The authors are aware that the number of individuals per group in the present trial may limit the accuracy of the data; however, the same individuals were simultaneously used to analyze the kinetics of protein synthesis to obtain a complete picture of nutrient metabolism in pigs [
18]. The whole process required surgical catheterization and confinement of pigs in metabolic cages; therefore, the experimental design restricted the available number of experimental animals.
This study aimed to compare specific aspects of the fat metabolism of two types of pigs generally used in southern Europe for different productive purposes. The 3W crossbred pigs come from intensive selection to enhance lean carcass and feed conversion; these leaner pigs are slaughtered at lighter weights to take advantage of their high growth potential. However, heavy pigs intended for high-quality pork (e.g., specific Duroc lines or Iberian pigs) are slaughtered at heavier weights and males are usually castrated to avoid boar taint and improve fat deposition [
27]. Thus, the term PT used throughout the manuscript is employed because it includes both genotype and castration traits, as Duroc barrows were surgically castrated shortly after birth to meet commercial conditions. In addition, within the heavy Duroc line, pigs were genotyped and selected to be homozygous for the
SCD_T and
SCD_C alleles to exploit their differential Δ9-desaturase activity and oleic acid synthesis capability [
15]. This study was performed in two GP (28.42 ± 0.861 kg BW and 87.40 ± 1.256 kg BW for growing and fattening phases, respectively), in which growing pigs showed differential rates of protein synthesis [
18] and also of fat metabolism, with a subsequent accretion that may be altered by selection strategies exerted on the genotypes used.
Fatty acids are deposited in adipose tissues through two mechanisms: direct FA incorporation, mostly from the diet [
28], but also from mobilization from other fat depots; and through de novo synthesis processes, using different precursors at different rates. The biological diversity of FA in adipose tissues and the variety of precursors make fat accretion a complex process. To address this topic, the authors administered D-labeled stearic acid (d
35-C18:0), with molecular hydrogens of the hydrocarbon chain that were labeled with deuterium, to monitor direct FA incorporation. Likewise, labeled oleic acid (d
33-C18:1
c9), with all hydrogens of the hydrocarbon chain D-labeled, was considered a valid index of endogenous FA de novo synthesis, since it derives directly from the desaturation of dietary d
35-C18:0 by the action of SCD.
The validity of the FIR relies on the rate between d
35-C18:0 incorporation into adipose tissues and available d
35-C18:0 in plasma. Thus, the robustness of the FIR depends on (i) the stability of plasma d
35-C18:0 enrichment and (ii) the absence of d
35-C18:0 recycling. Regarding the former point (i), plasma d
35-C18:0 enrichment leveled off after 72 h of its dietary administration, and consistent detection was obtained in adipose tissues 72 h thereafter. Considering that pigs were fed ad libitum and that digestion and intestinal absorption may buffer the discontinuity of discrete meals, plasma d
35-C18:0 enrichment was arguably constant. In relation to the last point (ii), the analytical protocol allowed the identification of d
35-C18:0 in plasma and tissues, so the possibility of background contamination or endogenous return in plasma was negligible. Following the same principle, the appearance of d
33-C18:1
c9 in tissues should be associated with d
35-C18:0 availability, without any possible interference with exogenous sources. Nevertheless, d
33-C18:1
c9 was only consistently detected in SC with our analytical approach, since SCD activity is significantly higher in this tissue [
29,
30].
4.1. Production Data and Differential Growth Intensity
In line with previous findings [
31], Duroc pigs homozygous for the
SCD_T allele presented lower feed consumption than Durocs carrying the
SCD_C allele, and tended to show lower performance in the fattening phase.
Regarding allometric coefficients, no abnormalities were observed in the growth profile during the experimental period [
32]. Considering that
SCD genotype variation only affects FA composition but not fat content [
15,
33], datasets from both Duroc genotypes (TT and CC) were pooled and used as a single group. As previously described [
34], pigs showed a high degree of maturity in the liver and a proximal-distal gradient in the hindlimb bones, all in negative allometry. Adipose tissue accretion was in positive allometry, whereas intermuscular fat differed between PTs. These differences in fat metabolism could be attributed to divergence maturity of adipose tissue between fatty and lean pigs [
14,
34]. The 3W pigs had a higher allometry coefficient in intermuscular fat, and a high tendency in semimembranosus muscle IMF. These results are consistent with an elevated ratio of intermuscular to SC fat, particularly in the Pietrain breed and to a lesser extent in purebred or crossbred Duroc, as well as a higher ratio in boars than in barrows [
32].
