High Trehalose Concentration in Tris-Based Egg Yolk Extender has Detrimental Effect on Post-Thaw Semen Quality in Nili-Ravi Buffalo Bull

Abstract

Background/Aim: The use of artificial insemination with cryopreserved semen is potential tool for breed improvement in buffalo production. However, the fertility with the use of cryopreserved semen is approximately 40% in buffaloes. It is the general opinion that a substantial number 50% of sperm damaged or dead during freezing and thawing process, is the potential cause of low fertility. The aim of the present study was to determine the effect of high trehalose concentration in Tris-based egg yolk extender on post-thawed semen quality in buffalo bull. Material and Method: Semen ejaculates n=04 collected from two mature buffalo bulls were extended with E1 Tris-citric acid-fructose TCF with 7% glycerol , E2 TCF plus 236 mM trehalose or E3 E2 plus 7% glycerol . All the extenders contained egg yolk 20%, v/v . After dilution, samples were cooled to 4°C and equilibrated for 2 hrs at 4°C. Samples were packed in 0.5 ml straws, frozen and stored in liquid nitrogen. Frozen straws were then thawed at 37°C for 20 sec. Motility was observed after dilution, equilibration and post-thawing. Plasma membrane integrity and percent live sperm were determined after thawing by using hypo-osmotic swelling assay and eosin-nigrosin staining, respectively. Results and Conclusion: The results showed that the motility was decreased p

Keywords

• Buffalo semen,Trehalose,Glycerol,Post-thaw semen quality.

Tris-Yumurta Sarısı Sulandırıcısına Yüksek Düzeyde Trehaloz İlavesi Nili-Ravi Manda Spermalarında Çözüm Sonu Parametreleri Olumsuz Etkiler

Öz

Özbilgi/Amaç: Dondurulmuş sperma ile suni tohumlamanın manda yetiştiriciliği ve üretiminde kullanımı önemli bir yöntemdir. Buna karşın, mandalarda dondurulmuş sperma ile fertilite oranı yaklaşık %40 dolayındadır. Fertilitedeki düşüş genel olarak dondurma ve çözdürme sırasında spermanın %50’sinin hasar görmesine yorumlanmıştır. Bu çalışmada yüksek trehaloz yoğunluklarının mandalarda çözüm sonu sperma kalitesi üzerine etkisinin belirlenmesi amaçlanmıştır. Materyal ve Metot: Toplam iki baş erişkin manda boğasından alınan sperma n=04 örnekleri E1 Tris-sitrik asit-fruktoz+%7 gliserol TCF , E2 TCF+236mM trehaloz veya E3 E2+%7 glycerol sulandırıcılarıyla sulandırıldı. Bu üç sulandırıcı da %20 v/v oranında yumurta sarısı içermekteydi. Daha sonra örnekler 4C°’ye kadar soğutuldu ve bu sıcaklıkta iki saat süre ile ekilibre edildi. Sperma örnekleri 0,5 ml lik payetlere çekildi ve sıvı azot içerisinde dondurularak saklandı. Payetler 37C°’de 20 saniyede çözdürüldü. Motilite değerleri sulandırma, ekilibrasyon ve çözüm sonrasında belirlendi. Plazma membran bütünlüğü ve canlı spermatozoon oranları, sırasıyla, hipoozmotik şişme testi ve eosin-nigrosin boyama yöntemiyle belirlendi. Bulgular ve Sonuç: Sulandırma, ekilibrasyon ve çözüm sonu motilite değerleri E2 ve E3 sulandırıcılarında E1’e göre daha düşük olarak belirlendi P

Anahtar Kelimeler

• Manda sperması,Trehaloz,Gliserol,Çözüm sonu sperma kalitesi.

