Ameliorative Effect of Ascorbic Acid and Glutathione in Combating the Cryoinjuries During Cryopreservation of Exotic Jack Semen

Journal of Equine Veterinary Science 81 (2019) 102796

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Original Research

Ameliorative Effect of Ascorbic Acid and Glutathione in Combating the Cryoinjuries During Cryopreservation of Exotic Jack Semen Prashant Kumar, Rabindra Kumar, Jitendra Singh Mehta, Ashok Kumar Chaudhary, Sanjay Kumar Ravi, Sharat Chandra Mehta, Mohd. Matin Ansari, Ram Avtar Legha, Bupendra Nath Tripathi, Thirumala Rao Talluri* Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner, Rajasthan, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 May 2019 Received in revised form 25 July 2019 Accepted 5 September 2019 Available online 11 September 2019

The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L), GSH (2.5 mM), or combination of both (AA 0.9 g/L þ GSH 2.5 mM), and another aliquot was kept as control without adding the antioxidants. The jack semen underwent cryodamage, which was evident by the observation of significant (P < .05) decline in the seminal quantitative parameters at various stages of cryopreservation process. Prefreeze and postthaw semen evaluation revealed that the values of plasma membrane, acrosome integrity, and chromatin integrity were found to be significantly higher (P < .01) in the group of samples supplemented with the combination (0.9 g/L AA þ2.5 mM GSH) than AA- and GSH-alone or control groups. Supplementation of antioxidants to the freezing extender improved jack prefreeze and postthaw semen quality with the superiority of GSH over AA alone. From the present study, it was inferred that, exotic jack spermatozoa are susceptible to injuries because of cryopreservation, but these cryo-induced damage can be ameliorated significantly (P < .05) with the use of antioxidants and contribute to the improvement of semen cryopreservation procedures. © 2019 Elsevier Inc. All rights reserved.

Keywords: Jack semen Antioxidants HOST DNA integrity Cryopreservation Oxidative stress

1. Introduction Today, most of the world donkey breeds either lack a population census or are endangered or critically endangered [1]. In several regions of the world, jacks are mated with mares to produce mule foals. However, not every donkey will be interested in breeding mares. Jacks usually need to have grown up with mares to show libido when in their presence. Artificial insemination (AI) using

Animal welfare/ethical statement: All procedures were approved by the Institute Animal welfare committee and Use Committee at ICAReNational Research Committee, Hisar, Haryana. Conflict of interest statement: The authors of the present manuscript declare that there are no conflicts of interests exist among them. * Corresponding author at: Thirumala Rao Talluri, Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner, Rajasthan 334001, India. E-mail address: [email protected] (T.R. Talluri). https://doi.org/10.1016/j.jevs.2019.102796 0737-0806/© 2019 Elsevier Inc. All rights reserved.

fresh, cooled, or frozen semen of jacks is the alternative option for achieving the conceptions in such cases. Cryopreservation of equine spermatozoa has created a new dimension for the equine breeding industry by making the possibility of the preservation of this biological material for an indefinite period and its worldwide distribution [2]. Advantages associated with the use of frozen-thawed jack semen are diverse including, elimination of intimidation of mares by the sexual behavior of jacks during natural service and mating, overcoming the physical limitations of a small jack servicing large breed mares, and easier movement or transport of valuable genetic resources as semen instead of live animals [3]. On the another hand, the process of cryopreservation of semen provokes cold shock reaction, a phenomenon known to induce negative impacts on lipid composition of spermatozoal membranes, deleterious effects on motility, membrane integrity, cell function, and also interfering with the fusion of male and female gametes, which ultimately results in

