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Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats
Published on: August 5, 2011
Cryopreservation of Dog Spermatozoa Using Essential and Non-Essential Amino Acids Solutions in An Egg Yolk-Free
N A H Talha1, Y Jeon2, I J Yu3
1Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan, Republic of Korea. Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology, Khartoum, Sudan.
Background:
Amino acids (AAs) have been indicated to have cryoprotective and antioxidative effects on sperm freezing using egg yolk (EY)-based extender. However, EY-based extender is difficult to be standardized for the effect of amino acids because the EY composition varies with the animal's diet.
Objective:
To test the effect of AAs in EY-free polyvinyl alcohol (EY-free PVA) extender and develop a chemically defined extender for dog sperm cryopreservation.
Materials And Methods:
In the first experiment (E1), dog spermatozoa (1ｘ108 sperms/mL) were frozen with EY-free PVA extender without AAs or supplemented with essential (EAAs, 50 x: 1, 2, 4 %) or non-essential amino acids (NEAAs, 100 x: 1, 2, 4 %). In the second experiment (E2), spermatozoa were frozen with EY-free PVA extender supplemented with 0, 0.5, 1 or 2 % of an EAA-NEAA mixture. Motility, viability and acrosome integrity were evaluated after thawing in E1 and E2. In the third experiment (E3), spermatozoa were frozen using an extender supplemented with 2 % EAAs, 2 % NEAAs or a 0.5 % EAA-NEAA mixture. Reactive oxygen species (ROS) and phosphatidylserine (PS) translocation were assessed. Expression of genes for motility-related sperm mitochondrial-associated cysteine-rich protein (SMCP), apoptosis-related B-cell lymphoma 2 (BCL2) and BCL2 associated X protein (BAX) was measured.
Results:
Addition of EAAs, NEAAs or an EAA-NEAA mixture to EY-free PVA extender significantly increased sperm motility without affecting viability. Only 1 % NEAAs significantly increased the acrosome membrane. EAA-NEAA mixture (0.5 %) significantly increased SMCP, BCL2 and BAX expression compared to the control group without significant effect on PS translocation or ROS.
Conclusion:
EAAs and NEAAs addition in EY-free PVA extender improved sperm motility, with limited effect on acrosome integrity and gene expression of SMCP, BCL2 and BAX during dog sperm cryopreservation.
