Effect of Moringa leaf flavonoids on the production performance, immune system, and rumen fermentation of dairy cows

Abstract Background Unreasonable use of antibiotics in animals is a major concern and will remain so, thus affecting people's health. However, the application of plant extracts can better solve this problem. Objectives The purpose of this study was to study the effect of Moringa leaf flavonoids on the production performance, immunity, and rumen fermentation of dairy cows. Methods Nine Holstein multiparous cows (average weight: 550 kg; days of lactation: 150 ± 6 days) were used in the experiment, using a 3 × 3 Latin square design. Cows were divided into three groups, each of which was supplemented with 0, 50, or 100 mg/body weight (BW) Moringa oleifera leaf flavonoids. Each experimental period consisted of three periods of 21 days, and the prefeeding period lasted 15 days. Results Our results indicated that supplementation with Moringa leaf flavonoids increased the protein content and decreased the number of somatic cells in milk; had little effect on the biochemical indices of blood, the rumen fermentation, and serum biochemical indicators; and improved the activity of antioxidant enzymes, the antioxidant capacity, and immunity. Conclusions Addition of 50 mg/BW Moringa leaf flavonoids to cow enhanced the antioxidant and immunity capacity in dairy cows but did not affect physiological levels of common biochemical parameters in blood or fermentation parameters in rumen.

Flavonoids are polyphenolic compounds commonly found in plants as secondary metabolites. Flavonoids have proven abilities in enhancing immunity in animals, through their anti-pathogenic, anti-oxidation, and anti-inflammatory functions. Flavonoids can affect rumen metabolism and rumen microbial fermentation (Linville et al. 2018) and promote the circulation of blood in dairy cows, thereby enhancing their metabolism, promoting the absorption of nutrients, and increasing milk production (Zhan et al., 2017). Flavonoids also have a weak estrogenic effect, which regulates the secretion of growth hormone and promotes breast development (Liu et al., 2020). Flavonoids can promote nitrogen metabolism in the rumen of dairy cows and can reduce methane production (Leake & Rankin, 1990).
Moringa is rich in nutrients and biologically active compounds, and thus has great potential to be used as a supplement in livestock feeds.
The leaves, seeds, and bark of Moringa can be readily consumed by cows, sheep, goats, pigs, chickens, and rabbits (Radványi et al., 2013).
Consumption of Moringa has been proven to improve the health, growth performance, milk production, and meat quality of livestock (Nardone & Valfrè, 1999). Moringa contains various flavonoids in the leaves, roots, flowers, and seed coats, and the contents of flavonoids vary depending the geographic origins of Moringa. The most common flavonoids in Moringa leaves are kaempferol, quercetin, isorhamnetin, and apigenin (Milugo et al., 2013).
So far, there have been few reports of the effects of Moringa leaves extracts rich in flavonoids on dairy cows. The aim of this study was to evaluate the effects of Moringa leaves flavonoids extract on the production performance, immune responses, and ruminal fermentation of dairy cows.

Materials
Nine Holstein cows from Wonderson pasture in Harbin were used in the experiment. The cows were in milk for 150 ± 6 days, with average body weight (BW) of 550 ± 25 kg. The experiment was conducted in a 3 × 3 Latin square design, consisting of three 21-day experimental periods, with 15 days for adaptation. Cows were divided into three groups and fed a total mixed ration (TMR) (

Testing of milk samples
The milk samples were stored at 4 • C and then submitted to Heilongjiang DHI Testing Center for analysis with a multifunctional dairy

Blood biochemical test
On the 20th day of each phase of the test, blood was collected from the tail vein with a coagulation vacuum tube (containing inert separating gel) before and 2 h after ingestion. The blood was allowed to stand for 1 h at room temperature, followed by centrifugation to separate the serum, which was then aliquoted and stored at −20 • C until further analysis.

Volatile acid determination
The temperature of the inlet and detector was 220 • C. The oven temperature scheme started with initial temperature at 120 • C for 3 min, and then increased to 180 • C at 10 • C/min. The carrier gas was highpurity nitrogen. The port pressure was maintained at 90 kpa; the hydrogen flow rate was 40 ml/min, the airflow rate was 400 ml/min, and the makeup flow rate was 45 ml/min.

Determination of antioxidant and immune indicators
The immunoglobulin content (IgG, IgM, and IgA) was determined by radioimmunoassay, which was completed by the Beijing Huaying

3.5.1
Effect of Moringa leaf flavonoids on dairy cow performance, blood biochemistry, and rumen fermentation

Effect of Moringa leaf flavonoids on dairy cow performance
The protein content in the milk was increased by the diet supplemented with 50 mg/BW Moringa leaf flavonoids compared to the control (p

Effect of Moringa leaf flavonoids on blood biochemical indicators
The blood total protein, ALB, GLB, low-density lipoprotein (LDH), and GLU content increased with the increase of the amount of Moringa leaf flavonoids with some noticeable variations. The content of HDL increased as the amount of the Moringa leaf flavonoids increased, and HDL was the highest in the diet supplemented with 100 mg/BW. However, no significant differences were observed among treatments. The above data indicate that Moringa leaf flavonoids had no significant effect on blood biochemical indicators.

Effect of Moringa leaf flavonoids on the volatile acids of milk
The content of acetic acid, propionic acid, and butyric acid in milk was lower in the treatments supplementation with Moringa leaf flavonoids than the control. The content of valeric acid, isobutyric acid, and isovaleric acid was the highest in the treatment with 50 mg/BW Moringa leaf flavonoids, but no significant differences were observed among diets.

