Effects of early intermittent maternal separation on cognitive development of the prefrontal cortex in piglets

doi:10.21203/rs.3.rs-2237920/v1

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Effects of early intermittent maternal separation on cognitive development of the prefrontal cortex in piglets

https://doi.org/10.21203/rs.3.rs-2237920/v1

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In actual production, because increase of sows litter size, the management of piglets in split-suckling leads to intermittent neonatal maternal separation (NMS). Early lactation is a critical period of cognitive development in the piglets' brain. The experiment hypothesizes that intermittent NMS may affect piglets' neurological development and cognitive ability. To determine the extent of effects of NMS on the piglets' prefrontal cortex cognitive development, the control group (Con group, n=53) was set up with no NMS, the experimental group (MS group, n=53) was set up with MS for 4 h/day, and the piglets were milk supplemented during the separation period and weaned until 35d. Twelve piglets at postnatal day (PND) 35 were selected from each group for cognitive assessment. Three PND35 piglets from each group were slaughtered, and collected prefrontal cortex samples for reference transcriptome sequencing. Following bioinformatics analysis, GO enrichment and KEGG enrichment analysis as well as candidate gene screening were performed for differentially expressed genes. Finally, quantitative reverse transcription PCR (qRT-PCR) was used to verify the sequencing data were accurate. The results showed that, in the spatial holeboard discrimination task, the working memory and reference memory of piglets in the MS group were significantly lower than those in the Con group (P < 0.01). In the new object recognition task, the MS group was significantly lower than the Con group in the number of exposure to new objects and discrimination index (P < 0.01). In the prefrontal cortex of the MS group, 349 up-regulated, 151 down-regulated differential genes, and 584 significant GO entries were discovered. Screening for 9 genes associated with cognition at large differential multiples. qPCR validation results showed that MC4R, GRP, CYFIP2, NMB, CPEB1, ADCYAP1 were significantly downregulated and MAG, PLP1, CPEB4 were significantly upregulated. Light microscopy results showed changes in the morphological structure of neuronal cells and microglia. In summary, intermittent NMS may impair partial cognitive function in piglets by damaging the structure of the prefrontal cortex.

Piglets

Intermittent neonatal maternal separation

Prefrontal cortex

Cognitive development

With the progress of production technology and feeding management technology, the number of piglets born in the litter of sows gradually rises, and there are often many piglets that unable to grab the teats. Cross-fostering, split-suckling and artificial lactation are applied in production to solve this difficulty. Many modern pig farms choose to nurse these piglets with split-suckling, resulting in piglets suffering some degree of neonatal maternal separation (NMS), which has important implications for offspring neurodevelopment and disease risk (Loewy et al., 2019). Newborn piglets' brain development during lactation is crucial, and early stress due to various social and environmental changes may have an impact on the piglets' neurodevelopmental brain, cognition, and social behavior.

Studies in mice have shown that the early life stress from NMS caused some impairment in brain development, which exhibit fear and anxiety-like behaviors in behavioral tests, and had a significant impact on cognitive abilities (Wei et al., 2020). Siegford et al.(2008) demonstrated that piglets weaned on PND12 while MS for a long period time were limited in their ability to reach the hidden-platform task (spatial task) in the Morris water maze, suggesting that the piglets' cognitive abilities were limited. However, there were fewer studies on the cognitive effects of intermittent NMS on piglets, so this experiment chose the spatial holeboard discrimination task and new object recognition to assess piglets' cognitive abilities. Both behavioral tests require the direct involvement of the prefrontal cortex.

In the number of mature neurons in the prefrontal cortex in rats, NMS24 hours could cause a reduction in prefrontal cortex thickness (Crombie et al., 2021). Fabricius et al. (2008) found that mice undergoing MS on PND9, the dentate gyrus neurons significant reduction. This suggests that MS can affect the number of hippocampal neurons in mice by decreasing neurogenesis or increasing neuronal apoptosis. Therefore, this experiment performed piglets prefrontal cortex pathological tests to observe the changes in neurons in it.

