Effects of in ovo Injection of Nano Zinc Oxide on the Hatchability, Immunity and Antioxidant Responses, and Relative Gene Expressions of Interleukin 2 and 12 in Broiler Chickens

Document Type : Research Articles

Authors

Department of Animal Science, Faculty of Animal Science and Fishery, Sari Agricultural Science and Natural Resources University, Sari, Iran

Abstract

This experiment was conducted to examine the effects of in ovo injection of nano zinc oxide (Nano-ZnO) on hatchability, immunity responses, and performance in broiler chickens. A total of 432 fertilized eggs (Ross 308) were assigned to a completely randomized design with four treatments by four replications and 27 eggs in each replicate. Treatments included: 1) negative control (no injection), 2) positive control- no treatments with injection of solutions (20 µL saline), 3) 20 µL of Nano-ZnO (0.5 mg of Nano-ZnO in 50 mL saline), and 4) 20 µL of Nano-ZnO (0.6 mg of Nano-ZnO in 50 mL saline). Results showed that hatchability (P=0.002) and weight of hatched chicks (P=0.0001) decreased in treatments two and three compared to negative control. However, embryonic mortality were increased in early (≤7 d) and late (17-21 d) stages in comparison with the negative control (P=0.008). Fourteen days after vaccination, antibody titers of avian Influenza (P=0.025) and Newcastle (P=0.002) disease were increased in chickens that received Nano-ZnO compared to control groups. Relative gene expression levels of interleukin-2 (IL-2) (P=0.002) and 12 (P<0.05) were greater in treatment 3 compared to other treatments. Although in ovo injection of Nano-ZnO reduced hatchability at first glance, this was the result of injection, but it did not reduce the hatchability and chickens’ weight compared to positive control. In ovo injection of Nano-ZnO increased embryonic mortality, and improved humoral and cellular immune responses and weight of the spleen, which indicates the positive effects of injections and confirms the hypothesis of the study.

Keywords


INTRODUCTION

Zinc plays an important role in many vital functions of the body (Suttle, 2010). Zinc requirements in poultry influenced with increasing in growth and production or presence of limiting factors in the diet in zinc stores in the body, therefore, zinc storage in body can easily be reduced. Its deficiency in breeders reduces hatch rates, increases mortality, and decreases chicken growth (Rossi et al. 2007). Addition of zinc to the diet of breeders increases fertility and egg production, and decreases mortality in various embryonic stages (Favero et al. 2013). Nutrition plays a key role during embryonic period through affecting the development and survival of birds; hence, lack of attention to it causes delays and decreases growth leading to economic loss. Suitable nutrition is crucial for breeders to ensure adequate nutrient transfer to eggs and nutrient supply for natural embryonic growth. In adequate or excessive nutrients in eggs cause embryonic death. Therefore, properly implemented chick’s nutrition programs can improve production and reproductive performance. The content of minerals in the yolk significantly was reported to reduce during the incubation of broiler embryos resulting in complications of nutritional deficiencies (Yair and Uni, 2011). Nano particles have higher surface area to volume ratio than larger particles, thus, rendering a higher absorption capacity. These materials are rapidly distributed to most organs and tissues after injection into the body, with a very high cellular absorption (Salata, 2004). Nanoparticles can bypass conventional physiological ways of nutrient distribution and transport across tissue and cell membranes, as well as protect compounds against destruction prior to reaching their targets. In ovo administration of nanoparticles, may be seen as a new method of nano-nutrition, providing embryos with an additional quantity of nutrients. Based on the research hypothesis, an experiment was conducted to evaluate the effects of in ovo enjection of Nano-ZnO on the hatchability, immune responses, oxidative enzymes, and relative genes expression levels of interleukin 2 and 12 in broiler chickens.

