4.1 VC serum concentration
VC is one of the most important vitamins with direct functions as anti-oxidant in animals [22]. It regulates oxidative stress through participating in various enzyme reactions associated with the synthesis of amino acids, cholesterol and carnitine [22]. It has been demonstrated that VC injection before transportation increases its concentration in cattle liver and improves the post transit performance [23]. Injections of this vitamin also led to an increase of VC in serum in humans and mice [24-25]. Although ruminants have the capacity to synthesize VC using glucose with the assistance of L-gulonolactone oxidase in liver, the VC level in tissue and blood decreases during stress [26-27]. Thus, it seems plausible that the capacity to synthesize VC in ruminants is diminished during stress conditions. Although serum glucose content did not differ between VC_CON and VC in this study, the fact that VC concentrations increase due to injection highlighted the usefulness of exogenous VC supplementation in the context of the antioxidant response in yak after transportation.
4.2 Health status of post-transit yak after VC injections
Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin (Bil) are important biomarkers to evaluate hepatocyte damage [28]. An increase of blood ALT, AST and T-Bil were observed in dairy cows with postpartal endometritis and mastitis [29]. Oxidative stress is one of the factors that can injure hepatocytes leading to high blood concentrations of ALT and AST [30], and combined with high T-Bil concentrations, these responses suggest the existence of oxidative stress [31]. Thus, it is plausible that the decrease of ALT, AST and T-Bil concentrations with VC injection reflected an improvement in liver function in post-transit yak.
High concentrations of creatinine (CREA) and UREA are normally used to evaluate kidney injury [32]. Creatinine is used most commonly to assess kidney function, thus the increasing of CREA in blood with VC injection in our study suggested that VC might lead to kidney dysfunction in post-transit yak. But the decreasing of serum UREA level was also observed in our study with VC injection [33]. Moreover, VC supplementation at 100 mg/kg led to lower serum concentrations of CREA and UREA in rats [34]. Given the same supplemented dose of VC with our study, we speculated that VC injection could partly improve renal function, but more work should be performed to elucidate the relationship between VC and the serum level of CREA in yak. C-reactive protein (CRP) is confirmed as a biomarker of inflammation [35]. A positive association between oxidative stress and CRP was reported recently [36]. Thus, it seems plausible that the VC injection reduced inflammation status through the regulation of oxidative stress in yak after transportation.
4.3 Oxidative stress state with VC injection in post-transit yak
LPO, MDA and ROS are normally used to evaluate the state of oxidative stress in animals and humans. Both LPO and MDA are biomarkers of lipid peroxidation [37]. MDA is the final product of lipid peroxidation and can cause cell damage [38]. There is a positive relationship between serum level of MDA and intensity of oxidative stress [37]. Thus, our data of higher serum level of MDA after VC injection suggested that it may have promoted oxidative stress in post-transit yak through stimulating lipid peroxidation. However, no significant difference in LPO was observed in the present study, which is the primary oxidation production once the peroxidation reaction begins [39]. Furthermore, it is noteworthy that MDA is the product of reducing hydrogen peroxide (H2O2) to water using VC with the assistance of ascorbate peroxidase [40]. It has been demonstrated that MDA not only causes cell damage during oxidative stress, but also functions as the biologically active compound [41]. Furthermore, no difference in ROS was observed in our study. Thus, combined with the higher serum level of VC, we speculated that VC injection had the ability to potentially alleviated oxidative stress state through regulating MDA synthesis in post-transit yaks.
4.4 VC improves the anti-oxidant capacity in post-transit yaks
Oxidative stress is induced because of the imbalance between ROS accumulation and antioxidant defenses. To further evaluate the influence of VC injection on anti-oxidant capacity in post-transit yak, the determination of serum levels of antioxidants was performed. H2O2 is a central redox molecule for the reduction to produce H2O and O2 with the assistance of GSH-PX and CAT [42]. Thus, our data of greater serum level of GSH-PX and CAT suggested that VC injection could promote anti-oxidant capacity in post-transit yaks.
Activity of SOD is the first line of defense against oxidative stress through catalyzing superoxide anion radical (O2•–) to H2O2 [43]. The decreased serum level of SOD after VC injection in the present study suggested that it might have been insufficient to stimulate the activity of the enzyme. Interestingly, VC injection led to greater serum level of POD, which could directly promote the generation of H2O2 without the oxidative reaction of O2 to superoxide anion radical (O2•–) [44]. Given that the same product (H2O2) of the catalyzing reaction of SOD and POD, it seems plausible that competition between the SOD and POD systems occurred relative to the generation of H2O2. Similar results were also observed in previous studies [45-46]. Thus, we speculated that VC injection could improve the anti-oxidant capability of post-transit yak through increasing the serum level of POD to regulate H2O2 metabolism.
4.5 Metabolomics
This study has demonstrated that VC injections can cause significant changes in metabolic pathways, with amino acid metabolism being the most stimulated by the VC injection. The present study has also demonstrated that VC injections can alter the synthesis of GSH through regulation of glucose metabolism.
4.5.1 Glutathione Metabolism
Glutathione plays an important role in the protection of cells from oxidative damage, a process which can cause cell death [47]. Glutathione is a non-protein sulfhydryl tripeptide compound formed by the condensation of glutamic acid, cysteine, and glycine through peptide bonds [48]. Thus, changes in the blood concentrations of these three amino acids can affect the concentration of glutathione. Glutamate is located at the center of ammonia-nitrogen exchange and is a major carrier of most non-essential amino acids such as ornithine and citrulline. It is an important precursor for the synthesis of nucleotides, amino sugars, and nicotinamide adenosine dinucleotides. Increased levels of glutamine can be catabolized to compensate for the loss of alpha-ketoglutarate and replenished into the TCA cycle, maintaining adenosine triphosphate and glutathione levels in response to oxidative stress [49-50]. The injection of VC increased glutamate concentrations, and there was an association between glutamate concentrations and antioxidants. In addition, the concentration of glutamate was negatively correlated with SOD, GSH-ST, and TPX, and positively correlated with CAT, POD, and GSH-PX, with the greatest correlation with CAT. Thus, together, these data indicated that an increase in glutamate concentrations could have been responsible for the increase in CAT. The results of the analysis are consistent with the changes in antioxidant concentrations we tested, thus, we speculated that the injection of VC enhanced antioxidant capacity through glutamate.
