1. Introduction
Heat stress leads to metabolic disorders in lactating dairy cows and reduces dry matter (DM) intake, milk production, and animal welfare [
1,
2,
3]. In addition, it has been reported that cows exposed to heat stress showed decreased antioxidant enzyme activity which resulted in a higher level of free radicals and lower unsaturated fatty acid (UFA) and polyunsaturated fatty acid (PUFA) concentrations in their milk than non-stressed cows [
4]. Thus, inflammatory diseases, such as mastitis and metritis, may be found in heat-stressed cows [
5]. Additionally, milk containing lower levels of UFA (
cis9,
trans11-conjugated linoleic acid (CLA), 18:2n-6, and 18:3n-3) are prone to higher lipid peroxidation, which is associated with lower antioxidant capacities in milk [
6]. Thus, it is necessary to consider how to maintain mammary gland health and milk stability in lactating cows under heat stress.
The antioxidant capacity of milk products is an important index for the dairy industry, as oxidation would induce deterioration of the milk nutritional quality [
7]. Higher milk antioxidant content can efficiently prevent lipid peroxidation and extend the shelf life of milk [
8]. Oxidation also leads to sensory quality deterioration, which can affect milk marketability [
9]. Milk antioxidant capacity depends on various factors such as fatty acid compositions [
6,
10] and concentrations of other antioxidant substrates, such as phenolics and flavonoids [
11]. Increasing dietary phenolic and flavonoid substrates induced by propolis extracts or
Agaricus blazei mushrooms could enhance the milk antioxidant capacity of dairy cows, indicating that dietary intervention is useful in modifying milk antioxidant capacities [
12,
13]. Moreover, it has been reported that feeding lactating cows with dry distiller’s grains with solubles, which have high UFA proportions, improved milk UFA concentrations, but not milk antioxidant capacity [
14,
15,
16]. This information indicated that the dairy rations, including microbiological products, may impact milk antioxidant capacity.
As a traditional Chinese alcoholic beverage, over one million tons of yellow wine lees (YWL) were produced from rice and wheat in 2015 [
17,
18]. We previously found that the antioxidant activity of the YWL can be improved when fermented with
Candida utilis and
Bacillus subtilis [
19]. In our preliminary in vitro study, when the ratio of soybean meal (SBM) to unfermented (UM) or fermented YWL mix (FM) in the simulated ration was at 1:1 (DM basis), the gas production parameters and volatile fatty acids were optimized and functioned similarly to the rations with SBM as the main protein resource [
20]. Moreover, we observed that total mixed rations (TMR) with partial replacement of SBM with UM/FM did not affect feed efficiency and milk compositions, indicating YWL as a potential protein source in dairy rations [
21]. We hypothesized that milk antioxidant capacity is affected by the dietary application of UM/FM. To validate our hypothesis, this study was conducted to compare fatty acid profiles, antioxidant capacity-related indices in feed, blood, and milk, and hematological parameters in heat-stressed dairy cows fed with different daily rations.
3. Results
Throughout the whole experiment, the average THI value was above 75 (
Figure 1), indicating that all the experimental cows experienced heat stress throughout the experiment. Compared with control, the rectal temperature of cows fed UM and FM were similar in the morning (0700 h), however, they were lower in the afternoon (1300 h,
p = 0.02,
Table 1). Moreover, cows consuming UM and FM had lower respiratory rates relative to the control animals (
p = 0.02,
Table 1).
The phenolic and flavonoid contents, as well as the antioxidant activities of SBM, UM, and FM, are presented in
Table 2. The UM and FM had greater total phenolics and total flavonoids relative to SBM (
p < 0.01). Compared with UM, FM was higher in terms of total phenolic and flavonoid contents (
p < 0.05). Reducing power, superoxide radical scavenging ability, and hydroxyl radical scavenging ability were greater in the UM and FM, compared to that of SBM (
p < 0.01). Moreover, FM had similar reducing power of UM, but was greater in terms of superoxide radical scavenging ability and hydroxyl radical scavenging ability than UM (
p < 0.01).
