Effect of various hesperidin and naringin addition levels on specific sperm indicators and the integrity of their DNA as measured by the comet assay in aged broiler breeder males

In broiler breeder males, the generation of semen decreases with age, and the body produces more free radicals when aging symptoms start to appear. Certain citrus wastes are a potentially sustainable source of high-biological value compounds with antioxidant properties that can enhance the performance of the male reproductive system. Hesperidin and naringin are two examples of these substances. Each treatment was given to the roosters separately, and their impacts on DNA integrity and semen parameters were later investigated using the comet assay. in contrast to the treatment used as the negative control. Most of the sperm characteristics (ejaculate volume, overall sperm motility, progressive sperm motility, sperm concentration, percentage of dead sperm, and percentage of abnormal sperm) were significantly and meaningfully improved by the hesperidin and naringin treatment. The percentage of genetic material that remains intact increased in the comet assay in favor of the treatments that also contained naringin and hesperidin. Finally, this study showed that hesperidin and naringin are sustainable substances led to improved semen qualities of elderly broiler breeder males and significantly increased the proportion of genetic material that was intact.


Introduction
One of the biggest issues impeding the production of broiler chickens is the issue of decreased fertility in broiler breeder flocks [1].With 342 million tons of meat produced worldwide, including 119.7 million tons of chicken, broiler chickens are the main source of poultry meat [2].A gap in food security results from the inability to supply enough poultry to satisfy the world's demand.In fertility is caused by a number of reasons, but all research [3,4] have stressed how important males are in this regard.Male broiler breeders age prematurely, which frequently causes them to gain weight and produce fewer sperm or sperm that are infertile.To address this problem, research has changed during the last 20 years.In order to address this situation, research has changed.Genetics have been the focus of some investigations [5].Whereas others have looked at food patterns [6].The inclusion of certain vitamins, minerals, and nanoparticles has also become popular [7,8].Around the world, citrus fruits are widely grown and consumed.Large volumes of waste are produced as a result of the industrial processes that employ 18% of all citrus cultivars.Waste citrus could be a source of compounds with high biological value, [9] The utilization of medicinal plants and their extracts has also been studied [10].Utilizing numerous active chemicals from plants, fruits, and leaves has become more popular recently.Hesperidin, which is one of the biologically active flavonoid molecules, is one of these substances.Citrus fruits like oranges and lemons contain it [11].Hesperidin, according to [12], possesses antioxidant properties, inhibits ROS (reactive oxygen species), preserves cell membranes, protects their vital functions, and increases the activity of several antioxidant enzymes, such as GSH (glutathione), CAT (catalase), and SOD (superoxide dismutase).It has been proposed that hesperidin's antioxidant activity functions similarly to vitamin E in defending cells from free radicals [13].Due to its antioxidant qualities, it has been shown to be beneficial against viruses, inflammation, allergies, some types of cancer cells, neurological and cardiovascular illnesses, and it has been shown to strengthen the immune system in the body [14].Some of its effects, such as lowering cholesterol and glucose levels, have been documented in studies [15].The citrus fruit known as grapefruit is a rich source of naringin, a flavonoid molecule that is a member of the flavanones subclass [16].It has a wide range of pharmacological and therapeutic qualities, including those that lower fat and cholesterol, have anti-inflammatory effects, act as an antioxidant to scavenge free radicals, reduce MDA levels, have anti-aging properties, and have anti-cancer activities [17].Thus, this study aimed to assess the extent of genetic damage and investigate the effects of naringin and hesperidin on the physiological markers and semen characteristics of broiler breeder roosters.The volume of seminal plasma, which is rich in nutrients, decreases in bird spermatozoa.This decrease has an impact on their capacity to be preserved in frozen and refrigerated environments [18].The majority of seminal fluid properties deteriorated along with the storage of avian spermatozoa at refrigerator temperature for 72 hours, according to several studies.Hesperidin and naringin have been added to semen extenders for bulls and rams, and this practice has been found to be effective [19,20].In order to better understand how hesperidin and naringin affect the qualities of semen as well as the degree of genetic damage in broiler breeder rooster sperm, this study was created.

Research Framework
Thirty Ross-308 broiler breeder males who were fifty weeks old were utilized in this study.The ethical committee of animal research of the department of animal production permitted all experimental protocols Tikrit University (No. TU3.7.139).These roosters were available for purchase from the Taktak Poultry Company.The birds were introduced to their environment and their food over the course of ten days of preparation.During this time, the roosters received instruction in semen collection methods.Five roosters per treatment were randomly assigned to one of the six treatments, each of which had five replications (one rooster per duplicate).The following were the treatment options: 1. T1: Control Treatment: Feeding without any additives 2. T2: Positive Control Treatment: Supplementing feed with 20 IU Vitamin E /(Kg of feed) 3. T3: Hesperidin addition of 250 mg per kilogram of feed.4. T4: Hesperidin addition of 500mg per kilogram of feed. 5. T5: Naringin addition of 250 mg per kilogram of feed.6. T6: Naringin addition of 500 mg per kilogram of feed.

