## 4 Discussion  
The objective of this study was to assess the effectiveness of OF alone or in combination with 2'-FL at altering recognition memory, brain structure, and hippocampal gene expression. Dietary intake of OF alone produced effects differential to that of OF + 2'-FL concerning recognition memory and hippocampal gene expression, yet both increased the relative volume of the olfactory bulbs compared to controls. An important limitation of the experimental design is the use of different doses for each source of oligosaccharidesacharide and lack of a full two-way ANOVA design, thus confounding the ability to delineate between dose and source effects. This choice was made to replicate the doses used from  
Figure 7: Significant correlations by diet or study-wide linear regression between the recognition index after a 1 h delay and genes affected by diet. No significant relationships were found with the recognition index after a 48 h delay. (**A**) A negative correlation for all diets was found between GABBR1 and the recognition index. Linear regression demonstrated the presence of a significant relationship—for every increase in 1 standard deviation of GABBR1 expression, the recognition index decreased by 0.13. (**B**,**C**) Some dietary groups showed a correlation between the recognition index and either HDAC5 or CHRM2. Overall, linear regression revealed significant negative relationships between mRNA expression and the recognition index. Abbreviations: _GABBR1_, GABA type B receptor subunit 1; _HDAC5_, histone deacetylase 5; _CHRM2_, cholinergic receptor muscarinic 2; CON, control group; OF, pigs fed oligofructose; OF + 2’-FL, pigs fed oligofructose and 2′ fucosyllactose. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{2}{c}{**CON**} & \multicolumn{2}{c}{**OF**} & \multicolumn{2}{c}{**OF + 2’-FL**} & \\ \hline
**Measure 2** & **N** & **Mean** & **N** & **Mean** & **N** & **Mean** & **SEM** & _p-Value 3_ \\ \hline _CHRM2_4 & 12 & 0 1  & 12 & 0 12 & \(-0.75\)  & 12 & 0 1 & \(-0.47\)  & 12 & 0 1 & \(-0.47\)  & 12 & 0 1 & \(-0.47\)  & 12 & 0 1 & \(-0.47\)  & 12 & 0 0 12 & \(-0.47\)  & 12 & 0 0 12 & \(-0.47\)  & 12 & 0 0 12 & \(-0.47\)  & 12 & 0.67  & 12 & 0 12 & \(-0.52\)  & 12 & 0 \(-0.45\)  & 12 & 0.42 & 0.32 & 0.037 \\ _HDAC5_ & 11 & 0 1 & 12 & \(-0.45\) [FOOTNOTE:1]Footnote 1: Abbreviations: CON, control group; OF,previous clinical trials, however [11; 31; 32; 33]. Here, the analyzed concentration of OF in each group was 3.41-3.62 g/L, and 2\({}^{\prime}\) FL was included in the combination at 1.12 g/L. This is in comparison to previous clinical trials investigating OF in infant formula at ranges from 3 to 5 g/L [31; 32] and 2\({}^{\prime}\) FL at ranges from 0.2 to 1.0 g/L [11; 33]. Thus, the aim was to assess if there is additional benefit to the addition of 2\({}^{\prime}\) FL in a formula already containing OF, using historically relevant doses for each OS. Still, this study adds to a growing body of literature suggesting that OS can alter behavior and neurobiology. However, the mechanisms of action remain unclear. While many OS have been cited as having such effects [44], it is becoming clear that not all OS act similarly or with the same efficacy. We chose to use the NOR task using two different delays to assess short-to-intermediate (1 h delay) and long-term (48 h delay) object recognition memory. While CON pigs failed to exhibit recognition memory after both the 1 and 48 h delays, pigs fed OF exhibited recognition memory after a 1 h delay, whereas pigs fed OF + 2\({}^{\prime}\)-FL exhibited recognition memory after a 48 h delay. Curiously, pigs fed the combination did not show an improvement in recognition memory after a 1 h delay, demonstrating specificity of the combination to improve type-specific recognition memory. This is potentially due to the requirement of the perirhinal cortex for short- but not long-term memory, and the role of the hippocampus in only long-term recognition memory [45].