Haematological responses of suckling piglets to combined multi-strain probiotic and multi-mineral supplementation

Introduction

Piglet diarrhoea is a recurring issue in swine farms and a major contributor to increased morbidity and death in new-born pigs. (Zhou et al., 2023) [24] . In pig farming, diarrhoea within the first seven days of life causes subpar performance and substantial financial loss. (Farmer and Edwards, 2022) [6] . Piglets must have a healthy gut microbiota and an intact gut barrier in their early years in order to be protected against intestinal illnesses. New-born piglets' gut is usually thought to be sterile, but because of their early exposure to the outside world, nursing piglets are susceptible to colonisation by dangerous infections, which increases the risk of diarrhoea. (Wang et al., 2021) [20] . New-born piglets' survival depends on their mother's passive immunity, which consists of three elements at minimum: There are three main forms of humoral immunity: systemic humoral immunity, local humoral immunity and maternal immunocompetent cells that transmit cellular immunity through mammary secretions (Salmon, 2000) [16] . Previous research has shown that mammary gland secretions, which are mostly colostrum, contain live immune cells in addition to antibodies. These cells are mainly lymphocytes, macrophages, neutrophils, and epithelial cells. Colostrum have the ability to transfer both humoral and cell-mediated immunity, according to Nechvatalova et al. (2011) [13] . The bulk of leukocytes in the blood of new-born pigs are colony-stimulating leukocytes, which are absorbed via the GI tract and affect the immune system of the piglets. We hypothesised that MSP supplementation and mineralization in early life of piglets would affect the haemoglobin and total erythrocyte count positively. The aim of the present study was to evaluate the effect of co administration of MSP and multi-minerals to piglets in early life on hemogram until weaning.

Cross streaking was done to check synergistic activity of all strains on MRS agar. Multi-minerals (MM) in injectable form (Stimvet, each mL contained 5 mg selenium, 40 mg zinc, 15 mg copper and 10 mg manganese) was procured from Wellcon Animal Health Private Limited, Agra, Uttar Pradesh, India.

Animals and experimental design

The present study was conducted in the experimental pig production farm of the Institute. Animal Ethics Committee of Institute approved the experiment protocol (approval no IAEC/L3). For this work, eight sows were randomly divided into four groups; control, multimineral (MM), multi-strain probiotic (MSP) and, MM + MSP groups. In control group, 17 piglets born from 2 sows received 1.0 mL of sterile normal saline (NSS) orally daily for seven days after birth; 22 piglets born from two sows were given 0.3 mL of MM injection in intramuscular (IM) route on days 0 and 7 of birth in group MM; 15 piglets borne from two sows in MSP group received MSP in ratio 1:1:1:1:1 at a dose rate of 10 9 CFU/piglet for first 7 days from birth, while in MM + MSP group, 23 piglets born from two sows received 10 9 CFU of MSP per piglet once daily first 7 days of birth along with 0.3 mL of MM injection IM route on day 0 and day 7. The piglets were given unlimited access to their respective sows' colostrum.

Collection of blood sample

Blood samples (1.0 mL) of piglets were collected in vial with anticoagulant (di-potassium ethylene diamine tetra acetic acid) from anterior vena cava on day 0 (prior initiation of intervention) and 7, 14 and 21 of birth.

Haematology

Haematology was performed in haematology analyser to measure haemoglobin (Hb) concentration, packed cell volume (PCV), total leukocyte count (TLC) and total erythrocyte count (TEC) in blood. Differential leukocyte count (DLC) was performed from blood smear that was stained with Giemsa stain (1:10 dilution in distilled water) manually. Total 100 cells were counted. The absolute neutrophil, lymphocyte, monocyte and eosinophil counts were derived from TLC.

Statistical analysis

The repeated measures ANOVA were applied to examine the effect of interventions on hemogram parameters. The multiple comparisons between groups, days and their interaction effects were done by using Tukey HSD test. An individual piglet served as the experimental unit. The GraphPad Prism 6 Statistics Guide was used for the date analysis. The results were presented as the mean and the standard error of the mean (SEM). Significant differences among mean values were accepted at p < 0.05.

Results

Hb, TEC and PCV

Statistically, a significant interaction was found between supplementations and days for TEC (p = 0.002) but, such interaction was not found for Hb (p = 0.080) and PCV (p = 0.066). The mean Hb concentration on day 14 (p < 0.05) and 21 (p < 0.05) and mean PCV value on day 14 (p < 0.05) were significantly lower from day 0 in control group. The mean Hb concentration on day 21 in MM group was significantly (p < 0.05) lower as compared to day 0 value. In MSP group, the mean Hb concentration from day 7 to 21, mean PCV value on day 14 and 21 and mean TEC on day 7 were significantly (p < 0.05) lower as compared to day 0 value. The mean Hb concentration on day 21, mean PCV value on day 7 and 21 and mean TEC on day 7 were significantly lower (p < 0.05) as compared to day 0 value in MM + MSP group. When the mean Hb, PCV and TEC values were compared among the control and supplementation groups, it was noted that the mean Hb and PCV values on day 14 and mean TEC on day 7 in MM, MSP and MM + MSP groups and mean Hb on day 21 in MSP group were statistically higher (p < 0.05) as compared to control group (Table 1).

