INFLUENCE OF ANTIOXIDANTS ON DNA INTEGRITY, MITOCHONDRIAL FUNCTION AND FERTILIZING POTENTIALS OF CRYOPRESERVED BUFFALO SPERMATOZOA

Artificial Insemination and Embryo Transfer Department,

Animal Reproduction Research Institute, Al Haram, Giza, Egypt.

Influence of antioxidants on DNA integrity, mitochondrial function and fertilizing potentials of cryopreserved buffalo spermatozoa

(With One Table, 6 Figures and 2 Photoes)

By

M.R. Badr; S.M. Abd el Hafez*

and Eman M. Abd el Fatah** 

* Unit of Immunobiology and Immunopharmacology, Animal Reproduction Research Institute, Al Haram, Giza, Egypt.

**Biotechnology Research Unit, Animal Reproduction Research Institute,

 Al Haram, Giza, Egypt.

(Received at 22/11/2008)

دراسة تأثير بعض مضادات الأکسدة علي قابليه حيامن الجاموس للتجميد وسلامة الحامض النووي للحيامن وکفاءتها الاخصابية معمليا وحقليا

مجدي رمضان بدر ، سلامة عبد الحافظ ، ايمان عبد الفتاح

تهدف الدراسة الحالية إلي دراسة تأثير التجميد وکذالک  تأثير اضافة بعض مضادات الاکسدة الي ممدد السائل المنوى علي قابلية حيامن الجاموس للتجميد وعلي الحامض النووى  للحيامن وکذا قدرتها الاخصابية معمليا ومعدل العشر حقليا. تم تجميع عينات السائل المنوي من ستة طلائق جاموسي وبعد تقيم السائل المنوي معمليا تم تمديده في ممدد التريس فقط (المجموعة الضابطة) أو المضاف الية ترکيزات مختلفة منبعض مضادات الأکسدة مثل الجلوتاثيون المختزل؛ کاتاليز؛ حمض الفا ليبوک وسوبر أوکسيد ديسميوتيز. بعد تبريد وتجميد الحيامن الجاموسي بالنظام الفرنسي تم تقيمه من حيث نسبه الحرکه اﻷماميه والحيويه وتشوهات القلنسوه وکذا اثر التجميد على الميتوکوندريا والحامض النووي للحيامن وکذالک  قدرته الاخصابيه معمليا وحقليا. حيث أوضحت نتائج الدراسة الحالية أن تجميد حيامن الجاموس في ممدد التريس (المجموعة الضابطة) أدي إلي تأثير سلبي کبير علي الحرکه اﻷماميه والحيويه وتشوهات القلنسوه (3.303±43.3 % ،24.4±86.67 و3.85±27.33% علي التوالي) وکذالک على وظائف الميتوکوندريا (±0.051.88) کما أدي الي ارتفاع تشظي الحامض النووي (4.42±71.67%) وکذالک قلل قدرته الاخصابيه معمليا وحقليا (46.58 و 51.67% علي التوالي ) بينما أظهرت نتائج الدراسة الحالية أن إضافة مضادات الأکسدة إلي ممدد التريس لة تأثير ايجابي کبير علي تحسن وظائف السائل المنوي الجاموسي المجمد بناء علي الترکيز المضاف به الي الممدد. حيث أوضحت نتائج الدراسة الحالية أن إضافة 10ملليمول من الجلوتاثيون المختزل أو 100 وحدة/مللي من کاتاليز أو 15 ملليمول من حمض الفا ليبوک أو 50 وحدة/مللي من سوبر أوکسد ديسميوتيز إلي الممدد قد أدي إلي زيادة معنوية کبيرة في نسبه الحرکة اﻷماميه للمني بعد الاساله (2.89 ±65.00 ، 1.66 ±66.67 ،1.66 ± 61.67  و3.34±63.33%  علي التوالي) وحيويه المني(10.12±167.50 ، 3.01±166.67  ، 4.65± 150.83  و10.1 ±168.33 علي التوالي) کما أدي إلي انخفاض معنوي کبير في نسبه تشوهات القلنسوة ( 4.05±11.66 ،2.91 ±11.33  ، 3.93±12.33  و2.34±10.33% علي التوالي) کما حسن من وظائف الميتوکوندريا حتي بعد مرور ثلاث ساعات من الاسالة (0.05±3.42 ، ±0.163.29  ،0.09±2.81 و 0.08±3.31علي التوالي) کما حافظ علي سلامة الحامض النووي للحيامن (4.33±37.67  ، 2.61 ±36.33 ، 5.05± 43.33  و 5.04 ±36.00% علي التوالي).  کما أوضحت نتائج الاخصاب المعملي إن إضافة 10ملليمول من الجلوتاثيون المختزل أو 100 وحدة/مللي من کاتاليز أو 50 وحدة/مللي من سوبر أوکسد ديسميوتيز إلي الممدد قد أدي إلي زيادة معنوية کبيرة في النمو إلي طور البلاستوسيست (11.86 ،11.54  و 14.20 % علي التوالي ) ٬ کما أظهرت نتائج التلقيح الحقلي أن إضافة 10ملليمول من الجلوتاثيون المختزل أو 100 وحدة/مللي من کاتاليز أو 50 وحدة/مللي من سوبر أوکسد ديسميوتيز إلي الممدد أدي الي زيادة کبيرة في معدل العشر بعد الجس (62.79٬62.11 و 67.74 % علي التوالي). ومن خلال نتائج الدراسة الحالية يمکن أن نستنتج أن للتجميد تأثير سيئ علي محتوي الحامض النووي والقدرة الاخصابية للسائل المنوي الجاموسي في حين أن إضافة مضادات الأکسدة الجلوتاثيون المختزل ؛کاتاليز حمض الفا ليبوک أو سوبر أو کسيد ديسميوتيز إلي ممدد التريس يلعب دورا هاما وکبيرا في تحسن وظائف السائل المنوي الجاموسي المجمد بناء علي الترکيز المضاف به إلي الممدد ويبدو ذلک  من خلال قدرتها علي مقاومة التأثير الضار لعمليات الأکسدة أثناء التجميد علي سلامة الحامض النووي للحيامن.

