The objectives were to determine the incorporation of dietary encapsulated fats differing in n-6:n-3 ratio into milk fat, plasma, and various ovarian compartments and to examine the effects on ovarian follicular status, preovulatory follicle characteristics, and oocyte quality. Twenty-four multiparous Israeli Holstein cows, averaging 114 d in milk, were assigned to 1 of 3 treatment groups: 1) control (n=7), in which cows were fed a lactating cow diet; 2) E-FLAX (n=8), in which cows were fed a lactating cow diet that consisted of 1kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% flaxseed oil, providing 242.2g of C18:3n-3 (low n-6:n-3 ratio); or 3) E-SUN (n=9), in which cows were fed a lactating cow diet that consisted of 1kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% sunflower oil, providing 260.0g of C18:2n-6 (high n-6:n-3 ratio). Ovaries were monitored by ultrasonography for follicular status, and after synchronization, follicles >7mm were aspirated and evaluated. Ovum pickup was performed (19 sessions for the control and E-FLAX groups and 11 for the E-SUN group), and in vitro maturation and oocyte fertilization were conducted. The E-FLAX treatment increased the proportions of C18:3n-3 (5.8 fold), C20:5n-3, and C22:5n-3 (approximately 4-fold) in milk fat as compared with the other 2 treatments. The proportion of C18:3n-3 fatty acid in plasma increased dramatically with the E-FLAX treatment, from 1.43 and 1.49% in the control and E-SUN groups, respectively, to 7.98% in the E-FLAX group. Consequently, the n-6:n-3 ratio in plasma was reduced from approximately 42 in the control and E-SUN groups to 6.74 in the E-FLAX group. Proportions of C18:3n-3 in follicular fluid and granulosa cells were approximately 5-fold higher in the E-FLAX group than in the other 2 groups. The percentage of C18:2n-6 in cumulus-oocyte complexes of cows in the E-SUN group was 54% higher than that in the E-FLAX group and was 2.4-fold higher than that in the control group; the proportion of C18:3n-3 in the E-FLAX group was 4.73% and was not detected in the other groups. The average numbers of 2- to 5-mm follicles on d 5 and 9 of the cycle were higher in the E-FLAX group than in the E-SUN group, whereas the average numbers of follicles > or =10mm on d 5, 9, and 13 were higher in the E-SUN group than in the other 2 groups. The estrous cycles of the cows were synchronized and PGF(2alpha) was injected on d 16 to 17 of the cycle. The interval from PGF(2alpha) injection to behavioral estrus was longer in the E-FLAX group than in the E-SUN group, and the beginning of the luteal phase of the subsequent cycle was delayed. Concentrations of estradiol in follicular fluid of the preovulatory follicles were higher in the E-SUN group than in the E-FLAX group. The number of follicles aspirated by ovum pickup was higher in the E-FLAX group than in the control group, and the cleavage rate in the E-FLAX group was higher than in the control group, but not the E-SUN group. In conclusion, dietary n-3 fatty acids influenced the follicular status and increased the cleavage rate of oocytes as compared with those of control cows. These findings could be related to modifications of the fatty acid composition in plasma and ovarian compartments in response to dietary supplementation.