The plasma fatty acid compositions of cows in the three diet groups are presented in
Table 3. Compared to control, UM and FM were higher in concentrations of C18:1
cis-9 (
p = 0.02) and C18:1
cis-11 (
p = 0.01), respectively. The concentrations of C22:4 and C22:5 n-3 in UM and FM were lower than in control (
p = 0.01). Other fatty acid compositions did not differ across the three diets.
The milk fatty acid profiles of cows fed control, UM, and FM are presented in
Table 4. Compared with control, cows fed UM or FM had greater proportions of C10:0, C18:0, C18:3 n-3, C20:0, and CLA (
cis-9
trans-11) in their milk (
p < 0.05). However, cows fed UM or FM had lower contents of C12:0, C14:0, C16:0, C16
cis-9, C17:0, C18:1
trans-6, C18:1
cis-9, and C19:0, compared to the control animals (
p < 0.05). The short-chain fatty acids were not different among the treatments (
p > 0.05), medium-chain fatty acids were lower, and long-chain fatty acids were higher in UM and FM animals than the control cows (
p < 0.01). The SFA was lower in cows fed UM and FM, while UFA (mono- and poly-) was higher than the control cows (
p < 0.01).
The plasma metabolites of experimental cows fed different diets are listed in
Table 5. The concentrations of total protein, glucose, cholesterol, NEFA, and β-hydroxybutyric acid (BHBA) of plasma samples in the experimental cows were not affected by the treatments. The albumin concentration of cows tended (
p = 0.07) to be affected by diet treatments. However, the numbers of the three groups were still close.
The whole blood variables of lactating cows fed different diets are listed in
Table 6. In brief, cows fed FM and UM were lower in levels of WBC (
p = 0.05) and neutrophil (
p = 0.02) in their blood relative to the control animals. Similarly, blood lymphocyte concentrations tended to be lower in cows fed FM and UM relative to control (
p = 0.07). Other hematological variables were similar among cows from the three treatment groups.
The antioxidant-related indices of the milk and plasma of cows from the three treatment groups are presented in
Table 7. Cows fed with UM and FM had higher total phenolic and total flavonoid concentrations in milk than the control cows (
p < 0.01). Higher levels of total phenolic and total flavonoid in milk and plasma of FM-fed animals were observed, compared with UM-fed ones (
p < 0.05). Milk MDA concentration was lower in the UM- and FM-fed cows compared with control (
p < 0.05). In contrast, milk DPPH concentrations were higher in cows with UM and FM compared to control (
p < 0.01). In terms of the plasma, cows consuming UM and FM had higher total phenolic and total flavonoid concentrations relative to the control (
p < 0.01). Concentrations of SOD (
p = 0.03), GSH-PX (
p = 0.04), and T-AOC (
p = 0.01) were greater in UM- and FM-fed animals compared with control cows. In contrast, plasma MDA concentrations were lower in cows with UM and FM than in control cows (
p = 0.05).
4. Discussion
Hydroxyl radicals and superoxide radicals are the most active forms of free oxygen in mammals [
33]. Increasing the concentrations of antioxidant substrates, such as phenolics and flavonoids in the diet, could help improve antioxidant capacity of the animals and their milk [
11]. Pretreatment with
Bacillus has been reported to enhance total flavanol and phenolic acid concentrations in the soybean feed, which ultimately improves the DPPH radical scavenging ability of the fermented soybean feed [
34]. Thus, a higher antioxidant capacity of UM and FM relative to SBM may be attributed to greater phenolic and flavonoid contents in TMR of the current study.
