Heat stress as a potential threat to dairy cows during the summer months is globally well recognized. Environmental temperature and humidity are both critical factors for evaluating the comfort of dairy cows, and THI 68 is widely used to determine the margins of the thermoneutral zone for dairy cows [26]. Compared with May, July had significantly higher temperature and humidity. Moreover, THI was above 80, indicating that the cow herds to be tested were subjected to a state of continuous heat stress. In the current study, other heat tolerance related indicators were all major contributors among the first four principal components retained, except for potassium ion content. HSPs are a group of proteins rapidly synthesized by the animal body under heat stress conditions, and play a major role in biological heat tolerance. For instance, when environmental changes generate stress, HSP70 can play a major role in modulating inflammation through enhancing the tolerance of cells to the next harmful injury [27]. Cortisol is related to metabolism, and shows continuous changes, so it is mostly used as a reference indicator of heat stress [26]. The alteration in milk yield of dairy cows, as the most practical indicator for measuring heat resistance, is affected by many aspects such as management practice and lactation number of dairy cows, so it has critical limitations [28]. Apparently, HS animals are known to have increased energy expenditure for heat loss mechanisms via panting and sweating, as shown by the elevation of RR and RT [29]. In our trial, only physiological measures related to heat tolerance were significantly different between the two groups. Estimates of RR and RT in the HT cows were lower than the HS cows, indicating that the HT cows had superior thermoregulatory capacity and less response to heat challenge, which confirm that our adopted grouping strategy was considerably accurate.
Heat stress affects the microbial environment in the rumen, resulting in an increase in pathogenic microorganisms, slowing down of adaptation pathways to the environment, and disorders of immune response pathways and metabolic pathways [30]. The concentration of rumen VFA depends on microbial fermentation efficiency, and host absorption rate [31]. In the current study, there were significant differences in acetic, propionic and butyric acid levels between the two groups, even A:P, indicating that the rumen fermentation pattern was distinct between both HT and HS cows. Rumen as an important nutrient digestion and absorption site in ruminant animals, the resulting products of fermentation performed by the rumen microorganisms are VFAs (mainly acetic, propionic and butyric acids), which directly associated with epithelial absorption by the animal [32]. Among them, acetic acid is an important substrate that has a greater impact on milk fat synthesis; whereas propionic acid is involved in lactose synthesis in milk through the liver, as a major substrate for gluconeogenesis [33]. Moreover, since propionate is the predominant glucose precursor in ruminants, elevations in glucose stimulate milk protein synthesis [34]. We speculate that the differential basic metabolism of VFAs in the HT and HS cows would cause variation in milk quality. Overall, such changes in VFA concentration demonstrate that difference in heat resistance may affect rumen fermentation by shifting the structure and function of the microbial communities within the rumen.
In order to figure out the possible mechanism of rumen parameters whether impacts the heat resistance, we measured the microbiota community via 16S rDNA sequencing technology. For the differential abundance comparison analysis at the phylum level, the ratio of Firmicutes to Bacteroidetes was found to be positively correlated with milk fat in dairy cows, as reported by Jami et al[35]. Firmicutes showed higher abundance with Bacteroidetes in both HS cows and HT cows, which is in agreement with most previous studies, but the difference between the two groups was not significant. Meanwhile, alpha diversity results obtained in this study indicated no significant difference in the species richness of the two groups. It could be, therefore, speculated that although the change of rumen bacteria in the HT and HS cows is closely related to the change of warm environment, the different adaptability of the two groups of cows to ambient environmental changes may be largely caused by the alteration in some rumen microorganisms, rather than the change in the overall bacterial diversity in the rumen.
