The prevalence of heat-stressed conditions in dairy cows is projected to become more frequent and severe due to global warming (Thornton et al., 2021). Advancing our knowledge of the underlying physiology is important for designing nutritional strategies targeting the alleviation of the negative effects of heat stress (Wheelock et al., 2010; Ruiz-González et al., 2023). Studies showed that the drop in dry matter intake (DMI) can only explain up to 50% of the decrease in milk yield, thus the negative metabolic effects caused by the heat stress could be partially responsible for the remaining drop in production performance (Rhoads et al., 2009; Wheelock et al., 2010).

Unlike the metabolic and endocrine regulation during the transition period where the DMI deficiency and negative energy balance are associated with decreased insulin sensitivity and upregulated lipolysis, the DMI drop during heat stress is linked with downregulated lipolysis and decreased glucose use in the mammary gland (as reviewed by Baumgard and Rhoads, 2013). These metabolic changes support the increased energy demand of the activated immune system. The immune activation is mainly due to the increased lipopolysaccharides (LPS) load as a consequence of local hypoxia and impaired integrity of the intestinal epithelial barrier (‘leaky gut’) (Lambert et al., 2002; Pearce et al., 2013). In response, muscle protein catabolism is enhanced supporting the increased hepatic and intestinal AA utilization, while AA availability for the mammary gland decreases (Rhoads et al., 2009; Kvidera et al., 2017a). These metabolic effects triggered by heat stress are parity-dependent as multiparous cows usually show more pronounced changes than primiparous cows (Bernabucci et al., 2014; Chen et al., 2022).

One of the main metabolic responses to heat stress is the enhanced labile protein catabolism, however, an increased supply of metabolizable protein (MP) was found to be unsuccessful in modulating muscle catabolism (Kaufman et al., 2018). Under thermoneutral conditions, dietary supplementation of limiting AA such as Met, Lys and His improved N use efficiency in low-protein diets concomitant with unchanged milk and milk protein synthesis (Lee et al., 2012; Räisänen et al., 2021). Under heat-stress conditions, milk yield and oxidative status of dairy cows was improved by increased Met supply, rather than MP (Mesgaran et al., 2022). In vitro studies on hyperthermic mammary gland epithelial cells indicated that increased Met supply can lead to a higher synthesis of the antioxidants taurine and glutathione and stimulate protein synthesis through the direct activation of the mechanistic target of rapamycin (mTOR) pathway or indirectly by increased branched-chain AA (BCAA) availability (Dong et al., 2018; Lopreiato et al., 2020; Coleman et al., 2022). However, the improved productive performance of heat-stressed cows after dietary Met supplementation was not consistent across the few reported studies (Pate et al., 2020; Mesgaran et al., 2022) and the mechanisms and pathways remain to be understood (Lopreiato et al., 2020; Mesgaran et al., 2022). Preliminary results of our team showed that increasing Met, Lys and His supply during heat stress alleviated the rise in body temperature and inflammation status, and improved milk protein content of high-yielding dairy cows (Ruiz-González et al., 2022a). Increased Met and Lys supply effects have been widely studied in other physiological circumstances (e.g. during the transition period). However, their potential to counteract heat stress effects remains to be comprehensively evaluated in dairy cows. Thus, the metabolomics analysis of blood plasma and milk could uncover the possible metabolic pathways by which increased Met, Lys and His supply contribute to heat stress alleviation.

We hypothesized that these alleviating effects of increased dietary Met, Lys and His availability are associated with increased antioxidant synthesis (e.g. taurine) and decreased AA catabolism, contributing to increased availability of other EAA during heat stress. This could be reflected in the plasma and milk metabolome profiles, with more prominent changes in multiparous cows. Hence, the aims of this study were: 1) to elucidate the potential alleviating effects of increased dietary Met, Lys and His supply on the metabolomic changes triggered by heat stress with particular focus to AA-related metabolism, and 2) to explore the possible interactions between the dietary strategy and parity on the metabolomic response to heat stress.

The metabolomic profiling led to the effective quantification of 212 and 164 out of 404 possible metabolites in plasma and milk, respectively. A list of the measured compounds and their abbreviations is available in Supplementary Table S1.

Our plasma and milk metabolomics data suggested that the heat stress-related depletion of the circulating PC pool was reverted by the elevated dietary Met supply. This can be explained by enhanced one-carbon metabolism in response to increased Met availability. In addition, the improved milk protein content in the HS+AA treatment was likely modulated by the increased Val and Trp availability as a consequence of the additional Lys supply. The main findings were linked to increased dietary Met or Lys supply, rather than His, as the increase in His availability was minor relative to the increase in the availability of Met and Lys. Interpretive schemes integrating the main results related to Met and Lys availability are presented in Figures 3, 4, respectively.