SUMMARY

The objective of the current study was to evaluate the effect of cryopreservation and fortification of freezing extender with some antioxidants on the DNA integrity and the fertilizing potentials of cryopreserved buffalo spermatozoa. Buffalo semen was collected, evaluated and extended in Tris-based extender supplemented with different concentrations of reduced glutathione (GSH), catalase (CAT), alpha lipoic acid (ALA) and superoxide dismutase (SOD). Semen was examined post-thawing to evaluate, freezability, mitochondrial function, DNA integrity and the natural and assisted fertilizing potentials. The main findings emerging from the present study were that cryopreservation decreased (P<0.01) significantly sperm freezability, mitochondrial activity, DNA integrity and fertilizing potentials of frozen-thawed buffalo spermatozoa. Meanwhile, in vitro provision of freezing extender with GSH, catalase, ALA and SOD had a beneficial effect on the function of the cyropreserved buffalo spermatozoa, in a dose dependent trend. Fortification of semen extender with 10 mM GSH, 100 U/ml catalase, 15 mM ALA or 50 U/ml SOD increased (P<0.01) significantly post-thawing progressive sperm motility (65.00±2.89, 66.67±1.66, 61.67±1.66 and 63.33±3.34%, respectively); viability indices (167.50±10.12, 166.67±3.01, 150.83±4.65 and 168.33±10.1, respectively) and maintained the acrosomal integrity (11.66±4.05, 11.33±2.91, 12.33±3.93 and 10.33±2.34%, respectively). Moreover, improved the mitochondrial function (3.42±0.05, 3.29±0.16, 2.81±0.09 and 3.31±0.08, respectively); DNA integrity (37.67±4.33, 36.33±2.61, 43.33±5.05 and 36.00±5.04%, respectively). Additionally, in vitro provision of freezing extender with 10 mM GSH, 100 U/ml catalase or 50 U/ml SOD increased in vitro embryo development to the blastocyst stage (11.86, 11.54 and 14.20 %, respectively) and augmented the natural pregnancy rate (62.11, 62.79 and 67.74%, respectively). In conclusion, cryopreservation promotes DNA fragmentation in buffalo spermatozoa that can be counteracted by the addition of GSH, catalase, ALA and SOD. These antioxidants appear to play an important role in sperm antioxidant defense strategy in a dose dependent trend and could be of significant benefit in improving the freezability, DNA integrity and the natural and assisted fertilizing capacity of the cryopreserved buffalo spermatozoa.

