Abstract #96

# 96
Y. Liu1, H. S. Pedersen*1, L. Foldager1, H. Callesen1, M. T. Sørensen1, 1Department of Animal Science, Aarhus University, Tjele, Denmark.

To assess reprotoxicity of pesticides and other xenobiotics, rodents are often used with natural mating and litter size as end-points. However, because a male mouse can produce normal-size litters even with >50% reduced sperm count (Ecotox. Env. Saf. 73, 1092), this model could approve some chemical substances despite their reprotoxic effect. Thus, we aimed to establish a mouse IVF system to evaluate reprotoxicity of xenobiotics, illustrated by 2 pesticides [vinclozolin (vin), chlormequat (ccc)]. Experimental mice were given 2 pesticide doses by mixing into the feed: low (L) or high (H), equivalent to no or lowest observed adverse effect level, respectively (Env. Health Perspect. 117, 1272). To produce experimental males, mated NMRI females were fed low dose (vinL, cccL), high dose (vinH, cccH), or no pesticide from 1-cell stage through birth and until weaning, from where experimental males continued pesticide feeding. Each experimental male at 8 to 10 weeks old was naturally mated with 2 females having normal feed for 5 days, then continued with pesticide or control feed for at least 35 days before use for IVF. Naturally mated females had normal feed until birth to evaluate litter size. For IVF, oocytes were collected after superovulation of 3- to 4-week-old C57BL/6J females (2–5/male). Based on our previous experiment (Anim. Reprod. 13, in press), 25,000 sperm/mL was within the responsive range and selected for IVF (Theriog. 65, 1716). The IVF embryos were in vitro cultured for 4 days. Pronuclei were evaluated 9 h after IVF start (Day 0), and 2-cell/blastocyst were evaluated on Day 1/4. Mean litter size was estimated by normal linear mixed-effects model, and mean pronuclei, 2-cell, and blastocyst rates were estimated by binomial generalized linear mixed-effects models with identity link function, included a male-subject random intercept accounting for correlation between multiple fertilizations by same male, and a factor defining groups as fixed effect. Preliminary results (Table 1) are based on current data from half of our full experiment. No signs of different litter sizes after natural mating were observed between pesticide groups and control. Compared with control, rates of pronuclei, 2-cell, and blastocyst tended to decrease in vinH and increase in vinL group. No clear differences between cccL, cccH, and control groups were found. These results could indicate a pesticide effect (vinH) on mouse male reproductive system that can be detected in an IVF system but not by natural mating. Our data show a large variation in IVF results between individual males and females, so our full dataset is required before conclusions are made. Table 1. Preliminary results 
Group1No. of malesNatural mating2IVF2
Litter size3 (no. of litters)No. of oocytes (no. of females)Pronuclei3 (%)2-cells3 (%)Blastocysts3 (%)
Control1311.7 ± 0.4 (22)1094 (39)45.4 ± 4.647.0 ± 5.035.8 ± 5.3
cccL712.8 ± 0.6 (13)523 (22)46.5 ± 6.450.7 ± 6.940.7 ± 7.6
cccH812.5 ± 0.5 (15)752 (26)39.6 ± 5.942.9 ± 6.433.1 ± 7.6
vinL611.5 ± 0.6 (12)731 (22)62.0 ± 6.764.2 ± 7.248.5 ± 8.3
vinH812.5 ± 0.5 (15)797 (30)33.6 ± 5.836.7 ± 6.420.2 ± 6.3
1L = low dose or H = high dose, equivalent to no or lowest observed adverse effect level, respectively. ccc = chlormequat; vin = vinclozolin. 2No statistical differences are shown 3Mean ± SEM.