3.3. Lambs’ Slaughter Performance and Carcass Traits
The parameters related to slaughter performance and carcass traits are reported in
Table 4.
The feeding plan, defined by different slaughter age of lambs and feeding level, affected significantly the weight of lambs at slaughter; indeed, both the slaughter body weight (22.9 vs. 26.3 and 27.9 kg for 90L, 120R and 120L lambs, respectively) and empty body weight (20.1 vs. 23.1 and 24.8 for 90L, 120R and 120L lambs, respectively) were obviously higher in 120 day-old lambs, especially when fed ad libitum, although the 120L and 120R plans did not have statistically dissimilar results. As a consequence, the weight of the entire (12.5 vs. 14.2 and 15.2 kg for 90L, 120R and 120L lambs, respectively) and half carcasses (5.05 vs. 5.74 and 6.16 kg for 90L, 120R and 120L lambs, respectively) were significantly lower for the younger lambs in comparison with the 120-day-old lambs. On average, the carcasses of 90L lambs weighed 12.5 kg, which is heavier than the carcasses produced from 130-day-old Comisana lambs fed with an analogous diet (11.3 kg) [
40], and comparable to those of 100-day Comisana lambs fed green forage of sulla (12.5 kg) [
43], and those of 100-day lambs of Barbaresca breed (from 12.1 to 13.1 kg) [
41]. Then, during the period from 90 to 120 days, the carcass weight increased on average by 1.7 kg and 2.7 kg with restricted and ad libitum feeding, respectively. On the basis of the carcass classification systems in the European Union (EEC 2137/92 and 461/93 regulations), the 90L carcasses were classified into class C (10.1–13 kg) with quality 2 (red meat or fatness score 1 or 4) of the scheme for lamb carcasses lighter than 13 kg [
44], whereas the 120-day carcasses were classified using the scheme for lamb carcasses weighing more than 13 kg and mainly scored as O (fair) for conformation and 2 (slight) for degree of fat cover (most of 120R carcasses) or as R (good) for conformation and 2 (slight) or 3 (average) for degree of fat cover (most of 120L carcasses).
Compared to the 120L lambs, the 90L lambs showed a higher incidence of head (8.11% vs. 7.57 %), a lower presence of perirenal and pelvic fat (2.15% vs. 2.68 %), and a tendency towards a lower incidence of the internal organs. These results can be attributed to the different growth rate of these body regions [
45], which is earlier for the head, due to its greater bone base, and later for the fat and internal organs; thus, the head showed a higher percentage in younger lambs, whereas the proportions of fat and organs increased in older lambs.
A tendency towards significance of the feeding plan also emerged for the incidence of the empty gastrointestinal tract and its content, which slightly increased in 120R lambs, and can be explained by the slower gastrointestinal transit associated to restricted feeding that would favor a longer permanence of ingested feeds in the digestive tract and, as a consequence, a slight increase recorded in its volume and content.
Regardless of the feeding plan, the carcasses of lambs fed DWB showed a greater incidence of head, which, also in this case, can be related to the greater development of the more precocious bone tissue [
45], favored by restricted or low energy diets. The DWB-based diet was also responsible for lower carcass yields for which, however, emerged a significant FP × diet interaction; indeed, the diet with DWB significantly reduced the carcass yields only in the restricted 120-day-old lambs. This result can be linked mainly to the higher incidences, although at a not significant level, of the empty gastrointestinal tract (+1.2%) and its contents (+1.2%) recorded in DWB20-120R than in DWB0-120R lambs, presumably due to the previously mentioned effect of restriction associated to a higher volume of the more fibrous DWB20 concentrate.
The sex of the lambs influenced the carcass yield expressed as a percentage of the slaughter body weight (SBW), which was higher in the females due to only their lower gastrointestinal content, since the same difference did not emerge for the carcass yield referred to the empty body weight (EBW). The carcasses of the females also showed a greater adiposity, as indicated by the higher content of perirenal and pelvic fat. In contrast, the males had a higher incidence of head, which denotes their greater skeletal development compared to the females. These expected results are in line with several findings on lambs from various breeds and weight ranges [
46] and are linked to the earlier maturity of females in comparison with males of same age; the precocity of female lambs is reflected in their greater tendency for fat deposition and lower incidence of bone and muscle tissue, and to the greater nitrogen retention of males, which develop more muscle than adipose tissue.
