4. Discussion
In all treatments, lambs showed DWG and FBW within the range reported in the literature for hair lambs of similar age fattened on high concentrate rations [
30]. The lambs fed with the HM1 diet were 6% heavier at the end of fattening compared to lambs fed with the CON diet. In addition, DMI was 11% higher in lambs fed with HM2 compared to lambs assigned to CON. In a similar study, Razo et al. [
31] examined the effects of HM (0, 1, 2, and 3 g kg
−1 DM for 60 days) based on
Withania somnifera,
Ocimum tenuiflorum,
Tinospora cordifolia, and
Emblica officinalis containing polyphenols and flavonoids, on lambs fed high-concentrate diets. In their investigation, lambs supplemented with low doses of polyphenols and flavonoids had higher DWG, DMI, and FBW compared to the other treatments. However, as the dose of polyphenols and flavonoids increased, the growth performance worsened, similar to the results observed in our study. In another study by Lobo et al. [
2], the effects of supplementing lambs were evaluated using leaf extracts from
Illex paraguariensis (0, 1, 2, and 4% for 53 days) containing phenolic compounds. It was observed that DWG, DMI, and FBW increased at doses up to 2% of the extract in the diet, however, these decreased at doses of 4%. These results suggest that the effects of phenols and flavonoids can improve the productive performance at low doses, but at high doses, they could affect the growth rate, probably due to toxic effects. The higher DMI observed in lambs that consumed HM1 was potentially associated with higher digestibility, as previously reported by Ma et al. [
32] in lambs supplemented with 0.25 mg d
−1 of resveratrol (a natural polyphenol). The inclusion of flavonoids in the diet can reduce the concentration of propionate in the rumen by changes in the fermentation carried out by the rumen microbiome [
3,
10]. Additionally, supplementation with flavonoids has the potential to modify gene expression involved in the regulation of DMI [
3]. Similar effects from the consumption of flavonoids seen in our study would partially explain a higher DMI in the lambs supplemented with 1 and 2 g of HM kg
−1 DM.
On the other hand, Du et al. [
12] reported that supplementation with flavonoids extracted from the plant
Allium mongolicum increased DWG by increasing the presence of bacteria (
Tenericutes and
Mollicutes) positively correlated with DWG in the rumen microbiome. It is suggested that the presence of flavonoids in the diet consumed by lambs stimulates the protein synthesis in muscle and inhibits proteolysis [
6], also increasing the duodenal flux of amino acids and microbial protein [
8]. In addition, a recent study in beef cattle showed that a 0.04% flavonoid dietary supplementation extracted from
Citrus aurantium improves rumen health [
3,
8]. This results in higher absorption of propionate through the rumen wall—a higher metabolic availability of nitrogenous compounds—which, in our case, explains the higher DWG in lambs supplemented with HM.
Results from other studies suggest that the addition of phenolic and flavonoid compounds in the diet acts by increasing the serum levels of growth hormone [
7], reducing the production of enteric methane [
9], increasing digestibility [
31], and improving energy utilization [
33]. These findings could explain the increases in DMI and DWG observed in lambs that consumed HM in the present study
Feed conversion ratio and BCS were similar for all treatments. These results are congruent with the findings of Lobo et al. [
2], who used leaf extracts of
Illex paraguariensis (0, 1, 2, and 4% for 53 days) containing phenolic compounds to feed lambs. In their study, CA and BCS were not affected by the supplementation of the extract in the diet. However, it was observed that TWG and carcass weight were higher in lambs supplemented with 2% of the extract. Additionally, Odhaib et al. [
10] also observed no differences in CA of lambs fed with high-concentrate diets supplemented with
Rosmarinus officinalis leaves,
Nigella sativa seeds and a combination of both plants, containing 12.35, 19.08, and 34.86 mg of total polyphenols kg
−1 DM, respectively. Although DWG increased significantly in lambs that consumed HM, in our study, DMI also increased, which explains the absence of significant changes in CA.