Key words: Semen, artificial insemination, cryopreservation

Introduction

Semen cryopreservation and artificial insemination (AI) offer many advantages to the livestock industry, particularly in conjunction with genetic evaluation and selection programs (Johnson et al., 2000). However, the biggest obstacle to exploiting cryopreserved semen of many species is damage of sperm membrane structures during freezing and thawing, which leads to fewer viable and motile cells post-thawing (Hammerstedt et al., 1990). Consequently, fertility following AI is poorer than with fresh semen in most species (Holt, 2000). Cryopresevation of spermatozoa is associated with an increase in reactive oxygen species (ROS) generation and lipid peroxidation (Linfor and Meyers, 2002 and Zalata et al., 2004); thereby, cryopreservation of spermatozoa may subject sperm cells to oxidative stress and potential DNA damage (Sanocka and Kurpisz, 2004). Spermatozoa are sensitive to oxidative stress because they lack cytoplasmic defenses (Saleh and Agarwal, 2002).  Excessive ROS formation by spermatozoa during the cryopreservation process has been associated with a detrimental effect on sperm DNA(Tominaga et al., 2004) and sperm fertilizing potentials (Ball et al., 2001). It is difficult to block the OS-induced injury to cells because ROS are continuously produced by cellular aerobic metabolism (Aitken and Krausz (2001)). However, oxidative DNA damage in mammalian sperm suspensions can be counteracted by the addition of seminal plasma that contains the predominant source of antioxidant protection (Potts et al., 2000). Unfortunately, sperm preparation for cryopreservation involves the removal of the seminal plasma and consequently the main source of sperm protection.  Moreover, the process of cryopreservation is associated with an alteration in antioxidant defense systems; including a decrease in intracellular glutathione (Bilodeau et al., 2000 and Gadea et al., 2004) and superoxide dismutase (SOD) content (Nair et al., 2006). So, one obvious way to overcome the detrimental effects of ROS on the cryopreserved sperm performance could be the addition of antioxidant compounds to the freezing extender to block or prevent oxidative stress. A variety of antioxidants has been examined to either scavenge ROS directly or counter the effects of ROS toxicity in the semen of mammalian species (Rossi et al., 2001). Therefore, the goal of the present study was to evaluate the effect of cryopreservation and the fortification of freezing extender with some antioxidants as reduced GSH, catalase, alpha lipoic acid (ALA) and superoxide dismutase (SOD), on buffalo semen freezability, mitochondrial function, DNA integrity, natural and assisted fertilizing potentials of the cryopreserved buffalo spermatozoa.

MaterialS and Methods

Semen collection and cryopreservation:

Semen samples were collected from six buffalo bulls of proven fertility, with the use of an artificial vagina. Only semen samples of at least 70 % initial motility and 800.00X10⁶sperm cells/ml were used.  Immediately after collection, semen samples were pooled and diluted at a 1:8 ratio at 30°C with a commercial Tris-egg yolk–based extender (Optidyl®; Bio-Vet France). Diluted semen samples were split into 13 portions as follows: control (extender only); GSH (5, 10 and 20 mM); catalase (100, 200 and 300 U/ml); ALA (10, 15 and 20 mM) and SOD (50, 100 and 200 U/ml). Immediately after dilution, the extended semen of each treatment was evaluated then, it was cooled to 5°C over 60 minute in a cold cabinet. The cooled semen was loaded into 0.25 ml French straws (IMV, l'Aigle, France) sealed with polyvinyl acid and then lowered into liquid nitrogen vapor inside foam box. The straws were then immersed into liquid nitrogen and stored for at least two weeks before analysis.  Frozen semen was thawed by plunging straws into a 37°C water bath for 30 seconds. Classical semen quality tests (motility, viability and acrosomal integrity) were assessed according to Mohammed et al. (1998) after thawing.

