This study is part of a multiyear research project. The protocol was approved by the Animal Care and Use Committee of Airlangga University (number 520/HRECC.FODM/VII/2019).

Dried green tea ( Camellia sinensis. Kuntze) leaves was obtained from Perkebunan Nusantara XII Malang, East Jawa Indonesia. Briefly, EGCG was isolated from C. sinensis using a thin-layer chromatography method and verified by a comparison with epigallocatechin gallate hydrate (Tokyo Chemical Co., Ltd., Japan). ²⁰ Subsequently, 5 mL of 0.1% chitosan solution [containing low molecular weight chitosan (Sigma-Aldrich) in 1% acetic acid] was added to 50 mL of EGCG solution [0.05% EGCG (Sigma-Aldrich) in distilled water], and the mixture was stirred at room temperature. Next, 0.5 mL of triphosphate (TPP) solution [0.025% TPP (Merck) in distilled water] was added drop-wise with stirring for 3 h at 112 × g. This solution was centrifuged at 21,952 × g for 10 min using a MC-10K centrifuge (Bio-Gener, Hangzhou, China) and washed three times with deionized water to obtain the EGCG CNPs, which were freeze dried for 48 h and stored at 4°C. ²¹ The particle size of EGCG CNPs was measured using a Zetasizer Nano ZS (ZEN 3600, Malvern Instruments Ltd., Worcestershire, UK). A helium–neon ion laser at a wavelength of 633 nm was used as the incident beam at 25°C with a 90° angle. ²²

Samples were collected from three of Kacang bucks aged two–three years and weighing 35–40 kg. These bucks were (owned by The Artificial Insemination Center, Airlangga University) fed approximately four kg of forage and 3.5 kg of concentrate (16%–18% crude protein) daily and provided with drinking water ad libitum. Semen was collected from the bucks using an artificial vagina twice per week to obtain six ejaculate samples to process as frozen semen.

Skim milk powder (15 g; 115338; Merck) was dissolved in distilled water to a volume of 150 mL, heated for 10 min to 92°C–95°C, and then cooled to room temperature (25°C). Egg yolk (5 mL; derived from laboratory chicken eggs) was added to 95 mL of skim milk solution, then added with 1 IU/mL of penicillin (Meiji Seika Pharma, Tokyo, Japan) and 1 μg/mL of streptomycin (Thermo Fisher Scientific, Singapore). ⁷ The solution was divided into five equal volumes without addition of EGCG CNPs for the control group (T0) and with addition of 0.5, 1.0, 1.5, and 2.0 μg of EGCG CNPs/mL extender for T1, T2, T3 and T4 groups, respectively.

Each SM–EY extender group was divided into two equal volumes. The first volume was added to fresh semen to obtain 480 million spermatozoa/mL. The second volume was added with glycerol up to 16% concentration, which was in turn added to the first mixture to obtain 240 million spermatozoa/mL. The cooling procedure was carried out by placing the semen diluted in extender into 15 mL Falcon tubes, which were then kept in a refrigerator (4–5 °C). The samples were placed in the refrigerator door to avoid direct exposure to cold airflow, ensuring a gradual reduction in temperature from 25 °C to 5 °C over a period of approximately 1 hour, then filled in 0.25 ml French straws (I.M.V., France) and sealed. The filled and sealed straws were chilled in liquid nitrogen vapor from 5°C to − 140°C for 10 min, and immediately stored in liquid nitrogen (−196°C) for 24 hours before evaluation were conducted. ⁷

The straws allocated to each group were thawed in sterile water for 30 s at 37°C. Six replicates randomly were used to assess sperm MPI, living cells, progressive motility, MDA levels, catalase levels, and DPPH scavenging, respectively, according to methods reported in a previous study. ⁷