Regarding the carcass characteristics, the results of the present study agree with those reported by Simitzis et al. [
34] in lambs that consumed diets with a high proportion of concentrate, supplemented with a pure flavonoid (hesperidin) at dietary concentrations of 1500 and 3000 mg kg
−1 of feed for 35 days. In their study HCW, CCW, and HCY were similar among treatments, perhaps as a consequence of the low impact of flavonoids supplementation on the FBW of the lambs and on the nutritional composition of the diet consumed, similar to what was observed in this study. Cimmino et al. [
11] also observed no differences in HCW, CCW, HCY, and CCY of goat kids fed with high-concentrate diets and supplemented with polyphenols (3.2 mg d
−1 for 78 days) taken from residual water after the olive oil extraction. However, polyphenols had positive effects on the fatty acid profile of the meat. The similarity of BFT in carcasses across all treatments could also partially explain the lack of changes in the carcass performance observed in the present study.
BFT and LMA values were also similar among treatments. Similar results were reported by Valero et al. [
35] in bulls supplemented with 0.162 g d
−1 of flavonoids extracted from propolis. On the contrary, Lobo et al. [
2] used incremental doses (0, 1, 2, and 4% for 53 days) of extracts from
Illex paraguariensis leaves, which contain phenols, in a 60% concentrate diet. Their results showed that BFT decreased as the extract dose increased. However, LMA was higher in lambs receiving the 2% extract dose, resulting in a higher lean muscle production, thus suggesting that the effects of phenols are dose-dependent. The mechanism of action of phenols and flavonoids on lipogenesis has not been studied in lambs. However, in beef cattle fed with high concentrate diets, the inclusion of isoflavone in the diet increased BFT, and it enhanced the synthesis of triglycerides on subcutaneous adipocytes by changing the differential expression of genes involved in lipid metabolism [
36]. In the present study, BFT was not affected by the inclusion of HM despite the fact that it contained flavonoids, which indicates that the source of flavonoids has an influence on the changes in BFT. Although genotype, age, sex, and weight of lambs show positive correlations with carcass fat deposition [
37] and with carcass physical and chemical characteristics [
38,
39], the homogeneity of these characteristics in the lambs used in the treatments partially explains the absence of changes in BFT and LMA.
Similar results on body morphometry and carcass compactness index were previously reported by Lobo et al. [
2] in lambs supplemented with extracts (0, 1, 2, and 4%) from
Illex paraguariensis leaves, containing phenolic compounds. However, there is limited information about the effects of phenolic and flavonoid compounds derived from herbal mixtures on morphometric measurements of ruminant carcasses. Perhaps, slaughter weight, cold carcass weight, muscle mass, and adipose tissue deposition are related to these variables [
24].
Regarding the organs, similar results were observed by Simitzis et al. [
13,
34] in lambs fed with high concentrate diets enriched with 2500 mg of purified flavonoids (hesperidin or naringenin) and with 1500 and 3000 mg of hesperidin kg
−1, respectively. Information about the effects of phenol and flavonoid consumption on the size and weight of internal organs of lambs is still limited, which complicates the explanation of the results observed in this and other studies. However, according to Riley et al. [
40], differences in weight and size of internal organs in ovines are influenced by genotype, sex, and age of the animals, and by the dietary restrictions [
41]. In addition, the tendency for liver and heart weights to increase in lambs supplemented with HM could be explained by their higher feed consumption [
42].
The average values of the chemical composition of meat are in the range reported in the literature for lambs fed high-concentrate diets [
30]. Similar results were previously reported by Qin et al. [
6] on lambs fed with pomace (0, 7.8, and 16% for 80 days) obtained from the
Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids, respectively. In their study, the dose of flavonoids did not affect the moisture, protein, and ash content of the meat. However, the high dose of flavonoids increased the fat content, which according to the authors, could result in a juicier and softer meat. Cimmino et al. [
11] also observed higher fat content, but similar moisture content, protein, ash, and collagen in meat from goat kids fed with high-concentrate diets supplemented with polyphenol extracts. (3.2 mg d
−1 for 78 days) taken from residual water after the olive oil extraction. These results suggest that the effects of phenols and flavonoids on the composition in lamb meat may be dependent on the dose, the botanical origin, and the duration of the experimental phase. On the other hand, considering that the chemical composition of meat varies according to the feeding regime [
43] and by the slaughter weight [
39], in our study, it is likely that the lack of effect on the chemical composition of meat was because HM had minimal effect on the nutritional composition and the FBW of the lambs.
