Research Report
Effects of nonenzymatic and enzymatic antioxidants in cryopreserved canine semen extended with docosahexaenoic acid
Roberto Rosa Filho*, Nívea Vieira*, Maíra Brito, João Losano, Giulia Kawai, Camilla Mendes, Bruno Rui, Ken Nagai, Álvaro Alves, Henrique Frias, Raphaela Sousa, Fábio Sanches, Mayra Assumpção, Marcilio Nichi
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil
Abstract
Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), could be an alternative to improve cryopreserved canine sperm by increasing membrane fluidity. However, PUFAs also increase sperm susceptibility to lipid peroxidation. Supplementation of the extender with DHA and antioxidants could be an alternative. We evaluated supplementation of antioxidants (vitamin E and catalase) of cryopreserved canine semen extended with DHA. In Experiment 1, ejaculates from 8 dogs were divided into 4 aliquots, with increasing concentrations of DHA in the extender (0, 1, 5, and 10 µM) and cryopreserved. Sperm motility increased in 5 µM DHA and it was used for Experiment 2; ejaculates from 8 dogs were divided into 4 groups: control (5 µM DHA), vitamin E + DHA, catalase + DHA, and vitamin E + catalase + DHA. Samples were evaluated for susceptibility to lipid peroxidation, sperm kinetics, mitochondrial function, DNA integrity, and plasma and acrosomal membrane integrity. Vitamin E + DHA group had beneficial effects on the characteristics of sperm kinetics whereas the catalase + DHA group was harmful to sperm mitochondria. Vitamin E + catalase + DHA group decreased oxidative stress and damage to the plasma membrane and had low mitochondrial membrane activity. Addition of DHA + vitamin E in canine semen extender improved sperm quality after cryopreservation.
Keywords: Lipid peroxidation, oxidative stress, dog, sperm kinetic, polyunsaturated fatty acid
Citation: Clinical Theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/CT.v17.12690
Copyright: © 2025 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Published: 01 August 2025
Competing interests and funding: None to declare.
CONTACT: Marcilio Nichi mnichi@usp.br
Introduction
Growing interest in increasing reproductive efficiency and societal value of dogs has led to a greater search for reproductive biotechnologies. Thus, male gamete preservation is a valuable tool since semen cryopreservation enables and facilitates its transport, storage for prolonged periods, and cost reduction.1
During spermatogenesis, sperm plasma membrane and other specific structures are prepared to react adequately to female genital tract and oocyte.2 Sperm membrane is composed of a bilayer of lipids, mainly polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA) that are of great importance to sperm, as they provide necessary fluidity characteristics for events associated with sperm permeability and fertilization,3 such as motility and acrosomal reaction.4 Plasma membrane fluidity depends on many factors, such as temperature, cholesterol content, and the degree of saturation of the carbon bonds.2 Saturated fatty acids guarantee greater rigidity whereas unsaturated ones provide greater fluidity to the membrane due to double bonds,5 an essential characteristic for reproductive events. In addition, lipid composition of sperm membrane in several species influences resistance to cold shock during refrigeration and cryopreservation.6 Therefore, polyunsaturated fatty acids are also directly involved in protecting the membrane during cryopreservation.7,8 An alternative would be to supplement the samples with PUFAs during cryopreservation, protecting sperm against cryoinjury.9 However, the high percentage of PUFAs makes sperm susceptible to oxidizing agents, leading to lipid peroxidation (due to their large amount of unsaturation, they are more easily oxidized).5 Additionally, for canine semen preservation, it is necessary to remove seminal plasma, and with that important antioxidants are removed.10 When reactive oxygen species (ROS) act on the unsaturated fatty acids’ double bonds, a lipid peroxidation chain reaction begins and, if not interrupted, leads to loss of membrane permeability and, consequently, sperm function.11 Based on these assertions, sperm treatment with PUFAs during cryopreservation could provide ambiguous results, but if PUFAs are incorporated into the sperm membrane, this would make it even more susceptible to ROS attack.