The effects of MS on cognitive development in the brain are regulated by various mechanisms and are mainly found in two important brain regions, the hippocampus and prefrontal cortex. It has been suggested that the MS affects hippocampal myelination and anxiety-like behavior through inhibition of the GABA/glutathione pathway (Aksic et al., 2013). However, there were fewer studies on the molecular level of prefrontal cortex effects in piglets. This study conducted a transcriptomic study on the piglets prefrontal cortex of to screen for mediated differential genes.

Therefore, this study used pathological assays, transcriptomic and quantitative reverse transcription PCR (qRT-PCR) techniques to evaluate the effects of intermittent NMS on the cognitive development of the prefrontal cortex in piglets at the molecular level. Meanwhile, proposed to construct a model of intermittent NMS, aiming to investigate the adverse effects of intermittent NMS on the cognitive development of piglets using lactating piglets as an animal model.

Experimental animals

In this experiment, conducted 12 litters of crossbred piglets from healthy and similar body condition Du Min (Duroc × Min-pig) sows (the same bloodline was used to ensure the same genetic background of the pigs used in the test), and the mating boars were Large White pigs (American Large White). Transfer the sows to the farrowing pens one week before farrowing. Performed experiments in loose farrowing and nursery pens (5.2 m × 1.6 m × 1.1m), feed troughs, and water spouts at the front of the pens. Maintained temperature and humidity in farrowing and nursery pens at a comfortable and constant level. Controlled the environment by an automatic ventilation system and the piglet rest area insulation heating device, with an average temperature of 26.2°C, an average humidity of 68.3%, natural lighting, and good ventilation. The sows and piglets were fed and raised in strict accordance with the production process requirements. The sows were fed daily at 6:00, 11:00 and 17:00, and the piglets had free access to food and water. Piglets received iron supplements at PND3 and were fed with creep feed from PND7. The pens were kept clean and hygienic and were regularly disinfected, the piglets were vaccinated. All piglets were not severed tail and castrated in the experiment to reduce stress. At PND35, three piglets in good condition were selected for slaughter in each of two groups (Con group, n = 6 litters; MS group, n = 6 litters). Afterward, the brain tissue was quickly removed and the 1 × 1 cm size prefrontal cortex brain region was collected and fixed in 4% paraformaldehyde for HE staining. The remaining prefrontal cortex brain region was collected in RNAase-free EP tubes and stored at -80°C for subsequent reference transcriptomic analysis and quantitative real-time fluorescence PCR (qRT-PCR).

Spatial holeboard discrimination task

The test method was shown in Fig. 1, and randomly selected 12 piglets of PND36 in each of the two treatments for the test. Let the selected piglets enter the field every day for a week in advance to get used to it. Before the start of the experiment, placed the piglets in the field of 3.2 m × 3.2 m 5 minutes in advance to familiarize themselves with the environment. Divide the space evenly into 4×4 squares, and fix the clean slatted floor in the site. The venue was disinfected with 75% alcohol after the behavioral test of each piglet to minimize the effect of odor on the test. Installed the camera directly above the test site to record the whole process. The first three days of the experiment began as an acclimatization period, with chocolate placed daily above the fixed four-slatted floors so that piglets could eat consistently. When the experiment was formally conducted, 16 slatted floors were disinfected and none of them without food. Spatial working memory and reference memory measures could be obtained by observing the number of visits and revisits of the piglets to the original baited and original unbaited areas within 5 min for scoring.

Working memory (%) = (number of first visits to the decoy area)/(number of visits and revisits to the decoy area) × 100%.

Reference memory (%) = (number of visits and revisits to decoy areas)/(number of visits and revisits to all areas) × 100% (Arts et al., 2012).

The new object recognition task

The Fig. 2 showed the test method, randomly selected 12 piglets of PND36 in each of the two treatments for the test. Let the selected piglets enter the field every day for a week in advance to get used to it. Placed the piglets in the prepared 3.5 m×3.5 m pen 5 min before the start of the experiment to familiarize themselves with the environment. During the familiarization phase, placed two identical objects A at the same distance from the test piglets and allowed to explore them for 5 min. During the formal experiment, one of the A objects was replaced with an object B of a different shape, same material, and color after one hour, and its exploration of the familiar object A and the new object B was observed for 5 min. After each piglet behavior test, the site was disinfected with 75% alcohol and wiped with warm water on each of the two objects to maximize the effect of odor on the test. Installed the camera directly above the test site to record the whole process. The discriminative index of the piglets was obtained by observing the piglets' exploration of the two objects A and B within 5 min (exploration time and the number of time). Exploration was defined as the piglet facing the object and the distance between the nose and the object ≤ 2 cm.