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declining the fertilizing capability of the spermatozoa [4e8]. Thus, semen quality is a major determinant of success in equine breeding programs. Mammalian sperm cells contain a high proportion of polyunsaturated fatty acids, predisposing them susceptible to peroxidative damage [9]. Damages to spermatozoa during the cryopreservation and thawing occur mostly because of oxidative stress caused by the production of free radicals or excessive reactive oxygen species (ROS) [10,11]. Like all the living cells, spermatozoa under the aerobic condition produce ROS because of normal metabolism of oxygen [12]. In a controlled concentration, ROS facilitate capacitation of sperm, acrosomal reaction, and signaling processes to ensure fertilization, implantation, and early embryo development. However, the overproduction of ROS causes structural damage of sperm membranes [12,13]. Spermatozoa and seminal plasma are inherently rich ROS scavengers and enzymatic and nonenzymatic defense mechanisms against free radicals viz., glutathione peroxidase (GPx), GSH reductase, catalase, and superoxide dismutase are among enzymatic or natural antioxidants, whereas reduced GSH, urate, ascorbic acid (AA), vitamin E, carotenoids, ubiquinones, selenium, and zinc constitute nonenzymatic or synthetic antioxidants [14e17]. Routine process of sperm centrifugation used to remove seminal plasma and concentrate spermatozoa before freezing removes antioxidants present in the semen and exposes the spermatozoa to the free radicals. The imbalance of these antioxidant systems and ROS concentration leads to detrimental changes in sperm cells [18e20]. Such events facilitate the increased cell permeability, inactivation of the enzymes, and the production of potentially deleterious by-products and end products [21]. A way to improve sperm viability and minimize the damage to sperm function was successfully achieved in several mammalian species by the addition of antioxidants to the freezing media before cryopreservation [22,23]. Antioxidants play a key and vital role in sperm motility, integrity, metabolism, and function, protecting the cells against oxidative damage [24]. Their effects have been widely studied in mammalian spermatozoa [25]; however, little attention has been paid to jack sperm. Ascorbic acid (vitamin C) is a naturally occurring free scavenger, and as such, its presence assists various other mechanisms in decreasing numerous disruptive free radical processes and protects the spermatozoa against the ROS by inhibiting the peroxidation of membrane lipids and provides higher integrity to plasma membrane and mitochondria as well as better kinematics for sperm after cryopreservation [26,27]. Glutathione is a thiol tripeptide (ɣ-glutamyl cysteinyl glycine), which has an important role in the antioxidation process of endogenous and exogenous composts, as well as in the maintenance of intracellular redox conditions [2]. Glutathione is a natural reservoir of redox force, which can be quickly used to defend cells against oxidative stress [28]. Glutathione is synthesized from different amino acids viz., glutamate, cysteine, and glycine. Its reductive power is used to maintain thiol groups in intracellular proteins and other molecules. It acts as a cysteine physiological reservoir and is involved in the regulation of protein synthesis, cellular detoxification, and leukotriene synthesis [2,28]. The protection by GSH against oxidative damage is provided by its sulfhydryl group, which can be presented in reduced GSH and oxidized GSH forms. The GSH's attack against ROS is favored by the interaction with enzymes, such as GSH reductase and GPx [28]. Despite the excellent semen quality, fertility rates of frozen donkey semen in Jennies are still low (0%e36%) [29,30] compared with mares (33%e53%). The low fertility rates achieved in studies using frozen donkey semen in jennies indicate the necessity of further research to improve the semen cryopreservation

