Goat milk enhances memory of d-galactose-induced aging rats.

Background and aim: Goat milk is a food of high nutritional value and has been proved to possess strong antioxidant and anti-inflammatory properties. However, thus far, little is known of its possible effects on brain especially on memory during aging. The aim of this study was to assess the effect of goat milk supplementation on memory in d-galactose-induced aging rat model. Experimental procedure: Fifty-two male Sprague Dawley rats were randomly divided into four groups: 1) control group, 2) goat milk treated group, 3) d-galactose treated group, and 4) goat milk plus d-galactose treated group. Goat milk (1 g/kg orally) and/or d-galactose (120 mg/kg subcutaneously) were administered continuously for six weeks preceded and followed by novel object recognition and T-maze test. Results and conclusion: Prior to goat milk and d-galactose administration, there was no significant difference (p > 0.05) in memory between all groups. Goat milk administration alone significantly increased short- and long-term memory (p < 0.05) while d-galactose administration alone significantly decreased short-, long-term and spatial memory (p < 0.001). Goat milk treatment to d-galactose-induced rats managed to protect against memory decline as exhibited by significantly higher short-, long-term and spatial memory (p < 0.0001) when compared to the untreated d-galactose-induced rats. These results suggest that goat milk as a whole or due to the taurine or sialic acid contained in goat milk is effective in improving memory functions and may be useful in protecting against age-related memory deficits.

statistical package for the social sciences

analysis of variance

standard error of mean

N-methyl-d-aspartate receptor

instrumental activities of daily living

Ras homolog enriched in brain

mammalian target of rapamycin

Introduction

Physiological aging is characterized by a gradual loss of cognitive performance, expressed as a decline in memory, reasoning, mental capacity and spatial ability. According to a report by United Nations, in the next 35 years the number of people aged 60 and above around the world is projected to get more than double, reaching almost 2.1 billion people. With this increase in life expectancy, the incidence of Alzheimer’s disease and related neurodegenerative conditions is rising equally. A decline in memory function results in a poor quality of life adversely affecting instrumental activities of daily living (IADLs) and compliance with healthcare. This renders the older adults vulnerable to development of further diseases and thus, a poor health status. It has been estimated that by the year 2040, almost 81.1 million people will be affected by dementia. Thus, it is imperative to identify agents or supplements that may help to prevent or delay memory decline. Memantine, a partial antagonist of N-methyl-d-aspartate receptor (NMDAR), is a neuroprotective agent that has been approved for treatment of Alzheimer’s disease. In spite of the relatively modest nature of its adverse effects, memantine has been shown to provide only a moderate decrease in clinical deterioration of Alzheimer’s disease, hence efforts should be taken to discover a more potent intervention.

d-galactose-induced brain aging model has been widely used and is established to be beneficial for aging studies. d-galactose is a reducing sugar that occurs naturally in the body in small quantities. Excess amount of d-galactose may increase reactive oxygen species and advance glycation end products which causes considerable brain oxidative damage leading to memory impairment., Several other mechanisms have been suggested in d-galactose-induced brain aging whereby d-galactose increases oxidative stress and inflammation, decreases antioxidant enzymes and brain-derived neurotrophic factor levels, induces apoptosis and neuromodulation, activates astrocytes and microglia and down-regulates Ras homolog enriched in brain (Rheb) while up-regulating mammalian target of rapamycin (mTOR).

Goat milk is a food of high nutritional value and plays a vital role in human nutrition. It is known to contain proteins, vitamins and fatty acids that possess high biological merit., Goat milk possesses higher levels of calcium, potassium and phosphorus compared to both human and cow’s milk and a substantially higher protein concentration compared to human milk., It is hypoallergic and its small fat globules makes it easily digestible. Thus, due to its composition, it is widely used as a functional food ameliorating health status as well as reducing the risk of disease development. Moreover, goat milk contains several bioactive peptides with potent antioxidant capacity. Despite various benefits of goat milk, research on the effects of goat milk in memory and learning particularly during aging is limited. Thus, the aim of the present study was to determine the possible memory-enhancing effects of goat milk supplementation in normal rat as well as in d-galactose-induced aging rat model.

Methodology

Animals

Eight-week-old male Sprague-Dawley rats were obtained from Animal Research and Service Centre, Universiti Sains Malaysia. All rats were kept in standard polypropylene cages (40 × 25 × 16 cm) with commercial pine chip bedding, exposed to 12 h light/dark cycle, in a well-ventilated room maintained at a consistent temperature of 25 °C. The rats were maintained on a standard balanced diet and had free access to food and water. All rats were habituated to animal room conditions at the Physiology Laboratory, Universiti Sains Malaysia a week prior to experimental manipulation to acclimatize the animals to the new environment and experimenter. All animal research procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Universiti Sains Malaysia (USM/IACUC/2017/(109) (879). All efforts were made to minimize suffering.