Discriminant index (%) = [(exploration time for new object B) - (exploration time for familiar object A)]/(total exploration time for familiar object A and new object B) × 100% (Antunes et al., 2012).

Transcriptomics profiling technique

Total RNA was isolated and purified from total samples using TRIzol (Invitrogen, USA), followed by quality control of the quantity and purity of total RNA using NanoDrop ND-1000 (NanoDrop, USA). The fragmented RNA was synthesized into cDNA by reverse transcriptase (Invitrogen SuperScript™ II Reverse Transcriptase, No. 1896649, CA, USA), which was then used to form a cDNA library with a fragment size of 300 bp ± 50 bp. Transcriptome profiling steps were carried out by LianChuan-biotechnology Co., Ltd (Hangzhou, China). The sequencing data were screened and sorted using a HiseqTM sequencer, and genes with the | log2 (Fold Change) | greater than 1.5 and P value less than 0.05 were defined as differential genes. Finally, significant difference analysis was performed using the R package DESeq2, GO and KEGG enrichment analyses were performed.

qRT-PCR verification of DEGs

Total RNA was extracted from the prefrontal cortex of piglets with TRIzol reagent (Invitrogen, China) according to the instructions of the RNA Extraction Kit (Beijing Mei5 Biotechnology Co., Ltd, China). The concentration and purity of total RNA were determined by using Quick DropTM, an ultra-micro-analyzer (Valley Molecular, Inc., USA). The mRNA was reverse transcribed according to the reaction system (20 μL) of the Reverse Transcription Kit (Beijing Mei5 Biotechnology Co., Ltd, China). Using Primer Premier 5.0 design specific primers for the screened differential gene and the internal reference gene β-actin based on the gene sequences published in GenBank. Primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). The specific primer sequences of the screened differential and internal reference genes β-actin were shown in Table S1. qRT-PCR was performed using the LightCycler®96 (Roche, Switzerland) for quantitative real-time PCR (qRT-PCR). The relative expression of mRNA of each gene was calculated using 2^-ΔΔCT (Li et al., 2021).

HE staining and light microscopic observation

The prefrontal tissue fixed in 4% paraformaldehyde was trimmed, rinsed with water, dehydrated with different concentrations of alcohol, paraffin-embedded, and cut into 6µm slices by conventional methods. The slices were stained with hematoxylin-eosin (HE) and sealed with neutral resin. A multifunctional digital light microscope (DMS-651) was used to observe and record the microstructural changes.

Data-processing and statistical analysis

All experimental data were preliminarily counted in Excel 2016 and used SPSS 22.0 for cognitive behavioral tests and mRNA data analysis. First, testing data were for normal distribution by Shapiro-Wilk test method, and the Levene test was used for homogeneity of variance test, to ensure that all data from the same treatment group conform to the normal distribution, the homogeneity of variance > 0.05. Differences in cognitive behavioral tests between the Con and MS groups were analyzed using independent samples T-test after compliance, and data that did not conform to a normal distribution were analyzed using the nonparametric Mann-Whitney U test. The same method was used to analyze the variability of differential and endogenous gene expression data in the Con and MS groups. Data were expressed as mean ± standard error (means ± SEM), and P < 0.05 indicates a statistically significant difference.

Spatial holeboard discrimination task

The results of the spatial holeboard recognition task were shown in Table 1. There was no difference between the two groups of piglets in the total number of visits to the holeboard (P > 0.05). The number of visits and revisits to the decoy area was significantly lower in the MS group than in the Con group (P < 0.05). Working memory and reference memory were significantly lower in the MS group than in the Con group (P < 0.01).

The new object recognition task

The results of the new object recognition task were shown in Table 2. There was no significant difference in the total number of times and the total time that piglets were exposed to objects during the test period (P > 0.05). Piglets in the MS group were exposed to new objects for a significantly (P < 0.05) shorter time than those in the Con group. The number of exposures to the new object and the discrimination index were significantly (P < 0.01) lower in the MS group than in the Con group.