The pH value of meat was similar among treatments but only the pH value of meat from lambs fed with HM was in the considered normal range between 5.5 and 5.8 [
44]. These results suggest that supplementation with HM during the final fattening period could improve the meat quality compared to the meat of lambs not supplemented with HM. Our findings are largely congruent with the results of Qin et al. [
6] in lambs supplemented with incremental levels (7.8 and 16% for 80 days) of pomace from
Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids, respectively. In addition, with the results of Simitzis et al. [
13] in lambs supplemented with hesperidin or naringenin at dietary feed concentrations of 2500 mg kg
−1; and results of Cimmino et al. [
11] in goat kids supplemented with 3.2 mg d
−1 of polyphenol extracts taken from residual water after the olive oil extraction. A pH value below 5.8 is important for the preservation of meat during storage as it has a bacteriostatic effect, while higher pH values favor the growth of proteolytic microorganisms [
45]. This indicates that supplementation with HM during the final fattening period can increase the shelf life of lamb meat.
Additionally, in the present study, there were no effects on meat color attributes (L*, a*, b*, Chroma, and Hue°) associated with the inclusion of HM in the diet. Similar results were previously reported by Qin et al. [
6] on meat from lambs fed with pomace from
Hippophae rhamnoides fruits in the diet (7.8 and 16% for 80 days), containing 0.69 and 1.02% of flavonoids, respectively, and by Simitzis et al. [
13] in lambs supplemented with hesperidin or naringenin at dietary concentrations of 2500 mg kg
−1 of feed for 35 days. In addition, Muela et al. [
46] did not observe changes in meat color in lambs supplemented with a commercial extract (150 mg kg
−1 DM for 40 days) taken from whole fruits of
Citrus paradisi,
Citrus aurantium bergamia,
Citrus sinensis, and
Citrus reticulata, plants, containing 3.5% polyphenols and 0.8% bioflavonoids (naringenin, quercetin and rutin). These results suggest that the effects of phenolic and flavonoid compounds on the color of lamb meat are not dependent on the dose, the botanical origin, or the period of administration.
Color is an important attribute of meat quality because it is the first aspect that attracts consumers when choosing fresh meat [
11]. Color stability depends on the oxidation of myoglobin and the formation and accumulation of metamyoglobin [
11]. However, several studies [
11,
46] have reported that the inclusion of polyphenols and flavonoids in the diet does not affect myoglobin, oxymyoglobin, and metamyoglobin contents in lamb meat, which would explain the absence of changes in meat color attributes observed in the lambs that were fed with HM in the present study.
The evaluation and validation of a new feed additive require the assessment of the health status of the animals after its consumption. In our study, the hematological parameters of lambs were similar among treatments and showed values within the normal physiological range [
47]. In a similar study, Razo et al. [
31] were investigated the effects of supplementation with a polyherbal mixture (0, 1, 2, and 3 g kg
−1 DM for 60 days) containing polyphenols and flavonoids on blood metabolites in lambs that were fed high-concentrate diets. In their study, they observed the highest platelet concentration in lambs consuming 1 g of HM, however, platelets decreased as the dose of HM increased, thus indicating that phenols and flavonoids can stimulate the immune response at low dose, but at high doses they may depress the immune system. On the other hand, Morsy et al. [
48] evaluated the effects of supplementation with red propolis (3 g d
−1 for 21 days) containing 43% of isoflavonoids in pregnant ewes fed with 50% of roughage in the diet and observed that the presence of isoflavonoids improved the total leucocyte concentration, total protein, and globulin in the blood. These results suggest that flavonoids can stimulate the immune system regardless of the physiological stage of the animal or the source of the metabolites, even when flavonoids are administered for short periods.
