Effect of habituation and gene silencing on blood biochemical traits in local chicken indicative of their level of welfare

doi:10.21203/rs.3.rs-3840714/v1

Chickens respond to the signals of other chickens, and as the signals are repeated, the chicken's reactions adapt over time. In studies of chickens responding to repeated playbacks of recorded chicken signals, the word "habituation" has been used to describe both overt behaviours and behavioural responses during specific tests in both field and laboratory settings. Gene silencing, on the other hand, is the regulation of gene expression in a cell that inhibits a particular gene from expressing. The current study aimed to reduce the stress responses of a breed of local chickens by changing their behavioural or genetic factors, and assessed blood traits that reflect their level of welfare. The study used 120 hen chickens obtained at 22 weeks old, divided into six treatments, with four replicates for each treatment (20 chicks per treatment, 5 chicks per replicate) as follows: T1 was a negative control treatment consisting of a local Iraqi chicken breed; T2 was a positive control treatment consisting of a commercial breed of chickens; T3 involved the local breed with siRNA used to silence the corticosterone hormone pathway to the CYP11B2 and CYP11B1 genes by injection in the jugular vein at the age of 24 and 40 weeks; T4 consisted of mixed groups with 25% commercial chicken + 75% local chicks; whilst T5 consisted of 50% commercial chicken + 50% local chicken; and T6 consisted of 75% commercial chicken + 25% local chicken. The results showed a significant improvement (P≤0.05) in Glucose, Cholesterol, Total Protein, LDL, HDL, Globulin and Albumin for T3 and T5 compared with the other treatments; and a significant improvement (P≤0.05) in blood corticosterone in T3 immediately after the injection and two weeks later. The results show that there is an effect of habituation and gene silencing on blood traits, indicating an improved level of welfare for the T3 and T5 chickens.

Habituation

Gene Silencing

SiRNA

CYP11B2 and CYP11B1

Blood Parameters

Chicken Welfare

Iraqi Local Chicken.

Since the 1970s, the development and genetic improvement of Iraqi local chickens began with the aim of obtaining lines with high production efficiency, resulting in six genetic lines distinguished by their production and high adaptation to the Iraqi environment. (Ali et al., 2019).However, the occupation of Baghdad in 2003 resulted in the complete loss of these lines and has prompted a return to selecting chickens close to those lines with important production specifications, and other good qualities, to improve the production of those lines now available (Taha & Hasan Aljumaily, 2021). The main problem with the local chicken breeds is their lack of egg production due to their fierce nature (Abdul-Lateif & Abdulateef, 2012), which increases the secretion of stress hormones that affect their reproductive hormones. These local chicken breeds are also distinguished by their ease of rearing, tolerance to hot and cold environmental conditions and resistance to diseases, whilst one breed can also resist stress (Abu-Rekaiba et al., 2021).

The aim of the current study was to find new methods that modify undesirable traits in a breed of local chickens to make them calmer; less active; have less social contact with other chickens; show less aggression towards would-be predators; be less stressed; and less prone to seek out food sources, than their wild counterparts (Farhan Musleh Al-Enzy & Tarek Mohammed, 2015). One of these methods is the use of different rearing methods, for example, the use of habituation and utilization of mixed groups with domestic chickens so that local chickens gain some of their good behavioral traits. Another method is the use of gene silencing to reduce the activation of corticosterone, thus reducing fear and stress responses in local chickens (Shihab & Nafea, 2022). Studies have also shown that Iraqi chicken has the genetic potential to be used as commercial laying hens as they carry the same chromosomal dependencies as those available in existing commercial laying chicken strains that are responsible egg production(F. H. Esaa & J. K. Kassim, 2021). However, there is an imbalance in the sex hormones responsible for regulating egg production in the birds, resulting in a defect in laying eggs (El-Labban et al., 2011).

Thus, this imbalance in the sex hormones is likely to occur due to the current fear and stress responses in Iraqi local chickens (Al-Maksousi et al., 2019). Therefore, there is a need to find novel solutions to mitigate the stress response in local chickens. The critical period when the chickens hatch can be used to change and modify their behaviour, which is called imprinting (Aoki et al., 2015), as well as gene silencing or SiRNA techniques to inhibit the expression of genes that control the corticosterone hormone in the cell(F. I. Al-Bazy et al., 2022); meaning the stress hormone response decreases (H. S. Chang et al., 2004). The objective of this study was to investigate the effect of habituation and gene silencing on blood biochemical traits and corticosterone levels in local chickens, with the aim of increasing the welfare of the local chickens and thus increasing their egg production capabilities.

