Edited by: Carina Soares-Cunha, University of Minho, Portugal
Reviewed by: Bárbara Coimbra, University of Minho, Portugal; Laurianne Canario, Institut National de la Recherche Agronomique de Toulouse, France
†These authors have contributed equally to this work
This article was submitted to Behavioral Endocrinology, a section of the journal Frontiers in Behavioral Neuroscience
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Early-life adversity may have programming effects on the psychological and physiological development of offspring. Domestic pigs (
In mammals, the lactation period is a time during which many ontogenetic processes, such as brain maturation, immune system development, and mother-infant bonding, are still ongoing (
Stress during the lactation period of pigs, resulting in elevated cortisol concentrations in the blood and milk, can disrupt the sow-piglet relationship and lead to piglet losses (
Disruptions in the sow-piglet relationship may result from psychological stress to piglets in the early-postnatal period of life. In laboratory animals and primates, psychosocial stress in the postnatal period leads to changes in stress adaptation and can have sustained adverse effects on immune responses (
Husbandry challenges in pigs have both physical and psychological components. These challenges include sows’ and littermates’ deprivation at weaning and mixing with unfamiliar conspecifics. These challenges may have acute and long-term effects on the immunocompetence of pigs (
We hypothesize that repeated maternal deprivation of piglets affects the mother-offspring relationship and stresses the sow. For piglets, we predict that the deprivation of mother and littermates in a group of familiar conspecifics is perceived as being less stressful than experiencing the same stressors alone. Piglets were separated from their mothers and littermates alone or with familiar conspecifics for 2 h daily over 2 weeks to test these hypotheses. During that time, maternal behavior, the neuroendocrine response of sows to separation, and sow-offspring interactions were observed. The plasma cortisol and immune parameters of piglets were also determined. One day and 25 days after the deprivation period, the piglets were behaviorally challenged by an open-field/novel-object (OF/NO) test to assess the effects of different psychosocial treatments on their behavioral reactivity in the short and long-term. Moreover, we investigated changes in gene expression in those brain areas involved in regulating the HPA axis.
All procedures involving animal handling and treatment were performed according to the German Animal protection law and were approved by the local authorities (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany; LALLF M-V/TSD/7221.3-1.1-003/18).
A total of 200 piglets from 20 sows of the German Landrace (
Milk samples were collected from sows on the second day of lactation before the piglets were separated (at 7:00) and immediately after the piglets had returned (at 10:00). On lactation days 9 and 15, milk samples were only collected immediately after the return of the piglets because taking milk before separation would have been an additional stress factor for the piglets. Blood samples of piglets were taken on PND 5 at 7:00 and PND 16 before (at 7:00) and immediately after the open-field/novel-object (OF/NO) test (8:00–12:00). The OF/NO test was repeated on PND 40. Two piglets (one male, one female) from each group and litter were sacrificed on PND 20 for brain tissue analysis.
Weaning of the piglets was performed after 28 days by transferring the deprivation and control litters to a common weaning pen with controlled light and temperature conditions and commercial feed from an automatic feeder. Feed and water were available to the animals
Maternal behavior and sow-piglet interactions were determined by continuous, direct observation of piglets and sows over three successive suckling bouts. The observation started after the return of all piglets from deprivation (at 10:00) simultaneously for the control and deprivation litters.
Immediately before any suckling events, the occurrence of the following behaviors was counted, and relative frequencies were calculated based on all litters and suckling events. These were, (1) “grunt”: sow initiated suckling by emitting grunts to call the piglets; (2) “piglet calls”: piglets initiated suckling by calling for attention; (3) “pre-lying behavior”: sow exhibited pre-lying behavior, such as scratching with one front leg, sniffing the ground, counting/sorting piglets, nest-building behavior (digging in straw; looking for material); (4) “lying down”: sow shows controlled and slow bending of front and hind legs; turning to the side is also controlled and slow; (5) “piglet contact”: sow reacts to piglets near her head by contact and sniffing; (6) “response to human”: sow reacts very nervously or aggressively to people nearby; (7) “response to screams”: sow shows restless behavior at piglet screams and looks for them; and (8) “suckling intervals”: time interval between nutritive suckling bouts.
