Open Veterinary Journal, (2025), Vol. 15(7): 3080-3086

Research Article

10.5455/OVJ.2025.v15.i7.19

Green tea extract addition to egg yolk–milk diluent affects the quality and antioxidant content of Gaok chicken semen freezing

Tatik Hernawati^1*, Suherni Susilowati¹, Erma Safitri¹, Sri Mulyati¹, Tita Damayanti Lestari¹, Imam Mustofa¹, Nurhusien Yimer Degu², Ary Setya Hernanda³, Aswin Rafif Khairullah⁴, Aldin Akbar Rachmatullah³, Riza Zainuddin Ahmad⁴, Nashihah Nashihah⁵ Ulvi Fitri Handayani⁶, Adeyinka Oye Akintunde⁷ and Sotya Balqis Maharani⁵

¹Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

²Department of Veterinary Sciences, School of Medicine, IMU University, Bukit Jalil, Malaysia

³Master Program of Reproductive Biology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁴Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁵Profession Program of Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁶Research Center for Animal Husbandry, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁷Department of Agriculture and Industrial Technology, Babcock University, Ilishan Remo, Nigeria

*Corresponding Author: Tatik Hernawati. Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, East Java, Indonesia. Email: tatik-h [at] fkh.unair.ac.id

Submitted: 13/03/2025 Revised: 16/06/2025 Accepted: 18/06/2025 Published: 31/07/2025

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ABSTRACT

Background: Gaok chicken spermatozoa that is diluted and stored at freezing temperatures will reduce spermatozoa motility. The specific target to be achieved by this research is to increase the population by improving the quality of frozen semen with the addition of green tea extract (GTE) to egg yolk–milk diluent. Epigallocatechin-3-gallate contained in GTE acts as an antioxidant in the cooling and freezing process of chicken semen.

Aim: This study examined the addition of GTE to fructose egg yolk–milk diluent to improve chicken spermatozoa quality before and after freezing, with the goal of enhancing the efficacy of artificial insemination programs to increase the Gaok chicken population.

Methods: This study consisted of four groups: T0, the control group: fructose egg yolk–milk diluent without GTE + semen; TI: fructose egg yolk–milk diluent + GTE dose 0.05 mg/100 ml diluent + semen; T2: fructose egg yolk–milk diluent + GTE dose 0.10 mg/100 ml diluent + semen; and T3: fructose egg yolk–milk diluent + extract green tea dose 0.15 mg/100 ml diluent + semen.

Results: Analysis of variance (p < 0.05) showed no significant effects at T0, T1, and T3, indicating that T2, the treatment group supplemented with extract green tea (Camellia sinensis), produced the most notable results. This group is maintained better than another group on parameters sperm motility, viability, plasma membrane integrity, and minimized necrosis, alongside increased catalase enzyme and superoxide dismutase activity.

Conclusion: The addition of green tea leaf extract reduced the malondialdehyde level. The addition of GTE at a dose of 0.10 mg/100 ml of egg yolk–milk diluent can act as an antioxidant in maintaining the quality of post-thawing Gaok chicken spermatozoa.

Keywords: Antioxidant levels; Conservation; Freezing; Gao chicken; Semen quality.

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Introduction

The Gaok chicken, a native Indonesian breed originating from Puteran Island in Sumenep Madura Regency, East Java, is valued for its rapid growth and is commonly used for its meat and eggs. However, the population of Madura has been declining. Therefore, conservation based on community needs to be undertaken to prevent the extinction of the Gaok chicken. Artificial insemination is a promising method to boost the population, although its success depends on several factors, including the diluent level, semen storage time, and temperature (Santoso et al., 2021). Dilution is crucial for increasing semen volume while maintaining viability and fertilization capability, but chicken semen is notably vulnerable to free radicals (Zong et al., 2023). The quality of semen, particularly after cold storage (5°C) or freezing (−196°C), is a critical factor in artificial insemination (Diskin, 2018).

Cooling and freezing reduce sperm motility due to temperature shifts from the chicken’s natural 37°C, adversely affecting semen quality (Hamad et al., 2023). Post-thaw, semen often shows reduced motility and vitality due to structural damage to spermatozoa, leading to fertilization failure (Hughes and da Silva, 2022). This sensitivity is associated with membrane instability, which is worsened by the freezing process, which disrupts membrane function and structure, elevates osmolarity, and introduces cryoprotectant toxicity (Murray et al., 2024). Chickens have a high ratio of unsaturated fatty acids, making them especially susceptible to the formation of reactive oxygen species (ROS) and the resulting production of malondialdehyde (MDA). Antioxidants are essential for neutralizing free radicals and safeguarding spermatozoa (Chen et al., 2016).

