This study evaluated the effects of non-neuronal cholinergic system (NNCS) and cholinergic antagonists on sperm parameters during cold seminal preservation to know if cholinergic agents can control sperm motility and recovery. Semen samples were collected from ten fight roosters by dorso-abdominal massage twice-weekly. Atropine used as a muscarinic acetylcholine receptor antagonist, atracurium as a nicotinic acetylcholine receptor antagonist, acetylcholinesterase, and N-ethylmaleimide a choline acetyltransferase inhibitor, and acetylcholine applied to recover sperm motility. The optimal concentration for each cholinergic agent then was determined and added to the diluted semen and preserved for adequate period of time. Consequently, to obtain the best sperm motility and quality in the evaluation testis, the optimal concentration of acetylcholine was added to the rooster semen liquid storage prepared for artificial insemination. In the performed pretests, 10mM acetylcholine, 10mM N-ethylmaleimide, 100mM and 100pM atropine, 1mM atracurium and 50nM acetylcholinesterase were determined as the optimal concentration (P<0.05). 24h preserved semen of the experimental groups were recovered by adding 10mM acetylcholine. in which it significantly increased the total and progressive motility of the sperm except for N-ethylmaleimide treated group (P<0.05). Some other Sperm parameters such as viability, acrosome, and plasma membrane integrity, and malondialdehyde (MDA) were not changed by the acetylcholine treatment, while the sperm fertility was lowered in the experimental groups as evaluated by in vitro sperm egg interaction. Rooster semen treated by indirect anticholinergic agents, such as acetylcholinesterase and N-ethylmaleimide showed lower sperm motility. Cholinergic agent; acetylcholine, added to the sperm after one or two days of cold preservation recovered the sperm motility to nearly 75% and 60%, respectively. We conclude that motility of the rooster sperm diluted in Lake extender can be recovered after one or two days at 4oC by 10mM of acetylcholine before artificial insemination.
