The Effect of Adding Different Levels of Reduced Glutathione to Extender on the Quality of Cooled Ring-Necked Pheasant Semen

Abstract

Aim:

Artificial propagation of ring-necked pheasant through semen preservation is of significance, as this species is facing enormous threats in its natural habitat. Semen preservation inevitably induces oxidative stress, and exogenous antioxidants need to be investigated for the preservation of ring-necked pheasant semen. Therefore, the current study was conducted to investigate the role of glutathione (GSH) in extender on the liquid storage of ring-necked pheasant semen.

Materials and Methods:

Semen was collected from 10 sexually mature males, evaluated for sperm motility, and pooled. Pooled semen was aliquoted for dilution with Beltsville poultry semen extender (1:5) at 37°C having GSH levels of 0.0 mM (Control), 0.2, 0.4, 0.6, and 0.8 mM. Extended semen was gradually cooled to 4°C and stored in a refrigerator (4°C) for 48 hours. Semen quality, that is, sperm motility, membrane integrity, viability, acrosomal integrity, and DNA integrity, was assessed at 0, 2, 6, 24, and 48 hours.

Results:

Sperm motility (%), plasma membrane integrity (%), viability (%), and acrosomal integrity (%) were recorded higher (p < 0.05), whereas DNA fragmentation (%) was recorded lower in extender supplemented with 0.4 mM GSH up to 48 hours of storage compared with 0.2, 0.6, and 0.8 mM GSH concentrations and control.

Conclusion:

It is concluded that 0.4 mM GSH in extender improves sperm quality parameters of ring-necked pheasant during liquid storage up to 48 hours at 4°C.

Introduction

The ring-necked pheasant also called common pheasant (Phasianus colchicus) belongs to the family “Phasianidae,”¹ native to Eurasia,² and is considered a well-known game bird. In addition to its regional significance, it is the ancient one in the world and is listed as least concern under IUCN red list of threatened species.³ However, the ring-necked pheasant is one of the world's most hunted birds and is used for meat purpose in several countries.^4,5

Further, remarkable transformations in farming practices, excessive use of agriculture related chemicals, increased grazing pressures, spraying and mowing of highways, and utility rights-of-ways during the last half of the 20th century have contributed a lot toward reducing this bird's population in the wild. The ring-necked pheasant's high dependence on habitats and niches in or around croplands/agricultural landscapes is driving it toward risk of declining. The major factors include conversion of open, indigenous grasslands and other unused habitats to managed grasslands, removal of luxuriant hedgerows, placement of fence rows, and clean farming acts that lead to a loss of its nests and protective covers.^6,7

Conservation of species beforehand has key importance in the management of declining populations.⁸ To achieve this, ex-situ conservation in conjunction with in-situ conservation programs may prove useful specifically under current scenario of habitat degradation. Ex-situ in-vitro management practices include germ cell storage for future use in assisted reproductive technologies, more commonly the cryopreserved semen. However, the liquid storage of semen has its own advantages; specifically, it induces lesser damage to sperm compared with freezing procedures. Liquid stored semen (5°C) serves to acclimatize in-vitro sperm storage conditions, primarily suppress the metabolism for days, and lower sperm energy utilization to enable survival for a longer time span.^9,10

In addition, liquid storage can generate 10 times higher dose number of ejaculated semen compared with frozen storage, which improves the breeding capacity of animals and brings about the balance of genetic variability among propagated individuals. Hence, the requirement of a lesser number of semen ejaculates with higher dose rates reduces the burden of rearing many males. Further, the expense of processing liquid stored semen is much less compared with cryopreservation due to exclusion of some equipment and continuous supply of liquid nitrogen.¹⁰

The sperm of Galliformes have unique physiology and comprise narrower sperm head width and smaller volume of cytoplasm, which makes them more vulnerable to the damage during preservation.¹¹ Specifically, avian sperm is more vulnerable to per-oxidative damage due to the high content of poly unsaturated fatty acids in its membrane, which adds on to the pathological state of in-vitro stored sperm.^11–13

Naturally, the imbalance of reactive oxygen species (ROS) is prevented by a well-defined antioxidant system of avian semen¹⁴; however, during in-vitro storage, this system is affected and brings several damaging effects, including per-oxidative damage to plasma membrane and chipping of DNA strand in sperm.^15,16 It is found that the level of antioxidants is reduced due to dilution with the media.^17,18 This depletion of natural antioxidants could be managed through augmentation of exogenous alternates.^19,20

One of the key antioxidants of semen is glutathione (l-g-glutamyl-l-cysteinyl glycine; GSH), a tripeptide, serving an indispensable role as an intracellular defense tool versus oxidative stress especially in its reduced form.^16,21 GSH is viewed as a non-enzymatic antioxidant, protecting sperm by either lowering ROS synthesis or shielding the di-sulfide bonds.^22,23 Therefore, it is quite logical to consider GSH as a protector for sperm and enhancer of post-thaw motility as well as acrosomal integrity.¹⁸ The current study was designed to investigate the role of GSH in extender on the liquid storage of ring-necked pheasant semen.