techniques and AI protocols. In addition, AI with cryopreserved donkey semen has been restricted in donkeys because of poor fertility rates [31]. Indeed, the use of several reproductive strategies, including deep intrauterine uterine insemination, increasing or decreasing the number of spermatozoa per insemination dose or multiple inseminations, did not increase the fertility rate of donkey jennies [32,33]. In recent years, studies have been conducted on donkey semen freezing extenders, including cryoprotectants such as glycerol, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, and ethylene glycol, to enhance the postthaw quality of spermatozoa [29e33]. But, studies on addition of antioxidants and their effect were meager in the literature. Hence, with this background, the present study was undertaken with an aim to study the cryoinjuries during cryopreservation process and to analyze the ameliorative effect of antioxidant supplementation to the freezing extender with antioxidant components that are naturally present in seminal plasma, that is, AA and GSH in combination or alone to the jack semen. 2. Materials and Methods 2.1. Animals Four exotic Jacks (Poitou breed, Martina franca) aged between 6 and 9 years and weighing between 500 and 650 kg body weight being reared at Equine Production Campus, Indian Council of Agricultural ResearcheNational Research Centre on Equines, Bikaner, Rajasthan, were used for this study. The experimental site was located in an arid zone of Northern India, in the state of Rajasthan. Jacks were housed at a farm with uniform feeding and management schedule occurring for all animals, and no special light was provided and were maintained under natural light conditions having free access to feed and water. The experiments were carried out in accordance with the guidelines and approval of Institute Animal Ethics Committee. Unless otherwise mentioned, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO). All the analyses were performed in the farm laboratory under standard operative procedures and during the breeding season (April to July). 2.2. Semen Collection and Processing Initially, the jacks’ semen was collected for 4 days continuously to remove the degenerated cells from the cauda epididymis and to stabilize the sperm parameters. A total of 24 semen ejaculates from four jacks (six ejaculates from each) were used for the study. The semen samples were collected twice weekly using a prewarmed (43 C) and lubricated artificial vagina (AV) (Colorado model) on estrus jenny as dummy. Each jack was given one false mount before actual collection. The semen samples were collected directly into a clean dry graduated plastic bottle attached at the end to the latex cone of the AV. Immediately after collection, the gel fraction was removed and semen was filtered through a sterile gauze. Volume, color, and consistency of the ejaculate were recorded manually, whereas other parameters such as percentage of progressively motile sperm and sperm motion variables were determined using a computer-assisted semen analyzer (CASA) (HTB CEROS II, Version 1.3, Hamilton Thorne Research, Beverly, MA, USA). Only ejaculates with more than 60% motility were used for cryopreservation. 2.3. Addition of Antioxidants to the Freezing Media Gel-free semen was mixed with the centrifugation media (0.15 g glucose, 0.37 g ethylenediaminetetraacetic acid [EDTA], 2.6 g sodium citrate, 0.12 g sodium bicarbonate, 100,000 IU penicillin, and

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0.10 g streptomycin in 100 mL of distilled water) in the ratio of 1:1 and centrifuged at 600 g for 3 minutes as per the standardized procedures [34,35]. The supernatant was discarded, and sperm pellet was obtained. The sperm pellet was suspended in a clarified egg yolk mixed in a freezing extender (6.0 g glucose, 11.00 g Lactose, 0.37 g EDTA, 0.37 g sodium citrate, 0.12 g sodium bicarbonate, 10% egg yolk, 5% dimethyl formamide, 100,000 IU penicillin, and 0.10 g streptomycin) and divided into four equal aliquots corresponding to different treatments viz., without any supplementation (control); supplemented with AA in final concentrations of 0.9 g/L, and with GSH in final concentrations of 2.5 mM and AA þ GSH (AA 0.9 g/ L þ GSH 2.5 mM). The concentrations of the antioxidants were chosen as per the previously reported studies in equids [2,36e39]. 2.4. Cryopreservation of Jack Semen The semen with a final concentration of 150e200  106/mL was loaded into 0.5 mL polyvinyl chloride straws (IMV Technologies, France), sealed with an automatic filling and sealing machine (IMV Technologies, L'Aigle, France) and then cooled to 4 C over 2 hours as equilibration period. Freezing was performed by traditional method of freezing in liquid nitrogen vapors by spreading the straws on a straw stand at height of 4 cm and then the straws were taken out after 10-minute exposure and plunged into canisters of liquid nitrogen (196 C) containers till further analysis. The straws were thawed in a water bath at 37 C for 30 seconds immediately before semen analyses. 2.5. Sperm Quality Assessment 2.5.1. Sperm Motility For the motility assessments, aliquots (10 mL) of diluted semen samples were placed on a clean grease-free slide covered with a glass cover slide (20  20 mm) and examined under high power phase objective lens (10 magnification) equipped with an attached thermostatically controlled stage (37 C). Semen motion variables were analyzed using CASA system. The CASA analysis was set up at 60 Hz (frame per second), 45 frames, minimum contrast of 70, and minimum cell size of four pixels. Cells were considered progressive with at least 50 mm/second average path velocity and 75% straightness. All the samples were incubated in a water bath at 37 C and evaluated for motility within 5 minutes of removal from incubation. Concentration of the spermatozoa was estimated using a hemocytometer chamber. 2.5.2. Sperm Plasma Membrane Integrity The sperm viability was evaluated by the physical integrity of the sperm plasma membrane with eosin-nigrosin staining that was adapted for horses [40]. Briefly, 10 mL of each sperm suspension was mixed with 10 mL of 5% eosin B and 10% nigrosin, and a smear was made after 2- to 3-min incubation on a stage warmer at 37 C. Microscopic fields were chosen randomly in the slide, and sperms classified as intact membrane (viable) were not stained by eosin, whereas the nonintact membrane (non-viable) showed the pinkredestained nuclei. Likewise, minimum 300 cells were counted per slide in different fields per ejaculate/jack. Results were expressed as a percentage (%), performed by the single technician. 2.5.3. Sperm Plasma Membrane Functionality The plasmatic membrane integrity was evaluated by using hypoosmotic swelling test, which is based on swelling ability of functioning sperms after being exposed to a hypoosmotic solution [41]. This test was performed by mixing 0.1 mL of semen with 1 mL of a 150 mOsmol/L hypoosmotic solution (sodium citrate 7.35 gm, fructose 13.51 gm, and double distilled water up to 1000 mL) and

