Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen

Ethical approval

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).

Preparation of EGCG CNPs

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).¹³ 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.¹⁴ 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.¹⁵

Experimental animals

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.

SM–EY extender

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.

Frozen semen

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 extended semen was cooled from room temperature (25°C) to 5°C for 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

Evaluation of post-thawed sperm quality

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 IPM, viability, progressive motility, MDA levels, catalase levels, and DPPH scavenging, respectively, according to methods reported in a previous study.⁷

IPM

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 IPM was assessed for 100 sperm under a light microscope (Olympus BX-53, Tokyo, Japan) at 400× magnification. Sperm with an IPM showed a curved tail, whereas those with a damaged plasma membrane showed a straight tail.⁷

Viability

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. A semen sample (100 μL) and MDA kits (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL of malondialdehyde) were added to 550 μL of distilled water and 100 μL of 20% trichloroacetic acid. 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

A 30 mM H₂O₂ phosphate-buffered solution [1 mL; comprising 0.34 mL of 30% H₂O₂ diluted in fresh phosphate buffer (50 mM and pH 7)] was added to a semen sample (2 mL) at room temperature and assessed against a blank (not containing an enzyme) solution. The UV spectrophotometric absorbance method was used to measure catalase activity at a wavelength of 240 nm.¹⁶

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) × 100.¹⁷ IC₅₀ values were calculated using a relationship curve of RSA versus concentrations of the respective sample curve.¹⁸

Data analysis

ANOVA followed by the Tukey Honestly Significant Difference test were used to analyze these data with a significance level of p ≤ 0.05. SPSS (Version 23, IBM Corp., Armonk, NY, USA) was used for data analysis.

Antioxidant capacity

Semen has antioxidant enzymes, namely catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX), which maintain the oxidant–antioxidant balance²⁵ 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.²⁶ Indeed, catalase activates the decomposition of hydrogen peroxide into water and oxygen, thereby blocking the ROS-generating pathway and reducing oxidative stress.²⁷ The addition of catalase to a commercially medium increased the total motility, membrane integrity, and viability of postliquid goat semen²⁸ and ram semen.²⁹ Optimal catalase levels in the extender also reduce detrimental effects on post-thawed motility, viability, plasma membrane, and acrosome integrity.³⁰ In humans, catalase is used as a molecular target for diagnosing and monitoring male infertility³¹ and in strategies for optimizing sperm parameters.³² This study’s result is consistent with that of Papas et al.,³³ 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.³⁴ 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.³³

A higher value of DPPH in the T3 than those of T0 group represents more effective free radical scavenging.³⁵ A DPPH measurement has an acceptable reproducibility for determination of radical scavenging activity in several samples.³⁶ 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).³⁷

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,³⁸ and higher MDA levels indicate free radical attacks³⁹ and plasma membrane damage.⁴⁰ The lower MDA levels in post-tahwed semen of T3 group indicated the decreasing of oxidative stress, followed by an increase in membrane fluidity and a lower percentage of acrosomal damage owing to the rearrangement of membrane lipids and proteins.²⁷

Quality of post-thawed semen

Goat semen is sensitive to cryopreservation. Freezing semen leads to excessive ROS production⁴¹ followed by lipid peroxidation of the membrane, resulting in MDA production,⁴² which in turn markedly reduces sperm IPM, viability, motility, and DNA integrity.⁴³

Intactness of plasma membrane is essential for protecting the organelles of sperm and molecular transportation; thus, it is crucial for sperm viability and sperm motility.⁴⁴ Post-thawed semen previously frozen without antioxidant EGCG CNPs in the extender showed the lowest IPM, sperm viability, 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.⁴⁵ 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.⁴³ Lipid peroxidation also damages axonemal and mitochondrial proteins, resulting in the loss of sperm motility, even though the spermatozoa remain alive.⁴⁶ Membrane damage due to lipid peroxidation also results in higher MDA levels.⁴⁷ Furthermore, ROS cause the opening of bonds between disulfide chains in proteins, thereby destabilizing the DNA structure and leading to DNA fragmentation.⁴⁸ The ejaculate defense system, including antioxidant enzymes such as GPX, catalase, and SOD, deals with ROS.⁴⁹ However, the smaller volume of the sperm cytoplasm than those of commonly cells is the challenging for the antioxidant.⁵⁰ 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 viability, 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.⁵¹ 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.⁵² The presence of antioxidants reduces lipid peroxidation in the plasma membrane of spermatozoa, which in turn increases sperm viability, motility, and acrosome integrity.⁵³ Adding EGCG CNPs at 1.5 μg/mL of SM–EY extender improved semen quality compared with that of the control group. These results are consistent with those of previous studies in which adding ethanol green tea extract in extender maintained the motility, viability, IPM, and DNA integrity of Simmental bull sperm.⁵⁴