 

MATERIALS AND METHODS

Experimental design and diets

The present study was approved by the Animal Care Committee of the Sari Agricultural Sciences and Natural Resources University (SANRU), northern Iran. A total of 432 fertile eggs (Ross 308) was assigned to a completely randomized design with four treatments and four replications with 27 eggs in each replicate. Treatments included: 1) negative control (no injection), 2) positive control (20 µL saline), 3) 20 µL Nano-ZnO (0.5 mg of Nano-ZnO in 50 mL saline), and 4) 20 µL Nano-ZnO (0.6 mg of Nano-ZnO in 50 mL saline). In order to investigate the effects of different Nano-ZnO concentrations on the immunity and performance of broilers, nine male chicks from each replicate were randomly selected and assigned to four experimental groups with four replicates for 42 days. All birds were housed in pens and received feed and water ad libitum. The diet was formulated according to Ross 308 broiler nutrition specification handbook (Hosseini et al. 2016) (Table 1).

 

In ovo injection

On the 10th day of the experiment, in ovo injection was performed into the air sac. To this end, all the injection areas were disinfected with 70% ethanol. Then a hole was then punched using a 21-gauge needle and then 20 μL of (in ovo injection) solution was injected into the air sac using a 22-gauge needle. After injection, the injection area was sealed with paraffin tape and the eggs transferred to hatching baskets.

 

Hatch characteristics and embryonic mortality

On hatching days, the number of chicks was counted in each replicate to calculate the hatchability percentage. Then, the embryonic mortality, and fertile and infertile eggs were examined by breaking the unhatched eggs. All infertile eggs were opened and examined macroscopically for evidence of embryonic mortality. All unhatched eggs were analyzed for developmental stage of dead embryos. The time of embryonic death was assigned to one of four categories: early dead (≤7 d), mid-dead (8-16 d), late dead (17-21 d), and pips (Cobb, 2001). The percentage of embryonic mortality and hatching percentage were expressed based on the eggs placed into the incubator. The percentage of fertile eggs and efficiency of converting eggs to chick were presented as described by (Joshua et al. 2016).

 

Breeding and feeding

On hatching days, nine chicks were randomly selected from each replicate and transferred to the experimental pens for rearing. A control corn-soybean meal based diet formulated according to Ross308 broiler nutrition specification to meet all nutrient requirements  (Table 1).

 

General immunity

In order to investigate the immune response, vaccination against Newcastle disease and influenza were carried out in 28 d of the experiment. On days 35 and 42 after vaccination, two chickens from each treatment were randomly selected and blood samples (3 mL) were collected from the left brachial vein. The presence of Newcastle and influenza antibodies in sera samples was assessed by hem agglutination inhibition (HI) method (Poorghasemi et al. 2015).

 

Cell-mediated immunity

An inter-digital skin test was made to evaluate the cutaneous basophil hypersensitivity (CBH) at 28-d of age as described by (Corrier and Deloach, 1990).

 

RNA isolation and cDNA synthesis

On the first day of breeding, two chicks were selected from each replicate and the spleen samples were collected, washed in sterile phosphate buffer saline, and immediately snap frozen in liquid nitrogen. Samples were stored at -80 ˚C until RNA extraction. Total RNA was isolated from spleen samples using the RNXTM-Plus solution (CinnaGen, Tehran, Iran) according to the manufacturer’s instructions, and stored at -80 ˚C pending cDNA synthesis. For each spleen sample, cDNA synthesis was carried out using the Omni script reverse transcriptase kit (Qiagen Inc., Tehran, Iran). The cDNA synthesis reaction conditions were 42 ˚C for 30 min and 95 ˚C for 3 min.

 

Table 1 Chemical compositions and nutrient contents of the experimental diet

1 Composition (per kg): Mn: 75000 mg; Fe: 50000 mg; Cu: 8,000 mg; I: 750 mg and Se: 60000 μg.

2 Composition (per kg): vitamin A: 8000 IU; vitamin D3: 2000 IU; vitamin K3: 1800 mg; vitamin B1: 1800 mg; vitamin B2: 6000 mg; vitamin B6: 2800 mg; vitamin B12: 12000 μg; Pantothenic acid: 10000 mg; Niacin: 40000 mg; Folic acid: 1000 mg and Biotin: 0.3 mg/kg.

