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

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Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (MPI), living cells and motility analyses. Results Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm viability, sperm motility, IPM, and the highest levels of MDA. However, the addition of EGCG CNPs at doses of 1.5 µg/mL extender increased post-thawed catalase, DPPH, sperm IPM, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group. Conclusion This study was the first in which EGCG CNPs were used in SM–EY extender, and the addition of only 1.0 µg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen. 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F1000Research 2025, 12 :32 ( https://doi.org/10.12688/f1000research.127744.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] Imam Mustofa https://orcid.org/0000-0003-4543-1659 1 , Suherni Susilowati 1 , Tri Wahyu Suprayogi 1 , Adeyinka Oye Akintunde 2 , Yudit Oktanella 3 , Djoko Agus Purwanto 4 Imam Mustofa https://orcid.org/0000-0003-4543-1659 1 , Suherni Susilowati 1 , [...] Tri Wahyu Suprayogi 1 , Adeyinka Oye Akintunde 2 , Yudit Oktanella 3 , Djoko Agus Purwanto 4 PUBLISHED 05 Sep 2025 Author details Author details 1 Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, 60115, Indonesia 2 Department of Agriculture and Industrial Technology, Babcock University, Ilishan-Remo, Ogun State, Nigeria 3 Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang, East Java, Indonesia 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Airlangga University, Surabaya, East Java, 60115, Indonesia Imam Mustofa Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Writing – Original Draft Preparation Suherni Susilowati Roles: Conceptualization, Data Curation, Project Administration, Writing – Original Draft Preparation Tri Wahyu Suprayogi Roles: Data Curation, Investigation, Resources, Software Adeyinka Oye Akintunde Roles: Validation, Visualization, Writing – Review & Editing Yudit Oktanella Roles: Data Curation, Formal Analysis, Methodology Djoko Agus Purwanto Roles: Investigation, Supervision, Validation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Nanoscience & Nanotechnology gateway. This article is included in the Agriculture, Food and Nutrition gateway. Abstract Background and Aim The Kacang goat ( Capra hircus ) is an indigenous livestock species in Indonesia that is at risk of extinction due to cross-breeding. Artificial insemination (AI) techniques are expected to increase the population of these goats. This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. Materials and Methods Fresh Kacang buck semen was diluted in SM–EY without or with the addition of 0.5, 1.0, 1.5, or 2.0 µg of EGCG CNPs/mL extender. Extended semen was packaged in French mini straws, frooze, and stored in liquid nitrogen at −196℃ for 24 hours. Six replicates from each treatment group were thawed for catalase, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, malondialdehyde (MDA), sperm intact plasma membrane (MPI), living cells and motility analyses. Results Post-thawed semen that was previously frozen without EGCG CNPs in the extender (control group) exhibited the lowest levels of catalase, DPPH, sperm viability, sperm motility, IPM, and the highest levels of MDA. However, the addition of EGCG CNPs at doses of 1.5 µg/mL extender increased post-thawed catalase, DPPH, sperm IPM, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group. Conclusion This study was the first in which EGCG CNPs were used in SM–EY extender, and the addition of only 1.0 µg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen. READ ALL READ LESS Keywords catalase, extinction risk, radical scavenging, smallholder and farm, sperm motility Corresponding Author(s) Imam Mustofa ( [email protected] ) Close Corresponding author: Imam Mustofa Competing interests: No competing interests were disclosed. Grant information: The authors thank Universitas Airlangga, Indonesia for funding this study (contract number: 1405/UN3.1.6/PT/2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Mustofa I et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Mustofa I, Susilowati S, Suprayogi TW et al. Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.12688/f1000research.127744.3 ) First published: 09 Jan 2023, 12 :32 ( https://doi.org/10.12688/f1000research.127744.1 ) Latest published: 05 Sep 2025, 12 :32 ( https://doi.org/10.12688/f1000research.127744.3 ) Revised Amendments from Version 2 Abstract We add "(MPI)" after “sperm intact plasma membrane”, and change the "viability" with "living cells" Methods We add more detail in "Frozen semen": The cooling procedure was … approximately 1 hour We add more detail in "MDA levels": Semen samples (100 μL) were ….at the specified wavelength. We add more detail in "Catalase levels”: The semen samples were …. To maintain consistency across analyses. 23 Data analysis We change the narration with: All data were … using SPSS (Version 23, IBM Corp., Armonk, NY, USA). Results We change the narration before “(Table 3)”: The addition of …. with the control group (T0) (p < 0.05) (Table 3). We change the narration before “(Table 4)”: “The addition of … of the control group (T0) to be: Adding EGCG CNPs at … between the two doses (Table 4). Discussion Antioxidant capacity We change the narration “A higher value of DPPH … free radical scavenging. 42 ” to be: The higher DPPH … free radical scavenging by EGCG CNPs at these doses. 42 We change the narration “The lower MDA … protein rearrangement. 34 to be: MDA levels observed in post-thawed semen of …. the rearrangement of membrane lipids and proteins. 34 We change the narration “ 60 Adding EGCG CNPs … to the control group”.to be: 60 With the exception of MPI, … compared with the control group. Conclusion We change the narration “EGCG CNPs to the SM–EY extender …. of Kacang buck post-thawed semen”. To be: The addition of EGCG CNPs …. of Kacang buck semen. Abstract We add "(MPI)" after “sperm intact plasma membrane”, and change the "viability" with "living cells" Methods We add more detail in "Frozen semen": The cooling procedure was … approximately 1 hour We add more detail in "MDA levels": Semen samples (100 μL) were ….at the specified wavelength. We add more detail in "Catalase levels”: The semen samples were …. To maintain consistency across analyses. 23 Data analysis We change the narration with: All data were … using SPSS (Version 23, IBM Corp., Armonk, NY, USA). Results We change the narration before “(Table 3)”: The addition of …. with the control group (T0) (p < 0.05) (Table 3). We change the narration before “(Table 4)”: “The addition of … of the control group (T0) to be: Adding EGCG CNPs at … between the two doses (Table 4). Discussion Antioxidant capacity We change the narration “A higher value of DPPH … free radical scavenging. 42 ” to be: The higher DPPH … free radical scavenging by EGCG CNPs at these doses. 42 We change the narration “The lower MDA … protein rearrangement. 34 to be: MDA levels observed in post-thawed semen of …. the rearrangement of membrane lipids and proteins. 