Neutrophil phagocytic activity of piglets born from sows supplemented with organic or inorganic selenium 

Selenium is a trace element and essential supplement to the diet of various biological species (Moeini et al., 2011; Wu et al., 2011; Waseem et al., 2016; Bunglavan et al., 2018; Prakash et al., 2019). It is actively involved in the formation of selenoprotein-containing foetal tissues (Kohrl et al., 2000). This element is essential for the normal function of all body systems, including the immune system (Hawkes et al., 2001; Ferencik and Ebringer, 2003). It regulates the antioxidant processes (Karren et al., 2010), phagocytosis (Wuryastuti et al., 1993; Okpala et al., 2015), inflammation (Mattmiller et al., 2013). Seleinium is involved in thyroid gland metabolism (De Vito et al., 2011; Konecny et al., 2015), helps the formation of reserve in the mammary gland (Slavik et al., 2008). Newborns with better selenium status are less frequently affected with diseases, associated with selenium deficiency (myopathies, liver necrosis) and worsened innate resistance (Hefnawy and Tortoro-Perez, 2010). Inorganic selenium solves the problems with deficiency states, but in nature, it is found in plants bound to amino acids (selenomethionine, selenocysteine) (Schrauzer, 2000). That is why, the form in which selenium is found in feeds is important for its utilisation by the body (Mahan, 2000; Stewart et al., 2012).  

Nutritional immunity is an approach of improving body defense by optimisation of the feeding regimen (Buttgereit et al., 2000; Butcher and Miles, 2002; Chandra, 2002; Marcos et al., 2003; Lee et al., 2016; Valpotic et al., 2016). Despite the acknowledged positive relationship between feed quality during the pregnancy of sows and the immune defense of newborns (Wu et al., 2004; Abu-Saad and Fraser, 2010) there are issues related to the transfer of trace elements and nutrients from the diet to the foetus-newborn system that are still unclear. The evaluation is further impeded by the numerous factors influencing pregnancy and parturition related to individual features- age, functional status of genitals, endocrine status, cytokine profile (Daher et al., 2004; Lewis, 2004; Moffett and Loke, 2004; Wu et al., 2012). As macro- and micronutrients are regulators of metabolism of energy substrates during the foetal growth and pregnant animals could not produce them, they should be present in rations (Jobgen et al., 2006; Wu et al., 2012). Hence, the aim of this study was investigate the effects of inorganic or organic selenium supplementation of diets of pregnant sows on neutrophil phagocytic activity in their offspring.

Piglets used in the study - newborn (n=9); 20-day-old (n=12) and 40-day-old (n=12) were born from sows, crosses of Landrace and Large White. Sows were artificially inseminated and pregnancy was detected by examination with Aloka SSD 500 ultrasound (Dimitrov et al., 2003). Fifty days prepartum and 35 days postpartum (for a total of 85 days), the basic ration of sows (barley 35%, wheat 34%, sunflower meal 14%, bran 12%) was supplemented with 3-5% mineral premix for sows (Agrex Ltd Sofia, Bulgaria). The premix did not contain any selenium for control group of sows (n=3). Sows from Group I (n=6) received also organic selenium (Sel-Plex®, Alltech Inc., USA; 0.3 ppm/kg) to the premix and those from Group II (n=6) received sodium selenite (Alltech Inc., USA; 0.3 ppm/kg). The daily diet of every Se-supplemented sow was 3 kg feed, which contained either 0.9 ppm organic or 0.9 ppm inorganic Se. All sows were placed under uniform rearing conditions and the same immunoprophylaxis and antiparasitic applied at the pig farm (field experiment). Animal welfare and public health guidelines were strictly observed by the farm staff.

All procedures performed in this study were in accordance with the ethical standards of the Faculty of Veterinary Medicine, Stara Zagora, Bulgaria and national regulations with respect to living environment conditions - ventilation, temperature, humidity, lighting, noise, bedding, as well as the specific conditions for growing the individual age groups.
 
Sample collection
 
All blood samples of piglets were collected in the morning between 8.00-8.30 AM to avoid circadian rhythm influence. Collection was compliant with animal protection and welfare norms. For phagocytosis assays, heparinised blood (25-30 U/mL) was sampled from sinus ophthalmicus after sedation with àlkaine 0.5% eye drops, solution (S.A. ALKON-COUVREUR N.V., Belgium). For collection of serum, plain tubes were used, samples were left to clot at room temperature and then centrifuged for 10 min at 3,000´g.
 