In line with their EAA character, i.e. dietary-dependent availability (Schwab and Broderick, 2017), increased dietary supply of Met, Lys and His in HS+AA was reflected by increased plasma and milk concentrations of these AA. Indeed, while Met and Lys supply increased by 77% and 65%, respectively, His was only increased by 22% which was reflected by a less pronounced increase of plasma His in HS+AA, compared with TN or HS treatments. Despite the lack of a significant FDR, plasma His had a significant raw-P value for the treatment effect, since it was higher in HS+AA than in the other two treatments.

The general trend of decreased plasma concentrations of PC, and to a lesser extent some of the LysoPC in HS were likely associated with systemic inflammation as previously proposed (Hung et al., 2012; Liu et al., 2017). While the concentrations of some LysoPC were numerically lower in HS than TN, due to the lack of significant FRD values, the following interpretations aim to propose hypotheses in an exploratory approach for further testing. Phosphatidylcholines are precursors for the synthesis of LysoPC, which play signaling roles at the cellular level and participate in inflammatory response in mammals, including bovines (del Bas et al., 2016; Humer et al., 2018; Rico et al., 2021). Thus, its decreased concentrations in heat-stressed cows were likely related to the systemic inflammation (Kvidera et al., 2017b). In fact, decreased plasma LysoPC concentrations were found after LPS challenge in bovines under thermoneutral conditions (Humer et al., 2018; Javaid et al., 2022). According to the milk lipidomics-based findings of Liu et al. (2017), the decrease in LysoPC should be a structural metabolic shift in response to heat stress as heat stress-sensitive cows have been observed to suffer greater changes in LysoPC than tolerant cows. In the current experiment, PC rather than LysoPC showed more evident changes during heat stress, thus PC were likely further utilized to sustain LysoPC synthesis. In addition, the PC: phosphatidylethanolamine balance plays an important role in membrane response and thermal sensing, which are highly conserved functions across species (as reviewed by Balogh et al., 2013). In summary, the decreased plasma and milk PC concentrations likely indicated increased cell membrane fluidity during HS.

However, increased Met availability in HS+AA appeared to have improved plasma and milk PC concentrations. Six out of 9 plasma PCs, and 4 out of 8 milk PCs that decreased or tended to decrease in HS vs. TN (FDR ≤ 0.10 for the treatment effect) were improved in HS+AA either to similar concentrations as in TN or intermediate concentrations between HS and TN. Moreover, when considering the raw-P values ≤ 0.10, most of the plasma (33 out of 40) and milk (13 out of 17) PCs showed negative fold change in HS compared either to TN or HS+AA (Supplementary Figure S2; Supplementary Tables S2, S3). Thus, our results point to at least a partial recovery of PC synthesis in HS+AA. Interestingly, despite Met being a precursor of taurine, a potent antioxidant that commonly decreases during heat stress (Belal et al., 2018), plasma and milk taurine concentrations did not improve after the increased dietary Met supply in HS+AA. Instead, the increased Met supply likely stimulated the one-carbon metabolism, leading to increased plasma and milk concentration of PC. Met supply can upregulate the phosphatidylethanolamine methyltransferase (PEMT) pathway in bovine liver cells (Zhou et al., 2018) in interaction with folic acid and re-methylation cycles, leading to enhanced synthesis of PC (Myers et al., 2019; McFadden et al., 2020) (Figure 3). In fact, methyl-donor supplementation, through the stimulation of one-carbon metabolism improved the cytoprotective characteristics of heat-stressed polymorphonuclear leukocytes of lactating dairy cows (Lopreiato et al., 2020). In the current experiment, the increased AA supply was associated with a decrease in serum lipopolysaccharide binding protein (LBP) concentration, milk SCC, rectal temperature, and respiration rate during heat stress, suggesting a partial alleviation of the inflammation and oxidative stress (Ruiz-González et al., 2022a). Phosphatidylcholines play a central role in cellular integrity and thus the increased PC availability may help maintain intestinal barrier integrity and prevent the inflammatory consequences of a ‘leaky gut’. In summary, since heat stress appears to enhance PC and LysoPC turnover and utilization by immune activation (Humer et al., 2018), nutritional strategies favoring PC synthesis (including increased dietary Met supply) appear as a potential avenue to alleviate heat stress effects in high-yielding dairy cows.