Thus, a plausible alternative would be the association between PUFAs and antioxidants during sperm cryopreservation, aiming to prevent the enhancement of oxidative events.12 Enzymatic and nonenzymatic antioxidants have specificity for certain oxidizing agents; therefore, prior to antioxidant treatment, it is essential to detect which ROS the sperm is most susceptible to, in order to perform a specific antioxidant treatment to eliminate this ROS.13 Also, hydrogen peroxide and hydroxyl radical were the most harmful ROS for canine sperm.13 Therefore, for an efficient antioxidant treatment, an association between enzymatic and nonenzymatic antioxidants would quell the aforementioned ROS.13 Catalase and vitamin E are examples of antioxidants that are extremely effective in neutralizing hydrogen peroxide and hydroxyl radical, respectively.14–16
As the largest lipid-soluble antioxidant of sperm, vitamin E (α-tocopherol) acts on the plasma membrane;17 it protects the double bond of unsaturated fatty acids against chain oxidation, donating electrons to a hydroxyl radical.18 It is the most efficient antioxidant to break this chain, reacting with the peroxyl radical, thereby preventing the reaction of this radical with another unsaturated fatty acid.19 It can also act as a membrane stabilizing agent, forming complexes with its phospholipids.20 Because of its lipophilic and antioxidant properties, it has a vital role in protecting biological membranes from peroxidative damage.19 Catalase is part of an enzyme system that degrades hydrogen peroxide into oxygen and water.21 Hydrogen peroxide is an extremely toxic ROS due to its ability to cross the membrane freely and to inhibit the enzymatic activities of cellular functions, thus decreasing the antioxidant defenses of sperm.22,23
Objective of this study was to determine the ideal concentration of DHA, test it in combination in the extender containing 2 antioxidants (vitamin E and catalase) in cryopreserved canine semen, and to verify a possible synergistic effect of these substances in sperm oxidative homeostasis and functionality.
Material and methods
Study was approved by the Bioethics Committee of the School of Veterinary Medicine and Animal Science, University of São Paulo (Protocol number 7693020215). Reagents were purchased from Sigma–Aldrich (St. Louis, MO).
Animals
Eight dogs (7 Belgian Malinois and 1 Labrador Retriever) of proven fertility with a body weight of 28-32 kg and 2-6 years were used. Dogs belonged to Canine Reproduction and Distribution Center of the 2nd Army Police Battalion (Osasco, Sao Paulo - Brazil). Dogs were periodically collected for the assisted reproductive program. Dogs were fed Royal Canin Max Adult® twice a day.
Experiment 1: Effect of supplementation with polyunsaturated fatty acid (docosahexaenoic acid [DHA]) on canine semen extender
Eight ejaculates were collected and diluted in a cryopreservation medium to arrive at a final concentration of 100 x 106 sperm per ml. Each ejaculate was divided into 4 aliquots and allocated to control group (0 µM DHA) and 1 µM DHA, 5 µM DHA, and 10 µM DHA groups. After dilution, samples underwent one-step sperm cryopreservation using an automated machine.
Experiment 2: Effect of vitamin E and catalase supplementation in cryopreserved canine semen in extender containing DHA
Eight ejaculates were collected and diluted in a cryopreservation medium for dogs to arrive at a final concentration of 100 x 106 sperm per ml. Each ejaculate was divided into 4 aliquots and allocated to control group (without antioxidant) and antioxidant groups (0.6 mM vitamin E, 300 U/ml catalase, and 0.6 mM vitamin E plus 300 U catalase/ml1). After dilution, samples underwent one-step sperm cryopreservation using an automated machine.
Semen collection, cryopreservation, and thawing
Sperm-rich fraction was collected using the digital manipulation method. Each ejaculate was assessed for motility, vigor, and concentration patterns under light microscopy (Nikon, Eclipse E200, Tokyo, Japan) and for volume via a graduated tube. Sperm concentration (106 sperm/ml) was determined in the Neubauer chamber. Samples contaminated with urine or blood or with motility below 70% were excluded.