With regard to the tissue composition (
Table 4) that resulted from the right hind leg dissection, the feeding plan affected the weight of the hind leg, which increased passing from 90L to 120R and 120L lambs (1.43 vs. 1.92 and 2.02 kg), and the fat tissue incidence, which also increased in older lambs (5.63 vs. 8.62 vs. 11.28 % in 90L, 120R and 120L lambs, respectively). However, the increasing adiposity recorded in the hind leg of older lambs corresponded to a degree of fat cover scored as 2 (light) or 3 (average), which are the levels denoting lean carcasses. The meat incidence showed an opposite trend due to the feeding plan (66.9 and 65.3 vs. 62.6 % in 90L, 120R and 120L lambs, respectively), whereas no effect emerged for the bone incidence and the meat-to-bone ratio. The diet did not modify these parameters, whereas the sex of lambs influenced the fat and bone incidences; indeed, the hind leg of male lambs was higher in bone and lower in fat, in line with the incidences of head and the perirenal and pelvic fat mentioned previously.
3.4. Meat Evaluation
Similarly to the carcass traits, the parameters of physical quality of LD meat were also more affected by the feeding plan rather than by the other factors considered (
Table 5); nevertheless, these traits appeared to be mainly influenced by the slaughter age of lambs rather than by the feeding level to the 120-day-old lambs were submitted.
The meat of the lighter carcasses from the 90L lambs showed an ultimate pH (6.01), measured at 24 h after slaughtering, higher than that of meat from 120-day lambs (5.71 and 5.68 for 120R and 120L lambs, respectively). As known, a higher meat pH is linked to a lower post mortem acidification, which, in turn, could depend on the mobilization of the muscle reserves of glycogen to sustain an increasing energy demand [
5,
47]; in this study, the higher energy expenditure of the 90L lambs could be linked more presumably to the emotional stress induced by the pre-slaughter condition, due to fasting and transport, to which the younger animals could be more susceptible, rather than to the feeding regimen.
Despite its higher pH, which should be linked to an increasing water holding capacity [
43], the LD meat of 90L lambs showed greater thawing losses (7.95% vs. 5.52% and 5.96% for 90L, 120R and 120L lambs, respectively), whereas no differences emerged for the water losses due to the successive cooking. The lower water retention observed for the meat of 90L lambs is in agreement with Budimir et al. [
9] and Russo et al. [
44], who found a lower water holding capacity for lighter carcasses due to a higher drip loss, explained by the weaker ability of myofibrillar proteins of meat from younger lambs to hold water [
9].
The higher values of shear force recorded for the 90L cooked meat indicates a lower tenderness of meat from young lambs, especially in comparison with the 120R meat (4.09 vs. 2.65 kg/cm
2). This result is in line with D’Alessandro et al. [
5], who detected a higher collagen content, the main determinant of meat toughness, in the intramuscular connective tissue of younger lambs. Nevertheless, the same authors [
5] suggested that collagen shows low variation with age and also weak correlations with the toughness of cooked meat. Accordingly, in this study, the greater water losses after thawing could also have contributed to the lower tenderness of the 90L cooked meat.
The colorimetric parameters of both meat and fat showed differences between the 90L carcasses and those from the 120-day-old lambs. On the whole, the meat of older lambs was brighter and had a more intense red color, as indicated by the higher values of lightness, redness, and chroma. In contrast, the fat of lighter carcasses was less bright and with a more intense color, as expressed by the lower values of lightness and the higher redness, yellowness and chroma.
Color is the main attribute used to appreciate meat freshness, and consumers tend to prefer a pale or pink color for light lamb meat [
5], and accept darker meat from older lambs [
3]. In line with this study, other authors [
3,
7,
9] found an increased redness, related to the higher content of myoglobin [
1], in meat from older and heavier lambs. The levels of red color of meat from Valle del Belice lambs were within the commonly detected ranges; in this regard, the redness of the 90L meat was slightly higher than that observed in 100-day-old Comisana lambs [
43], and comparable to that found in Barbaresca lambs of both 100 [
41] and 130 days of age [
42], and in 5-month-old Fabrianese lambs [
3], whereas the darker meat from 120-day-old Valle del Belice lambs approached the redness values observed in 130-day-old Comisana lambs [
40] and also in 60-day-old Bergamasca light lambs [
9].
The diet only influenced the lightness of fat, which increased with the DWB20 diet, presumably as a consequence of the transfer and deposition to the fat tissue of carotenoid pigments contained in the DWB, including lutein, β-cryptoxanthin, zeaxanthin and β-carotene [
48].
No significant FP × diet interaction emerged, while sex affected the pH of meat, which was higher in females at a negligible level, and showed only tendencies towards a higher lightness and a lower chroma of fat in the carcasses of females.
