Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters
and Detect of Chromatin Condensation by Toluidine Blue Staining
in Frozen-Th awed Bull Sperm

ESİN, Burcu; CEVIK, MESUT; AKAR, MELİH; KAYA, Cumali

doi:10.9775/kvfd.2021.26950

Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm

Mehmet DEMİRCİ, (Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Dölleme ve Suni Tohumlama Anabilim Dalı, Samsun, Türkiye)

Melih AKAR, (Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Dölleme ve Suni Tohumlama Anabilim Dalı, Samsun, Türkiye)

Cumali KAYA, (Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Dölleme ve Suni Tohumlama Anabilim Dalı, Samsun, Türkiye)

Mesut ÇEVİK (Ondokuz Mayıs Üniversitesi Veteriner Fakültesi Dölleme ve Suni Tohumlama Anabilim Dalı, Samsun, Türkiye)

Kafkas Üniversitesi Veteriner Fakültesi Dergisi

3 2

Yıl: 2022 Cilt: 28 Sayı: 3 Sayfa Aralığı: 307 - 313 Metin Dili: İngilizce DOI: 10.9775/kvfd.2021.26950 İndeks Tarihi: 07-07-2022

Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm

Öz:

Th e purpose of this study was to observe the eff ect of diff erent thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining. A total of 20 frozen sperm straws from the same Holstein bull were used. While the 30 sec thawing protocol at 37°C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70°C constitutes our fast-thawing group (n=10). Th e motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated. Th ere was a significant diff erence (P<0.05) in sperm plasma membrane integrity, head defect, and total abnormal sperm morphology. Th e chromatin decondensation rate detected by TB in bull semen thawed in the slow thaw system, and the decondensation rate in the fast thaw system, diff ered significantly from each other in line with the literature data (P<0.05). According to the evaluations made in terms of chromatin decondensation rate, the rate obtained in slow thawing (7±0.39) shows an increase up to two times compared to the fast-thawing rate (3.3±0.33) (P<0.05). Th e TB staining procedure can be used to evaluate infertility and chromatin integrity, especially in cases that are suspicious and require rapid evaluation.

Anahtar Kelime:

Dondurulmuş-Çözdürülmüş Boğa Spermasında Hızlı ve Yavaş Çözdürmenin Spermatolojik Parametreler ve Toluidin Mavisi Boyama ile Kromatin Kondenzasyonunun Belirlenmesi Üzerine Etkisi

Öz:

Bu çalışmanın amacı, farklı çözdürme yöntemlerinin motilite ve morfoloji gibi sperma parametreleri ve toluidin mavisi boyası kullanılarak sperm kromatin bütünlüğü üzerindeki etkisini gözlemlemekti. Aynı Holstein boğasından toplam 20 adet donmuş sperma payeti kullanıldı. Dondurulmuş sperma payetlerinin çözdürülmesinde kullanılan 37°C’de 30 saniyelik çözdürme protokolü yavaş çözdürme grubumuzu (n=10) oluştururken, 70°C’de 6 saniyelik çözdürme protokolü hızlı çözdürme grubumuzu (n=10) oluşturmaktadır. Tüm çözdürülmüş spermaların motilitesi, canlılığı, morfolojisi, plazma membran bütünlüğü ve sperm kromatin yoğunlaşma parametrelerinin analizleri yapıldı. Karşılaştırılan iki grup arasında spermatozoon plazma membran bütünlüğü, baş ve toplam anormal spermatozoon morfolojisinde önemli bir fark gözlendi (P<0.05). Mevcut literatür verileri doğrultusunda TB boyaması ile saptanan kromatin dekondenzasyonu yavaş çözdürülen spermalarda hızlı çözdürülen spermalara göre birbirinden önemli ölçüde farklılık göstermiştir (P<0.05). Kromatin yoğunlaşma hızı açısından yapılan değerlendirmelere göre yavaş çözdürme ile elde edilen oran (7±0.39) hızlı çözülme hızına (3.3±0.33) göre iki kata kadar artış göstermektedir (P<0.05). Böylelikle TB boyama prosedürü, özellikle şüpheli ve hızlı değerlendirme gerektiren durumlarda infertilite ve kromatin bütünlüğünü değerlendirmek için kullanılabilir.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