Seminal plasma was removed by centrifuging semen at 500 x g for 5 minutes (Centribio®). Pellets were divided into 4 aliquots and suspended at a final concentration of 100 x 106 sperm/ml with 263 mM tris-yolk-fructose-citric acid (TRIS-hydroxy-methyl-aminomethane [Trizma-base; T-6791 Sigma]), 84.7 mM citric acid monohydrate (T-1909), 63 mM D-fructose (F-2543), egg yolk 20%, gentamicin 10 mg/ml (G-1264), and glycerol 5% and used for each treatment mentioned in the respective experiments.
After dilution, seminal aliquots were packed in 0.5 ml straws (IMV Technologies, France) and allocated in an automated cryopreservation machine TK3000® Compacta (TK Tecnologia em Congelação LTDA; Uberaba, MG, Brazil). Subsequently, the samples were refrigerated to 5ºC (0.25°C/minute); straws were then positioned 3 cm above liquid nitrogen (–20ºC) and immersed in liquid nitrogen. Samples were stored in liquid nitrogen cylinders and packed in racks. One week after cryopreservation and storage, samples were thawed at 37°C for 30 seconds and subjected to sperm evaluation.
Sperm evaluation
Sperm were evaluated for kinetics, plasma and acrosomal membrane integrity, mitochondrial function (activity and mitochondrial membrane potential), DNA integrity, and oxidative status.
Computerized analysis of sperm kinetic patterns
Kinetic patterns were assessed using Computerized Sperm Analysis (CASA; Ivos 12.3, Hamilton-Thorne®, Beverly, MA) with equipment settings for dogs (Table in Appendix). For the analysis, 6 µl of the sample was pipetted in a glass slide with a coverslip heated to 37ºC and ~ 1,000 sperm were evaluated. Following variables were considered: motility (%), progressive motility (%), VAP (velocity average pathway, µm/second), VSL (velocity straight line, µm/second), VCL (curvilinear velocity, µm/second), ALH (amplitude of lateral head displacement, µm), BCF (beat cross-frequency, Hz), STR (straightness %) and LIN (linearity %). Sperm speed was classified into 4 groups: fast (%), medium (%), slow (%) and static (%).24
Functional sperm tests
Flow cytometry (Guava EasyCyteTM Mini System, Guava® Technologies, Hayward, CA) was used to test sperm function with the concentration fixed at 25 x 106 sperm cells/ml after dilution with TRIS egg yolk. Gate details are provided (Figure in Appendix). A total of 10,000 events per sample were analyzed, and data corresponding to yellow (PM1 photodetector: 583 nm), red (PM2 photodetector: 680 nm), and green fluorescent signals (PM3 photodetector: 525 nm) were recorded after a logarithmic amplification. Data were analyzed by FlowJo® v10.2 software (Flow Cytometry Analysis Software, Tree Star Inc, Ashland, OR).25
Plasma and acrosomal membranes were evaluated with propidium iodide (PI) and FITC probes conjugated to Pisum sativum agglutinin (FITC-PSA), respectively. This association of fluorophores allocates the sperm population into 4 groups: intact membrane and intact acrosome (IMIA), intact membrane and damaged acrosome (IMDA), damaged membrane and intact acrosome (DMIA) and damaged membrane and damaged acrosome (DMDA). Samples were stained with 0.5 mg/ml PI in NaCl 0.9% and 100 mg/ml FITC-PSA (FITC-PSA L-0770) in a 10% sodium azide solution in DPBS. Samples were analyzed by flow cytometry after 10 minutes, excited at 488 nm and detected at 630-650 nm (PI) and 515-530 nm (FITC). Results were expressed as percentage.
Mitochondrial membrane potential was assessed by flow cytometry. Evaluation was made using JC-1 probe (5,5’,6,6’-tetrachloro-1,1’,3,3’ 201-tetraethyl-benzimidazolylcarbocyanine chloride; Invitrogen, Eugene, OR). For the assay, 0.5 μl of the fluorescent probe JC-1 (76.5 mM) was added to the samples and incubated at 37°C for 5 minutes before analysis on the flow cytometer, where there was excitation at 488 nm and detection at 583 nm. Samples were classified into percentages of sperm with high (JC-1 high), intermediate (JC-1 intermediate), and low (JC-1 low) mitochondrial membrane potential.