Materials and Methods

Ethical statement

The study was approved by the Ethics Committee of Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan, for the use of animals.

Animal management

Ten adult male Ring-necked Pheasants (P. colchicus) were managed at Avian Research Station, PMAS Arid Agriculture University Rawalpindi. Birds were reared under a natural photoperiod, that is, 16 hours of light and 8 hours of dark and temperature conditions in individual sand floor pens of 106.68 × 121.92 cm with roof sleeve. Each bird was provided with 100 g of commercially available poultry cock breeder feed daily, and fresh water was provided ad libitum during the experimentation period.

Semen collection and preliminary evaluation

Semen was collected in semen collection vials by massage technique during morning hours of the day.²⁴ Micropipette (μL) was used to measure semen volume. Each ejaculate's sperm motility was determined initially as described by Zemjanis.²⁵ Percentage of motile spermatozoa was assessed by placing a drop of semen sample on pre-warmed glass slide (37°C) and observed under a phase-contrast microscope at 400 × (Olympus BX20, Japan). Sperm concentration was measured by using 1 μL of semen and 200 μL of formal citrate solution (1 mL of 37% formaldehyde in 99 mL of 2.9% [w/v] sodium citrate) under a phase-contrast microscope (400 × ; Olympus BX20) through Neubauer's hemocytometer (Marienfeld, Germany).

Extender composition and sample processing

Beltsville Poultry Semen Extender (BPSE) was prepared by adding fructose (0.3 g), potassium citrate (0.0384 g), sodium glutamate (0.5202 g), magnesium chloride (0.0204 g), di-potassium hydrogen phosphate (0.7620 g), potassium di-hydrogen phosphate (0.039 g), sodium acetate (0.258 g), and N-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid (0.3170 g) to 100 mL of double-distilled water (pH 7.3, osmotic pressure 330 mOsm/kg). BPSE extender was supplemented with five different concentrations of reduced glutathione, that is, 0.0, 0.2, 0.4, 0.6, and 0.8 mM. Qualifying ejaculates (>60% motility) were pooled. Five semen aliquots were made and diluted 1:5 with BPSE. Extended semen was cooled in 2 hours from 37°C to 4°C and kept for 48 hours in a refrigerator (4°C). Hence, samples were assessed for sperm motility, sperm membrane integrity, viability, acrosomal integrity, and sperm DNA fragmentation at 0, 2, 6, 24, and 48 hours of liquid storage.

Semen quality parameters

Sperm motility

Percent sperm motility was measured on a scale of 0% to 100% by placing a droplet of diluted sample on a pre-warmed slide (37°C) that was covered by a cover slip. The slide was examined under a phase-contrast microscope (400 × ).²⁵

Sperm membrane integrity

Plasma membrane integrity of Ring-necked Pheasant spermatozoa was evaluated using Hypo-osmotic swelling test.²⁶ Hypo-osmotic swelling (HOS) solution was made by diluting 1 g of sodium citrate with 100 mL of distilled water. A 25 μL diluted semen sample was mixed with 500 μL of HOS solution (100 mOsm/kg) and was incubated at 37°C for 30 minutes. A droplet of this solution was fixed in buffered 2% glutaraldehyde on a pre-warmed glass slide. Spermatozoa with swollen heads and swollen plus coiled tails were considered normal with intact plasma membranes under a phase-contrast microscope (1000 × with oil immersion). A total of 200 spermatozoa were counted at five different fields on a slide.

