The Impact of Varying the Amount of Octacosanol Added to Laying Hens’ Productive Performance and Various Oxidative Tests with and Without Vitamin E

A total of 120 laying hens were employed in this investigation, with 20 hens per treatment and 10 repetitions (2 hens each replicate). The experimental treatments included the following: T1: Control group, which received a basil diet without the addition; T2: 20 milligrams of vitamin E per kilogram of feed; T3: 20 milligrams of octacosanol per kilogram of feed; T4: 40 milligrams of octacosanol per kilogram of feed; T5: 20 milligrams/Kg (mixed octacosanol + vitamin E). The findings indicate that the treatments including the addition of octacosanol significantly increased the percentage of eggs produced, improved feed conversion ratio, and increased the eggs mass. Compared to the first, second, and third treatments, the MDA concentration significantly decreasing during the fourth, fifth, and sixth treatments. There were no significantly variations in glutathione levels. The concentration of the GOT enzyme also decreased significantly across all treatments as compared to the control group. When compared to the control treatment, the third treatment significantly improved the concentration of the GPT enzyme. Estrogen levels were not significantly affected by any of the treatments.


Introduction
Global egg production reached 86 million metric tons by the start of the first two decades of the present century, an increase of nearly 26% over what was produced in the first decade, with rising egg consumption demand [1].Its production mostly depends on flocks of chickens that lay table eggs, with an average annual production rate of about 300 eggs [2].It was crucial to focus on genetic improvement initiatives, as well as feeding and management methods, in order to reach these high levels of productivity.The length of the peak production period, the appearance of various metabolic illnesses, and the possibility that these birds would be exposed to diseases were all negatively impacted by these procedures despite their usefulness as tools to improve production [3].According to several studies, the age of laying hens is one of the elements that determines output, and the decline in it is primarily caused by the reduction in the number of follicles from the ovary that have fully formed [4], which is attributed in turn to the decrease in the ovulation rate that is related to the sensitivity of the hypothalamus to positive feedback towards the hormone progesterone [5]  ).Indicators of oxidative stress such MDA, GSHPX, and CAT, as well as inflammation, significantly increased in older laying hens.The expression of antioxidant genes significantly decreased in the liver tissue of the old chicken in comparison to the young, which is associated with an increase in the production of active oxygen species and hydrogen peroxide that have a direct effect on the processes of fat oxidation and an increase in enzymes necessary for the pathways of cholesterol production and its absorption in liver cells [6].With 28 carbon atoms, octacosanol is a long-chain aliphatic alcohol.It can be found in some animal sources, wheat germ oil, rice germ oil, and the outer layer of plants.Octacosanol demonstrated a range of biological activity in trials, including antistress effects [7], antioxidant activities [8], cholesterollowering [9], and cytoprotective [10].In addition to a cycle in energy production pathways [11].It was found that this substance has a positive role in regulating levels of sex hormones and increasing egg production in laying hens by regulating the gene expression mechanisms of hormones responsible for egg production [12].Based on these findings, the concept for this study came from adding octacosinol to the diets of advanced laying hens along with vitamin E, a substance whose effect has been demonstrated in cases of oxidation, and demonstrating its impact on productive performance and some physiological measures over two productive periods.

Materials and Methods
This study was conducted in the poultry bird farm of the Animal Production Department/ Faculty of Agriculture -Tikrit University and the study was approved by the Ethical Committee [Approval letter No. UoM.Dent/A.L.66/21].No. Um.VET.2021.5.A total of 120 Hy-line Brown laying hens at the age of 72 weeks in the second production year were used in this experiment, raised in hierarchical cages 60 cages made of iron, and distributed randomly in the six experience transactions and by two hens For each cage, which is considered as one repeated and of 10 bis per treatment, and the drinking water system is represented in a niple type (nipple) in each cage, distributing feed within the longitudinal feedback and the number of lighting hours was 16 light and 8 darkness according to the education guide.

Productive Performance
The daily calculation of egg production was based on the number of hen days.Egg mass was calculated based on grams of egg/hen/day and daily egg weight records.The feed conversion ratio, which measures the amount of feed consumed per mass-produced egg.By deducting the residual feed, which was collected at the end of the month, from the total feed provided, the feed intake was calculated according to method [13].

Oxidation Parameters
Three hens were randomly chosen from each treatment at the conclusion of the two experiment periods, and blood samples were taken from the brachial vein using a 5-ml syringe into nonheparinized tubes and centrifuged at 3500 g for 20 min to extract sera.Individual serum samples were analyzed for the enzymatic concentration (GOT and GPT) was estimated according to the work method attached from the AGAPPE manufactured for several examination and the use of the Apel Spectrum Measurement device (PD-303).Thiobarbituric acid method (TBA) developed by the Guidet and Shah [14] was used to estimate the concentration of (MDA) Malondialdehyde.Fat peroxides, especially Manoldaldhyde with TBA.The concentration of Glotathion in the blood serum was measured using the Al -Hassani method [15] using Elleman's detector .The concentration of estrogen in the blood serum was measured using several ready -made analysis (Kit) manufactured by the Chinese company (Guanghou Wondfo Biotech).

Statistical Analysis
Data were analyzed using GLM procedure [16].Significant differences among treatments were determined using Duncan's multiple range test.