The daily THI throughout the experiment was above 68, which is the standard for exposure to heat stress for lactating dairy cows [
27]. The lower respiratory rate and rectal temperature (measured at 1300 h) in cows that consumed UM and FM suggest that YWL supplementation may be beneficial to relieving heat stress in lactating dairy cows. Reduced heat stress induced by YWL could be attributed to the greater phenolic and flavonoid concentrations in the diets. Cows under heat stress produce a high level of harmful free radicals, such as reactive oxygen species, and exhibit a lower activity of antioxidant enzymes, which may induce an inflammatory reaction in dairy cows and enhance rectal temperature and respiratory rate [
35]. Plant flavonoids, such as silymarin and quercetin, play important roles in improving antioxidant capacity in the CHO-K1 cell line [
36]. It has been reported that flavonoids reduced endoplasmic reticulum stress and hepatic inflammation in early lactating dairy cows [
37]. Moreover, polyphenols were reported to eliminate reactive oxygen species and improve the activity of antioxidant enzymes in dairy cows [
38]. Dietary phenolics can reduce the inflammatory response in intestinal epithelial cells [
39]. Thus, lower oxidative stress in cows fed UM and FM are partly attributed to increased polyphenol and flavonoid contents in the experimental feeds. Also, the higher inclusion of flavonoids and polyphenols in the FM diet caused a higher antioxidant component and capacity in the milk and plasma, compared with the UM diet. This is consistent with our previous production performance study, in which the DM intake, milk yield, and milk protein yield were higher in cows fed the FM diets than in cows fed the UM diet [
21]. Min et al. [
40] found that long-term heat stress can induce an inflammatory response in lactating cows, which can be induced by increased levels of WBCs, neutrophils, and lymphocytes in their blood. The relatively lower concentrations of WBC, neutrophil, and lymphocytes in the blood of cows fed with UM and FM suggested that the inflammation state of heat-stressed dairy cows had been relieved. In summary, partial replacement of SBM with UM or FM may benefit the health of dairy cows, especially in seasons or areas with a high THI.
Greater proportions of mono- and poly-UFA and lower proportions SFA in the milk of cows fed with UM and FM are related with greater UFA (such as C18:1
cis-9, C18:1
Cis-11, and C18:2) and numerically lower concentrations of some SFA (such as C16:0 and C20:0) in these animals, respectively. Secondly, phenolic components such as 4-methylcatechol can efficiently inhibit biohydrogenation in the rumen [
41]. Feeding beef cows with TMR containing
Broussonetia papyrifera L. silage improved PUFA in the meat, which is attributed to the inhibition of flavonoid-induced biohydrogenation in the rumen [
42]. These results suggest that both phenolics and flavonoids can inhibit rumen biohydrogenation, which can potentially improve UFA concentrations in both blood and milk. Thus, relative to the control, the greater proportion of UFA in the milk fatty acids profile of cows consuming UM and FM is partly attributed to favorable fatty acid profiles and greater antioxidant molecules (phenolics and flavonoids) in the feed substrates.
A previous study suggested that the dietary addition of grape residue silage (containing high levels of phenolics and flavonoids) in the feed improved PUFA proportion, the PUFA to SFA ratio, and antioxidant capacity in the milk of dairy cows, which ultimately improved milk quality [
43]. A recent finding also suggested that pomegranate peel addition, which contains high concentrations of polyphenols, can improve the milk antioxidant capacity of lactating ewes [
44]. These observations suggested that food byproducts containing higher levels of phenolics or flavonoids could be beneficial to milk fat quality and shelf life. A higher SFA proportion in milk is associated with improved milk oxidative stability, while a higher milk UFA proportion may cause a lower oxidative stability of milk [
4]. Aguiar et al. [
12] found that milk with higher phenolic concentrations had greater oxidative stability even when it had a higher UFA proportion. In summary, our observations suggested that feeding cows with diets containing YWL could be beneficial to the oxidative stability of milk produced by cows during hot seasons, and it was found that the oxidative stability of milk in FM group was even higher.