Plasma hydroxyproline is synthetized from Pro, which also contributes to membrane integrity through the formation of collagen, a key component for cellular thermal stability (Kubow et al., 2015). We detected decreased hydroxyproline concentrations during heat stress in the plasma and decreased Pro concentrations in the milk but not in the plasma. Heat stress enhances protein catabolism including collagen breakdown which is largely composed of Gly and Pro (Ríus, 2019); thus, unchanged Pro and lower hydroxyproline plasma concentrations should be indicative of preferential use of Pro for other processes rather than hydroxyproline synthesis (Figure 3). Pro can act as an antioxidant source (Krishnan et al., 2008) and its lower concentrations in the milk could suggest local utilization to counteract ROS at the mammary gland level. Alternatively, Pro could be channeled to other organs, e.g. the intestines which are particularly challenged by oxidative stress and inflammation during heat stress (Kvidera et al., 2017b). Based on heat-stressed porcine adipocytes, Qu and Ajuwon (2018) suggested that increased Pro catabolism should be linked to antioxidant action during thermal stress.

Lower plasma and milk Gly concentrations in HS and HS+AA are also in agreement with previous studies as this might be the consequence of its increased utilization for antioxidant synthesis and gluconeogenesis in response to heat stress (Ríus, 2019). Fan et al. (2019) reported lower concentrations of both Gly and Pro in the mammary gland of heat-stressed multiparous cows. Gly is involved in the synthesis of folic acid which interacts with the Met cycle and the transsulfuration pathway, hence serving as a precursor for glutathione together with Cys and Glu. In turn, the reduced form of glutathione is a potent antioxidant that typically decreases under heat-stressed conditions (Sordillo and Aitken, 2009; Kawano et al., 2022). In addition, Gly has been suggested to have a direct antioxidant activity in intestinal epithelial cells during heat stress (Wang et al., 2014), supporting the intestinal barrier integrity by regulating the gene expression of tight junction elements (Li et al., 2016). Gly supplementation was shown to alleviate whole-body protein degradation and inflammation in growing piglets challenged by LPS (Wu, 2015). Both of these phenomena (inflammation and labile protein catabolism) also occur in dairy cows in response to heat stress (Ríus, 2019). Thus, the decreased milk concentrations of Gly during HS could be the consequence of its primary use in support of the antioxidant system and counteracting inflammation.

Increased plasma and milk concentrations of Val and Trp in HS+AA likely reflected a combined effect of increased dietary supply and a lower oxidation of these AA due to higher Lys availability. In fact, the HS+AA treatment showed 20% and 27% higher plasma and milk Val concentrations, respectively, and 25% higher milk Trp concentration, compared with HS. However, the dietary supply of Val and Trp was estimated to be 11% higher compared to the other treatments due to the inclusion of blood meal, according to the NASEM system (NASEM, 2021; data not shown). Therefore, we hypothesize that preferential oxidation of Lys due to its increased availability led to downregulated oxidation of Val and Trp and therefore to an increased availability of these EAA. In fact, in the current study, isotopically labeled Leu experiments performed on the last experimental day (day 14) demonstrated that its increased fractional oxidation during heat stress was reversed in HS+AA, possibly due to a higher oxidation of Lys (Ruiz-González et al., 2022b). Our metabolomics data seem to confirm this hypothesis as Lys catabolic products (α-aminoadipic acid, acetyl-carnitine) decreased during heat stress but increased in HS+AA in both plasma and milk (Figure 4). Moreover, Trp which has a common catabolic pathway with Lys, was also increased after the AA increased supply. In particular, α-aminoadipic acid produced from Lys can be metabolized into 2-oxoadipic acid by kynurenine-α-aminoadipic acid transferase, an enzyme previously reported in cows (Deshmukh and Mungre, 1989; Leandro and Houten, 2020). Thus, this enzymatic connection between Trp and Lys could regulate preferential degradation of Lys instead of Trp. Downstream Lys degradation also overlaps with fatty acid degradation, with acetyl-CoA being the catabolic end product. Accordingly, decreased milk acetyl-carnitine in HS was reverted in HS+AA and butyryl-carnitine increased during heat stress and decreased after the increased AA supply, suggesting an enhanced flow of the Lys degradation pathway and improved mitochondrial fatty acid metabolism. In a similar experiment comparing thermoneutral and heat-stressed conditions in multiparous cows, we observed increased milk carnitine and butyryl-carnitine concentrations during heat stress (Jorge-Smeding et al., 2023), suggesting incomplete β-oxidation due to impaired mitochondrial metabolism (Kenéz et al., 2015; Huber et al., 2016; Kinoshita et al., 2018).

