The effects of dietary edible bird nest supplementation on learning and memory functions of multigenerational mice

Abstract Introduction Edible bird nest (EBN) is a natural food product produced from edible nest swiftlet's saliva which consists of glycoproteins as one of its main components; these glycoproteins contain an abundant of sialic acid. The dietary EBN supplementation has been reported to enhance brain functions in mammals and that the bioactivities and nutritional value of EBN are important during periods of rapid brain growth particularly for preterm infant. However, the effects of EBN in maternal on multigeneration learning and memory function still remain unclear. Thus, the present study aimed to determine the effects of maternal EBN supplementation on learning and memory function of their first (F1)‐ and second (F2)‐generation mice. Methods CJ57BL/6 breeder F0 mice were fed with EBN (10 mg/kg) from different sources. After 6 weeks of diet supplementations, the F0 animals were bred to produce F1 and F2 animals. At 6 weeks of age, the F1 and F2 animals were tested for spatial recognition memory using a Y‐maze test. The sialic acid content from EBN and brain gene expression were analyzed using HPLC and PCR, respectively. Results All EBN samples contained glycoprotein with high level of sialic acid. Dietary EBN supplementation also showed an upregulation of GNE, ST8SiaIV, SLC17A5, and BDNF mRNA associated with an improvement in Y‐maze cognitive performance in both generations of animal. Qualitatively, the densities of synaptic vesicles in the presynaptic terminal were higher in the F1 and F2 animals which might derive from maternal EBN supplementation. Conclusion This study provided a solid foundation toward the growing research on nutritional intervention from dietary EBN supplementation on cognitive and neurological development in the generation of mammals.


| INTRODUC TI ON
Edible bird nest (EBN) produced from the saliva of edible nest swiftlets (Aerodramus fuciphagus) which are commonly found in the South-East Asia countries including Malaysia. EBN has been reported worldwide as an effective health supplementation and food product as well as beauty enhancer (Hao & Rahman, 2016;Wong, 2013). The major nutrient components in the nest are glycoproteins, carbohydrates, and essential trace elements such as calcium, sodium, magnesium, zinc, and iron (Norhayati, Azman, & Wan Nazaimoon, 2010). However, it is established that the glycoprotein from EBN is found to contain sialic acid (Aswir & Nazaimoon, 2011;Norhayati et al., 2010;Xie et al., 2018). In fact, it has been reported that sialic acid from EBN can facilitate the development of brain and intelligence (Wang, 2009(Wang, , 2012Xie et al., 2018). Although sialic acid levels have been associated with the EBN, a more comprehensive study should be carried out to investigate the role of sialic acid from EBN especially in the improvement of learning and memory functions on the preterm infant.
The newborn infant's growth and development requires outstanding demands on the nutrients supply. Any deficit of nutrient has profound effects on somatic growth, organ structural and functional development, especially in the brain. One of the important nutrients is sialic acid (also known as N-acetylneuraminic acid), a nine-carbon sugar that is a structural and functional component of brain gangliosides (Wang, McVeagh, Petocz, & Brand-Miller, 2003). The gangliosides induced by sialic acid may play a role in the structural and functional establishment of neurotransmitter in the synaptic vesicle (Reigada et al., 2003) and altering existing synaptic morphology (Morgan, Kuyatt, & Fink, 1985;Morgan, Oppenheimer, & Winick, 1981;.
The mechanism as to how sialic acid mediates the brain function system is yet to be discovered; it is envisioned that the gene expression analysis may give additional information with regard to the possible involvement of sialic acid.
In such studies, ST8SiaIV mRNA expression was increased in both neonatal and adult's brain when they were supplemented with sialic acid (Kurosawa et al., 1997;Yoshida, Kojima, & Tsuji, 1995).
The relative mRNA expression levels of Solute Carrier Family 17 Member 5 (SLC17A5) gene were upregulated in the hippocampus of pigs after supplementation with milk rich in sialyllactose (Obelitz-Ryom et al., 2019). Moreover, maternal EBN supplementation during pregnancy and lactation periods enhanced the levels of sialic acid in maternal mouse milk, which was associated with an increased level of brain-derived neurotrophic factor (BDNF) gene among their generations (Xie et al., 2018).
An expression of some genes resulted from dietary sialic acid supplementation has a marked influence on the brain physiological processes such as cell adhesion and signal transduction toward to proper brain cognitive development. However, the upregulation of brain gene expression by EBN supplementation remains controversial since the content of sialic acid varied in the EBN sources from different localities. In addition, brain cognitive functions have been linked to different genetic alterations which can be transferred across generations early in life (Alese & Mabandla, 2019). The transfer process, also referred to as epigenetics changes, is involved with a chromatin network modifications and alteration of DNA methylation (Fagiolini, Jensen, & Champagne, 2009). Thus, the alteration of DNA methylation may interact with other epigenetic changes to regulate the expression of brain genes that are involved in a wide range of learning and memory functions.
The long generation gap in humans, as well as the relative difficulty in quantifying cognitive function development in human neonates, remains a major obstacle in studying the effects of dietary EBN supplementation associated with neural function development.
The content of sialic acid from EBN is critical under conditions of rapid growth of the infant brain, particularly during the neuron development in several generation mammals. The effects of sialic acid on brain cognitive function are well known; however, little is known about the cellular changes and the mechanism effects of sialic acid from EBN-supplemented diet associated with cognitive function in multigeneration mammals. Previously, we confirmed that cognitive deficits after multigenerational maintenance on omega-3 fatty acid-deficient diet can be prevented by 16 weeks of dietary repletion with omega-3 fatty acids (Hafandi et al., 2014). Thus, the findings arising from this study may affect the way in which dietary modifications such as addition of EBN supplementation so as to improve brain cognitive function in the next-generation mammals.