Differential gene expression and analysis

To investigate the changes in the genetic level of prefrontal cortex in early intermittent maternal separation piglets, the genetic level of prefrontal cortex was determined by transcriptome sequencing. Gene expression was compared between early intermittent maternal separation piglets and normally reared piglets, and a total of 500 differential genes were identified, including 349 up-regulated differential genes and 151 down-regulated differential genes (see Fig. 3).

Differential Gene GO Enrichment Analysis

The GO database was used to analyze differentially expressed genes in the prefrontal cortex, and GO enrichment analysis of differentially expressed genes resulted in 2537 GO entries (584 significant) for differentially expressed genes in the prefrontal cortex. All GO entries could be classified according to biological process, cellular component and molecular function (one entry could not be enriched to these three categories). The differential genes were enriched to 1726 GO entries (389 significant), 336 GO entries (89 significant), and 473 GO entries (105 significant) in biological process, cellular component, and molecular function, respectively. The top 25 most significantly enriched GO entries were selected for display, and the results of the enrichment bubble plot display were shown in Fig. 4a.

The first five GO entries in the biological process were GO:0045944 (positive regulation of transcription by RNA polymerase II), GO:0000122 (negative regulation of transcription by RNA polymerase II), GO:0006355 (regulation of transcription, DNA-templated), GO:0007165 (signal transduction), GO:0007155 (cell adhesion), the number of enriched genes were: 27, 25, 25, 21, 20. The first five GO entries in cellular component were: GO:0016020 (membrane), GO:001602 (integral component of membrane), GO GO:0005634 (nucleus), GO:0005737 (cytoplasm), GO:0005886 (plasma membrane), and the number of enriched genes were 148, 124, 105, 96, 83 respectively. The top 5 GO entries in molecular function were GO:0005515 (protein binding), GO:0046872 (metal ion binding), GO:0000166 (nucleotide binding), GO:0005524 (ATP binding), GO:0008270 (zinc ion binding), the number of enriched genes were: 4, 6, 5, 5, and 8, respectively. The first 25 GO entries in biological processes, 15 GO entries in cellular components, and the first 10 GO entries in molecular functions were shown in Fig. 4b.

Validation of differential gene screening and transcriptome sequencing results

Through the literature and the results of this experiment, it was found that cognition and behavior are related to neurons, neuropeptides, synapses, etc., Screening genes related to cognitive memory (such as neurons, neuropeptides, synapses, myelin, etc.), |log₂FC| > 1, and P < 0.05. Screened out 9 cognitive-related differential genes as candidate genes, using these candidate genes were verified by qRT-PCR, and the comparative analysis of transcriptome sequencing results and qRT-PCR results were shown in Table 3. qRT-PCR and transcriptome sequencing results maintained the same trend, indicating that the transcriptome sequencing results were reliable.

Morphological changes of prefrontal cortex

The morphological and ultrastructural changes of the prefrontal lobe were shown in Fig. 5. In the prefrontal cortex tissue of the MS group, more neurons were seen with mild edema around them, forming gaps (see black arrows in Fig. 5b and Fig. 5c), and a large number of neurons have deepened staining (see yellow arrows in Fig. 5b and Fig. 5c). At the same time, there was a mild proliferation of microglia (see red arrows in Fig. 5b and Fig. 5c), neuronal folds, deepened cell staining, and indistinct demarcation of the nucleus and cytoplasm (see black arrows in Fig. 5e and Fig. 5f), accompanied by an increase in the number of glial cells (see red arrows in Fig. 5e and Fig. 5f). Neurons in the prefrontal cortex tissue of the Con group were more numerous, structurally intact, with clear nucleoplasmic boundaries, neatly arranged, and mild edema around the neurons (see Fig. 5a and Fig. 5d).