A combined 10-min OF/NO test was used to investigate the influence of early-postnatal psychosocial stress on the behavioral reactivity of piglets. Testing took place in a separate, noise-attenuated room with a square test arena (2.80 × 2.80 × 1.25 m) and on two different days of life (PNDs 16, 40). Piglets were tested in random order regardless of sex. The piglets’ behaviors were recorded during the 10-min test period using the focal sampling method with Observer XT 13 software (Noldus Information Technology). The first 5 min represented the OF situation. Immediately afterward, a foreign object (PND 16: plastic shoe; PND 40: 1.5 kg medicine ball) was lowered from the ceiling and remained suspended approx. 10 cm above the floor in the arena until the end of the 10-min test period (NO situation). The arena was cleaned between the tested piglets. First, urine and feces were removed with dry cloths. Then the arena was washed thoroughly with soapy water. The OF/NO tests were always performed by the same persons. The behaviors listed below were recorded and analyzed in terms of duration, frequency, and latency. The observed behaviors were defined as (1) “standing”: no active locomotion, standing on at least three legs; (2) “lying”: the ground is touched with all four legs and the abdomen; (3) “locomotion”: active locomotion with at least with two steps; (4) “escape”: active attempt to leave the arena by jumping up the wall; (5) “defecation”: excretion of feces; (6) “urinating”: discharge of urine; (7) “object contact”: active touching or manipulation of the novel object with the snout; and (8) “vocalization”: active vocalizations (grunting, screaming) (
Milk was collected by gentle, manual teat massage at the start of a suckling bout. The milk was centrifuged at 40,000 × g for 1 h at 4°C. The phase in the middle was centrifuged again at 20,000 × g for 30 min at 4°C. The supernatant was stored at –20°C until analysis.
Blood samples were taken while piglets were in a supine position by anterior vena cava puncture. The whole procedure lasted approx. 1 min. The samples were transferred to ice-cooled polypropylene tubes containing EDTA solution and placed on ice. A blood sample of 100 μl was stored separately for differential blood counts (see section “Determination of the neutrophil to lymphocyte ratio from blood”). The rest was centrifuged at 2,000 × g at 4°C for 15 min. The resulting plasma was stored at –20°C until analysis.
On PND 20, one male and one female piglet from each group (DA, DG, C) and sow were anesthetized with Ursotamin® (100 mg/mL ketamine hydrochloride, Serumwerk Bernburg AG, Bernburg, Germany) and Stresnil® (40 mg/mL Azaperone, Elanco, Homburg, Germany) and euthanized by an intravenous injection of T61® (embutramide/mebezonium iodide/tetracaine hydrochloride, Intervet, Unterschleiβheim, Germany). The brains were quickly removed and the hypothalamus, hippocampus, amygdala, and prefrontal cortex (PFC) were dissected from both hemispheres and stored at –80°C until mRNA analysis. The spleen was transferred to 0.8% NaCl solution (Fresenius Kabi Deutschland GmbH, Langenhagen, Germany) until isolation of mononuclear cells (see section “Isolation of mononuclear cells from spleens”).
Milk and plasma cortisol concentrations were measured in duplicate using a commercially available ELISA kit for human samples (DRG Instruments, Marburg, Germany) according to the manufacturer’s instructions. The assay’s sensitivity was 3.4 ng/mL, and the intra- and inter-assay coefficients of variation (CV) were 6.2 and 9.4%, respectively. The assay has been validated for pig cortisol before (
Milk oxytocin was determined using a commercially available ELISA kit (Arbor Assays, Arbor, MI, USA) according to the manufacturer’s instructions. The assay’s sensitivity was 14.8 ng/mL, and the intra- and inter-assay coefficients of variation (CV) were 6 and 7.3%, respectively. Milk supernatants (see above) were diluted 1:10 in assay buffer for the assay.
Plasma concentrations of immunoglobulin A (IgA) were analyzed by porcine-specific enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Bethyl, Laboratories Inc., Montgomery, TX, USA). The assay’s sensitivity was 14.9 ng/mL, and the intra- and inter-assay CVs were <5 and <10%, respectively (
A drop of blood was smeared across a microscopic slide. Blood smears were stained with May-Grünwald solution and subsequently with Giemsa solution (both from Carl Roth, Karlsruhe, Germany) to determine relative leukocyte counts (lymphocytes, monocytes, basophils, eosinophils, neutrophils). At least 200 leukocytes were counted and identified in the smears, and the N/L ratio was calculated.
