Effect of in ovo Injection of Nicotonic Acid, Pantothenic Acid or Folic Acid on Immune System and Growth of Broiler Chickens

Introduction

The nutrients needed by the chicken em- bryo are met by hen, so the embryo only has access to the egg nutrient resevours after lay- ing (Jha et al, 2019). In recent decades, the use of in ovo injection has been considered for early feeding in birds (Dal Pont et al., 2019). In this process, nutrients are fed to the embryo within the egg at various stages of the embryo’s development.

Vitamins are nutrients, which are essential at very low levels. Pantothenic acid has an important role in coenzyme A, acyl carrier proteins synthesis (Lanska Douglas, 2012). Pantothenic acid is  also  involved in gener- al metabolism and the previous reports have confirmed its positive effect on the health and performance of birds (Wang et al., 2016). Folic acid has a critical role in one-carbon metabolism and is involved in DNA, RNA and protein methylation and DNA synthesis and maintenance (Leung et al., 2013). Folic acid also enhances the immunity (Delaney et

al., 2013; Feng et al., 2011).

Niacin is involved in electron transport for intracellular respiration and there are reports on the positive effects of niacin on growth of poultry (Jiang et al., 2014). This study was designed to evaluate the effects of in ovo in- jection of nicotonic acid, pantothenic acid or folic acid on the live weight, carcass param- eters and immune system of broilers.

Materials and Methods

In this study, 450 Ross 308 fertile eggs with 65 g average weight were obtained from breeder flock at 43 weeks of age. The eggs were individually weighed and divid- ed into five experimental groups including  4 replicates. The experimental treatments were the non-injected group, the group in- jected  with  1  ml  physiological  serum and

three groups injected with 1 cc of physio- logical saline solution containing 0.121 mg of nicotinic acid, 0.052 mg of pantothenic acid or 0.007 mg of folic acid. The select- ed vitamin levels were based 75% of the recommended level throughout the starter phase of the rearing period (Aviagen, 2014). To prepare injectable solutions, nicotonic acid (Merck, Swizerland, 99.9% purity), Pantothenic acid (LeSen, China, 99% puri- ty) and folic acid (DSM, Neiderland, 80% purity), in the amount of 0.211, 0.052    and

0.007 mg, were dissolved in 50 ml physio- logical serum, respectively.

In ovo injection of vitamins was carried out in the air cell of eggs  on the 14th  day  of incubation and after separation of broken eggs. Before injection, the broad end of the eggs was disinfected using 96% ethyl alco- hol. Disposable Insulin syringes with . 22 gauge needle were used for injection. In the wider part of the shell, eggs were pierced with a needle and the solutions were inject- ed into the air cell. The temperature of the hatchery was 37.8 °C and the humidity was 65%. After completion of injection, the place was disinfected with alcohol, blocked using melted paraffin, and then transferred to the incubation trays.

At the end of the incubation period, the hatching chicks after counting and weighing were transferred to the poultry house. The hatchability of eggs in different groups was comparable, between 68 and 71 percent. The chicks were grown for 42-day experimental period under the same environmental condi- tions and diet formulated based on dietary re- quirements of the Ross 308 broiler (Aviagen, 2014). The mortality rate in all the experi- mental groups was less than 5%. Birds were vaccinated at 9 days old by injection of    the

dual Newcastle and Avian influenza vaccine (Razi Institute, Karaj, Iran) and eye drop of dual bronchitis-B1 Newcastle virus vaccine, H120-B1 (bronchitis-B1 Newcastle virus ) (Razi Institute, Karaj, Iran). َ At 15 days old Gumboro Nobilis D78 vaccine (MSD, Neth- erlands) was administrated in drinking water. At 18 days of age, Newcastle VITAPEST L vaccine (Ceva Santé Animale,  France)  and at 25 days of age Newcastle Avinew (Boeh- ringer Ingelheim, Germany) were used in drinking water.

At the end of the experiment,  a  chick  was selected from each replicate and blood samples were taken from wing vein. Blood samples were  placed  in  tubes  containing  50 μl EDTA to count the lymphocytes and heterocytes. The counting of white blood cells was done by observation and counting by eye after gisma staining using an optical microscope (Olympus UK Ltd., Essex, UK) (Grass and Siegel., 1983).

