Freezing of Honamli Goat Buck Sperm with Commercial and Laboratory Extenders and Evaluation of In Vitro Spermatological Parameters

Abstract

This study aimed to evaluate the effectiveness of commercial versus laboratory-made extenders for cryopreserving Honamli buck semen. Semen was collected from three 2-3-year-old bucks and pooled. The pooled semen was divided into two portions: one with seminal plasma removed and diluted with Tris egg yolk-based extender (TEYBE+), and the other with seminal plasma retained, extended with commercial Bioxcell (B), Tris egg yolk-based extender (TEYBE-), and Tris lecithin-based extender (TLEBE). The extended semen was stabilized at +4 °C for 2 hours, cryopreserved by exposure to liquid nitrogen steam at -120 °C for 12 minutes, and frozen. After thawing, motility, viability, plasma membrane and acrosome integrity (PMAI), high mitochondrial membrane potential (HMMP), and mitochondrial reactive oxygen species (MROS) levels were evaluated. Group B exhibited the lowest post-thaw motility, viability, PMAI, and HMMP values (p < 0.001). The highest viability rate was recorded in the TEYBE+ group (p < 0.05). TEYBE- showed superior motility, PMAI, HMMP, and viability compared to B, whereas TLEBE did not outperform TEYBE+. The lowest MROS levels were observed in the TEYBE+ group (p < 0.05, compared to B). In conclusion, the findings indicated that the separation of the seminal plasma used in the freezing of buck semen had a beneficial impact on the freezing process. Conversely, the B extender, which is commercially employed for freezing bull semen, was ineffective in cryopreservation of the buck semen when compared to extenders containing lecithin and egg yolk.

Keywords

• Bioxcell
• Cryopreservation
• Flow cytometry
• Honamlı

Honamlı Teke Spermasının Ticari ve Laboratuvarda Hazırlanan Sulandırıcılar ile Dondurulması ve İn Vitro Spermatalojik Parametrelerinin Değerlendirilmesi

Abstract

Bu çalışmanın amacı Honamlı Teke spermasının kriyoprezervasyonunda ticari ve laboratuvar sulandırıcılarını etkinliğini değerlendirmektir. Çalışmada 2-3 yaşlarında üç Honamlı tekesinden spermalar toplandı ve birleştirildi. Birleştirilen sperma iki eşit parçaya bölündü; Bir kısım seminal plazma uzaklaştırıldı ve tris yumurta sarısı bazlı sulandırıcı (TYSBS+) ile sulandırıldı; diğer kısım seminal plazması uzaklaştırılmadı ve Bioxcell (B), tris yumurta sarısı bazlı sulandırıcı (TYSBS-) ve tris lesitin bazlı sulandırıcı (TLBS) ticari sulandırıcı ile sulandırıldı. Sulandırılan sperma örnekleri +4 derecede 2 saat boyunca ekilibrasyona ve sıvı nitrojen buharında (-120°C'de 12 dakika) kriyoprezervasyona tabi tutularak donduruldu. Çözdürme işleminden sonra spermaların motilite, canlılık, plazma membranı ve akrozom bütünlüğü (PMAI), yüksek mitokondriyal membran potansiyeli (HMMP) ve mitokondriyal reaktif oksijen türleri (MROS) seviyeleri değerlendirildi. B grubu, çözdürme sonrası en düşük motilite, canlılık, PMAI ve HMMP değerlerini göstermiştir (p < 0.001). En yüksek canlılık oranı TYSBS+ grubunda kaydedilmiştir (p<0.05). TYSBS-, B'ye kıyasla daha yüksek motilite, PMAI, HMMP ve canlılık gösterirken, TLBS, TYSBS+'den daha iyi performans sergileyememiştir. En düşük MROS seviyeleri TYSBS+'de gözlenmiştir (p < 0,05, B ile karşılaştırıldığında). Sonuç olarak, teke spermasının dondurulması sürecinde seminal plazmanın uzaklaştırılmasının dondurma başarısı üzerinde olumlu bir etkisi olduğu belirlenmiştir. Buna karşılık, boğa spermasının dondurulmasında yaygın olarak kullanılan ticari B sulandırıcısının, teke spermasının kriyoprezervasyonu açısından, yumurta sarısı ve lesitin içeren sulandırıcılara kıyasla etkisiz olduğu tespit edilmiştir.

