Buffalo milk is more effect to rescue fatigue than cow milk in young adult mouse model
Many cross-sectional studies infer that females have a higher capacity to resist fatigue than males [27]. Herein, we studied the anti-fatigue effects of buffalo milk in female and male mice, separately. In male mice, anti-fatigues effects of pasteurized buffalo milk (PBM) were compared with pasteurized cow milk (PCM) (Fig. 1). Taurine (Tau) can increase the exercise tolerance, and reduce exercise-induced fatigue by modulating pro-inflammatory factors [28], so we employed Taurine as a positive control. Bodyweight curve identified that PBM treated mice had a trend of the slight increase in body weight than control, PCM, and Tau groups (Fig. 1a). Forced swimming time and biomarkers such as hepatic glycogen, blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) concentration were used as indicators to evaluate anti-fatigue capacity [29, 30]. There was no significant difference in forced swimming time among the four groups of male mice (P > 0.05) (Fig. 1b). In male mice, LDL levels in PCM group is lower than Tau and PBM groups (Fig. 1c). BUN concentrations of PBM and Tau groups was significantly lower than that of control and PCM groups (P < 0.05) (Fig. 1d). Hepatic glycogen content of PBM groups were significantly increased in the Tau group when compared with the PCM group (P < 0.05) (Fig. 1e).
In female mice, the average bodyweight of the PBM group was slightly higher, but there were no significant differences with other groups (P > 0.05) (Fig. 2a). Compared with the control group, the forced swimming time in the Tau and PBM groups increased significantly (P < 0.05) (Fig. 2b). The serum LDH concentration of Tau, PBM, and PCM groups was elevated compared to the control group (P < 0.05) (Fig. 2c). The BUN concentration was significantly lower in all treatment groups than control and the PBM group exhibiting lowest BUN levels (Fig. 2d). Hepatic glycogen content among the four groups was non-significant (P > 0.05) (Fig. 2e). These results indicate that pasteurized buffalo milk had a better anti-fatigue effect on female mice.
Pasteurized buffalo milk has better antioxidant effects than high temperature sterilized buffalo milk in D-galactose induced aging mice model
Aging reduces the efficiency of anti-oxidative enzymes rendering older people more susceptible to free radical stress and oxidative insult [31]. Chronic exposure of Dgalactose (D-gal) is a well-established model to induce accelerated aging in mice [32]. Based on better antifatigue effects of PBM in in young mice model, we employed D-gal induced aging model to evaluate whether PBM or/and high temperature sterilized buffalo milk (PTBM) could rescue oxidative stress induced by aging. Antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) function as scavengers for superoxide and hydrogen to inhibit ROS-induced damage and serve as biomarkers of oxidative stress [33]. D-gal treatment significantly decreased SOD (P < 0.05) and GSH-Px (P < 0.05) levels in serum, whereas levels of MDA were increased (P < 0.05). Besides, the GSH-Px was also significantly downregulated, and MDA was upregulated in liver tissue (Fig. 3). These indicated the successful establishment of the D-gal induced aging mouse model. Vitamin C (Vc) which has potent antioxidant and scavenging ability of hydroxyl radical and superoxide anion [34], was used as a positive control. There was no difference between the average daily consumption between pasteurized and high temperature sterilized buffalo milk (Table S1). Nonetheless, the total body weight gains of D-PBM increased significantly than the D-gal group (P < 0.05) (Table 1), while other groups had no significant difference. These results indicated that PBM has more favorable bodyweight competence in D-gal induced aging mice.