TLC and DLC

The mean TLC and lymphocyte count on day 7 to 21, neutrophil count on day 7 and monocyte count on day 14 were significantly (p < 0.05) higher from day 0 value in control group. In MM group, the mean lymphocyte count on day 14 was significantly (p < 0.05) higher as compared to day 0. The mean TLC and neutrophil count on day 14 to 21 and lymphocyte count on day 21 were significantly (p < 0.05) lower from day 0 value in MSP group. In MM + MSP group, the mean TLC on day 14 to 21 and neutrophil count on day 7 to 21 were significantly (p < 0.05) lower while mean lymphocyte count on day 7 and 21 was significantly (p < 0.05) higher from day 0 value. Statistically, the mean eosinophil count in MM, MSP and MM + MSP groups and mean monocyte count in MM, MSP and MM + MSP did not vary significantly from day 0 to 21. The average monocyte count on day 14 in control was higher (p < 0.05) as compared to day 0 and 7. A significant interaction was found between supplementations and days for TLC (p < 0.001), neutrophil (p = 0.001), lymphocyte (p < 0.01) and monocyte (p < 0.001) but, such interaction was not found for eosinophil count (p = 0.396) (Table 2).

Discussion

In this study, a significantly lower concentration of Hb on day 14 and 21 and mean PCV on day 14 from day 0 in control group might be due to either insufficient iron (Fe) reserves or rapid growth in the critical age of early life and the finding agrees with earlier report on new borne pigs (Cruz et al., 2023) [2] . The insufficient translocation of Fe from sows to neonatal pigs and poor adaptation of the hematopoietic system of suckling piglets in their early life are the other attributing factors (Szudzik et al., 2018) [18] . Therefore, newborn pigs frequently require additional Fe supplementation to prevent anemia (Ding et al., 2020) [4] . The non-significant improvement of Hb concentration till day 14 in MM group could be due to either interference of MM with Fe absorption or unpredictable interactions among micronutrients in the body (Rossander-Hulten et al., 1991) [15] . Previous research also showed that supplementation of Fe alone is more effective than MM for raising Hb levels in anemic children and non-pregnant women (Gera et al., 2009) [7] . Significantly lower concentrations of Hb and PCV on day 7 to 21 in MSP group is in accordance with earlier study on suckling piglets where MSP supplementation did not produce any substantial effect on hemogram (Hansen et al., 2022) [8] . However, significantly (p < 0.5) higher the Hb concentration and PCV on days 14 and 21 in MSP group than control group agrees with earlier studies on grower pigs and calves fed lactic acid bacteria (LAB) including P. acidilactici FT28 (Dowarah et al., 2018) [5] . The LAB has a positive impact on dietary non-heme Fe absorption by enterocytes through increasing productions of mucin, organic acids and microbial by-product like phydroxyphenyl lactic acid which facilitate the conversion of ferric iron to the more accessible ferrous form at the gut surface (Hoppe et al., 2017) [10] . In our investigation, coadministrations of MM and MSP did not significantly affect the hemogram of suckling piglets, despite the fact that early life interventions with MM and probiotics are thought to have a major impact on piglet health (da Silva et al., 2021) [3] . Here, the significant rise (p < 0.05) of TLC and lymphocyte count on day 7 to 21 and neutrophil count on day 7 in control group may be the result of the piglets' varied levels of early-life antigen exposure (Rolinec et al., 2015) [14] . The activation and faster maturation of lymphocytes from the exposure to environmental antigens also attribute to the higher counts of absolute leukocyte and its subpopulations (Siddiqui et al., 2006) [17] . A transient increase of monocyte count on day 14 in control group might be a cause of temporary response to stress or exercise (Carter et al., 2018) [1] . A profound increase (p < 0.05) of mean lymphocyte count on day 14 from day 0 in MM group could be the result of enhanced cellular immunity of piglets (Weyh et al., 2022) [22] . The trace minerals augment the functioning of immune system through increased production of cytokines, proliferation of T cells including T helper cells and promoting the functions of various coenzymes (Wang et al., 2025) [21] . It is worthwhile to mention here that the effect of the trace minerals on the immune system is not adequately examined in suckling piglets as compared to other farm animals. In this study, a marked decrease of mean TLC, and neutrophil count on day 14 to 21 and lymphocyte count on day 21 in both MSP and MM + MSP groups suggest that early-life supplementation of MSP either alone or in combination with MM did not produce any additional effect in modulation of leukogram of sucking piglets.

Conclusion

The findings of the current study indicate that the MSP intervention in early life effectively improves haemoglobin until weaning whereas MM augments the functioning of immune system through increased production of cytokines, proliferation of T cells including T helper cells and promoting the functions of various coenzymes. However, more research including a large number of observations on multi-location farms is necessary before the intervention can be suggested as a set of practices in swine production.