Green tea extract (GTE), rich in epigallocatechin-3-gallate (EGCG), offers potent antioxidative activity, surpassing vitamins C and E (Prastiya et al., 2023), and has been shown to maintain sperm quality post-thawing in cattle (Susilowati et al., 2021). This study examined the addition of GTE to fructose egg yolk–milk diluent to improve chicken spermatozoa quality before and after freezing, with the goal of enhancing the efficacy of artificial insemination programs to increase the Gaok chicken population. This research posits that integrating GTE into the fructose egg yolk–milk diluent may significantly enhance semen quality both before and after freezing, offering a viable strategy for the long-term preservation and population recovery of the Gaok chicken.

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Materials and Methods

Animal experiment and semen collection

One Gaok chicken used in this study was 1–1.5 years old and weighed 2–4 kg. Gaok chickens are given food and drink ad libitum every day and then trained first to collect semen using the lumbar reflex method, which assists in inverting the copulatory papilla and then be gently massaged with the operator’s index finger and thumb while waiting for the reflex movement (Getachew, 2016). Semen samples were collected twice weekly.

Fresh semen evaluation

Fresh semen evaluation including volume, odor, consistency, pH, color, concentration, motility (mass and individual), and viability. The percentage of motility and viability must reach ≥70% to be continued for research.

Preparation of a fructose egg yolk–milk diluter

A total of 10 g of skim milk was added to 100 ml of distilled water and heated. The egg yolks are separated from the whites, added to milk at a ratio of 5% volume, and then added to 0.75% fructose, in addition to penicillin 1,000 IU and 1 mg/ml streptomycin diluent.

Treatment of diluents

The diluents were divided into four groups. Control group (T0): semen + egg yolk–milk + 0.75% fructose. Group II (T1): semen + egg yolk–milk + 0.75% fructose + GTE (0.05 mg/100 ml diluent). Group III (T2): semen + egg yolk–milk + 0.75% fructose + 0.10 mg GTE/100 ml of diluent. Group IV (T3): semen + egg yolk–milk + 0.75% fructose + 0.15 mg GTE/100 ml of diluent. Furthermore, freezing was performed with the stages of glycerolization, filling and sealing straws, prefreezing, and freezing (Susilowati et al., 2021). Before freezing, the quality of the spermatozoa was assessed.

Frozen semen

The semen was poured into diluters that had been divided into four treatment groups and then cooled at 5°C for 1 hour before the freezing process. The samples were mixed with N,N-Dimethylformamide as a cryoprotectant. The semen samples were placed into mini straws with a volume of 0.25 ml, sealed using polyvinyl chloride powder, and equilibrated at 5°C for 15 minutes. After equilibration, the semen samples were cryopreserved in liquid nitrogen (temperature −196°C) (Ratchamak et al., 2023).

Thawing

The straw is thawed by placing it in warm water at 37°C for 30 seconds, using tweezers to handle it (Wajdi et al., 2021). After thawing, the mini straw is dried with tissue, and a small cut is made in the middle to prevent it from breaking. The sample is then bent or attached to a glass slide, and one end is cut to release the liquid inside for examination.

Spermatozoa motility

A 10 µl sample of semen suspension was combined with 10 µl of physiological NaCl, mixed thoroughly, and placed on a glass slide. The sample was then covered with a cover slip, and the progressive (forward) movement of the spermatozoa was examined under a microscope at 400x magnification (Susilowati et al., 2021).

Spermatozoa viability

Fresh semen is dripped into the object glass, added with eosin-nigrosin, mixed until homogeneous, and a smear preparation is made and dried over a fast flame. The results of the review were examined using a microscope at a magnification of 400 times. Sperms that live with a transparent head. Spermatozoa with dead heads appear reddish (Susilowati et al., 2021).

Intact plasma membrane

Examination of the intact plasma membrane using the hypo-osmotic solution test consisting of 7.35 g sodium citrate and 13.52 g fructose. Spermatozoa with intact membranes show a bulging neck and a bent tail tip, whereas a damaged plasma membrane shows a bulging and straight neck to tail.

Spermatozoa necrosis

The percentage of dead spermatozoa was assessed by detecting nuclear changes, such as pyknosis, karyorrhexis, and karyolysis, through hematoxylin and eosin staining. Spermatozoa MDA levels were measured using a modified thiobarbituric acid test method, whereas superoxide dismutase (SOD) and catalase (CAT) levels were measured using a spectrophotometer (Alipour-Jenaghard et al., 2023). Analyses of motility, viability, intact plasma membrane, MDA levels, spermatozoa necrosis, and CAT and SOD levels were conducted using the F test, and differences were further analyzed using the least significant difference test (Wurlina et al., 2022).

Data analysis

The collected data were analyzed using ANOVA, and Duncan’s test was used to determine significant differences (p < 0.05), using the Statistical Program for Social Sciences (SPSS) version 27.0 for Windows.