Goat milk and d-galactose treatments

All rats were randomly and equally divided into four groups as follows: i) control group rats: orally and subcutaneously administered with normal saline, ii) goat milk treated rats: orally administered with goat milk and subcutaneously injected with normal saline, iii) d-galactose treated rats: orally administered with normal saline and subcutaneously injected with d-galactose and iv) goat milk and d-galactose treated rats: orally administered with goat milk and subcutaneously injected with d-galactose concomitantly. The goat milk used in this study was commercially available (Batch number catalogue: 9-555701-000978, Salic goat, Muazz Marketing, Malaysia) from that of Saanen goat breed (originating from Holland). The dosage used in this study was 1 g/kg of rat’s body weight. This dosage was established after conducting a pilot study in which 9 rats were divided into three different groups that were given 3 different dosages of goat milk of 0.5 g/kg, 1 g/kg and 2 g/kg along with administration of 120 mg/kg of D-gal. The dosages were chosen based on previous studies that employed goat milk., These 3 groups were tested using T-maze every week for 6 weeks and the dosage with best results was chosen for this study. The dosage of 1 g/kg per day provided the best results and was selected for the study. The d-galactose was from Sigma-Aldrich, USA and was administered at a dose of 120 mg/kg., The goat milk and d-galactose were administered continuously for six weeks preceded and followed by novel object recognition and T-maze test. The present study was conducted according to the experimental schedule described in Fig. 1.

Fig. 1

Experimental schedule.

Sample size determination

In order to determine an adequate sample size, power analysis was conducted in G*Power version 3.1 for a one-way ANOVA with four groups. Type 1 error probability (α) and the power of the study were set at 0.05 and 80%, respectively. Following the guidelines of Cohen, an effects size of 0.50 was chosen. The calculated sample size was 12 rats per group. A dropout of 10% was expected, therefore, the final sample size was 13 rats per group.

Learning and memory tests

Novel object recognition test

The principle of novel object recognition test is based on the innate preference of rats to explore novel objects as compared to familiar objects., The test was carried out in a transparent open apparatus (60 × 60 × 30 cm) made of transparent polyvinyl chloride (Fig. 2). The test began by placing the rat in the empty apparatus for 10 min on the first two days. On the third day, two similar objects (A1 and A2) were placed in the open field at an equal distance from each other and from the walls. The rat was placed inside the open field facing away from the objects for 5 min. Then, 2 h later, one of the familiar objects was replaced with a novel object (B) and the rat was placed back in the arena to explore for 5 min testing for short-term memory. The time spent to explore either object was recorded carefully with a stopwatch in presence of cameras. To test for long-term memory, the previous novel object (B) was replaced with a new novel object (C) and its place was swapped with the familiar object to eliminate biasness. Again, the rat was placed in the open field for 5 min and the time to explore both objects was recorded. In order to score for short- and long-term memory, discrimination index was calculated by dividing the difference of time used to explore novel and familiar object by the sum of time spent to explore both novel and familiar object for each rat. The discrimination index ranges from −1 to +1; −1 suggesting poor memory and +1 suggesting excellent memory. The test was repeated six weeks later to compare treatment effects within groups. When the test was repeated after six weeks, new set of objects were used to avoid practice effect. In order to avoid any discrimination due to olfactory cues, the objects and the chamber were cleaned with 70% alcohol after each test.

Fig. 2

Schematic representation of the novel object recognition test.

1. Day 1 and day 2; the rat explores empty arena.

2. Day 3; the rat explores two similar objects.

3. Day 3; the rat exploring the new object (B) after 2 h of familiarization.

4. Day 4; the rat exploring the other novel object (C) after 24 h of familiarization.

T-maze (spontaneous alternation protocol)

Spontaneous alternation protocol using a T-maze was utilized to test for spatial memory. The T-maze consisted of three arms made of polyvinyl chloride including the start arm (60 × 16.5 × 30 cm), right arm (50 × 16.5 × 30 cm) and left arm (50 × 16.5 × 30 cm). The maze was equipped with three guillotine doors. The test began with gently putting the animal in the start arm and allowing it to choose a goal arm (Fig. 3). Once the goal arm was chosen, the rat was confined to the chosen arm by quietly sliding guillotine door down for 30 s. After 30 s the animal was gently removed from maze and the guillotine door was lifted. Then, the animal was placed back in the maze facing away from goal arms and allowed to choose one of the goal arms. If the animal chose the other arm as compared to the arm chosen in the first time, the trail was considered successful and a score of 1 was given. If the rat entered the same arm as the one chosen initially, the trial was considered unsuccessful and a score of zero was given. A total of 5 trials were conducted for each rat and the score was given out of 5. The principle of alternation is that, because the animal prefers visiting the less recently visited arm, it is implied that it will need to recall which was the last arm it visited and thus choose the novel arm. After each trial for each rat, the floor of T-maze was cleaned using 70% alcohol to remove olfactory cues and to prevent the animal from losing interest.

