To the authors' best knowledge, the present study is the first study to investigate the simultaneous effect of the TP and induced feed restriction on the mRNA abundance of the hepatic genes related to lipogenesis and fat oxidationin fat-tailed sheep. According to the literature, energy precursors such as BHB and FFA are affected by TP plus feed restriction, and consequently, changing the hormonal array of milk biosynthesis could also affect animal performance (Zarrin et al., 2021). Investigating the mRNA abundance of the hepatic genes related to lipogenesis (FAS, ACC) before and after lambing proved that feed restriction at the time of sampling had no effect on these genes and was almost identical in both treatment groups.
The ACC, codenamed acetyl-CoA carboxylase, involves in the biosynthesis of long-chain fatty acids. This enzyme is responsible for catalyzing ATP-dependent carboxylation, which converts acetyl-CoA to malonyl-CoA. Malonyl-CoA is recognized as the raw material for synthesizing palmitic acid as the building block in the biosynthesis of long-chain fatty acids (Smit et al., 2003). Fatty acid synthase is an enzyme encoded by FAS. This enzyme catalyzes the synthesis of palmitate from malonyl-CoA and acetyl-CoA by the attendance of NADPH and ultimately produces long-chain saturated fatty acids (Leonard et al., 2004). Contrary to the results obtained in this study, Dervishi et al. (2011) stated that insufficient access to feed suppressed the activity of essential enzymes such as ACC and FAS in lipid syntheses by reducing their mRNA abundance. Zakariapourbahnamiri et al. (2018a, b) have also suggested that feed restriction reduces the mRNA load of crucial enzymes such as ACC and FAS, associated with lipid biosynthesis, in the subcutaneous adipose tissue in lambs.
The current study showed that the synthesis of fatty acids and lipogenesis was affected by lambing and significantly downregulated the studied genes compared to the prepartum period. In a study on subcutaneous adipose tissue genes in bovines during the TP, it has been reported that the mRNA abundance of the ACC and FAS genes in the first postpartum day was significantly lower compared to the prepartum period (Sadri et al., 2010). Sadri et al. (2011) described that the non-esterified fatty acid (NEFA) concentration on the first postpartum day was higher than those at other sampling times (week 8 before calving and week 5 after calving) in cattle. The same researchers also reported that the mRNA abundance of the ACSL, unlike the FAS and ACC genes, was not affected and showed no significant change (Sadri et al., 2011). Lack of effectiveness of feed restriction on the mRNA load of the desired genes before and after lambing in the current research might be partially linked to the energy level, which its indices did not show significant differences between the sampling days (Zarrin et al., 2021). In addition, the capacity of fat-tailed ewes to adapt to NEB requirements can also be acknowledged as one of the advantages of such breeds. Downregulation in the lipogenesis-related genes in this study following lambing may be due to NEB elongation during this session. Although there is still a lot more to be known, it has been suggested that decreased mRNA level of the FAS and ACC genes in the postpartum period has reduced the de novo synthesis of fatty acids, indicating decreased lipogenesis during the TP (Doepel et al., 2002; Sadri et al., 2011).
The mRNA quantity of both CPTI and CPTII genes was not affected by feed restriction, while lambing suppressed these two genes in the present study. Carnitine palmitoyltransferase1 is encoded by the CPTI B gene and counted as part of the mitochondrial transport system and a key enzyme controlling the beta-oxidation of long-chain fatty acids. This enzyme induces the oxidation of fatty acids in mitochondria (Bartelds et al., 2004). The CPT1 gene is a crucial regulator of fatty acid metabolism and is fundamental in transporting the fatty acyl-CoA into mitochondria (McGarry and Brown, 1997). The critical role of CPT enzymes in the metabolism of fatty acids and especially the transfer of fatty acyl-CoA into the mitochondria can also explain the reduction of mRNA abundance of other genes involved in beta-oxidation and fatty acid synthesis (van Dorland et al., 2009). In other words, a decrease in the mRNA concentration of these genes might lead to a reduction in the quantity of the mRNA of other understudied genes.
