Adding a bio-response modifier and zinc oxide to piglet weaner diets influences immunological responses to weaning Ujjwal Kumar De A,F , Reena Mukherjee A , Chandan Prakash B , Bhimnere Hanumanthagouda Manjunatha Patel C , Sukdeb Nandi B , Umesh Dimri A , Ashok Kumar Verma D and Med Ram Verma E A Division of Medicine, Indian Veterinary Research Institute, Izatnagar 243 122 (Uttar Pradesh), India. B CADRAD, Indian Veterinary Research Institute, Izatnagar 243 122 (Uttar Pradesh), India. C Livestock Production and Management Section, Indian Veterinary Research Institute, Izatnagar 243 122 (Uttar Pradesh), India. D Division of Animal Nutrition, Indian Veterinary Research Institute, Izatnagar 243 122 (Uttar Pradesh), India. E Division of Livestock Economics, Statistics and Information Technology, Indian Veterinary Research Institute, Izatnagar 243 122 (Uttar Pradesh), India. F Corresponding author. Email: ujjwalde@gmail.com Abstract. The effect of zinc oxide (ZnO) and a Mycobacterium smegmatis-derived bio-response modifier (BRM) supplementation on blood neutrophil functions, high-mobility group box 1 (HMGB1) protein and pro-inflammatory cytokine responses was studied in early weanling piglets. In total, 45 piglets were placed in the following five groups: basal diet only (I), supplemented with ZnO (II), supplemented with BRM (III), supplemented with ZnO plus BRM (IV) in basal diet and basal diet without weaning from dam (V). The phagocytic activity, superoxide anion and myeloperoxidase production in blood neutrophils and the concentrations of HMGB1, TNF-a, IFN-g and IL-1b in blood plasma were measured before and after weaning. The neutrophil functions were impaired and the concentrations of HMGB1, inflammatory cytokines, were elevated in piglets during the post-weaning period. The neutrophil functions were not improved until Day 7 of weaning (P > 0.05) and pronounced elevation (P < 0.05) in the concentration of pro-inflammatory cytokines and HMGB1 was observed until Days 14 and 21 respectively, in Groups II and III. The addition of BRM plus ZnO in basal diet improved superoxide anion and myeloperoxidase production on Day 2 (P < 0.05) and decreased the TNF-a and IFN-g concentrations on Day 7 (P < 0.05), with no significant change in the level of IL-1b and HMGB1 in Group IV. Finally, it is concluded that addition of ZnO plus BRM in the diet induced the neutrophil functions and reduced the inflammatory cytokine response much earlier to stimulate innate immunity than did ZnO or BRM alone. Additional keywords: HMGB1, Mycobacterium smegmatis, phagocytic activity, piglet, weaning. Received 19 May 2016, accepted 11 October 2017, published online 7 February 2018 Introduction In spite of potential advantages of early weaning in pig husbandry over the traditional practices, the piglets are exposed to substantial physical, social and emotional stress due to maternal separation, relocation, introduction to new social groups and the change of diet (Hohenshell et al. 2000; Lima et al. 2012). The deleterious effects of weaning include impaired function of neutrophils, natural killer cells, lymphocytes and upregulation of pro-inflammatory cytokines, and making the piglets vulnerable to infection (Bonnette et al. 1990; Campbell et al. 2013; Hu et al. 2013). To improve the overall health condition and performance of the piglets, suboptimal doses of antibiotic supplementation in the ration have become a routine practice over the years during the weaning transition to check the infection (Cromwell 2002). However, antibiotic residues in the meat and meat products are not easily accepted by the sensible consumers due to potential health hazards (Reijnders 2006). To circumvent this problem, there is an urgent need to find out the safe and viable alternatives such as bio-response modifiers (BRM) and micronutrients (Gallois and Oswald 2008; Wang et al. 2008). Mycobacterium species are known to have immunomodulatory activity in mammals when administered either as live organism, inactivated whole mycobacteria, cell-wall skeletal fraction, or as individual cell-wall components such as muramyl dipeptide or trehalose mycolate (Azuma et al. 1974; Adam and Lederer 1984). The mycobacterial cell wall has been shown to stimulate pro-inflammatory cytokines, activate macrophages and neutrophils, and generally upregulate the host’s immunologic system (Rogan et al. 2007). The cell-wall component of the mycobacteria CSIRO PUBLISHING Animal Production Science https://doi.org/10.1071/AN16332 Journal compilation Ó CSIRO 2018 www.publish.csiro.au/journals/an