Metabolisable energy (ME) was calculated according to Bourdillon et al. (1990).

 

Quantitative real-time PCR (qPCR)

Quantitative real-time polymerase chain reaction (qPCR) was implemented to determine the relative transcripts of interleukin 2 (IL2) and interleukin 12 (IL12) in the spleen tissue. Details of primer sequences are provided in Table 2. Expression of beta-actin transcript was used as an internal housekeeping gene. All transcripts were quantified using Quanti Fast SYBR Green PCR Kit (Cat. No. 204052; Qiagen, GmbH, Germany) in a 20 μL reaction volume containing 1 μL of single-strand cDNA, 10 μL of master mix, 0.5 μL of each forward and reverse primers, and 8 μL of distilled H2O in 20 μL by Rotor-Gene 6000 real-time PCR software (Corbett Research, Sydney, Australia). The program used for the amplification of genes consisted of a denaturing cycle of 5 minutes at 95 ˚C, followed by 40 cycles (95 ˚C for 15 seconds, annealing, and extension at 60 ˚C for 40 seconds). At the end of each PCR, a melting curve analysis was performed at arate of 0.1˚C/s for all genes to check the specificity of the products. Standard curves of each primer pair efficiency were determined with five series of 10-fold dilution of positive control cDNA as a template. The efficiency of the assays (E) was ≥ 95%, and standard curve R2 was ≥ 0.999. The relative levels of mRNA were analyzed by the 2-ΔΔCt method (Livak, 2001).

 

Gene expressions of interleukin 2 and 12

On the first day of breeding, two chicks were selected from each replicate and the spleen samples were collected, washed in sterile phosphate buffer saline, and immediately snap frozen in liquid nitrogen. Samples were stored at -80 ˚C until RNA extraction. Total RNA was extracted from spleen samples using the RNXTM-Plus solution (Cinna Gen, Tehran, Iran) according to the manufacturer’s instruction sand was then reverse transcribed in the presence of 1 µM oligo-primer and 4 U Omni script RTase (Qiagen Inc., Tehran, Iran). Real-time PCR reactions were carried out in a total volume of 20 ml with 1 ml of complementary DNA (50 ng/mL), 10 mL of SYBR green master mix (QuantiNova SYBR Green PCR Kit; Qiagen Inc), 0.5 mL of forward and 0.5 mL reverse primers (20 ng of each), and 8 Ml of nuclease-free H2O. Samples were run in duplicate and expressed relative to β-actin as housekeeping gene, which was stable under the culture conditions used.

 

Table 2 Primer sequences used for real-time quantitative PCR1

1 The listed oligonucleotides were used to analyze gene expression via real-time quantitative PCR.

2 IL-2: interleukin 2 and IL-12: Interleukin 12.

 

Data were normalized to a calibrator sample using the ΔCt method with correction for amplification efficiency (Livak, 2001).

 

Carcass quality

Chickens were slaughtered at 42 days of age and the weight of individual carcass components was evaluated and calculated as the percentage of each carcass component.

 

Antioxidant enzyme activities

In order to evaluate the antioxidant enzyme activity, two samples from each replicate were randomly selected on hatching days and blood samples were taken from their hearts and collected in plastic tubes containing ethylenediaminetetraacetic acid (EDTA). Samples were maintained on ice until plasma was separated by centrifugation (1500×g for 20 minutes) within 1h of collection. Plasma was harvested and stored at -20 ˚C until further analysis. Concentrations of glutathione peroxidase (GPX) and superoxide dismutase (SOD) were determined using commercial RANSEL and RANSOD kits (RANDOX Laboratories Ltd., London, UK) according to the manufacturer’s instructions.

 

Statistical analysis

This research was conducted in a completely randomized design with four treatments, four replicates, and 27 observations per replication using the Generalized Linear model (GLM) procedure of SAS (2003) software. Differences among treatment means were measured by Duncan’s multiple range test and considered significant at P < 0.05. Gene expression fold changes, standard error, and statistical significance were calculated by the software based on the formula developed by (Pfaffl and Hageleit, 2001). Data were normalized to a calibrator sample using the ΔC t method with correction for amplification efficiency (Livak, 2001). A P-value of less than 0.05 was considered as statistically significant.