MPI

A semen sample (0.1 mL) was added to 1 mL of a hypoosmotic solution [containing 7.35 g of sodium citrate (Sigma-Aldrich) and 13.52 g of fructose (Sigma-Aldrich) dissolved in distilled water to a volume of 1 L)] and incubated at 37°C for 30 min. The sperm MPI was assessed for 100 sperm under a light microscope (Olympus BX-53, Tokyo, Japan) at 400× magnification. Sperm with an MPI showed a curved tail, whereas those with a damaged plasma membrane showed a straight tail. ⁷

Living cells

A drop of semen sample and a drop of nigrosine (Sigma-Aldrich) were mixed and smeared on a glass slide, after which the slide was dried over a flame. The slide was then examined under a light microscope (Olympus BX-53, Tokyo, Japan) at 400× magnification to evaluate the percentage of live sperm in 100 spermatozoa. Live sperm were identified by their brightly transparent heads, whereas dead sperm were colored red. ⁷

Motility

An homogenate of a semen sample (10 μL) and a 0.9% (w/v) NaCl solution (1 mL) was dropped onto a glass slide and covered. The number of progressively motile sperm was counted for 100 sperm at 400× magnification under a light microscope (Olympus BX-53, Tokyo, Japan) equipped with Linkam Warming Stages set at 37°C–38°C (Meyer Instruments, Texas, USA). ⁷

MDA levels

MDA levels in semen samples were determined using the thiobarbituric acid (Sigma-Aldrich) method. Semen samples (100 μL) were mixed with 550 μL of distilled water and 100 μL of 20% trichloroacetic acid. The supernatant was then reacted with thiobarbituric acid according to the kit instructions. A standard curve was prepared using MDA standards (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL) to calculate the MDA concentration from the absorbance at the specified wavelength. These mixtures were then homogenized for 30 s, and 250 μL of HCl (1 N) was then added and homogenized. Subsequently, 100 μL of 1% sodium thiobarbiturate was added and homogenized. This mixture was centrifuged at 28 × g for 10 min, and the supernatant was incubated in a 100°C water bath for 30 min before being left to room temperature (25°C). The color absorption was determined at a wavelength of 533 nm using a spectrophotometer (Thermo Fisher Scientific). MDA levels (ng/mL) were determined by extrapolating the sample absorbance values using a standard MDA curve. ⁷

Catalase levels

The semen samples were diluted with the SM–EY extender to achieve a total volume of 2 mL for the catalase assay. Specifically, 0.5–1.5 mL of ejaculated semen was diluted to 2 mL with the extender at room temperature before adding 1 mL of 30 mM H ₂O ₂ phosphate-buffered solution. The catalase activity was then measured using UV spectrophotometry at 240 nm against a blank. All other parameters, including DPPH, MDA, sperm motility, viability, and IPM, were measured using the same diluted semen samples to maintain consistency across analyses. ²³

DPPH radical scavenging

A 5 mL of DPPH radicals (10 mM) in methanol were added to a cuvette containing 970 mL of mixed methanol. This mixture was incubated at 20°C for 3 min, and the absorbance was measured at 517 nm (A517) using a UV-Vis Spectrophotometer (Thermo Fisher Scientific). Next, 25 mL of each sample and 25 mL of an acetonitrile solution (9.5 M; used as negative control) were added and mixed, and the mixture was incubated at 20°C for 3 min. Subsequently, the A517 decrease related to DPPH radical decomposition was measured. All experiments were performed in duplicate, and the mean DPPH scavenging effect was calculated according to the following formula: DPPH scavenging effect (%) = (1 − A517 sample/A517 negative control) × 100. ²⁴ IC ₅₀ values were calculated using a relationship curve of RSA versus concentrations of the respective sample curve. ²⁵

All data were first tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. Parametric data were analyzed by one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) post hoc test to compare the effects of different EGCG CNPs doses on semen parameters (catalase, DPPH, MDA, motility, viability, and IPM). A significance level of p ≤ 0.05 was applied. All analyses were performed using SPSS (Version 23, IBM Corp., Armonk, NY, USA).