Evaluation of mitochondrial function of frozen-thawed spermatozoa:

Functional integrity of sperm mitochondria was assessed using a modified mitochondrial reduction assay according to Mosmann (1983).  For each treatment of the frozen–thawed semen samples, three wells were used. A 200 µl of semen sample and 10 µl of freshly prepared Tetrazolium salt (MTT, Sigma) [3-(4, 5-dimethylthiazol-2-VL)-2, 5- diphenyl tetrazolium bromide] stock solution were placed in ELISA plate. The reaction was left under 5% CO₂ at 37°C for four hours in darkness and then lyses buffer was added over night. The rate of reduction was determined using ELISA reader (versa max, USA); at wave length 470 nm and the reading was expressed at optical density.  This assay was performed for three successive hours post-thawing to obtain three optical densities for each treatment.

Assessment of sperm DNA integrity:

The DNA status of individual cells was determined by the neutral single cell gel electrophoresis (comet) assay according to (Boe-Hansen 2005). For this assay, frozen-thawed spermatozoa were diluted in phosphate buffer saline (PBS) then embedded in agarose, followed by cell lysis, DNA decondensation, electrophoresis, neutralization, and DNA staining with 50 µl of 20 µg/ml ethidium bromide (Sigma). The cells were then visualized by fluorescent microscopy. Intact nuclei in the comet assay appeared to have compact and brightly fluorescent heads; in contrast, strand breaks in damaged cells allow DNA migration during electrophoresis, and a tail of DNA could be seen behind the head, giving the appearance of a comet (Hughes et al., 1996). After subjecting spermatozoa to the comet assay, sperm nuclei were imaged with epifluorescent microscope (Green filter: N2.1 with Exitation filter: BP 515-560, Dichromatic Mirror: 580, Suppression filter: LP 590, Germany). Images of sperm nuclei were digitized with Leica (DFC 280, Cambridge, UK) camera. One hundred (100) sperm nuclei were scored for each sample. Sperm comets were visually scored according to Collins et al. (1995) into five grades from grade 0, no comet (no damage) to grade 4, large comet (extensive damage). The individual grade scores for 100 spermatozoa from each treatment were converted into a composite score by multiplying the number of sperm nuclei by the corresponding numerical score. Thus, the composite score could range from 0 (all undamaged) to 400 (all maximally damaged).

Evaluation of in vitro fertilizing potential of the treated buffalo semen:

Semen in vitro fertilizing ability was assessed using in vitro fertilization technology, according to Totey et al. (1992). Immature buffalo oocytes were collected from fresh ovaries just after slaughter at a local abattoir. Cumulus-oocyte complexes (COCs) were collected by aspiration of medium-sized (2-8 ml) ovarian follicles and were washed twice in modified Dulbecco's phosphate-buffered saline. Only oocytes with a homogeneous ooplasm and a complete and dense cumulus cells were selected for in vitro maturation. The selected oocytes were cultured in TCM-199 medium (Earl's salt, Sigma Chemical CO., St. Louis, Mo., USA) supplemented with 10% heat inactivated fetal calf serum (FCS, Gibco, 30 K-0351), 10 μg/ml Luteinizing hormone, 5 μg/ml  follicle stimulating hormone and 1 μg/ml estradiol-17β. The oocytes were cultured for 24 hour at 39°C in an atmosphere of 5% CO₂ in air with maximum humidity.

