Intrauterine growth restriction in piglets alters blood cell counts and impairs cytokine responses in peripheral mononuclear cells 24 days post-partum

Large litter sizes have resulted in more piglets being exposed to intrauterine growth restriction (IUGR). Growth restriction during fetal life is linked with lower growth efficiency and increased susceptibility to infections in postnatal life and IUGR may associate with an altered innate immune system. We investigated the haematological, thromboelastography and plasma biochemical profiles of IUGR and normal piglets as well as cytokine responses in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS) at 24 days of age. Piglets were classified at birth based on their head morphology as either IUGR or normal. The present study showed a modulation of the immune function of IUGR pigs, characterized by an increase in neutrophil percentage and fibrinogen levels but a decrease in CD4+ T-cells. A lower level of LPS-induced IL-1β production was evident in IUGR pigs, suggesting immunological hypo-responsiveness. Furthermore, higher levels of reticulocytes, MCV and MCH and lower levels of erythrocytes in IUGR pigs suggest altered bone marrow hematopoiesis. All together, the results suggest a moderate suppression of the immune response of IUGR piglets, which may have implications for resistance to pathogen challenges in the post-weaning period. Serum metabolites and blood clotting profile did not differ between IUGR and normal piglets.

results in pigs would identify abnormalities/differences and a disruption of hemostasis between the two groups of pigs. Our overall hypothesis was that IUGR piglets at the age of 24 days would have an altered immune phenotype compared to normal piglets.

Methods
Animals and experimental design. Forty piglets (20 IUGR and 20 normal) were selected from a 1,600sow (Danish Landrace × Danish Yorkshire) Danish piggery. The piglets were a subpopulation of pigs in a trial investigating growth, whole blood glucose and rectal temperatures of IUGR piglets compared to normal piglets 10 . Piglets were selected on the day of birth (day 0), before litter equalization, and ear tagged, and sex of the piglet, sow identification, litter size, and birth weights (BiW) were noted. Piglets were visually graded and categorized as either IUGR or normal by the modified characteristics from Hales et al. 5 and Chevaux et al. 11 . Piglets defined as IUGR pigs included the criteria: (a) a steep dolphin-like forehead, and one or more of the following characteristics: (b) bulging eyes, (c) wrinkles perpendicular to the mouth or (d) hair with no direction of growth. If none of the criteria applied, the piglet was defined as normal. For each IUGR piglet in a litter, one corresponding normal piglet (none of the criteria) was chosen from the same litter. The piglets were individually weighed on a scale (UWE, Bjerringbro Vaegte ApS, Bjerringbro, Denmark) and ear tagged for identification.
Housing, feeding and management. All experimental animals were managed according to general routines at the farm. While in the farrowing unit, piglets were housed with a sow in individual farrowing pens (1.67 m × 2.38 m), with slatted floor (1.3 m × 1.67 m) and a floor heated creep area (0.60 m 2 ). Litters for both sows and gilts were equalized to 16 piglets by cross-fostering piglets born within the same 12 to 24 hours. No castrations were performed in the production and the experiment therefore included entire males. The sows had ad libitum access to drinking water and were fed a commercially formulated mash diet according to Danish recommendations based on wheat, barley and soybean meal (Vilomix A/S, Mørke, Denmark). In addition, all farrowing pens were installed with automatic milk cups, where the piglets had access to milk replacer (Schils, Sittard, the Netherlands) from day one. Three to four times daily from day 14, the piglets received a handful dry feed from a commercial mix (Danish New Wean feed, Danish Agro, Karise, Denmark).
Blood and tissue sampling. On day 24, piglets were transported to research facilities at the University of Copenhagen and blood sampled within an hour after arriving to the facilities. In order to process the samples as quickly as possible (within an hour), piglets were brought in over four consecutive weeks (5 IUGR and 5 normal pigs per week). Five blood samples were taken from each piglet. The first sample was taken in a 1.8 ml citrat stabilized tube for (TEG) and fibrinogen analysis. The second and third samples were taken into heparinized tubes for the LPS challenge on blood, and phenotyping of peripheral leukocytes by flow cytometry. The fourth sample was taken in a 4.0 ml tube (EDTA) for hematology (CBC/Diff/Retic) and finally the fifth sample was taken in a 4.0 ml tube for serum, spun down and serum frozen for later analysis of IGF-1 and biochemistry. The tubes for TEG were taken to a nearby lab for immediate processing and likewise the tubes for the LPS challenge.
Blood sample analysis. The TEG analyses were performed with the same model of a computerized thromboelastograph (TEG 5000 Haemostasis Analyzer, Haemoscope Corporation, Niles, IL) according to the previously published method 12,13 . In brief, data were obtained continuously by electronic transfer to computer from the analyzer. The hematology profile was analyzed on an Advia 2120 Hematology System (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) and the serum analyses for biochemistry were assayed using an Advia 1800 Chemistry System (Siemens Healthcare Diagnostics, Tarrytown, NY, USA).

