| ObjectiveThis study was based on three generation toxicity study in order to evaluate the reproductive and the neurodevelopmental toxicity of offspring of transgenic Cry1Ab and epsps gene herbicide and insect resistant maize C0030.3.5(DBN9936)in SD rats.The body weight,food intake,blood index,organ and tissue pathology,reproductive system and offspring development were evaluated among three generations of SD rats.It further enriched the safety data of transgenic maize with Cry1Ab and epsps genes,and provided experimental basis for the reproductive and the neurobehavioral and cognitive ability of transgenic maize in multi-generational animal feeding trials.Methods1.A three generation study of genetically modified herbicide and insect resistant maize with Cry1Ab and epsps genes in SD rats1.1 Identification of transgenic Maize1.1.1 Qualitative detection of exogenous genes in transgenic Maize samplesIn this study,PCR technique was used to detect whether the transgenic maize contained exogenous genes Cry1Ab and epsps.1.1.2 Quantitative detection of exogenous proteins in transgenic Maize samplesIn this study,ELISA analysis was used to detect the contents of exogenous Cry1Ab and EPSPS proteins in transgenic maize.1.2 Nutrients compositional analysis of transgenic Maize and its parental MaizeThe protein,fat,carbohydrate,cellulose,amino acid,vitamin,mineral matter,ash and moisture of transgenic maize and parental maize were determined in accordance with Chinese standard methods.1.3 Three-generation reproductive toxicity study of transgenic Maize in SD ratsThe maximum amount of maize is mixed into feed under the premise of ensuring nutrition balance.The weaned SD rats were randomly divided into three groups,which contains parental maize control group(group 1),transgenic maize group(group 2)and AIN-93G control group(group 3),60 rats in each group,half male and female,five rats in each cage.Each group was fed with corresponding feed.Fed the subjects at least 70 days(considering that the rat sperm maturation period takes about 70 days).The F0 generation animals were mated in the same cage at the proportion of 1:1 between male and female until conception or maximum 2 weeks.After birth,the F1 offspring rats were examined for litter size,deaths,and macroscopic malformations,and were weighed on PND4.Adjust each litter to 8 offspring(half male and female),if one sex were less than 4 puppies,then another sex animal was added,the total number was 8 offspring.After F1 generation weaning,blood index,serum sex hormone,organ weight and pathology,the spermatozoa of F0 generation were tested.Randomly selected 30 males and 30 females from each group(at least one male and one female in each litter)to continue feeding the test material for 70 days.F1 generation were mated according to the above methods,and got F2 and F3 generation.After birth,the F3 offspring rats were examined for litter size,deaths,and macroscopic malformations,and were weighed on PND4.Adjust each litter to 10 offspring(half male and female),if one sex were less than 5 puppies,then another sex animal was added,the total number was 10 offspring.Randomly selected 20 litters of F3 generations per group to check the early physiological development index.After F3 generation weaning,blood index,serum sex hormone,organ weight and pathology,the spermatozoa of F2 generation were tested.20 litters(one male and one female each litter and totally 40 animals)were randomly selected for 90-day feeding trials.After 90 days,blood index,serum sex hormone,organ weight and pathology,the spermatozoa of F3 generation were tested.2.Three-generation study on neuroethology and cognitive ability of genetically modified maize with Cry1Ab and epsps genes on the SD ratsAfter three generations of breeding,20 litters of rats those from pre-weaning F3 generation were selected randomly to evaluate early postnatal developmental neurotoxicity(PND4).After weaning,10 pups were randomly selected from each litter(1 male or female per litter)for future experiments of motor activity,cognitive function and brain neuropathology as adult(PND64-70).Results1.A three generation study of genetically modified herbicide and insect resistant maize with Cry1Ab and epsps genes in SD rats1.1 Identification of transgenic Maize1.1.1 Qualitative detection of exogenous genes in transgenic Maize samplesThe target band was amplified from the transgenic maize which was consistent with the expected size,whereas the target band was not found from the parental maize.1.1.2 Quantitative detection of exogenous proteins in transgenic Maize samplesBy ELISA method,exogenous protein Cry1Ab and EPSPS were detected in transgenic maize with the contents of 0.480?g/g and 9.058?g/g,respectively.But no proteins above were detected in parental maize.1.2 Nutrients compositional analysis of transgenic Maize and its parental MaizeMost contents of main nutrients,vitamins,minerals and amino acids of transgenic maize were consistent with those of parental maize.Most results of nutritional components fell within the range of values reported by OECD.1.3 Three-generation reproductive toxicity study of transgenic Maize in SD ratsThere were no significant differences caused by transgenic subjects among three generations in each group for the body weight,food intake,organ coefficient,organ pathology,blood biochemical indexes and serum sex hormone levels.There were no significant differences caused by transgenic subjects in F0,F1,F1 female rats for body weight during pregnancy and lactation,reproductive index,embryo implantation number,reproductive organ coefficient(uterus and ovary)and organ pathology.The mating success rate of group 2 in F1 generation was lower than that in group 3(P<0.05).There were no significant differences in F0,F1,F2,F3 male rats in each group for total sperm count,sperm deformity rate,sperm motility,weight of reproductive organs(testis,epididymis,prostate,seminal vesicle)and relative organ coefficients.And there were no significant differences in F1.F2.F3 offspring in each group for reproductive index,body weight during lactation and early physiological development indexes.2.Three-generation study on neuroethology and cognitive ability of genetically modified maize with Cry1Ab and epsps genes on the SD ratsThere were no significant differences caused by transgenic subjects in F0,F1,F2,F3 rats in each group for early neurological development,neuroethology and cognitive ability.Conclusions1.The most contents of main nutrients,vitamins,minerals and amino acids of transgenic maize were consistent with those of parental maize.Most results of nutritional components fell within the range of values reported by OECD.2.Transgenic maize had no adverse effects on body weight,food intake,blood index,organ and tissue pathology,reproductive system and offspring development among three generations of SD rats.3.No adverse effects were found in neuroethology and cognitive ability on SD rats though the three generation study of genetically modified maize with Cry1Ab and epsps genes. |
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