In this study, we used 16S rRNA and LC–MS metabolomics to compare milk bacteria and metabolites, revealing that ART moderately increased milk production and milk fat content and tended to decrease the SCC at the end of the treatment. Although there was no significant difference in the bacterial diversity (Shannon and Simpson indexes) between the ART and CON groups, the bacterial community richness (ACE and Chao indexes) was significantly lower in the ART group. In addition, the phylum abundances showed that Firmicutes was significantly decreased, while Proteobacteria was higher after treatment with ART. The well-recognized functional data of the milk microbiota can be used not only to identify the quality of milk but also to judge the health status of dairy cow mammary glands (Mansor 2012; Sun et al. 2017a). Correlation analysis of the microbiota and metabolites in milk revealed changes in Aerococcus, Facklamia, and Staphylococcus-related metabolites. Those identified metabolites could cause the differences in milk components between the groups. Thus, we propose that ART activities affect the organism in terms of its milk microbiota and metabolites, in turn triggering milk fat changes.
In the present study, ART supplementation moderately increased milk yield and significantly increased milk fat rate. In line with a previous study, adding Artemisia annua to the cow diet increased milk production, which was attributed to the activity of phenols and flavonoids in Artemisia annua (Ferreira et al. 2011; Zhan et al. 2017). Furthermore, it has also been reported that plant flavonoids can increase the acetic acid concentration of dairy cows (Broudiscou et al. 2000). It is well known that acetic acid is the main precursor for milk fat synthesis; similarly, cow milk fat production can be significantly increased by intravenous acetic acid injection (Storry and Rook 1965). Therefore, milk fat increase might be caused by rumen acetate acid changes, which warrants further investigation in future studies.
In addition, the SCC tended to decrease in the ART group compared with the CON group. The SCC is one of the most useful and widely used tools to predict mammary gland health in bovines (Tong et al. 2019); thus, our results proved that ART could reduce mastitis susceptibility. Similar to the results of previous studies by Zhan et al. (Zhan et al. 2017), feeding 60 mg/kg alfalfa flavones could reduce the SCC, alter the composition of milk, improve antioxidant properties and affect immunity in dairy cows. In addition, members of the microbiota such as corynebacteria in milk are usually associated with low-SCC intramammary infections (IMIs) (Guan et al. 2014). Accordingly, the ART antimicrobial and anti-inflammatory properties function by inhibiting the synthesis of cell walls and membranes, interfering with enzymes in or on specific cells and inhibiting bacterial proliferation that causes mastitis in dairy cows (Cushnie and Lamb 2011), especially when modulating the rumen or gut microbiota (Amaretti et al. 2015; Zhan et al. 2017). Similar results were also obtained in our study; the relative abundance of Firmicutes was significantly decreased with ART supplementation, whereas that of Proteobacteria was significantly increased. These species are similar to members of the rumen microbiome (Wang et al. 2018), and we hypothesize that the bacteria can be transferred from the rumen to the mammary gland. Therefore, ART may be associated with a systemic and local immune responsiveness of the udder that is accompanied by microbiota changes.
Multivariate analysis of milk
16S rRNA sequencing and analysis provide a low-cost and high-yield method for the evaluation of milk microflora (Caporaso et al. 2011; Hélène et al. 2016). In the present study, milk microbial richness was significantly decreased in the ART group. Recent investigations of the bovine milk microbiota show that it usually has a variety of bioactive or probiotic properties to resist the defense mechanism of the udder (F et al. 2016). Additionally, the effect of plant bacteriostatic factors on the rumen microbiota or pathogens of mastitis in dairy cows (Durmic et al. 2008; Cushnie and Lamb 2011) may also be associated with milk microbiota changes in the present study. Similarly, antimicrobials could reduce the diversity of microbial ecosystems and, in alternative stable forms, accompany this with reduced species richness (Lozupone and Knight 2006). Henderson et al. (Henderson et al. 2013) also reported that sample extraction may also affect DNA quality and impact microbiota diversity. Moreover, previous research found that milk microbial diversity was related to mastitis (Hélène et al. 2016). Therefore, no difference in microbiota diversity was caused by the ART treatment, which will strengthen the use of ART as novel prophylactic or therapeutic product (or both) alternatives to antimicrobials in dairy cows.