Table 1
Effect of pasteurized and high temperature sterilized buffalo milk on body weight of ageing mice
Groups (n = 10) | Initial weight (g) | 0–4 weeks weight gain (g) | 4–8 weeks weight gain (g) | Total weight gain (g) | Weight gain compared to control (%) |
Control | 24.45 ± 0.57a | 6.93 ± 0.61ab | 2.93 ± 0.71ab | 9.86 ± 1.19ab | —— |
D-gal | 24.47 ± 0.51a | 6.89 ± 0.45ab | 1.57 ± 0.16a | 8.46 ± 0.45a | -14.20% |
D-Vc | 24.48 ± 0.60a | 6.45 ± 0.53a | 2.05 ± 0.21ab | 8.5 ± 0.57a | -13.79% |
D-PBM | 24.56 ± 0.56a | 8.44 ± 0.74b | 3.04 ± 0.73b | 11.47 ± 1.38b | 16.36% |
D-HTBM | 24.38 ± 0.49a | 7.54 ± 0.72ab | 2.75 ± 0.35ab | 10.28 ± 0.72ab | 4.28% |
Data are expressed as means ± standard error (n = 10), different letters in the same column indicate significant differences (ANOVA, LSD test) between samples (P < 0.05). |
We next assessed serum antioxidant effects of buffalo milk on biochemical parameters related to antioxidant competence in aging mice (Fig. 3). Compared with the D-gal group, the serum SOD activity of the D-Vc group, D-PBM group, and the D-HTBM group increased significantly (P < 0.05) in the serum (Fig. 3a). Vc, PBM, and HTBM treatments all diminished elevated MDA levels induced by D-gal (Fig. 3c). The serum MDA level of the D-PBM group was significantly lower than the D-Vc group (Fig. 3c). Compared with the D-gal group, the activity of GSH-Px in the serum of the D-gal induced aging mice supplied with Vc (D-Vc), PBM (D-PBM), or HTBM (D-HTBM) groups significantly increased (P < 0.05) (Fig. 3e). PBM and Vc treatment restored the GSH-Px to control levels (Fig. 3e). However, GSH-Px in the HTBM group was significantly lower than the control group (P < 0.05) (Fig. 3e).
We then detected the effects of buffalo milk related to oxidative stress in the liver in our aging mouse model. The liver SOD activity of the D-PBM group significantly increased when compared with all the other groups (P < 0.05) (Fig. 3b). Both Vc, PBM, and HTBM were able to decrease MDA concentrations to basal levels (Fig. 3d), indicating that Vc and buffalo milk can alleviate the age-induced increase of MDA. Compared with the D-gal group, liver GSH-Px activities of D-Vc, D-PBM groups were significantly increased (P < 0.05), but HTBM treatment failed to reinforce D-gal induced decline of GSH-Px (Fig. 3f).
These results showed that buffalo milk and Vc had boosted antioxidant effects on aging mice by increasing SOD, GSH-Px content, and decreasing MDA levels. More importantly, pasteurized buffalo milk has a better antioxidant effect than high-temperature buffalo milk.
Pasteurized and high-temperature sterilized buffalo milk boosted learning and memory in D-galactose induced aging mice
We next evaluated the effect of buffalo milk on learning and memory capacity by employing the D-galactose induced aging mice model. The physiological basis of animal learning and memory ability is a conditioned reflex. The main conditioned reflex tests include passive avoidance task, spontaneous novel object recognition, and object location recognition, and Morris water-maze procedure [35]. Results of object recognition test (ORT) (Fig. 4a, b), and object location test (OLT) (Fig. 4c, d) showed that the priority index (PID) and the discrimination index (DI) of the D-gal group were significantly lower than the control group (P < 0.05). Besides, the D-gal group had significantly lower GSH-PX, SOD, and higher MDA levels than the control group (P < 0.05) (Fig. 5), which confirmed the success of the aging mice model.
In the ORT experiment, the PID was significantly increased in D-gal induced aging mice supplied with AP (D-AP), D-PBM, and D-HTBM group (Fig. 4a). Consistently, the discrimination index of the D-gal group was also successfully rescued by AP, PBM, or HTBM treatment (Fig. 4b). In the OLT experiment, compared with the D-gal group, both the DI and PID of the D-AP, the D-PBM, and the D-HTBM groups increased significantly (P < 0.05) (Fig. 4c, d). These data suggested that buffalo milk could improve the decline in learning and memory capacity caused by D-gal induced aging.
Subsequently, the biochemical indicators (SOD, GSH-Px, and MDA) related to oxidation in the brain were examined. Compared with the D-gal group, the brain SOD and GSH-Px activities of the D-AP, D-PBM, and D-HTBM groups increased significantly (Fig. 5a, c), while MDA activity was significantly decreased in D-AP and D-PBM groups (P < 0.05) (Fig. 5b). Finally, we detected the brain protein content in different groups, the brain protein content of the D-gal group was significantly lower than that of the control group while a substantial increase was observed in D-AP (13.38%), D-PBM (16.20%), and D-HTBM (14.43%) compared to the D-gal group (P < 0.05) (Fig. 5d).