Parameters and assay methods
 
Serum Se concentrations were assayed by atomic absorption spectrophotometry (Buck Scientific 200A). Total leukocytes (109/L) were counted in the Bürker chamber. Differential leukocyte counts were determined on blood smears stained by Romanovski-Giemsa. Absolute segmented neutrophil count was obtained by multiplication of segmented neutrophil percentage and total leukocyte counts, divided to 100. Phagocytosis was assayed by immunofluorescence method of Samnaliev et al., (1995) using a XSZ N107E fluorescence microscope. This in vitro method used blood and serum. Cell nuclei were stained brown-red with ethidium bromide, while phagocytosis object was visualised by fluorescein isothiocyanate (FITC). Percentage of phagocytosis and phagocytic number were defined by counting 150 neutrophils on the smear.

Statistical analysis
 
Data (mean±SEM) were submitted to one-way ANOVA and LSD post hoc test. Kolmogorov-Smirnov test was used to evaluate the normal distribution of data in each sample.

Serum selenium concentration in piglets
 
Serum Se levels in 20-day-old and 40-day-old offspring of sows supplemented with Sel-Plex® (Group I), sodium selenite (Group II) and control sows (Group III) were statistically significantly higher (p<0.001) in piglets from Group I and II vs controls, in line with the report of Wu et al., (2012) for the existence of a relationship between feeding of pregnant animals and their offspring. In 20-day-old offspring of sows from Group I and II, blood Se levels were similar (0.145±0.006 ppm and 0.132±0.006 ppm respectively) (Table 1), allowing affirming that the transfer of this trace element from the feed of sows to offspring took place regardless of the supplement form (organic or inorganic). On the opposite, 40-day-old piglets from Group I exhibited significantly higher blood Se levels than those from Group II (p<0.001), showing that organic Se was a more appropriate form for storage of selenium in the body. The chosen approach for dietary supplementation with organic (Sel-Plex®) and inorganic (sodium selenite) to pregnant sows ensures the biological activity of the element and prevented its overdosing and toxicity in the offspring. The inconsistent data on the relationship between systemic selenium reserve and immune protection efficiency (Valenta et al., 2012) focuses the attention on the status of innate defense mechanisms as a first line of defense.


 
Phagocytosis and phagocytic parameters
 
Our data exhibited that newborn piglets had sufficient cellular factors of innate resistance (Table 2). Regardless of the group, absolute segmented neutrophil count varied from 3.56±0.92x109/L for Group I, 3.08±0.82x109/L for Group II and 3.38±0.28x109/L for Group III. In 20-day-old piglets, average counts decreased to 1.92±0.32x109/L (Group I), 2.44±0.31x109/L (Group II) and 1.96±0.26´109/L (Group III) and to 40 days of age, segmented neutrophil counts increased statistically significantly exceeding values in 20-day-old animals (Table 2). The described changes in 20-day-old piglets are possibly associated to weaning (Mike and Varley, 2001), while in 40-day-olds, changes could reflect the start of immunological maturation. Neutrophils are the most active mobile phagocytes (Mizgerd, 2002; Tizard, 2013), that is why their phagocytic activity has been determined. Phagocytosis percentages were high in neonates, not only in controls (p<0.01), but also in Group II (p<0.001) compared to 40-day-old piglets, in which phagocytosis percentages were lower both vs those in newborns and vs 20-day-old animals as well (Table 2). Phagocytic numbers were also high in neonates. Therefore, cellular factors of innate immunity were well manifested as early as after birth. Similar studies (Satwani et al., 2005; Jiang et al., 2009; Kollmann et al., 2009; Dowling and Levy, 2014) are focused on mechanisms of defense in newborns. Data however show clearly that the changes in cellular elements of innate resistance in newborn piglets and in piglets at 20 and 40 days of age were age-dependent and not related to selenium reserves of animals. That is why the knowledge of the main tendencies in cellular and humoral factors of innate immunity after birth is a reliable background for developing new approaches for immune response optimisation.

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High blood selenium concentration in the offspring of sows supplemented with either organic or inorganic selenium, did not correspond to phagocytic activity of neutrophils. Data provided evidence of highest absolute segmented neutrophil counts, phagocytosis percentage and phagocytic number in newborn piglets regardless of regardless of sows’ supplementation. The lowest values of parameters were found out in 40-day-old animals despite their substantial blood selenium reserve.

Many thanks to Mrs Daniela Ivanova, Faculty of Veterinary Medicine, for her technical assistance in preparing this manuscript.