In our current study, Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes were the major phyla in the milk of the two groups, and the predominant genera were Aerococcus, Corynebacterium_1, Staphylococcus and Enterococcus, which is consistent with previous studies (Zhang et al. 2017). Similar results were also obtained in the current study; Firmicutes was significantly decreased with ART supplementation, whereas Probacteria was significantly increased. These species are similar to those in the rumen microbiome (Jinjin et al. 2018; Wang et al. 2018). Furthermore, recent reports support this entero-mammary pathway via lymphatic and peripheral blood circulation in humans and mice (Rodríguez, 2014) via transfer from mothers to neonates by the gut-breast axis (Jost et al. 2014).
It has been shown that mastitis in cows or goats by Staphylococcus (Guan et al. 2014) causes serious losses in dairy products and to the animal husbandry industry (Tong et al. 2019). Increases in the relative abundances of Corynebacterium boris, Aerococcus and Staphylococcus increase the SCC in the milk of dairy cows (Hogan et al. 1988; Sun et al. 2017b). Furthermore, the contents of Corynebacterium, Facklamia and Aerococcus were higher in dairy cows with a history of clinical mastitis than in healthy dairy cows (Hooman et al. 2018). These pathogens can be found in healthy dairy cows because they are an important cause of mastitis when the proportions of bacteria are out of balance (Guan et al. 2014). In our study, the Staphylococcus, Corynebacterium and Aerococcus abundances were extremely reduced in the milk from the ART group; these genera were the most commonly identified genera omnipresent in the dairy environment and gained great attention as the leading bacteria in IMI (Pyrl and Taponen 2009; Vanderhaeghen et al. 2014; Hooman et al. 2018). Our study also detected that Pseudomonas was negatively correlated with most metabolites associated with the ART treatment. (Figure 6). It has been reported that Pseudomonas causes milk deterioration by producing lipase and proteolytic enzymes, and the quality of milk can be maintained by controlling its growth (Chikage et al. 2018). When the lipase content increased, the hydrolysis of triglycerides in dairy cows and long-chain fatty acid production increased. These mechanistic insights into the microbiota response to ART may have important implications for understanding how the milk microbiota participates in biosynthesis regulation and in relation to udder health and mastitis.
Differences in milk metabolites
Metabolomics is an emerging area of research involving organisms; its methods detect small molecular metabolites in samples and enable a comprehensive understanding of biological processes (Sun et al. 2017a). We used LC–MS to analyze the milk metabolite response to ART supplementation. In total, 35 different metabolites were identified between the ART and CON groups. Some metabolites were upregulated, such as glycerolipids (MG(0:0/14:0/0:0)), flavonoids (isovitexin 7-(6’’’-sinapoylglucoside)4′-glucoside and 6′’-p-coumaroylprunin), carboxylic acids and their derivatives (5-aminopentanamide), and vitamins (pantothenic acid). MG is a monoacylglyceride product of triglycerides that regulates liver development and activity (Coleman and Haynes, 1984). In our study, MG was significantly increased (by 2.67-fold) in the ART group, which strengthens the idea that ART could trigger the mammary gland response to milk biosynthesis, and a remarkably negative correlation was found between MG and Aerococcus. Meanwhile, MG is absorbed more easily than other fatty acid derivatives in the intestine; this absorption increases the content of docosahexaenoic acid and increases the antioxidant activity of mouse plasma (Cho et al. 2009). A plausible hypothesis, similar to one previously reported, is that the mechanisms underlying the effects of nutrition on the potential involvement of the microbiota in the microbiome-gut-brain axis (Cryan and O’Mahony, 2011) are worthy of further study.
In the present study, we found that the flavonoids increased the most of all the metabolites, followed by isovitexin 7-(6′’’-sinapoylglucoside)4′-glucoside (by 3.81-fold) and 6′’-p-coumaroylprunin (by 2.52-fold), in the ART group. These metabolites were negatively correlated with Staphylococcus. Recent studies have shown that flavonoids have antibacterial and antioxidant properties that promote the production and quality of animal products (Zhan et al. 2017). Furthermore, flavonoids increase the activity of antioxidant enzymes to enhance antioxidant capacity and protect tissues and cells from free radical-mediated damage (Xiao-Shuang et al. 2015). These findings may partially explain the moderate SCC decrease induced by ART supplementation. Interestingly, we detected that many metabolites were significantly correlated with members of the microbiota that were potential pathogens, such as Staphylococcus, Facklamia, and Aerococcus. Our findings collectively highlighted the observed microbiota and metabolic changes and provided further insight into the performance of specific ART-related functions in milk profiles that can be used to analyze the differences in milk synthesis between the groups. We speculated that this discrepancy might be associated with the decrease in the SCC resulting from ART supplementation.