The collected spleen pieces were transferred into gentleMACS tubes (Miltenyi Biotec, Bergisch-Gladbach, Germany) with 6 ml PBS solution each and disrupted using gentleMACS dissociator (Miltenyi Biotec). Subsequently, the cell suspension obtained was filtered through a cell strainer (70 μm) with 18 ml PBS (phosphate-buffered saline, Sigma-Aldrich/Merck, Darmstadt, Germany), the PBMCs were separated by density gradient centrifugation, and the erythrocytes were lysed [1 ml H2O for 15 s, osmolarity reinstalled with 108 μl NaCl solution (8.8 %)]. The cell suspension was then filtered again through a cell strainer (50 μm). The cells were counted using a cell counter (Multisizer™ 3 Coulter Counter, Beckmann Coulter, Krefeld, Germany) and the cell number was adjusted to 2 × 106 cells/ml in culture medium (RPMI-1640, PAN-Biotech, Aidenbach, Germany; 10% FBS, Biochrom, Berlin, Germany; 2 mM glutamine, 50 μg/ml gentamycin, 0.05 mM mercaptoethanol, Sigma-Aldrich/Merck) for the proliferation assay.
Splenic mononuclear cells were seeded at 1 × 106 cells/ml density in culture medium either without (unstimulated control) or with the mitogens concanavalin A (ConA; 25 μg/ml) or lipopolysaccharide (LPS; 12.5 μg/ml; Sigma-Aldrich/Merck) and incubated for 72 h at 37°C and 5% CO2 in a 96-well plate (200 μl per well). The plate was centrifuged at 220 × g for 10 min at room temperature and 100 μl of supernatant was taken from each well. Then, 10 μl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich/Merck; 5 mg/ml in PBS) was pipetted into each well. After incubation for 4 h at 37°C and 5% CO2, 100 μl of a pre-warmed (37°C) SDS solution (sodium dodecyl sulfate, Sigma-Aldrich/Merck) was added and incubated overnight at 37°C and 5% CO2. The metabolic activity of the cells was measured by determining the optical density (550 nm; reference 690 nm) using a micro-plate reader (Spectrostar nano, BMG Labtech, Ortenberg, Germany). The results were expressed as proliferation index (PI), which could be calculated according to the following formula:
PI values of ≥1.4 were considered proliferation.
RNA extraction of brain samples was performed using the RNeasy Lipid Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The RNA concentration was determined at 260 nm by using a NanoPhotometer™ (Implen, Munich, Germany) and the purity and integrity were determined by calculating the 260/280 nm ratio. Furthermore, mRNA expression was monitored by reverse transcription (RT) of 750 ng of RNA using the iScript cDNA synthesis kit (Bio-Rad, Munich, Germany) according to the manufacturer’s guidelines. The resulting cDNA was amplified by real-time PCR (iCycler, Bio-Rad) for the following genes: NR3C2 (mineralocorticoid receptor; MR), NR3C1 (glucocorticoid receptor; GR), CRHR1 (corticotropin releasing hormone receptor 1), CRHR2 (corticotropin releasing hormone receptor 2), AVPR1 (arginine vasopressin receptor 1a), OXTR (oxytocin receptor), BDNF (brain-derived neurotrophin receptor), ACTB (actin beta), and TBP (TATA-box binding protein). One microliter of the RT reaction solution was added to 6 μL of iQ SYBR Green Supermix (Bio-Rad) and 4 μL of primer mix with gene-specific oligonucleotides (TIB Molbiol, Berlin, Germany). All the reactions were performed in triplicate. Primers were designed corresponding to the gene sequences of the NCBI database. Whenever possible, primers were designed to span the exon-exon junctions and to anneal between 57° and 61°C. The oligonucleotide sequences of the primers are summarized in
PCR was performed using a hot start (3 min, 94°C; 30 s, 60°C; 45 s, 70°C), 39 cycles (10 s 94°C; 30 s 60°C; 45 s 70°C with 5 s of time extension per cycle) and a final cycle (10 s 94°C; 30 s 60°C; 7 min 70°C, 1 min 94°C), corresponding to denaturation, annealing, and elongation, respectively. The specificity of the products was assessed using melting point analysis (60°–90°C, 1°C per 10 s), and agarose gel electrophoresis (3.5%). The oligonucleotide structure was verified by sequencing in a subset of the experiments. The relative quantification was calculated using the quantification module of the CFX Manager Software™ version 2.1 (Bio-Rad). Data for mRNA expression of the investigated genes are presented as relative expression ratios normalized to ACTB (beta-actin) and TBP (TATA-box binding protein).