Sperm viability

Sperm viability of Ring-necked Pheasant spermatozoa was obtained using water soluble eosin (1 g) and water-soluble nigrosine (5 g), which was added to Lake's glutamate solution.²⁷ Lake's glutamate solution was made by adding potassium citrate (0.00128 g), sodium glutamate (0.01735 g), magnesium chloride (0.000676 g), and sodium acetate (0.0085 g) to 100 mL distilled water. Then, 12 drops of stain were homogenized with 1 droplet of sample; smear was formed and air dried. Smear was observed under a phase-contrast microscope (1000 × with oil immersion). Observed spermatozoa were classified into two categories: alive (physically normal and unstained) and dead (totally or partially stained). A total of 200 spermatozoa were observed at five separate fields on a slide.¹⁸

Sperm acrosomal integrity

Sperm acrosomal integrity of Ring-necked Pheasant was evaluated via Giemsa stain.^8,28 Stain was made by mixing Giemsa (3 g) and phosphate-buffered saline (PBS) at pH 7.0 (2 mL) into 35 mL water. Five microliters of semen sample was used to make smear on a glass slide and then air dried and fixed by neutral formal saline (5% formaldehyde) for 30 minutes. Fixed slides were immersed in Giemsa stain for 1.5 hours. Sperm having normal acrosome were evenly stained, whereas sperm having abnormal acrosomes were unevenly stained and sperm with ruptured acrosomes remained unstained. A sum of 200 spermatozoa was observed in five different fields under a phase-contrast microscope ( × 400; Olympus BX20) at 1000 × with oil immersion.

Sperm DNA fragmentation

Sperm DNA fragmentation was determined by aniline blue stain. Staining solution was prepared by mixing 5 g of aniline blue to 100 mL of PBS. Then stain was filtered, and its pH was adjusted to 3.5 with the addition of 2% glacial acetic acid. Ten microliters of semen was smeared on a glass slide and air dried. Slides were stained for 5 minutes with aniline blue stain and then rinsed with distilled water and air dried. A total of 200 spermatozoa was observed using a phase-contrast microscope (1000 × with oil immersion). The spermatozoa stained dark blue were regarded as having fragmented DNA.²⁹

Statistical analysis

Data are presented as mean ± SEM. The data on different concentrations of reduced glutathione in extender during liquid storage of Ring-necked pheasant semen were analyzed through two-factor factorial analysis of variance in MSTAT-C^® software (version 1.42; Michigan State University, East Lansing, MI) in a completely randomized design. When F-ratio was found significant (p < 0.05), a comparison of means was done by fisher-protected least-significant difference test.

Results

Effect of GSH on sperm motility during liquid storage

The data on the effect of different concentrations of GSH in extender on sperm motility at different hours (0, 2, 6, 24, and 48) of liquid storage are presented in Figure 1. Addition of GSH in extender at the concentration of 0.4 mM was found to maintain significantly higher sperm motility (p < 0.05) at all the storage hours compared with other concentrations of GSH, that is, 0.2, 0.6, 0.8 mM, and control. At all hours, lower concentrations of GSH, that is, 0.2 and 0.4 mM were found to increase sperm motility, whereas higher concentrations of GSH, that is, 0.6 and 0.8 mM resulted in a gradual decrease of sperm motility though it remained higher compared with the control.

FIG. 1.

Effect of GSH concentrations (0.0, 0.2, 0.4, 0.6 and 0.8 mM) on sperm motility (%) of Ring-necked pheasant semen at different hours of liquid storage. Bars with different letters show significant differences (p < 0.05) throughout the storage hours. GSH, glutathione.

Effect of GSH on sperm membrane integrity during liquid storage

The data on the effect of addition of different concentrations of GSH in extender on plasma membrane integrity of spermatozoa at different hours (0, 2, 6, 24, and 48) of liquid storage are presented in Figure 2. At all storage hours, sperm plasma membrane integrity was improved with the supplementation of GSH at all levels compared with control. However, GSH supplementation at lower levels, that is, 0.2 and 0.4 mM were found to improve plasma membrane integrity compared with further higher concentrations of GSH, that is, 0.6 and 0.8 mM. Sperm plasma membrane integrity (%) was found to be significantly higher (p < 0.05) with the addition of GSH at the concentration of 0.4 mM in the extender in comparison to other concentrations (0.2, 0.6, 0.8 mM) and control at all hours of liquid storage.

FIG. 2.

Effect of GSH concentrations (0.0, 0.2, 0.4, 0.6, and 0.8 mM) on plasma membrane integrity (%) of ring-necked pheasant spermatozoa at different hours of liquid storage. Bars with alphabetical letters show significant differences (p < 0.05) throughout the storage hours.