34 We change the narration “ 60 Adding EGCG CNPs … to the control group”.to be: 60 With the exception of MPI, … compared with the control group. Conclusion We change the narration “EGCG CNPs to the SM–EY extender …. of Kacang buck post-thawed semen”. To be: The addition of EGCG CNPs …. of Kacang buck semen. See the authors' detailed response to the review by Maria Madalena Pessoa Guerra See the authors' detailed response to the review by Muhammed Duman READ REVIEWER RESPONSES Introduction Smallholder farmers raise Kacang goats ( Capra hircus ) to increase their financial income, reduce poverty, and prevent malnutrition. However, the pure breed of this indigenous livestock species in Indonesia is at risk of extinction due to cross-breeding and must be protected. 1 Artificial insemination (AI) techniques involving freeze–thawed semen are expected to increase the population of these goats. However, goat sperm is sensitive to cold shock. 2 Indeed, >60% sperm death was detected in post-thawed goat semen previously frozen in skim milk–egg yolk (SM–EY) extender without antioxidants, 3 which does not meet the minimum requirements for AI, i.e., motility must be >40%. 4 High sperm death and low motility occurs because the freezing and thawing process leads to an excess of reactive oxygen species (ROS) production, which damages the polyunsaturated fatty acids (PUFAs) in the plasma membrane of spermatozoa, increasing malondialdehyde (MDA) levels and ultimately reducing sperm living cells and motility. 5 Therefore, antioxidants are needed to counteract the effects of ROS and thereby increase post-thawed semen quality. 6 In our previous studies, green tea extract was used to improve semen quality and decrease nucleotide mutations in mtDNA 3 and protein-encoded mtDNA, 7 presumably due to an increase in the antioxidant capacity. The freeze–thawing process in goat semen causes lipid peroxidation in the spermatozoa membrane, which reduces semen quality. 8 Semen contains endogenous antioxidants that help maintain the oxidant–antioxidant balance. 9 However, the excessive production of ROS due to the freeze–thawing process cannot be overcome by endogenous antioxidants owing to the limitations of the spermatozoa cytoplasm and decreased antioxidant levels due to the addition of extenders. 10 Several studies to improve the quality of post-thawing goats semen have been carried out, including adding extenders with egg yolk omega-3, 11 fish semen plasma, 12 , 13 curcumin, 14 butylated hydroxytoluene, 15 combination of myo-inositol and melatonin, 16 and L-carnitine. 17 Epigallocatechin-3-gallate (EGCG) is a powerful antioxidant extracted from green tea. 18 The nanoparticle extract has a large surface area to volume ratio, so it is expected to exhibit increased penetration into cells, 19 including sperm cells, and improve the quality of post-thawed semen. The addition of EGCG chitosan nanoparticles (CNPs) to the extender when freezing Kacang goat semen has not yet been studied. Thus, in the present study, the addition of EGCG CNPs to SM–EY extender and their ability to increase antioxidant capacity was investigated by assessing catalase and 2,2-diphenyl-1-picrylhydrazyl (DPPH) levels. The aim of this study was to obtain the best possible quality of post-thawed Kacang buck semen for AI according to MDA levels, sperm membrane plasma integrity (MPI), living cells, and motility. Methods The study was conducted in February to August 2022, at The Artificial Insemination Center of Airlangga University, Tanjung village, Kedamean, Gresik District, East Java, Indonesia, at coordinates 7° 19′ 25′′ S and 112° 32′ 54′′ E. 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). 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 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 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 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 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 2 O 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 Data analysis 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). Results The diameters particles of EGCG CNPs was in range 41.31 – 388.36 nm with the averages as presented in Table 1 and size distribution curves of EGCG CNPs as seen in Figure 1 . The progressive motility of sperm indicates the quality of fresh semen. Based on the criteria for the motility of individual spermatozoa of >70%, the obtained semen ejaculate of the Kacang goats met the requirements for freezing ( Table 2 ). Table 1. Size (nm) and distribution of EGCG CNPs. Range Averages Percentage 10–100 47.87 ± 6.56 98.6% 100–1000 333.9 ± 54.46 1.4% Figure 1. Size distribution curves of EGCG CNPs (Zetasizer Ver. 7.01 (MAL1061025, Malvern Instruments Ltd, Worcs., UK)). Table 2. Macroscopic and microscopic parameters of fresh Kacang buck semen. Parameter Value Volume (mL) 1.37 ± 0.06 Color Creamy white (normal) pH 6–7 Consistency Thick (normal) Concentration (million/mL) 1933.67 ± 51.03 Mass motility +++ (normal) Progressive motility (%) 83.33 ± 2.89 Living cells (%) 88.33 ± 1.52 Post-thawed semen that was previously frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited the lowest levels of catalase and DPPH and the highest levels of MDA (p < 0.05). The addition of EGCG CNPs at 1.5 and 2.0 μg/mL in the extender resulted in significantly higher catalase activity and DPPH scavenging activity, and lower MDA levels compared with the control group (T0) (p < 0.05) ( Table 3 ). Table 3. Post-thawed sperm malondialdehyde (MDA; nmol/mL), catalase (× 10 −3 U/mg), and 2,2-diphenyl-1-picrylhydrazyl (DPPH; %) levels in Kacang buck semen extended in skim milk–egg yolk (SM–EY) with or without the addition of epigallocatechin-3-gallate nanoparticles (EGCG CNPs). Data were analyzed using ANOVA followed by the Tukey Honestly Significant Difference test at a significance level of p ≤ 0.05. Groups Catalase DPPH MDA T0 43.07 ± 1.75 a 68.37 ± 3.88 a 2096.28 ± 45.16 b T1 50.28 ± 1.87 b 74.55 ± 3.29 a 1779.51 ± 55.10 b T2 61.40 ± 2.44 c 82.16 ± 1.35 b 1381.17 ± 65.70 a T3 67.68 ± 2.36 d 86.04 ± 1.28 c 1250.22 ± 43.06 a T4 69.62 ± 2.82 d 87.39 ± 1.48 c 1356.74 ± 62.41 a Semen that was frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited lowest sperm MPI, living cells, and motility both in the pre-freezing and post-thawed conditions (p < 0.05). Adding EGCG CNPs at 1.5 and 2.0 μg/mL to the SM–EY extender improved semen quality compared with the control group (T0) (p < 0.05), with no significant difference between the two doses ( Table 4 ). Table 4. Pre-freezing and post-thawed sperm living cells, progressive motility, and membrane plasma integrity (MPI) of Kacang buck semen extended in skim milk–egg yolk (SM–EY) with or without the addition of epigallocatechin-3-gallate nanoparticles (EGCG CNPs). Data were analyzed using ANOVA followed by the Tukey Honestly Significant Difference test at a significance level of p ≤ 0.05. Groups MPI Living cells Motility Pre-freezing Post-thawed Pre-freezing Post-thawed Pre-freezing Post-thawed T0 47.17 ± 2.56 a 30.33 ± 3.08 a 58.33 ± 2.58 a 33.33 ± 2.66 a 54.33 ± 3.50 a 30.00 ± 4.47 a T1 53.50 ± 4.85 a 31.17 ± 3.06 a 60.83 ± 3.76 a 36.67 ± 2.58 a 57.83 ± 4.58 a 33.50 ± 3.21 a T2 57.67 ± 3.50 a 34.33 ± 2.25 a 65.00 ± 3.16 ab 40.83 ± 2.04 ab 61.67 ± 3.61 ab 38.17 ± 2.40 ab T3 65.17 ± 5.64 ab 38.00 ± 2.61 ab 72.50 ± 2.74 b 42.50 ± 2.74 b 67.83 ± 3.43 b 40.33 ± 2.25 b T4 64.67 ± 4.37 ab 40.17 ± 4.67 b 75.00 ± 4.47 b 44.17 ± 2.04 b 71.83 ± 4.02 b 43.50 ± 4.81 b Discussion Of the EGCG CNPs added to the SM-EY extender in the study, 98.6% were 10-100 nm in diameter, and the remaining percentage was 100-1000 nm. Nanotechnology techniques have been used to produce particles with a size scale of 0.1–1000 nm. 26 The smaller particle size of nanoparticles than the microparticles, causes the NPs have larger surface areas to volume ratio and the opportunities for chemical reactions and biological activities also increase. Bioavailability is the ability of NPs to penetrate cells. The effect of NPs on the target site depends on their chemical composition, shape, surface structure, surface charge, catalytic properties, and aggregation ability with other materials. 