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incubated in a water bath at 37 C for 1 hour. After incubation, a drop of well-mixed solution was taken on a clean dry glass slide and covered with a coverslip. Different types of curling patterns of sperm tail were recorded as an effect of swelling due to influx of water. A total of 300 spermatozoa were counted in different fields at 400 magnification under a phase-contrast microscope and were expressed in %. 2.5.4. Acrosome Integrity Analysis Giemsa stain was used to assess the acrosome integrity of jack spermatozoa as per Watson (1975) [42]. Diluted semen drop was kept on a clean grease-free slide, and a thin smear was prepared. After air drying the smear, the slide was fixed in methanol for 15 minutes and then after washing, the fixed slide was kept in working solution of Giemsa for 90 minutes. Excess stain was removed by washing under gentle stream of water. It was dried in air and examined under the bright field 100 oil immersion objective of the phase-contrast microscope. Around 300 spermatozoa were assessed in different fields of a slide, and the same were expressed in percentage. 2.5.5. DNA Intactness For detecting the DNA intactness of spermatozoa, the method described by Soni et al. (2018) [35] was adopted. Briefly, a small aliquot (20 mL) of sperm suspension was glass smeared. The smear was air-dried and then fixed overnight in Carnoy's solution (methanol/acetic acid, 3: 1). Once rinsed and air-dried, the slides were stained for 5 minutes with freshly prepared acridine orange stain. After washing and drying, the slides were examined using a fluorescent microscope (Nikon, Japan; excitation of 450e490 nm). Two hundred sperms per sample were counted under the epifluorescence microscope (480/550 nm). Sperm heads with intact chromatin showed green fluorescence and those with denatured (nonintact DNA) chromatin had orange-red or yellow fluorescence [43]. The percentage of spermatozoa with single-stranded DNA was calculated from the ratio of spermatozoa with red, orange, or yellow fluorescence to all spermatozoa counted per sample. 3. Statistical Analysis A detailed analysis of the various seminal parameters recorded for fresh, cooled, and frozen-thawed semen was performed for determining the uniform distribution and statistical validity (R version: 3.1.3). As repeated collection of semen was performed on the same jack at different time intervals, a repeated measure ANOVA was carried out to partition the variability attributable to differences between treatments and individual variation among jack in treatment groups. Data pertaining to fresh, equilibration, and postthaw stages of cryopreservation were analyzed by one-way ANOVA using Tukey’s test for normality for all. The factorial model included the effect of AA and reduced GSH as independent variables and percent postthawed progressive motility, live sperm count, acrosome-intact sperm, hypoosmotic swelling positive sperm, and DNA-intact sperm as dependent variables. Data were subjected to ANOVA, using the post hoc procedure from Statistical software package, version 20 (SPSS 20). For fresh, unextended semen, the paired t-test model was used to see the variability between jacks. All results are expressed as the mean ± standard error and were considered significant at either P  .05 or P  .01. 4. Results and Discussion Cryopreservation of spermatozoa has immense potential in preserving elite and endangering genetic materials in both humans and livestock [44]. Unfortunately, during this procedure, sperm

