Influence of 2'-Fucosyllactose and Bifidobacterium longum Subspecies infantis Supplementation on Cognitive and Structural Brain Development in Young Pigs.

Development of the gut-brain axis during early-life is an important contributor of brain structural and functional development. Human milk oligosaccharides and gut microbiota have potential beneficial effects on various aspects of development; however, the effects of 2'-fucosyllactose (2'-FL) and Bifidobacterium longum subsp. infantis Bi-26 (Bi-26) administration during infancy separately and combined are still not clear. Therefore, we investigated the effects of early administration of dietary 2'-FL and Bi-26 on brain structural and functional development in the young pig. From postnatal day (PND) 2-34 or 35, fifty-two intact male pigs were randomly assigned to treatment groups in a 2 × 2 factorial arrangement and provided ad libitum access to a nutritionally adequate milk replacer without or with 1.0 g of 2'-FL/L of reconstituted liquid. Pigs within each diet group were further stratified to receive a daily oral dose of glycerol stock without or with Bi-26 (109 CFU). Pigs were subjected to the novel object recognition (NOR) task from PND 27-31 to assess recognition memory and subsequently underwent magnetic resonance imaging procedures at PND 32 or 33 to assess brain macrostructure and microstructure. Pigs that received Bi-26 had smaller absolute brain volumes for 9 of 27 brain regions of interest, and smaller relative volumes for 2 regions associated with kinesthesia (P < 0.05). Synbiotic administration of 2'-FL and Bi-26 elicited interactive effects (P < 0.05) on several microstructural brain components, where dual supplementation negated the effects of each test article alone. Behavioral outcomes indicated that pigs did not express novelty preference, regardless of treatment group, demonstrating no effects of 2'-FL and Bi-26 on recognition memory when supplemented alone or in combination. Interactive effects (P < 0.05) were observed for the number of all object visits, latency to the first object visit, and number of familiar object visits. Pigs that did not receive Bi-26 supplementation exhibited less time interacting with the familiar object in total (P = 0.002) and on average (P = 0.005). In conclusion, supplementation of 2'-FL and/or Bi-26 elicited some alterations in object exploratory behaviors and macro/micro-structures of the brain, but changes in recognition memory were not observed. Specifically in brain microstructure, synbiotic administration of 2'-FL and Bi-26 appeared to negate effects observed when each dietary article was supplemented separately.

Influence of Dietary Polar Lipid Supplementation on Memory and Longitudinal Brain Development.

Polar lipids, which are found in human milk, serve essential functions within biological membranes, hence their importance in brain development and cognition. Therefore, we aimed to evaluate the longitudinal effects on brain macrostructural and microstructural development and recognition memory of early-life polar lipid supplementation using the translational pig model. Twenty-eight intact (i.e., not castrated) male pigs were provided either a control diet (n = 14) or the control diet supplemented with polar lipids (n = 14) from postnatal day 2 until postnatal week 4. After postnatal week 4, all animals were provided the same nutritionally-adequate diets until postnatal week 24. Pigs underwent magnetic resonance imaging at 8 longitudinal time-points to model brain macrostructural and microstructural developmental trajectories. The novel object recognition task was implemented at postnatal weeks 4 and 8 to evaluate recognition memory. Subtle differences were observed between groups in hippocampal absolute brain volumes and fractional anisotropy, and no differences in myelin water fraction developmental patterns were noted. Behavioral outcomes did not differ in recognition memory, and only minimal differences were observed in exploratory behaviors. Our findings suggest that early-life dietary supplementation of polar lipids has limited effect on brain developmental patterns, object recognition memory, and exploratory behaviors.

Young Domestic Pigs (Sus scrofa) Can Perform Pavlovian Eyeblink Conditioning.

Introduction: Pigs have been an increasingly popular preclinical model in nutritional neuroscience, as their anatomy, physiology, and nutrition requirements are highly comparable to those of humans. Eyeblink conditioning is one of the most well-validated behavioral paradigms in neuroscience to study underlying mechanisms of learning and memory formation in the cerebellum. Eyeblink conditioning has been performed in many species but has never been done on young pigs. Therefore, our aim here was to develop and validate an eyeblink conditioning paradigm in young pigs. Method: Eighteen intact male pigs were artificially reared from postnatal day 2-30. The eyeblink conditioning setup consisted of a sound-damping box with a hammock that pigs were placed in, which allowed the pig to remain comfortable yet maintain a typical range of head motion. In a delay conditioning paradigm, the conditional stimulus (CS) was a 550 ms blue light-emitting diode (LED), the unconditional stimulus (US) was a 50 ms eye air-puff, the CS-US interval was 500 ms. Starting at postnatal day 14, pigs were habituated for 5 days to the eyeblink conditioning setup, followed by 5 daily sessions of acquisition training (40 paired CS-US trials each day). Results: The group-averaged amplitude of conditioned eyelid responses gradually increased over the course of the 5 days of training, indicating that pigs learned to make the association between the LED light CS and the air-puff US. A similar increase was found for the conditioned response (CR) probability: the group-averaged CR probability on session 1 was about 12% and reached a CR probability of 55% on day 5. The latency to CR peak time lacked a temporal preference in the first session but clearly showed preference from the moment that animals started to show more CRs in session 2 and onwards whereby the eyelid was maximally closed exactly at the moment that the US would be delivered. Conclusion: We concluded that 3-week-old pigs have the capability of performing in a cerebellar classical conditioning task, demonstrating for the first time that eyeblink conditioning in young pigs has the potential to be a valuable behavioral tool to measure neurodevelopment.