2.1. Ethical Approval

The study was conducted in accordance with the protocol authorized by the University of Anbar, Ethics Committee, Iraq. The Agricultural Research Directorate/Ministry of Agriculture/Iraq provided local, day-old laying chickens.

2.2. Study Period and Location

This study was conducted in the fields of the College of Agriculture University of Anbar, Department of Animal Production, for the period from 15/08/2022 to 28/02/2023. This study is complementary to a previous study, in which siRNA was also used and the local chicks were mixed with broiler chicks at one-day-old.

2.3. Experimental Study

The study used 120-hen chickens obtained at 22 weeks old. The chicks were divided into six treatments with 4 replicates for each treatment (20 chicks per treatment, 5 chicks per replicate) as follows: T1 – a negative control treatment consisting of local chicken; T2 – a positive control treatment consisting of commercial chicken; T3 – siRNA was used for silencing of the corticosterone hormone pathway to the CYP11B2 and CYP11B1 genes by injection in the jugular vein at the ages of 24 and 40 weeks; T4 – consisted of 25% commercial chicken + 75% local chicks; T5 – consisted of 50% commercial chicken + 50% local chicken; and T6 – consisted of 75% commercial chicken + 25% local chicken.

2.3.1. Chickens were obtained as follows:

The commercial chickens from ISA Brown were obtained from an Iraqi poultry production company, whilst the Iraqi wild-type chickens were obtained from local breeders.

2.3.2. The study took place

This study was conducted at the Department of Animal Production College of the Agriculture University of Anbar, Iraq, with data collection occurring for the period from 15/08/2022 to 28/02/2023.

2.3.3. Housing System Set-Up

housing close system

2.3.4. Stocking Density

20 chicks per treatment, 5 chicks per replicate, each replicate was kept in a 2.5 × 3 m area.

2.3.5. Feed Composition

The feed composition was according to the approved recommendation from the NRC.

2.3.5. Lighting Routine

The light regime was according to the approved recommendation from the ISA Brown index.

2.4. Habituation

Since the commercial layer hen (from ISA Brown) is relatively docile, it was chosen for training the wild-type, non-hybrid, local Iraqi type chickens.

For the experiment, 60 chicks (30 of each breed type) were housed in small groups with different proportions of each breed from immediately after hatching until the age of 22 weeks. This was to exploit the critical period when imprinting occurs, through which the chicks acquire behavioural characteristics from observing the other birds, according to Konrad Lorenz's theory. This process of imprinting is considered to be part of early learning, during the critical period, and is limited to the first few days after hatching, and is part of early learning to help birds remain stable (Tzschentke & Plagemann, 2006).

2.4.1. Procedure for habituation

Newly hatched wild local breed chicks were mixed with ISA Brown cross-bred commercial chickens in different ratios, to evaluate the extent to which the wild-type chickens acquired calm traits from the commercial chickens; or conversely that the commercial chickens acquired aggressive traits from the wild-type chickens. The proportion of each chicken type for each treatment groups were as follows:

T4: 25% commercial chicken + 75% local chicks

T5: 50% commercial chicken + 50% local chicks

T6: 75% commercial chicken + 25% local chicks

The focus was on the first days of hatching and taking advantage of the critical period, after which habituation procedures were carried out by excluding the ISA Brown commercial chicken birds for a week. Then they are returned to a recurring stimulus that repeats the habituation process, meaning that whenever the stimulus decreases, the stimulus is gone, as the motivation of trained chickens decreases, commercial chickens are reintroduced, which is a strong incentive for habituation is brought back again to complete habituation used to it stronger according to Pavlov's theory (Çevik, 2014). This condition continued until 21 weeks, after which the trained chickens were introduced to commercial chickens to begin the experiment at 22 weeks.