1. Rodriguez‐Martinez H: Laboratory semen assessment and prediction of fertility: Still utopi? Reprod Domest Anim, 38 (4): 312-318, 2003. DOI: 10.1046/j.1439-0531.2003.00436.x
2. Sharma V: Sperm storage for cancer patients in the UK: A review of current practice. Hum Reprod, 26 (11): 2935-2943, 2011. DOI: 10.1093/ humrep/der281
3. Grötter LG, Cattaneo L, Marini PE, Kjelland ME, Ferré LB: Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post‐thaw quality optimization. Reprod Domest Anim, 54 (4): 655- 665, 2019. DOI: 10.1111/rda.13409
4. Nur Z, Dogan I, Soylu MK, Ak K: Effect of different thawing procedures on the quality of bull semen. Rev Med Vet, 154 (7): 487-490, 2003.
5. Rodriguez OL, Berndtson WE, Ennen BD, Pickett BW: Effect of rates of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J Anim Sci, 41 (1): 129-136, 1975. DOI: 10.2527/jas1975.411129x
6. Correa JR, Rodriguez MC, Patterson DJ, Zavos PM: Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity. Theriogenology, 46 (3): 413-420, 1996. DOI: 10.1016/ 0093-691X(96)00163-X
7. Dhami AJ, Sahni KL: Evaluation of different cooling rates, equilibration periods and diluents for effects on deep-freezing, enzyme leakage and fertility of taurine bull spermatozoa. Theriogenology, 40 (6): 1269-1280, 1993. DOI: 10.1016/0093-691X(93)90297-I
8. DeJarnette JM, Barnes DA, Marshall CE: Effects of pre-and postthaw thermal insults on viability characteristics of cryopreserved bovine semen. Theriogenology, 53 (6): 1225-1238, 2000. DOI: 10.1016/S0093- 691X(00)00267-3
9. Hernández M, Roca J, Gil MA, Vázquez JM, Martínez EA: Adjustments on the cryopreservation conditions reduce the incidence of boar ejaculates with poor sperm freezability. Theriogenology, 67 (9): 1436-1445, 2007. DOI: 10.1016/j.theriogenology.2007.02.012
10. Lyashenko A: Effect of different thawing procedures on the quality and fertility of the bull spermatozoa. Asian Pac J Reprod, 4 (1): 17-21, 2015. DOI: 10.1016/S2305-0500(14)60051-8
11. Agarwal A, Allamaneni SS: The effect of sperm DNA damage on assisted reproduction outcomes. Minerva Gynecol, 56 (3): 235-245, 2004.
12. Monachesi NE, Neild D, Carretero M: Dog sperm DNA: Raw semen evaluation with Toluidine blue stain. Reprod Domest Anim, 54 (8): 1078-1084, 2019. DOI: 10.1111/rda.13490
13. Oliva R: Protamines and male infertility. Hum Reprod Update, 12 (4): 417-435, 2006. DOI: 10.1093/humupd/dml009
14. Córdova-Izquierdo A, Oliva JH, Lleó B, García-Artiga C, Corcuera BD, Perez-Gutierrez JF: Effect of different thawing temperatures on the viability, in vitro fertilizing capacity and chromatin condensation of frozen boar semen packaged in 5 ml straws. Anim Reprod Sci, 92 (1-2): 145-154, 2006. DOI: 10.1016/j.anireprosci.2005.05.011
15. Souza ET, Silva CV, Travençolo BAN, Alves BG, Beletti ME: Sperm chromatin alterations in fertile and subfertile bulls. Reprod Biol, 18 (2): 177- 181, 2018. DOI: 10.1016/j.repbio.2018.04.001
16. Hammadeh ME, Zeginiadov T, Rosenbaum P, Georg T, Schmidt W, Strehler E: Predictive value of sperm chromatin condensation (aniline blue staining) in the assessment of male fertility. Arch Androl, 46 (2): 99-104, 2001. DOI: 10.1080/01485010117363
17. Ribas‐Maynou J, García‐Peiró A, Fernández‐Encinas A, Abad C, Amengual MJ, Prada E, Navarro J, Benet J: Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay. Andrology, 1 (5): 715-722, 2013. DOI: 10.1111/j.2047-2927.2013.00111.x
18. Cortés‐Gutiérrez EI, Dávila‐Rodríguez MI, Cerda‐Flores RM, Fernández JL, López‐Fernández C, Aragón Tovar AR, Gosálvez J: Localisation and quantification of alkali‐labile sites in human spermatozoa by DNA breakage detection-fluorescence in situ hybridisation. Andrologia, 47 (2): 221-227, 2015. DOI: 10.1111/and.12250
19. Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M: Toluidine blue cytometry test for sperm DNA conformation: Comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod, 19 (10): 2277-2282, 2004. DOI: 10.1093/humrep/deh417
20. Gilmore A, Hitit M, Ugur MR, Dinh TTN, Tan W, Jousan D, Nicodemus M, Topper E, Kaya A, Memili E: Functional variables of bull sperm associated with cryotolerance. Kafkas Univ Vet Fak Derg, 27 (3): 371-379, 2021. DOI: 10.9775/kvfd.2021.25653