27 The NPs size causes the active compound to spread in the medium and reach the target with increased accuracy. 28 One of the safest materials used in NP encapsulation technology is chitosan. 29 EGCG possesses metal-chelating properties that provide antioxidant functions. The two structures that give EGCGs metal chelation properties are the ortho-3′,4′-dihydroxy moiety and the 4-keto, 3-hydroxyl, or 4-keto and 5-hydroxyl moiety. Catechins prevent the generation of potentially damaging free radicals via the chelation of metal ions. Through their ability to chelate transition metal ions, flavonoids can complex and inactivate iron ions, thereby suppressing the superoxide-driven Fenton reactions that are thought to be a crucial route to forming ROS. Electron transfer from catechins to ROS-induced radical sites on DNA and the formation of stable semiquinone free radicals are other mechanisms by which catechins exert their antioxidant effects, 30 which are more pronounced than those of vitamins C and E. 31 Antioxidant capacity 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 Quality of post-thawed semen 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 Conclusion In this study, EGCG CNPs were used for the first time in SM–EY extender to improve the antioxidant capacity and quality of post-thawed semen. The addition of EGCG CNPs at 1.5 and 2.0 μg/mL to the SM–EY extender enhanced the antioxidant capacity and improved the post-thawed quality of Kacang buck semen. Future studies are required to validate this finding for AI under farm conditions. Author’s contributions Imam Mustofa (IM), Suherni Susilowat I (SS), Tri Wahyu Suparyogi (TWS), Adeyinka Oye Akintunde (AOA), Yudit Oktanella (YO), Djoko Agus Purwanto (DAP) IM, SS and TWS conceived the idea, designed the mainframe of this study, and conceived in detail the manuscript. IM, TWS collected, prepared extender and freezing semen process. DAP: extracted EGCG from green tea leaves. YO: processing EGCG nanoparticles and measuring the particle size of extenders. SS, YO: evaluated semen quality. IM: interpreted the data and statistical analysi. AOA, YO, DAP: read critically and revised the manuscript for intellectual content. All authors read and approved the final manuscript. Data availability Biostudies. Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang Goat semen. DOI: https://www.ebi.ac.uk/biostudies/studies/S-BSST924 62 This project contains the following data: - This study aimed to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI Reporting guidelines ARRIVE checklist. Figshare. DOI: https://doi.org/10.6084/m9.figshare.21531213.v1 63 Acknowledgments The authors thank Dikky Eka Mandala Putranto, DMV, M.Sc., the Chairman of the Insemination Center, Airlangga University, Subchan Aziz, and Agus Purwanto for technical support. Approval of support_EGCG CNPs, DOI: 10.6084/m9.figshare.21532326 . https://figshare.com/articles/poster/Approval_of_support_EGCG_CNPs/21532326 . References 1. Suswono SDecree of the Minister of Agriculture of the Republic of Indonesia Number 2840/Kits/LB.430/8/2012.2012. Accessed June 17 2022. Reference Source 2. Hahn K, Failing K, Wehrend A: Effect of temperature and time after collection on buck sperm quality. BMC Vet. Res. 2019; 15 : 355. PubMed Abstract | Publisher Full Text | Free Full Text 3. Mustofa I, Susilowati S, Wurlina W, et al. : Green tea extract increases the quality and reduced DNA mutation of post-thawed Kacang buck sperm. Heliyon. 2021; 7 : e06372. PubMed Abstract | Publisher Full Text | Free Full Text 4. 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Publisher Full Text Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 09 Jan 2023 ADD YOUR COMMENT Comment Author details Author details 1 Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, 60115, Indonesia 2 Department of Agriculture and Industrial Technology, Babcock University, Ilishan-Remo, Ogun State, Nigeria 3 Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang, East Java, Indonesia 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Airlangga University, Surabaya, East Java, 60115, Indonesia Imam Mustofa Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Writing – Original Draft Preparation Suherni Susilowati Roles: Conceptualization, Data Curation, Project Administration, Writing – Original Draft Preparation Tri Wahyu Suprayogi Roles: Data Curation, Investigation, Resources, Software Adeyinka Oye Akintunde Roles: Validation, Visualization, Writing – Review & Editing Yudit Oktanella Roles: Data Curation, Formal Analysis, Methodology Djoko Agus Purwanto Roles: Investigation, Supervision, Validation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The authors thank Universitas Airlangga, Indonesia for funding this study (contract number: 1405/UN3.1.6/PT/2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 05 Sep 2025, 12:32 https://doi.org/10.12688/f1000research.127744.3 version 2 Revised Published: 04 Apr 2023, 12:32 https://doi.org/10.12688/f1000research.127744.2 version 1 Published: 09 Jan 2023, 12:32 https://doi.org/10.12688/f1000research.127744.1 Copyright © 2025 Mustofa I et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Mustofa I, Susilowati S, Suprayogi TW et al. Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.12688/f1000research.127744.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 3 VERSION 3 PUBLISHED 05 Sep 2025 Revised Views 0 Cite How to cite this report: Eidan SM. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r416962 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-416962 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 14 Oct 2025 Sajeda Mahdi Eidan , University of Baghdad, Baghdad, Iraq Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.187380.r416962 The terminology should be standardized throughout manuscript according to the first abbreviation. For instance, MPI should be used consistently instead of IPM, EGCG CNPs should replace with EGCG chitosan nanoparticles (CNPs), and viability should replace with living ... Continue reading READ ALL The terminology should be standardized throughout manuscript according to the first abbreviation. For instance, MPI should be used consistently instead of IPM, EGCG CNPs should replace with EGCG chitosan nanoparticles (CNPs), and viability should replace with living cells. Don’t use brackets next to each other “(T0) (p < 0.05) (Table 3) “, combine them to one bracket & separate the references with semicolons “(T0;p < 0.05; Table 3)” The equilibrium period for glycerol during the cooling stage is one hrs. which is a short duration. Please justify this with an appropriate reference or clarify the basis for this statement. Kindly check The correct reference for plasma membrane integrity is Jeyendran et al., (1984). Please confirm this reference ‘ Jeyendran, R,S., H.H. Vander van, M. Pelaez, B.G. Crabo and L.J.D.1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship other semen characteristics. J. Reprod. Fertil.,70:219-228.’’ Kindly check the MDA statistical analysis (Table 3), as it seems that no significant differences exist among treatments. “Semen that was frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited lowest sperm MPI, living cells, and motility both in the pre-freezing and post-thawed conditions (p < 0.05). Adding EGCG CNPs at1.5and2.0 μg/ mL to the SM–EY extender improved semen quality compared with the control group(T0)(p<0.05),with no significant difference between the two doses (Table 4).” Referring to Table 4, no significant differences are observed among treatments. All means are marked with ‘a’ except T3&T4 which have both a & b. Please verify and correct any inconsistencies in the statistical analysis and notation. To provide more comprehensive evaluation of the treatments effects, it would be beneficial to include data on fertility outcomes of goats inseminated with treated semen, especially this study involves an endangered goat breed (Kacang goats) as stated in introduction. Following addition of data on acrosome integrity, sperms abnormalities, sperm DNA damage, and fertility percentages. The conclusion should be reformulated to reflects these findings Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Semen cryopreservation, metabolic biomarkers, Fertility of males and females, molecular biology related to fertility , Nutrition related to fertility I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Eidan SM. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r416962 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-416962 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Berean D. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r412691 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-412691 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 16 Sep 2025 Daniel Berean , University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca, Romania Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.187380.r412691 Congratulations on your study. Improving sperm cell characteristics after the thawing process is not easy to achieve and requires meticulous and hard work. The importance of such research is continuously increasing, particularly in the small ruminant sector, where sperm conservation ... Continue reading READ ALL Congratulations on your study. Improving sperm cell characteristics after the thawing process is not easy to achieve and requires meticulous and hard work. The importance of such research is continuously increasing, particularly in the small ruminant sector, where sperm conservation techniques are not as well developed as in large ruminants or the equine sector. The article is well written and has a logical structure; however, there are some gaps that need to be addressed. I will enumerate them below. Where it is no longer possible to address certain points, I suggest kindly including them in the limitations of the study as a final paragraph in the discussion section. I suggest revising the title to make it more concise and readable, for example: "Epigallocatechin-3-gallate Chitosan Nanoparticles Enhance Antioxidant Capacity and Post-Thaw Quality of Kacang Goat Semen." In the results section of the abstract, it appears that only this concentration (1.5/ml) improved the parameters. Please clarify this point or specify that 1.5 µg/mL was the optimal concentration. In the conclusion of the abstract, the value of 1 µg/mL is already mentioned; I suggest here including the most effective concentration and, in the results section, providing a summary of the effects of the other concentrations. Please verify that the introduction section addresses the characteristics and importance of all the sperm parameters investigated. I suggest presenting this information in a chronological and logical order rather than providing an amalgam of details, which forces the reader to discern what is important. Additionally, a brief discussion on sperm evaluation methods and how these parameters are assessed should be included. A clearer and simpler description of the dilutions is needed, as the current form does not clearly explain what happened after semen collection. For example, were the samples mixed or kept separate? The dilution rate should also be specified. Additionally, information about the bucks is missing, were they collected prior to estrus, and was a doe in estrus used for sperm collection? The main gap in the Materials and Methods section is the use of classical, non-automated methods for assessing sperm characteristics. This limitation should be explicitly acknowledged and categorized as an important limitation of the study. In some parts of the article, the authors refer to motility, while in other parts they mention progressive motility. It should be clarified which parameter was actually assessed, or, if both were measured, the text should be revised to clearly distinguish and describe each. In the discussion section, more data from your own study should be included and developed in comparison with other studies. In its current form, the authors refer extensively to literature data while only occasionally discussing their own results. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes References 1. Berean D, Bogdan L, Opris P, Cimpean R: Economical implications and the impact of gonadotropin-releasing hormone administration at the time of artificial insemination in cows raised in the extensive system in North Romania. Frontiers in Veterinary Science . 2023; 10 . Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Small ruminants reproduction, sperm cryopreservation I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Berean D. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r412691 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-412691 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 04 Apr 2023 Revised Views 0 Cite How to cite this report: GANGWAR C. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r318492 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-318492 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 12 Sep 2024 CHETNA GANGWAR , ICAR-Central Institute for Research on Goats, Uttar Pradesh, India Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.146355.r318492 1. Authors used different nomenclature at different places like MPI and IPM. 2. In Preparation of EGCG CNPs - Author used this statement - This solution was centrifuged at 21,952 × g for 10 min. How this can accurately done. ... Continue reading READ ALL 1. Authors used different nomenclature at different places like MPI and IPM. 2. In Preparation of EGCG CNPs - Author used this statement - This solution was centrifuged at 21,952 × g for 10 min. How this can accurately done. 3. In estimation of MDA levels - author write this - A semen sample (100 μL) and MDA kits (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL of malondialdehyde) were added, again this incorrect. 