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Table 1 Quantitative seminal parameters (mean ± SE) of Poitou jacks' fresh semen (n ¼ 24). Poitou Jack

Total Ejaculate Volume (mL)

1 2 3 4 Overall

105.83 41.66 55.33 55.33 63.20

± ± ± ± ±

Gel Volume (mL)

18.04a 7.03b 7.49b 7.49b 7.39

25.83 11.50 12.00 10.00 14.83

± ± ± ± ±

8.79a 3.64b 2.38b 1.82b 2.68

Gel-Free Volume (mL) 70.00 30.16 43.33 40.00 45.87

± ± ± ± ±

Sperm Concentration (106/mL)

pH

11.69a 5.13b 8.13b 6.95b 4.95

7.80 7.66 7.63 7.83 7.73

± ± ± ± ±

0.08 0.06 0.05 0.14 0.04

288.16 278.16 285.16 318.83 292.58

± ± ± ± ±

4.12a 10.89a 5.46a 6.28b 4.65

Means with different superscripts in the same row indicate significant differences.

experiences various degrees of damage, which may result in perturbations to the sperm organelles and changes in membrane fluidity and enzymatic activity that subsequently result in retardation of the sperm motility, diminishing the viability and fertilizing ability [45]. The process of cryopreservation is associated with an increase in ROS production and a decrease in the naturally existing antioxidant levels, which in turn increases the susceptibility of the frozen/thawed spermatozoa to the lipid peroxidation [46]. In stallions, multiple studies have been conducted to neutralize or minimize the effects of ROS by adding various antioxidants (e.g., superoxide dismutase, catalase, GPx, AA, a-tocopherol, AA, quercetin, butylated hydroxytoluene, L-carnitine) to semen extenders or the freezing media with results that are variable [47e53]. However, this kind of studies lack in jacks, and there are only few studies about jack semen cryopreservation [35,54,55]. Seminal plasma naturally harbors the ROS scavengers that prevent oxidative-induced sperm damage [56]. Cryopreservation process of donkey sperm typically requires removal of most if not all seminal plasma, which results in loss of naturally occurring antioxidant systems, and increases the susceptibility of spermatozoa to damage by free radicals. Hence, to overcome the oxidative stress and to ameliorate the quality of various spermatic characteristics, the present study was conducted. The standard semen parameters such as total volume, gel-free semen volume, pH, and sperm concentration in fresh semen ejaculates of the four exotic jacks used in this study were assessed, and the results are presented in Table 1. The color of the semen appeared to be milky white, and the consistency varied from thick to thin. The semen was free from blood and urine. Significant differences (P < .01) were observed in the total volume of fresh semen and the gel-free semen obtained from the jacks during the semen collection process, and it varied from 38 mL to 127 mL and 32 mL to 89 mL respectively. The pH of the fresh semen varied from 7.59 to 7.88. The spermatozoal concentration in the fresh semen ranged from 267 to 326  106/mL. All these values are observed to be in the standard range and correlating with the earlier studies [57,58]. A significant decrease (P  .05) in the progressive motility and percentage of sperm with an intact plasmatic membrane in postthaw semen was observed as compared with fresh, prefreeze, and frozen-thawed jack semen (Table 2). This infers the direct effect of cryopreservation on diminishing the progressive motility and

Table 2 Seminal parameters of Poitou Jacks at various stages of cryopreservation (n ¼ 24). Seminal Parameter (%) Motility Livability HOST reactive sperm Acrosome integrity DNA intactness