21. Padrik P, Hallap T, Kaart T, Bulitko T, Jaakma Ü: Relationships between the results of hypo-osmotic swelling tests, sperm motility, and fertility in Estonian Holstein dairy bulls. Czech J Anim Sci, 57 (10): 490-497, 2012. DOI: 10.17221/6349-CJAS
22. Beletti ME, Mello MLS: Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology. Theriogenology, 62 (3-4): 398-402, 2004. DOI: 10.1016/j.theriogenology.2003.10.016
23. Mazur P: Freezing of living cells: Mechanisms and implications. Am J Physiol, 247 (3 Pt 1): C125-C142, 1984. DOI: 10.1152/ajpcell.1984.247.3.C125
24. Watson PF: Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev, 7 (4): 871-891, 1995. DOI: 10.1071/rd9950871
25. Correa JR, Heersche G, Zavos PM: Sperm membrane functional integrity and response of frozen-thawed bovine spermatozoa during the hypoosmotic swelling test incubation at varying temperatures. Theriogenology, 47 (3): 715-721, 1997. DOI: 10.1016/S0093-691X(97)00029-0
26. Holt WV: Basic aspects of frozen storage of semen. Anim Reprod Sci, 62 (1-3): 3-22, 2000. DOI: 10.1016/S0378-4320(00)00152-4
27. Curry MR, Watson PF: Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology, 31 (1): 39-46, 1994. DOI: 10.1006/cryo.1994.1005
28. Foote RH: Artificial insemination to cloning: Tracing 50 years of research. Internet-First University Press, 1998.
29. Nur Z, Ileri IK, Ak K: Effects of different temperature treatments applied to deep stored bull semen on post-thaw cold shocked spermatozoa. Bull Vet Inst Pulawy, 50 (1): 79, 2006.
30. Yilmaz E, Ak K, Baran A: Effect of different thawing time and high temperature on frozen thawed bull semen traits. J Anim Vet Adv, 18 (7): 239- 245, 2019. DOI: 10.36478/javaa.2019.239.245
31. Rota A, Manuali E, Caire S, Appino S: Severe tail defects in the spermatozoa ejaculated by an English bulldog. J Vet Med Sci, 70 (1): 123-125, 2008. DOI: 10.1292/jvms.70.123
32. Molnár A, Sarlós P, Fáncsi G, Rátky J, Nagy S, Kovács A: A sperm tail defect associated with infertility in a goat - Case report. Acta Vet Hung, 49 (3): 341-348, 2001. DOI: 10.1556/004.49.2001.3.11
33. Vishwanath R, Shannon P: Storage of bovine semen in liquid and frozen state. Anim Reprod Sci, 62 (1-3): 23-53, 2000. DOI: 10.1016/S0378- 4320(00)00153-6
34. Martins MC, Gonçalves LM, Nonato A, Travençolo BAN, Alves BG, Beletti ME: Sperm head morphometry and chromatin condensation are in constant change at seminiferous tubules, epididymis, and ductus deferens in bulls. Theriogenology, 161, 200-209, 2021. DOI: 10.1016/j. theriogenology.2020.12.004
35. Kazerooni T, Asadi N, Jadid L, Kazerooni M, Ghanadi A, Ghaffarpasand F, Zolghadr J: Evaluation of sperm’s chromatin quality with acridine orange test, chromomycin A3 and aniline blue staining in couples with unexplained recurrent abortion. J Assist Reprod Genet, 26 (11): 591- 596, 2009. DOI: 10.1007/s10815-009-9361-3
36. Rastegarnia A, Shahverdi A, Topraggaleh TR, Ebrahimi B, Shafipour V: Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo (Bubalus bubalis) spermatozoa. Cell J, 14 (4): 306-313, 2013.
37. Dutta S, Henkel R, Agarwal A: Comparative analysis of tests used to assess sperm chromatin integrity and DNA fragmentation. Andrologia, 53 (2):e13718, 2021. DOI: 10.1111/and.13718
38. Banaszewska D, Andraszek K, Biesiada-Drzazga B: Evaluation of sperm chromatin structure in boar semen. Bull Vet Inst Pulawy, 59 (2): 271- 277, 2015. DOI: 10.1015/bvip-2015-0040
39. Muratori M, Tamburrino L, Marchiani S, Cambi M, Olivito B, Azzari C, Forti G, Baldi E: Investigation on the origin of sperm DNA fragmentation: Role of apoptosis, immaturity and oxidative stress. Mol Med, 21 (1): 109-122, 2015. DOI: 10.2119/molmed.2014.00158
40. Ajina T, Ammar O, Haouas Z, Sallem A, Ezzi L, Grissa I, Sakly W, Jlali A, Mehdi M: Assessment of human sperm DNA integrity using two cytochemical tests: Acridine orange test and toluidine blue assay. Andrologia, 49 (10):e12765, 2017. DOI: 10.1111/and.12765
41. Hao SL, Ni FD, Yang WX: The dynamics and regulation of chromatin remodelling during spermiogenesis. Gene, 706, 201-210, 2019. DOI: 10.1016/ j.gene.2019.05.027
42. Lange-Consiglio A, Antonucci N, Manes S, Corradetti B, Cremonesi F, Bizzaro D: Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds. J Small Anim Pract, 51 (12): 624- 627, 2010. DOI: 10.1111/j.1748-5827.2010.01001.x