4. Moreover, many microscopic evaluation can be done to assess the NPs effect. 6. In Catalase test- A 30 mM H 2 O 2 phosphate-buffered solution [1 mL; comprising 0.34 mL of 30% H 2 O 2 diluted in fresh phosphate buffer (50 mM and pH 7)] was added to a semen sample (2 mL) , whether this sample is diluted or neat. Generally bucks give 0.5 to 1.5 ml semen. If you used whole semen, how you did other parameters. 5. No complete detail in statistical analysis is included. 6. There are many mistakes in English. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Andrology, Buck semen cryopreservation I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT GANGWAR C. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r318492 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-318492 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Duman M. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r168663 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-168663 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Apr 2023 Muhammed Duman , Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Bursa Uludag University, Bursa, Turkey Approved VIEWS 0 https://doi.org/10.5256/f1000research.146355.r168663 The revised version meets all criteria mentioned by reviewers. The ... Continue reading READ ALL The revised version meets all criteria mentioned by reviewers. The authors have done all suggested sections based on reviewer reports. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Semen quality and additives I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Duman M. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r168663 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-168663 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 09 Jan 2023 Views 0 Cite How to cite this report: Guerra MMP. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r160298 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-160298 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 17 Feb 2023 Maria Madalena Pessoa Guerra , [email protected] , University Federal Rural of Pernambuco (UFRPE), Recife, Brazil Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.140281.r160298 The article aims to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. The work is well written and can make ... Continue reading READ ALL The article aims to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. The work is well written and can make a great contribution to the freezing of goat semen. However, you need to make a lot of corrections before it's indexed. The term "viability" is not appropriate, as it does not define well what was evaluated in the spermatic cell. I suggest replacing it with "living cells" With the exception of IPM, the addition of EGCG CNPs at doses of 1.5 and 2.0 μg/mL extended post-thawed catalase, DPPH, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group. The conclusion of the abstract is not consistent with the results, as 1.5 μg/mL and 2.0 μg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen. What is the methodology used to cool semen from room temperature (25°C) to 5°C for 1 hour? Results: Part of the first paragraph can transfer to the discussion. This statement “The addition of EGCG CNPs in extender resulted in higher catalase and DPPH levels and lower MDA levels (p < 0.05) in optimum dose of 1.5 μg/mL of extender (T3) than those in the T0 group (p < 0.05) (Table 3). “ is not correct, because the significant difference occurred at doses 1.5 and 2.0 μg/mL. This statement “The addition of EGCG CNPs at 1.5 μg/mL of extender (T3) was optimum dose for increasing of sperm viability, motility and IPM (p < 0.05) than those in the T0 group (Table 4).” also does not agree with the results, since there was a difference between analyses: MPI was better from T4 (2.0 μg/mL), while live and motility were better with T3 (1.5 μg/mL), and T4 (2.0 μg/mL). The discussion needs to be modified. Little of the results obtained were discussed with the literature data. Looks like a literature review. This statement “The lower MDA levels in post-thawed 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.” should be modified, because groups T2, T3 and T4 did not differ from each other and were inferior to the T) (control). The statement that "A higher value of DPPH in the T3 than those of T0 group represents more effective free radical scavenging." should also be modified, since T4 did not differ from T3. The statement that “Adding EGCG CNPs at 1.5 μg/mL of SM–EY extender improved semen quality compared with that of the control group.” should be modified, as T3 and T4 did not differ from each other. The statement of the conclusion that “Indeed, the addition of 1.5 μg/mL EGCG CNPs to the SM–EY extender increased the antioxidant capacity and quality of Kacang buck post-thawed semen.” must be modified. The title of the tables should explain the analyses performed, in the same order in which the results are presented below. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: - I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Guerra MMP. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r160298 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-160298 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 04 Apr 2023 Imam Mustofa , Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia 04 Apr 2023 Author Response Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, ... Continue reading Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers. Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers. Competing Interests: We have no competing interest Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 04 Apr 2023 Imam Mustofa , Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia 04 Apr 2023 Author Response Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, ... Continue reading Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers. Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers. Competing Interests: We have no competing interest Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Duman M. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r161557 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-161557 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 07 Feb 2023 Muhammed Duman , Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Bursa Uludag University, Bursa, Turkey Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.140281.r161557 I would like to suggest some additions to the manuscript, including the introduction and discussion sections. When I read the article, it sounds much more scientific and understandable. I do not see any methodological error, but I think additions for ... Continue reading READ ALL I would like to suggest some additions to the manuscript, including the introduction and discussion sections. When I read the article, it sounds much more scientific and understandable. I do not see any methodological error, but I think additions for improving post-thawed semen quality in goats are popular subjects. In this updated area, the authors need to add some updated additives to improve semen quality in the introduction section and need to discuss them in the discussion. Why Epigallocatechin-3-gallate chitosan nanoparticles are well comparing other antioxidants and freezer protectors in goat semen. I am suggesting some minor points below: Introduction should be enlarged There are some other studies about additions increase post-thawed semen quality such as fish seminal plasma, egg yolk, curcumin, inositol and carnitine. Authors should mention other articles in the introduction section some samples given below Information about post-thawed semen quality in goats is very critical subject and authors need to mention what is the updated researches on this subject. For example: https://doi.org/10.1016/j.anireprosci.2015.11.017 https://doi.org/10.1016/j.smallrumres.2009.11.015 http://zsp.com.pk/pdf47/119-124%20(16)%20PJZ-1915-14%2011-12-14%20page%20proof%20PJZ.pdf http://www.pvj.com.pk/pdf-files/34_3/347-350.pdf https://doi.org/10.1111/and.13555 The mentioned studies in introduction section, should be discussed here The reference (56) is not relevant to the study and should be discarded After revisions, the manuscript will be high impact and mostly cited because of comprising an updated subject. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Ustuner B, Alcay S, Toker MB, Nur Z, et al.: Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender. Anim Reprod Sci . 2016; 164 : 97-104 PubMed Abstract | Publisher Full Text 2. Bucak M, Sarıözkan S, Tuncer P, Sakin F, et al.: The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research . 2010; 89 (1): 24-30 Publisher Full Text 3. Iqbal Z, Ijaz A, Aleem M, Shahzad AH, et al.: Effect of butylated hydroxytoluene on post-thawed semen quality of beetal goat buck, Capra hircus. Pakistan Journal of Zoology . 2015; 47 : 119-24 Reference Source 4. Yimer N, Noraisyah AH, Rosnina Y, Wahid H, et al.: Comparison of cryopreservative effect of different levels of omega-3 egg-yolk in citrate extender on the quality of goat spermatozoa. Pakistan Veterinary Journal . 2014; 34 (3): 347-350 Reference Source 5. Alcay S, Ustuner B, Aktar A, Mulkpinar E, et al.: Goat semen cryopreservation with rainbow trout seminal plasma supplemented lecithin-based extenders. Andrologia . 2020; 52 (4): e13555 PubMed Abstract | Publisher Full Text Competing Interests: No competing interests were disclosed. Reviewer Expertise: Semen quality and additives I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Duman M. Reviewer Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r161557 ) The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-161557 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 04 Apr 2023 Imam Mustofa , Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia 04 Apr 2023 Author Response Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender ... Continue reading Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added. Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added. Competing Interests: We have no competing interest Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 04 Apr 2023 Imam Mustofa , Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia 04 Apr 2023 Author Response Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender ... Continue reading Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added. Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added. Competing Interests: We have no competing interest Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 3 VERSION 3 PUBLISHED 09 Jan 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 5 Version 3 (revision) 05 Sep 25 read read Version 2 (revision) 04 Apr 23 read read Version 1 09 Jan 23 read read Muhammed Duman , Bursa Uludag University, Bursa, Turkey Maria Madalena Pessoa Guerra , University Federal Rural of Pernambuco (UFRPE), Recife, Brazil CHETNA GANGWAR , ICAR-Central Institute for Research on Goats, Uttar Pradesh, India Daniel Berean , University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca, Romania Sajeda Mahdi Eidan , University of Baghdad, Baghdad, Iraq Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Eidan S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 Oct 2025 | for Version 3 Sajeda Mahdi Eidan , University of Baghdad, Baghdad, Iraq 0 Views copyright © 2025 Eidan S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The terminology should be standardized throughout manuscript according to the first abbreviation. For instance, MPI should be used consistently instead of IPM, EGCG CNPs should replace with EGCG chitosan nanoparticles (CNPs), and viability should replace with living cells. Don’t use brackets next to each other “(T0) (p < 0.05) (Table 3) “, combine them to one bracket & separate the references with semicolons “(T0;p < 0.05; Table 3)” The equilibrium period for glycerol during the cooling stage is one hrs. which is a short duration. Please justify this with an appropriate reference or clarify the basis for this statement. Kindly check The correct reference for plasma membrane integrity is Jeyendran et al., (1984). Please confirm this reference ‘ Jeyendran, R,S., H.H. Vander van, M. Pelaez, B.G. Crabo and L.J.D.1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship other semen characteristics. J. Reprod. Fertil.,70:219-228.’’ Kindly check the MDA statistical analysis (Table 3), as it seems that no significant differences exist among treatments. “Semen that was frozen without antioxidant EGCG CNPs in the extender (T0 group) exhibited lowest sperm MPI, living cells, and motility both in the pre-freezing and post-thawed conditions (p < 0.05). Adding EGCG CNPs at1.5and2.0 μg/ mL to the SM–EY extender improved semen quality compared with the control group(T0)(p<0.05),with no significant difference between the two doses (Table 4).” Referring to Table 4, no significant differences are observed among treatments. All means are marked with ‘a’ except T3&T4 which have both a & b. Please verify and correct any inconsistencies in the statistical analysis and notation. To provide more comprehensive evaluation of the treatments effects, it would be beneficial to include data on fertility outcomes of goats inseminated with treated semen, especially this study involves an endangered goat breed (Kacang goats) as stated in introduction. Following addition of data on acrosome integrity, sperms abnormalities, sperm DNA damage, and fertility percentages. The conclusion should be reformulated to reflects these findings Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Semen cryopreservation, metabolic biomarkers, Fertility of males and females, molecular biology related to fertility , Nutrition related to fertility I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Eidan SM. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r416962) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-416962 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Berean D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 16 Sep 2025 | for Version 3 Daniel Berean , University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Cluj-Napoca, Romania 0 Views copyright © 2025 Berean D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Congratulations on your study. Improving sperm cell characteristics after the thawing process is not easy to achieve and requires meticulous and hard work. The importance of such research is continuously increasing, particularly in the small ruminant sector, where sperm conservation techniques are not as well developed as in large ruminants or the equine sector. The article is well written and has a logical structure; however, there are some gaps that need to be addressed. I will enumerate them below. Where it is no longer possible to address certain points, I suggest kindly including them in the limitations of the study as a final paragraph in the discussion section. I suggest revising the title to make it more concise and readable, for example: "Epigallocatechin-3-gallate Chitosan Nanoparticles Enhance Antioxidant Capacity and Post-Thaw Quality of Kacang Goat Semen." In the results section of the abstract, it appears that only this concentration (1.5/ml) improved the parameters. Please clarify this point or specify that 1.5 µg/mL was the optimal concentration. In the conclusion of the abstract, the value of 1 µg/mL is already mentioned; I suggest here including the most effective concentration and, in the results section, providing a summary of the effects of the other concentrations. Please verify that the introduction section addresses the characteristics and importance of all the sperm parameters investigated. I suggest presenting this information in a chronological and logical order rather than providing an amalgam of details, which forces the reader to discern what is important. Additionally, a brief discussion on sperm evaluation methods and how these parameters are assessed should be included. A clearer and simpler description of the dilutions is needed, as the current form does not clearly explain what happened after semen collection. For example, were the samples mixed or kept separate? The dilution rate should also be specified. Additionally, information about the bucks is missing, were they collected prior to estrus, and was a doe in estrus used for sperm collection? The main gap in the Materials and Methods section is the use of classical, non-automated methods for assessing sperm characteristics. This limitation should be explicitly acknowledged and categorized as an important limitation of the study. In some parts of the article, the authors refer to motility, while in other parts they mention progressive motility. It should be clarified which parameter was actually assessed, or, if both were measured, the text should be revised to clearly distinguish and describe each. In the discussion section, more data from your own study should be included and developed in comparison with other studies. In its current form, the authors refer extensively to literature data while only occasionally discussing their own results. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes References 1. Berean D, Bogdan L, Opris P, Cimpean R: Economical implications and the impact of gonadotropin-releasing hormone administration at the time of artificial insemination in cows raised in the extensive system in North Romania. Frontiers in Veterinary Science . 2023; 10 . Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Small ruminants reproduction, sperm cryopreservation I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Berean D. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.187380.r412691) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v3#referee-response-412691 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 GANGWAR C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Sep 2024 | for Version 2 CHETNA GANGWAR , ICAR-Central Institute for Research on Goats, Uttar Pradesh, India 0 Views copyright © 2024 GANGWAR C. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 1. Authors used different nomenclature at different places like MPI and IPM. 2. In Preparation of EGCG CNPs - Author used this statement - This solution was centrifuged at 21,952 × g for 10 min. How this can accurately done. 3. In estimation of MDA levels - author write this - A semen sample (100 μL) and MDA kits (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL of malondialdehyde) were added, again this incorrect. 4. Moreover, many microscopic evaluation can be done to assess the NPs effect. 6. In Catalase test- A 30 mM H 2 O 2 phosphate-buffered solution [1 mL; comprising 0.34 mL of 30% H 2 O 2 diluted in fresh phosphate buffer (50 mM and pH 7)] was added to a semen sample (2 mL) , whether this sample is diluted or neat. Generally bucks give 0.5 to 1.5 ml semen. If you used whole semen, how you did other parameters. 5. No complete detail in statistical analysis is included. 6. There are many mistakes in English. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Andrology, Buck semen cryopreservation I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (0) GANGWAR C. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r318492) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-318492 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Duman M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Apr 2023 | for Version 2 Muhammed Duman , Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Bursa Uludag University, Bursa, Turkey 0 Views copyright © 2023 Duman M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The revised version meets all criteria mentioned by reviewers. The authors have done all suggested sections based on reviewer reports. Competing Interests No competing interests were disclosed. Reviewer Expertise Semen quality and additives I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Duman M. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.146355.r168663) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v2#referee-response-168663 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Guerra M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 17 Feb 2023 | for Version 1 Maria Madalena Pessoa Guerra , [email protected] , University Federal Rural of Pernambuco (UFRPE), Recife, Brazil 0 Views copyright © 2023 Guerra M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The article aims to determine the addition of epigallocatechin-3-gallate chitosan nanoparticles (EGCG CNPs) to skim milk–egg yolk (SM–EY) extender to obtain the best possible quality of post-thawed Kacang buck semen for AI. The work is well written and can make a great contribution to the freezing of goat semen. However, you need to make a lot of corrections before it's indexed. The term "viability" is not appropriate, as it does not define well what was evaluated in the spermatic cell. I suggest replacing it with "living cells" With the exception of IPM, the addition of EGCG CNPs at doses of 1.5 and 2.0 μg/mL extended