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The term “genetically modified organisms (GMOs)” refers to plants, microbes, and animals with genes transferred from other species in order to produce certain novel characteristics (for example resistance to pests, or herbicides), and produced by recombinant DNA technology. There are two standard methods, which are generally used to introduce new DNA (genes) into a plant cell, which is going to be modified. Neither is perfect and both could not guarantee that the rest of the plant genome remains unchanged. Therefore the safety of the GM crops created with the help of these methods cannot be guaranteed for human and animal health, nor for the environment (Ho and Tappeser, 1997; Kuznetsov et al., 2004; Wilson et al., 2004; Ermakova, 2005). Four main sources of the hazards of GMOs are now accepted by scientists worldwide: 1) those due to the new genes, and gene products introduced; 2) unintended effects inherent in the technology; 3) interactions between foreign genes and host genes; and 4) those arising from the spread of the introduced genes by ordinary cross-pollination as well as by horizontal gene transfer (World Scientists Statement, 2000).

GM crops contain material which is not present in them under natural conditions and they form a part of our daily diet. To understand what effect they can have on us and on our animals and whether their risks may outweigh the benefits, it is vitally important to study the influence of these GM plants on different organisms for several generations. At present, the studies pertaining to the issue are lacking from the scientific literature. Also, several detrimental effects of GM crops had been shown on the metabolism of animals. The hazard of GMOs was shown for animals and the environment in extensive investigations (Traavik, 1999; Ho and Tappeser, 1997; Pusztai, 1998 and 2001; Kuznetsov et al., 2004 and others). Earlier it was shown that the consumption of GM foods by animals led to the negative changes in their organisms. Experiments, conducted by Pusztai showed that potatoes modified by the insertion of the gene of the snowdrop lectin (an insecticidal protein) stunted the growth of rats, significantly affected some of their vital organs, including the kidneys, thymus, gastrocnemius muscle and others (1998) and damaged their intestines and their immune system (Ewen and Pusztai, 1999).Similar effect of GM potatoes on rats was obtained at the Institute of Nutrition in Russia (Ermakova, 2005).

It is put forward in the risk assessment documents that the GM components of transformed plants are completely destroyed in the digestive tract¹⁵ of humans and animals, together with the other genetic material found in them. However, foreign DNA plasmids¹⁶ are steadier against the digestion than it was originally believed. Plasmid DNA and GM DNA were found in microorganisms¹⁷ of the intestine and in saliva¹⁸ (Mercer et al, 1999; Coghlan, 2002). Experimental research in mice showed that ingested foreign DNA can persist in fragmented form in the gastrointestinal tract¹⁹, penetrate the intestinal wall, and reach the nuclei²⁰ of leukocytes²¹, spleen²² and liver cells (Schubbert et al., 1994). In another research of Schubbert et al. (1998), foreign DNA, orally ingested by pregnant mice, was discovered in blood (leukocytes), spleen, liver, heart, brain, testes²³ and other organs of fetuses²⁴ and newborn animals. They considered that maternally ingested foreign DNA could be potential mutagens²⁵ for the developing fetus. However, Brake and Evenson (2004) analyzing the testis in mice as a sensitive biomonitor of potential toxin, didn’t find adverse effects of transgenic soybean diet on fetal²⁶ development. From the literature review, there seems a lack of investigations on the influence of GM crops on mammals²⁷, especially on their reproductive function. Therefore, the objective of the study we undertake is to see the effect of the most commonly used GM crops on the birth rate, mortality and weight gain of rat pups, whose mothers were fed with diets supplemented with Roundup Ready soya, a kind of GM foods.

Wistar rats were used as the subjects in the experiment. The animals were brought up to sexual maturity on laboratory rat feed. When their weight reached about 180-200
G, the female rats were divided into 3 groups, housed in groups (3 rats / cage), and kept under normal laboratory conditions. The feeding scheme was as follows. Females in every cage daily received dry pellets from a special container placed on the top of their cage. Those rats receiving soya flour supplement were given the soya flour in a small container placed inside their cage (20 g x 40 ml water for 3 rats), so 5-7 g flour for each rat every day.

One group of female rats of 180-200 g weight (6, in 2 cages) was allocated to the experimental group and received 5-7 g soya flour / rat / day prepared from Roundup Ready soya added to the rat feed for 2 weeks. Another group of females (3) was allocated to the control group but their diet was supplemented with the same amount of soya flour prepared from the traditional soya in which only traces (0.08 + 0.04%) of the GM construct were present, most likely resulting from cross-contamination. We also introduced a positive control group (6, in 2 cages), which had not been exposed to soya flour. Therefore, the females only got the standard laboratory feed without any supplementation.