Table 5 reports the chemical composition, polyphenols content and antioxidant capacity of LD meat.
The chemical composition of LD meat was not affected by the feeding plan, in accordance with studies in which the slaughter age or carcass weight did not influence the chemical composition of lamb meat [
5,
9]. Accordingly, the intramuscular fat content, which represents the later fat deposition, did not increase from 90L to 120R and 120L meat, as occurred for the incidence of earlier fat depots, such as perirenal and pelvic fat in the carcasses and the separable fat tissue in the hind leg. Thus, when prolonging the growth period by 30 days, the lamb meat did not show differences in fat infiltration. In contrast, the diet with DWB reduced the fat content of meat, balanced by an increase of protein, although only at a tendency level. This result indicates the effect of DWB inclusion in the concentrate in reducing the level of intramuscular fat, which emerged with both a restricted and an ad libitum feeding level. Moreover, the calculated amount of total lipid in fresh meat obtained with the DWB inclusion (5.60% and 6.26% with DWB20-120R and DWB20-120L diet, respectively) only was slightly higher than the levels (<5%), indicating lean meat, according to the Food Advisory Committee [
49].
The sex of lambs strongly influenced the composition of the LD meat; indeed, the females, due to their earlier maturity, showed a more consistent intramuscular fat deposition to which the reduction of the other components corresponded.
The content of total polyphenols and the antioxidant capacity of LD meat, detected exclusively for the male lambs, were strongly correlated (r = 0.64;
p < 0.0001), and were both influenced by feeding plan, diet and their interaction. The significance of the interaction shows that a higher presence of polyphenols in the meat, and the consequent improvement of the meat antioxidant capacity, occurred only in the 120-day-old lambs fed with DWB, regardless of the feeding level. Presumably, this result can be linked to the higher DWB intake of 120-day lambs, but also to a longer accumulation in the tissues of the compounds with antioxidant activity contained in the ingested DWB, such as phenolic acids, especially ferulic acid [
19], together with carotenoids [
48]. Therefore, the inclusion of DWB in the diet contributed advantageously to increasing the content of phenolic compounds and the antioxidant activity of lamb meat. This result further confirms that ingested polyphenols could move into the muscles, as suggested by Moñino et al. [
50], who observed a great presence of polyphenols in the meat of lambs suckling from ewes fed with rosemary extracts. On the other hand, the study of Soberon et al. [
51] provided evidence of the possible tissue uptake of free ferulic acid dosed orally in lambs. A higher polyphenol content and antioxidant capacity were also detected in cheeses obtained from milk of cows fed a diet supplemented with 3 kg/day of DWB [
20]. Accordingly, the DWB seems to have the potential to enrich the meat of bioactive compounds, consisting of phenolic acids and carotenoids, which are able to improve its oxidative stability and health properties.
In the triangle tests, the panelists were able to detect sensorial differences between samples of cooked meat due to the presence of DWB in the diet and the feeding level, restricted or ad libitum, although these differences were always perceived at a moderate level. In particular, the assessors discriminated significantly the diet in the meat of 90L (DWB0-90L vs. DWB20-90L, 66.7% correct answers, p < 0.05) and 120R lambs (DWB0-120R vs. DWB20-120R, 66.7% correct answers, p < 0.05), but not in the meat of 120L lambs (DWB0-120L vs. DWB20-120L, 33.3% correct answers). Instead, the effect of the feeding level was perceived significantly only in the meat from lambs fed the diet without the DWB (DWB0-120R vs. DWB0-120L, 83.3% correct answers, p < 0.001), and not in the meat from lambs fed the DWB20 diet (DWB20-120R vs. DWB20-120L, 50% correct answers).
3.5. Meat Fatty Acid Profile
The FA composition of intramuscular fat of LD meat samples taken from 120-day-old male lambs (
Table 6) was mainly influenced by the presence of DWB in the diet, whereas the significant effect of the feeding level emerged especially in the interaction with the diet.
Oleic acid (OA, C18:1
c9), recognized for its hypolipidemic effect, which is important for human health to reduce plasma cholesterol and triglycerides [
52], has been confirmed as the prevalent FA in lamb meat [
2,
3]. In this study, the OA was not affected by the feeding treatments; since the most common FA in the dietary components was linoleic acid (LA, C18:2 n-6) (
Table 1), the OA derived by the endogenous desaturation of stearic acid originated, in turn, from the biohydrogenation of mainly LA in the rumen [
2,
40,
53].
