1. Can Changes In Daily Body Weight Be Used For Early Pregnancy Detection In C57BL/6 Mice? Nicole Libal, BS, RLAT; Ling Zhang, MD; Sarah L. Mader, BS; Stephanie J. Murphy, VMD, PhD, DACLAM Anesthesiology and Perioperative Medicine, Oregon Health & Science University, Portland, OR, USA Early pregnancy detection in timed-mating studies offers several advantages like advanced notice of how many timed pregnant mice will be available weekly for experiments and earlier reuse of nonpregnant females in timed-mating protocols. We have previously shown that increases in body weight at 1 and 2 weeks after timed male exposure are a more reliable early pregnancy indicator than plug detection in genetically engineered mice on a C57BL/6 background. Our current study objective was to determine if changes in daily body weight could indicate pregnancy sooner than 1 week after timed male exposure. Since our lab is studying the effects of ethanol exposure during pregnancy on developing mouse brain, in this study, C57BL/6 female mice were given daily intraperitoneal ethanol (0.2 g/kg) or saline (vehicle) injections 1 week before timed-mating and during gestation. Estrous cycles were synchronized by exposing group- housed females to male urine soaked bedding for 4 days, thus taking advantage of the Whitten effect. Timed matings were set up by placing 2 synchronized females with a male for 48 hours. We weighed females just before timed male exposure and then daily after removal of males for 2 weeks. Pregnancy was confirmed by the birth of a litter. Litter size and pup birth weights were comparable between saline and ethanol groups. In the saline-treated mice, % baseline body weight was significantly (p<0.05) increased with pregnancy (107 + 1%; n=10) as early as 6 days after timed male exposure compared to nonpregnant mice (103 + 1%; n=22). In the ethanol-treated mice, % baseline body weight was significantly (p<0.05) increased with pregnancy (106 + 1%; n=15) as early as 3 days after timed male exposure compared to nonpregnant mice (100 + 1%; n=24). Our findings suggest that changes in daily body weight may reliably indicate pregnancy within days of timed male exposure and may be affected by perinatal treatments. ABSTRACT INTRODUCTION STUDY OBJECTIVE METHODS RESULTS CONCLUSIONS  Early pregnancy detection in timed mating studies offers several advantages  Advanced notice of how many timed mated mice will be available weekly for experiments  Earlier reuse of nonpregnant females in timed mating protocols  We have previously shown that increases in body weight at 1 and 2 weeks after timed male exposure are a more reliable early pregnancy indicator than plug detection in 2 genetically engineered mouse strains on a C57BL/6 background  Mader SL, Libal NL, Pritchett-Corning K, Yang R, Murphy SJ. 2009. Refining Timed Pregnancies In Two Strains Of Genetically Engineered Mice. Lab Animal 38(9):305-310.  Strategies are needed to further optimize timed mating protocols To determine if changes in daily body weight could reliably indicate pregnancy sooner than 1 week after timed male exposure in mice  C57BL/6 female mice (Charles River Laboratories, Wilmington, MA, USA), 8 to 14 weeks of age and weighing 20 to 25 g were used  Female mice were weighed between 10:30 and 11:30 AM each day just before receiving intraperitoneal injections of either 2% ethanol solution (0.2 g/kg) or saline (vehicle control) 1 week before, during and up to 3 weeks after timed mating  Estrous cycles were synchronized by exposing pair housed females to male urine soaked bedding 4 days before timed mating (Whitten effect)  Timed mating was initiated by placing 2 synchronized females with a male for 48 hours  Experimental variables were evaluated and averaged for each experimental group using excel spread sheets  Daily female body weights (g)  Litter size  Individual pup (P0) birth weights (g)  Daily female body weight (% baseline body weight) was subjected to two-way ANOVA with post-hoc Newman-Keuls test  Differences in litter size and birth weight were determined by t-test  Values are expressed as mean + SEM  Statistical significance is p<0.05  Our findings suggest that increases in daily body weight in female mice following timed male exposure may be:  Used to reliably indicate pregnancy days after timed mating  Affected by perinatal treatments  Based on our observations, we will further refine our protocols by:  Limiting male exposure to 24 to 48 hours for timing pregnancies  Using daily body weight measurements up to 1 week after male removal for early pregnancy confirmation Supported by the Portland Alcohol Research Center Pilot Grant Nicole Libal, libaln@ohsu.edu