Keywords

• Bioxcell
• Kriyoprezervasyon
• Akış sitometrisi
• Honamlı

References

1. Aires VA, Hinsch K-D, Mueller-Schloesser F, Bogner K, Mueller-Schloesser S, Hinsch E, 2003: In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology, 60 (2), 269-279.
2. Akhter S, Ansari MS, Rakha BA, Andrabi SMH, Iqbal S, Ullah N, 2010: Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell® extender. Theriogenology, 74 (6), 951-955.
3. Alçay S, Berk Toker M, Gokce E, Ustuner B, Tekin Onder N, Sagirkaya H, Nur Z, Soylu MK, 2015: Successful ram semen cryopreservation with lyophilized egg yolk-based extender. Cryobiology, 71 2, 329-333.
4. Anand M, Yadav S, Shukla P, 2014: Cryoprotectant in semen extender: From egg yolk to low density lipoprotein(LDL). Indian J Small Rumin, 20, 84-53.
5. Büyükleblebici S, Tuncer PB, Taşdemir U, Özgürtaş T, Durmaz E, Büyükleblebici O, 2014: The comparison of three different cryoprotectants in cryopreservation of angora goat semen. Kafkas Univ Vet Fak Derg, 20 (4), 613-619.
6. Çevik M,Tuncer PB, 2005: Evcil hayvanlarda seminal plazmanın fiziko-kimyasal yapısı ve üreme fonksiyonları üzerindeki etkileri. Lalahan Hay Araşt Enst Derg, 45 (2), 63-72.
7. Daşkın A, Kulaksız R, Akçay E, Erol H, 2011: The effect of different dilution rates of Angora buck semen frozen with Bioxcell® extender on the post-thaw quality. J Fac Vet Med Erciyes, 8 (1), 23-26.
8. de Paz P, Esteso MC, Alvarez M, Mata M, Chamorro CA, Anel L, 2010: Development of extender based on soybean lecithin for its application in liquid ram semen. Theriogenology, 74 (4), 663-671.