Cysteine is an amino acid that is commonly found in living organisms. Methionine and cysteine are used as precursors for S-adenosylmethionine, taurine, sulfuric acid, and glutathione [51]. In the present study, VC injection caused an increase in cysteine levels, suggesting that VC promoted activity of the one-carbon metabolism pathway to enhance the metabolism of methionine to homocysteine, which was then rapidly converted to cystathionine. Subsequently, this molecule was converted to taurine and glutathione via the transculturation pathway to mitigate oxidative stress due to transportation. Glycine is a non-essential amino acid that is used in the endogenous production of glutathione, and is often supplemented exogenously when stress occurs in the organism [52]. In this study, VC injection caused an increase of glycine levels suggesting that glycine levels play an important role in the alleviation of oxidative stress.
Besides the increase in the concentrations of glutamate, cysteine, and glycine, and the results of KEGG pathway annotation, VC injections increased glutathione concentrations. Thus, we hypothesized that a key effect of VC injection is to trigger various mechanisms to enhance glutathione synthesis to restore antioxidant capacity during transportation stress.
4.5.2 Phenylalanine, Tyrosine and Tryptophan Metabolism
Phenylalanine can be metabolized to tyrosine via the enzyme phenylalanine hydroxylase. This increase in essential amino acid content induces oxidative stress [53]. The reported effect of phenylalanine on the expression or activity of enzymes participating in metabolic pathways known to be responsive to redox signaling might be mediated through oxidative stress [54]. Therefore, we hypothesize that the increase in phenylalanine levels is related to the transport stress status of yaks. At the same time, the increase in phenylalanine levels also caused an increase in tyrosine levels. Despite the overwhelming evidence for an association between phenylalanine and oxidative stress, it remains unclear whether VC injection is a determinant of the rise in phenylalanine levels and further development of research in this area is needed.
Tryptophan plays (an essential amino acid) an important role in the metabolism, development and growth of animals [55], and it also participates in regulating immunity of livestock [56]. Tryptophan is a precursor of active molecules such as melatonin, a metabolite with antioxidant effects. In addition, 2-Oxoadipic acid, a key metabolite of tryptophan, during the metabolism of tryptophan, the content of 2-Oxoadipic acid was significantly increased in the VC injection group. This is consistent with the higher serum concentration of 2-Oxoadipic acid determined based on VIP analysis. In the correlation analysis, 2-Oxoadipic acid, which is involved in tryptophan metabolism as a member of the amino acid metabolic process, showed positive correlation with SOD and POD, indicating a close relationship between tryptophan metabolism and antioxidants. This conclusion is consistent with the study of Yao et al. who showed that tryptophan and its metabolites (e.g., serotonin (5-hydroxytryptamine, 5-HT) and melatonin)) can regulate feed intake, reproduction, immunity, neurological function, and anti-stress responses [57]. Therefore, it is reasonable to speculate that the increased tryptophan metabolism induced by VC injection is effective against oxidative stress.
4.5.3 Alanine and Aspartate Metabolism
Transamination plays an important role in the degradation of alanine, aspartate and glutamate. This process results in the production of pyruvate, oxaloacetate, and alpha ketoglutarate, which can serve as carbon sources for the tricarboxylic acid cycle and a source of adenosine triphosphate for the synthesis of purine and pyrimidine nucleotides [58]. Thus, the increased concentrations of glutamine, citrate, and aspartate after VC injection indicated that transamination of alanine, aspartate, and glutamate was increased to provide a carbon source for the TCA cycle.
The correlation analysis revealed a relationship between choline and various antioxidants. It shows a negative correlation with SOD, GR, and TPX, and a positive correlation with POD, CAT, GSH-PX, GSH, and GSH-ST, suggesting that an increase in choline concentration caused a decrease in SOD, GR, and TPX concentrations and an increase in the concentrations of other antioxidants that were positively correlated with choline. The changes caused by choline were less pronounced because the correlation was not very strong, which is consistent with the results of our tests on the changes in antioxidant content. Thus, it can be hypothesized that VC injection caused an upregulation of alanine concentration, which in turn led to an increase in pyruvate and choline concentrations to regulate antioxidant levels.
The concentration of asparagine was increased after VC injection. The results of the correlation analysis showed that asparagine also had relationships with various types of antioxidants; it had a significant negative correlation with SOD, GR, and TPX, and a significant positive correlation with POD, CAT, and GSH-PX, and when the concentration of asparagine increased, the concentration of POD, CAT, and GSH-PX also increased, and conversely, the concentration of SOD, GR, and TPX decreased. This is consistent with the results of the tests we did on the variation of antioxidant concentration. Thus, it can be inferred that the injection of VC caused the change of aspartate metabolism, which led to the upregulation of asparagine concentration and finally caused the change of antioxidant content. In our study, changes in aspartate content could modulate the antioxidant content, a finding consistent with that of Sivaperumal et al [59]. In their study, aspartic acid could regulate lipid peroxidation as well as antioxidants. This suggests that the injection of VC can protect against oxidative stress by affecting the metabolism and thus the oxidative stress.