 

RESULTS AND DISCUSSION

Data of hatchability, embryonic mortality, and relative chick weights are presented in Table 3. The highest andlowest values for hatchability and fertile hatchability were obtained in T1 and T4, respectively. In ovo injection of 20 µg of Nano-ZnO (T4) resulted in increased embryonic mortality in an early stage. The results showed that in ovo injection reduced hatchability and increased early and late embryonic mortality compared to the negative control (P<0.05), but this difference was not significant compared to the positive control (T2). Titers against influenza and Newcastle disease viruses measured at 35 and 42-d after vaccination are presented in Table 4. There were no significant differences in influenza and Newcastle antibody titers between treatments (P<0.05) seven days after vaccination. However, both antibody titers increased significantly 14 days after vaccination, indicating that Nano-ZnO increased blood immunity. The data showed that cutaneous basophil hypersensitivity was not affected by the treatments (Table 4). The relative gene expression levels of interleukin 2 and 12 are presented (Table 5) in T3 were significantly higher than those of other treatments (P<0.05). Also, increasing doses of injection T4 reduced IL-2 and 12 gene expressions compared to T3, indicating that T3 has the most appropriate concentration of Nano-ZnO to increase the expression of IL 2 and 12. The concentrations of antioxidant enzymes (SOD and GPX) and the percentages of organs that involved in immunity response (spleen, bursa, and liver) are presented in Table 6. Table 7 shows that SOD and GPX activities were not influenced by the treatments. Mean concentrations of GPX and SOD in plasma was not influenced by the treatments, but the spleen relative weight increased in T4 (P=0.023). The percentages of carcass components are shown (Table 7) did not differ between treatments (P>0.05). Adding Nano-ZnO to the diet did not affect (P>0.05) body weight and feed conversion ratio, but feed intake decreased in all treatments compared to T1 (Table 8) (P=0.030). In the current study, in ovo injection of Nano-ZnO reduced percentage of hatchability, but it is not only reason for the increased embryo mortality, because in ovo injection of saline also increased embryo mortality in this stage.

 

Table 3 Effects of in ovo injection of zinc oxidnano particles on hatchability, embryonic mortality, and body weight on the 1thd and relative body weight of broiler chickens

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

BW: body weight.

* (chicken weight / egg weight) × 100.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 4 Effects of in ovo injection of zinc oxidnano particles on humoral [anti-log2] and cellular immunity responses

1 Antibody titers against avian influenza [log2] after 7 days (*), and 14 days (†) of vaccination. Antibody titers against Newcastle disease [log2] after 7 days (#) and 14 days (§) of vaccination. Cutaneous basophil hypersensitivity after12 h () and 24 h (¥). Hypersensitivity test after 24 hours.

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 5 Effects of in ovo injection of zinc oxidenanoparticles on relative gene expressions of interleukin-2 and 12

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

IL-2: interleukin 2 and IL-12: Interleukin 12.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 6 Effects of in ovo injection of zinc oxidenanoparticles on concentrations of antioxidant enzymes and relative immunity organs of broiler chickens

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

SOD: super oxide dismutase and GPX: glutathion peroxidase.

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 7 Effect of in ovo injection of zinc oxidenanoparticles on relative weight of carcass components

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Table 8 Effect of in ovo injection of zinc oxidenanoparticles on chickens’ performance at 42 days of age

T1: no injection; T2: injection of 20 µL of saline; T3: injection of 20 µL of Nano-ZnO (0.5 g/mL of Nano-ZnO in 50 mL of saline) and T4: injection of 20 µL of Nano-ZnO (0.6 g/mL of Nano-ZnO in 50 mL of saline).