Productive Performance
Through the results of table (1), we note the existence of significant increase (P ≤ 0.05) in egg production in treatments with octacosanol alone or with vitamin E compared to the control treatment.With a significant increase (P ≤ 0.05) in the efficiency of food conversion in these treatments compared to the first treatment.With regard to the rate of egg weight, the treatments had no significant effect, all treatments of adding octacosanol and vitamin E were recorded individually or together did not have any significant effect (P ≤ 0.05) In Egg mass compared to the control group.• abc ... different letters indicate that there are significant differences T1: control treatment T2: 20 mg vitamin E / kg feed T3: 20 mg octacosanol / kg feed T4: 40 mg octacosanol / kg feed T5: 20 mg octacosanol / kg feed+ 20 mg vitamin E / kg feed T6: 40 mg octacosanol / kg feed + 40 mg vitamin E / kg feed.
There is a group of common factors in egg production operations, including what is hormonal through an infrastructure hypothalamus-pituitary -ovaries, including what is related to the work of the digestive system and its ability to provide nutrients.It was noted that the hormones is negatively affected by aging in hens.Aging causes a decrease in estrogen and progesterone receptors in the hypothalamus and pituitary [17] also changes in the nature of the microbial and tissue composition of the older hens [18] as well as a decrease in the activity of Hydroxylase -α1 and the effective form of vitamin D (1.25 DHCC) All of these changes are associated with increasing the production of effective oxygen varieties and free radicals [19,20].Long and others [12] noted that treating laying hens at the age of 67 weeks using different levels of octacosanol improved the production of eggs, which resulted in a significant increase in Mature and well-developed follicles, which mainly depends on what is produced from the hormone FSH [21] The hormone LH which is responsible for completing the development of these follicles and its ovulation and that this rise in the level of these hormones has resulted in the increase in the genetic expression of the FSH and LH in the ovary cells.On the other hand, we note from the results of our current study that the treatments caused a significant improvement in the efficiency of food conversion, and this is consistent with the results of Long and others [12], and this may indicate an improvement in the efficiency of digestion and absorption of nutrients in the intestine by improving the microscopic environment or by improving the environment of the Intestine histological structure [22].Perhaps this is related to the improvement observed in oxidative stress indicators in the chicken groups treated with octacosanol and vitamin E in our current study.

Biochemical Properties of Blood
From the results of Table (2), we note that there was a significant decrease in MDA levels for the fourth, fifth, and sixth treatments with the control treatment compared to the second, third, and third treatments.The addition and combination of octacosanol and vitamin E between treatments had no significant effect on serum glutathione levels compared to the control treatment.From the results of Table (2), we note that there was no significant difference in the serum estrogen concentration of Hy-line brown laying hens treated with different levels of octacosanol and vitamin E and their combinations.The lack of significant differences in estrogen concentration may be due to the relative stability in Glutathione levels, which gives a preliminary indication of the stability of redox states [23].
Table 2. Effect of adding octacosanol and vitamin E and their mixtures on the levels of Malondialdehyde, Glutathione and Estrogen hormones in serum of Hy-line brown laying hens.• abc ... different letters indicate that there are significant differences T1: control treatment T2: 20 mg vitamin E / kg feed T3: 20 mg octacosanol / kg feed T4: 40 mg octacosanol / kg feed T5: 20 mg octacosanol / kg feed+ 20 mg vitamin E / kg feed T6: 40 mg octacosanol / kg feed + 40 mg vitamin E / kg feed.

Concentration of GOT and GPT Enzymes in Blood Serum
The results in table (3) showed a significant decrease in the GPT enzyme concentration in blood serum for all treatments to which octacosanol and vitamin E and their mixtures were added, compared with the control treatment.And supplementation of vitamin E and octacosanol alone at 20 mg/kg feed concentration (third treatment, with and without vitamin E (T5 and T6), fourth treatment (octacosanol 20 mg/kg feed with vitamin E) .And there was a significant decrease in GOT in the blood serum in the third treatment compared to the first, fourth and sixth treatment (table 3).The rest of the treatments did not register significant differences compared to the control treatment.
Table 3.Effect of adding octacosanol and vitamin E and their mixture on GPT and GOT enzyme levels in the serum of Hy-line brown laying hens.Treatment with octacosanol led to a significant decrease in the level of some oxidation markers such as MDA and GOT enzyme in blood serum.This improvement may be due to the role of octacosanol in stimulating the activity of some antioxidant enzymes.Lee et al [20] noticed a significant improvement in the activity of SOD and GSH-PX enzymes.The first of these enzymes works to inhibit the production of free oxygen radicals [24], while the second works to convert them.Glutathione from the reduced form to the oxidized form to limit the production of active oxygen species and free radicals.In addition, increased production of free radicals is often associated with damage to cell membranes through the oxidation of fatty acids in them [25].Taha [23] found a positive, significant correlation coefficient between levels of fatty acidity indicators.and GOT levels, namely octacosanol, is a protector from free radical damage to cell membranes, as evidenced by the reduction in GOT level.

Conclusion
In this study, it can be concluded that supplementing diet of laying hens with with octacosanol alone or with vitamin E led to a significant improvement in Egg production in aged laying hens, and the addition of octacosanol improved the redox and this improvement increased in the presence of vitamin E.
120 hens were used, randomly distributed on 6 experimental transactions, by 20 chickens per treatment that included 10 bis (2 hens/bis), and the experiment transactions were as follows:  The first treatment T1: Treatment of control without addition  The second treatment T2: Adding 20 mg vitamin E / kg feed. The third treatment T3: Adding 20 mg Octacosanol/ kg feed. Fourth treatment T4: Adding 40 mg Octacosanol / kg feed+ 20 mg vitamin E / kg feed  Fifth treatment T5: Adding 20 mg Octacosanol / kg feed + 20 mg vitamin E / kg feed  The sixth treatment T6: Adding 40 mg Octacosanol / kg feed + 20 mg vitamin E / kg feed 2.2.Studied Qualities (Williams and Sharp,  1259 (2023)012068 2 1978

Table 1 .
The effect of adding octacosanol, vitamin E, and their mixture on productive performance of Hy-line brown hens.