Fresh Semen 80.05 86.79 57.82 89.20 94.28

± ± ± ± ±

c

1.48 1.92c 1.54c 1.90c 0.21b

Prefreeze Semen 70.46 74.37 39.08 79.45 91.23

± ± ± ± ±

b

2.47 1.25b 1.47ab 0.98ab 0.06b

Postthaw Semen 42.11 57.43 30.11 76.27 84.20

± ± ± ± ±

1.77a 2.81a 1.19a 1.47a 0.07a

Abbreviation: HOST, hypoosmotic swelling test. Data are presented as the mean ± SEM, n ¼ 24. Means with different superscripts in the same row indicate significant differences between fresh, prefreeze, and postthaw semen within each parameter, P < .05.

viability of spermatozoa (Fig. 1). Cryopreservation had a significant adverse effect on the other qualitative seminal parameters such as sperm plasma membrane functionality and acrosome membranes. There was a significant (P  .05) reduction in the number of spermatozoa with functional plasma membrane at fresh and postthaw stage (57.82 ± 1.54 vs. 43.42 ± 2.51), and this difference seems to be narrowed at 2 hours of incubation during the prefreeze stage (57.82 ± 1.54 vs. 56.70 ± 1.21). A significant decline (P  .05) in the number of spermatozoa with intact acrosome was recorded at fresh, prefreeze, and postthaw stages of jack spermatozoa (Table 1), and this difference was found to be nonsignificant from the prefreeze stage to the postthaw stage. Similar kind of observations was made in case of the DNA intactness of the jack spermatozoa. A decrease in almost all qualitative seminal parameters was observed because during freezing, sperm is exposed to severe osmotic, thermal, and oxidative stress, which damaged the plasma membrane and other spermatic structures such as acrosome and impaired the chromatin structure [33]. Process of semen cryopreservation, however, increased susceptibility of equine sperm to lipid peroxidation because of higher production of ROS [43,56,59], and this damage might further increase with the removal of seminal plasma during semen processing because much of the antioxidant capacity in semen resides with seminal plasma [60,61]. The observations regarding the effect of cryopreservation on sperm quality of the present study are consistent with previous studies indicating that freeze-thawing procedures damage different membranes of spermatozoa [62e64]. The effect of supplementation of semen extender with different antioxidants, that is, AA and GSH alone or in combination on sperm progressive motility, viability, and integrity of various sperm membranes of cooled and frozen-thawed Poitou jack spermatozoa is presented in Table 3. A significant increase in the motility and the number of live spermatozoa was recorded in the semen samples supplemented with AA, GSH, or AA þ GSH (P  .05). Among the antioxidants, combination of both the antioxidants (AA þ GSH) groups showed marked increase in the motility and viability of exotic jack semen compared with the AA or GSH alone, and at the same time GSH over the AA proved to be a better antioxidant in increasing the motility KINETIC PROPERTIES OF SPERMATOZOA AT PRE-FREEZE STAGE Control

AA

GSH

AA+GSH

180 160 140 120 100 80 60 40 20 0 VSL

VAP

VCL

Fig. 1. Kinetic properties of jack spermatozoa at various stages of cryopreservation.

P. Kumar et al. / Journal of Equine Veterinary Science 81 (2019) 102796

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Table 3 Effect of addition of antioxidants on Poitou jack semen quality at different stages of cryopreservation (n ¼ 24, each sample). Stage of Cryopreservation

Group

Motility (%)

Prefreeze

Control AA GSH AA þ GSH Control AA GSH AA þ GSH

70.46 76.76 78.28 81.32 42.11 48.55 52.99 56.43

Postthaw

± ± ± ± ± ± ± ±

Sperm Livability (%)

2.47A 1.52B 1.46BC 1.54C 1.77A 3.30B 3.05C 2.93D

74.37 78.39 83.90 82.21 57.43 55.51 63.82 65.44

± ± ± ± ± ± ± ±

1.25A 1.89AB 1.22B 1.79B 2.81B 2.13A 1.54CD 2.97D

HOST Reactive Sperm (%) 39.08 45.70 54.01 56.70 30.11 37.00 41.95 43.42

± ± ± ± ± ± ± ±

1.47A 1.49B 1.25C 1.21C 1.19A 1.64B 1.74C 2.51C

Acrosome Integrity (%) 79.45 82.57 81.52 82.18 76.27 80.07 78.34 83.50

± ± ± ± ± ± ± ±

0.98 0.84 1.25 2.04 1.47A 1.56B 1.58AB 0.80B

DNA Intactness (%) 91.23 92.11 92.51 93.14 84.20 85.04 85.68 86.60

± ± ± ± ± ± ± ±

0.06A 0.03A 0.04A 0.04B 0.07A 0.07A 0.05A 0.07B

Abbreviations: AA, ascorbic acid; GSH, glutathione; HOST, hypoosmotic swelling test. Data are presented as the mean ± SEM. Different superscripts indicate significant differences between the control and treated semen within each parameter: P < .05.

