The basic safety assessment of genetically modified (GM) crops is based on the principle of "substantial equivalence" concept, but the "substantial equivalence" principle is a starting point rather than an endpoint (König et al., 2004; Muccilli et al., 2020). Giving the randomly inserted trait and the potential synergism with extrinsic factors of action mode of Bt protein, the expression level and recombination mode of Bt protein in different parts of the plant is not the same (Then, 2010). In any safety assessment of GM crops case, we all need novel and comprehensive methods, concepts and ideas to probe into the intended and/or unintended effects of GM technology and crops on target and/or non-target organisms, for the GM technology and crops need to be based on a scientific foundation. Thus, it has also become crucial to apply the case-by-case approach to assess the toxicity of GM plants, especially Bt rice, which plays an significant role in human health. Considering that the straw is the winter livestock feed for cattle in southern China, the unintended effects of Bt rice straw on mammalian especially on its reproductive system should also be valued despite much of the previous literature involving the safety assessment of Bt rice on mice,.
When compared with the control mice, the dietary intake, drinking and daily behavior of ICR mice in the Cry1Ab rice straw fed group were normal and no toxicity or mortality was recorded during the 90-d experimental period. In a similar study, no adverse effects of Cry1Ab protein (Bt rice) on animal behavior or weight increasing on Wistar rats were found (Schrøder et al., 2007). The physiological changes of the blood can directly reflect the health condition of animals. Apoptosis is an important indicator of animal toxicology and pathology (Zeng et al., 2015). Calcium ion has an extremely close relationship with apoptosis and participates in signal transduction regulation of apoptosis pathway (Clapham, 2007). This study demonstrated that no advert effects of Cry1Ab protein exist on the hemogram (Table 1), blood biochemistry (Table 2), apoptosis rate and calcium ion concentration of the blood lymphocytes (Fig. 1-2), and CAT, GSH-Px, POD, SOD, AchE activities of serum (Fig. 3). The results showed that the Bt rice straw had no significant influence on the blood biochemistry of mice. These results are consistent with the previous research in a 90-d study of Sprague Dawley rats fed with transgenic T1C-1 rice expressing Cry1C protein (Tang et al., 2012).
Organ index indicates the ratio of the organ weight to animal weight, which can directly reflect the status of animal disease or organ suffered from toxic effect. In the 90 d Bt rice feeding study, we found that the organ index of ICR mice has no significant differences between the test and control groups (Fig. 4). These results are consistent with previous studies, which showed Cry1Ab does not affect weight gain of Sprague-Dawley rat and Wistar rat (Schrøder et al., 2007; Wang et al., 2002). The antioxidants, including CAT, GSH-Px, POD and SOD that present widely in aerobic organisms can eliminate the free radicals (Jones, 2008; Sharma et al., 2018). In addition, AchE is closely related to neuronal development and regeneration (Fossati et al., 2015). In our study, we detected some enzyme activities related with reproduction in ICR mice organs in the test group, which showed that CAT, GSH-Px, POD, SOD, and AchE enzyme activities had no significant differences compared with those in the control group (Fig. 5). This phenomenon suggested that the growth and development of ICR mice organs didn’t be influenced by taking in Bt rice straw powder for a long period (90 d). And Wang et al. (2013b) showed that there were no significant differences in rat organ weight and enzyme activities between the Cry1Ab protein in test group and the control group in a 90 d study.
The sperm density is the most basic parameter in the reproductive safety assessment, and sperm motility is the main indicator of sperm quality. Sperm abnormalities have long been associated with male infertility and sterility, and sperm density and quality play a substantial role in fertilization and pregnancy outcomes (Chenoweth, 2005). As shown in Fig. 6 and Table 3, the sperm density was in the normal range and was not significantly different between the test and control groups. This finding also showed that Cry1Ab protein exerted no detectable adverse effects on mice sperm vitality, sperm motility rate, sperm abnormalities and sperm acrosome intact rate, which are concordant with several previous studies (Ning et al., 2000; Wang et al., 2002). The safety assessment of Cry1C protein from genetically modified rice also showed that the Cry1C protein had no significant effect on the incidence of sperm abnormality in mice (Cao et al., 2010). In addition, it was reported that there was no negative effect of Bt rice on testis of Sprague-Dawley rats (Wang et al., 2004). Another research also pointed out that Bt corn had neither long-term effects nor multi-generational dominant toxic effects on the development of germ cells and testes in the mice reproductive system, yet the Cry 1Ab have no association with the testes development of rats (Brake et al., 2004; Betz et al., 2000). The differences in ovarian organ index and follicle number at different levels were insignificant (Fig. 7). The granule cells were complete, and the corpus was visible in the test and control groups of female mice. The ovarian slices texture and structure were clear, and the morphology of follicles was no significant differences between the test and control groups (Fig. 8). The results showed that no adverse effects of Bt rice straw on the ovary and follicle of mice. The results were consistent with no significant difference in CAT, GSH-Px, POD, SOD and AchE in testes and ovary and corroborated each other. Thus, all results showed no adverse effects of Bt rice straw on the reproductive safety of female and male mice in the 90-d study. Our findings are supported by a study on three generations of mice fed with transgenic rice, which reported no potential adverse effect on the male reproductive axis, copulation index, fertility index, gestation length, live-birth rate and litter number (Zhou et al., 2012).
Cry proteins have high species-specific toxicity against certain insects. The mode of action in the target insect is through specific receptors in the gut, which is highly alkaline, with binding of the toxin resulting in pore-formation, osmotic imbalance, cell lysis and subsequent death of the insect (Palma et al., 2014). Due to species-specificity, the Cry proteins are regarded harmless or non-toxic to mammals, including humans. Hitherto, numerous data from toxicity studies have showed that no significant adverse effects of Cry proteins on body weight and clinical observations. The Bt insecticidal toxin-encoding gene has a long history of safe use in biotechnology (Mendelsohn et al., 2003). Animal feeding trials is an important assessment method for potential adverse effects of GM crops, and a 90 d rodent feeding study with GM food is suitable and valid (Poulsen et al., 2007). Nevertheless, animal's complex physiological structure and function sometimes could bring unforeseen difficulties in the experiment process and results (Zhou et al., 2012). We need to evaluate possible potential effects from transgenic crops through a large number of comprehensive analysis assessments.