Three straws from each treatment were thawed in a water bath at 37°C for 30 sec. The most motile spermatozoa were separated by swim up technique in the fertilization medium, modified Tyrode's Albumin-Lactate-Pyruvate (TALP) containing 6 mg/ml bovine serum albumin (BSA), for 1 hour (Parrish et al., 1988). The uppermost layer of the medium containing the most spermatozoa was collected and washed twice by centrifugation at 2000 rpm for 10 minutes. The sperm pellet was resuspended in the fertilization TALP medium containing 10 μg/ml heparin. After appropriate dilution, 2 μl of sperm suspension was added to the fertilization drops, containing the matured oocytes, at a final concentration 2 X10⁶ sperm cell/ml. Gametes were co-incubated in the fertilization drops under sterile mineral oil for 18 hour at 39°C in an atmosphere of 5% CO₂ in air with maximum humidity. At the end of gametes co-incubation, some inseminated oocytes were examined for signs of fertilization and the other oocytes were in vitro cultured for further embryo development. The inseminated oocytes were freed from cumulus cells and attached spermatozoa by gentle pipetting and then cultured in TCM-199 medium with Hepes modification for seven days at 39°C in an atmosphere of 5% CO₂ in air with maximum humidity. The proportional of cleaved oocytes was recorded 48 hour after insemination and those developed to the morula and blastocyst stages were recorded at 5-7 day post-insemination.

Fertility Study:

A preliminary fertility trial was performed in a private farm to compare between control semen and antioxidants treated semen at their best concentration, as shown from freezability, mitochondrial function and DNA integrity, results. Buffaloes (3.5 - 4 years) were randomly assigned to one of five treated groups: group 1 (120 buffaloes) was inseminated using control semen; group 2 (95 buffaloes) was inseminated using GSH treated semen, group 3 (86 buffaloes) was inseminated using catalase treated semen, group 4 (98 buffaloes) was inseminated using ALA treated semen and group 5 (93 buffaloes) was inseminated using SOD treated semen. Pregnancy diagnosis was performed 45 days post-insemination by transrectal palpation.

Statistical analysis:

All data were analyzed by using Costat Computer Program (1986)Cottort Software, and were compared by the least significant difference least (LSD) at 1% and 5% levels of probability. The results were expressed as means ± S.E.M.  Pearson correlation coefficients were used to calculate the relationships between DNA integrity and sperm quality tests. In vitro fertilization rate, embryo development and total pregnancy rate were analyzed by chi-square analysis (X²).

Results 

Data presented in table 1 revealed that, fortification of freezing extender with antioxidants improved the freezability of buffalo semen compared to the control semen in a dose-dependent trend.  Addition of 10 mM GSH, 100 U/ml catalase, 15 mM ALA or 50 U/ml SOD to semen extender, appeared to be the best concentrations that improved (P<0.01) significantly the post-thawing sperm motility (65.00±2.89, 66. 67±1.66, 61.67±1.66 and 63.33±3.34%, respectively); viability indices (167.50±10.12, 166.67±3.01, 150.83±4.65 and 168.33±10.1, respectively) and maintained acrosomal integrity (11.66±4.05, 11.33±2.91, 12.33±3.93 and 10.33±2.34%, respectively) compared to the control semen.  

Table 1: Effect of antioxidants addition to the freezing extender on the buffalo semen freezability.

GSH: Reduced glutathione   CAT: Catalase   SOD: Superoxide dismutase   ALA: Alpha lipoic acid

       Values with different superscript letters in the same columns are significantly different at least (P<0.05).

Data regarding the effect of antioxidants addition to the freezing extender on the mitochondrial function of the cryopreserved semen are illustrated in Fig. 1. In vitro provision of buffalo semen extender with antioxidants significantly (P<0.01) increased the mitochondrial function of the frozen-thawed buffalo semen in a dose dependent trend. The present data showed that, addition of 10 mM GSH, 100 U/ml catalase, 15 mM ALA or 50 U/ml SOD, to the semen extender were the best concentrations that enhanced (P<0.01) significantly the mitochondrial activity of the frozen-thawed buffalo spermatozoa after 3 hour of thawing (3.42±0.05, 3.29±0.16, 2.81±0.09 and 3.31±0.08, respectively) compared to the control semen (1.88±0.05). The current results showed strong positive correlation between mitochondrial activity and in vitro fertilization rate and pregnancy rate (R=0.97 and 0.82, respectively, P<0.01).  

Fig. 1: Effect of antioxidants addition to the freezing extender on the mitochondrial activity of the cryopreserved buffalo spermatozoa.