Effect of GSH on sperm viability during liquid storage

The data on the effect of various concentrations of GSH in extender on sperm viability at different hours (0, 2, 6, 24, and 48) of liquid storage are presented in Figure 3. Addition of GSH in extender at 0.4 mM was recorded to maintain higher (p < 0.05) sperm viability at all the storage hours compared with other concentrations of GSH (0.2, 0.6, 0.8 mM) and control. The trend for sperm viability remained the same as for both sperm motility and plasma membrane integrity. The sperm viability was improved with the addition of GSH at lower concentrations (0.2 and 0.4 mM) compared with higher concentrations (0.6 and 0.8 mM), though it remained higher compared with control at all concentrations throughout the storage period.

FIG. 3.

Effect of GSH concentrations' (0.0, 0.2, 0.4, 0.6, and 0.8 mM) supplementation on sperm viability of ring-necked pheasant spermatozoa at different hours of liquid storage. Bars with various letters represent significant differences (p < 0.05) throughout the storage hours.

Effect of GSH on sperm acrosomal integrity during liquid storage

The data on the effect of supplementing different concentrations of GSH in extender on acrosome integrity of spermatozoa at different hours (0, 2, 6, 24, and 48) of liquid storage are displayed in Figure 4. Addition of GSH in extender at the concentration of 0.4 mM was found to maintain acrosomal integrity significantly higher (p < 0.05) compared with other concentrations of GSH (0.2, 0.6, 0.8 mM) and control at all hours. Improvement in acrosomal integrity was evident at lower concentrations of GSH, that is, 0.2 and 0.4 mM; however, higher concentrations of GSH (0.6 and 0.8 mM) resulted in decreased acrosome integrity at all storage hours though it remained higher compared with control.

FIG. 4.

Effect of different concentrations of GSH (0.0, 0.2, 0.4, 0.6, and 0.8 mM) on sperm acrosomal integrity of ring-necked pheasants at several hours of liquid storage (4°C). Error bars with letters depict significant differences (p < 0.05) throughout the storage hours.

Effect of GSH on sperm DNA fragmentation during liquid storage

The data on the effect of supplementing various concentrations of GSH in extender on sperm DNA fragmentation at different hours (0, 2, 6, 24, and 48) of liquid storage are presented in Figure 5. Addition of GSH in extender at the concentration of 0.4 mM was found to reduce (p < 0.05) DNA fragmentation compared with other concentrations of GSH (0.2, 0.6, 0.8 mM) and control at all observed hours. It was viewed that with the lower GSH levels there was a decrease in DNA fragmentation, that is, 0.2 and 0.4 mM but further higher concentrations of GSH; that is, 0.6 and 0.8 mM were found to gradually increase the DNA fragmentation at all storage hours. However, DNA fragmentation remained lower than the control.

FIG. 5.

Effect of supplementation of GSH concentrations (0.0, 0.2, 0.4, 0.6, and 0.8 mM) on sperm DNA fragmentation of ring-necked pheasant. Error bars along with letters depict significant variations (p < 0.05) throughout the storage hours.

Discussion

Glutathione is an intracellular defense tool that is used for combating oxidative stress, especially in its reduced form. Many studies have reported the positive impact of GSH supplementation in liquid stored^30–33 mammalian sperm, whereas fewer studies are available on liquid-stored avian semen. Further, among avian species, the impact of antioxidants is mainly studied in liquid-stored semen from different chicken breeds.^34–36 Therefore, the full potential of avian semen to tolerate conditions of liquid storage is yet to be explored, as avian sperm has a long head and little cytoplasm, making it more sensitive to low temperatures compared with the sperm of other species.³⁷

In-vitro manipulation of avian semen for successful implementation of assisted reproductive techniques requires dilution with suitable media and appropriate storage temperatures, which could provide sperm with a minimum shelf-life of 2 to 4 days.¹⁰ In the present study, liquid storage of ring-necked pheasant semen was performed for 48 hours at 4°C and time-dependent decline was observed in semen quality at 0, 2, 6, 24, and 48 hours with maximum semen quality recorded at initial storage hours. Extended storage hours and days for liquid preservation of semen severely affect sperm characteristics due to oxidative stress³⁸ and decline of antioxidants.³⁹

In such cases, supplementation of exogenous antioxidants is required to maintain the semen quality for maximum utility of liquid-stored semen.^40,41 Reduced glutathione is one of the major antioxidants in semen and can be supplemented in media to replenish its level during in-vitro semen preservation.^42–44 According to current results, semen quality is observed maximum at 0.4 mM GSH and found to decline above this level; however, it remained higher compared with the control. Structurally, reduced glutathione is a natural molecule in sperm cells,⁴⁵ composed of cysteine, glutamate, and glycine amino acids. It is utilized to maintain membrane proteins and nucleic acids due to its redox power.^46–48