Statistical analysis was performed using SAS software for Windows, version 9.4 (Copyright 2002–2012, SAS Institute Inc., Cary, NC, USA). Descriptive statistics and tests for normality were calculated with the UNIVARIATE procedure of the Base SAS software.
A multifactorial analysis of variance (ANOVA) was used to analyze normally distributed data (suckling interval and milk cortisol) using the SAS procedure MIXED. Fixed effects included trial (levels: 1–10) and treatment (levels: deprivation litter, control litter). Repeated measurements were accounted for by using the statement “repeated” to determine the block diagonal structure of the residual covariance matrix.
Non-normally distributed data were analyzed using the SAS procedure GLIMMIX. Here, the ANOVA model for the analysis of the brain samples included the fixed effects trial (levels: 1–10), treatment (levels: DA, DG, C), sex (levels: male, female), and the interaction “treatment × sex.” The ANOVA model for the blood samples, and OF/NO duration data also included the fixed effect time (levels: PND 16, PND 40) and the interactions “treatment × time” and “treatment × time × sex.” The sow was included as a random effect and measurement replicates on the same animal with the option “random residual,” to determine the block diagonal structure of the residual covariance matrix.
For the analysis of behaviors characterizing the sow-piglet interactions and OF/NO frequency data, a Poisson model was used to determine discrete characteristics. Fixed effects and interactions corresponded to the model above.
Results are presented as least squares means (LSM) and standard errors (SE) for all fixed effects of the above models. Multiple pairwise comparisons of these LS means were performed with the Tukey-Kramer procedure, with the significance level α chosen as 0.05 (test results with
We hypothesized that deprivation would be a psychosocial stressor for both mother and offspring. Therefore, we studied the behavioral and neuroendocrine changes in sows and piglets.
To assess the acute stress response of sows to deprivation treatment, milk samples were collected on the second lactation day before piglet withdrawal and after the return of the piglets and were analyzed for cortisol and oxytocin. There was a significant increase in cortisol in the milk of sows of the deprivation litters compared to sows of the control litters after the first deprivation treatment (
Cortisol and oxytocin concentrations in the milk of sows on the second lactation day before and after deprivation treatment.
Treatment group |
|||
Parameter | Deprivation | Control | Treatment |
Cortisol (ng/ml) | |||
Before | 22.99 ± 3.05 | 35.43 ± 2.93 | 0.077 |
After | 31.19 ± 2.57 | 26.35 ± 2.92 | 0.905 |
Difference (after-before) | 7.62 ± 3.70 | –7.26 ± 3.70 | <0.05 |
Oxytocin (pg/ml) | |||
Before | 776.38 ± 202.92 | 944.43 ± 191.08 | 0.999 |
After | 477.17 ± 166.26 | 549.14 ± 191.08 | 1.000 |
Difference (after-before) | –269.38 ± 452.74 | –274.38 ± 452.74 | 0.994 |
Results are presented as LSM ± SE of the sows of deprivation and control litters. Tukey-Kramer test;
Behavioral interactions between sows and piglets were observed immediately after the reunion of sows and piglets. While maternal behaviors did not show any significant differences between deprivation and control litters, piglets of deprivation litters vocalized more than piglets of control litters. We interpreted the piglet calls as a request for milk because we observed the sows to assume a nursing position (
Relative frequencies (%) of behaviors of sows and piglets preceding the first three suckling acts after the end of the deprivation procedure.