Mitochondrial activity of 100 sperm were determined using the DAB technique (3’3 diaminobenzidine) in phase contrast microscopy at 1,000 x magnification26 and classified into 4 classes, according to the percentage of stained midpiece: completely stained, indicating high mitochondrial activity (DAB I); more than 50% colored intermediate piece, indicating average activity (DAB II); more than 50% unstained intermediate piece, indicating low activity (DAB III); and intermediate piece completely unstained, indicating absence of mitochondrial activity (DAB IV). Results were expressed as a percentage (%) and these classes were used to provide an index of mitochondrial activity.
Chromatin stability was tested by sperm chromatin structure assay (SCSA),27 modified28 for dogs. Samples were incubated with 50 µl of TNE buffer (0.01 M Tris-HCl, 0.15 M NaCl, 1 mM EDTA and distilled water, pH 7.4) and 100 µl acid detergent (0.08 M HCl, 0.15 M NaCl, Triton X-100 0.1% in distilled water, pH 1.2). After 30 seconds, acridine orange (6 µg/ml stock solution) was added, and each sample was analyzed on flow cytometry after 5 minutes of incubation at 37ºC, excited at 488 nm and detected at 630-650 nm (yellow) and 515-530 nm (green). Debris were excluded using a green fluorescence/direct dispersion filter; then the percentage of sperm with DNA alteration (AO+) were selected in an αT histogram. To determine the AO positive (AO+) population, a positive control was incubated with hydrochloric acid (1.2 M in acid detergent, pH 0.1) for 5 minutes to induce DNA fragmentation in almost 100% of sperm.25
Evaluation of sperm resistance to oxidative stress
Susceptibility to lipid peroxidation was assessed by the thiobarbituric acid [TBA] reactive substance (TBARS) test that was adapted.29 Samples were incubated with iron sulfate (4 mM) and vitamin C (20 mM) at 37ºC for 90 minutes. After these inductions, 10% trichloroacetic acid (600 ml) was added. Samples were centrifuged at 20,800 x g for 15 minutes at 5ºC to isolate proteins and debris. Subsequently, 800 µl of the supernatant was removed and transferred to cryotubes. Then, 1% TBA (800 µl) was added to the tubes, which were incubated at 95ºC in a water bath for 15 minutes. In this reaction, malondialdehyde (MDA; the primary product of lipid peroxidation) and TBA produce a pink-colored complex. This staining was quantified on the spectrophotometer (Ultrospec 3300 Pro® Amersham Biosciences, Woburn, MA) at a wavelength of 532 nm. Results were expressed in nanograms of TBARS/106 sperm.
To detect ROS, a penetrating fluorescent probe was used, which, when oxidized by intracellular free radicals, binds to DNA and emits a higher intensity of green fluorescence (CellROX® green, Molecular Probes, Eugene, OR). Samples were stained with CellROX® green (final concentration of 5 µM) for 30 minutes at 37ºC, and PI was added (final concentration, 6 µM) in the last 10 minutes. Samples were analyzed by flow cytometry, excited at 488 nm, and detected at 630- 650 nm (PI) and 515-530 nm (CellROX® green).
Sperm data were analyzed for negative oxidative stress and intact plasma membrane (NOIM), negative oxidative stress and damaged plasma membrane (NODM), positive oxidative stress and intact plasma membrane (POIM), and positive oxidative stress and damaged plasma membrane (PODM), and expressed as a percentage.
Data analysis
Data were analyzed by the SAS System for Windows (SAS, Institute Inc., Cary, NC, 2000). Treatment effects were tested for normality of residues (Gaussian distribution) and homogeneity of variances using the Guided Data Analysis application. Means were compared using the ANOVA method applying the Least Significant Difference test for multiple comparisons. Results were presented as means with their respective standard errors of the mean; significance was set at p < 0.05.
