The effect of maternal diet, gavage stress, corn oil dosing vehicle and data analysis on the outcome and interpretation of reproductive development of mouse offspring. Zacharewski T.R., Fielden M.R., Haslam S.Z.

The effect of maternal diet, gavage stress, corn oil dosing vehicle and data analysis on the outcome and interpretation of reproductive development of mouse offspring. Recent reports of unconfirmed in vivo responses in mice following in utero exposure to endocrine disruptors prompted us to test the hypothesis that factors related to experimental protocol and data analysis were sufficient to alter the outcome and interpretation of reproductive development of male and female B6D2-F1 offspring. The effect of 1) a maternal diet high in phytoestrogens, 2) maternal stress (mock gavage), 3) maternal corn oil (~3ml/kg gavage) and 4) diethylstilbestrol (DES) (1 µg/kg in corn oil gavage) from gestational day 12 to postnatal day (PND) 21 was examined. Neonatal body weight and anogenital distance (AGD) were measured on PND 7 and 21 and subsequently examined on PND 49 for body weight, testes and seminal vesicle weight, and female mammary gland growth, proliferation and differentiation. A diet high in phytoestrogens (containing 245 µg total genistein per g feed, 243 µg total diadzein per g feed, and 254 µg total glycitein per g feed) decreased male AGD/cube root of body weight ratio on PND 20 but not PND 7. Maternal stress due to gavage decreased male AGD/cube root of body weight ratio on PND 7 and decreased female AGD/body weight ratio on PND 20. DES increased male AGD/cube root of body weight ratio on PND 7 and 20. Corn oil had no significant effects on reproductive development, except for an increase in male body weight on PND 7 and a decrease in absolute testis weight. Only DES induced a statistically significant affect on mammary gland growth and alveolar bud formation. No factors effected relative testis The effect of maternal diet, gavage stress, corn oil dosing vehicle and data analysis on the outcome and interpretation of reproductive development of mouse offspring. Recent reports of unconfirmed in vivo responses in mice following in utero exposure to endocrine disruptors prompted us to test the hypothesis that factors related to experimental protocol and data analysis were sufficient to alter the outcome and interpretation of reproductive development of male and female B6D2-F1 offspring. The effect of 1) a maternal diet high in phytoestrogens, 2) maternal stress (mock gavage), 3) maternal corn oil (~3ml/kg gavage) and 4) diethylstilbestrol (DES) (1 µg/kg in corn oil gavage) from gestational day 12 to postnatal day (PND) 21 was examined. Neonatal body weight and anogenital distance (AGD) were measured on PND 7 and 21 and subsequently examined on PND 49 for body weight, testes and seminal vesicle weight, and female mammary gland growth, proliferation and differentiation. A diet high in phytoestrogens (containing 245 µg total genistein per g feed, 243 µg total diadzein per g feed, and 254 µg total glycitein per g feed) decreased male AGD/cube root of body weight ratio on PND 20 but not PND 7. Maternal stress due to gavage decreased male AGD/cube root of body weight ratio on PND 7 and decreased female AGD/body weight ratio on PND 20. DES increased male AGD/cube root of body weight ratio on PND 7 and 20. Corn oil had no significant effects on reproductive development, except for an increase in male body weight on PND 7 and a decrease in absolute testis weight. Only DES induced a statistically significant affect on mammary gland growth and alveolar bud formation. No factors effected relative testis or seminal vesicle weight. Correcting organ weight for body weight, and AGD for body weight or cube root of body weight affected the magnitude, direction and statistical significance of the measured responses in comparison to absolute values. When the data was analyzed using the individual animal as the experimental unit, the level of significance (i.e. P value) for many of the effects had decreased to significant levels when compared to the analysis using the litter as the experimental unit. The results illustrate how differences in protocol and data analysis can alter the response and interpretation of reproductive endpoints commonly used to study endocrine disruption in vivo.or seminal vesicle weight. Correcting organ weight for body weight, and AGD for body weight or cube root of body weight affected the magnitude, direction and statistical significance of the measured responses in comparison to absolute values. When the data was analyzed using the individual animal as the experimental unit, the level of significance (i.e. P value) for many of the effects had decreased to significant levels when compared to the analysis using the litter as the experimental unit. The results illustrate how differences in protocol and data analysis can alter the response and interpretation of reproductive endpoints commonly used to study endocrine disruption in vivo.