Despite the current results, Loor et al. (2005) has reported an increase in the CPTI mRNA abundance on the first day after calving in transition cows. The inconsistency between the current study and the study by Loor et al. (2005) might be described by several pieces of evidence, including different sampling times (one day after delivery and three weeks after delivery), different body reserves, and different species. On the other hand, based upon the preliminary data (Zarrin et al., 2021), the concentration of fatty acids in postpartum at the time of sampling might not be high enough to enhance the changes in the mRNA abundance of the selected genes. Based on the comprehensive studies conducted by different researchers, the load of different genes related to metabolism should be considered all in one and their associations together. It has been substantiated that the activity of some genes is relevant to the activity of other ones (van Dorland et al., 2009). These researchers investigated the extensive correlations of various genes involved in energy metabolism pathways, especially gluconeogenesis and fat metabolism in cows with high BHB and NEFA levels. They also showed that various genes involved in these metabolic processes positively correlate with the genes, metabolites, and hormones altered by changing the BHB and NEFA concentrations under different conditions and negative correlations with other genes. Among the positive correlations of the studied genes, the correlation of the ACSL with CPTI and CPTII genes at different times of the TP could be pointed out (van Dorland et al., 2009).
One of the most critical criteria in measuring lipogenesis is a reconstitution of triglycerides from fatty acids, in which the ACSL enzyme plays a pivotal role. The enzyme converts long-chain fatty acids to fatty acyl-CoA esters, which ultimately play an essential role in lipid biosynthesis. The ACSL gene catalyzes the first step in the fatty acid metabolism, which is the activation of fatty acids to acyl CoA in the cytosol (Voet and Voet. 2004). There was no difference in the mRNA level of ASCL before and after lambing between the Restriction and Control groups in the present study. However, the mRNA content of the ASCL gene in the postpartum period showed a significant reduction compared to the prepartum duration.
It had been hypothesized that the increase in the concentration of the FFA and BHB concentrations in the Restriction group before lambing would boost the ACSL gene, and its mRNA abundance would be higher than the Control, but the observation did not reinforce the assumption. The findings on the ACSL gene in the present study corroborate the research results on dairy cattle with NEB and high circulatory BHB in the fourth week of lactation (van Dorland et al., 2009). Lack of significant differences between the circulatory energy metabolism indices, including FFA, glucose, and BHB concentrations (Zarrin et al., 2021), between the treatment modalities during the weeks of liver biopsy lead the authors to expect untouched ASCL after parturition. The concentration of such metabolites declined after lambing compared to the prepartum period. Differences between the treatments might be attributed to the metabolic status of the animals at the time of sampling, which in this case, lack of difference could be explained as equilibria in a state of energy that the animals necessitated no beta-oxidation of long-chain fatty acids (C18: 0 to C12: 0) to cover the NEB (Loor et al., 2005; van Dorland et al., 2009). However, the precise mechanism remains unclear that offers opportunities for further study. Since the mRNA abundance of the desired genes was not affected by feed restriction, it can be inferred that fat-tailed ewes have evolved to cope with the period of feed scarcity, relying on deposited fat around the tail-head, phenomenal fat metabolism, and liver capacity.
It would be reasonable to conclude that the force of lambing on mRNA abundance of the hepatic genes during prepartum can be attributed to the augmented demands of energy as an outgrowth of fetal growth during the last months of pregnancy. Although we should stress that these results are only provisional, the reduction in mRNA plentitude of the FAS, ACC, CPTI, CPTII, and ACSL1 genes compared to the prepartum period probably indicates the transfer of colostrum and milk compounds, including fatty acids mobilized from the dam's circulatory system to the udder tissue. The findings presented in this study provide a starting point for further examination regarding the evolutionary benefit of fat-tail, particularly in tropical regions and impoverished pastures. Moreover, if the results are reproducible in other studies, a deeper understating of the mechanism behind fat metabolism during the TP might be attainable in their light.