THE INFLUENCE OF FEEDING GMO-PEAS ON GROWTH OF ANIMAL MODELS

Introduction of genetically modified (GM) food or feed into the commercial sale represents a very complicated process. One of the most important steps in approval process is the evaluation of all risks on the health status of people and animal models. Within our project the genetically modified peas was breeded that showed significant resistance against Pea seedborne mosaic virus and Pea enation mosaic virus. Preclinical studies have been conducted to found out the effect of GMO peas on animals rats of outbreeding line Wistar. In a total, 24 male, specific pathogen free Wistar rats were used in the experiment. At the beginning of the experiment, the animals were 28 days old. The three experimental groups with 8 individuals were created. The first group of rats was fed with GMO peas, the second group of rats consumed mix of pea cultivar Raman and the third group was control without pea addition (wheat and soya were used instead of pea). In the present study we focused our attention on health, growth and utility features of rats fed with GM pea. All characteristic were observed during the experiment lasting 35 days. Consumed feed was weighted daily and the weight of the animals was measured every seven days. The average values were compared within the groups. The aim of the experiment was to verify if resistant lines of pea influence the weight growth of animal models. The results of our experiment showed that even a high concentration (30% of GM pea) did not influence growth rate of rats to compare with both rats fed with pea of Raman cultivar and control group. We did not observe any health problems of animal models during the experiment.


INTRODUCTION
Crop that have been genetically modified include plants with changes in DNA structure through genetic engineering (Morisset et al., 2008).Genetic modifications have been considered as modern plant breeding methods in biotechnologies and depend on spontaneous processes in nature.It does not mean creating and transfer of artificial genes.GMO crops have some specific features such as resistance to harmful conditions including pests and diseases (Han and Jung 2013), low temperatures (Sakamoto et al., 2004), drought (Lawlor, 2013) etc., or tolerance to affusion against non-selective herbicides used to kill ineligible plants (Gryson, 2010; Ujhelyi et al., 2012).Genetic engineering facilitates the transfer of desired characteristics into other plants, which is not possible through conventional plant breeding (Ahmad et al. 2012).Pea (Pisum sativum L.) is attacked by wide range of pests and pathogens.Some of them can cause economically significant diseases and losses.The pea is sensitive to a large number of viruses transferred by pea weevil.More than 120 species of viruses that are able to infect a pea has been noticed.Only some of them occurred in such rate that they could be considered as economically significant (Larsen et al., 2007;Pflughoft et al., 2012).
Recently researches detected that only two of the viruses -Pea enation mosaic virus and Pea seed-borne mosaic virus had economic effect on pea production.The Pea enation mosaic virus (PEMV) is unique within plant viruses.In fact, it is occurred in the form of two viruses in obligatory symbiosis -PEMV-1 (Enamovirus) and PEMV-2 (Umbravirus).The presence of both viruses is necessary for induction of wild type infection (Hodge and Powell 2010).Pea seed-borne mosaic virus (PSMV) is a typical representant of genus Potyvirus.The disease was first described in the former Czechoslovakia by Musil (Musil, 1966), and a year later in Japan (Inouye, 1967) and two years later it was recorded in the USA (Stewenson and Hagedorn, 1969).Typical symptoms of infection with this virus are pea leaf roll leaves, shortening of internodes, degree of stunting of infected plants, further deformation of the flowers and development of small deformed pods.Transfer by seeds is a reason for its easy spreading as it has been noticed in worldwide important crops as pea, lentic or broad bean.The spreading by seeds up to 30% in sensitive pea seeds has been noticed, but there were also cases in which 90% of commercial seed was infected.Nowadays these two previously mentioned species of viruses are extended in leguminous plants worldwide (Safarova et al., 2008) et al., 2003).
The aim of the present experiment was to study the effect of GM pea resistant to PEMV and PSMV viruses on morphological parameters and weight gain in rat models.