The acrosome and sperm membranes are considered significant predictors of fertility^49–51 and these are the primary sperm structures affected due to altered physiology of in-vitro stored semen.^50,52 Lipid peroxidation and protein denaturation in plasma membrane is associated with the loss of sperm motility,^47,53 whereas antioxidants provide longevity and acrosomal intactness to spermatozoa by preventing ROS buildup until acrosomal reaction.^53,54

The present study reported maintenance of motility, intact acrosome, and plasma membrane altogether with GSH fortification of extender for liquid storage of ring-necked pheasant semen at all stages. The avian sperm are more sensitive to cryodamage due to their elongated shape; hence, the anti-oxidative impact of GSH on avian sperm amplifies during initial storage time,¹⁸ whereas for mammalian sperm GSH is reported to show significant effects after temperature decline/freezing.⁵⁵ Still there are minor reports of immediate GSH influence on mammalian semen in terms of livability and intact acrosome.⁵⁶

Further, GSH was reported to exert beneficial effects on diluted/cooled cockerel semen in terms of semen quality parameters regardless of the prolonged storage period (72 hours) compared with control.⁵⁴ It has previously been reported that without exogenous antioxidants imbalance of saturated to unsaturated fatty acids ratio and loss of cholesterol⁵⁷ results in the leakage of cell constituents.

The viable sperm with intact DNA succeed toward infundibulum and fuse with the ovum^8,58,59; however, the ROS react with normal metabolic components in the absence of anti-oxidative checkpoints and cause telomere reduction, DNA epigenetic changes, and Y chromosomal micro-deletions⁶⁰ with serious repercussions on post-fertilization development.⁶¹ It is well known that GSH protects against ROS and electrophiles by its sulfhydryl group, which is a strong nucleophile.⁶² The current results showed that sperm viability and DNA integrity consistently remained higher with 0.2 and 0.4 mM GSH from 0 to 48 hours.

It indicates that GSH maintained sperm physiology and DNA structure in its original state soon after dilution and during liquid storage. Previous studies reported reduced DNA damage and higher fertility rates due to GSH addition in liquid storage of cockerel,⁵⁴ turkey,³⁷ rooster,⁶³ and buffalo semen.^64,65 The DNA damage is correlated with ROS levels^37,66 and the role of GSH as antioxidant for in-vitro stored semen has been reported.

Possibly, the natural GSH in control was exhausted in the current study, resulting in a faster decline of sperm DNA integrity compared with GSH-treated groups. However, excessive GSH may disrupt the normal metabolic needs of cell and produce ill-effects due to the absence of physiological levels of ROS.¹⁸ This study reports the efficiency of glutathione for liquid storage of ring-necked pheasant semen and its inclusion at 0.4 mM of extender was identified best for storage up to 48 hours. These findings pave a way toward investigation beyond cooling and for evaluation of reduced glutathione in extender in terms of quality and fertility of cryopreserved ring-necked pheasant semen.

Conclusion

It is concluded that 0.4 mM of GSH in extender enhances motility, membrane integrity, viability, and acrosomal integrity whereas it lowers DNA fragmentation of ring-necked pheasant sperm during liquid storage at 4°C.

Footnotes

Authors' Contributions

S.Z.: Management of Study Animals (lead), Investigation (lead), Formal Analysis (lead), Methodology (lead), Writing-Original draft (lead). B.A.R.: Conceptualization (lead), Project administration (lead), Methodology (lead), Supervision (lead). S.A.: Resources (lead), Visualization (lead), Validation (lead), Writing-Review and editing (equal). M.S.A.: Data Curation (lead), Validation (supporting), Writing-Review and Editing (equal) K.W.: Management of Study Animals (Supporting), Formal Analysis (Supporting).

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

References

Jobling

JA.

The Helm Dictionary of Scientific Bird Names. Christopher Helm: London, LDN; 2010.

, Liu

, Bao

, et al. Phylogeography of the ring-necked pheasant (Phasianus colchicus) in China. Mol Phylogenet Evol, 2009; 52(1):125–132; doi: https://doi.org/10.1016/j.ympev.2009.03.015

The IUCN Red List of Threatened Species, Phasianus colchicus. 2016. Available from: https://www.iucnredlist.org/en [Last accessed: November 11, 2022].

Robertson

PA.