and protecting the membranes (Fig. 2). The present study results are in accordance to that of previous studies carried out on GSH supplementation to the horse semen [20,38,65e67] and boar semen [68,69]. However, Baumber et al (2005) [61] did not observe an improvement in total and progressive motility after the addition of 10 mM GSH. A protective effect of AA for membrane integrity was observed, but a deleterious effect on progressive motility was reported [36]. The supplementation of AA or GSH to the freezing extender led to higher postthaw hypo osmotic solution-reacted spermatozoa than the control group spermatozoa. A significant increase (P  .05) in swelling, coiling, and hypo osmotic solution-reacted sperms was found in AA, GSH, and AA þ GSH groups in comparison to the control group at the cooling and postthaw stage. Acrosome integrity of the jack spermatozoa deteriorated during the freeze-thaw process. After incubation, the antioxidant group compared with the control group successfully protected the spermatozoal acrosome integrity at the prefreeze and postthaw stages (P < .05), which is in corroboration with the earlier findings [2,33,36,56,61]. Evaluation of DNA integrity of spermatozoa was also assessed to establish the magnitude of damage at the prefreeze and freezingthawing processes and how this damage changes after incubation and thawing. Supplementation of the freezing media with amino acids (AA þ GSH) had showed significant (P < .05) increase in the stability and maintaining the DNA integrity when compared with the control group semen samples both at incubation and after thawing process. The percentages of sperm with intact DNA did not significantly differ among the control, AA, and GSH groups at both prefreeze and postthaw stages and ranged from 90.11% to 94.87% at the cooling incubation stage and 83.26%e87.64% in frozen-thawed semen. There was a significant drop in values of sperm membrane (plasmatic and acrosome) integrity, which is expected after freezing. High salt concentration due to ice crystal formation can

KINETIC PROPERTIES OF SPERMATOZOA AT POST-THAW STAGE Control

AA

GSH

AA+GSH

140 120 100 80 60 40 20 0

VSL

VAP

VCL

Fig. 2. Kinetic properties of jack spermatozoa at various stages of cryopreservation supplemented with antioxidants.

dehydrate sperm cells, deforming them and causing damages in the acrosome and plasmatic membrane, reduction in metabolism, and loss of intracellular components [70,71]. However, the addition of 0.9 g/L AA and 2.5 mM GSH was highly efficient for preservation of viability and membrane plasmatic integrity compared with the control group. The similar observations were made in the semen cryopreserved with GSH, which improved the total and progressive motility, plasma membrane integrity, and functionality in Caspian miniature stallions [72]. Addition of antioxidants (AA and GSH) alone or in combination has significantly provided the cryoprotective effect to the spermatozoa during the cryopreservation process, which is evident from the data presented in Table 3. Membrane functional integrity yielded higher values in antioxidant groups than the control group (P < .05). Among the two antioxidants, GSH over AA proves to be a better antioxidant, as use of AA might be controversial, based on the previous studies, which stated that it may also act as a prooxidant in the presence of transition metals and it may make free radicals highly reactive and more destructive, thus generating more free radicals [73,74]. There is ambiguity in the literature for the protective effect of antioxidants added to semen extenders. Some studies reported their positive effects, whereas others documented no benefit of addition of antioxidants [30,33,75,76]. In contrast, Aurich et al [36] observed that AA had a protective effect on sperm membrane integrity of diluted stallion sperm. Likewise, the addition of GSH to freezing extender improved postthaw sperm quality in bulls [77,78]. The discrepancy in the results within species may be due to variations in age, animal breed, diluent components, semen conservation procedures, doses, and combinations of antioxidants. Oxidative damage is then well recognized as a major factor in sperm cryodamage and therefore justifies the use of antioxidants to improve the outcomes of sperm cryopreservation. The addition of antioxidants to the semen extender or freezing media can minimize the detrimental effect of ROS on sperm function and hence improve post-thaw semen quality and fertility with use of donkey spermatozoa. Supplementation of AA and GSH to the freezing extender ameliorated the sperm susceptibility to the cryopreservation conditions. Supplementing of freezing media with 0.9 g/L AA and 2 mM GSH not only does have a positive impact on the chromatin stability and membrane integrity of jack sperm to withstand freeze-thawing procedures but also increase in overall sperm motility, viability at postthawing reported in previous studies [2,30,33,36,37,56,61] is related to the structure of sperm subpopulations. Although the present study was conducted on a limited number of animals, it can be suggested that supplementation of antioxidants like AA and GSH should be considered when semen from individual jacks is needed to be cryopreserved for long term in liquid nitrogen. A greater understanding of the mechanisms involved in the equine semen cryopreservation and dependent cryoinjuries is required to