Fig. 3

Schematic representation of the T-maze.

1. The rat is placed in the start arm.

2. The rat chooses one arm of the T-maze and is confined in the respective arm for 30 seconds.

3. The rat is placed back in start arm and facing the opposite wall.

4. The rat selects the other arm compared to the first choice and alternates successfully.

Statistical analysis

Statistical analysis for all the data was performed using SPSS version 24. Repeated measures ANOVA with pairwise comparison with Bonferroni correction was used to determine any changes in the memory pre- and six weeks post-intervention/treatment within groups. The factor for within-subject analysis was time with two levels i.e. pre- and post-treatment and the factor for between-subject analysis was the treatment group with four levels i.e. control, goat milk treatment, D-gal treatment and D-gal + goat milk treatment. Before applying the test, assumptions of normality, homogeneity of variances and compound symmetry were checked and were fulfilled. One-way ANOVA with post-hoc Tukey test was used to analyze any significant changes in memory between groups after six weeks of d-galactose and/or goat milk treatment. Before applying the test, the data was tested for normal distribution using the normality test while homogeneity of variance was measured using Levene’s test. Results were presented as mean and standard error of means (S.E.M). Probability values that were less than 5% (p ˂ 0.05) were considered as statistically significant.

Results

Short-term memory

To assess short-term memory performance, analysis of discrimination index scores of all groups was performed using repeated measures ANOVA. The factor for within-subject analysis was time with two levels i.e. pre- and post-treatment and the factor for between-subject analysis was the treatment group with four levels i.e. control, goat milk treatment, D-gal treatment and D-gal + goat milk treatment. Repeated measures ANOVA revealed significant main effect of time on the mean short-term memory within the treatment groups (F (1, 46) = 7.77, p < 0.01). The analyses were followed by pairwise comparison with confidence interval adjustment by Bonferroni correction. The pairwise comparison revealed a significant effect of d-galactose treatment on short-term memory pre- and post-treatment (F (1, 12) = 214.405, p < 0.001) (Fig. 4). There was a significant improvement in short-term memory among normal rats after six weeks of goat milk administration (F (1, 11) = 5.235, p < 0.05). There was no significant difference in short-term memory between pre- and post-treatment in d-galactose rats treated with goat milk, suggesting the protective effect of goat milk against harmful effects of d-galactose (F (1,12) = 0.920, p > 0.05). One-way ANOVA revealed that short-term memory of the group treated with goat milk alone was significantly higher (p < 0.05) compared to the control group whereas, short-term memory of the group treated with d-galactose alone was significantly lower (p < 0.0001) than the control group. The d-galactose treated rats supplemented with goat milk exhibited significantly higher mean short-term memory than the d-galactose group (p < 0.0001).

Fig. 4

Comparison of mean short-term memory discrimination index before and after six weeks of treatment and between different experimental groups.

Data are presented as mean ± SEM.

#P < 0.05, ###P < 0.001, significant difference between pre- and post-treatment within group.

*P < 0.05, ****P < 0.0001, significantly different from control group.

$$$$P < 0.0001 significantly different from d-galactose group.

Long-term memory

To assess long-term memory performance, analysis of discrimination index scores of all groups was performed using repeated measures ANOVA. The factor for within-subject analysis was time with two levels i.e. pre- and post-treatment and the factor for between-subject analysis was the treatment group with four levels i.e. control, goat milk treatment, D-gal treatment and D-gal + goat milk treatment. Repeated measures ANOVA revealed significant main effect of time on the mean long-term memory within the treatment groups (F (1, 46) = 7.53, p < 0.01). The analyses were followed by pairwise comparison with confidence interval adjustment by Bonferroni correction. The pairwise comparison revealed a significant effect of d-galactose treatment on long-term memory pre- and post-treatment (F (1, 12) = 109.787, p < 0.001) (Fig. 5). There was a significant improvement in long-term memory among normal rats after six weeks of goat milk administration (F (1, 11) = 4.696, p < 0.05). There was no significant difference in long-term memory between pre- and post-treatment in d-galactose rats treated with goat milk, suggesting the protective effect of goat milk against harmful effects of d-galactose (F (1,12) = 0.139, p > 0.05). One-way ANOVA revealed that long-term memory of the group treated with goat milk alone was significantly higher (p < 0.05) compared to the control group whereas, long-term memory of the group treated with d-galactose alone was significantly lower (p < 0.0001) than the control group. The d-galactose treated rats supplemented with goat milk exhibited significantly higher mean long-term memory than the d-galactose group (p < 0.0001).