The means within the same column with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

 

Hence many of reasons related to drop in hatchability such as type of injection; location and time of injection is well visible. But hatchability was not significantly different in the treatments administrated with Nano-ZnO. El-Rayes et al. (2019) has been reported that, in ovo zinc injection at a proper level has no adverse effect on developing chicken embryo or hatchability. Meanwhile, high levels of zinc showed a reduction in the hatchability and this may be attributed to the imbalance of amnion minerals content that interfered with embryogenesis during the late incubation or the toxicity of zinc nano-form due to its high availability (Jose et al. 2018). The amount of Nano-ZnO and injection site might be the factors that increased mortality. Injection of nutrients into the air sac reduces hatchability and leads to the sensitivity of the chorioallantois membrane, interruption of respiration, and embryonic death (Ohta et al. 1999). Salmanzadeh et al. (2012) was conducted an experiment with glucose injection, the results of this experiment showed that hatchability had been decreased. They assumed that a lower hatchability of in ovo injection of glucose attributed to a possible allergic reaction in air sac that prevents the respiration of the embryo, however, it must be confirmed that such type of allergic reaction occurs with regard to in ovo injection of nanoparticles of trace minerals. Biria et al. (2020) indicated that in ovo injection of nano ZnO particles had positive effects on early embryo mortality rate in broiler chicken. Optimal consumption of Zn is very important in the development and maintenance of the immune system and its related cells. Sunder et al. (2008) reported that cellular and hum oral immune responses in broilers supplemented with 80 mg/kg of Zn were significantly higher than those received lower levels of Zn. By activating the thymus, Nano-ZnO causes maturation of T lymphocytes and increases the functioning of B lymphocyte leading to secretion of macrophages. Interleukin 2 is a type of cytokine signaling molecule in the immune system that stimulates the growth and differentiation of T lymphocytes and plays a vital role in the functions of B lymphocyte, natural killer cells (NK cells), macrophages, and oligodendrocytes (Kidd, 2003). Interleukin 12 is one of the most important cytokines that improves cellular immunity to respond to viral infections (Takei et al. 2008). Immunologists' believe that interleukin 12 is a key point to create a balance between Thelper-1 and Thelper-2 (Romani, 2008). The toxicity of nano-selenium is 7 times lower than that of inorganic selenium and 3 times lower than that of organic selenium (Peng et al. 2007). Trace minerals are important nutritional components for imparting immunity and in ovo enrichment can be a way for improving the immune system of the birds (Hassan, 2018). El-Bahr et al. (2020) reported that inclusion of ZnO-NPs, particularly 60 mg/kg, in the diet of Japanese quails to improve antioxidant and immune status. In the current study, the relative expression levels of IL-2 and IL-12 genes in T3 were significantly higher than other treatments. Obminska-Mrukowicz and Szczypka, (2005) reported that the use of sodium di-ethyldithiocarbamate with zinc sulfate in human increased interleukin 2, and subsequently promoted bodily immunity, which is the same as the current research. Goel et al. (2012) reported that injection of Zn (0.5 mg) into eggs caused a significant increase in interleukin 2 and 12 concentrations. Prasad et al. (1988) reported that secretion of interleukin-12 was affected by various concentrations of Zn. Similarly, increasing doses of injection in T4 reduced IL-2 and 12 gene expressions compared to T3 in here, suggesting that T3 has the most appropriate concentration of Nano-ZnO to increase the expressions of ILs 2 and 12. Goel et al. (2012) stated that in ovo injection of Nano-ZnO could increase cellular immunity and attributed this to the role of zinc in the proliferation and differentiation of lymphocytes and increased expression of IL-2 gene. A bird fed with zinc-rich diet was reported to have more active macrophages than a bird fed with Zn-poor diet (Kidd, 2003). As zinc plays an important role in the structure of transcriptional enzymes (DNA polymerase and RNA polymerase), it probably increases gene expression of interleukin 2 receptors as a result of increased secretion of interleukin 2. Jose et al. (2018) reported that Nano Zn-administered group showed a non-significant