After 2 weeks on the diets all the females were mated with 2 healthy males of the same age, which had never been exposed to soya flour supplement. In order to avoid infection of females, the sperm count and quality had not been determined. We carried on feeding the respective diets to all females during mating and pregnancy. Upon delivery, all females were transferred to individual cages, and the amount of soya flour supplement was increased by an additional gram for every pup born. Lab feed and water was available for all animals during the experimental period. When the rat pups opened their eyes and could feed themselves (from 13-14 days of age), the daily dose of soya supplement was increased to 2-3 g for every pup, although all rats had free approach to the soya. All rats ate their soya portions well. After the experiment was finished the organs of some pups were taken out and weighed. The level of mortality was analyzed by the one-way ANOVA, using the Newman-Keuls test for share distribution. The pups' weight and its distribution were checked by Mann-Whitney test and Chi-square in StatSoft Statistica v6.0 Multilingual (Russia).

By the end of the experiment, from the 15 females included in the experiment, 11 gave birth and produced a total of 132 rat pups. The 4 rats who became pregnant from 6 females on the positive control diet gave birth to 44 pups (an average of 11 pups / female), while the 4 females, from the 6 on GM-soya flour supplemented groups gave birth to 45 (11.3 pups / female), and the 3 females from the traditional soya group—33 pups (11 pups / female).

Supplementation of the diet of the females with GM soya led to the death of 25 pups, out of the 45 born by the end of the third week of lactation²⁸, while during the same period only 3 pups born to the females on the traditional soya supplemented diets died from 33.The mortality in the positive control group was also 3, but from the larger number of pups born, as seen in Table 1. High pup mortality was generally characteristic of every litter from the females fed with the GM soya flour (Table 2). Among the pups from the females fed with the positive control diet, 2 pups died during the first week, and 1 during the second week after delivery. All those pups that died from the females fed with traditional soya flour did so during the first week after birth. However, pups from the females fed with the GM soya flour supplemented diet kept dying during lactation period as it is evident from Table 3.

In 2 weeks after their birth the weight of the pups from the GM soya supplemented group was less (23.95 g ± 1.5 g) than that of the pups of the positive control group (30.03 g ± 1.1 g; p < 0.005), or from the traditional soya flour supplemented group (27.1 g ± 0.9 g; p < 0.1). Since the number of surviving pups was so different, the weight distribution of the pups was compared in Table 4. From the data it is evident that 36% of the pups from the GM soya fed group weighed less than 20
G, in comparison with 6% in the positive control group, and with 6.7% found in the traditional soya supplemented diet group (Table 4). The study of pups’ organ mass showed that the organs of small pups from GM soya fed group were tiny in comparison with the same of the other groups except the brain mass (Table 5). This fact indicated that changes occurred with the development of internal organs. Slight negative effect was found in the group which received the traditional soya, but this effect was not significant. No mortality of females and survived young pups eating the GM soya flour supplemented diet was observed.

The reproductive behaviour of female rats fed on standard laboratory feed supplemented with soya flour prepared from either genetically modified soya or traditional soya was studied to see the effect of the diet on pregnancy, lactation and the growth of the rat pups. Since it is well established that raw soybean contains a number of anti-nutrients²⁹ (Pusztai, 1998), and female hormone-like³⁰ substances, it was thought to be necessary to compare these data with those from a positive control group when animals were not exposed to any soya flour supplementation.

In order to understand the mechanism of how this widely consumed GM crop exerts its influence on the reproductive performance of mammals and their offspring, it would be necessary to perform complex research, including histological³¹, genetic and embryo-toxicological³² investigations. However, we had to restrict our experiments only to a short time span, and start to feed the female rats 2 weeks before mating. However, unlike in the experiments of Schubbert et al. (1998) or Brake and Evenson (2004), who started to feed pregnant mice, in our experiments the diets supplemented with GM or traditional soya flour were already given to the female rats 2 weeks before mating, and we continued to treat them with their respective diet until the pups were weaned.

Upon delivery, very unexpectedly a very high rate of pup mortality (55.6%) was observed in the group of females whose diet was supplemented with the GM soya flour in comparison with the pups of both the positive control (6.8%) and the traditional soya flour supplemented (9%) groups. Also, in this group the pups continued to die over the period of lactation, which occurred only in the GM soya fed group. At the same time, the weights of the surviving rat pups were also lower. Since the amount of the pups was smaller, about half, more milk should have been available for the individual pups. They should have a better chance to grow optimally, unless the amount and / or the quality of the milk were affected by consuming the GM soya flour.

Our data allow us to speculate and presume that the negative effect of GM soya on the newborn pups could be explained by two possible factors. Firstly, it can be the result of the transformation and the insertion of the foreign genes, which could penetrate into the sexual / stem cells, or / and into cells of the fetus, as it was observed by Schubbert et al. (1998). Secondly, the negative effect could be caused by the accumulation of the GM soya residues. However, no mortality was observed with the female rats, nor with the young pups survived, although they also began to eat the GM soya. It is supposed that the effect could be caused by the first factor.

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