We observed some genus of Firmicutes, including Ruminococcaceae_UCG-014, and Prevotella_1 has an enrich relative abundance in the HS cows compared with the HT cows, whereas Ruminococcaceae_NK4A214_group, and Christensenellaceae_R-7_group have higher abundance in the HT group than the HS cows. These genus were related to eating habits [36]. In particular, Ruminococcaceae taxa plays an important role in cellulose and hemicellulose degradation as well as rumen biohydrogenation pathways, where the enhancement of hemicellulose degradation can improve feed utilization and ultimately improve animal performance [37]. Therefore, both HT and HS cows under heat stress conditions may have significant differences in feed intake and uptake of nutrients such as fat and protein. Such specific mechanisms of action during heat stress needs further investigation. In Chinese sheep, Mi [38] found that a negative correlation between Succiniclasticum and methane emissions. Not only Succiniclasticum, but also Prevotella_1 and Rikenellaceae_RC9_gut_group were able to reduce the methanogenesis rate. Importantly, Succiniclasticum can produce propionic acid by succinic acid deacidification compete with methanogens for hydrogen and contribute to the attenuation of methanogenesis [39]. Rikenellaceae_RC9_gut_group was involved in the VFA production and the scavenging of H2 [40]. The HT cows may also inhibit rumen fermentation and reduce methane emission in the rumen environment by inhibiting protein and fiber degrading bacteria represented by Prevotella_1 [41]. In addition, Rikenellaceae_RC9_gut_group, which belong to the Bacteroidetes phylum, is closely related to the metabolism of thyroxine [42]. Rikenellaceae_RC9_gut_group was negatively correlated with RR and RT measured in the present study; whereas it correlated positively with propionic acid that had the greatest effect on the bacterial species distribution in the HT cows as shown by the RDA analysis. This is consistent with the fact that propionic acid, which is a substrate for gluconeogenesis, activates gluconeogenic gene expression to maintain energy homeostasis [43]. It is speculated that Rikenellaceae_RC9_gut_group plays an essential role in heat production and body temperature regulation in the HT cows, and promotes the recovery of the metabolic rate in heat stress environment. The results mentioned above indicate that HT cows can reduce methane emission and decrease heat production. Briefly, these differential genera play a crucial role in the basic metabolic process in the HT cows.
Metabolites reflect alterations in the metabolism of dairy cows, which can enable a comprehensive understanding of an organism’s physiological and biochemical status. High-yielding dairy cows under the heat stress conditions noticeably consume large amounts of glucose for fat mobilization and are mostly in a negative energy balance state. According to previous reports, cows can alleviate the negative energy balance through the activity of fatty acid oxidation, and glycerol catabolism pathways [44]. In the current study, we found that the contents of maltose, glycerol, and mannitol in the HT cows increased compared with the HS cows, where these metabolites were mainly involved in carbohydrate metabolism. It is worth noting that glycerol, as an important precursor of gluconeogenesis, provides energy to cells through glycolysis in the liver [45]. Simultaneously, glycerin and long-chain fatty acids form triglycerides, which are important component involved in milk fat synthesis [46]. Agreed with that reported by Wheelock [47], we speculate that the HT cows may minimize the adipose tissue triglyceride mobilization allowing for a stronger ability to alleviate the negative energy balance they suffer from. According to Robergs and Griffin [48], glycerol intake contributes to improved thermoregulation and heat tolerance ability when humans are exposed to hot environments. Similarly, Kim [14] has reported that Holstein cows had higher RT and RR than Jersey cows under the heat stress condition of THI of 87.5, providing that the last breed is less susceptible to heat stress, and has more carbohydrate-related metabolic pathway genes. Consistent with previous reports, our data demonstrate that differential metabolites (glycerol, mannitol, and maltose) were negatively correlated with RR, since lower RR values indicated to the thermoregulatory ability of the HT cows, which were more advantageous than the HS cows in terms of body heat production and heat dissipation, and showed better adaptability to harsh environments. Furthermore, maltose and mannitol were positively correlated with Rikenellaceae_RC9_gut_group. It is worth noting that Rikenellaceae_RC9_gut_group can degrade structural carbohydrates and starch in the rumen of dairy cows and plays a key role in carbohydrate metabolism [49]. This would imply that a shift of the metabolic pathways of microbes may be related to carbohydrate degradation. Under the same dietary level and feeding conditions, HT cows may convert microorganisms in the rumen into VFAs through carbohydrate metabolism [50], in order to obtain the carbon skeleton of gluconeogenesis and improve their own heat resistance and by providing metabolic energy required for rumen microbiota.