Pheasants. Voyageur Press: Minnesota, MN; 1997.

Dos Santos Schmidt

, Paulillo

, Santin

, et al. Hematological and serum chemistry values for the ring-necked pheasant (Phasianus colchicus): Variation with sex and age. Int J Poult Sci, 2007; 6:137–139; doi: 10.3923/ijps.2007.137.139

Smith

, Stewart

, Gates

. Home ranges, habitat selection and mortality of ring-necked pheasants (Phasianus colchicus) in north-central Maryland. Am Midl Nat, 1999; 141(1):185–197.

Pauly

, Runia

, Solem

, et al. Ring-necked pheasant nest success and habitat selection in central South Dakota. Gt Plains Res, 2018; 28(1):39–50.

Rakha

, Ansari

, Hussain

, et al. Comparison of extenders for liquid storage of Indian Red Jungle Fowl (Gallus gallus murghi) spermatozoa. Avian Biol Res, 2016; 9(3):207–212; doi: 10.3184/175815516X14679871861418

Vishwanath

, Shannon

. Storage of bovine semen in liquid and frozen state. Anim Reprod Sci, 2000; 62(1–3):23–53; doi: 10.1016/S0378-4320(00)00153-6

10.

Yang

, Standley

, Xu

. Application of liquid semen technology under the seasonal dairy production system in New Zealand. Anim Reprod Sci, 2018; 194:2–10; doi: 10.1016/j.anireprosci.2018.01.004

11.

Donoghue

, Wishart

. Storage of poultry semen. Anim Reprod Sci, 2000; 62(1–3):213–232; doi: 10.1016/S0378-4320(00)00160-3

12.

Dimitrov

, Atanasov

, Surai

, et al. Effect of organic selenium on turkey semen quality during liquid storage. Anim Reprod Sci, 2007; 100(3–4):311–317; doi: 10.1016/j.anireprosci.2006.07.007

13.

Partyka

, Lukaszewicz

, Niżański

, et al. Detection of lipid peroxidation in frozen-thawed avian spermatozoa using C11-BODIPY581/591. Theriogenology, 2011; 75(9):1623–1629; doi: 10.1016/j.theriogenology.2011.01.002

14.

Partyka

, Lukaszewicz

, Niżański

. Lipid peroxidation and antioxidant enzymes activity in avian semen. Anim Reprod Sci, 2012; 134(3–4):184–190; doi: 10.1016/j.anireprosci.2012.07.007

15.

Aitken

, De Iuliis

, Finnie

, et al. Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria. Hum Reprod, 2010; 25(10):2415–2426; doi: 10.1093/humrep/deq214

16.

Masoudi

, Sharafi

, Shahneh

, et al. Effects of reduced glutathione on the quality of rooster sperm during cryopreservation. Theriogenology, 2019; 128:149–155; doi: 10.1016/j.theriogenology.2019.01.016

17.

Ansari

, Rakha

, Ullah

, et al. Effect of exogenous glutathione in extender on the freezability of Nili-Ravi buffalo (Bubalus bubalis) bull spermatozoa. Anim Sci Pap Rep, 2010; 28(3):235–244.

18.

Ansari

, Rakha

, Akhter

, et al. Effect of glutathione on pre and post-freezing sperm quality of Indian red jungle fowl (Gallus gallus murghi). Theriogenology, 2021; 172:73–79; doi: 10.1016/j.theriogenology.2021.06.008

19.

Gadea

, García-Vazquez

, Matás

, et al. Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function. J Androl, 2005; 26(3):396–404; doi: 10.2164/jandrol.04155

20.

Bucak

, Sarıözkan

, Tuncer

, et al. The effect of antioxidants on post-thawed Angora goat (Capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Rumin Res, 2010; 89(1):24–30; doi: 10.1016/j.smallrumres.2009.11.015

21.

Irvine

. Glutathione as a treatment for male infertility. Rev Reprod, 1996; 1(1):6–12; doi: 10.1530/revreprod/1.1.6

22.

Alvarez

, Storey

. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res, 1989; 23(1):77–90; doi: 10.1002/mrd.1120230108

23.

Yeste

, Flores

, Estrada

, et al. Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze–thawing by stabilising disulfide bonds. Reprod Fertil Dev, 2013; 25(7):1036–1050; doi: 10.1071/RD12230

24.

Burrows

, Quinn

. The collection of spermatozoa from the domestic fowl and turkey. Poult Sci, 1937; 16(1):19–24; doi: 10.3382/ps.0160019

25.