Results
Experiment 1
Groups treated with 5 or 10 µM DHA had higher percentage of sperm with progressive motility (Figure 1A) and rapid speed (Figure 1B) compared to 1 µM group but were similar to control group. The group treated with 10 µM DHA had lower percentage of sperm with slow movement (Figure 1C) and was not different from control. There was a lower percentage of static sperm in 5 µM DHA (Figure 1D). Other sperm kinetic (Table 1) and function (Table 2) variables were not different. There was no difference among groups in lipid peroxidation rates from TBARS and oxidative stress from CellROX® (Table 3).
Figure 1. Effect of various concentrations of docosahexaenoic acid (DHA) on sperm kinetics variables a,bWithin an end point, columns without a common superscript differed (p ≤ 0.05)
Thus, based on the results, 5 µM DHA was selected for Experiment 2 to test the effect of antioxidants vitamin E and catalase in samples of cryopreserved canine semen in an extender containing DHA.
Experiment 2
The group treated with vitamin E had higher velocity average pathway (VAP - Figure 2A), velocity straight line (VSL - Figure 2B), total sperm motility (Figure 2C), progressive sperm motility (Figure 2D), and rapid sperm movement speed (Figure 2E) compared to control group. Additionally, the group treated with vitamin E did not differ from other treatments for these variables, except for progressive sperm motility, where the percentage of sperm in the group treated with catalase was significantly lower (Figure 2D). Other kinetic variables were not different among groups (Table 4).
Figure 2. Effect of vitamin E and catalase and their combination on sperm kinetics variables in an extender containing DHA a,bWithin an end point, columns without a common superscript differed (p ≤ 0.05)
There was no difference in mitochondrial function between control and vitamin E group in any variable (Figures 3A-3D). However, catalase and vitamin E + catalase groups had lower percentage of high mitochondrial activity (DABI) compared to control and vitamin E groups (Figure 3A) and a higher percentage of low mitochondrial activity (DABIII) compared to control group (Figure 3C). Catalase group also had a higher percentage of mitochondrial inactivity (DABIV) compared to control group (Figure 3D). Control group had higher mitochondrial activity index than vitamin E + catalase and catalase group, vitamin E had higher index than catalase group (Figure 3E). No other sperm functional test had a difference among groups (Table 5).
Figure 3. Effect of vitamin E and catalase and their combination on sperm mitochondrial function in an extender containing DHA a-cWithin an end point, columns without a common superscript differed (p ≤ 0.05)
The vitamin E + catalase group had higher percentage of sperm with negative oxidative stress and intact plasma membrane (NOIM) compared to control group (Figure 4A). Positive oxidative stress and damaged plasma membrane (PODM - Figure 4B), positive oxidative stress and intact plasma membrane (POIM - Figure 4C), negative oxidative stress and damaged membrane (NODM - Figure 4D) and lipid peroxidation (TBARS - Figure 4E) were not different among groups.
Figure 4. Effect of vitamin E and catalase and their combination to sperm oxidative stress features
Discussion
Synergistic effects of PUFAs and enzymatic and nonenzymatic antioxidants added to the canine semen extender were evaluated to prevent exacerbated lipid peroxidation caused by the addition of these fatty acids.30 In Experiment 1, 5 µM of DHA had the lowest percentage of static sperm, with same progressive and rapid motility as control group. Motility parameters analyzed by CASA have high potential in clinical applications since there were significant relationships between aspects of sperm movement and cervical mucus penetration, fertilization in vitro and in vivo.31 Also, sperm with fast and linear movements had greater resistance to cryopreservation and are more likely to overcome female reproductive tract barriers to enable fertilization.32