Treatment group |
|||
Behavior | Deprivation | Control | Treatment |
Lying down | 96.00 ± 22.44 | 94.48 ± 30.5 | 0.557 |
Piglet contact | 26.54 ± 3.86 | 31.33 ± 4.22 | 0.403 |
Grunting | 44.65 ± 4.84 | 58.12 ± 5.22 | 0.083 |
Piglet calling | 59.65 ± 4.79 |
43.46 ± 5.28 |
< 0.05 |
Lying down (sow showed controlled lying down behavior), piglet contact (sow responded to piglets near her head with contact/sniffing), grunting (the sow initiated suckling by calling), piglet calling (piglets initiated suckling by calling). Results are given as LSM ± SE and
The intervals between the first and second, as well as second and third suckling bouts, were averaged over lactation days 2, 5, 7, 9, 12, 14, and 19 to determine the effect of deprivation treatment on the intervals between suckling bouts after the piglets returned to the sow. Here, statistical analysis revealed a significant treatment effect on the mean interval length [
Average interval lengths between suckling bouts of deprivation and control litters. Results are presented as LSM ± SE. Significant differences are marked with asterisks (*
Blood samples were collected on PNDs 5 and 16 to assess the effects of maternal deprivation on the baseline stress level of piglets and potential effects on their immune system. There was no significant effect of treatment, sex, or the interaction of treatment × sex on plasma cortisol concentrations, plasma IgA and neutrophil/lymphocyte (N/L) ratio of piglets on PNDs 5 and 16. However, cortisol concentrations tended to be affected by sex on PND 16 (
Cortisol concentrations and immunological parameters in the blood of piglets.
Treatment group |
||||||
Parameter | DA | DG | K | Treatment | Sex | Treatment × sex |
PND 5 | 33.53 ± 3.21 | 33.34 ± 3.22 | 26.46 ± 3.15 | 0.234 | 0.725 | 0.155 |
PND 16 | 22.78 ± 3.04 | 23.09 ± 3.08 | 17.29 ± 3.01 | 0.360 | 0.084 | 0.265 |
PND 5 | 2.23 ± 0.32 | 2.07 ± 0.32 | 2.02 ± 0.32 | 0.637 | 0.695 | 0.881 |
PND 16 | 0.12 ± 0.02 | 0.10 ± 0.02 | 0.10 ± 0.02 | 0.229 | 0.226 | 0.451 |
PND 5 | 2.05 ± 0.18 | 1.97 ± 0.18 | 2.11 ± 0.18 | 0.835 | 0.900 | 0.925 |
PND 16 | 0.82 ± 0.24 | 0.79 ± 0.24 | 0.99 ± 0.24 | 0.818 | 0.764 | 0.147 |
Results are presented as LSM ± SE of the three treatment groups DA (deprivation alone), DG (deprivation in a group with littermates), C (controls, no deprivation), and the
Lymphocyte proliferation was tested on spleen mononuclear cells on PND 20. We found no significant treatment effects on the proliferation capacity of spleen cells after ConA or LPS stimulation (
One day after the end of the deprivation period, we exposed the piglets to an open-field/novel-object (OF/NO) test to check how repeated maternal deprivation would affect piglets’ behavioral reactivity to the challenge of brief isolation in an unfamiliar environment. The OF/NO test was repeated on PND 40.
Blood samples from all piglets on PND 16 before and after the OF/NO test were analyzed for cortisol to assess the influence of maternal deprivation on piglets’ cortisol concentrations when exposed to a challenge. We found that the type of deprivation treatment significantly affected the piglets’ cortisol concentrations after the OF/NO test [
Increases in plasma cortisol concentrations of
Statistical analysis of OF/NO behavior revealed that the type of treatment had a significant effect on the latency of object contacts [
Pairwise comparisons of piglets’ behavior in the OF/NO test showed differences between PND 16 and PND 40 (
Behavior in the open-field/novel-object test. Latency
When confronted with a novel object, the latency to object contact was higher in DA and DG piglets than in C piglets on PND 16. On PND 40, however, DA piglets showed a shorter latency than DG piglets and compared to PND 16 (
In terms of acoustic signals, DA piglets showed a higher latency to vocalization (
Male and female piglets showed the same OF/NO behavior for all of the above parameters except for latency to locomotion, latency to vocalization, and duration of contact. Female DA piglets had a higher latency to locomotion and vocalization than female DG and C piglets on PND 16 (
Behavior in the open-field/novel-object test on PND 16. Latency of locomotion
Interestingly, C piglets showed a shorter duration of contact on PND 40 than on PND 16 (
Psychosocial stress elicits a response from the limbic system and the PFC. Therefore, we studied the expression of genes involved in HPA axis function and regulation in the hypothalamus, amygdala, hippocampus, and PFC.