Influence of Rearing Environment on Longitudinal Brain Development, Object Recognition Memory, and Exploratory Behaviors in the Domestic Pig (Sus scrofa).

INTRODUCTION: Over the last 40 years, the domestic pig has emerged as a prominent preclinical model as this species shares similarities with humans with regard to immunity, gastrointestinal physiology, and neurodevelopment. Artificial rearing of pigs provides a number of advantages over conventional rearing (i.e., true maternal care), including careful control of nutrient intake and environment conditions. Yet there remains a gap in knowledge when comparing brain development between sow-reared and artificially reared domestic pigs. Thus, our research sought to model brain development and assess recognition memory in a longitudinal manner by directly comparing rearing environments. METHODS: Forty-four intact (i.e., not castrated) male pigs were artificially reared or sow-reared from postnatal day 2 until postnatal week 4. After postnatal week 4, all pigs were housed in a group setting within the same environment until postnatal week 24. Magnetic resonance imaging was conducted on pigs at 8 longitudinal time-points to model developmental trajectories of brain macrostructural and microstructural outcomes. Additionally, pigs behavior were tested using the novel object recognition task at postnatal weeks 4 and 8. RESULTS: Throughout the 24-week study, no differences between rearing groups were noted in weekly body weights, average growth and feed intake patterns, or feed efficiency. Whole brain, gray matter, white matter, and cerebrospinal fluid growth patterns also did not differ between pigs assigned to different early-life rearing environments. Moreover, minimal differences in regional absolute volumes and fractional anisotropy developmental trajectories were identified, though artificially reared pigs exhibited higher initial rates of myelination in multiple brain regions compared with sow-reared pigs. Furthermore, behavioral assessment at both PNW 4 and 8 suggested little influence of rearing environment on recognition memory, however, an age-dependent increase in object recognition memory was observed in the sow-reared group. CONCLUSION: Our findings suggest that early-life rearing environment influences the rate of development in some brain regions but has little influence on overall brain growth and object recognition memory and exploratory behaviors in the domestic pig. Artificial rearing may promote maturation in certain brain areas but does not appear to elicit long-term effects in outcomes including brain structure or object recognition memory.

High-resolution magnetic resonance imaging-based atlases for the young and adolescent domesticated pig (Sus scrofa).

BACKGROUND: Neurodevelopmental studies utilize the pig as a translational animal model due to anatomical and morphological similarities between the pig and human brain. However, neuroimaging resources are not as well developed for the pig as they are for humans and other animal models. We established a magnetic resonance imaging-based brain atlas at two different ages for biomedical studies utilizing the pig as a preclinical model. NEW METHOD: Twenty artificially-reared domesticated male pigs (Sus scrofa) and thirteen sow-reared adolescent domesticated male pigs (Sus scrofa) underwent a series of scans measuring brain macrostructure, microstructure, and arterial cerebral blood volume. RESULTS: An atlas for the 4-week-old and 12-week-old pig were created along with twenty-six regions of interest. Normative data for brain measures were obtained and detailed descriptions of the data processing pipelines were provided. COMPARISON WITH EXISTING METHOD: Atlases at the two different ages were created for the pig utilizing newer imaging technology and software. This facilitates the performance of longitudinal studies and enables more precise volume measurements in pigs of various ages by appropriately representing the neuroanatomical features of younger and older pigs and accommodating the proportion differences of the brain over time. CONCLUSION: Two high-resolution MRI brain atlases specific to the domesticated young and adolescent pig were created using defined image acquisition and data processing methods to facilitate the generation of high-quality normative data for neurodevelopmental research.

Early-Life Supplementation of Bovine Milk Osteopontin Supports Neurodevelopment and Influences Exploratory Behavior.

INTRODUCTION: Osteopontin (OPN) is a whey protein found at high concentration in human milk and is involved in processes such as bone cell proliferation and differentiation. Milk OPN has shown to be involved in various aspects of development, including the immune system and gut health. However, the influence of dietary bovine milk OPN inclusion on brain and cognitive development has not been studied extensively until recently. This research examines whether dietary supplementation of bovine milk OPN supports brain and cognitive development in the translational pig model. METHODS: From postnatal day (PND) 2 to 34, twenty-one intact male pigs were provided ad libitum access to one of two dietary treatments, a standard soy protein isolate-based milk replacer to serve as a control diet (n = 11) and the same base diet supplemented with bovine milk OPN to serve as a test diet (n = 10). In addition to growth and health outcomes, recognition memory was tested using the novel object recognition (NOR) task from PND 28 to 32, and magnetic resonance imaging was conducted at PND 34 to evaluate brain development. RESULTS: No dietary effects were observed for growth performance or health indices. For the behavioral analysis, pigs that received the test diet exhibited shorter (p < 0.05) latency to the first object visited compared with pigs fed the control diet. Although the control group exhibited novelty preference, there was no difference in recognition index between dietary groups. Neuroimaging outcomes revealed increased (p < 0.05) relative brain volumes of the corpus callosum, lateral ventricle, left and right internal capsule, left and right putamen-globus pallidus, and right hippocampus, and right cortex in the test group. Diffusion tensor imaging revealed higher (p < 0.05) radial diffusivity in the corpus callosum and lower (p < 0.05) fractional anisotropy in pigs provided the test diet. CONCLUSION: Dietary supplementation of bovine milk OPN increased the relative volume of several brain regions and altered behaviors in the NOR task. Underlying mechanisms of bovine milk OPN influencing the development of brain structures and additional behaviors warrant further investigation.