Statistical analysis.
Data was analyzed in the statistical program SAS (GLM procedure of SAS; SAS Inst. Inc., Cary, NC) according to the following model: www.nature.com/scientificreports www.nature.com/scientificreports/ where Y ij is the dependent variable measured (cells, blood characteristics, cytokines, blood parameters), μ denotes the overall mean, α i denotes the effect of classification (i = IUGR, normal), β j denotes the effect of batch (j = 1, 2, 3, 4), (αβ) ij is the interaction between classification × batch, and ε ij describes the random error term. The interaction between classification × batch was only included when significant. Means were separated using the PDIFF option and presented as Least Square Means ± SEM and considered significant when P < 0.05 and a tendency when P < 0.10. ethics approval and consent to participate. The experiment was carried out with respect to animal experimentation, and with the approval of the Danish Animal Experimentation Inspectorate (2016-15-0201-01018).

Results
Blood analysis. The birth weight and body weight at day 24 are presented in Table 1. On average the normal piglets weighed 1.51 ± 0.05 kg at birth and 6.84 ± 0.31 kg at day 24 of age and an IUGR piglet weighed 0.77 ± 0.02 kg and 4.53 ± 0.17 kg at day 24 of age (Table 1; P < 0.001). There was a difference between the groups on number of erythrocytes and percentage of lymphocytes (P < 0.05) with lower levels found in IUGR piglets compared to normal piglets. Further, there were differences between MCH, MCV, percentage neutrophils and fibrinogen (P < 0.05), with lower levels in normal pigs compared to IUGR pigs. IGF-1 levels were similar between groups, with 61.3 ng/ml vs. 67.6 ng/ml (SEM 2.64 ng/ml; P = 0.151) in IUGR and normal pigs respectively. The TEG analysis is presented in Table 2. Similar TEG values were obtained between IUGR and normal pigs for most parameters, however IUGR pigs tended to reach lysis at 60 min, but a lower clot lysis at 30 min, compared to normal pigs. No differences were observed in the blood biochemistry of piglets at day 24 of age (Table 3).
cytokine and lymphocyte concentrations. The concentrations of cytokines released from LPS-stimulated and mock-treated cells are shown in Table 4. There was a higher concentration of IL-1β following LPS stimulation in PBMC from normal piglets compared to cells from IUGR piglets (17.3 ± 2.55 vs. 9.9 ± 1.75 ng/