Both treatment and the interaction of treatment × sex significantly influenced CRHR1 mRNA expression in the hypothalamus. It was significantly higher in DA piglets than in DG piglets (
Relative mRNA expression of HPA-related parameters in the hypothalamus, amygdala, hippocampus and PFC of piglets of the different treatment groups.
Treatment group |
||||||
Parameter | DA | DG | C | Treatment | Sex | Treatment × sex |
MR | 1.01 ± 0.11 | 0.90 ± 0.11 | 0.89 ± 0.11 | 0.687 | 0.352 | 0.487 |
GR | 1.05 ± 0.14 | 1.10 ± 0.14 | 1.27 ± 0.14 | 0.523 | 0.055 | 0.087 |
MR/GR ratio | 1.22 ± 0.17 | 1.02 ± 0.17 | 0.75 ± 0.17 | 0.182 | 0.829 | 0.284 |
CRHR1 | 1.02 ± 0.11 | 0.567 | ||||
CRHR2 | 1.27 ± 0.14 | 1.07 ± 0.14 | 1.11 ± 0.14 | 0.551 | 0.495 | 0.250 |
AVPR1A | 1.21 ± 0.16 | 1.42 ± 0.16 | 1.12 ± 0.16 | 0.437 | 0.552 | 0.378 |
OXTR | 0.97 ± 0.21 | 1.29 ± 0.21 | 1.27 ± 0.21 | 0.472 | 0.697 | 0.715 |
MR | 1.05 ± 0.15 | 0.82 ± 0.15 | 1.24 ± 0.15 | 0.078 | 0.297 | 0.162 |
GR | 0.89 ± 0.10 | 0.96 ± 0.10 | 0.85 ± 0.10 | 0.721 | 0.866 | 0.428 |
MR/GR ratio | 1.26 ± 0.35 | 1.35 ± 0.36 | 1.78 ± 0.35 | 0.580 | 0.070 | 0.704 |
CRHR1 | 1.09 ± 0.13 | 0.82 ± 0.13 | 1.16 ± 0.13 | 0.122 | 0.645 | 0.290 |
CRHR2 | 1.22 ± 0.18 | 0.99 ± 0.18 | 1.20 ± 0.18 | 0.474 | 0.568 | 0.157 |
AVPR1A | 1.11 ± 0.16 | 1.04 ± 0.16 | 1.12 ± 0.16 | 0.882 | 0.134 | 0.379 |
OXTR | 1.09 ± 0.11 | 0.87 ± 0.11 | 0.79 ± 0.11 | 0.127 | 0.428 | 0.658 |
MR | 0.79 ± 0.09 | 0.452 | 0.248 | |||
GR | 0.93 ± 0.08 | 0.99 ± 0.08 | 0.92 ± 0.08 | 0.823 | 0.358 | 0.293 |
MR/GR ratio | 1.32 ± 0.14 | 0.93 ± 0.15 | 0.93 ± 0.14 | 0.108 | 0.117 | 0.304 |
AVPR1A | 1.37 ± 0.28 | 1.90 ± 0.29 | 1.69 ± 0.28 | 0.315 | 0.566 | 0.529 |
OXTR | 1.24 ± 0.15 | 1.31 ± 0.15 | 1.28 ± 0.15 | 0.946 | 0.250 | 0.596 |
BDNF | 1.13 ± 0.13 | 1.54 ± 0.13 | 1.38 ± 0.13 | 0.100 | 0.914 | |
MR | ||||||
GR | ||||||
MR/GR ratio | 1.23 ± 0.08 | 1.29 ± 0.08 | 1.17 ± 0.08 | 0.507 | 0.954 | 0.083 |
CRHR1 | 0.067 | 0.067 | ||||
CRHR2 | 0.70 ± 0.10 | 0.67 ± 0.10 | 1.02 ± 0.10 | 0.070 | 0.377 | 0.724 |
AVPR1A | 1.05 ± 0.14 | 1.05 ± 0.14 | 1.31 ± 0.14 | 0.350 | 0.738 | 0.277 |
OXTR | 0.93 ± 0.06 | 0.85 ± 0.06 | 1.00 ± 0.06 | 0.184 | 0.212 |
DA (deprivation alone), DG (deprivation with a group of littermates), and C (control, no deprivation); MR (mineralocorticoid receptor;
Gene expression in the hypothalamus of piglets on PND 20. CRHR1 (corticotropin releasing hormone receptor 1) mRNA expression of DA (deprivation alone), DG (deprivation with a group of littermates) and C (control, no deprivation) piglets. Data are expressed as arbitrary units after normalization to ACTB and TBP mRNA expression as endogenous reference genes and represent the LS means ± SE. Significant differences are indicated by asterisks (*
