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The objective of this study was to assess the phenotypic and genetic variability of production traits and milk fatty acid (FA) contents throughout lactation. Genetic parameters for milk, fat, and protein yields, fat and protein contents, and 19 groups and individual FA contents in milk were estimated for first-parity Holstein cows in the Walloon Region of Belgium using single-trait, test-day animal models and random regressions. Data included 130,285 records from 26,166 cows in 531 herds. Heritabilities indicated that de novo synthesized FA were under stronger genetic control than FA originating from the diet and from body fat mobilization. Estimates for saturated short- and medium-chain individual FA ranged from 0.35 for C4:0 to 0.44 for C8:0, whereas those for monounsaturated long-chain individual FA were lower (around 0.18). Moreover, de novo synthesized FA were more heritable in mid to late lactation …