In general of this study, adding EGCG CNPs in SM-EY extenders resulted in higher catalase and DPPH and lower MDA than those of SM-EY extenders without EGCG CNPs. Previous study reported that adding 2.5 mM curcumin in a Tris-based extender did not decrease lipid peroxidation, and malondialdehyde formation on Angora buck semen compared to inositol and carnitine supplementation. ¹⁴ Another study reported that lipid peroxidation can be lowered with supplementation of combined myo-inositol and melatonin, ¹⁶ or 5 mM L-carnitine in the plant-based extenders. ¹⁷ Semen has antioxidant enzymes, namely catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX), which maintain the oxidant–antioxidant balance ³² and play fundamental roles in protecting biological systems against free radical attacks. The scavenging activity of SOD is accomplished via catalase, which reduces hydrogen peroxide to water and molecular oxygen. ³³ Indeed, catalase activates the decomposition of hydrogen peroxide into water and oxygen, thereby blocking the ROS-generating pathway and reducing oxidative stress. ³⁴ The addition of catalase to a commercially medium increased the total motility, membrane integrity, and living cells of postliquid goat semen ³⁵ and ram semen. ³⁶ Optimal catalase levels in the extender also reduce detrimental effects on post-thawed motility, living cells, plasma membrane, and acrosome integrity. ³⁷ In humans, catalase is used as a molecular target for diagnosing and monitoring male infertility ³⁸ and in strategies for optimizing sperm parameters. ³⁹ This study’s result is consistent with that of Papas et al., ⁴⁰ who demonstrated that the specific activities of catalase, GPX, and glutathione reductase during stallion semen cryopreservation were similar between effective and ineffective freezing of ejaculates. However, SOD activity was found to be higher in ejaculate following effective freezing than in ejaculate subjected to poor freezing power. ⁴¹ In stallions, the total and specific activity of catalase in seminal plasma is high; however, no correlation was observed between total catalase activity in stallion seminal plasma and sperm kinematic parameters. ⁴⁰

The higher DPPH values observed in T3 and T4 groups compared with the control (T0) indicate more effective free radical scavenging by EGCG CNPs at these doses. ⁴² A DPPH measurement has an acceptable reproducibility for determination of radical scavenging activity in several samples. ⁴³ The DPPH assay, a popular method for evaluating the kinetics and stoichiometry of antioxidative reactions, is used widely because it is easy to use, rapid, and sensitive. The assay is based on the reduction of the purple chromogen DPPH· via a hydrogen atom or electron transfer from the scavenging molecule, i.e., an antioxidant, which causes the formation of pale yellow hydrazine (i.e., DPPH). ⁴⁴

The highest levels of MDA in the control group indicate highest lipid peroxidation. Oxidative stress may result in an imbalance between ROS generation and endogenous antioxidant activities. Higher ROS levels cause cell damage through the peroxidation of PUFAs in the sperm plasma membrane. Lipid peroxidation generates toxic lipid aldehyde species, including MDA, ⁴⁵ and higher MDA levels indicate free radical attacks ⁴⁶ and plasma membrane damage. ⁴⁷ The lower MDA levels observed in post-thawed semen of T2, T3, and T4 groups compared with the control (T0) indicate a decrease in oxidative stress, which may contribute to improved membrane fluidity and reduced acrosomal damage owing to the rearrangement of membrane lipids and proteins. ³⁴