In the amygdala, we did not find any significant effects between treatment groups (
Gene expression in the amygdala of piglets on PND 20. MR (mineralocorticoid receptor; NR3C2) mRNA expression of DA (deprivation alone), DG (deprivation with a group of littermates), and C (control, no deprivation) piglets. Data are expressed as arbitrary units after normalization to ACTB and TBP mRNA expression as endogenous reference genes and represent the LS means ± SE. Significant differences are indicated by asterisks (*
We found a significant treatment effect on MR mRNA expression (
Gene expression in the hippocampus of piglets on PND 20.
Also, we found a significant sex effect on BDNF mRNA expression (
The strongest effects could be observed in the PFC. Treatment had a significant effect on MR, GR, and CRHR1 mRNA expression. All three receptors and in tendency also CRHR2 were more weakly expressed in DA and DG piglets compared to C piglets (
Both male and female DA piglets showed a lower expression of GR mRNA (
Gene expression in the PFC of piglets on PND 20.
The OXTR mRNA expression showed a significant sex effect (
This study addressed the consequences of repeated maternal deprivation of piglets during lactation with and without a group of conspecifics. Analysis of cortisol in milk showed an increase during the deprivation procedure in sows of the deprivation litters. One explanation for the tendency of higher cortisol levels on the second day of lactation in the control group compared to the deprivation group before the start of deprivation treatment could be the increase in cortisol during parturition. The experiment started at the same time for all sows, but the interval to farrowing varied individually by several hours. Sows were randomly assigned to the deprivation or control group on the second day of lactation. Therefore, the differences between the two groups of sows before treatment were due to random individual differences. Nevertheless, to compare the groups, we calculated the difference in cortisol levels between the end and the beginning of the first deprivation treatment on the second day of lactation. While cortisol levels should naturally decrease during the course of the deprivation treatment as observed in the control sows, the deprivation sows showed an increase in cortisol levels due to stress.