| Edible bird nest (EBN) collection and preparation
A total of three raw unprocessed EBN samples from three different states of Malaysia (e.g., North and South Peninsular Malaysia, and Borneo Sabah) were collected during the breeding season of edible nest swiftlet from April to August 2017. The samples were manually cleaned from gross dirt, and the visible feathers were removed using forceps. The commercial EBN was purchased from a retail outlet (Dingshen Imperial Birdnest). The EBN was finely grounded using an electrical grinder. Briefly, 10 mg/kg of dried EBN was immersed in the distilled water for 3 hr. Then, the soaked EBN in the water was boiled for 30 min with the solid-liquid ratio at 1:10 (g/ml). This solution was used to feed the individual animals. In addition, the sialic acid content from EBN was determined at SIRIM Berhad, Malaysia, using the high-performance liquid chromatography (HPLC).

| Animals
CJ57B/6 breeder's mice at 4 weeks of age (n = 60, 20 males and 40 females) were purchased from the Biosystem Corporation, Singapore. These breeders were considered as F0 generation and were used to produce the first (F1)-and second (F2)-generation of mice ( Figure 1). One week before mating, the breeder mice were di- given continuously until the female mice were pregnant. It was estimated that the female mice gave birth to approximately eight female pups of F1 generation in each treatment group and a control group (n = 40). After weaning at week 3, the breeder mice were removed from the cage and the F1 female mice were given free access pellets and water ad libitum. At 6 weeks of age, the F1 female mice were tested on spatial recognition memory using a Y-maze test. Our previous finding reported that 3 weeks of dietary with omega-3 fatty acid supplementation improved cognitive function in mice (Nur Farhana et al., 2015). Therefore, in this current study, at 6 weeks of age is sufficient time to evaluate the mice on cognitive performance. Then, at 8 weeks of age, the F1 female mice were mated to produce the F2 generation of mice. At 6 weeks of age, the F2 female mice were tested on spatial recognition memory similar to F1 female mice. At the end of the test, the F1 and F2 female mice were euthanized and their brains were processed for histological and gene expression study.