When it is necessary to remember spatial information, the brain will preferentially mobilize a common bilateral frontoparietal cortex network, especially the prefrontal cortex among them (Walter et al., 2003). Therefore, the spatial holeboard recognition task is very suitable for evaluating the cognitive development of the prefrontal cortex. Studies have shown that memory loss have a negative impact on working memory and reference memory (Van et al., 1990). Stress can also lead to deficits in spatial learning ability in animals (Cazakoff et al., 2010), and the results of the cognitive ability test in this study were consistent with this. In the spatial holeboard recognition task in this experiment, the piglets' spatial working memory and reference memory after maternal separation were significantly lower than those of the Con group piglets. This finding may suggest that piglets' short- and long-term memories after separation stress were significantly diminished, which is crucial. It may be related to the regulation of genes related to synapses and neurons in the prefrontal cortex, but this needs further experiments to verify. According to studies found that the prefrontal cortex is involved in non-spatial learning and memory processes (Blasi et al., 2014). Tests in rats found that damage to the prefrontal lobe reduced the memory of visual objects (Ragozzino et al., 2002), and the novel object recognition (NOR) task is widely used to assess the animals' non-spatial learning and memory. In the NOR task, it was found that a single prolonged stress exposure impaired the organism's preference for object novelty and to impair memory for object recognition (Eagle et al., 2013). And, interestingly, discovered that mice separated from their fathers had effects on NOR test results and cognitive function (Agarwal et al., 2020), which suggested that both parental and maternal separation have an impact on offspring. In this study, it was also found that the discrimination index, time and frequency of exposure to new objects were significantly lower in the Con group piglets than the control group piglets. This may indicate that piglets have a reduced preference for exploring new objects after separation, and there may be damage to the prefrontal cortex, adversely affects cognition.

The prefrontal cortex is a brain region susceptible to stressful changes, and early stressful stress may lead to impaired prefrontal cortex neuronal development. In this experiment, tests of cognitive function revealed that piglets who experienced early intermittent maternal separation may be under stress, which resulted in cognitive deficits in piglets. Therefore, we found that these related GO items were significant through the screening of transcriptome sequencing GO items, and screened out 9 differential genes: MC4R, NMB, GRP, ADCYAP1, CYFIP2, MAG, CPEB1, CPEB4, PLP1, these genes are all related to the recognition related to memory. Neuromedin B (NMB) exerts its function through the NMB receptor. Studies have found that stress can affect the NMB receptors deficiency and impair memory (Yamada et al., 2003), indicating that NMB and its receptors are closely related to cognitive memory. Therefore, the maternal separation piglets' memory impairment is related to the down-regulation of NMB gene, but this needs further experiments to verify. Gastrin-releasing peptide (GRP) and its receptors are widely distributed in various brain regions, and GRP plays a key role in synaptic power, which is critical for cognitive function (Yang et al., 2017). GRP is a marker of neuroprotective neurons, and studies have found that a lack of GRP may lead to elevated neurogenesis and impaired neuronal development (Walton et al., 2014). In this study, the GRP gene showed a tendency to be downregulated in maternal-separated piglets, and GRP downregulation ken makes the neuronal protection not work well, resulting in impaired neuronal development and cognition, and neuronal damage was also clearly observed in pathological observations.

It is well known that the melanocortin receptor (MCR) in the brain is related to cognitive function, and MCR can improve animals learning and memory ability (McLay et al., 2001). Studies have found that neuroinflammation can reduce the melanocortin system (MC4R and α-melanocyte-stimulating hormone) expression (Flores-Bastías et al., 2020), which is similar to our research results. Early stress may induce neuroinflammation in piglets and make MC4R expression decrease. MC4R can also control food motivation related to the brain reward system (Yoon et al., 2015), which is consistent with the results of our spatial holeboard recognition task. MC4R downregulation affects cognitive recovery and is also related to the brain reward system of the spatial hole recognition task.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a neurotransmitter or neuromodulator, promotes neuronal survival, and plays a role in various forms of stress (Condro et al., 2016). Whereas PACAP knockout mice showed cognitive impairment in the novel object recognition test (Takuma et al., 2014), which is similar to our study results. The piglets after the maternal separation also showed cognitive impairment in the novel object recognition test. The expression of the PACAP gene was also reduced. According to the above research, it is speculated that the down-regulation of this gene affects the cognitive function of piglets, but this needs further experiments to verify.

