Abstract
The objective of the present study was to investigate the effect of different freezing rates on post-thaw sperm motility, acrosome defect, and sperm chromatin structure and apoptotic activity in ram semen. Collected semen was diluted at 1:5 (semen/extender) with Bioxel® (IMV technologies France) at 30ºC and then cooled to 5ºC within 1h. Cooled semen was subjected to the equilibration for 2 hours. Equilibrated semen was frozen in 0.25 ml straw at two different cooling rates (slow: 0.5°C/min from 5 to -20°C and fast: 5°C/min from 5 to -20°C). Both groups were frozen from -20 to -120°C at 25°C/min and stored in liquid nitrogen until use. Post-thaw (37°C/30 min) sperm motility, defected acrosome (Pisum sativum agglutinin fluorescein conjugate, FITC PSA), sperm chromatin structure determined by Acridin Orange (AO) and apoptotic activity using terminaldeoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) were evaluated. Post-thaw sperm motility, acrosome defect, AO and TUNEL for slow frozen semen were 42.8±8.8%, 31.6±12.9%, 2.9±2.4% and 2.8±1.6%, and for fast frozen semen were 36.5±9.9%, 24.7±11.1%, 3.3±2.2% and 6.3±3.4%, respectively. Post-thaw semen analyses showed that there was no significant difference between two freezing curves in terms of acrosome defect, sperm chromatin damage (AO). However, a significant difference was found for postthaw semen motility between two groups (P<0.05). In conclusion, while the slow freezing procedure improved post-thaw sperm motility, acrosome and chromatin integrities and apoptotic index in ram spermatozoa did not show any significant difference between freezing rates
Keywords
References
- Abdelhakeam AA, Graham EF, Vazquez IA, Chaloner KM (1991): Studies on the absence of glycerol in unfrozen and frozen ram semen. Development of an extender for freezing: effects of osmotic pressure, egg yolk levels, type of sugars and the method of dilution. Cryobiology, 28, 43- 49.
- Abdelhakeam AA, Graham EF, Vazquez IA (1991): Studies on the absence of glycerol in unfrozen and frozen ram semen: Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol. Cryobiology, 28, 36-42.
- Byrne GP, Lonergan P, Wade MJ, Duffy P, Donovan A, Hanrahan JP, Boland MP (2000): Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci, 62, 265-275.
- Chohan KR, Griffin JT, Carrell DT (2004): Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation. Andrologia, 36, 321–326.
- De Leeuw FE, De Leeuw A M, Den Daas JHG, Colenbrander B, Verkleij AJ (1993): Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling. Cryobiology, 30, 32–44.
- Duran EH, Morshedi M, Taylor S, Oehninger S (2002): Sperm DNA quality predicts intrauterine insemination outcome – a prospective cohort study. Hum Reprod, 17, 3122-3128.
- Fiser P, Fairfull RW (1984): The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology, 21, 542-551.
- Gunay U, Dogan I, Nur Z, Manolov I, Sagırkaya H, Soylu MK, Kaptan C, Akpınar L (2006): Influence of bull seminal plasma on post-thaw ram semen parameters and fertility. Bull Vet Inst Pulawy, 50, 503-507.
- Hafez ESE (1993): Semen evaluation. 405-423. In: Reproduction In Farm Animals. edited by E.S.E. Hafez, Philadelphia, Lea and Febiger.
- Hammadeh ME, Askari A, Gerorg T, Rosenbaum P, Schmidt W (1999): Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl, 22, 155–162.
- Hammadeh ME, Dehn CH, Hippach M., Zeginiadou T, Stieber M, Georg T, Rosenbaum P, Schmidt W (2001): Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men. Int J Androl,24, 66–72.
- Kawakami E, Morita Y, Hori T, Tsutsui T (2002): Lectin-binding characteristics and capacitation of canine epididymal spermatozoa. J Vet Med Sci, 64, 543–49.
- Li X, Darzynkiewicz Z (1995): Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif, 28, 571–579.
- Martí E, Pérez-Pé R, Muiño-Blanco T, Cebrián-Pérez JA (2006): Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa. J Androl, 27, 746–53.
- Maxwell WMC, Landers AJ, Evans G (1995): Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes. Theriogenology, 43, 1201-1210.
- Nur Z, Zik B, Ustuner B, Sagirkaya H, Ozguden CG (2010): Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology, 73, 1267-1275.