Management of birds and their nutrition
Individual birds were raised in galvanized iron floor cages that were 85 cm x 85 cm x 80 cm in size.Each cage contains a 3 liter inverted drinker, a hanging feeder, and a 5 cm thick layer of wood shavings serving as the lining.The building's LED lights all had a 40 watt power rating and were distributed evenly at a height of 2 meters.Mercury and electrical instruments were used to measure the temperature and humidity within the building.Taking the Ross308 (2020) company guide's recommendations, the birds' diet contained 2700 kcal of energy per kilogram of feed and an 11.5% crude protein content.Water was available to the birds at all times during the experiment.The concentrations of vitamin E, hesperidin, and naringin in the meal increased after the birds' periods of adaptation and training.Over a period of two weeks, with a one-week interval in between, three collections of semen were made each week.There were operations for two months.

The semen collection process
It was run three times a week for two weeks in a row.Every day at 10:00 AM, this procedure was carried out while taking into account the need to deprive the birds of food and water for at least six hours before collecting.This was done to ensure that a clean, free of contamination semen sample was obtained.The procedure described by [21] was used to collect rooster semen.

Characteristics of the studied semen
A delicate electronic balance was used to measure ejaculation volume the [22].Approach was used to determine the sperm's total motility.Using the grading system offered by [22] A drop of semen was placed on a glass slide to measure the motility of individual sperm.This examination focused particularly on the progressive forward movement of sperm.Using a mixture of Eosin-Nigrosine stain that had been made beforehand and according to the directions provided by [23] the percentage of dead and aberrant sperm was calculated.On the slide, at least 200 sperm were counted in various locations using an x1000 magnification.[24] described a method for measuring packed sperm volume (PCV%), and [25] suggested a method for determining sperm concentration.

An assessment of comet assay for sperm
Is carried out in order to measure the degree of genetic damage in the sperm of broiler breeder roosters.Each rooster's fresh semen samples receive individualized treatment in accordance with [26] method.

The statistical analysis
Was following the collection and analysis of the study's data with version 9 of the SAS statistical analysis program.The data that had been collected was analyzed using a completely random design.[27] The significance of differences in the means of the qualities under investigation was evaluated using a significance level of P≤0.05.Similar letters in a column.mean that, at the probability level (p≤0.05),no significant differences exist.T1= negative control without addition, T2= positive control (IU20 E), T3= adding (250 mg hesperidin /kg feed), T4= adding (500 mg hesperidin /kg feed), T5= adding (250 mg naringin / kg of feed), T6= adding (500 mg naringin /kg feed).