PS and PE are important components of biomembrane structure (Cole et al. 2012). The phospholipids in milk are mainly PE, PC and SM (Shoji et al. 2006). PC, which plays a key role in lipid metabolism, can synthesize very low density lipoprotein (VLDL) and be used for the export of triacylglycerol (TAG) in the liver (Cole et al. 2012). Furthermore, aggregation of ATG in the liver may increase fatty liver production (Elke et al. 2016). The current study revealed that PC and PS production was downregulated and PE production was upregulated in the ART group compared with the CON group. Furthermore, the mammary gland utilizes blood PC as a cellular energy source for the production of glycerophosphocholine and free fatty acids and can synthesize milk triglycerides and phospholipids (Easter et al. 1971). Thus, this might explain the mechanism by which the milk fat content was significantly increased in the ART group; specifically, we revealed that glycerophospholipid metabolites were significantly reduced by ART treatment. However, the content of PC was lower in the CON group because when the PC breakdown rate decreased, which is closely associated with negative energy balance (NEB) and fat mobilization, causing metabolic disorders and ketosis (Klein et al. 2012). In conclusion, our results indicate that the glycerol phospholipid metabolic pathway may be the main component of the mechanism by which ART affects milk quality.
Key differential metabolic pathways between the two groups
Based on a comprehensive analysis of important metabolic pathways that identified 33 differential milk metabolites between the two groups, lipid metabolism may be the most important pathway for milk quality improvement induced by ART. An unavoidable NEB accompanies various metabolic disorders and affects the immune system, and the energy and lipid metabolism of dairy cows is abnormal and vigorous, causing many diseases (Bouvier-Muller et al. 2018). However, the conversion of PC into milk fat could protect free blood fatty acids, thus reducing the mobilization of body fat (Klein et al. 2012). It is reasonable to conclude that ART protects the health of the body. Furthermore, ART supplementation significantly increased β-alanine metabolism relative to that of the control group. β-Alanine metabolism mainly occurs in muscle and brain, and its final metabolite is acetic acid (Griffith 1986). Meanwhile, β-alanine can improve the antioxidant capacity of the body(Smith et al. 2012), which may be the reason for the improvement in milk quality. In addition, pantothenate and CoA biosynthesis was also significantly upregulated by ART. Pantothenic acid is a water-soluble vitamin. When it is transformed into CoA or bound to acyl carrier protein (ACP) in vivo, it mainly participates in the metabolism of fatty acids, carbohydrates and energy and significantly reduces the levels of cholesterol and triglycerides (Smith and Song 1996). This may reduce abnormalities in lipid and energy metabolism in dairy cows. Therefore, our data indicate that glycerophospholipids and glycerolipids could be potential biomarkers in the milk response to ART feed in dairy cows, which further supports the functional link between ART and milk changes. Taken together, the results in our study support the assumption that ART changes substances in milk by maintaining lipid metabolism in the mammary gland.
In summary, LC–MS and 16S rRNA was used to analyze milk metabolomics and bacterial community profiles for dairy cows, revealing that ART supplementation increased milk fat, decreased the SCC and may affect the structures of bacterial communities, metabolites and metabolic pathways. Moreover, ART decreased the relative abundances of Corynebacterium_1, Aerococcus, Staphylococcus and Facklamia. Our results also revealed that some of the 33 metabolites that changed significantly in milk after ART supplementation were potential biomarkers that respond to ART. In addition, glycerophospholipids and glycerolipids could be potential biomarkers in the milk response to ART feed in dairy cows, and ART changes substances in milk by protecting lipid metabolism in the mammary gland. This study has provided further insights into the mechanisms at the metabolic level that can improve understanding of the effects of ART addition. Overall, our findings provide new strategies for improving milk quality with the use of ART; however, they warrant further investigations for identification of potential immune regulation mechanisms underlying the effects of ART on dairy cows.