Zemjanis

Diagnostic and Therapeutic Techniques in Animal Reproduction. William & wilkins, 2nd ed.. Philadelphia, PA; 1970.

26.

Uysal

, Bucak

. Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Vet Brno, 2007; 76(3):383–390; doi: 10.2754/avb200776030383

27.

Bakst

, Cecil

. Techniques for Semen Evaluation, Semen Storage, and Fertility Determination. 3. Sperm Acrosomal Integrity. I. Nigrosin/Eosin Stain for Determining Live/Dead and Abnormal Sperm Counts. Savoy, IL: The Poultry Science Association, Inc., 1997; pp. 29–34.

28.

Liu

, Zhang

. Methods and effects of Hongshan cock spermatozoa cryopreservation. Wuhan Univ J Nat Sci, 2006; 11(2):447–450.

29.

Ansari

, Rakha

, Akhter

, et al. Effect of cryopreservation on lipid peroxidation, antioxidant potential, chromatin integrity, and mitochondrial activity of Indian red jungle fowl (Gallus gallus murghi) semen. Biopreserv Biobank, 2019; 17(4):288–295; doi: 10.1089/bio.2018.0111

30.

Zhang

, Liu

, Wang

, et al. Effects of glutathione on sperm quality during liquid storage in boars. Anim Sci J, 2016; 87(10):1195–1201.

31.

Bucak

, Tekin

. Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rumin Res, 2007; 73(1–3):103–108; doi: 10.1016/j.smallrumres.2006.12.001

32.

Shi

, Jin

, Hu

, et al. Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5°C. Small Rumin Res, 2020; 189:106139l; doi: 10.1016/j.smallrumres.2020.106139

33.

Mata-Campuzano

, Álvarez-Rodríguez

, Tamayo-Canul

, et al. Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: Effect of temperature, extender and storage time. Anim Reprod Sci, 2014; 151(3–4):137–147; doi: 10.1016/j.anireprosci.2014.10.006

34.

Iswati

, Susilawati

. Effect of addition of glutathione in diluent ringer's on spermatozoa quality of domestic chicken during cold storage. AJMBES, 2018; 20(1):12–20.

35.

Siudzińska

, Łukaszewicz

. Effect of semen extenders and storage time on sperm morphology of four chicken breeds. JAPR, 2008; 17(1):101–108; doi: 10.3382/japr.2007-00048

36.

Gopi

, Beulah

, Prabakar

, et al. Polyphenols as an antioxidant agent improves chicken sperm qualities during cold storage. JAPS, 2020; 30(6):1653–1658; doi: 10.36899/JAPS.2020.6.0186

37.

Izanloo

, Soleimanzadeh

, Bucak

, et al. The effects of varying concentrations of glutathione and trehalose in improving microscopic and oxidative stress parameters in Turkey semen during liquid storage at 5°C. Cryobiology, 2021; 101:12–19; doi: 10.1016/j.cryobiol.2021.07.002

38.

Johnson

, Weitze

, Fiser

, et al. Storage of boar semen. Anim Reprod Sci, 2000; 62(1–3):143–172; doi: 10.1016/S0378-4320(00)00157-3

39.

Sikka

. Oxidative stress and role of antioxidants in normal and abnormal sperm function. Front Biosci, 1996; 1:e78–e86; doi: 10.2741/a146

40.

Zhang

, Yi

, Chen

, et al. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. Anim Reprod Sci, 2012; 132(3–4):123–128; doi: 10.1016/j.anireprosci.2012.05.009

41.

Chankitisakul

. Supplementation of a commercial extender (BTS) with melatonin to improve quality of boar semen in cooling storage at 15 C. Khon Kaen Agr J, 2014; 42(4):617–626.

42.

Funahashi

, Sano

. Select antioxidants improve the function of extended boar semen stored at 10°C. Theriogenology, 2005; 63(6):1605–1616; doi: 10.1016/j.theriogenology.2004.06.016

43.

Kaeoket

, Tantiparinyakul

, Kladkaew

, et al. Effect of different antioxidants on quality of cryopreserved boar semen in different breeds. Thai J Agri Sci, 2008; 41(1–2):1–9.

44.

Clarkson

, Thompson

. Antioxidants: what role do they play in physical activity and health?. Am J Clin Nutr, 2000; 72(2):637S–646S; doi: 10.1093/ajcn/72.2.637S

45.

