Abstract. The purpose of this study was to estimate environmental effects on semen production of artificial insemination (AI) Holstein bulls managed under Moroccan conditions. A total of 4 046 ejaculates collected from 34 Holstein bulls in the years 2009–2013 were analysed. Studied variables were volume, concentration, total number of spermatozoa, mass motility, individual motility and post-thawing motility. Data were analysed by REML method using the mixed model including the random effect of bull and the fixed effects of age at collection, season of collection, year of collection, interval between two collections and ejaculate order. The effect of age of bulls was significant for all studied variables, except for mass motility. Spring and winter were the best seasons for sperm production and quality. Bulls’ ejaculates collected once a day at one day interval produced monthly 30 %, 86 %, 156 %, 183 % and 185 % more motile spermatozoa than those collected once a day at 2, 3, 4, 5 and 6 days interval, respectively. Moreover, bulls’ sperm collected twice a day at 1, 2, 3, 4, 5 and 6 days interval produced monthly (sum of motile spermatozoa obtained at 1st and 2nd ejaculates) 77 %, 70 %, 65 %, 68 %, 84 % and 91 % more than those collected once a day at the same interval, respectively. Repeatability estimates for semen traits were medium to high. They varied from 0.157 for mass motility to 0.411 for ejaculate volume. It was concluded that environmental factors clearly contribute to semen production in Holstein bulls and short intervals between collections and two collections per day are maximising sperm production.