Data regarding the effect of antioxidants addition to the freezing extender on the DNA integrity of the frozen-thawed buffalo spermatozoa are illustrated in Figs. 2 and 3. The present data indicated that, semen cryopreservation increased (P<0.01) significantly the DNA damage and the composite score (71.67±4.42% and 127.67±17.04, respectively) as shown in photograph, 1. However, the increase in DNA fragmentation and composite score associated cryopreservation was significantly reduced (P <0.01) by the addition of GSH, catalase, ALA and SOD in a dose dependent manner.  Addition of 10 mM GSH, 100 U/ml catalase, 15 mM ALA or 50 U/ml SOD to the semen extender were the best antioxidant concentrations that maintained the DNA integrity (37.67±4.33, 36.33±2.61, 43.33±5.05 and 36.00±5.04%, respectively) and significantly (P<0.01) decreased the composite score of the frozen-thawed semen (66.00±9.06, 65.67±8.30, 87.33±5.82 and 64.67±5.51, respectively), compared to the control frozen semen. The current results showed a significant negative correlation between DNA damage and post-thawing motility (R=- 0.95, P<0.01), viability (R=- 0.97, P<0.01), mitochondrial function (R=- 0.95, P<0.01).   There was also a strong negative correlation between DNA damage and the in vitro fertilization rate and pregnancy rate (R=- 0.96 and -0.77, respectively, P<0.01).

Fig. 2: Effect of antioxidants addition to the freezing extender on the DNA integrity of the cryopreserved buffalo spermatozoa.

Fig. 3: Effect of antioxidants addition to the freezing extender on the DNA composite score of the cryopreserved buffalo spermatozoa.

Data regarding the effect of replenishing of semen extender with antioxidants on the in vitro fertilizing potentials and embryo development are presented in Figs. 4 and 5. The current results revealed that, addition of antioxidants to the freezing extender had a positive effect on the in vitro fertilization rate in a dose-dependent manner (P < 0.05) compared with the control semen. When 10 mM GSH, 100 U/ml catalase or 50 U/ml SOD was added to the freezing extender, a higher proportion of decondensed sperm heads was observed inside the oocyte (65.06, 67.95 and 68.67%, respectively) compared with the control semen (46.58 %).

Fig. 4: Effect of antioxidants addition to the freezing extender on the in vitro fertilizing ability of the cryopreserved buffalo spermatozoa.

Moreover, the results presented in Fig. 5 clarified that, addition of antioxidants to the freezing extender prior to freezing significantly increased (P < 0.05) the cleavage rate, the morula and the blastocyst development, in a dose dependent manner. Addition of 10 mM GSH, 100 U/ml catalase or 50 U/ml SOD to the freezing extender, significantly (P< 0.05) increased the cleavage rate (50.58, 46.55 and 48.21%, respectively), the morula (18.64, 19.23 and 19.64%, respectively) and the blastocyst development (11.86, 11.54 and 14.20%, respectively) compared to the control semen (36.99, 6.85 and 2.74%, respectively) as shown in photograph, 2. The current results demonstrated that, addition of ALA to the freezing extender had a non beneficial effect on the in vitro fertilizing ability and embryo development of the cyopreserved buffalo spermatozoa. Moreover, addition of higher antioxidant concentrations to the semen extender resulted in a detrimental effect on the fertilizing potential and embryo development in vitro.

Fig. 5: Effect of antioxidants addition to the freezing extender on the embryo development in vitro.

Data regarding the effect of fortification of semen extender with antioxidants on the pregnancy rate is demonstrated in Fig. 6. The results showed that addition of 10 mM GSH, 100 U/ml catalase or 50 U/ml SOD to the semen extender significantly (P< 0.05) augmented the pregnancy rate (62.11, 62.79 and 67.74%, respectively) compared with the control semen (51.67%). However, the pregnancy rate among the antioxidants treated groups were not statistically significant (P > 0.05).

Fig. 6: Effect of antioxidants addition to the freezing extender on the pregnancy   rate.

