The effect of different levels of raffinose and different extenders on characteristics of Dalagh ram semen after freezing and thawing

Abstract

Abstract

Background and objectives: One of the pivotal technology for genetic resorviors preservation is the freezing of semen. As freezing causes a reduction in sperm fertility, adding some components to semen to preserve its ferility would be important. Since beginning the time of artificial insemination technique, different diluents have been used to preservation semen in liquid and frozen condition. Therefore, the goal of this stady was to investigate the effect of adding different levels of raffinose and milk, Tris-chicken egg yolk and Tris-soybean lecithin extenders on ovine semen characteristics after freezing and thawing.
Materials and methods: Samples were collected from four mature Dalagh rams and extended by different extenders and then raffinose was added. Semen samples were aspirated into 0.5 ml straws and cooled to 4°C within 150 min and then frozen in liquid nitrogen Frozen straws were transferred to a tank containing liquid nitrogen. After thawing spermatozoa quality parameters including percentage of sperm motility, viability, membrane integrity and sperm normality were evaluated. This study was performed in a 4×3 factorial experiment with a completely randomized arrangement. Treatments were four levels of raffinose (0, 60, 70 and 80 mmol) and also three kinds of extenders (milk, tris-egg yolk and tris-soy lecithin). The obtained data from this study were analyzed by ANOVA procedure of Statistical Analysis System SAS (version 9.1). Comparison of data means were performed by Tukey test at the level of %1 error.
Results: Results showed that adding raffinose had a significant effect on sperm viability and mobility (P<0.01), but did not significantly affect on sperm memberane integrity and normal morphology percentages. Effect of extender on percentages of motility, viability, membrane integrity and normal morphology of sperms was significant (P<0.01). The interaction between raffinose levels and extenders on percentages of motility, membrane integrity and normal morphology of sperms was significant while it was not for viability percentage. The greatest sperm mobility (35.00±2.09%) was in tris-egg yolk extender containing 60 mmol raffinose while that greatest sperm viabilty (74.40±1.88%) was in tris-egg yolk extender containing 80 mmol raffinose but also the most percentages of membrane integrity (66.40±2.60%) was in Tris-chicken egg yolk containing 0 mM raffinose. The greatest sperm normal morphology (74.80±2.82%) was observed in tris-soy lecithin extender containing 60 mmol raffinose.
Conclusion: Findings of the study showed that sperm characteristics including mobility and normal morphology percentages in level 60ml raffinose, membrane integrity percentages in level 0ml raffinose wihle for viability percentage were better preserved in 80ml level. In the other hand, tris-egg yolk extender had more prominant effect on preservation of sperm characteristics.