To determine the antibody titer of the bird against sheep red blood cells (SRBC), sheep blood was centrifuged for 10 min at a rate  of 2500 rpm and each time the upper fluid was discarded and a same amount of chlo- ride solution 0.9% was added. The washed red blood cells were diluted with sodium chloride solution so that a solution of 5% red blood cells was obtained. Injection of 0.1 ml SRBC at 18 and 35 days of rearing period (injection in the chest muscle) was carried out (Grasman, 2010) and blood  samples were collected from left wing vein at 22 and 42 days of age (Grasman, 2010). The hemag- glutination inhibition (HI) test using ELISA with a Newcastle virus (NDV) mono-spe- cific antiserum was used to detect the titer against NDV (OIE, 2012). To measure the immunoglobulin G, Mercaptoethanol was used to create sediment. Immunoglobulin  M

was obtained by subtracting immunoglobu- lin G from total immunoglobulin (Cheema et al., 2003).

At the end of the rearing period, after re- cording live weight, 10 birds were slaugh- tered from each treatment and the weights of carcass, bursa of Fabricius, Thymus, Heart, Liver, Spleen and abdominal fat were re- corded. Statistical analysis was performed using SAS 9.1 software and GLM proce- dure. The comparison of the meanings was done using Duncan's multiple range test and the 5% probability level was considered as a significant level.

Results

Tables 2 and 3 show the effect of in ovo injection of Nicotonic acid,  Pantothenic  acid, and Folic acid vitamins on antibody titers against SRBC and Newcastle Virus (NDV), IgG and IgM in serum at 18 and 35 days of age. At 18 days of age, in ovo in- jection of pantothenic acid and nicotinic acid resulted in an increase in the antibody titre against Newcastle Virus (NDV) compared to the control group and the folic acid injected group (P<0.05), but this parameter was not affected by experimental treatments  at  age 35 days. Antibody titers against sheep red blood cells were also influenced by exper- imental treatments. At the age of 18 days, injection of folic acid and pantothenic acid reduced the titer in comparison with the neg- ative control group (P<0.05), and at 35 days of age, nicotinic acid and folic acid groups had a lower titer than the negative control group (P<0.05).

At   18 days,  the level of IgG in serum   of chicks in  ovo  injected  with  vitamins  was lower than the negative control group (P<0.05), and a similar relationship was ob- served  for  IgM  in  chickens  of pantothenic

acid group (P<0.05). At 35 days of age, there was no significant difference in serum IgG levels  in different  groups,  but the IgM  lev-

els in the nicotinic acid and folic acid groups were lower than the negative control group (P<0.05).

Table 1. Ingredients and nutrient composition of basal diets fed (as-fed)

1 Provided the following (per kg of diet): vitamin A 8,800 IU; vitamin D3 2,500 IU; vitamin E 11 IU; vitamin K3 2.2 mg; thiamine

1.5 mg; riboflavin 4 mg; pyridoxine 2.5 mg; pantothenic acid 8 mg; nicotinic acid 35 mg; folate 0.48 mg; cyanocobalamin, 0.01 mg; choline chloride 200 mg.

2 Provided the following (per kg of diet): Mn (MnSO4, H2O) 75 mg; Zn (ZnO) 64 mg; Fe (FeSO4, H2O) 75 mg; Cu (CuSO4, 5H2O) 6 mg; I (KI) 0.86 mg; and Se (Na2SeO3) 0.2 mg.

Table 2. Effect of in ovo injection of Nicotonic acid, Pantothenic acid, and Folic acid on serum antibody titers and at 18 days of age (log2)

a-bMeans in the same column without a common superscript differ significantly (P <0.05).

1 The titers are for SRBC injected birds.

Table 3. Effect of in ovo injection of Nicotonic acid, Pantothenic acid, and Fo- lic acid on serum’s antibody titers and at 35 days of age (log2)

a-cMeans in the same column without a common superscript differ significantly (P <0.05)

1 The titers are for SRBC injected birds.

Table 4 shows the effect of in ovo injection of vitamins on white blood cells. The high- est heterphils to lymphocytes (H/L) ratio was observed in the pantothenic acid group and the lowest levels were seen in the folic acid group (P<0.05). The highest levels of lym- phocyte and heterophile were recorded in folic acid and nicotinic acid groups, respec- tively  (P<0.05). The total  number  of  white

blood cells counted in the folic acid group was higher than the negative control group (P<0.05).