Result and Discussion
According to Table 1's findings, compared to all other addition treatments and both positive and negative control treatments, the addition of hesperidin and naringin to the ejaculate volume of ROSS 308 broiler breeder males significantly outweighed the effect of the high-concentration hesperidin treatment.Except for the positive control treatment.Compared to the negative control treatment, the ejaculate volume was significantly increased by treatments T3, T5, and T6.As compared to the negative control treatment and low dosage treatments (250 mg/kg feed), the table (1) shows a significant improvement in mass and individual sperm motility in T4 and T6 treatments when hesperidin and naringin were introduced at a high concentration (500 mg/kg feed).There were no discernible differences between these treatments.When compared to T2 (IU20 E), Sperm motility significantly decreased in the treatment group.Moreover, treatments T3 and T5, which included the addition of hesperidin and naringin at low concentrations (250 mg/kg feed), considerably increased sperm motility in comparison to the negative control treatment.We find that the fourth treatment (Hesperidin 500mg/kg feed) significantly outperforms the control treatment, the other treatments that added naringin and low concentrations of Hesperidin (250mg/kg feed), in terms of sperm PVC% and sperm concentration.However, there was no discernible distinction between the treatment and the positive control treatment (IU20 E).We further note that the third and sixth treatments exhibited a definite superiority over the control treatment.Furthermore, when compared to the negative control and the treatments of sperm concentration, there was no discernible difference between the treatments of adding naringin and low concentrations of Hesperidin (250 mg/kg feed) and the positive control treatment with vitamin (IU20 E) and the T4 (Hesperidin 500 mg/kg feed).Additionally, there was a noticeable benefit to the third, fifth, and sixth treatments over the control treatment.All Hesperidin and Naringin treatment groups significantly reduced the percentages of aberrant and dead sperm in broiler breeder roosters when compared to the negative control group.The T4 and T6 treatments, which had higher concentrations of hesperidin and naringin than the T3 and T5 treatments, had a significantly lower percentage of dead and abnormal sperm.However, there were no discernible improvements when compared to the positive control treatment.Moreover, no appreciable difference was observed between the T3 and T5 treatments.When contrasted with the positive control treatment, they showed a significantly lower percentage of dead sperm.The impact of varying hesperidin and naringin concentrations on % DNA damage by comet assay: Similar letters in a column mean that, at the probability level (p≤0.05),no significant differences exist.T1= negative control without addition, T2= positive control (IU20 E), T3= adding (250 mg hesperidin /kg feed), T4= adding (500 mg hesperidin /kg feed), T5= adding (250 mg naringin / kg of feed), T6= adding (500 mg naringin /kg feed).Figure 1 shows the addition of different levels of hesperidin and naringin in the sperm of aged broiler breeder males in the Comet assay genetic damage test.Comparing the addition treatments to the control treatment (T1), an improvement in the genetic material's integrity ratio is seen.The comet assay also showed that treatment T4 greatly exceeded the other additional treatments in terms of the integrity of the genetic material in the head.However, the other addition treatments demonstrated a reduction in genetic material integrity in comparison to the positive control treatment, with the exception of the positive control treatment (Vit E), which did not differ from T4.The other addition treatments, in contrast to the positive control treatment (Vit E), revealed a loss of genetic material integrity.Vit E was the only treatment that did not differ from T4, though.Comparing all additive treatments to the negative control treatment, a considerable drop in the proportion of damaged material is seen in favor of all additive treatments.With a far lower percentage of genetic material that was destroyed than with the other additive treatments, the treatments T2 and T4 both produced the best results.The images illustrate the impact of varying hesperidin and naringin concentrations on the comet assay for DNA in the sperm of broiler breeder males.
Normal DNA is shown by the circle.Damaged DNA is represented by the rectangular shape.
T1= negative control without addition, T2= positive control (IU20 E), T3= adding (250 mg hesperidin /kg feed), T4= adding (500 mg hesperidin /kg feed), T5= adding (250 mg naringin / kg of feed), T6= adding (500 mg naringin /kg feed).By comparing all treatments involving hesperidin, naringin, and vitamin E to the negative control treatment, statistical analysis of the Comet assay reveals an increase in DNA integrity and a decrease in its damage.However, the T2 and T4 treatments showed the biggest effects in maintaining DNA integrity and reducing damage.Studies have shown that additives such hesperidin, naringin, and antioxidant vitamin E protect cells from the risk of ROS (reactive oxygen species), which are produced by metabolism and other variables.This impact can be linked to the additives' participation in this.Due to their antioxidant capabilities, flavonoid compounds have been shown to protect DNA from free radicals, stop DNA mutations, and support genetic stability [28].According to studies, hesperidin's antioxidant activity is due both to its capacity to strengthen cellular antioxidant defense mechanisms and to its ability to scavenge free radicals [29].Hesperidin has Fe2+ chelating properties and is effective in eliminating hydrogen and hydroxyl radicals as well as hydrogen peroxide when compared to other natural and synthetic antioxidants like tocopherol, ascorbic acid, butylated hydroxytoluene (BHT), and Trolox.Thus, hesperidin provides DNA with a stronger defense against genetic mutations and damage .[30].In the testes of male rats with diabetes mellitus31] discovered that the rate at which hesperidin was administered-200 mg/kg per day-was associated with a noteworthy drop in the amounts of DNA damage and 8-OHdG.Additionally, there was a significant increase in sperm motility and a significant decrease in the rate of abnormal and dead sperm.Naringin has been shown by [32] to be able to prevent the DNA damage that daunorubicin (Dau) causes in mouse liver and heart cells.
Naringin was added at rates of (50, 250, and 500 mg/kg feed), and it showed its ability to scavenge free radicals, decrease cholesterol, and affect the CYP3A4 and cyp1a4 enzymes.The outcomes of the genetic comet assay demonstrated the importance of naringin in maintaining the integrity of genetic material, showing, for liver and heart cells, respective values of 71.3% and 51.1%.

Conclusions
This study demonstrated the environmental friendliness of hesperidin and naringin, two sustainable supplements made from leftover citrus peels.enhanced the sperm qualities of older broiler breeder males and, according to the sperm DNA comet assay, resulted in a notable rise in the percentage of intact genetic material and a fall in the percentage of damaged genetic material

Figure 1 .
Figure 1.Different levels of hesperidin and naringin in the Comet assay genetic test conducted on the sperm of aging broiler breeder males.

Figure 2 .
Figure 2. The effect of adding different levels of hesperidin and naringin in the comet assay for DNA in the sperm of broiler breeder males.

Table 1 .
The impact of varying hesperidin and naringin concentrations on markers of semen quality in male broiler breeders.