166 IN VIVO MODEL TO EXAMINE THE LONG-LASTING EFFECTS OF ACUTE DI-(2-ETHYLHEXYL) PHTHALATE (DEHP) EXPOSURE ON OVARIAN FUNCTION IN BOVINE

Abstract

Di-(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer. Its metabolites have been shown to have adverse effects on reproduction and development in laboratory animals. However, the mechanisms by which they induce infertility remain elusive. We established an experimental model in which lactating Holstein cows were synchronized (GnRH–PG–GnRH) and tube-fed with DEHP (100 mg kg^–1 per day; n = 4) or water (n = 5), for 3 days. Urine and plasma samples were collected before (Day 0), during (Days 2 and 4), and after (Days 11, 19, and 24) treatment initiation. For each group, and on each day, samples were pooled and analysed to determine DEHP metabolite concentrations (MEHP, 5OH-MEHP, 5oxo-MEHP, 2cx-MMHP, 5cx-MEPP) by liquid chromatography–mass spectrometry/mass spectrometry. Incorporation of DEHP resulted in relatively high metabolite concentrations on the exposure days (acute phase; Days 1–4), which decreased dramatically through the subsequent period (chronic phase; Days >5). For example, the average plasma MEHP concentration in the control group was 0.012 ± 0.0017 µM throughout the experimental period. In the treated group, it increased to 47.7 ± 8.9 µM in the acute phase, then decreased to 0.045 ± 0.02 µM. To examine the effects on ovarian function, cows were resynchronized during the chronic phase, and monitored by ultrasonography scanner (SSD-900, Aloka, Tokyo, Japan; 7.5 MHz) to classify ovarian follicular dynamics. Follicular fluids of the dominant follicles were aspirated with an ultrasonic scanner connected to a vaginal sector transducer (Pie Medical Imaging BV, Maastricht, the Netherlands; 7.5 MHz). Data were analysed using JMP-7 software (SAS Institute Inc., Cary, NC, USA, 2004). Differences among treatments were analysed by one-way ANOVA followed by Student's t-test. Findings revealed the pronounced effect of DEHP on the dominant follicles. The diameter (15.7 ± 1.8 v. 10.4 ± 1.8 mm) and growth rate (0.88 ± 0.15 v. 0.43 ± 0.17 mm day^–1) of the first-wave dominant follicle were higher in the control cows (P < 0.05). The developmental pattern of the second-wave dominant follicle differed between groups, with a higher growth rate during the follicular phase (1.99 ± 0.19 v. 0.46 ± 0.29 mm day^–1; P < 0.02) and a larger diameter of the preovulatory follicle (14.48 ± 0.35 v. 9.67 ± 1.85 mm; P < 0.01) in the control group. The pattern of corpus luteum growth and regression also differed between groups, expressed by higher volume (4.37 × 10³ ± 0.27 v. 2.91 × 10³ ± 0.31 mm³; P < 0.05) in the control group. The proportion of dominant follicles that developed to follicular cysts (> 25 mm) tended to be higher (75 v. 20%; P < 0.09) in the treated group. The average oestradiol concentration in the follicular fluid of the preovulatory follicle was lower in the DEHP-treated group (361.6 ± 130.5 v. 832.6 ± 109.7 ng mL^–1; P < 0.05). However, the average progesterone concentration in the plasma during the luteal phase were normal (4.5–5.0 ng mL^–1) and did not differ between groups. The findings reveal the potential risk of DEHP exposure and its long-lasting effects on bovine ovarian function. These impairments are suggested to adversely affect fertility.