Post-thawed semen quality in goats is a very critical subject. Several previous studies have reported the following results. Post-thawed of goat semen frozen in a citrate extender with 10% egg yolk omega-3 showed higher live sperm and sperm motility. ¹¹ Supplementation of an egg yolk extender with 1% rainbow trout plasma semen resulted in a higher of post-thawed sperm living cells of ram semen. ¹² Post-thawed Saanen goats frozen semen in a soy lecithin-based extender with 8% rainbow trout seminal plasma showed higher sperm motility, acrosome integrity, plasma membrane integrity, and mitochondrial function. ¹³ Adding 2.5 mM curcumin in a Tris-based extender resulted in higher post-thawed sperm motility of Angora buck semen compared to inositol and carnitine supplementation. ¹⁴ Beetal buck semen cryopreserved in tris egg yolk with butylated hydroxytoluene showed increased acrosome integrity but was not significantly different on sperm living cells, even decreased sperm motility. ¹⁵ The combination of myo-inositol and melatonin improved post-thawed sperm living cells, sperm motility, and plasma membrane integrity. ¹⁶ Supplementation of 5 mM L-carnitine in the plant-based extenders improves sperm living cells, sperm motility, and membrane integrity. ¹⁷

In the recent study, adding of EGCG CNPs in the SM-EY extender increased the MPI, the living cell sperm, and sperm motility compared to those of the SM-EY extender without EGCG CNPs. Goat semen is sensitive to cryopreservation. Freezing semen leads to excessive ROS production ⁴⁸ followed by lipid peroxidation of the membrane, resulting in MDA production, ⁴⁹ which in turn markedly reduces sperm MPI, living cells, motility, and DNA integrity. ⁵⁰

Intactness of plasma membrane is essential for protecting the organelles of sperm and molecular transportation; thus, it is crucial for sperm living cells and sperm motility. ⁵¹ Post-thawed semen previously frozen without antioxidant EGCG CNPs in the extender showed the lowest MPI, sperm living cells, and sperm motility levels, whereas adding EGCG CNPs to the extender increased each of these levels. Damage to the plasma membrane of the spermatozoa reduces the quality of post-thawed semen because the integrity of this membrane is essential for the survival and motility of sperm. ⁵² Excessive ROS production in semen due to the freezing–thawing process causes lipid peroxidation in the sperm membrane, ⁸ disrupting the structure and function of this membrane and leading to the death of the spermatozoa. ⁵⁰ Lipid peroxidation also damages axonemal and mitochondrial proteins, resulting in the loss of sperm motility, even though the spermatozoa remain alive. ⁵³ Membrane damage due to lipid peroxidation also results in higher MDA levels. ⁵⁴ Furthermore, ROS cause the opening of bonds between disulfide chains in proteins, thereby destabilizing the DNA structure and leading to DNA fragmentation. ⁵⁵ The ejaculate defense system, including antioxidant enzymes such as GPX, catalase, and SOD, deals with ROS. ⁵⁶ However, the smaller volume of the sperm cytoplasm than those of commonly cells is the challenging for the antioxidant. ⁵⁷ The addition of semen extender might also lead to decreases of endogenous antioxidants. Insufficient antioxidant levels to combat oxidative stress during cryopreservation can have multiple negative effects, including decreased sperm living cells, sperm motility, and plasma sperm integrity. Thus, the addition of antioxidants to the extender prior to semen freezing is necessary.

The EGCG is an antioxidant that can reduce lipid peroxidation, protein carbonylation, and sperm DNA damage. ⁵⁸ The NPs form of EGCG increases the ratio of surface area of membrane to volume of particles, which allows EGCG to penetrate sperm cells efficiently. ⁵⁹ The presence of antioxidants reduces lipid peroxidation in the plasma membrane of spermatozoa, which in turn increases sperm living cells, motility, and acrosome integrity. ⁶⁰ With the exception of MPI, the addition of EGCG CNPs at doses of 1.5 and 2.0 μg/mL significantly improved post-thawed catalase activity, DPPH scavenging activity, sperm motility, and the percentage of living cells, while decreasing MDA levels (p < 0.05) compared with the control group. These results are consistent with those of previous studies in which adding ethanol green tea extract in extender maintained the motility, living cells, MPI, and DNA integrity of Simmental bull sperm. ⁶¹