Effects of addition of different sugars and antioxidants to extender on quality of Afshari ram frozen semen — ASN Events
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Effects of addition of different sugars and antioxidants to extender on quality of Afshari ram frozen semen (#509)

Dariush Ebrahimi 1 , Behnam Rostami 1 , Hassan Sadeghipanah 2 , Reza Masoumi 1 , Mohammad Hosein Shahir 1 , Mehrdad Meamar 3 , Sona Zargari 1 , Parisa Taghilou 1
  1. Department of Animal Science, University of Zanjan, Zanjan, Iran
  2. Animal Science, ASRI (Animal Science Research Institution of Iran), Karaj, Iran
  3. Department of Animal Science, University of Yasouj, Yasouj, Iran

Spermatozoa are susceptible to lipid peroxidation during cryopreservation and thawing (1). Cryopreservation also produces physical and chemical stress on the sperm membrane, associated with oxidative stress and ROS generated by dead spermatozoa and atmospheric or molecular oxygen of the environment, inducing decreases in the motility, membrane integrity and fertilizing potential of spermatozoa for AI (2). Antioxidant vitamins act as a cellular stabilizer of unsaturated lipids against oxidative deterioration, thus maintaining structural and functional integrity at the subcellular level (3).

This study was conducted in order to determine the influence of the addition of certain sugars (trehalose, sucrose and raffinose) and antioxidants (vitamin E, C and taurine) in tris and egg yolk based extender during cryopreservation on standard semen parameters after thawing.

Ejaculates collected from five Afshari rams, a native breed of sheep of Iran, were evaluated and pooled at 33 C. Semen samples were diluted with a Tris- egg yolk based extender and were treated with different levels of sugars and antioxidants in 3 experiments. Extender containing no additives was served as control.

In experiment 1, different levels of trehalose (0, 50, 100 mM) were tested with different levels of antioxidants ((taurine; 0, 25, 50 mM), and vitamin E and C; 0,1, 2 mM)).

In experiment 2, different levels of sucrose (0, 60, 80 mM) were tested with different levels of antioxidants ((taurine; 0, 25, 50 mM), and vitamin E and C; 0,1, 2 mM)).

In experiment 3, different levels of raffinose (0, 5, 10 mM) were tested with different levels of antioxidants ((taurine; 0, 25, 50 mM), and vitamin E and C; 0,1, 2 mM)).

In experiment 4, the best extenders of experiments 1, 2 and 3 were compared.

Samples were cooled to 5 C and frozen in 0.25 ml French straws, being stored in liquid nitrogen. Frozen straws were thawed individually at 37 C for 20 s in a water bath for evaluation. Sperm parameters including motility and progressive motility were assessed by a Computer Assisted Sperm Analyzer (CASA). Viability, membrane integrity and abnormality were assessed by HOST, Eosin-Nigrosin and Hankok tests respectively (4).

Addition of  100 mM trehalose plus 2 mM vitamin E in experiment 1, 60 mM sucrose plus 2 mM vitamin E in experiment 2 and 10 mM raffinose plus 2 mM vitamin E in experiment 3 significantly (P<0.05) increased motile sperm cells in compare to controls (62.41, 59.52 and 58.33 and %46 respectively). Percentage of progressive motile cells were significantly different (P<0.05) in compare to controls ( 57.18, 57.49, 55.03 and %41.2 respectively). Results of experiment 4 showed that between 3 best extenders of treatments1, 2 and 3 trehalose concentration of 100 mM plus 2 mM of vitamin E  resulted in highest motility, progressive motility and viability (P<0.05).

In conclusion, the addition of 100 mM trehalose plus 2 mM vitamin E to ram semen extender significantly improved sperm motility and viability in this experiment.

  1. Câmara, D. R., S. V. Silva, F. C. Almeida, J. F. Nunes, and M. M. P. Guerra. 2011. Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen. Theriogenology 76(2):342-350.
  2. Purdy, P. H., E. Mocé, R. Stobart, W. J. Murdoch, G. E. Moss, B. Larson, S. Ramsey, J. K. Graham, and H. D. Blackburn. 2010. The fertility of ram sperm held for 24 h at 5 °C prior to cryopreservation. Animal Reproduction Science 118(2–4):231-235.
  3. Silva, S. V., A. T. Soares, A. M. Batista, F. C. Almeida, J. F. Nunes, C. A. Peixoto, and M. M. P. Guerra. 2013. Vitamin E (Trolox) addition to Tris-egg yolk extender preserves ram spermatozoon structure and kinematics after cryopreservation. Animal Reproduction Science 137(1–2):37-44.
  4. Bucak, M. N., A. Ateşşahin, Ö. Varışlı, A. Yüce, N. Tekin, and A. Akçay. 2007. The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: Microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology 67(5):1060-1067.