MATERIAL AND METHODOLOGY
The experiment was conducted at Department of animal nutrition and forage production of Agronomic faculty of Mendel University in Brno (in according with animal cruelty law No. 246/1992 Sb).
In a total, 24 male, specific pathogen free Wistar rats (Biotest, Konarovice, Czech Republic) were used in the experiment.At the beginning of the experiment, the animals were 28 days old and differences in body weight were in a range ±5 g.The animals were kept in an airconditioned room with stable temperature 23 ±1 °C, light period 12 hours and humidity 60%.Photoperiod was based on pattern 12 hour/day and 12 hour/night with maximum intensity 200 lx.The monitored air content of CO 2 was -max.0.25% and NH 3 max.0.0025%.Food and water was provided ad libitum.
The experiment started after 8 days of quarantine period.The animals were divided into three groups, each of 8 animals.The experiment lasted 35 days.One group served as a control and these rats had not been fed with GM peas.Instead of pea the mixture of whey and soya was used.The remaining two groups were supplemented with 30% of GM pea or Raman pea according to the following scheme: Composition of feed mixture is given in Table 1.Feed mixture KS1 was used as a negative control, the second KS 2 contained GMO pea and the third KS 3 contained was created by pea cultivar Raman.
The data were processed using MICROSOFT EXCEL® (USA) and STATISTICA.CZ Version 10.0 (Czech Republic).Differences with p-value ≥0.05 (α = 5%) were considered significant and were determined by T-test, which was applied for means comparison.
Following parameters were monitored and calculated individually in groups of rats during the experiment: net intake of feed, conversion of feed, weight increment and health (were studied anatomical pathology and bacteriology parasitological virological indicators) status of animals.
The animals were treated and fed every day and once a week they were weighted.
The conversion of feed mixture was calculated according to the following formula: Conversion = Feed consumption / (Final weight -Starting weight)

RESULTS AND DISCUSSION
During the last years the research and breeding activities have been focused on problems of pea viruses (Safarova et al., 2008).This concern was caused by frequent and repeating pea viruses occurrence (Jeger et al., 2012; Soylu and Dervis 2011).It has been confirmed that only using of cultivars resistant to economically significant viruses represent effective measures against their negative influence on pea production and its seeds.

Changes in body weight of experimental animals
The changes in body weight were observed daily during the period of 35 days.The weights of the animals in three experimental groups are recorded in tables 2, 3 and 4.
The average weight values of the three groups were compared in.In the first group (rats feeded by mixture 1, negative control) was noticed the average weight 343 g at the end of experiment.In the second group (experiment utilized GMO pea as feeding mixture), the average weight reached up 331 g and in the third group (positive control) it was 348 g.There have not been noticed any statistically significant differences among compared groups.Standard deviations between groups were less than 3 %.Average values of weights at the end of experiment (after 35 days) are presented in Figure 1.
All experimental animals were in good health condition without any differences in growth and changes in behaviour.The average values of weight were compared in three groups.In the first group (rats fed mixture 1, negative control) was noticed the average weight 343 g at the end of experiment.In the second group (experiment fed GMO pea), the average weight reached up 331 g and in the third group (positive control) it was 348 g.There have not been noticed any statistically significant differences among compared groups.Standard deviations between groups were less than 3%.Average values of weights at the end of experiment (after 35 days) are presented in Figure 1.
All experimental animals were in good health condition without any differences in growth and changes in behaviour.

Study of feeding conversion
Feed conversion is defined as ratio between total increase of animal to feed consumption.It is calculated difference between weight at the begging and end of experiment.This total increase is rated by feed consumption during experimental period.All necessary values of 24 experimental animals are expressed in table 5.
Results of feeding conversion showed that there were no statistically significant differences between observed groups.Values reached up from 3.61 up to 3.81.Results of feeding conversion are given at Figure 2.
Although the genetic transformation of leguminous plants has been considered as very difficult and published protocols are reproducible with difficulties, there have been created new GM materials with declared resistance to biotic factors.From studied legumes -soybean, bean, pea, chickpea, peanut and vigna was released only soybean (Huyghe, 1998; Wang and Brummer 2012).Precising and acceleration of breeding process has been possible by rapid development of information and techniques.It has been realised thank to adequate DNA markers in binding to genes of resistance, in ideal cases by direct detection of responsible genes (Gilliland et al., 2003).The results of our experiment suggest that feeding GM pea to rats does not have detrimental effect on their health.There is potential possibility of GM peas utilisation and ranking GM pea into the List of approved GM crops.

Table 2
Increase of weight of rats fed mixture 1

Table 3
Increase of weight of rats fed mixture 2

Table 4
Increase of weight of rats fed mixture 3 The average increases in weight of rats in three experimental groups