2.5. Small interfering RNA (siRNA)

Silencing of CYB11B1 and CYB11B2: The cDNA of each gene involved in the corticosterone manufacture pathway was obtained from NCBI. The coding sequence from the five ends at cap direction was revised to its opposite strand to be complementary for mRNA, then two siRNAs were delivered from Macrogene company (South Korea) in lyophilized form in high concentration with the following sequences:

CYP11B2f 5-acacctctgcctttgccctgagtgccat-3

CYP11B1r 5-ctaaagtcaaactcagccccacattcat-3

The siRNAs were dissolved in molecular-grade distilled water to get a final concentration of 500 pmol/ul . Each hen chicken in T3 of the study was injected into the jugular artery with 125 ul of the siRNA solution at 24 and 40 weeks of age. Procedures were according to the manufacturer's recommendations (Macrogene company; South Korea).

2.6. Blood Parameters

Blood samples were collected from three chickens from each replicate the from jugular vein at the age of 42 weeks and placed in heparinized (0.5 mL), EDTA (1 mL), and nonadditive (3 mL) blood collection tubes. After collection, blood samples were then placed in a tube containing Ethglene Diamine Tetra Acetic Acid Anticoagulant (EDTA). The remaining blood samples were placed in a centrifuge device at 3,000 rpm for 15 minutes for the purpose of plasma separation, then kept in the freezer at - 21°C to estimate the blood parameters of interest (Glucose, Cholesterol, Triglycerides, Total Protein, LDL, HDL, Globulin, Albumin and VLDL); as stated on testing kits purchased from Nanjing Jiancheng Institute of Bioengineering (Jiangsu, China) (SHEARER, 1979).

2.7 Hormone Concentration

After separating the serum from the cellular part of the blood, the plastic tubes containing the blood serum were transported, via a cool box, to the Al-Kak Specialized Scientific Laboratory, to conduct an ELISA test to measure the concentration of corticosterone in the blood serum using a testing kit. The ELISA test kit was produced by SunLong Biotech Co.,LTD, and was used according to the leaflet directions accompanying the kit (Rettenbacher et al., 2004).

2.8. Statistical analysis

This experiment was carried out using a Complete Randomized Design (CRD) and the data were analyzed by using the SAS program for statistical analysis (SAS, 2004). The means for each treatment were compared by using Duncan’s polynomial, using 0.05 and 0.01 significance levels to determine significant differences between the averages (Duncan, 1955).

Table 1. shows the effect of the treatment group on blood biochemical traits. Significant differences were recorded in glucose levels, with T3 and T5 showing significantly higher glucose levels (P≤0.05) than the other treatments. Also, T3, and T5 recorded significantly higher (P≤0.05) glucose levels than T6, whilst they did not differ significantly from T5.

The results obtained show that the level of glucose in T1, the control group of local wild-type chickens, was not within the normal range for local chickens, which is between (200-450 mg / 100 ml) of blood plasma. The explanation for this is that glucose is affected by the environmental, nutritional, and health status of the birds (Zhao et al., 2022). The reason for the rise in glucose levels above the normal range in T1 may be due to the stress the birds were exposed to, which works to release the hormone corticosterone from the adrenal cortex (A.R. Alkhateeb et al., 2022). This hormone works to supply glucose to the body from non-carbohydrate sources in a process known as gluconeogenesis, enabling the body to carry out its functions that require energy (Rengaraj et al., 2012).

In T3, glucose was recorded at a normal level. Although T3 involved the wild-type breed in normal stressful conditions, the reason for the normal blood glucose level could be the use of siRNA technology. This is believed because this technique inhibits the stress hormone corticosterone by inhibiting the manufacture of proteins in the cell. Thus, gene expression does not occur from the RNA molecules (Mishra et al., 2020) due to the destruction of the mRNA code carrier, with this process known to inhibit expression of the gene controlling production of the corticosterone hormone (Alkubaisy et al., 2021). When no gene expression occurs (A.J. A. Sahib et al., 2022), this will lead to a halt or a decrease in the level of corticosterone, and the bird will feel less stressed and more calm (A.J. A. Sahib et al., 2022). Corticosterone works to supply the body with glucose from non-carbohydrate sources as part of the stress response to face the emergency (Shakeer et al., 2020), which is the reason for the high level of glucose in the blood (Zhang et al., 2021).

For the level of total protein, T3 recorded a significantly higher level than all other treatment groups (P≤0.05). The reason for the high level of total protein in T3 may be attributed to inflammatory diseases, immune disorders (Bagno et al., 2021), or hepatitis. High total protein in the blood plasma could be the result of an increase in the concentration of immunoglobulins in the plasma (Abdulateef et al., 2021), which occurs as a result of pathological infection. The analysis of total protein in the blood compares the amount of albumin to globulin, whilst globulin constitutes 40% of the proteins in the blood, and albumin constitutes 60% of the total proteins (Onyenweaku et al., 2022).