With regard to the effect of DWB, the DWB20 diet resulted in a decrease of n-6 FA, especially due to the reduction of LA and arachidonic acid (AA), and an increase of n-3 FA for the main contribution of the eicosapentaenoic acid (EPA); these results corresponded to the favourable strong reduction of the n-6/n-3 ratio to values strictly close to the threshold (≤5) recommended by the FAO/WHO [
54] in the human diet for the prevention and treatment of chronic diseases. AA and long-chain n-3 FA, especially EPA and docosahexaenoic acid (DHA), are of interest for infant nutrition, as they are essential for optimal neonatal growth and development; in this regard, it can be noticed that their levels recorded with the DWB20-120L were strictly close to those that Nudda et al. [
55] found in fresh meat from suckling lambs, which were higher than those detected in commercial lamb-based infant foods.
However, EPA and DHA are recognized for their multiple health benefits, mediated by their anti-inflammatory actions [
56,
57] and especially for their role in reducing the risk of cardiovascular disorders in humans [
58]. Therefore, their presence should encourage lamb meat consumption to reach adequate intakes and reduce the n-6/n-3 ratio of the diet.
On the contrary, the diets with DWB were responsible for the tendency for a lower level of rumenic acid (RA), the main isomer of the conjugated linoleic acids (CLA), known for its health benefits [
59,
60]; this effect occurred independently of the feeding level, and despite the precursor of the RA, the
trans vaccenic acid (VA) only showed a reduction with the restricted DWB20 diet.
The VA is an intermediate of the biohydrogenation of dietary polyunsaturated FA to stearic acid performed by the micro-organisms in the rumen, whereas the RA is produced by the endogenous desaturation of VA in the tissues due to the Δ9-desaturase [
61]. Thus, the reduction of both VA and RA recorded with DWB-based diets, could be linked to a favourable effect towards a the complete biohydrogenation process in the rumen, more pronounced with lower intakes, as occurred with the restricted level, and confirmed by the higher incidence of stearic acid in the meat fat with the DWB20-120R diet. In this regard, an adverse effect of DWB or its phenolic compounds on the successive VA desaturation can be excluded by the comparable values of the desaturase index of VA obtained among feeding treatments (
Table 6).
Significant interactions with the FL × diet emerged for the total of saturated FA (SFA) and unsaturated FA (UFA), and then for their PUFA/SFA and UFA/SFA ratios. The effect of DWB on the level of saturated FA was opposite in relation to the feeding level, since the byproduct induced a decrease of saturated FA, especially palmitic (C16:0) and stearic (C18:0) acids, when offered ad libitum. This result could be a confirmation of the effect of restricted DWB20 diet in favouring the complete biohydrogenation process in the rumen. The levels of saturated FA with DWB20-120L and DWB0-120R diets (37–38 g/100 g FA) resulting from this study were lower than those recorded in meat from light lambs of the Leccese breed (from 50 to 55 g/100 g FA) [
2,
4] and the Bergamasca breed (51–53 g/100 g FA) [
9], and comparable to those of heavier 130-day-old Comisana lambs (36–39 g/100 g FA) [
40] and Fabrianese lambs of 2 and 5 months of age (40–42 g/100 g FA) [
3]. These comparisons are in agreement with the increase in the unsaturation of fat depots observed with increasing age at slaughter [
2].
Compared to the DWB0-120L diet, the DWB20 diet offered ad libitum was responsible for the rise in unsaturated FA, which occurred as a consequence of the reduced saturated FA, and despite the reduction of α-linolenic acid (ALA). Since ALA represents the precursor of the long-chain n-3 FA, its reduction could be linked to the major biosynthesis of EPA, DPA and DHA that emerged with the DWB20-120L diet. Significant interactions also emerged for the health indexes used to assess the health value of meat fat; indeed, when the lambs were fed the DWB20 diet ad libitum, the trombogenic index and the h/H ratio improved by decreasing, whereas the health promoting did not decrease, as occurred with the DWB20 restricted diet.
Moreover, the DWB20-120L diet led to improvements in the PUFA/SFA and UFA/SFA ratios, approaching the level recorded with the DWB0-120R. In particular, the ratio PUFA/SFA of both DWB0-120R and DWB20-120L diets reached closer values to that recommended for human health (0.45) [
62].
On the whole, these results show that the presence of DWB in the diets of lambs increased the level on n-3 FA in the meat, especially EPA and DHA, reducing the n-6/n-3 ratio; moreover, when the DWB was offered in a greater amount, as obtained when the DWB20 concentrate was fed ad libitum, the byproduct was able to further improve the health profile of meat FA by reducing the saturated FA, and increasing the more beneficial unsaturated FA and the PUFA/SFA and UFA/SFA ratios.