Cytoplasmic FMR1-interacting protein 2 (CYFIP2) plays a critical role in regulating actin dynamics and neuronal excitability in primary neurons in the medial prefrontal cortex (mPFC) (Zhang et al., 2020). Although the in vivo neurobiological mechanisms underlying CYFIP2-related brain diseases are not well understood, CYFIP2 protein expression was significantly reduced in Alzheimer's disease cases, and cognitive behavioral tests found that reduced CYFIP2 expression also prevented mice from being able to space the water maze (Tiwari et al., 2016), affecting the body spatial memory function. This shows that the gene down-regulation is likely to be related to the piglets cognitive decline in the spatial hole recognition task in this study. Therefore, the CYFIP2 gene down-regulation of piglets prefrontal cortex may affect neuron function and lead to cognitive deficits.

Abnormal axonal regeneration after CNS injury is partly due to the presence of myelin-associated molecules that inhibit axonal growth, including myelin-associated glycoprotein (MAG), which is immunologically increased in brain tissue following brain injury (Thompson et al., 2006). Studies have found that MAG in the body may lead to the central nervous system lacks regeneration in mammals after injury (Tang et al., 1997). Similar findings were also found in rodents, where MAG was significantly increased in poison-stressed mouse brain tissue (Li et al., 2015). In this study, piglets MAG also showed an up-regulation trend after the intermittent MS. The MS may caused piglets brain damage, inhibited axonal growth, hindered the recovery of cognitive memory, and damaged the nervous system.

Cytoplasmic polyadenylation element-binding protein (CPEB) exists in neurons and glial cells. CPEB affects learning and memory, and affects neuron morphology and synaptic plasticity, and is a key mediator of important biological processes such as learning and memory (Bestman et al., 2009; Ivshina et al., 2014). CPEB1 and CPEB4 belong to this family of proteins, but they play different roles in regulating the normal development of the brain. Studies have found that CPEB1 is down-regulated and CPEB4 is up-regulated in glioblastoma (Skubal et al., 2016; Zhijun et al., 2017), leading to abnormalities in the organism's brain development. Interestingly, some studies have found that CPEB4 knockout mice were normal, but only neonatal animals expressing CPEB4 show impaired activity and neuronal developmental defects (Shinet al., 2016), indicating that CPEB4 has certain effects on brain neurodevelopment, its absence did not have much effect on neuronal development. In contrast, the CPEB1 regulatory mechanism is still unclear, but the level of CPEB1 is decreased in schizophrenia patients (Xia et al., 2017), and the knockdown of CPEB1 expression inhibits the activation of astrocytes (Kim et al., 2011), which indicates that the CPEB1 downregulation can lead to immune function exception. In this study, it was found that early intermittent MS also led to the down-regulation of CPEB1 and the up-regulation of CPEB4 in the prefrontal cortex. This study is similar to the above-mentioned findings. The changes in CPEB1 and CPEB4 may have affected immune function and neuronal development, with some negative effects on brain development.

Proteolipid protein 1 (PLP1) is one of the leading candidate genes for brain white matter structure in and is essential for cognition and behavior. Studies have found that PLP1 overexpression can lead to progressive leukodystrophy and cognitive decline (Li et al., 2019). In animal models of PLP1 overexpression, the organism exhibits anxiety-like behavior, spatial learning deficits and working memory deficits (Tanaka et al., 2009). This study found that piglets undergoing early intermittent MS had deficits in spatial learning ability and working memory, which is similar to the above-mentioned findings. It may be related to brain damage such as leukodystrophy caused by the up-regulation of PLP1 gene.

Numerous studies have found that stress-induced dendrites in the medial prefrontal cortex undergo rapid changes, as well as changes in the spatial arrangement of neurons and glial cells (Brown et al., 2005; Noorafshan et al., 2015), and early MS will make the prefrontal cortex tree decreased branching (Gibb et al., 2005), suggesting that stress leads to neuronal structure changes. This study found that the neurons in the piglet prefrontal cortex with early intermittent MS were shrinking, mild edema around the neurons, and darkened cell staining, indicating that the neuronal cell function was not active. Microglia play a key role in a mature and healthy brain, providing nutrient support for neurons, regulating neuronal activity, and altering synaptic connections and plasticity (Wu et al., 2015). This study found that microglia hyperplasia in the prefrontal cortex of piglets, a brain lesion, is a characteristic symptom of brain injury-induced reactive gliosis, which generally occurs in the damaged central nervous system. The increase in the number of glial cells indicates that the body may be in a state of inflammatory stimulation, and neurons in the prefrontal cortex may be damaged.