Epididymal bull sperm response to DHA addition was good9,33 and DHA has a role in sperm plasma membrane stabilization in many domestic species.34 Human semen incubated with various concentrations of DHA (7.2, 14.4, 28.8, 57.6, or 115.2 µM) had no significant impact on sperm viability, with the exception of the highest concentration tested (115.2 µM) that was cytotoxic.35 As in the present study, DHA supplement to semen extender also increased progressive motility of boar sperm.36
In Experiment 2, sperm kinetics were significantly different; the vitamin E group had superior results compared to control group. Vitamin E helps to maintain the structural integrity of sperm architecture and prevents mitochondrial dysfunction by inhibiting oxidative stress, preventing reproductive dysfunctions;37 it is an effective antioxidant and has a very important role in protecting the integrity of sperm membrane structure and the structural integrity of mitochondria and tails.37
Oral supplementation of vitamin E improved semen quality in dogs with low seminal plasma superoxide dismutase.38 Vitamin E addition to extender in cattle semen at a concentration of 1 mg/ml provided protection to sperm plasma membrane from bulls with good quality semen, preserved metabolic activity and cell viability.39 Similarly, in boars, a protective effect on post thaw sperm motility and viability was observed when vitamin E was added to the diluter (concentrations of 200 µM1-1) to the extender.40 In dogs, higher values for total motility, rapid steady forward and viability were obtained when vitamin E was added at a concentration of 0.3 mM.1
Treatment groups had lower levels of oxidative stress compared to control group, but catalase was most harmful to canine sperm. A reductive stress effect on mitochondria caused by excess of antioxidants may increase ROS and induce mitochondrial malfunction,41 and it may be the cause for lower levels of progressive motility and mitochondrial activity index in catalase group and higher percentage of high mitochondrial activity and low percentage of low mitochondrial activity in vitamin E + catalase group. Both oxidative stress and reductive stress conditions have detrimental effects on male fertility, resulting in alteration of semen parameters such as concentration, motility, or normal sperm morphology.42 In a recent study, the same authors demonstrated that the establishment of a reductive environment due to high antioxidant concentration has a deleterious impact on sperm motility, vitality, and mitochondrial activity.42 Addition of 20 IU catalase/ml to bull sperm extender improved sperm quality; higher sperm motility, lower sperm DNA fragmentation and ROS production, a higher mitochondrial membrane potential, and higher percentage of sperm with intact acrosome and plasma membranes,43 suggesting that the concentration used in this study was too high. In fact, lower concentrations of antioxidants improved human sperm parameters and chromatin quality when used at a concentration of 100 IU/ml.44
Enzymatic and nonenzymatic antioxidants are efficient and have improved post thaw semen quality. However, for many antioxidants, their function or mechanism of action at the cellular level are not clearly known.45 For this reason, the antioxidant treatment of semen is still a field that needs to be studied. We identified the beneficial effects of vitamin E and the deleterious effects of high concentrations of catalase. In conclusion, the addition of 5 µM DHA and 0.6 mM vitamin E to canine semen extender improved semen quality after cryopreservation.
References
| 1. | Michael A, Alexopoulos C, Pontiki E, et al: Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa. Theriogenology 2007;68:204-212. doi: 10.1016/j.theriogenology.2007.04.053 |
| 2. | Flesch FM, Gadella BM: Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta 2000;1469:197-235. doi: 10.1016/S0304-4157(00)00018-6 |
| 3. | Mandal R, Badyakar D, Chakrabarty J: Role of membrane lipid fatty acids in sperm cryopreservation. Adv Androl 2014;2014:1-9. doi: 10.1155/2014/190542 |