Keywords

Main Subjects


1.Agca, Y., Gilmore, J., Byers, M., Woods, E., Liu, J., and Critser, J.K. 2002.
Osmotic Characteristics of mouse spermatozoa in the presence of extender and sugers. Biology of Reproduction. 67: 1493-1510.
2.Amirat, L., Tainturier, D., Jeanneau, L., Thorin, C., Gerard, O., Courtens, J.L., and Anton, M. 2004. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with optidyl, a commercial egg yolk . Theriogenology. 61: 895–907.
3.Ari, U.Ç., Kulaksiz, R., and Ӧztϋrkler, Y.
2011. Freezability of Tushin ram semen extended with goat or cow milk based extenders. Reproduction in Domestic Animals. 46: 975–979.
4.Baily, J.F., Bilodeau, J.F., and Cormier, N. 2000. Semen cryopreservation in domestic animals. Andrology. 21: 1-7.
5.Barbas, J., and Mascarenhas, R.D. 2009. Cryopreservation of domestic animal sperm cells. Cell and Tissue Banking. 10: 49-62.
6.Bucak, M.N., Tuncer, P.B., Sarıozkan, S., Bas-pınar, N., Tas-pınar, M., Coyan, K.,
Bilgili, A., Akalın, P.P., Buyukleblebici, S., Aydos, S., and Ilgaz, S. 2010. Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage. Cryobiology. 61: 248–253.
7.Dolati Doorbash, P., Moghaddam, Gh., Daghighkia, H., Taghizadeh, A. and Rafat, S.A. 2015. The effect of adding different raffinose concentrations in the diluents in semen cryopereservation of different breeds of ram at the reproductive season. Animal Science Researches. 25: 121-132. (In Persian)
8.Evans, G., and Maxwell, W.M.C. 1987. Salamons artificial insemination of sheep and goats. Butterworths. Sydney. 194.
9.Fiser, P.S., Ainsworth, L., and Fairfull, R.W. 1987. Evaluation of new diluent and different processing procedures for cryopreservation of ram semen. Theriogenology. 28: 599-607.
10.Fouladvand, F., Karimi, K., Zhandi, M. and Tofighi, K. 2013. Comparison the effects of lecitine, egg yolk and milk as extenders on preservation of ram semen in cool condition. Scientific Research Journal of Animal Environment. 6: 83-90. (In Persian).
11.Jafaroghli, M., Khalili, B., Farshad, A., and Zamiri, M.J. 2011. The effect of supplementation of cryopreservation diluents whit sugars on the post-thawing fertility of ram semen. Small Ruminant Reserch. 96: 58-63.
12.Hancock, J. 1956. The morphology of boar spermatozoa. Journal of the Royal Microscopical Society. 76: 84-97.
13.Kasimanickam, P., Kasimanickam, V., Tibaryc, A., and Pelzer, K. 2011. Effect of semen extenders on sperm parameters of ram semen during liquid storage at 4 ◦C. Small Ruminant Research. 99: 208-213.
14.Linford, E., Glover, F.A., Bishop, C., and Stewart, D.L. 1976. The relationship between semen evaluation methods and fertility in the bulls. Reproduction and Fertility. 47: 283-291.
15.Lio, Z., Foote, R.H., and Brockett, C.C.
1998. Survival of bull sperm frozen at different rates in media varying in osmolality. Cryobiology. 37: 219-230. 16.Martin, I.C.A. 1963. The freezing of dog spermatozoa to -79°C. Research in Veterinary Science. 4: 304.
17.Medeiros, C.M.O., Forell, F., Oliveira, A.T.D., and Rodrigues, J. 2002. Current status of sperm cryopreservation: why isn't it better. Theriogenology. 57: 327-344.
18.NRC, 1985. Nutrient Requirements of Sheep. National Research Council. Academy Press, USA.
19.Papa, F.O., Felicio, G.B., Melo-Ona, C.M., Avarenga, M.A., De.Vita, B., Trinque, C., Puoli-Filhob, J.N., and Dell Aqu, J. 2011. Replacing egg yolk with soyban lecithin in the cryopreservation of stallion semen. Animal Reproduction Science. 129: 73-77.
20.Rasul, Z., Anzar, M., Jalali, S., and Ahmad, N. 2003. Effect of buffering system postthaw motion characteristics, plasma membrane integrity, acrosome morphology of buffalo spermatozoa. Animal Reproduction Science. 59: 31-4.
21.Revell, S., and Mrode, R. 1994 .An osmotic resistance test for bovine semen. Animal Reproduction Science.
36:1- 77-86.
22.Safdarian, M. 2007. Artificial insemination of sheep and goats. The first Edition. Institute Applied Science Agriculture. 232p. (In Persian).
23.Salamon, S., and Maxwell, W.M. 1995. Frozen storage of ram semen. Cause of low fertilitafter cervical insemination and metod of improvement. Animal Reproduction Science. 38: 1-36.
24. Salamon, S., and Maxwell, W.M.C.
2000. Storage of ram semen. Animal Reproduction Science. 62:77-111.
25. Sarıözkan, S., Bucak, M.N., Canturk, F., Özdamar, S., Yay, A., Tuncer, P.B., Ozkan, S., Sorgucu, N., and Caner, Y. 2012. The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4° C. Cryobiology. 65: 93-97.
26. Sharafi, M., Eghbalsaied, S., Nili, N., and Nasr-Esfahani, M.H. 2009. Ram semen in vitro fertility cryopreservation
using soybean lectin and egg yolk based extenders. 13th Annual Conference on Reproduction in Domestic Animals. 44: 90-95.
27. Storey, B., Noiles, E.E., and Thompson, K.A. 1998.Comparison glycerol, other
polyols, trehalose and raffinose to provide defined cryoprotectant medium for mouse sperm cryopreservation.
Cryobiology. 37: 46-58.
28. Tuncer, P.B., Bucak, M.N., Sarıözkan, S., Sakin, F., Yeni, D., Çiğerci, İ.H., Ateshahin, A., Avdatek, F., Gundoghan, M., and Büyükleblebici, O. 2010. The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircusancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities. Cryobiology. 61: 89-93.
29. Vishwanath, R., and Shannon, P. 2000. Storage of bovine semen in liquid and frozen state. Animal Reproduction Science. 62: 23-53.
30. Woelders, H., Matthijs, A., and 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.