Fig. 5

Comparison of mean long-term memory discrimination index before and after six weeks of treatment and between different experimental groups.

Data are presented as mean ± SEM.

#P < 0.05, ###P < 0.001, significant difference between pre- and post-treatment within group.

*P < 0.05, ****P < 0.0001, significantly different from control group.

$$$$P < 0.0001 significantly different from d-galactose group.

Spatial memory

To assess spatial memory, analysis of alternation scores of all groups was performed using repeated measures ANOVA. The factor for within-subject analysis was time with two levels i.e. pre- and post-treatment and the factor for between-subject analysis was the treatment group with four levels i.e. control, goat milk treatment, D-gal treatment and D-gal + goat milk treatment. There was no significant main effect of time on the mean spatial memory within the treatment groups (F (1, 46) = 0.272, p > 0.05). However, there was a significant time and groups interaction (F (3, 46) = 10.53, p < 0.0001). Thus, the analyses were followed by pairwise comparison with confidence interval adjustment by Bonferroni correction. The pairwise comparison revealed a significant effect of d-galactose treatment on spatial memory of rats pre- and post-treatment (F (1, 12) = 28.444, p < 0.001) (Fig. 6). There was no significant difference between pre- and post-treatment in d-galactose rats treated with goat milk (F (1, 12) = 0.806, p > 0.05). One-way ANOVA revealed that the d-galactose group exhibited significantly lower mean spatial memory compared to the control group (p < 0.0001), whereas the d-galactose rats supplemented with goat milk exhibited significantly higher mean spatial memory than the d-galactose group (p < 0.0001).

Fig. 6

Comparison of mean number of correct alternations before and after six weeks of treatment and between different experimental groups.

Data are presented as mean ± SEM.

###P < 0.001, significant difference between pre- and post-treatment within group.

****P < 0.0001, significantly different from control group.

$$$$P < 0.0001 significantly different from d-galactose group.

Discussion

According to the results of our study, goat milk supplementations successfully improved short- and long-term memory of the normal rats. In addition, goat milk was able to protect against memory decline in the d-galactose-treated rats. This is demonstrated by significant difference in memory between the d-galactose group and the d-galactose treated with goat milk group in which the memory score of the latter group was significantly higher. Interestingly, the memory of the d-galactose rats treated with goat milk was comparable to that of the normal control rats, suggesting the goat milk’s ability to normalize memory functions. To our knowledge this is the first study reporting the memory enhancing ability of whole goat milk. Previous studies have reported memory enhancing ability of goat milk-based products. Xu et al. (2015) reported that goat milk-based formula resulted in superior cognitive and spatial ability in weaned Sprague-Dawley rats as compared to control rats and rats supplemented with cow milk-based formula. Medeiros et al. (2018) made use of goat whey, which is a by-product of cheese curd production from goat milk, to test memory in moderately malnourished male pups and reported that goat whey was able to enhance their memory as compared to the pups in control group.

There is a dearth of literature regarding any specific memory enhancing constituents of goat milk however, it was reported that the memory-enhancing effects of dried goat whey are attributed to taurine, an amino acid found in goat milk in abundance. In addition, taurine when added to drinking water resulted in recovery of learning and memory in mouse model of Alzheimer’s disease. A few studies have reported the beneficial effects of sialic acid, which is another component abundantly present in goat milk, on memory. The sialic acid profile of goat milk closely matches that of human milk. A positive correlation between sialic acid present in food source and greater cognitive development in animals has also been reported. Research demonstrated that rat pups performed better at memory and learning tests when consumed sialic acid. Furthermore, piglets administered sialic acid from a food source exhibited improved learning and memory compared to controls as assessed by an 8-arm radial maze. Therefore, it is hypothesized that the goat milk used in this study may have possessed taurine or sialic acid which exerted the memory-enhancing effects as seen in the goat milk-treated group.

World-wide, several health and nutritional organizations stress upon the importance of daily consumption of dairy products to attain optimal health and strongly recommend adding dairy products in diet. Our current findings suggest that goat milk was able to protect against memory impairment that is caused by d-galactose. However, the results of our study explored the effects of whole goat milk rather than its individual constituents. The use of whole goat milk renders it ambiguous as to which component of goat milk was responsible for memory preservation. Moreover, the mechanism by which goat milk caused improvement in memory function was not explored. According to the results of a separate study conducted at our laboratory, goat milk was able to suppress oxidative stress in the brains and enhanced the brain neurotrophic factors of rats treated with d-galactose. There are various other factors which may be the mechanisms of action of goat milk such as antioxidant, anti-inflammatory or neuroprotective activities. This possibility is currently being investigated in our laboratory.

Conclusion

The present study demonstrated that the administration of goat milk may improve short- and long-term memory in normal rats. Interestingly, goat milk may also ameliorate aging-induced memory deficits.

Declaration of competing interest

None.