For skeletal muscles, the semimembranosus was in negative allometry, indicating a higher degree of maturity than other muscles in the ham. Likewise, the allometric growth rate of this muscle indicates how a greater degree of maturity may advance IMF metabolism, which is the last fat depot to develop [
35].
4.2. Apparent Ileal Digestibility
The results showed a clear effect of GP on the mechanisms regulating fat absorption and deposition; moreover, the effect of GP was not homogeneous and interacted with PT. The AID of EE was higher and homogenous in fattening pigs, whereas in growing pigs EE-AID was impacted by PT, being significantly reduced in 3W pigs. Such interaction also was observed in several FA fractions.
The link between animal development and the rate of FA digestibility was previously suggested by Powles et al. [
36], who revealed that the physicochemical structure of FA effectively alters the digestibility process. Digestibility is favored by the increase of the unsaturation rate and the reduction of the hydrocarbon chain length through micelle formation [
37,
38], although such effect (saturated/unsaturated FA ratio) was found more pronounced in young than in old pigs [
36]. Our findings also confirmed that saturated FA were the least digested, followed by monounsaturated and polyunsaturated FA, with mean AID coefficients of 63.59%, 73.87%, and 84.95%, respectively.
It should also be pointed out that dietary EE content and FA profiles differed between diets, with fattening diets containing a higher proportion of EE (60%) than the growing ones. In this regard, previous studies suggested that fat AID may be increased with fat inclusion level [
39] or may differ with FA composition [
38]. In any case, a differential dietary composition in EE may have masked the effect of age on fat digestibility.
Regarding pig PT, growing 3W pigs showed lower AID for DM, CP, and EE, coinciding with lower rates of AID in saturated and monounsaturated FA, although these coefficients converged in the fattening phase. These findings agree with previous studies [
40,
41] in which fatty genotypes appeared to use dietary nutrients more efficiently at earlier ages than leaner ones, due to both earlier development of their digestive tract and higher enzyme activity.
4.3. Fractional Incorporation Rate of Stearic Acid
Incorporation of dietary fat into tissues differed between liver and adipose tissues, and semimembranosus IMF was the depot with the lowest FIR, as corresponds to a mature organ with a low allometry coefficient. The liver was much more active during the fattening phase in all PTs, whereas IMF of skeletal muscles had higher FIR in the growing phase, showing PT-dependence for each GP. Distinct tissues play different roles in fat metabolism [
11,
42]; while in pigs adipose tissue is the most active for FA synthesis [
11], the liver is mainly involved in lipid oxidation and long-chain PUFA synthesis. Age also affects lipid metabolism on a large scale. Duran-Montgé et al. [
42] established that lipogenic gene expression was higher in adipose tissue at 60 kg BW, whereas at 100 kg BW it was greater in the liver. In this regard, it was reported that adipose tissue reached the highest lipogenic activity at 120 days of age, decreasing gradually thereafter [
43]. Therefore, both liver and adipose tissue may change their role in fat metabolism over time.
Differences between PTs in SC-FIR showed up mainly in the fattening phase, when fat deposition is predominant [
32]. While Duroc pigs maintained their FIR stable throughout age, 3W pigs significantly increased it, along with an improvement in their FA-AID, which suggests an increase of dietary fat incorporation with age. However, in IMF, the FIR of 3W pigs showed a different development than in SC. The longissimus dorsi FIR was higher in 3W than in Duroc pigs at both GP, but experienced the greatest decrease in the fattening phase. This decrease in FIR could explain the lower IMF content in this leaner genotype compared to Duroc [
6,
8]. Dietary fat incorporation in 3W pigs may be more centered on other fat depots, such as SC or intermuscular adipose tissue, since this latter showed a higher relative growth rate than Duroc.
The lower rates of d
35-C18:0 incorporation recorded in Duroc pigs may be explained by the dietary FA dilution with saturated and monounsaturated FA coming from endogenous synthesis and/or desaturation. Several authors have described the increased lipolytic activity of Duroc pigs during their growth [
35], and the higher adipogenic and lipogenic gene expression in adipose tissues of fatty pigs [
44] in comparison with leaner breeds. Consequently, fat incorporation in Duroc pigs appears to be less dependent on the availability of dietary FA in contrast to the leaner 3W genotype. However, processes associated with lipid mobilization, lipolysis, and extracellular matrix formation are upregulated in lean pigs [
22,
45,
46].