In summary, early intermittent MS may impair some of the cognitive functions of piglets by damaging the structures of the prefrontal cortex.

Acknowledgements The authors would like to thank Dr. Houjuan Xing and Qi Han for their help in the successful completion of this study. We thank Jane Charbonneau, DVM, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Author contributions Zheng Cheng: Conceptualization, Formal analysis, Writing- Original draft preparation, Visualization. Sitong Zhou: Investigation, Software, Methodology. Xiaohong Zhang: oftware, Writing - Review & Editing. Qin Fu: Methodology, Writing - Review & Editing. Yue Yang: Investigation, Validation. Wenbo Ji: Conceptualization, Resources, Writing - Review & Editing. Honggui Liu: Conceptualization, Resources, Writing - Review & Editing, Funding acquisition.

Funding The study was supported by China Agriculture Research System of MOF and MARA (CARS-35).

Data and model availability statement None of the data were deposited in an official repository.

Declaration of interest None.

Ethics approval All procedures were described in this study had been approved by the Ethical and Animal Welfare Committee of Heilongjiang Province, China.

Consent for publication All authors consent to the publication of the manuscript.

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Table 1 Effects of MS on the spatial holeboard discrimination task of piglets (n=12).

	Con group	MS group	F（df1,df2）	P-value
Total number of visits (n)	6.67 ± 0.89	6.33^ns ± 0.59	0.195	P = 0.663
Number of visits and revisits to decoy areas (n)	3.08 ± 0.41	2.08^* ± 0.32	7.402	P = 0.024
Working memory (%)	69.72 ± 8.44	40.28^** ± 10.64	9.399	P = 0.002
Reference memory (%)	50.10 ± 4.73	33.15^**± 5.00	12.131	P = 0.002

Results are expressed as mean ± standard error (means ± SEM). ns = Not significant (P > 0.05); * = P < 0.05; ** = P < 0.01. The Con group: control piglets; MS group: piglets in intermittent neonatal maternal separation.

Table 2 Effects of MS on the novel object recognition test of piglets (n=12).

	Con group	MS group	F（df1,df2）	P-value
Total number of object contacts during the test period (n)	3.33 ± 0.40	2.83^ns ± 0.38	1.623	P = 0.219
Total time in contact with the object during the test period (s)	8.92 ± 2.11	8.08^ns ± 1.56	0.202	P = 0.799
Number of contacts with new objects (n)	2.17 ± 0.34	1.17^** ± 0.16	14.143	P = 0.002
Working memory (s)	5.83 ± 1.69	3.33^* ± 0.66	3.814	P = 0.033
Reference memory (%)	28.28 ± 9.53	-17.21^**± 7.23	28.932	P < 0.001

Table 3 Comparison analysis of the RNA-Seq and qRT-PCR results (n=6).

Gene name	RNA-seq			qRT-PCR
Gene name	Fold change	log₂FC	P值	Fold change	log₂FC	P值
MC4R	0.17	-2.58	P < 0.01	0.03	-5.11	P < 0.01
GRP	0.32	-1.66	P < 0.01	0.08	-3.59	P < 0.01
CYFIP2	0.36	-1.49	P < 0.01	0.07	-3.82	P < 0.01
NMB	0.42	-1.26	P = 0.01	0.13	-2.95	P < 0.01
CPEB1	0.46	-1.13	P < 0.01	0.09	-3.43	P < 0.01
ADCYAP1	0.46	-1.11	P < 0.01	0.10	-3.27	P < 0.01
MAG	2.62	1.39	P < 0.01	4.71	2.24	P = 0.04
PLP1	2.64	1.40	P < 0.01	3.24	1.70	P = 0.02
CPEB4	2.71	1.44	P = 0.04	2.86	1.52	P < 0.01

No competing interests reported.

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Effects of early intermittent maternal separation on cognitive development of the prefrontal cortex in piglets

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