Bucak

, Ateşşahin

, Yüce

. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze–thawing process. Small Rumin Res, 2008; 75(2–3):128–134; doi: 10.1016/j.smallrumres.2007.09.002

46.

Luberda

. The role of glutathione in mammalian gametes. Reprod Biol, 2005; 5(1):5–17.

47.

Dickinson

, Forman

. Cellular glutathione and thiols metabolism. Biochem Pharmacol, 2002; 64(5–6):1019–1026; doi: 10.1016/S0006-2952(02)01172-3

48.

Gadea

, Molla

, Selles

, et al. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology, 2011; 62(1):40–46; doi: 10.1016/j.cryobiol.2010.12.001

49.

Jones

, Mann

. Damage to ram spermatozoa by peroxidation of endogenous phospholipids. Reproduction, 1977; 50(2):261–268; doi: 10.1530/jrf.0.0500261

50.

Barthelemy

, Royere

, Hammahah

, et al. Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation. Arch Androl, 1990; 25(1):29–40; doi: 10.3109/01485019008987591

51.

Esteves

, Verza Jr

. Relationship of in vitro acrosome reaction to sperm function: An update. Open Reprod Sci J, 2011; 3(1):72–84; doi: 10.2174/1874255601103010072

52.

Liu

, Dong

, Ma

, et al. Effects of pH during liquid storage of goat semen on sperm viability and fertilizing potential. Anim Reprod Sci, 2016; 164:47–56; doi: 10.1016/j.anireprosci.2015.11.011

53.

Maxwell

, Stojanov

. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod Fertil Dev, 1996; 8(6):1013–1020; doi: 10.1071/RD9961013

54.

Shamiah

, El-Karim

, Ragaa

, et al. Effect of glutathione supplementation to semen extender on cockerel sperm characteristics in semen stored at 5°C for different periods. EJAP, 2017; 54(1):41–46; doi: 10.21608/ejap.2017.93288

55.

Sinha

, Sinha

, Singh

, et al. The effect of glutathione on the motility, enzyme leakage and fertility of frozen goat semen. Anim Reprod Sci, 1996; 41(3–4):237–243. doi: 10.1016/0378-4320(95)01450-0

56.

Solouma

GMA

. The influence of adding glutathione on semen characteristics of sohagi rams. EJSGS, 2013; 8(2): 61–68; doi: 10.21608/ejsgs.2013.26776

57.

Falchi

, Galleri

, Zedda

, et al. Liquid storage of ram semen for 96 h: Effects on kinematic parameters, membranes and DNA integrity, and ROS production. Livest Sci, 2018; 207:1–6; doi: 10.1016/j.livsci.2017.11.001

58.

Salisbury

, Hart

. Gamete aging and its consequences. Biol Reprod, 1970; 2:1–13; doi: 10.1095/biolreprod2.Supplement_2.1

59.

Fernández-López

, Garriga

, Casas

, et al. Predicting fertility from sperm motility landscapes. Commun Biol, 2022; 5(1):1–11; doi: 10.1038/s42003-022-04060-x

60.

Bui

, Sharma

, Henkel

, et al. Reactive oxygen species impact on sperm DNA and its role in male infertility. Andrologia, 2018; 50(8):e13012; doi: 10.1111/and.13012

61.

Aitken

, Gordon

, Harkiss

, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod, 1998; 59(5):1037–1046; doi: 10.1095/biolreprod59.5.1037

62.

Levy

, Anderson

, Meister

. Transport of glutathione diethyl ester into human cells. Proc Natl Acad Sci U S A, 1993; 90(19):9171–9175; doi: 10.1073/pnas.90.19.9171

63.

Masoudi

, Sharafi

, Pourazadi

, et al. Supplementation of chilling storage medium with glutathione protects rooster sperm quality. Cryobiology, 2020; 92:260–262; doi: 10.1016/j.cryobiol.2019.10.005

64.

El-kon

, Darwish

. Effect of glutathione (GSH) on microscopic parameters and DNA integrity in Egyptian buffalo semen during liquid and frozen storage. J Reprod Infertil, 2011; 2(3):32–40.

65.

Ansari

, Rakha

, Ullah

, et al. Glutathione addition in tris-citric egg yolk extender improves the quality of cooled buffalo (Bubalus bubalis) bull semen. Pak J Zool, 2011; 43(1):49–55.

66.

Kadirvel

, Kumar

, Kumaresan

. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Anim Reprod Sci, 2009; 114(1–3):125–134; doi: 10.1016/j.anireprosci.2008.10.002