causes HDL, LDL, and VLDL levels to rise because both triglycerides and cholesterol in plasma are contained in lipoproteins. Therefore, increases in serum cholesterol concentrations reflect higher concentrations of cholesterol-rich lipoproteins (e.g., LDL and HDL) (Latimer, 2011).
In T4 (SC + 0% GS), significant and negative correlations were observed between glucose and protein (r = -0.93), albumin (r = -0.79), and globulin (r = − 0.97), which can be attributed to the action of the yeast. SC lowers glucose levels in a very significant way as shown in the correlation between the SC-supplemented group and the control group, which could explain this inverse correlation. That is, since blood glucose did not increase, the combined effect of glycosylation and protein oxidation did not occur.
Due to the action of SC, a highly significant correlation of total protein and albumin with total cholesterol was also found. Although T4 did not have GS as an additional source of protein, the yeast generated an increase in plasma protein, attributable to the abovementioned effect (in regard to glucose), which produced bioavailability of proteins and amino acids in the bloodstream to participate in the formation of apoproteins needed for transporting lipids. This effect was reflected in the high correlation found between total cholesterol and serum proteins, which in turn explains the correlation between total cholesterol and VLDL and LDL lipoproteins and triglycerides. This is also consistent with the highly significant correlations of VLDL, LDL, and triglycerides with albumin and total proteins, since maturation of these particles requires numerous protein factors mediating the binding of lipids with apoproteins (Carvajal, 2014).
T5 broilers (SC + 10% GS) exhibited a highly significant correlation between total cholesterol and low-density cholesterol (LDL), which comprises 60–70% of serum total cholesterol. That is, SC supplementation and GS replacement at 10% increased protein availability, which in turn promoted production of apoproteins needed for transport of total cholesterol by low-density lipoproteins.
It is worth noting that GS contains secondary compounds with anti-nutritional properties. One of the most important secondary metabolites are condensed tannins, which can have positive and negative effects on digestibility of proteins, carbohydrates, and fibers in animal feeds (Romero, et al., 2000). However, some authors have suggested that adding exogenous enzymes to feeds can help minimize the harmful effects of these secondary metabolites in non-ruminant animals (Oloruntola et al., 2018).
In this research, the various types of enzymes found in SC cells — namely, vacuolar protease (e.g., serine, aspartyl, and metalloprotease), pectinase (Ferreira, et al., 2010), amylase, maltase, sucrose, lactate dehydrogenase, proteinase, polypeptide, dipeptidase, deaminase, transaminase, lipase, phospholipase, phosphatase, and phytase (Çalışlar and Kanat, 2021) — may have counterbalanced the anti-nutritional effects of GS and, as a result, promoted the development of the specimens under investigation.
T6 (SC + 15% GS) showed effects of GS on serum glucose as there were four highly significant and positive correlations with total cholesterol (r = 0.91), including the fractions VLDL (r = 0.92), LDL (r = 0 .87), and triglycerides (r = 0.92), indicating that an increase in serum glucose causes the different fractions of lipoproteins to increase as well.
A similar result was found for LDL, VLDL, and triglycerides, which contribute to increasing total cholesterol, with positive and highly significant correlations. Triglycerides exhibited strong and positive correlations with VLDL (r = 1), LDL (r = 0.86), and total cholesterol (r = 0.90). This result can be attributed to GS percentage in the diet, which despite possible degradative effects of Saccharomyces enzymes, may not have been sufficient to counterbalance the total anti-nutritional effects of GS present in the diet. As this forage contains coumarols, which confer a bitter flavor to it, cyanogenic glycoside, tannin, saponin, and cell wall content that makes it less useful for monogastric animals (Ogungbesan et al., 2013). Tannins are heat-stable and reduce protein digestibility in animals and humans, probably by making protein partially unavailable or by inhibiting digestive enzymes and increasing fecal nitrogen.
Present in food products, tannins inhibit the action of trypsin, chymotrypsin, amylase, and lipase, lower the quality of food proteins, and interfere with iron absorption. In addition, tannins are responsible for reducing feed intake, growth rate, feed efficiency, and protein digestibility in experimental animals. For their part, saponins reduce intestinal absorption of certain nutrients (e.g. glucose and cholesterol) through intraluminal physicochemical interaction, which is why hypocholesterolemic effects have been reported (Tadele, 2015).
Oloruntola and colleagues (2018) supplemented a 15%-GS rabbit diet and found that the anti-nutritional properties of this forage can be counterbalanced by using exogenous enzymes. They also showed that triglyceride levels drop when the amount of GS in the diet is raised. They attributed this finding to the presence of bioactive compounds in this forage, which reduce fat absorption and, consequently, plasma depletion. Likewise, Agbede and Aletor (2003), who found a decrease in cholesterol and LDL levels in rabbits fed a 50%-GS diet, consider the effect of Gliricidia sepium on triglyceride reduction beneficial to consumers, especially people predisposed to heart diseases.
T6 also exhibited negative correlations of HDL with protein and globulin, possibly due to the increase in Apo A-I apoproteins, whose concentration can rise in response to the increase in protein availability promoted by GS, thus increasing HDL and decreasing serum protein and globulins. Needed for transporting lipoproteins, apoproteins bind to VLDL and HDL and are released from the liver into the bloodstream. Endothelial cells release LP-lipase, which metabolizes VLDL into LDL. In this study, this function of lipoproteins was evidenced by the strong and positive correlation found between VLDL and LDL (r = 0.86).