        Grade 0                                                               Grade 1 

Grade 2                                                     Grade 3

Grade 4

Photograph 1: Epiflourescent images of buffalo spermatozoa after the single cell gel electrophoresis (comet) assay and staining by ethedium bromide. Relative changes in DNA fragmentation are represented by an increasing amount of DNA present in the comet tail and are scored from grade 0 (no comet tail) to grade 4 (maximum damage). Magnification 40 ×.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

Photograph 2: (a) Fetilized oocyte showing male pronucleus formation, (b) compact morula stage and             (e) blastocyst stage that resulted from insemination of matured buffalo oocytes with antioxidant-treated semen.

Discussion

Reactive oxygen species (ROS) generation during sperm processing (cryopreservation/thawing), accompanied by low scavenging and antioxidant levels in sperm-processing media will induce a state of oxidative stress (Hellstrom et al., 1994). The authors further added that, high levels of ROS (superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) endanger sperm motility, viability, and function by interacting with membrane lipids, proteins and mitochondrial DNA. These observations  may explain the results of the current study, that emphasized that, cryopreservation of buffalo spermatozoa is associated with damage of sperm function affecting those processes required for the successful fertilization. These results are in consistent with Bilodeau et al. (2000). The significant decrease in the cryopreservsed sperm motility, viability and acrosomal integrity may be attributed to extensive chemical-physical damage to the extracellular and intracellular membranes of the sperm that resulted in changes in the lipid phase transition and/or increased lipid peroxidation during cryopreservation (Ball et al., 2001). After cryopreservation, the production of ROS leads to increased lipid peroxidation (Zalata et al., 2004) which leads to a significant loss of sperm motility (O’Connell et al., 2003).  Moreover, the link between ROS and reduced motility may be due to a cascade of events that result in a decrease in axonemal protein phosphorylation and sperm immobilization, both of which are associated with a reduction in membrane fluidity (de Lamirande and Gagnon, 1995).  

Sperm DNA integrity is important for the success of natural or assisted fertilization, including normal development of the embryo (Lopes et al., 1998). The current results revealed that cryopreservation is associated with a drastic effect on DNA integrity and mitochondrial function. This may be true particularly in light of a recent report by Baumber et al. (2003); Sanocka and Kurpisz (2004) and Slowińska et al. (2008) who showed that sperm DNA integrity and mitochondrial function deteriorated after cryopreservation. Sperm DNA damage induced by cryopreservation may be attributed to the oxidative stress, which is detrimental to sperm DNA integrity (Fraser and Strzeźek, 2005). Recently, Ward and Ward (2004) provided evidence that mammalian spermatozoa contain a mechanism by which they can digest their own DNA when exposed to stressful environment. Thus, increased DNA damage of the cryopreserved spermatozoa could be attributed to the activation of endonucleases released from the deteriorated plasma membrane of the frozen-thawed spermatozoa (Fraser and Strzeźek, 2005). The highly significant inverse associations between DNA damage and post-thawing sperm motility and viability indices indicating that as the percentage of buffalo spermatozoa with good motility declined, susceptibility to chromatin denaturation rose. These results are supported by other studies with bull(Januskauska et al., 2001).In addition, Januskauska et al. (2001) reported negative relationships between DNA damage and sperm viability and speculated that abnormal chromatin structure might impede the survival of bull spermatozoa during freezing and thawing. Also, there was a strong negative correlation between DNA damage and the percentage of spermatozoa with dysfunctional mitochondria in the cryopreserved buffalo semen. This may explain that sperm motility is associated with healthy mitochondria and that mitochondrial damage may result in a reduction in sperm movement (Donnelly et al., 2000). Moreover, a strong inverse association has been observed in the present study between DNA damage and the capacity of sperm to fertilize. These results are in accordance with Rosenbusch (2000), who indicated that human spermatozoa that have incomplete chromatin condensation fertilize a very low percentage of ova in vitro or fail to fertilize, even after direct injection of spermatozoa into the ovum.  Therefore, the consistent association found in the current study between DNA integrity and sperm parameters, suggest that detection of DNA deterioration by comet assay might be a useful index for the assessment of sperm function and fertility.

