Artificial insemination with chilled sperm was introduced as an efficient assisted reproduction technique to increase male breeding value in animal species [1]. The cooling process preserves sperm quality by reducing sperm metabolism, but cold shock can have some adverse effects on spermatozoa. Many studies have been conducted to maintain the quality of stored rooster semen longer than 24 h, but fertility performance was not desirable when hens were inseminated with sperm stored for longer than 6 h [2]. It is well documented that the higher content of oxidizable polyunsaturated fatty acids (PUFAs) in the membrane of avian spermatozoa stimulates the formation of reactive oxygen species (ROS) and increases the susceptibility of rooster sperm to oxidative injury [3]. During cold storage, PUFAs undergo lipid peroxidation in the presence of ROS and produce lipid peroxyl radicals that have negative effects on sperm membrane and reduce fertility performance [4]. To overcome this problem, the use of antioxidants could be an additional protective system to protect sperm during the cooling process since the amount of endogenous antioxidants is not enough to neutralize produced ROS [5]. Several studies have described the protective effects of antioxidants such as glutathione [6,7] and CoQ10 [8] during sperm cooling process in rooster.

Mitoquinol (MitoQ), a mitochondria-targeted antioxidant, is produced by the combination of CoQ10 and triphenylphosphonium (TPP+) cation. The lipophilicity of TPP+ transfers MitoQ across biological membranes and leads to accumulation in mitochondria at higher concentrations (100–1000 times) than non-targeted derivatives [9]. It is a chain-breaking antioxidant by donating a hydrogen atom from one of its hydroxyl groups to the lipid peroxyl radical. Therefore, this action leads to a reduction in lipid peroxidation within the mitochondrial inner membrane [10].

MitoQ reduces mitochondrial ROS production and protects cells from oxidative damage in various pathological conditions such as vascular dysfunction [11] cardiac ischemia-reperfusion injury and diabetic kidney disease [12]. It also effectively increases testicular weight, maintains the integrity of the blood-testicular barrier, protects testicular tissue microstructure and sperm morphology by inhibiting oxidative stress [13]. On the other hand, in the case of sperm biology, the use of MitoQ in the semen extender preserved the quality of thawed sperm in different species such as humans [14,15] buffalo [10], rams [16] and yellow catfish [17].

No study has yet been conducted to evaluate the effects of MitoQ on chilled semen quality and fertility potential in birds. Therefore, this report aimed to assess the effects of supplementing cooling medium with MitoQ on motility, mitochondrial activity, viability, membrane integrity, and lipid peroxidation, and investigate the fertility potential of rooster sperm during cold storage periods.