DISCUSSION
The addition of appropriate amount of Moringa leaf flavonoids did not increase milk production in dairy cows. Some studies have shown that the addition of flavonoids in the form of grape pomace powder, green tea, and turmeric extracts does not increase milk production (Williamson et al., 2005). The results of our study are consistent with these findings. However, previous work has shown that the addition of propolis flavone as a feed additive has no effect on the number of somatic cells in the milk of dairy cows, which is inconsistent with the results of our study showing that the addition of Moringa leaf flavonoids decreases the number of somatic cells. This inconsistency can be explained by the different sources, types, and structures of flavonoids used (Zicker & Wedekind, 2005). Elevated somatic cells in milk indicate poor breast health and milk quality, which is a major problem in modern animal husbandry. Cows with elevated somatic cells produce less milk than healthy cows, which results in major economic losses to the dairy industry. Approximately 70% of subclinical mastitis is related to a temporary or permanent decrease in milk production, which mainly stems from inflammatory damage to breast tissue (Eckersall et al., 2006). In general, nutritional factors play a key role in enhancing resistance to breast infections, and excessive amounts of micronutrients with effective antioxidant and immuneenhancing properties added to the diet such as plant extracts have been reported to enhance breast health and reduce the somatic cell count (Scaletti et al., 2003). Milk production is lower in cows with higher initial somatic cell levels. Previous studies have shown that breast infections may significantly reduce milk production (Tikofsky et al., 2003). Previous work indicates that damage to secretory tissue and fibrous tissue can lead to breast infection, which causes a temporary or permanent decrease in milk production (Lauzon et al., 2006). The leaf flavonoids showed no effect on serum total protein, including GLB, ALB, HDL, GLU, and LDL.
Rumens are unique digestive organs of ruminants that contain a large number of microorganisms, which can obtain nutrients from feed through fermentation processes. Regulation of the rumen can improve the utilization rate of feed and promote the production performance of ruminants. The volatile acids produced by rumen fermentation of dairy cows are important energy sources for dairy cows, among which acetic acid, propionic acid, and butyric acid are the three main volatile acids accounting for more than 80% of the total volatile acids (Foiklang & Toburan, 2011). Volatile fatty acids are the main product of carbohydrate degradation in the rumen, the main energy source for ruminants, and the main raw material for synthetic milk fat. Xi et al. (2007) found that the addition of vegetable oils significantly reduced total volatile fatty acid (TVFA) production. Using in vitro culture methods and hay as the fermentation substrate, Vasta et al. (2008) showed that cinnamon oil has no significant effect on the concentration of volatile acids and the ratio of acetic acid and propionic acid. Kwekkeboom et al. (1993) studied the effect of capsaicin on the rumen fermentation of lactating dairy cows and showed that these plant essential oils had no effect on TVFAs, their composition, and ammonia nitrogen. The findings of our study were similar to their results, as Moringa leaf flavonoids did not affect the rumen fermentation index.
Free radicals play an important role in immunity and signal transduction, but excessive free radicals can lead to the lipid peroxidation of cell membranes (Turner et al., 2004). Endogenous antioxidant enzymes, including SOD, CAT, and GSH-Px, neutralize oxidative stress, which is the main form of intracellular defence (Manor et al., 1999). The antioxidant effect is achieved by the conversion of oxygen-free radicals into weakly oxidized forms (Kamel, 2005). Supplementation of flavonoids can improve antioxidant capacity, improve non-specific immunity, and reduce oxidative stress by increasing SOD and GSH-Px activity while reducing the concentration of MDA. The underlying mechanism involves flavonoids acting as reducing agents and hydrogen donors to neutralize oxygen-free radicals and remove hydrogen peroxide and superoxide ions (Kahkonen, 1999).
In of previous research, we speculate that Moringa leaf flavonoids stimulate mitochondrial adenosine triphosphate production, which may be mediated by active oxidants and enhance cellular/mitochondrial antioxidant status. However, the specific mechanism by which Moringa leaf flavonoids enhance immune function requires further study (Leonard et al., 1984). The addition of Astragalus extract in the diet can reduce the plasma T 3 level, which may lead to a decrease in the basal metabolic rate of heat-stressed broilers and increase the energy required for broiler growth. These findings differ from the results of this experiment. This may be explained by the difference in the number of stomachs between ruminants and other animals. There was no difference in the concentration of T 4 among the groups in this experiment. Cooke et al. (2011) showed that T 3 in dairy cows is higher in diets containing tea pollen; however, tea pollen has no effect on the T 4 concentration (Taraghijou et al., 2012). In this study, T 3 increased significantly in dairy cows fed with 50 mg/BW Moringa flavonoids, which is consistent with the results of previous studies.

CONCLUSIONS
In conclusion, the addition of 50 mg/BW Moringa leaf flavonoids to cow diet increased the protein content in milk, but had no effect on milk yield/day, mild fat, total solids, or other quality traits. Additionally, Moringa leaf flavonoids enhanced the antioxidant and immunity capacity in dairy cows but did not affect physiological levels of common biochemical parameters in blood or fermentation parameters in rumen. the key technology of improving the quality of corn flour with microbial enzymes (spkf202012). We would like to thank Jianyang Liu, who works in Virginia Tech, for helping us revise the manuscript.

CONFLICTS OF INTEREST
The authors declare no conflict of interest.

ETHICS STATEMENT
All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this article.