- O’Neill DJ (1998): Studies on the cryopreservation of ram spermatozoza. MSc(Agr.) Thesis, National University of Ireland.
- Pontbriand D, Howard JG, Schieve MC, Stuart LD, Wildt DE (1989): Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa. Cryobiology, 26, 341-354.
- Salamon S, Maxwell WMC (1993): Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim Reprod Sci, 38, 1–36.
- Sanches-Partida LG, Maxwell WMC, Pateg LG, Setchell BP (1992): Proline and glycine betaine in the cryoprotective diluents for ram spermatozoa. Reprod Fertil Dev, 4, 113-118.
- Soylu MK, Nur Z, Ustuner B, Dogan I, Sagirkaya H, Gunay U, Ak K (2007): Effects of various cryoprotective agents and extender osmolality on post-thaw ram semen. Bull Vet Inst Pulawy, 51, 241–246.
- Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B, Suchanek E (2000): Comparison of protective media and freezing techniques for cryopreservation of human semen. Eur J Obstet Gynecol Reprod Biol, 91, 65–70.
- Varisli O, Uguz C, Agca C, Agca Y (2008): Motility and acrosomal integrity comparisons between electro- ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures. Anim Reprod Sci, 110, 256–268.
- Watson PF (1995) : Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev, 7, 871-891.
- Woelders H, Matthijs A, Engel B (1997): Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology, 35, 93–105.
- Wulster-Radcliffe MC, Wang S, Lewis GS (2004): Transcervical artifcial insemination in sheep: effects of a new transcervical artifcial insemination instrument and traversing the cervix on pregnancy and lambing rates. Theriogenology, 62, 990–1002.
- Yildiz C, Kaya A, Aksoy M, Tekeli T (2000): Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology, 54, 579–585.
Abstract
Bu çalışma, farklı dondurma hızlarının, eritme sonrası motilite, akrozom bütünlüğü, kromatin yapısı ve apoptozis üzerine olan etkilerini araştırmak amacıyla gerçekleştirildi. Toplanan sperma örnekleri 30ºC’de 1/5 oranında (sperma/sulandırıcı) Bioxel® (IMV, Fransa) ile sulandırıldı ve 1 saatte +5ºC’ye düşürülerek 2 saat süre ile ekilibrasyona bırakıldı. Ekilibre edilen sperma 0.25 ml’lik payetlere çekilerek iki farklı soğutma hızında (yavaş: +5°C’tan -20°C’a 0.5°C/dak ve hızlı: +5°C’tan -20°C’a 5°C/dak) donduruldu. Her iki grup -20°C’tan -120°C’a 25°C/dak hızla donduruldu. Eritme sonrası (37°C/30 dak) aşamada; akrozom bozukluğu Pisum sativum agglutinin fluorescein conjugate ( FITC PSA) ile kromatin yapısı Acridin Orange (AO) ile ve apoptozis ise terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling tekniği (TUNEL) kullanarak belirlendi. Motilite muayeneleri ısıtma tablalı faz kontrast mikroskop ile gerçekleştirildi. Eritme sonrası motilite, akrozom bozukluğu, AO ve TUNEL değerleri sırasıyla yavaş dondurma için %42.8±8.8, %31.6±12.9, %2.9±2.4 ve 2.8±1.6 hızlı dondurma için ise %36.5±9.9, %24.7±11.1, %3.3±2.2 ve %6.3±3.4 olarak saptandı. Spermanın eritme sonrası bulgularının değerlendirilmesinde akrozom bozukluğu ve sperma kromatin yapısı (AO) yönünden iki soğutma hızı arasında fark bulunmazken, eritme sonrası motilitede gruplar arasında istatistiksel fark bulundu (P<0.05). Sonuç olarak, koç spermasında yavaş soğutma prosedürü ile dondurma, eritme sonrası motiliteyi iyileştirirken, akrozom ve kromatin bütünlüğünü ve apoptotik indeksi soğutma oranları arasında önemli bir fark göstermemiştir
Keywords
References
- Abdelhakeam AA, Graham EF, Vazquez IA, Chaloner KM (1991): Studies on the absence of glycerol in unfrozen and frozen ram semen. Development of an extender for freezing: effects of osmotic pressure, egg yolk levels, type of sugars and the method of dilution. Cryobiology, 28, 43- 49.
- Abdelhakeam AA, Graham EF, Vazquez IA (1991): Studies on the absence of glycerol in unfrozen and frozen ram semen: Fertility trials and the effect of dilution methods on freezing ram semen in the absence of glycerol. Cryobiology, 28, 36-42.