post-thawed catalase, DPPH, viability, and sperm motility and decreased MDA levels (p < 0.05) than those of control group. The conclusion of the abstract is not consistent with the results, as 1.5 μg/mL and 2.0 μg/mL of EGCG CNPs in this extender increased the antioxidant capacity and post-thawed quality of Kacang buck semen. What is the methodology used to cool semen from room temperature (25°C) to 5°C for 1 hour? Results: Part of the first paragraph can transfer to the discussion. This statement “The addition of EGCG CNPs in extender resulted in higher catalase and DPPH levels and lower MDA levels (p < 0.05) in optimum dose of 1.5 μg/mL of extender (T3) than those in the T0 group (p < 0.05) (Table 3). “ is not correct, because the significant difference occurred at doses 1.5 and 2.0 μg/mL. This statement “The addition of EGCG CNPs at 1.5 μg/mL of extender (T3) was optimum dose for increasing of sperm viability, motility and IPM (p < 0.05) than those in the T0 group (Table 4).” also does not agree with the results, since there was a difference between analyses: MPI was better from T4 (2.0 μg/mL), while live and motility were better with T3 (1.5 μg/mL), and T4 (2.0 μg/mL). The discussion needs to be modified. Little of the results obtained were discussed with the literature data. Looks like a literature review. This statement “The lower MDA levels in post-thawed 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.” should be modified, because groups T2, T3 and T4 did not differ from each other and were inferior to the T) (control). The statement that "A higher value of DPPH in the T3 than those of T0 group represents more effective free radical scavenging." should also be modified, since T4 did not differ from T3. The statement that “Adding EGCG CNPs at 1.5 μg/mL of SM–EY extender improved semen quality compared with that of the control group.” should be modified, as T3 and T4 did not differ from each other. The statement of the conclusion that “Indeed, the addition of 1.5 μg/mL EGCG CNPs to the SM–EY extender increased the antioxidant capacity and quality of Kacang buck post-thawed semen.” must be modified. The title of the tables should explain the analyses performed, in the same order in which the results are presented below. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No Competing Interests No competing interests were disclosed. Reviewer Expertise - I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 04 Apr 2023 Imam Mustofa, Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have revised the narration in the Abstract, Introduction, Results, Discussion, and Conclusion based on data and suggestion of reviewers. View more View less Competing Interests We have no competing interest reply Respond Report a concern Guerra MMP. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r160298) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-160298 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Duman M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 07 Feb 2023 | for Version 1 Muhammed Duman , Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Bursa Uludag University, Bursa, Turkey 0 Views copyright © 2023 Duman M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I would like to suggest some additions to the manuscript, including the introduction and discussion sections. When I read the article, it sounds much more scientific and understandable. I do not see any methodological error, but I think additions for improving post-thawed semen quality in goats are popular subjects. In this updated area, the authors need to add some updated additives to improve semen quality in the introduction section and need to discuss them in the discussion. Why Epigallocatechin-3-gallate chitosan nanoparticles are well comparing other antioxidants and freezer protectors in goat semen. I am suggesting some minor points below: Introduction should be enlarged There are some other studies about additions increase post-thawed semen quality such as fish seminal plasma, egg yolk, curcumin, inositol and carnitine. Authors should mention other articles in the introduction section some samples given below Information about post-thawed semen quality in goats is very critical subject and authors need to mention what is the updated researches on this subject. For example: https://doi.org/10.1016/j.anireprosci.2015.11.017 https://doi.org/10.1016/j.smallrumres.2009.11.015 http://zsp.com.pk/pdf47/119-124%20(16)%20PJZ-1915-14%2011-12-14%20page%20proof%20PJZ.pdf http://www.pvj.com.pk/pdf-files/34_3/347-350.pdf https://doi.org/10.1111/and.13555 The mentioned studies in introduction section, should be discussed here The reference (56) is not relevant to the study and should be discarded After revisions, the manuscript will be high impact and mostly cited because of comprising an updated subject. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Ustuner B, Alcay S, Toker MB, Nur Z, et al.: Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender. Anim Reprod Sci . 2016; 164 : 97-104 PubMed Abstract | Publisher Full Text 2. Bucak M, Sarıözkan S, Tuncer P, Sakin F, et al.: The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Ruminant Research . 2010; 89 (1): 24-30 Publisher Full Text 3. Iqbal Z, Ijaz A, Aleem M, Shahzad AH, et al.: Effect of butylated hydroxytoluene on post-thawed semen quality of beetal goat buck, Capra hircus. Pakistan Journal of Zoology . 2015; 47 : 119-24 Reference Source 4. Yimer N, Noraisyah AH, Rosnina Y, Wahid H, et al.: Comparison of cryopreservative effect of different levels of omega-3 egg-yolk in citrate extender on the quality of goat spermatozoa. Pakistan Veterinary Journal . 2014; 34 (3): 347-350 Reference Source 5. Alcay S, Ustuner B, Aktar A, Mulkpinar E, et al.: Goat semen cryopreservation with rainbow trout seminal plasma supplemented lecithin-based extenders. Andrologia . 2020; 52 (4): e13555 PubMed Abstract | Publisher Full Text Competing Interests No competing interests were disclosed. Reviewer Expertise Semen quality and additives I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 04 Apr 2023 Imam Mustofa, Division of Veterinary Reproduction, Faculty of Veterinary Medicine, Airlangga University, Surabaya, 60115, Indonesia Thank you the suggestions for improvement of our article from the reviewers. We have corrected this article as follows. We have added some other studies about additions substances in extender to increase post-thawed goats semen quality in the Introduction and Discussion section. The relevant references were added. View more View less Competing Interests We have no competing interest reply Respond Report a concern Duman M. Peer Review Report For: Epigallocatechin-3-gallate chitosan nanoparticles in an extender improve the antioxidant capacity and post-thawed quality of Kacang goat semen [version 3; peer review: 1 approved, 2 approved with reservations, 2 not approved] . F1000Research 2025, 12 :32 ( https://doi.org/10.5256/f1000research.140281.r161557) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-32/v1#referee-response-161557 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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