For the level of globulin, T3 recorded a significantly higher level than all other treatment groups (P≤0.05). The reason for the high level of globulin in T3 may be attributed to inflammatory diseases (Tóthová et al., 2019), infection, or immune disorders, due to the important role of globulins in liver function, blood clotting, and fighting infection. Globulin helps to transport thyroxine and third iodide thyronine in the blood stream, which is important in increasing the amount of energy available in the body, as well as carrying out metabolism processes in the cells, the exchange of sugars and proteins, and stimulating blood circulation. Furthermore, globulin is the main carrier of the stress hormone (corticosterone) in the blood plasma to prepare the body in response to any stressful conditions that the bird is exposed to (Mousa, 2018).

Table 1.

Table 2. shows the effect of the habituation and gene silencing treatments on lipid profile. There were no statistically significant differences between the treatments in the concentration of triglycerides or cholesterol. The results in the current study differed from what was reached (Kamil et al., 2020) in a previous study comparing Iraqi local chicken breeds, where the results obtained were almost within the normal limits for cholesterol in blood plasma. Also, the current study differed from a previous study which indicated that high cholesterol and triglycerides are associated with liver disease (Omer et al., 2020). The results of that study differed from what was concluded (Khan et al., 2021) in a previous study, stating that there is an inverse relationship between the levels of lipoproteins (HDL and LDL), which is a natural result of reversing their functions. That study was consistent with what was found in the current study where high cholesterol levels are correlated with high levels of LDL (Vella, 1988).

For the level of VLDL concentration, T3 recorded a significantly lower level (P≤0.05) than T1, T2, T4, T5, and T6. The reason for the improvement for T3 may be attributed to very low-density lipoprotein (VLDL) levels in the body required for exporting triglycerides to the target sites, mainly adipose tissue or egg cells (W. H. Chang et al., 2019), which are required by the mature chicken to complete the process of producing eggs as it is used to form yolk fats. VLDL is derived from the liver when the bird is exposed to stress, where stress works to break down the fat stores in the body to produce the energy that the bird needs to face the stress, and thus the level of triglycerides in the blood rises.

Table 2.

Figure 2 shows the effect of using SiRNA technology on the level of blood corticosterone. There was a significant decrease (P≤0.01) in the level of corticosterone after the first injection at 24 weeks of age, with the quantity of corticosterone decreasing from 2.5 ng/100 ml to 0.210 ng/100 ml of blood. At the age of 40 weeks, it was observed that there was a significant decrease (P≤00.01) in corticosterone after the second injection, from 0.912 ng/100 ml to 0.236 ng/100 ml blood.

Figure 1.

The reason for the significant decrease in the concentration of corticosterone may be attributed to the use of SiRNA technology, which inhibits gene expression in RNA molecules due to the destruction of specific molecules in the mRNA vector, preventing its translation. Any gene can be targeted by SiRNA since only the correct nucleotide sequence along the target mRNA needs to be identified (Janas et al., 2019). SiRNA is processed from double-stranded RNA. This double-stranded RNA is cleaved by Dicer-dependent ribonuclease, into 21-23 nucleotide long fragments. This SiRNA is then loaded onto another protein, called Argonaute. Argonaute contains four different domains (N-terminal, PAZ, Mid, and PIWI). Its PIWI domain contains RNase activity that enables Argonaute to cleave the target mRNA. The Argonaute-SiRNA complex then binds to the helix and other proteins to form an RNA inhibition complex called RISC to inhibit future mRNA (Kim et al., 2019).

A double-stranded RNA molecule, called small interfering RNA (siRNA), can be utilized to silence particular genes. It is an effective method for researching gene function and creating novel treatments. In birds. the adrenal glands emit the glucocorticoid hormone corticosterone in reaction to stress (H. S. Chang et al., 2004). It affects a number of physiological systems, such as immunological function, inflammation, and metabolism. Studies have demonstrated that siRNA may be used to mute the genes that control and produce corticosterone in chickens. As an illustration, one study found that silencing the gene CYP11B1, which codes for the enzyme that transforms cholesterol into corticosterone, significantly reduced the amount of corticosterone in chickens(Gong et al., 2019). In another study, siRNA silencing of the glucocorticoid receptor gene NR3C1 decreased the sensitivity of hens to corticosterone (H. S. Chang et al., 2004). According to these studies, siRNA may be utilized to create novel treatments for chicken stress-related diseases like feather picking and cannibalism. SiRNA may also be utilized to lower corticosterone levels in birds reared for meat or eggs. This might enhance the well-being of the birds and result in meat and eggs that are less laden with stress hormones (Gong et al., 2019).