Ethical approval

This research was registered with ethical clearance with number KEH.071.05.2023.

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Results

Cooling semen analysis

The effects of GTE on the cooling cement quality parameters are displayed in Table 1 (availability data and reasonable request data set). The progressive motility, viability, and plasma membrane integrity in the egg yolk diluent addition GTE T2 dose 0.10 mg/100 ml diluent groups were significantly higher than those in the T1 dose 0.05 mg/100 ml diluent and T3 dose 0.15 mg/100 ml diluent (p < 0.05). The groups for spermatozoa necrosis (%) with a significant difference (p < 0.05) lower percentage was found in T2 (2.90d ± 0.41) and T1 (3.10b ± 0.65) and T3 (3.40c ± 1.14).

Post-thaw semen analysis

The effects of GTE on post-thaw semen parameters are presented in Table 2. The results indicated that semen quality was significantly (p < 0.05) decreased after freezing compared with that after cooling. Progressive motility, viability, and plasma membrane integrity in egg yolk diluent addition GTE post-thawing T2 were significantly (p < 0.05) higher than those in T1 and T3.

The result of necrosis post-thawing was significantly different (p < 0.05) where T2 was lower than T1 and T3. The MDA (p < 0.05) showed significant differences where T2 was lower than T1 and T3. The results of SOD and CAT showed significant differences (p < 0.05) where the antioxidant enzymes were higher in group T2 than in groups T1 and T3.

Motility and viability

Post-thawing progressive motility and viability group T0 (without addition of GTE) showed a low percentage; there was a significant increase (p < 0.05) in the treatment groups T1, T2, and T3, with the best progressive motility and viability in group T2 dose 0.10 mg/100 ml diluent (Fig. 1). This effect can be caused by green tea’s high content of potential antioxidant compounds, especially catechin compounds, which can reduce ROS. Several previous studies showed that the addition of antioxidants to cement thinners could improve the quality of frozen semen produced by preventing acrosome damage (Bebas et al., 2023; Qamar et al., 2023).

Plasma membrane integrity

Plasma membrane integrity showed a significant difference (p > 0.05) increase in post-thawing groups T1, T2, and T3 compared with the control group (T0). However, in treatments, T2 on plasma membrane integrity was higher than that of the other groups (Fig. 2). This is in accordance with the fact that green tea (Camellia sinensis) can reduce ROS production by complementing unpaired electrons, thereby reducing damage to proteins, lipid membranes, and nucleic acids (Zhang et al., 2024).

Necrosis and CAT enzyme

Post-thaw necrosis and CAT enzyme results show significant difference (p > 0.05), explained that it has lower necrosis and the higher CAT enzyme in group T2 dose 0.10 mg/100 ml diluent (Fig. 3). This is in accordance with previous research, that is, the addition of antioxidants to diluents has quite good benefits, including preventing free radical activity from damaging spermatozoa cell membranes, which affects the viability and fertility of spermatozoa.

MDA

T1 and T3 levels did not experience a significant decrease from T0 (p > 0.05), but then at T2 dose 0.10 mg/100 ml diluent, there was a significant decrease with increasing concentrations of green tea leaf extract (Camelia sinensis) (Fig. 4). The antioxidant potential of green tea polyphenols, especially catechin compounds, is directly related to the combination of aromatic rings and hydroxyl groups. Green tea polyphenols encourage the detoxification activity of xenobiotic components and can also increase the levels of metal ions, such as iron, which results in oxygen free radicals.

Table 1. Effects of green tea leaf extract (Camelia sinensis) on the semen quality of chickens before freezing.

Table 2. Effects of green tea leaf extract (Camelia sinensis) on the semen quality of chicken after freezing.

Fig. 1. Diagram showing the addition of green tea extract to egg yolk–milk diluent. (A) Progressive motility (%) and (B) viability (%) after thawing the semen of Gaok chicken.

Fig. 2. Diagram of the addition of green tea extract to egg yolk–milk diluent plasma membrane integrity (%) after thawing the semen of Gaok chicken.

SOD

The post-thaw semen Gaok chicken analysis of variance (p < 0.05) showed a significant effect in treatment group T2 (Fig. 5) than in other groups which experienced a high increase so that SOD, which functions as the most important antioxidant enzyme that is already present in spermatozoa cells as a defense which is responsible for damage caused by radicals and ROS, is free.

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Discussion

To prevent high ROS levels in cells, antioxidants, which act as free radical scavengers, are needed (Appiah et al., 2019). Natural sources of antioxidants are herbs as antioxidant derivatives which are associated with improving the quality of frozen semen (Rakha et al., 2023). The EGCG contained in green tea (Camellia sinensis) has antioxidant activity (Roychoudhury et al., 2017). The best result was found to be 0.10 mg of GTE per 100 ml of diluter, according to the measured characteristics. However, adding more GTE than that (0.05 mg/100 ml and 0.15 mg/100 ml) would result in a reduction in the quality of the cryopreserved semen from the Gaok chicken. Previously, anti-free radical compounds have become pro-oxidants or chemicals that stimulate the production of free radicals. Oxidative stress can arise from this, and cells can be harmed (Zahra et al., 2024).