In our study, it was observed a quadratic reduction in the concentration of segmented neutrophils in lambs that consumed 1 and 2 g of HM. Similar results were reported by Molosse et al. [
32] in suckling lambs supplemented with curcumin, and by Odhaib et al. [
10], where lambs were fed high-concentrate diets supplemented with 1% of
Rosmarinus officinalis leaves,
Nigella sativa seeds, or a combination of both plants containing 12.35, 19.08, and 34.86 mg of total polyphenols kg
−1 DM, respectively. Their results also showed that polyphenols improved the immune response without affecting hematological parameters or blood biochemistry in all treatments. Generally, the reduction of neutrophils occurs in the presence of bacterial infections, however, there are other factors that can decrease the number of neutrophils in healthy individuals [
49].
The blood concentration of lymphocytes often increases in animals under stress, excitement, and fear [
50] or during bacterial infections [
47]. Lobo et al. [
2] observed no changes in the concentration of lymphocytes in lambs supplemented with 1, 2, and 4% extract of
Illex paraguariensis leaves as a source of phenols. On the other hand, in the present study, the concentration of lymphocytes increased in lambs supplemented with HM. However, lambs supplemented with HM showed no signs of disease during the experimental phase and even had better productive performance compared to lambs assigned to the control treatment.
According to Braun et al. [
51], biochemical parameters in ovines are mainly used for the diagnosis of liver, muscle, and nutritional disorders. In the present study, the concentration of these blood metabolites was in the normal range for ovines [
52], suggesting that the HM used did not cause liver, muscle, or nutritional disorders in the lambs. Similar results were previously reported by Lobo et al. [
2] on lambs supplemented with 1, 2, and 4% extract of
Illex paraguariensis leaves containing phenols; by Qin et al. [
6] on lambs supplemented with 7.8 and 16% of pomace from
Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids; and by Razo et al. [
31], where lambs were supplemented with incremental doses (0, 1, 2, and 3 g kg
−1 DM for 60 days) of a polyherbal mixture of Indian plants containing polyphenols and flavonoids, and observed that the blood chemistry values of all treatments were within the ranges reported as normal for ovines.
Regarding serum uric acid and urea concentrations, similar results were previously reported by Zhong et al. [
53] in lambs supplemented with polyphenols extracted from the green tea plant at concentrations of 2, 4, and 6 g kg
−1 of feed for 56 days, and by Razo et al. [
31], in lambs supplemented with a polyherbal mixture (1, 2, and 3 g kg
−1 DM for 60 days) containing polyphenols and flavonoids. Their results showed that uric acid rose as the dose of polyphenols and flavonoids increased, however, the urea was similar among treatments. It has also been reported an increased amino acid and microbial protein duodenal flux in ruminants supplemented with flavonoids [
8], which would partially explain the increase in urea and serum uric acid in lambs supplemented with HM.
In our study, lambs supplemented with HM showed low serum creatinine levels, but these were in the normal range for healthy ovine [
52], while concentrations of that blood metabolite in the lambs assigned to the control group were up to 27% above the normal range. By contrast, Lobo et al. [
2] reported an increase of serum creatinine in lambs as the dietary level of phenolic compounds was increased. On the other hand, it is known that serum creatinine increases in cases of chronic and acute renal failure [
54], which suggests that the HM used in the present study did not affect renal health.
There is limited information about the use of herbal mixtures and their effects on the mineral status of ruminants. Calcium and phosphorus in blood serum are valuable indicators of the nutritional status of animals due to the low variability of its concentration in the blood [
55]. The serum concentrations of calcium and phosphorus in our study were similar among treatments (
p > 0.05) and were in the normal range for ovines [
53], indicating that the consumption of HM did not affect the mineral balance in the diet or the nutritional status of the lambs. However, in a similar study, Razo et al. [
31] investigated the effects of supplementation with a polyherbal mixture (0, 1, 2, and 3 g kg
−1 DM for 60 days) containing polyphenols and flavonoids on blood metabolites of lambs fed high-concentrate diets. In that study, serum calcium was also not affected by the dose of polyphenols and flavonoids, however, serum phosphorus concentration increased as the dose of polyphenols and flavonoids increased. These results suggest that the effects of HM containing polyphenols and flavonoids on the mineral status of lambs depend on the dose used and the botanical origin.