References

1. Aboagla EME, Terada T (2003). Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing. Biology of Reproduction, 69, 1245-1250.
2. Ahmad E, Aksoy M (2012). Trehalose as a cryoprotective agent for the sperm cells: A mini review. Animal Health, Production and Hygiene, 1(2), 123 – 129.
3. Ahmad E, Ahmad N, Naseer Z, Aleem M, Khan MS, Ashiq M, Younis M (2011). Relationship of age to body weight, scrotal circumference, testicular ultrasonograms, and semen quality in Sahiwal bulls. Tro- pical Animal Health and Production, 43, 159-164.
4. Aisen E, Quintana M, Medina V, Morello H, Venturino A (2005). Ultra- microscopic and biochemical changes in ram spermatozoa cryop- reserved with trehalose-based hypertonic extenders. Cryobiology, 50, 239-249.
5. Aisen EG, Medina VH, Venturino A (2002). Cryopreservation and post- thawed fertility of ram semen frozen in different trehalose concent- rations. Theriogenology, 57, 1801-1088.
6. Badr MR, Abd El-Malak MG, Hassan MH (2010). Effect of trehalose on cryopreservation, oxidative stress and DNA intregity of buffalo spermatozoa. Journal of Reproduction and Infertility, 1(2), 50-57.
7. Bucak MN, Ateşşahin A, Varışlı Ö, Yüce A, Tekin N, Akçay A (2007). The influence of trehalose, taurine, cysteamine and hyaluronanon ram semen: Microscobic and oxidative parameters after freze-thawing process. Theriogenology, 67, 1060-1067.
8. Chen Y, Foote RH, Brockett CC (1993). Effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm. Cryobiology, 30, 23–31.

9. Chhillar S, Singh VK, Kumar R, Atreja SK (2012). Effects of Taurine or Trehalose supplementation on functional competence of cryopre- served Karan Fries semen. Animal Reproduction Science, 135, 1-7.
10. Hu JH, Zan LS, Zhao XL, Li QW, Jiang ZL, Li YK, Li X (2010). Effects of trehalose supplementation on semen quality and oxidative stress variables in frozen-thawed bovine semen. Journal of Animal Scien- ce, 88, 1657-1662.
11. Jafaroghli M, Khalili B, Farshad A, Zamiri MJ (2011). The effect of supp- lementation of cryopreservation diluents with sugars on the post- thawing fertility of ram semen. Small Ruminant Research, 96, 58- 63.
12. Khalili B, Farshad A, Zamiri MJ, Rashidi A, Fazeli P (2009). Effects of sucrose and trehalose on the freezability of Markhoz goat sperma- tozoa. Asian-Australasian Journal Animal Science, 22, 1614- 1619.
13. Kumar R, Singh V, Chhillar S, Atreja S (2013). Effect of supplementation of taurine or trehalose in extender on immunolocalization of tyro- sine phosphoproteins in buffalo and cattle (Karan Fries) cryopreser- ved spermatozoa. Reproduction in Domestic Animal, 48, 407–415.
14. Liu Z, Foote RH, Brockett CC (1998). Survival of bull sperm frozen at different rates in media varying in osmolarity. Cryobiology, 37, 219–230.
15. Molinia FC, Evans G, Maxwell WMC (1994). Effects of monosacchari- des and disaccharides in tris-based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. Animal Reproduction Science, 36, 113–122.
16. Quan GB, Hong QH, Lan ZG, Yang HY, Wu SS (2012). Comparison of the effect of various disaccharides on frozen goat spermatozoa. Biopre- servation and Biobanking, 10, 439-445.
17. Reddy NSS, Mohanarao GJ, Atreja SK (2010). Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation. Animal Repro- duction Science, 119, 183-190
18. Uysal O, Bucak MN (2009). The role of different trehalose concentra- tions and cooling rates in freezing of ram semen. Ankara Üniversiti Veteriner Fakultesi Dergisi, 56, 99-103.
19. Watson PF (1979). The preservation of semen in mammals. In: Finn CA (ed.), Oxford Rev Rep Biol, vol. 1. Oxford: Clarendon Press, 283–350.
20. Watson PF (2000). The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science, 60–61, 481–492.