With reverence to the effect of cryopresevation on the fertilizing potentials of fozen-thawed buffalo spermatozoa in vitro, the present study clarified that cryopreservation of buffalo spermatozoa decreased significantly the in vitro embryo development. These results are in accordance with Host et al. (2000) and Aitken and Krausz (2001). and this may be attributed to intrinsic generation of excess ROS that resulted in increased oxidative stress and increased DNA fragmentation. Sperm DNA damage induced by oxidative stress will be associated with collateral peroxidative damage to the sperm plasma membrane, consequently the plasma membrane loses the fluidity and integrity that is necessary for sperm-oocyte fusion and can therefore compromise the fertilization potential of the sperm or resulting in a low rate of embryonic development and early pregnancy loss (Ahmadi and Ng 1999 and Sakkas et al., 2002). 

The current results clarified that, addition of antioxidants to the freezing extender prior to cryopreservation significantly improved the freezability and the natural and assisted fertilizing potential of the frozen-thawed buffalo spermatozoa. This protective effect on sperm function was dose dependent. These results are in consistent with Gadea et al. (2005) and Kawakami et al. (2007). GSH could be an important regulator of the scavenging system and one of the most important non-enzymatic antioxidants in sperm cells (Griveau and Le Lannou, 1997) as, GSH probably affects plasma membrane lipid packing and sulfhydryl group content in membrane proteins in sperm (Gadea et al., 2005). Therefore, the beneficial effect of GSH on the sperm freezability and fertilizing potentials may be attributed to its ability to protect sperm against oxidative damage and reduction of oxidative stress-induced DNA oxidation and DNA fragmentation (Agarwal and Said, 2003 and Funahashi and Sano, 2005). Moreover, GSH has a likely role in sperm nucleus decondensation and may alter spindle microtubule formation in the ovum and protect ova and embryo during in vitro fertilization, thus affecting the outcome of pregnancy (Gadea et al., 2004 and Sikka, 2004).

The advantageous effect of SOD on the sperm function may be attributed to the protection of cells from the toxic effect of superoxide anions via dismutates (O₂^-) to form O₂ and H₂O₂ and also could counteract the NADPH-induced oxidative stress in sperm  (Mennella and Jones, 1980). The main function of SOD is to protect spermatozoa against spontaneous O₂ toxicity and lipid peroxidation (Alvarez et al., 1987). A significant correlation has been found between the malondialdehyde production and SOD activity was found in the bovine spermatozoa (Beconi et al., 1991). The presence of high SOD endogenous content delays the onset of lipid peroxidation and the loss of motility as the SOD level is positively correlated with the sperm motility (Alvarez et al., 1987). 

The useful effect of catalase on semen freezability and fertilizing potentials that observed in the current study may reside in its ability to convert H₂O₂ to water and oxygen. This might cause inhibition of anaerobic glycolysis with a subsequent reduction in fructose utilization by spermatozoa (O’flaherty et al., 1997). It is well recognized that H₂O₂ is highly toxic to mammalian spermatozoa and the oxygenation of semen is deleterious to sperm viability (Jeulin et al., 1989). Therefore, the existence of an efficient hydrogen peroxide scavenging mechanism in spermatozoa is necessary for sperm survival

The favorable effect of ALA on the sperm freezability may be due to, alpha lipoic acid fights free radical damage in both fatty and watery regions of cells and helps to recycle other antioxidants in what is described as "antioxidant synergism" (Selvakumar et al., 2006).

However, the results of the present study clarified that, inclusion of increasing levels of antioxidants in the semen extender induced a drastic effect on sperm function. These results are in accordance with Aitken et al. (1998) who indicated that, high levels of antioxidants are associated with impaired sperm function and this may be due to the spermatozoa become more susceptible to the cytotoxic effect of H₂O₂ or the removal of the O₂ which is an important mediator of normal sperm function.

In conclusion, cryopreservation promotes DNA fragmentation in buffalo spermatozoa. DNA fragmentation can be counteracted by the addition of antioxidants, GSH, catalase, ALA and SOD. The addition of these antioxidants to the freezing extender appear to play an important role in sperm antioxidant defense strategy in a dose dependent trend  and could be of significant benefit in improving the freezability, DNA integrity and the natural and assisted fertilizing capacity of the cryopreserved buffalo spermatozoa

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