| 4. | Rooke JA, Shao CC, Speake BK: Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen. Reproduction 2001;121:315-322. doi: 10.1530/rep.0.1210315 |
| 5. | Andrade ER, Melo-Sterza FA, Seneda MM, et al: Consequências da produção das espécies reativas de oxigênio na reprodução e principais mecanismos antioxidantes. Rev Bras Reprod Anim 2010;34:79-85. Available from: http://www.cbra.org.br/pages/publicacoes/rbra/v34n2/p79-86.pdf [cited 24 March 2024]. |
| 6. | Parks JE, Lynch DV: Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 1992;29:255-266. doi: 10.1016/0011-2240(92)90024-V |
| 7. | Lenzi A, Gandini L, Picardo M, et al: Lipoperoxidation damage of spermatozoa polyunsaturated fatty acids (PUFA): scavenger mechanisms and possible scavenger therapies. Front Biosci 2000;5:1-15. doi: 10.2741/lenzi |
| 8. | Agarwal A, Saleh RA, Bedaiwy MA: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829-843. doi: 10.1016/S0015-0282(02)04948-8 |
| 9. | Losano JDA, Angrimani DSR, Rui BR, et al: The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls. Zygote 2018b;26:199-206. doi: 10.1017/S0967199418000096 |
| 10. | Lecewicz M, Strzezek R, Kordan W, et al: Effect of extender supplementation with low-molecular-weight antioxidants on selected quality parameters of cryopreserved canine spermatozoa. J Vet Res 2018;62:221-227. doi: 10.2478/jvetres-2018-0032 |
| 11. | Aitken RJ, Krausz C: Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001;122:497-506. doi: 10.1530/rep.0.1220497 |
| 12. | Losano JDA, Angrimani DSR, Dalmazzo A, et al: Effect of vitamin E and polyunsaturated fatty acids on cryopreserved sperm quality in Bos taurus bulls under testicular heat stress. Anim Biotechnol 2018a;29:100-109. doi: 10.1080/10495398.2017.1322973 |
| 13. | Vieira NMG, Losano JDA, Angrimani DSR, et al: Induced sperm oxidative stress in dogs: susceptibility against different reactive oxygen species and protective role of seminal plasma. Theriogenology 2018;108:39-45. doi: 10.1016/j.theriogenology.2017.11.020 |
| 14. | Nordberg J, Arner ES: Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 2001;31:1287-1312. doi: 10.1016/S0891-5849(01)00724-9 |
| 15. | Halliwell B, Gutteridge JM: Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246:501-514. doi: 10.1016/0003-9861(86)90305-X |
| 16. | Bjorneboe A, Bjorneboe GE, Drevon CA: Absorption, transport and distribution of vitamin E. J Nutr 1990;120:233-242. doi: 10.1093/jn/120.3.233 |
| 17. | Mates JM: Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 2000;153:83-104. doi: 10.1016/S0300-483X(00)00306-1 |
| 18. | Bendich A: Antioxidant micronutrients and immune responses. Ann NY Acad Sci 1990;587:168-180. doi: 10.1111/j.1749-6632.1990.tb00144.x |
| 19. | Perly B, Smith IC, Hughes L, et al: Estimation of the location of natural alpha-tocopherol in lipid bilayers by 13C-NMR spectroscopy. Biochim Biophys Acta 1985;819:131-135. doi: 10.1016/0005-2736(85)90203-2 |
| 20. | Bradford A, Atkinson J, Fuller N, et al: The effect of vitamin E on the structure of membrane lipid assemblies. J Lipid Res 2003;44:1940-1945. doi: 10.1194/jlr.M300146-JLR200 |
| 21. | Griveau JF, Le Lannou D: Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 1997;20:61-69. doi: 10.1046/j.1365-2605.1997.00044.x |
| 22. | Aitken RJ, Buckingham D, Harkiss D: Use of a xanthine-oxidase free-radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441-450. doi: 10.1530/jrf.0.0970441 |
| 23. | Griveau JF, Dumont E, Renard P, et al: Reactive oxygen species, lipid-peroxidation and enzymatic defense systems in human spermatozoa. J Reprod Fertil 1995;103:17-26. doi: 10.1530/jrf.0.1030017 |
| 24. | Verstegen J, Iguer-Ouada M, Onclin K: Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002;57:149-179. doi: 10.1016/S0093-691X(01)00664-1 |
| 25. | de Castro LS, de Assis PM, Siqueira AFP, et al: Sperm oxidative stress is detrimental to embryo development: A dose-dependent study model and a new and more sensitive oxidative status evaluation. Oxid Med Cell Longev 2016;1:8213071. doi: 10.1155/2016/8213071 |
