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 groups13. Type 1 error probability (α) and the power of the study were set at 0.05 and 80%, respectively. Following the guidelines of Cohen14, an effects size of 0.50 was chosen. The calculated sample size was 12 rats per group. A dropout of 10% was expected, and therefore, the final sample size was 13 rats per group.

Figure 1

The principle of novel object recognition test is based on the innate preference of rats to explore novel objects as compared to familiar objects15. The protocol used to perform NORT was based on the guidelines provided by previous research studies16. The test was carried out in a transparent open apparatus (as shown in Figure 1). The test began by placing the rat in the empty apparatus for a duration of 10 minutes 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 minutes. Then, 2 hours 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 minutes as a test for short-term memory (STM). The time spent to explore either objects was recorded carefully. To test for long-term memory (LTM), after 24 hours, the previous novel object (B) was replaced with a new novel object (C) and its place was swapped with the familiar object to eliminate bias. Again, the rat was placed in the open field for 5 minutes and the time to explore both objects was recorded. In order to score for STM and LTM, discrimination index (DI) was calculated by dividing the difference of time used to explore novel and familiar objects by the sum of time spent to explore both novel and familiar objects for each rat. The value for DI ranged from -1 to +1, with -1 representing poor memory and +1 representing excellent memory.

In order to test for spatial memory performance, a spontaneous alteration protocol using a T-maze was followed17. The test began with gently putting the animal in the start arm and allowing it to choose a goal arm (Figure 2). Once the goal arm was chosen, the rat was confined to the chosen arm by quietly sliding the guillotine door down for 30 seconds. After 30 seconds, the animal was gently removed from the maze and the guillotine door was lifted. Then, the animal was placed back in the maze facing away from the 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 was conducted for each rat and the score was given out of 5. The principle of alternation is based on the fact that the animal prefers visiting the less recently visited arm, thus implying that it would need to recall which was the last arm it visited and thus choose the novel arm18.

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 performance pre- and six-weeks post- intervention/treatment within the groups. 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 performance between the groups after six weeks of D-gal 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 mean (S.E.M). Probability values that were less than 5% (p˂0.05) were considered as statistically significant.

Figure 2

Table 1

Time	Treatment group	Mean STM	95% Confidence interval
Pre-treatment	Control	.478	.360 - .595
	Goat milk	.472	.354 - .589
	D-galactose	.512	.400 - .625
	D-gal+Goat milk	.521	.408 - .633
Post-treatment	Control	.447	.358 - .535
	Goat milk	.625*	.537 - .713
	D-galactose	-.272*	-.357 - -.187
	D-gal+Goat milk	.511$	.426 - .596

Note: If the mean of one group does not overlap with corresponding confidence interval of another group, the mean STM is significantly different between groups in that particular time of measurement

There was a significant difference in mean STM within the treatment groups based on time (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-gal on STM pre- and post-treatment (F (1, 12) = 214.405, p ˂0.001). There was a significant improvement in STM among normal rats after six weeks of goat milk administration (F (1, 11) = 5.235, p˂0.05). There was no significant difference in STM between pre- and post-treatment in D-gal rats treated with goat milk, thereby suggesting the protective effect of goat milk against harmful effects of D-gal (F (1,12) = 0.920, p ˃0.05) (Table 1). One-way ANOVA revealed that the mean STM of the goat milk group was significantly higher (p ˂ 0.05) compared to the control group, whereas the mean STM of the D-gal group was significantly lower (p ˂ 0.0001) than that of the control group. The D-gal treated rats supplemented with goat milk exhibited significantly higher mean STM than the D-gal group (p < 0.0001) (Figure 3).

Figure 3

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-gal group

Considering the results of this study and those from previous reported studies, we conclude that the addition of goat milk to the diet- early on or during old age- may result in protection from memory decline. Thus, goat milk supplementation to diet may eventually lead to better memory performance and, thus, better quality of life.

D-gal: D-galactose

ROS: Reactive oxygen species

AGE: Advanced glycation end products

NORT: Novel object recognition test

STM: Short-term memory

LTM: Long-term memory

DI: Discrimination index

SEM: Standard error of mean

IADL’s: Instrumental activities of daily living

SA: Sialic acid

CLA: Conjugated linoleic acid

The authors declare no conflicts of interest.

A.S. carried out the experiments, analysed the data and wrote the paper with input from all authors. K.F.A. designed and supervised the project. R.Z. helped supervise the project. C.B.A.A. partially funded the project. U.R. helped with the experiments.