down regulation of MUC2 gene. Fathi et al. (2016) reported that SOD activity in the serum increased in broiler chickens received 20 mg/kg of Nano-ZnO. Zinc is a cofactor and involved in more than 240 enzymes and can influence oxidative processes. Zinc is also necessary for the structure and function of Cu-Zn-SOD, which contains 90 percent of the total SOD and protects tissues from the oxidative lesion (Noor et al. 2002). In agreement with our findings, Liu et al. (2015) reported that dietary supplemental Zn increased Cu-Zn-SOD activities in the breast and thigh muscles of broilers. These different results might be due to the different ways of Zn injection and Zn-adequate eggs used in the present study. Another insignificant reason that can be that the enzyme concentration was evaluated on the hatch day, which might have been better to be evaluated at the middle or end of the experiment as the relative weights of the spleen and the liver were affected by Nano Zn oxide injection at these two time points. Shokraneh et al. (2020) reported that injection of Nano-Se and Nano-ZnO increased antioxidant activity and reduced oxidative stress in broiler hatchlings. It has been shown that Zn sources can substantially enhance antioxidant capacity and resistance against oxidative stress in developing embryos and hatchlings (Zhang et al. 2018). Zhu et al. (2017) also indicated that maternal dietary zinc could protect chick embryos against maternal heat stress by elevating antioxidant capacity. Oxidative stress induced upregulation of SOD-1 gene expression, which is usually followed by upregulation of the expression of CAT and GPX genes (Ahmad et al. 2012). The significant upregulation of antioxidant enzymes because of ZnO-NP supplementation has been confirmed in broilers (Ahmad et al. 2012). Nanoparticles have been reported to be more efficient for animals than larger particles at low doses due to the easy absorption and biological availability as well as appear to interact better with other materials due to the significance of the active surface (El-Basuini et al. 2017). Liu et al. (2011) examined the effect of different zinc supplementation levels on carcass characteristics and meat quality in broiler chickens and reported that various levels of zinc increased breast weight. Sahin et al. (2005) stated that dietary supplementation with zinc sulphate could increase carcass weight in Japanese quail. MyoD expression may increase in the breast tissue as in the embryonic tissue and possibly, zinc has a direct effect on the expression of this gene. Liu et al. (2011) stated that addition of zinc in the diet reduced breast and leg fats of broiler chickens, which is not in agreement with the results of this research. In the present study adding Nano-ZnO to the diet did not affect (P>0.05) body weight and feed convert ratio (FCR), which may be related to the amount or the form of Nano-ZnO injection, which is consistent with previous studies (Sunder et al. 2008). Zinc deficiency in poultry causes weight loss, therefore, dietary supplementation with zinc often causes a positive response in the performance of broiler chicks (Blamberg et al. 1960). Łukasiewicz et al. (2020) reported an absence of effects on dressing percentage and the carcass content of breast muscle and giblets at 42 days. Several reports show enhanced growth performance (Fathi et al. 2016; Sahoo et al. 2016; Hassan, 2018), but others show no effects of Zn nanoparticles on BW (Asheer, 2017; Bami et al. 2018). After absorption in the intestine, nanoparticles can enter the bloodstream and be stored in various organs and be distributed independently of the blood circulation (Anjum et al. 2016). Badawi et al. (2017) also indicated that the chickens supplemented with NanoZnO had a higher body gain and better feed conversion rate (FCR) than birds without Zn supplement.

 

CONCLUSION

As a conclusion, our findings showed that although in ovo injection of Nano-ZnO reduced hatchability at first glance, this was the result of injection effect, because it did not reduce the hatchability and chickens’ weight compared to positive control. In ovo injection of 20 µl of Nano-ZnO increased embryo mortality, and improved humoral and cellular immune responses and weight of the spleen, which indicates the positive effects of injections and confirms the hypothesis of the study.

 

ACKNOWLEDGEMENT

The authors acknowledge the managers and staff of Ghatre Tala parent stock farm and also Ghatre Tala Agricultural and Animal Husbandry Company for providing bolus supplements.

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