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overcome the cryoinduced sperm damage and such an approach will be of immense importance to semen cryopreservation techniques. This would allow a more efficient use of cryopreserved donkey semen for insemination and the establishment of sperm banks. It is a preliminary study and subsequent fertility testing is necessary to be conducted to evaluate in vivo fertility rates of frozen-thawed donkey spermatozoa supplemented with these antioxidants. 5. Conclusion The cryopreservation has an adverse effect on the different qualitative parameters of the exotic jack spermatozoa. The supplementation of AA and GSH to the freezing extender not only reduced the oxidative stress provoked by cryopreservation of jack spermatozoa but also ameliorated, preserved, and improved various seminal qualitative parameters. A combination of antioxidants than the single antioxidant supplementation proved to be significantly efficient in preserving the sperm from the cryodamage effect. Acknowledgment The authors thank the director, ICAR-NRC on Equines, Hisar, for providing financial assistance and necessary infrastructure facilities for carrying out the study. The authors also thank the incharge, Equine Production Campus, for his kind cooperation. References [1] Food and Agricultural Organization (FAO). Commission on genetic resources for food and agriculture: status and trends of animal genetic resources. Available at, http://www.fao.org/3/a-mm278e.pdf. [Accessed 18 February 2018]. [2] de Oliveira RA, Wolf CA, de Oliveira Viu MA, Gambarini ML. Addition of glutathione to an extender for frozen equine semen. J Equine Vet Sci 2013;33: 1148e52. [3] Madison RJ, Evans LE, Youngs CR. The effect of 2-hydroxypropyl-b-cyclodextrin on post-thaw parameters of cryopreserved jack and stallion semen. J Equine Vet Sci 2013;33:272e8. [4] Aitken RJ. Pathophysiology of human spermatozoa. Curr Opin Obstet Gynecol 1994;6:128e35. [5] Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci 1996;42:55e65. [6] Bakst MR, Sexton TJ. Fertilizing capacity and ultrastructure of fowl and Turkey spermatozoa before and after freezing. J Reprod Fertil 1979;55:1e7. [7] Hammerstedt RH. Maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: a review of the effect on design of storage preservation systems. Reprod Fertil Dev 1993;5:675e90. [8] Yanagimachi R. Mammalian fertilization. Phys Reprod 1994;1:189e317. [9] Alvarez JG, Storey BT. Spontaneous lipid peroxidation in rabbit epididymal spermatozoa: its effect on sperm motility. Biol Reprod 1982;27:1102e8. [10] Tariq M, Khan MS, Shah MG, Nisha AR, Umer M, Hasan SM, Rahman A, Rabbani I. Exogenous antioxidants inclusion during semen cryopreservation of farm animals. J Chem Pharm Res 2015;7:2273e80. [11] Papa FO. Effect of BHT (Butylated Hydroxytoluene) addition in cooling and freezing extenders on equine sperm freezability. Faculdade de Medici
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