Our results disagreed with the findings of Kassube et al. (2017), where Met, Lys and BCAA jugular infusions did not affect milk protein synthesis during heat stress. On the contrary, our previously published results demonstrated that the decreased milk protein content in heat stress was reversed in the HS+AA treatment (Ruiz-González et al., 2022a). Not only did milk protein improve but also milk urea decreased in HS+AA treatment, indicating improved AA utilization efficiency. The increased Met and Lys availability likely also contributed to improved milk protein content as these are usually the most limiting AA (Bequette et al., 1998). Besides, the increased concentrations of other EAA, in particular BCAA, likely stimulated protein synthesis through the mTOR pathway (Doelman et al., 2015). Decreased availability of individual AA and downregulated activity of mTOR, a master regulator of protein synthesis stimulated by BCAA and Lys (Doelman et al., 2015; Lin et al., 2018), were suggested to explain the decreased protein synthesis in the mammary gland during heat stress (Ríus, 2019). Heat stress downregulated the expression of genes involved in AA utilization, AA transport, and mTOR kinase activity in bovine mammary epithelial cells (Kaufman et al., 2018; Dado-Senn et al., 2021). Through a different pathway, insulin also activates downstream mTOR pathways but it is dependent on BCAA availability (Shimobayashi and Hall, 2016; Kenéz et al., 2019). Protein synthesis stimulation in the mammary gland through the mTOR pathway is associated with increased mammary gland insulin/IGF1 sensitivity during lactation (Bionaz and Loor, 2011). In the current experiment, we found increased plasma insulin concentrations during heat stress which was reverted in HS+AA by the end of the experimental period along with the recovery of milk protein content and decrease of milk urea (Ruiz-González et al., 2022a). Lastly, Met was also suggested to activate the mTOR pathway in the mammary gland, however, the exact mechanisms are not yet known (Coleman et al., 2022). Our results indicated that improved milk protein content in HS+AA was associated with the increased availability of EAA, however, the exact interactions between Lys, BCAA, Met and the mTOR pathway are yet to be fully elucidated.

Changes in the concentrations of Phe and Tyr were part of the metabolic response to heat stress, which was independent of Met and Lys availability. Previous heat stress studies reported a parity-dependent increase of Phe and Tyr concentrations, highlighting significant differences only in primiparous cows (Chen et al., 2022). In contrast, our metabolic pathway analysis found the Phe and Tyr metabolism differed only in the multiparous cows. Dado-Senn et al. (2021) observed increased plasma concentrations of Phe after heat stress pointing to links with immune and heat shock response. Phe metabolism interacts with T-cell immune response suppression (Yang et al., 2012) and its supplementation increased HSP70 expression (Plakidou-Dymock and McGivan, 1994). Additionally, Phe is directly catabolized to Tyr by phenylalanine hydroxylase (Crompton et al., 2018). In turn, Tyr can be either oxidized or used to synthesize thyroid hormones (T3, T4) which play a central role in thermal homeostasis and regulation of the basal metabolic rate (Aleena et al., 2016). It has been suggested that decreased blood flow to the thyroid gland due to vasoconstriction during heat stress could lead to downregulated secretion of thyroid hormones in rabbits (Mustafa et al., 2008). Thus, increased concentrations of the Phe and Tyr could reflect downregulated secretion of thyroid hormones leading to the accumulation of its precursors as observed previously (Magdub et al., 1982; Weitzel et al., 2017; Pontiggia et al., 2023).

Heat stress caused a shift in the plasma and milk metabolic profiles and some of these changes were restored due to the increased dietary Met, Lys and His availability. Our metabolomics findings herein suggested that this was associated with an increase of the circulating phosphatidylcholine pool. Further, the increased Lys availability associated with the greater availability of the overall BCAA pool can likely explain the increase in milk protein synthesis. Our results suggest that increasing dietary supply of Met, Lys and His can be useful to counteract the negative metabolic effects of heat stress; however, our results pointed to stronger links with Met and Lys, likely due to their higher supplementation rate relative to His. Further studies are warranted to validate these results on a larger independent cohort of cows and to establish specific nutritional recommendations considering parity, genetic merit, and degree of heat stress tolerance.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

The animal study was approved by The Animal Care Committee of the Centre de Recherche en Science Animales de Deschambault. The study was conducted in accordance with the local legislation and institutional requirements.

EJ-S: Writing – original draft, Formal Analysis, Data curation. AR-G: Writing – review & editing, Investigation. YL: Writing – original draft, Investigation. AT: Writing – review & editing, Supervision. AA: Writing – review & editing, Supervision. DO: Writing – review & editing, Resources, Conceptualization. HL: Writing – review & editing, Resources, Conceptualization. DR: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. ÁK: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.