The only carcass and organs parameter affected by experimental treatments was the relative weight of the liver that increased in the nicotinic acid group compared with the control groups (P <0.05) (Table 5). In ovo in- jection of nicotinic acid and pantothenic acid

caused weight loss in chicks during the first and second weeks of rearing period com- pared to the positive and negative control groups (P<0.05) (Fig. 1). However, the body

weight at the 6th week of rearing period in the nicotinic acid injected group was signifi- cantly higher than other groups (P<0.05) ex- cept for the negative control group (Fig.  1).

Table 4. Effect of in ovo injection of Nicotonic acid, Pantothenic acid, and Folic acid on white blood cells

a-dMeans in the same column without a common superscript differ significantly (P <0.05)

Table 5. Effect of in ovo injection of Nicotonic acid, Pantothenic acid, and Folic acid on the relative weights of organs of broilers

a-bMeans in the same column without a common superscript differ significantly (P <0.05)

Figure 1. Effect of in ovo injection of water-soluble vitamins on the live weight of broilers   chickens.

Discuusion

The Heterophil-to-lymphocyte ratio (H/L) is known as an index to stress (Stefaniak et al., 2019). Stressors, such as fasting or water deprivation and environmental extremes in- crease the H/L ratio (McFarlane et al., 1989). Then the lower H/L ratio in birds from eggs in ovo injected with folic acid could be a pos- itive indicator of bird’s health. There are not many reports on the effects of in ovo injec- tion of B-complex vitamins on the immune system of poultry. One of the few reports is available from Li et al. (2016) who showed that in ovo injection of folate injection im- proved immunity by increasing plasma lyso- zyme activity and plasma IgG and IgM con- centrations, and changes in  the  expression of immune-linked genes. These researchers reported the best effects within ovo injection of 150 ng folic acid. In the present study, the reduction of blood immunoglobulin levels was observed by vitamins injection, which seems inconsistent with the previous report and further studies need to clearify the mech- anism of the observerd effects.

In this study, the relative weight of the liv- er that increased in the nicotinic acid group was compared with the control groups. Liver is the main source of lipid synthesis in birds and changes in the liver weight could be due to increased fat content. Folic acid is a co- factor of coenzyme A that plays a vital role  in lipid metabolism and fatty acid synthesis (Feng et al., 2011). The liver, as the main source of metabolism in the body, plays an important role in the production of IGF-2 in the bloodstream (Liu et al., 2016). There are also reports that there is a positive relation- ship between liver weight and IGF-2 gene expression in ducks (Jianmin et al.,  2014).

The finding  of this  study  showed  a  neg-

ative effect of in ovo injection of nicotinic acid and pantothenic  acid  on  weight  gain in chicks during the first and second weeks  of rearing period compared to the positive and negative control groups. However, the body weight at the 6th week of rearing pe- riod in the nicotinic acid injected group was increased. In a study by Wang et al. (2016), consumption of about 26 mg of pantothen-  ic acid improved the weight gain of geese. Bootwalla and Harms (1991) indicated that the level of 4.8 mg of pantothenic acid in the diet was the best level to increase the weight of broiler chicks. Qi et al. (1998) reported that dietary supplementation with pantothen- ic acid increased the metabolism  of  calci- um and phosphorus, which results in higher body weight gain. The researchers suggested 10 mg / kg pantothenic acid for best perfor- mance. Positive effects of in ovo injection of folic acid on the weight gain of chicks have also been reported.

Liu et al. (2016) reported that in ovo in- jection of 150 μg folic acid increased the weight of the chicks compared to the control group. In that study, IGF-2 expression was increased. The correlation between plasma IGF-2 levels and chick embryo weight has already been reported (Lu et al., 2007). Fo- lic acid is a potent antioxidant agent (Lete- lier et al., 2010) and plays a major  role  in the immune system (Duthie et al., 2010) and this can help the bird to cope with oxidative stress. In the study of Jiang et al. (2014), the body weight of broiler chickens fed 60 mg/ kg nicotinic acid was improved, an observa- tion in line with the higher final body weight of chickens in the nicotinic acid in ovo in- jected group in the present study. In the pres- ent study, the in ovo injection of folic acid had the highest effect on the weight of the chicks.