- Byrne GP, Lonergan P, Wade MJ, Duffy P, Donovan A, Hanrahan JP, Boland MP (2000): Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci, 62, 265-275.
- Chohan KR, Griffin JT, Carrell DT (2004): Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation. Andrologia, 36, 321–326.
- De Leeuw FE, De Leeuw A M, Den Daas JHG, Colenbrander B, Verkleij AJ (1993): Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling. Cryobiology, 30, 32–44.
- Duran EH, Morshedi M, Taylor S, Oehninger S (2002): Sperm DNA quality predicts intrauterine insemination outcome – a prospective cohort study. Hum Reprod, 17, 3122-3128.
- Fiser P, Fairfull RW (1984): The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology, 21, 542-551.
- Gunay U, Dogan I, Nur Z, Manolov I, Sagırkaya H, Soylu MK, Kaptan C, Akpınar L (2006): Influence of bull seminal plasma on post-thaw ram semen parameters and fertility. Bull Vet Inst Pulawy, 50, 503-507.
- Hafez ESE (1993): Semen evaluation. 405-423. In: Reproduction In Farm Animals. edited by E.S.E. Hafez, Philadelphia, Lea and Febiger.
- Hammadeh ME, Askari A, Gerorg T, Rosenbaum P, Schmidt W (1999): Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl, 22, 155–162.
- Hammadeh ME, Dehn CH, Hippach M., Zeginiadou T, Stieber M, Georg T, Rosenbaum P, Schmidt W (2001): Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men. Int J Androl,24, 66–72.
- Kawakami E, Morita Y, Hori T, Tsutsui T (2002): Lectin-binding characteristics and capacitation of canine epididymal spermatozoa. J Vet Med Sci, 64, 543–49.
- Li X, Darzynkiewicz Z (1995): Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif, 28, 571–579.
- Martí E, Pérez-Pé R, Muiño-Blanco T, Cebrián-Pérez JA (2006): Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa. J Androl, 27, 746–53.
- Maxwell WMC, Landers AJ, Evans G (1995): Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes. Theriogenology, 43, 1201-1210.
- Nur Z, Zik B, Ustuner B, Sagirkaya H, Ozguden CG (2010): Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology, 73, 1267-1275.
- O’Neill DJ (1998): Studies on the cryopreservation of ram spermatozoza. MSc(Agr.) Thesis, National University of Ireland.
- Pontbriand D, Howard JG, Schieve MC, Stuart LD, Wildt DE (1989): Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa. Cryobiology, 26, 341-354.
- Salamon S, Maxwell WMC (1993): Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim Reprod Sci, 38, 1–36.
- Sanches-Partida LG, Maxwell WMC, Pateg LG, Setchell BP (1992): Proline and glycine betaine in the cryoprotective diluents for ram spermatozoa. Reprod Fertil Dev, 4, 113-118.
- Soylu MK, Nur Z, Ustuner B, Dogan I, Sagirkaya H, Gunay U, Ak K (2007): Effects of various cryoprotective agents and extender osmolality on post-thaw ram semen. Bull Vet Inst Pulawy, 51, 241–246.
- Stanic P, Tandara M, Sonicki Z, Simunic V, Radakovic B, Suchanek E (2000): Comparison of protective media and freezing techniques for cryopreservation of human semen. Eur J Obstet Gynecol Reprod Biol, 91, 65–70.
- Varisli O, Uguz C, Agca C, Agca Y (2008): Motility and acrosomal integrity comparisons between electro- ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures. Anim Reprod Sci, 110, 256–268.
- Watson PF (1995) : Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev, 7, 871-891.
- Woelders H, Matthijs A, Engel B (1997): Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology, 35, 93–105.
- Wulster-Radcliffe MC, Wang S, Lewis GS (2004): Transcervical artifcial insemination in sheep: effects of a new transcervical artifcial insemination instrument and traversing the cervix on pregnancy and lambing rates. Theriogenology, 62, 990–1002.
- Yildiz C, Kaya A, Aksoy M, Tekeli T (2000): Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology, 54, 579–585.
Details
| Primary Language | English |
|---|---|
| Subjects | Veterinary Surgery |
| Other ID | JA76UT89BC |
| Journal Section | Research Article |
| Authors | |
| Publication Date | December 1, 2011 |
| Published in Issue | Year 2011Volume: 58 Issue: 4 |