4.4. Endogenous Oleic Acid Synthesis: Oleic/Stearic Ratio
Since stearic acid is the main substrate of the SCD enzyme [
16], the C18:1
c9/C18:0 ratio has been largely used as a measure of apparent SCD activity. In this regard, significant increases in apparent SCD activity with adipose tissue maturity have been reported [
33,
35]. However, in the present study, apparent FUR was significantly lower in the fattening than in the growing phase; possible reasons will be discussed further below.
Moreover, the authors are unaware of any previous in vivo experimental models using labeled FA as an index of real SCD activity in pigs; the existent bibliography comprises only in vitro models [
29,
30]. In this approach, (
14C) oleic acid is obtained through the incubation of tissues with (
14C) stearic acid, and in line with our results, significant increases in real SCD activity were reported with age in animals between 51 and 95 kg BW [
28], and despite this, activity remained unchanged or even decreased up to 128 kg BW. Conversely, previous expression studies described decreases in
SCD gene expression in adipose tissues as animals fattened, between 60 and 100 kg BW [
42]. However, some authors have found no direct correlation between gene expression level and FA profile [
47], and these differences could rely on the poorly predicted post-transcriptional regulation of
SCD among tissues by their mRNA levels [
48].
Concerning PT, it is worth mentioning that animals were genotyped for the rs80912566
SCD polymorphism, and in the 3W group, five growing and seven fattening pigs were homozygous for the
SCD_T allele, with the rest (three growing pigs and one fattening pig) being heterozygous (CT). The
SCD_T allele is almost fixed in some pig breeds, including Landrace and Pietrain, although in Duroc it segregates at intermediate frequencies [
15].
Following the present results, both Duroc TT and 3W pigs showed higher apparent and real FUR than Duroc CC pigs, although these differences in real FUR were only found in the fattening phase. Such differences mostly agree with patterns previously reported [
15,
33], demonstrating that the
SCD_T allele enhances FA desaturation, with Duroc TT and Duroc CT pigs having 2% and 1% more monounsaturated FA, and 2% and 1% less saturated FA than Duroc CC pigs, respectively [
3]. This favorable effect of the
SCD_T allele has also been confirmed in other Duroc crossbreds such as Duroc × Iberian [
15]. Although their pigs were between 95 and 130 kg BW, the authors suggested that the variation in SCD activity was maintained throughout the growing–fattening period.
In our work, the significant decrease of apparent FUR related to animal maturity may be due to a stagnation in the C18:1
c9/C18:0 ratio with age, which makes the rate of desaturation per day lower, through (i) lower oleic acid incorporation or (ii) greater stearic acid incorporation into SC. The high availability (8.27 g/kg DM), high digestibility (85.76%), and improved de novo synthesis (245.02%; real FUR) of oleic acid in the diet during the fattening phase suggests that the first possibility (i) should be neglected. In relation to the second possibility (ii), 3W and Duroc genotypes appeared to behave differentially. The increase in the concentration of stearic acid in the SC of fattening 3W pigs is explained by the increase of FIR in this depot during the fattening phase, which also increases the substrate of the SCD enzyme, promoting de novo synthesis [
49]. However, the same explanation cannot be applied to the Duroc pigs since FIR remained stable in Duroc TT pigs or slightly decreased in Duroc CC pigs during the growing–fattening period. If exogenous FA incorporation cannot explain the increase in stearic acid pool, then de novo synthesis may account for such increase, as about 80% of total FA deposition comes from biosynthesis [
13]. This assumption is also supported by the fact that the SC allometry coefficient in Duroc pigs increased with the same intensity as that of 3W pigs, despite the lower FIR and similar FUR of Duroc pigs compared to 3W pigs in the fattening phase. In that sense, the increased content of unsaturated FA in SC with animal maturity is consistent with previous studies using similar animals [
35]. In that case, the reduction in apparent FUR in Duroc pigs is compatible with the increased de novo synthesis of oleic acid, since real FUR accounts for the increase in d
33-C18:1
c9 per unit of d
35-C18:0; therefore, the incorporation of de novo synthesized unlabeled stearic acid in SC would not alter the d
33-C18:1
c9/d
35-C18:0 ratio.