All animal testing was approved by the Institutional for Animal
Care and Use Committee (IACUC), Universiti Putra Malaysia (UPM/ IACUC/AUP-R092/2017).

| Learning and memory functions by the Y-maze test
The learning and memory functions of mice were tested using the Y-maze which was described previously (Hafandi et al., 2014). The Y-maze had three identical arms of equal size: the start arm, in which the mouse is first placed (always open); the familiar arm (always open); and the novel arm, which was blocked during the first trial but opened during the second trail ( Figure 2). Different visual cues were placed on the wall at the end of each arm of the maze. The Y-maze testing consisted of two trials separated by an interval of 1 hr. The first trial was 10 min in duration and allowed the mouse to explore only two arms (the start and familiar arms) of the maze, with the third arm (novel arm) blocked. After 1 hr, the second trial was conducted; mice were placed in the same starting arm as in trial 1, with free access to all three arms for 5 min. Trials were recorded by using a ceiling-mounted camera.

| Transmission Electron Microscopy (TEM) brain neuron histological changes
The brain hippocampus was prepared of about 1 mm 3 in diameter and fix with 2.5% glutaraldehyde for 24 hr. Then, the samples were

| RNA extraction and real-time RT-PCR
Tissue samples of hippocampus (~20 mg) were collected and extracted by using RNeasy Mini Kit Qiagen, German. The quality and quantity of extracted RNA were analyzed using Thermo Scientific NanoDrop2000c. The final concentration of cDNA 50 ng/ µl was produced from the total RNA from each sample of using One Taq ® RT-PCR kit (New England Biolabs) according to manufacturer instructions. The qRT-PCR assays for relative quantification of the respective gene expression (e.g., GNE, SLC17A5, ST8SiaIV, and BDNF) in each sample were performed using an iTaq Universal SYBR Green Supermix from Bio-Rad. The PCR primers set for the all genes were adapted from the previous study (Duncan, Raymond, Fuerholz, & Sprenger, 2009;Wang et al., 2007;Xie et al., 2018) and were synthesized by first base according to the cDNA sequences obtained from the Gene Bank TM database for mouse. The relative expression ratios were normalized to β-actin and GADPH, and the sequences of primers are presented in Table 1.
During initial optimization, the exact primer concentrations and PCR conditions were determined. Following optimization experiments, the assays of total volume of 20 µl reaction mix consisting of

| Statistical analysis
All data were expressed as mean ± SEM (standard error) and analyzed with the statistics software package (SPSS) for Windows version 25.
The assumption of normality and homogeneity of variances were assessed by Shapiro-Wilk's test and Levene's test, respectively. Oneway ANOVA was used to compare the effects of different maternal EBN supplementation on the cognitive performance of F1 and F2 animals. The significance of the difference between comparisons was determined by using Tukey's HSD test. All statistical tests were performed at 95% confident level, and p < .05 was considered to be significant.

| Sialic acid content of EBN
The retention time and HPLC analysis of sialic acid content from different EBN sample as shown in