Discussion
There is a high mortality in IUGR piglets compared to normal piglets during the suckling period in Danish piggeries 5 . In addition, most pigs are challenged at weaning as they are removed from the passive immune protection from the milk of the sow, thereby increasing their susceptibility to enterotoxigenic Escherichia coli (E. coli) infection and other bacterial, viral and parasitic pathogens 14,15 . We hypothesized that, due to their compromised growth pattern, IUGR piglets would be more susceptible to disease at weaning than normal piglets and this would be measurable in an ex vivo blood challenge. The IUGR piglets weighed 4.5 kg at the end of the experiment (day 24 of age) compared to 6.8 kg for the normal pigs. Over the last decade, weaning age has been dramatically reduced and as a result pigs are smaller and have less mature immune and digestive systems at weaning, making them more susceptible to problems in  www.nature.com/scientificreports www.nature.com/scientificreports/ the early postweaning period 7 . IUGR pigs were approximately one week behind growth wise at day 24 although their fractional growth rate is in fact increased compared to normal pigs 8 . The difference in the immune system between IUGR and normal piglets at day 24 of age was tested using an ex vivo LPS challenge model with PBMC to evaluate differences in the ability of the two groups of piglets to respond to an acute inflammatory stimulus. LPS is an integral component of the outer membrane of gram-negative bacteria, which, by stimulating immune cells to synthesise cytokines such as TNFα, IL-1 and IL-6, induce marked changes in behaviour, metabolic and neuroendocrine systems 16 . These cytokine-mediated events are part of a general homeostatic reaction and therefore serve as the first line of defense of the organism against infection 17 .
The majority of both anti-and proinflammatory cytokines were similar for IUGR and normal pigs, with a lower IL-1β in the IUGR group as the only exception. The efficiency of the innate response is crucial for survival and for an optimum priming of acquired immunity 16 . A lower IL-1β response in piglets is probably not beneficial as this would likely mean a slower or diminished response to pathogens such as E. coli. The acute phase response is characterized by a dramatic increase in the production of a group of proteins by the liver. Bacterial LPS is an endotoxin, a potent inducer of the acute phase response and systemic inflammation. This response is induced by the production of TNFα, IL-1β, and IL-6 from activated monocytes and neutrophils in response to inflammatory stimuli 18 . This suggests that the immune system of IUGR pigs is not as able as that of normal pigs to mount an innate inflammatory immune response.  Table 5. Percentages of total T-cells (CD3 + ), γδ T-cells, B-cells (CD79α + ), and CD4 + and CD8 + cells within the peripheral blood mononuclear cell population, and percentages of CD4 + and CD8 + T-cells (CD3 + CD4 + and CD3 + CD8 + ). *Effect of batch No interactions between classification and batch were observed. (2020) 10:4683 | https://doi.org/10.1038/s41598-020-61623-w www.nature.com/scientificreports www.nature.com/scientificreports/ For the majority of lymphocyte subsets values were similar for IUGR and normal piglets with only a tendency toward lower percentage for CD3 + CD4+ in IUGR pigs, while IUGR pigs had a significantly lower percentage of total lymphocytes compared to normal piglets. This is most likely due to a drop in CD4+ cells in IUGR pigs compared to normal pigs. A study by Kanitz et al. 19 , found that weaning suppresses lymphocyte function, causes changes in neuroendocrine regulation and has a substantial effect on behavioural and endocrine responses to acute peripheral LPS challenge. The results from the current experiment suggest that IUGR piglets are even more susceptible to disease than normal pigs at weaning. Consequently, it could impair health and welfare by increased disease susceptibility in newly weaned piglets 19 . Our hypothesis is therefore partly confirmed, as there was a minor modulation of the immune function in IUGR pigs at day 24 of age, most likely due to a drop in CD4+ t-cells compared to normal piglets.
Total erythrocyte counts were lower in IUGR pigs compared to normal pigs. Erythrocyte levels were found to be lower and levels of pro-inflammatory cytokines in plasma elevated in humans with periodontitis, potentially suppressing erythropoiesis 20 . Disease is considered to be a major contributing factor associated with poor performance of pigs 7 . Additionally, the weaning transition involves complex social, environmental, and nutritional changes for piglets, and it is a stressful event 6 and therefore, the results together suggest that IUGR piglets might be more challenged at weaning than normal piglets. Overall, however, the immune phenotype of the IUGR piglets was still broadly similar to the normal piglets, with a few notable differences. A number of other factors are also likely to contribute to the poor health and disease susceptibility of small piglets post-weaning, such as delayed gut development 21 , poor mucosal barrier function 22 , and altered hepatic metabolism 23 .
IUGR pigs had numerically higher levels of reticulocytes and a significant higher level of mean cell volume. Furthermore, the IUGR pigs had fewer erythrocytes but higher levels of MCH compared to normal pigs. Both findings suggest macrocytic anemia in IUGR pigs compared to normal pigs possibly as a compensatory approach in the IUGR pig. In addition, the significant lower levels of IL-1β in IUGR pigs compared to normal pigs suggest that the IUGR pigs are hyporesponsive and cannot produce the same amount of IL-1β as normal pigs. This could suggest that the IUGR pigs are immune compromised and further being hypo responsive is associated with altered bone marrow hematopoiesis. Taken together, the hematological profile of the piglets suggests that IUGR pigs show signs of changes in the bone marrow function compared to normal pigs.
The biochemistry profile was similar between IUGR and normal piglets, suggesting similar protein turnover, enzyme activity and growth in both phenotypes. This further supports growth data from the same farm 10 where the same growth pattern was found, even though IUGR piglets are 6 days behind compared to normal piglets, mostly due to the smaller starting point. In addition, IGF-1 concentrations were similar between groups 10 . However, Lynegaard et al., (2019) did find that the morphometric profile of IUGR piglets at day 24 of age differed from normal piglets. IUGR piglets were found to have relatively larger brain, liver, lungs and adrenal glands at day 24 of age compared to normal piglets 10 . In addition, Huting et al. 24 , has also recently shown that morphometric characteristics at birth also affect pig lifetime growth performance. Taken together with the haematological results IUGR piglets are at risk of being delayed both in terms of growth but also due to a slightly suppressed immune response increasing the risk of disease transmission compared to normal piglets.
Only small changes were observed in the TEG analysis. The TEG analysis is a whole-blood, point of care coagulation assay that provides information on the kinetic and mechanical properties of a clot as it forms, matures, and undergoes fibrinolysis 25 . It was hypothesized that TEG results in pigs would identify abnormalities/differences between the groups of pigs and would disrupt hemostasis. In horses TEG performed at admission identified abnormalities associated with inflammatory lesions, systemic inflammatory response syndrome (SIRS), the development of diarrhea, thrombophlebitis, laminitis, and fatality in horses 25 . Although only minor differences were found in the measured TEG parameters, differences were observed in an acute phase protein, fibrinogen, the precursor to fibrin, with higher levels in IUGR pigs.

conclusions
In conclusion, a minor modulation of the immune function of IUGR pigs was found at day 24 of age, most likely due to a drop in CD4+ t-cells compared to normal piglets. In addition, a lower level of IL-1β was found suggesting that IUGR pigs are hyporesponsive compared to normal pigs. Furthermore, higher levels of reticulocytes and MCV as well as lower levels of erythrocytes and higher levels of MCH in IUGR pigs suggest altered bone marrow hematopoiesis compared to normal pigs. All together the results suggest that the immune response of IUGR piglets is slightly suppressed compared to normal piglets at day 24 of age. No differences were observed in serum biochemistry supporting a similar growth pattern between IUGR and normal piglets.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.