APA	ESİN B, AKAR M, KAYA C, CEVIK M (2022). Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. , 307 - 313. 10.9775/kvfd.2021.26950
Chicago	ESİN Burcu,AKAR MELİH,KAYA Cumali,CEVIK MESUT Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. (2022): 307 - 313. 10.9775/kvfd.2021.26950
MLA	ESİN Burcu,AKAR MELİH,KAYA Cumali,CEVIK MESUT Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. , 2022, ss.307 - 313. 10.9775/kvfd.2021.26950
AMA	ESİN B,AKAR M,KAYA C,CEVIK M Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. . 2022; 307 - 313. 10.9775/kvfd.2021.26950
Vancouver	ESİN B,AKAR M,KAYA C,CEVIK M Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. . 2022; 307 - 313. 10.9775/kvfd.2021.26950
IEEE	ESİN B,AKAR M,KAYA C,CEVIK M "Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm." , ss.307 - 313, 2022. 10.9775/kvfd.2021.26950
ISNAD	ESİN, Burcu vd. "Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm". (2022), 307-313. https://doi.org/10.9775/kvfd.2021.26950

APA	ESİN B, AKAR M, KAYA C, CEVIK M (2022). Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 28(3), 307 - 313. 10.9775/kvfd.2021.26950
Chicago	ESİN Burcu,AKAR MELİH,KAYA Cumali,CEVIK MESUT Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. Kafkas Üniversitesi Veteriner Fakültesi Dergisi 28, no.3 (2022): 307 - 313. 10.9775/kvfd.2021.26950
MLA	ESİN Burcu,AKAR MELİH,KAYA Cumali,CEVIK MESUT Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, vol.28, no.3, 2022, ss.307 - 313. 10.9775/kvfd.2021.26950
AMA	ESİN B,AKAR M,KAYA C,CEVIK M Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2022; 28(3): 307 - 313. 10.9775/kvfd.2021.26950
Vancouver	ESİN B,AKAR M,KAYA C,CEVIK M Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm. Kafkas Üniversitesi Veteriner Fakültesi Dergisi. 2022; 28(3): 307 - 313. 10.9775/kvfd.2021.26950
IEEE	ESİN B,AKAR M,KAYA C,CEVIK M "Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm." Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 28, ss.307 - 313, 2022. 10.9775/kvfd.2021.26950
ISNAD	ESİN, Burcu vd. "Th e Eff ects of Fast and Slow Th awing on Spermatological Parameters and Detect of Chromatin Condensation by Toluidine Blue Staining in Frozen-Th awed Bull Sperm". Kafkas Üniversitesi Veteriner Fakültesi Dergisi 28/3 (2022), 307-313. https://doi.org/10.9775/kvfd.2021.26950