Fig. 3. Diagram showing the addition of green tea extract to egg yolk–milk diluent in (A) necrosis spermatozoa and (B) catalase enzyme (U/ml) post-thawing semen of Gaok chicken.

Fig. 4. Diagram addition of green tea extract to egg yolk–milk diluent and MDA (U/ml) after thawing the semen of Gaok chicken.

Fig. 5. Diagram showing the addition of green tea extract to egg yolk–milk diluent and superoxide dismutase (U/ml) after thawing the semen of Gaok chicken.

The percentage of progressive motility is a crucial parameter for assessing semen quality, as such motility enables spermatozoa to traverse the female reproductive tract and reach the infundibulum for successful ovum penetration (Elbashir et al., 2018). The semen dilution and freezing processes cause oxidative stress in spermatozoa, which can form ROS. This finding is corroborated by other researchers who have indicated that semen from various male types exhibits differing levels of resistance to extreme conditions (Vašíček et al., 2022). The freezing process damages the function and structure of the membrane and the ability of spermatozoa to maintain life (Swelum et al., 2022). The plasma membrane of spermatozoa functions as a critical barrier that regulates the exchange of intraextracellular substances essential for metabolic processes (Roca et al., 2022). Previous studies have reported that the main cause of sperm freezing damage is due to an imbalance between cellular antioxidant defenses and the production of ROS (Zong et al., 2023). As shown in the table, the addition of green tea leaf extract significantly increased the levels of motility, viability, and plasma membrane integrity in the T2 group. One of the contents of green tea in the form of flavonoids acts as an oxidative chain reaction breaker by providing H-electrons so as to reduce oxidative stress and make the compound more stable (Wajdi et al., 2021).

Susceptibility to cold temperatures is also associated with a high ratio of unsaturated fatty acids to saturated fats so that the ROS formed is also high, the end result of which is MDA (Mohan et al., 2013). High MDA levels experienced a significant decrease in the T2 group, and SOD levels also experienced a significant increase in the T2 group. Elevated ROS levels lead to increased oxidative stress, which is reflected by elevated MDA concentrations, which are markers of lipid peroxidation and membrane damage. Other than that, SOD, CAT, glutathione peroxidase, and glutathione reductase are the major antioxidant enzymes present in mammalian seminal plasma and are involved in the scavenging of ROS (Papas et al., 2019).

After freezing, the damage increases the intracellular electrolyte concentration, which in turn promotes ice crystal formation. In severe cases, this damage can extend to the nuclear membrane (Neila-Montero et al., 2024). On the other hand, spermatozoa physiologically require low levels of ROS to maintain optimal function (Wagner et al., 2017). Disruptions in ROS and antioxidant levels can lead to redox imbalances. Excessive ROS levels or an overabundance of antioxidants can disturb this balance and adversely affect sperm health (Zahra et al., 2024). Specifically, high doses of GTE in the T3 group result in diminished ROS levels, which impaired normal sperm functionality. This phenomenon underscores the antioxidant paradox, in which excessive antioxidant levels can paradoxically compromise male fertility (Majzoub and Agarwal, 2018). Concurrently, the body’s antioxidant defenses, including SOD and CAT, attempt to mitigate this oxidative damage. However, if ROS levels overwhelm these antioxidant systems, the resultant oxidative stress can impair sperm function and viability. Thus, maintaining an optimal balance between ROS and antioxidant activity is crucial for preserving spermatozoa health and fertility.

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Conclusion

The addition of green tea leaf extract subsequently reduced MDA levels. The addition of GTE at a dose of 0.10 mg/100 ml of egg yolk–milk diluent can act as an antioxidant in maintaining the quality of post-thawing Gaok chicken spermatozoa.

Acknowledgments

The authors thank Universitas Airlangga.

Conflict of interest

The authors declare no conflict of interest.

Funding

The authors thank the Directorate of Research and Community Service and Faculty of Veterinary Medicine, Airlangga University, for the PUF Fiscal Year 2018 (number: 1722/UN3.1.6/LT/2018).

Author’s contributions

Laboratory work and data collection: TH, SS, AOA, and AAR. Field sampling: SBM, NN, and ASH. Data analysis and manuscript writing: ES, SM, TDL, and IM. Research concepts: ARK, NYD, RZA, and UFH. All authors have read and approved the final manuscript.

Data availability

All data supporting the findings of this study are available in the manuscript, and no additional data sources are required.

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