Polygonatum sibiricum polysaccharide affects growth performance and immune response in cyclophosphamide-induced immunosuppressed chickens CURRENT STATUS: POSTED

Background: This study aims to identify the effects of dietary Polygonatum sibiricum polysaccharide (PP) on growth performance and blood parameters in immunosuppressed chickens induced by cyclophosphamide (CY). Results: The results showed that PP enhanced the growth performance and immune response in chickens treated with cyclophosphamide characterized by increasing relative weight of thymus, total antioxidant capacity (T-AOC), superoxidase dismutase (SOD), albumin (ALB), immunoglobulin M (IgM), and antibody titers of Newcastle disease (ND) and avian influenza (H9), and decreasing feed conversion ratio (FCR) and malondialdehyde (MDA) level. Conclusions: PP enhanced chickens’ growth performance via improving antioxidant ability and alleviating CY-induced immunological stress. study to elucidate the effects of PP on growth performance, immune and biochemical traits of immunosuppressed chickens induced by CY.


Background
Infectious diseases and mycotoxins induce immunosuppression, a disorder of immune system in broilers, which reduces immune response to vaccines, increases morbidity and mortality of flocks [1,2] and causes higher feed conversions and lower average daily weight gains [3,4].Cyclophosphamide (CY) is an alkylating agent widely used to build immunosuppression and stress animal models via intramuscular [5,6] or intraperitoneal injection.It inhibited both humoral and cellular immunity, causing B cell damage irreversibly [5] and depressed both antibody formation and immunoglobulin synthesis [7].Strategies to minimize immunosuppressed diseases are mainly via vaccination and stress control [2].However, inactivated vaccine cannot produce sufficient antibody, attenuated vaccine induces reactivation and infection [8].Therefore, new methods to control stress are in great demand.
Polygonatum sibiricum polysaccharide (PP) is a recently isolated water soluble component of rhizome polygonatum, which has medicinal efficiency in enhancing immune function [9], vital essence replenishment, dryness removal, and promotion of fluid secretion .In laboratory animals, PP has the potential as a treatment of diabetes and ocular complications, an alternative to estrogen to treat osteoporosis, a neuroprotective agent, a prevention for osteoporosis, and a remedy for inflammatory disorders [10][11][12][13].Liu had shown that the polysaccharide from Polygonatum sibiricum played an important role in the protection of the Cy-treated immunosuppressed mice and could be used as a potential immunostimulant agent [14].However, its protective effect against immunosuppression in chickens is poorly understood.Therefore, the objective of the present study was to elucidate the effects of PP on growth performance, immune and biochemical traits of immunosuppressed chickens induced by CY.

Growth Performance
Growth performance indices were affected by CY and PP during the overall period.From d 21 to d  1).

Serum Antioxidant Indices
PP group had the highest T-AOC and SOD and the lowest MDA among all groups at all time points.CY group had the lowest T-AOC and SOD, and the highest MDA at all time points, compared with other groups.PP+CY had lower MDA compared with CY group on d 14 and 21 (p < 0.05) (Table 2).

Blood Biochemical Indices
PP group had higher TP (p < 0.05) than CY and PP+CY groups on d 14.CY group had lower ALB than other groups on d 14 (p < 0.05).PP group had higher ALB compared with other groups on d 28 (p < 0.05) (Table 2).

Immune Organ Relative Weights
For spleen, PP groups were higher than CY and PP+CY groups on d 14 and 28 (p<0.05).For relative weights of thymus, the Control and PP groups were higher than CY and PP+CY on d 14 (p<0.05).
PP+CY group was higher than CY group on d 21 (p<0.05).For bursa, the Control and PP groups were higher than CY and PP+CY groups on d 14, 21, and 28 (p<0.05)(Table 3).

Effect of PP on Immunoglobulin
IgG levels had no difference among four groups.For IgM, the Control was the lowest on d 14 (p<0.05).
PP group was higher than CY and PP+CY group on d 28 (p < 0.05) (Table 4).