Maternal behavior such as controlled laying down to avoid piglet crushing and responses to piglets near the sow’s head by touching or sniffing showed no significant differences between mothers of deprivation and control litters. This was a bit surprising, as studies in rats have shown increased maternal behavior such as licking and grooming after the pups were returned from an isolation treatment (
Contrary to our expectations, we did not find more effects of deprivation on maternal behavior. Of note, these sows were in the second to fourth parity. Therefore, they had already experienced husbandry-related separations from their very young offspring and could have become habituated to such stressors. In addition, lactation has a dampening effect on the activity of the HPA axis (
Analysis of piglets’ blood samples on PNDs 5 and 9 revealed no deprivation-induced effects, neither on cortisol nor on IgA concentrations and N/L ratios. A previous article showed increased plasma IgA after stress in pigs (
The N/L ratio reflects glucocorticoid-mediated effects on the immune system and is therefore often used as a proxy for stress-induced immunomodulation because cortisol increases neutrophil proliferation and reduces lymphocyte proliferation (
The behavior of DA piglets in the OF/NO test on PND 16 could be interpreted as less aroused than that of DG and C piglets. They had a higher latency to vocalization and they vocalized less, which was attributed by
This habituation to solitude can be compared to the situation when an OF/NO test was repeated the next day (
When comparing the first and the second OF/NO test, all piglets showed reduced locomotion counts in the OF/NO test on PND 40, but only DG and C piglets displayed a shorter duration of locomotion in the second OF test. At this time, DA piglets exhibited a significantly longer duration of locomotion than DG and C piglets and no difference from their own behavior on PND 16. This is rather unusual as repeated OF/NO tests have been shown to reduce locomotion (
Behavioral differences between DA and DG piglets suggest that social support from a group of familiar conspecifics can compensate for maternal deprivation. Even the presence of a single conspecific during isolation without any physical interaction has been shown to blunt the behavioral responses of isolated piglets (
Expression of HPA-associated genes in stress-related brain areas was affected by deprivation treatment. CRHR1 mRNA expression in the hypothalamus was higher in DA than in DG piglets. Male rats, exposed to prenatal hypoxia stress, had a higher CRHR1 mRNA expression in the paraventricular nucleus of the hypothalamus than male control rats and showed higher anxiety. In contrast, females had a reduced CRHR1 mRNA expression and did not show changes in anxiety-like behavior. This suggests local positive feedback of CRH production of hypothalamic neurons, which may lead to increased anxiety (
In the hippocampus, MR mRNA expression was higher in DA than in DG piglets. This is surprising as a number of studies have shown that chronic stress and depressive behavior are correlated with decreased MR mRNA expression in the hippocampus, and decreased MR expression is generally linked to increased basal and stress-induced HPA axis activity (
BDNF is a neurotrophin that contributes to synaptic plasticity and neurite outgrowth and is important in controlling learning behavior and memories. It is co-expressed with GR and MR in hippocampal neurons. The cross-talk between glucocorticoids and BDNF shapes HPA-axis development in early life. During this period, high BDNF and low glucocorticoids are necessary for neurons. There is plenty of evidence that early-life adversity shifts the BDNF-glucocorticoid balance and may cause long-term stress vulnerability (
In the PFC, both DA and DG piglets showed lower expression of
AVP and OXT have been shown to influence HPA activity (
The behavior of female piglets in the OF/NO test differed from that of male piglets in the parameters “latency to locomotion” and “latency to vocalization” on PND 16, indicating lower arousal than in male piglets. Although DA piglets of both sexes showed low cortisol responses to this OF/NO test, only male DA piglets showed an upregulated hypothalamic CRHR1 mRNA expression compared to male DG and C piglets and a hippocampal MR mRNA expression that was higher than in male DG piglets. Increased hypothalamic CRHR1 mRNA expression was also found exclusively in male rats after prenatal stress, which was attributed to differential epigenetic modifications and accompanied by higher anxiety (
With regard to the C piglets, several genes were higher expressed in females than in males. These were
Furthermore,
Early-life adversity by repeated maternal deprivation of domestic piglets stresses both sows and piglets. While maternal behavior is not affected, piglets show strong and sustained alterations in OF/NO behavior and changes in gene expression in limbic areas and the PFC, suggesting an altered stress regulation system. The baseline immune parameters of the piglets were not affected, but the possible occurrence of stress-induced immunomodulation may be better assessed during a real immunological challenge. Social support effects, as well as sex-specific stress effects, could be seen in OF/NO behavior and gene expression in the brain.
The original contributions presented in this study are included in the article/
The animal study was reviewed and approved by Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Germany; LALLF M-V/TSD/7221.3-1.1-003/18.
EK and MT contributed to the conception and design of the study. MT, EK, and RB performed the experiments and collected and analyzed the data. AT performed the statistical analyses. UG, RB, and EK wrote the manuscript. All authors interpreted the data.
The publication of this article was funded by the Open Access Fund of the FBN.
We thank the staff of the Institute of Behavioral Physiology for their excellent technical assistance, and the experimental pig facility for providing animals and support.
RB is currently employed by the company EUROIMMUN Medizinische Labordiagnostika AG. During the study he was employed by the FBN. The Service Group Statistical Consulting is not a company but an organizational unit of the FBN. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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