| 26. | Hrudka F: Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress. Int J Androl 1987;10:809-828. doi: 10.1111/j.1365-2605.1987.tb00385.x |
| 27. | Evenson D, Jost L: Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22:169-189. doi: 10.1023/A:1009844109023 |
| 28. | Lucio CF, Regazzi FM, Silva LCG, et al: Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs. Theriogenology 2016;85:1568-1575. doi: 10.1016/j.theriogenology.2016.01.016 |
| 29. | Nichi M, Goovaerts IGF, Cortada CNM, et al: Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C. Theriogenology 2007;67:334-340. doi: 10.1016/j.theriogenology.2006.08.002 |
| 30. | Strzezek R, Koziorowska-Gilun M, Stawiszynska M: Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa. Pol J Vet Sci 2012;15:721-726. doi: 10.2478/v10181-012-0109-0 |
| 31. | Mortimer ST: A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997;3:403-439. doi: 10.1093/humupd/3.5.403 |
| 32. | Martins MIM, Souza AK, Trautwein LGC: Subpopulações espermáticas. Rev Bras Reprod Anim 2017;41:243-247. Available from: http://www.cbra.org.br/portal/downloads/publicacoes/rbra/v41/n1/p243-247%20(RB653).pdf [cited 24 March 2024]. |
| 33. | Kaka A, Haron W, Yusoff R, et al: Effect of docosahexanoic acid on quality of frozen-thawed bull semen in BioXcell extender. Reprod Fertil Dev 2017;29:490-495. doi: 10.1071/RD15089 |
| 34. | Chanapiwat P, Kaeoket K, Tummaruk P: Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen. Asian J Androl 2009;11:600-608. doi: 10.1038/aja.2009.40 |
| 35. | Aitken RJ, Wingate JK, De Iuliis GN, et al: Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. J Clin Endocrinol Metab 2006;91:4154-4163. doi: 10.1210/jc.2006-1309 |
| 36. | Kaeoket K, Sang-urai P, Thamniyom A, et al: Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds. Reprod Domest Anim 2010;45:458-463. doi: 10.1111/j.1439-0531.2008.01239.x |
| 37. | Wang N, Qian HY, Zhou XQ, et al: Mitochondrial energy metabolism dysfunction involved in reproductive toxicity of mice caused by endosulfan and protective effects of vitamin E. Ecotoxicol Environ Saf 2012;82:96-103. doi: 10.1016/j.ecoenv.2012.05.014 |
| 38. | Kawakami E, Kobayashi M, Hori T, et al: Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma. J Vet Med Sci 2016;77:1711-1714. doi: 10.1292/jvms.15-0294 |
| 39. | Beconi MT, Francia CR, Mora NG, et al: Effect of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841-851. doi: 10.1016/0093-691X(93)90219-U |
| 40. | Pena FJ, Johannisson A, Wallgren M, et al: Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci 2003;78:85-98. doi: 10.1016/S0378-4320(03)00049-6 |
| 41. | Angrimani DSR, Nichi M, Brito MM, et al: The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs. Arq Bras Med Vet Zootec 2018;70:419-428. doi: 10.1590/1678-4162-9684 |
| 42. | Panner Selvam MK, Agarwal A, Henkel R, et al: The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med 2020;152:375-385. doi: 10.1016/j.freeradbiomed.2020.03.008 |
| 43. | Arslan HO, Herrera C, Malama E, et al: Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm. Cryobiology 2019;91:40-52. doi: 10.1016/j.cryobiol.2019.10.200 |
| 44. | Mohammadzadeh M, Ramazani V, Khalili MA, et al: Medium containing different concentrations of catalase as a strategy for optimising sperm parameters and chromatin in normospermic persons. Andrologia 2019;51:1-6. doi: 10.1111/and.13231 |
| 45. | Kumar A, Prasad JK, Srivastava N, et al: Strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreserv Biobank 2019;17:603-612. doi: 10.1089/bio.2019.0037 |
Appendix
Figure. A gate referred to as ‘spitz’ was defined based on green fluorescence intensity and particle size, using a logarithmic scale, to encompass the main sperm population (A), when both green and red fluorescence signals were simultaneously applied to the same population 4 distinct subpopulations were defined (B); IAIM (intact acrosome/intact plasma membrane), IADM (intact acrosome/damaged plasma membrane), DAIM (damaged acrosome/intact plasma membrane), and DADM (damaged acrosome/damaged plasma membrane).