Effect of PP on Antibody Assay
For ND, CY group was the lowest on d 14 (p<0.05).PP+CY group was higher than CY at all time points (p<0.05).The Control was higher than CY at all time points (p<0.05).For H9, CY was lower than PP and PP+CY on d 14 (p<0.05).CY group was the lowest on d 21 (p<0.05).PP was the highest and CY group was the lowest compared to other treatments on d 28 (p<0.05)(Table 5).

Discussion
In the present study, we built the model of immunosuppression chickens induced by CY in order to study PP's function in growth promoting and immune booster in broilers.The results indicated that the growth performance, relative weights of bursa, Spleen and thymus, antibody titers to ND and H9 of CY group decreased (p<0.05)compared to the Control, which were consistent with the immunosuppressed model in other studies with bursa atrophy that showed depression of ND and HI antibody production [15].
Studies have indicated that the deficiency of immune system resulted in poor growth performance, low DFI and DWG and high FCR [16].Consistent with this finding, CY lowered DWG and DFI from d 1 to d 35 compared to Control group in our study (p<0.05).However, PP group had higher DWG and DFI from d 1 to d 35 and lower FCR from d 7 to d 21 compared with PP+CY group (p<0.05), which indicated that PP displayed positive effect on chicken growth and promoted the feed conversion efficiency of birds.In consistent with other studies, Astragalus polysaccharide, and 2 g/kg polysaccharide extracts of Lentinus edodes inclusion increased broilers' BWG and decreased FCR [17,18].T-AOC is an integrate and sensitive antioxidant index that identifies oxidative status in vivo [19].MDA is the product of lipid peroxidation and tissue damage manifestation [20].CY caused overproduction of reactive oxygen species (ROS) [21], which is an inclusion of superoxide anion (O Polysaccharides extracted from Platycodon grandiflorum [23], Rhododendron aganniphum [24], Pleurotus eryngii [25] , and Glycine soja [26] have great antioxidant capacity in elevating SOD and decreasing MDA concentration in pheochromocytoma cells and showing good radical scavenging activity for 1,1-diphenyl-2-picrylhydrazyl radicals in vitro.
IgG is the principle antibody in immune response for anti-infection, and IgM arises in initial stage of anti-infection immune response [2].The levels of IgG and IgM are indicators of antiviral response capacity [33].In the current study, PP group had higher IgM on d 28 than PP+CY group and on d 21 than Control group (p < 0.05), which indicated that PP enhances anti-infection and anti-virus ability of chickens.Similar results have been reported in previous studies.Polysaccharides extracted from Astragalus membranaceu, Astragalus, sulfated epimedium, Tremella fuciformis, and Lentinus edodes increased production of IgG and IgM in chickens [34].
Antibody titers manifest humoral immune response mediated by B lymphocyte.The results showed PP+CY had higher antibody titer of ND and H9 compared with CY group during the whole period (p<0.05).This indicates that PP resisted the decreased antibody formation and immunoglobulin synthesis induced by CY, which confirms immunomodulatory effects of polysaccharides.

Conclusions
PP at a dose of 800 mg/kg in diets enhanced immune response in a local meat-type chicken induced by CY and improved its growth performance and antioxidant levels.PP may be a potential feed additive to mount a successful defense against immunodeficiency diseases in chickens and other animals as well.