In conclusion, the habituation and gene silencing treatments resulted in an improvement in the biochemical traits of blood glucose, total protein and globulin, as well as an improvement in the lipid profile traits for HDL, LDL, and VLDL, plus decreased corticosterone levels. Therefore, these treatments are expected to improve the level of welfare and production of the chickens.

Supplementary Materials:

No Supplementary Materials.

Author Contributions:

S. M. Abdulateef and O. K. Mahmood; methodology, writing—original draft preparation, H. M. Alnori; writing—review and editing, S. M. Abdulateef and A. Tatar; paraphrasing. All authors have read and agreed to the published version of the manuscript.

Funding:

This research received no external funding.

Institutional Review Board Statement:

The study was conducted in accordance with the protocol authorized by the University of Anbar, Ethics Committee, Iraq.

Informed Consent Statement:

No Informed Consent Statement.

Data Availability Statement:

No Data Availability Statement.

Conflicts of Interest:

The authors declare no conflict of interest.

Acknowledgments:

The authors are thankful for the help of the Animal Resources Field Manager, The College Dean, and the Head of the Animal Production Dept. in The College of Agriculture, University of Anbar, Iraq. We would also like to thank the undergraduate students for their valuable help and technical assistance in conducting this research.

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Table 1. The effect of habituation and gene silencing on the biochemical blood traits.

Treatments	Glucose mg/d	Total Protein g/dl	Albumin g/dl	Globulin g/dl
T1	c 118.4	8.21 c	a2.30	c 5.91
T2	147.2 c	9.01 bc	a2.29	c 6.71
T3	221.2 a	a 13.98	b1.04	a 11.69
T4	198.1 b	10.57 b	a1.83	b 9.53
T5	217.6 a	11.4 b	a1.47	b 9.57
T6	139.9 bc	10.91 b	a1.58	b 9.44
LSD	116.7 ^N.S	^N.S7.17	1.096^N.S	7.62 ^N.S

T1 was a negative control treatment consisting of local chicken; T2 was a positive control treatment consisting of commercial chicken; T3 used siRNA for silencing of the corticosterone hormone pathway to the CYP11B2 and CYP11B1 genes), injected in the jugular vein at the age of 24 and 40 weeks; T4 consisted of 25% commercial chicken + 75% local chicks; T5 consisted of 50% commercial chicken + 50% local chicken; and T6 consisted of 75% commercial chicken + 25% local chicks.

* Different lowercase letters within a column indicate significant differences at the level of probability (P <0.05) Average ± standard error.

Table 2. The effect of habituation and gene silencing on the lipid profile traits of blood.

Treatments	Triglycerides mg/dl	Cholesterol mg/dl	HDL mg/dl	LDL mg/dl	VLDL mg/dl
T1	216	286	a 1..8	a 223	a 69.2
T2	247	290	a 79.5	a 212	a 58
T3	253	229	c 46.5	c 102	c 38.4
T4	231	292	b 72.4	b 194	b 42.4
T5	245	298	b 71.9	b 195	b 39.6
T6	221	298	b 72.7	b 197	bc 45.6
LSD	351^N.S	201.5^N.S	37.22	163.38	66.23

T1 was a negative control treatment consisting of local chicken; T2 was a positive control treatment consisting of commercial chicken; T3 used siRNA for silencing of the corticosterone hormone pathway to the CYP11B2 and CYP11B1 genes), injected in the jugular vein at the age of 24 and 40 weeks; T4 consisted of 25% commercial chicken + 75% local chicks; T5 consisted of 50% commercial chicken + 50% local chicken; and T6 consisted of 75% commercial chicken + 25% local chicks.

* Different lowercase letters within a column indicate significant differences at the level of probability (P <0.05) Average ± standard error.

No competing interests reported.

Effect of habituation and gene silencing on blood biochemical traits in local chicken indicative of their level of welfare

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Abstract

Figures

1. Introduction

2. Materials and Methods

3. Results and Discussion

4. Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1