2.2. Rooster and semen collection

This study was performed on ten adult Ross 308 broiler breeder roosters) 30 weeks old) which were kept individually in cages (diet compositions included: 12% crude protein and 2,750 kcal maintenance energy/kg). The experiment was performed in five replicates on 5 collection days with a frequency of twice per week. Ejaculates were collected using the dorso-abdominal massage method and collection was always performed by the same person and under the same condition [16]. Semen samples from each rooster were analyzed individually. The samples that had the standard criteria motility of >80%, concentration of >3 × 10⁹ sperm/mL (sperm concentration was determined using a hemocytometer (HBG, Berlin, Germany)) and volume of >0.2 mL were used in the study. Over all replicates, 2 ejaculates (of all 50 ejaculates) were rejected. The pooled semen samples were then split according to the number of treatments (seven equal aliquots).