of EBN Linearity
Correlation

| D ISCUSS I ON
The current study showed the content of sialic acid in 100 g of  Norhayati et al., 2010).
The content of sialic acid in all EBN groups in this study was found to improve learning and memory functions in the F1 and F2 animals.
This was shown by the higher number of entries and time spent in the novel arm of the Y-maze task performance compared to the control animals. This was probably due to the maternal mice were given EBN which contained high sialic acid that might had positive effect on the cognitive performance of their F1 and F2 offspring. These results indicated that the sialic acid from EBN was synthesized through the placenta and maternal milk which contributed to the neuronal transmission and synaptic plasticity development in their offspring (Oliveros et al., 2018;Wang et al., 2003;Xie et al., 2018). In addition, the synthesis of sialic acid in the F1 and F2 offspring improved the concentration of ganglioside bound and protein bound in the brain, suggesting that an increased sialylation on gangliosides mediates the learning and memory responses (Karim & Wang, 2006). In fact, the metabolism of sialic acid from EBN produced several neuraminic acids (e.g., N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetylneuraminate) that are integral structural and functional components of the nervous system (Karim & Wang, 2006;Khalid et al., 2019;Yu-Qin et al., 2000). Therefore, the ability of F1 and F2 mice to complete the Y-maze learning task suggested that the intake of exogenetic sialic acid from EBN is an essential micronutrient for optimal brain metabolism and cognitive development in multigeneration mice.
Dietary EBN supplementation for maternal mice showed an increase in the densities of synaptic vesicle in the pre-and postsynaptic CA1 hippocampus region on their F1 and F2 generations.
This finding is consistent with previous studies that showed the numbers of neurons on CA1, CA3, and DG regions of the hippocampus were significantly higher in mice treated with EBN rich in sialic acid (Xie et al., 2018). The higher densities of synaptic vesicles as shown in our study were in parallel with an improvement in the Y-maze task performance especially in the F1 and F2 animals. A possible explanation is that the sialic acid in EBN plays an important role at the synapses which can modulate the neurotransmission from the vesicle, via interactions calcium ion channel and chemical messages (Palmano, Rowan, Guillermo, Guan, & McJarrow, 2015;Rahmann, 1995;Wang, 2012). In fact, the synthesis of sialic acid in the neurons leads to altered synaptic plasticity required for memory consolidation (Foley et al., 2003;Wartman & Holahan, 2014). Thus, this probably indicated that EBN supplementation promotes the development of hippocampal neurons, which in turn affects the number and quality of synaptic vesicles connections especially during learning and memory processes. on NCAM during the period of high sialic acid demand such as during brain growth and development. Previous findings had also reported that the regulation of brain gene expression by dietary omega-3 fatty acids is due to the interactions of fatty acids with the ligands that bind to response factors acting on cis-regulatory elements of the gene, which finally turn on or off mRNA synthesis (Kitajka et al., 2004;Nur Farhana et al., 2015).
The current study showed EBN supplementation in maternal mice increased the BDNF gene in both generations due to the ability of EBN compounds to activate the extracellular signal-regulated kinase and signaling pathway. This pathway may lead to the activation of the cyclic adenosine monophosphate response element binding protein, which is a transcription factor that can increase the expression of a neurotrophins such as BDNF (Meeusen, 2014).
Indeed, BDNF promotes neurons survival, maturation, and synaptic functions and involved in several aspects of learning and memory functions (Bathina & Das, 2015;Buchman et al., 2016;Fernandes et al., 2011). It is suggested that the BDNF can induce neural plasticity and mitochondrial biogenesis, and hence has the ability to improve the hippocampal neurogenesis during pre-and postnatal development. Hence, the improvement in learning and memory functions in the Y-maze task in F1 and F2 mice as shown in this study was due to the synthesis of BDNF gene resulted from the dietary EBN supplementation in maternal mice especially during pregnancy and lactation periods.
Our finding also suggested that an upregulation of BDNF gene in the hippocampus was due to the changes in DNA methylation during learning and memory formation of Y-maze task in the F1 and F2 generations. In fact, DNA methylation is critical toward maternal effects on gene expression and generate phenotypic differentiation of offspring which resulted in behavioral changes across generations (Champagne & Curley, 2009)

. Previous cellular and
animal studies also reported that changes in neurons are related to an epigenetic alterations that may contribute to the higher variations of DNA methylation within neurons (Iwamoto et al., 2011;McCoy et al., 2019). Moreover, the unique changes in BDNF methylation were also associated with the different pattern of exon IV in the memory formation (Day & Sweatt, 2011). Therefore, our  In conclusion, the improvement learning and memory functions of F1 and F2 mice in the Y-maze task were due to the synthetized EBN supplementation from their maternal, and this result correlated with an increase in some brain gene expression and synaptic vesicle densities. In addition, it was shown here that the brain gene expression was involved in sialic acid biosynthesis during period of maternal EBN supplementation and transmitted to their generations. Further studies are required to compare sialic acid content in the F1-and F2-generation animals. This may explain how much of the sialic acid content from the maternal EBN supplementation can truly be considered as transmitted across several generation offspring.

ACK N OWLED G M ENTS
The authors would like to thank the staff of the Laboratory of Physiology Faculty of Veterinary Medicine, SIRIM and ABI for their assistance during the study. The authors wish to acknowledge the contribution of all Malaysian EBN farmers in the completion of this study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N
HA and SAA designed the experiments. OM, MAPR, MJE, and NMH performed the experiments and contributed to the collection, analysis, and interpretation of the data. HA and OM drafted the manuscript, and revised by MHMN, HAH, and SAA. All authors provided the approval for publication of the manuscript.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1817.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.