9. Dorado J, Hidalgo M, Muñoz A, Rodríguez I, 2009: Assessment of goat semen freezability according to the spermatozoa characteristics from fresh and frozen samples. Anim Reprod Sci, 112 1-2, 150-157.
10. Dönmez H, İnanç ME, 2024: Spermatological Parameters In Vitro of Holstein Bulls and Relationship with Male Fertility. Acta Sci Vet, 52 (1), 2-3.
11. Emamverdi M, Zhandi M, Shahneh AZ, Sharafi M, Akhlaghi A, Motlagh MK, Dadkhah F, Davachi ND, 2015: Flow Cytometric and Microscopic Evaluation of Post-Thawed Ram Semen Cryopreserved in Chemically Defined Home-Made or Commercial Extenders. Anim Reprod Sci, 55 (4), 551.
12. Fernández-Novo A, Santos-Lopez S, Barrajon-Masa C, Mozas P, Mercado Ed, Cáceres E, Garrafa A, Gonzalez-Martin J-V, Pérez‐Villalobos N, Oliet A, Astiz S, Pérez-Garnelo SS, 2021: Effects of Extender Type, Storage Time, and Temperature on Bull Semen Parameters. Biology, 10 (7), 630.
13. Ferreira VdS, Mello MRB, Fonseca CEMd, Dias ÁCF, Cardoso JM, Silva RBB, Júnior WPM, 2014: Effect of seminal plasma and egg yolk concentration on freezability of goat semen. R Bras Zootec, 43, 513-518.
14. Forouzanfar M, Sharafi M, Hosseini SM, Ostadhosseini S, Hajian M, Hosseini L, Abedi P, Nili N, Rahmani H, Nasr-Esfahani MH, 2010: In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen. Theriogenology, 73 4, 480-487.
15. Gangwar C, Kharche SD, Kumar S, Sk J, 2016: Cryopreservation of goat semen: status and prospects. Indian J Small Rumin, 22, 1-10.
16. Gholami M, Faraji Z, Zamiri MJ, 2012: Effect of Egg Yolk of Four Avıan Specıes On The Cryopreserved Ram Spermatozoa. Iran J Vet Res, 13, 23-27.
17. Inanc ME, Guzel FN, Gungor S, Herdogan M, Mart FN, Kahraman D, Cay HA, Ata A, 2023: The interaction between seminal plasma biochemical constituents and short‐term semen preservation in Honamli and hair goats. Reprod Domest Anim, 58 (3), 387-395.
18. Kaka A, Wahid H, Rosnina Y, Yimer N, Khumran AM, Sarsaifi K, Behan AA, Kaka U, Memon AA, Ebrahimi M, 2017: Effect of Semen in BioXcell Extender. Reprod Fertil Dev, 29 (3), 490.
19. Khatun A, Fazili MuR, Malik AA, Shah RA, Khan HM, Choudhury AR, Naikoo M, Lone FA, Malik AA, 2021: In vitro Assessment of Tris Egg Yolk and Soybean Lecithin Based Extenders for Cryopreservation of Crossbred Ram Semen. Cryo letters, 42 2, 73-80.
20. Kulaksiz R, Ari U, Daşkin A, Üner AG, 2013: The effect of different glycerol concentrations on freezability of semen from Angora, Kilis and Saanen goats. Slovak J Anim Sci, 46 (2), 39-44.
21. Layek SS, Mohanty TK, Kumaresan A, Parks JE, 2016: Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Anim Reprod Sci, 172, 1-9.
22. Madrigali A, Rota A, Panzani D, Castellani S, Shawahina M, Hassan AW, Iacovo FD, Rossignoli C, Camillo F, 2021: Artificial Insemination in Sheep With Fresh Diluted Semen: Comparison Between Two Different Semen Extenders and Management Protocols. Trop Anim Sci J, 44 (3), 255-260.
23. Masoudi R, Sharafi M, Zareh Shahneh A, Towhidi A, Kohram H, Esmaeili V, Shahverdi A, Davachi ND, 2016: Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin. Cryobiology, 73 1, 69-72.
24. Mohamed A, Moustafa MZ, 2017: Privilege of Gum Arabic Inclusion in Semen Extender Compared With Egg Yolk on the Herri Ram’s Subsequent Fertility Outcomes. Int J Anim Res. 1 (1), 15-21.
25. Nethenzheni L, Mphaphathi M, Madzhie L, Negota N, Kalonji P, Nedambale T, Barry D, 2021: Effect of Bioxcell® and Triladyl® Extenders and Removal of Seminal Plasma on Equilibrated and Cryopreserved Semen From South African Unimproved Indigenous Bucks. J Bacteriol Mycol, 8 (1), 1164.
26. Purdy P, 2006: A review on goat sperm cryopreservation. Small Rumin. Res., 63 (3), 215-225.
27. Salamon S, Maxwell WMC, 2000: Storage of ram semen. Anim Reprod Sci, 62 (1-3), 77-111.
28. Sarıözkan S, Bucak M, Tuncer P, Taşdemir U, Kinet H, Ulutaş P, 2010: Effects of different extenders and centrifugation/washing on postthaw microscopic-oxidative stress parameters and fertilizing ability of Angora buck sperm. Theriogenology, 73 (3), 316-323.
29. Sen CC, Tekin K, 2015: Effect of Egg Yolk and Removal of Seminal Fluid on Semen Cryopreservation in Norduz Goat. Harran Üniv Vet Fak Derg, 4 (2), 64-67.
30. Sharma A, Sood P, 2019: Cryopreservation and fertility of frozen thawed Chegu goat semen. Indian J Anim Res, 53 (11), 1414-1419.
31. Sun L, Fan W, Wu C, Zhang S, Dai J, Zhang D, 2020: Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation. Cryobiology, 92, 146-150.
32. Üstüner B, Aktar A, Yılmaz M, Mülkpınar E, Gökçe E, Boz E, Alçay S, Uçar Ş, 2020: Kollajenin Saanen Teke Spermasının Dondurulabilirliği Üzerine Etkisi. J Res Vet Med, 39 (2), 101-105.
33. Watson PF, 2000: The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci, 60-61, 481-492.
34. Zhao J, Xiao G, Zhu W-L, Fang D, Li N, Han C-M, Gao Q, 2020: Trehalose Addition to a Tris-Fructose Egg Yolk Extender on Quality of Ram Sperm Preserved at 0 °C. R Bras Zootec, 49.