References

1. Carter CM. Alterations in blood components. In: Comprehensive Toxicology. 2017:249-293.
2. Cruz TA, Muro BB, Lima EMC, Moreira VS, Carvalho JCC, Garbossa CAP, et al. Enhancing the performance, carcass characteristics, diet digestibility, and economic viability of pigs in the growing and finishing phases through aspartic protease supplementation: a comprehensive evaluation. Research Square. 2023. doi:10.21203/rs.3.rs-3482660/v1.
3. da Silva CA, Bentin LA, Dias CP, Callegari MA, Facina VB, Dias FT, et al. Impact of zinc oxide, benzoic acid and probiotics on the performance and cecal microbiota of piglets. Anim Microbiome. 2021;3(1):86.
4. Ding H, Yu X, Feng J. Iron homeostasis disorder in piglet intestine. Metallomics. 2020;12(10):1494-1507.
5. Dowarah R, Verma AK, Agarwal N, Singh P, Singh BR. Selection and characterization of probiotic lactic acid bacteria and its impact on growth, nutrient digestibility, health and antioxidant status in weaned piglets. PLoS One. 2018;13(3).
6. Farmer C, Edwards SA. Improving the performance of neonatal piglets. Animal. 2022;16:100350.
7. Gera T, Sachdev HP, Nestel P. Effect of combining multiple micronutrients with iron supplementation on Hb response in children: systematic review of randomized controlled trials. Public Health Nutr. 2009;12(6):756-763.
8. Hansen SV, Nørskov NP, Nørgaard JV, Woyengo TA, Poulsen HD, Nielsen TS. Determination of the optimal level of dietary zinc for newly weaned pigs: a doseresponse study. Animals (Basel). 2022;12:1552.
9. Hao Y, Xing M, Gu X. Research progress on oxidative stress and its nutritional regulation strategies in pigs. Animals (Basel). 2021;11:1384.
10. Hoppe M, Önning G, Hulthén L. Freeze-dried Lactobacillus plantarum 299v increases iron absorption in young females-double isotope sequential singleblind studies. PLoS One. 2017;12.
11. Jiang Z, Yang M, Su W, Mei L, Li Y, Guo Y, et al. Probiotics in piglet: from gut health to pathogen defense mechanisms. Front Immunol. 2024;15:1468873.
12. Jin S, Xu H, Yang C. Regulation of oxidative stress in the intestine of piglets after enterotoxigenic Escherichia coli (ETEC) infection. Biochim Biophys Acta Mol Cell Res. 2024;1871:119711.
13. Nechvatalova K, Kudlackova H, Leva L, Babickova K, Faldyna M. Transfer of humoral and cell-mediated immunity via colostrum in pigs. Vet Immunol Immunopathol. 2011;142(1-2):95-100.
14. Rolinec M, Bíro D, Šťastný P, Kanka T, Gálik B, Šimko M, et al. Development of leukocyte count in blood of neonatal pigs after colostrum intake. J Cent Eur Agric. 2015;16:399-409.
15. Rossander-Hultén L, Brune M, Sandström B, Lönnerdal B, Hallberg L. Competitive inhibition of iron absorption by manganese and zinc in humans. Am J Clin Nutr. 1991;54:152-156.
16. Salmon H. Mammary gland immunology and neonate protection in pigs: homing of lymphocytes into the MG. In: Biology of the Mammary Gland. Boston (MA): Springer US; 2000. p. 279-286.
17. Siddiqui M, Ristow K, Markovic SN, Witzig TE, Habermann TM, Colgan JP, et al. Absolute lymphocyte count predicts overall survival in follicular lymphomas. Br J Haematol. 2006;134:596-601.
18. Szudzik M, Starzyński RR, Jończy A, Mazgaj R, Lenartowicz M, Lipiński P. Iron supplementation in suckling piglets. Pharmaceuticals (Basel). 2018;11:128.
19. Tian S, Wang J, Gao R, Wang J, Zhu W. Galactooligosaccharides reduce oxidative stress in piglets. J Anim Sci Biotechnol. 2022;13:70.
20. Wang K, Hu C, Tang W, Azad MAK, Zhu Q, He Q, et al. The enhancement of intestinal immunity in offspring piglets by maternal probiotic or synbiotic supplementation is associated with the alteration of gut microbiota. Front Nutr. 2021;8:686053.
21. Wang SQ, Peng Z, Sun H, Han YM, Zhang B, Pineda L, et al. Evaluating the impact of an organic trace mineral mix on the redox homeostasis, immunity, and performance of sows and their offspring. Biol Trace Elem Res. 2025;203:1798-1807.
22. Weyh C, Krüger K, Peeling P, Castell L. Role of minerals in immune function. Nutrients. 2022;14:644.
23. Xu RJ, Wang F, Zhang SH. Postnatal adaptation of gastrointestinal tract in pigs. Livest Prod Sci. 2000;66:95-107.
24. Zhou X, Liang J, Xiong X, Yin Y. Amino acids in piglet diarrhea. Anim Nutr. 2023;16:267-274.