Animals Husbandry and Immunosuppressed Model Design
The objective of the present study was to elucidate the effects of PP on growth performance, immune and biochemical traits of immunosuppressed chickens induced by CY.
Four hundred and eighty one-day-old Erlang mountainous chickens (line SD02) were provided by Poultry Breeding Farm of Sichuan Agricultural University and had free access to water and basal diets which met the NRC requirements [35].And the formulation and nutrient levels were according to the National Research Council and shown in Table 6.Chickens were raised in cages in a ventilated house maintained at 50% relative humidity.The photoperiod was 24 h for the first 14 days and 20 h for the subsequent days.Temperature was 34℃ initially and decreased by 2℃ per week to 24℃, which was maintained till the end of the experiment.
Polygonatum sibiricum polysaccharide at 0 or 800 mg/kg of the diet and CY at 0 or 80 mg/kg were used in a 2 × 2 factorial designed with 4 randomized complete blocks consisting of initial BW of 37.83±3.45gchickens.They were allocated into 4 groups: 1) non CY-induced control (CON; birds fed basal diets and treated with sterile saline); 2) CY-induced control (CY; birds fed basal diets and treated with 80 mg/kg CY); 3) non CY-induced group + PP (PP; birds fed basal diets supplemented with 800 mg/kg PP and treated with sterile saline); 4) CY-induced group + PP (PP + CY; birds fed basal diets supplemented with 800 mg/kg PP and treated 80 mg/kg CY).There were four replicates (30 birds per replicate) randomly assigned into each of 4 diet groups.CY and sterile saline were delivered through intramuscular injection on d 7, 8, and 9, respectively with a dose of 80 mg/kg of body weight according to the previous report.Our previous study showed basal diet supplemented with 800 mg/kg PP had better effect than 200 mg/kg, 400 mg/kg, and 1200 mg/kg on increasing daily weight gain (DWG), daily feed intake (DFI), relative weights of bursa, spleen, thymus and SOD, TP, ALB, T-AOC, and decreasing feed conversion ratio (FCR) and MDA.

Growth Performance
Feed intake was monitored for each cage every week after a 12 h fast.Average DWG and DFI were weighed on d 1, 7, 14, 21, 28, and 35.FCR was calculated as DWG/DFI.These growth performance parameters were adjusted by mortality.On d 14, 21, and 28, five chickens close to mean body weight were euthanized by injecting pentobarbital sodium intravenously at a dose of 100 mg/kg without any fear and agony, as pentobarbital sodium can inhabit the ascending reticular activating system of brainstem, leading to the death of animals.And tissues were collected and stored at -80℃ for further analysis.

Biochemical Parameter Assay
On d 14, 21, and 28, 1mL of blood was obtained from the brachial vein and centrifuged at 2,000 x g for 5min.Supernatant liquid of blood serum was collected and stored at -20°C for further analysis.
The contents of T-AOC, SOD, MDA, TP, and ALB were measured using ELISA kits according to manufacturer's instructions.

Immune Organ Relative Weight
Spleen, thymus, and bursa were collected and weighed on d 14, 21, and 28.Immune organ relative weights were expressed as the ratio of organ weight to body weight (mg/g).

Serum Immunoglobulin and Antibody Assays
Blood samples of 2.0 mL were extracted from the main brachial vein of chicken and clotted at 37 °C for 2 h.To collect serum, the blood samples were centrifuged at 2,000 x g for 3 min.IgG and IgM were measured using ELISA kits according to manufacturer's instructions.The serum antibody titers of subtype avian influenza viruses H9 (H9) and Newcastle disease virus (ND) were assessed by hemagglutination assay (HA) and hemagglutination inhibition (HI) assay.The concentrations of titer were recorded as log 2 value.The reciprocal of the highest dilution was the complete agglutination.
a-c Means in a row without the same superscripts differ significantly (p < 0.05).

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Table 1
Effect of Polygonatum sibiricum polysaccharide on growth performance in chickens (Mean

Table 2
Effect of dietary PP on serum biochemical indices of chickens (Mean ±SE)

Table 3
Effect of PP on relative weights of spleen, thymus, and bursa of chickens (Mean ±SE) ab Means in a row without the same superscripts differ significantly (p<0.05).

Table 4
Effect of PP on serum globulin of chickens (Mean ±SE) -c Means in a row without the same superscripts differ significantly (p <0 .05).The antibody titers were shown as log 2 value of the reciprocal of the highest dilution.
ab Means in a row without the same superscripts differ significantly (p < 0.05).a#
a b Nutrient levels are calculated value.