Janczick et al.12

Modification of potatoes, by either recombinant DNA

technology or conventional breeding, affects their

nutritional value for the rat

Pawel Janczyk1

, Carola Wolf3

, Anja Hartmann2

, H. Junghans4

,

M. Schwerin2,5

, W. B. Souffrant1*

1

Research Units Nutritional Physiology “Oskar Kellner”,

2

Research Group Functional

Genomics, Research Institute for the Biology of Farm Animals Dummerstorf, 18196

Dummerstorf; Germany;

3

Office for Veterinary and Foodstuff Examination of the

Mecklenburg-Vorpommern (LVL), Thierfelderstr. 18, 18059 Rostock, Germany;

4

NORIKA Ltd., 18186 Groß Luesewitz, Germany;

5

Institute of Farm Animal Sciences

and Technology, University of Rostock, 18059 Rostock, Germany.

ABSTRACT

The present study was conducted to compare the composition and

nutritional value of two genetically modified potato lines, Desiree

35Svp60SEK-6 (Des-VP60) and Albatross GBSSctxBvp60SEK (Alb-VP60),

and their conventional parental counterparts in a feeding experiment in rats.

Both transgenic potatoes express the VP60 viral antigen (a major structural

protein of the Calicivirus causing the rabbit hemorrhagic disease) as the ‘novel’

protein. The nutritional value of the diets was investigated based on weight

gain, nitrogen balance, protein and amino acids digestibility, and changes in gut

microbiota of rats fed for 14 days on a semi-synthetic diet supplemented with

potato. The amount of potato to be in the diet was calculated on N basis. 15% N

of diet protein was of potato origin.

The nitrogen balance study revealed differences in the nutritive values of

both parental potato lines Des and Alb, these differences were also seen in the

transgenic counterparts. The increased nutritive value of the Desiree lines in

comparison with the Albatros lines is possibly due to the higher protein and

lower starch content in Desiree relative to Albatros,. Genetic modification of the

two parental potato lines differentially affected the nutritive value of the

transgenic potatoes. The genetic modification of the Desiree potato led to a

significant improvement of N-balance and the total tract apparent crude protein

digestibility, whereas N-balance and the other nutritional characteristics remain

unchanged in the Alb-VP60 potato when compared with the parental Alb

potato. The increased nutritional value of the genetically modified (GM) Des-

VP60 potato compared to its parent strain was probably due to alterations in

chemical composition of the potatoes that are caused by pleiotrophic metabolic

effects related to insertion of a ‘foreign’ DNA sequences into the potato

genome, rather than by biological effects of the expressed VP60 protein.

Archiva Zootechnica vol. 10, 2007 13

Keywords: genetically modified organisms (GMO), nutritional value of

protein, rat, transgenic plant, VP60

INTRODUCTION

During the last few years many studies have determined the nutrient value

of genetically modified (GM) feeds compared to their conventional counterparts

(reviewed by Flachowsky et al. 2005). Beside the possible influence of the

‘novel’ protein in the digestive tract and tissues of food-producing animals, and

on animal health and product quality, insertion of the ‘foreign’ DNA sequences

into the plant genome may disrupt, modify or silence active genes or activate

silent genes, which may result in the formation of either new metabolites or

altered concentrations of existing metabolites and by this the composition of

macro- and micro-nutrient (Ewen & Pusztai, 1999). The results available to date

are suggest that there are few significant differences in the nutritional value of

feedstuffs containing material derived from genetically modified (GM) plants

(those with unchanged crude composition) when compared to non-GM varieties

(e. g., Hammond et al. 1996; Aulrich et al. 2001; Boehme et al. 2001;

Chrenkova et al. 2002; Murai et al. 2002; Reuter et al. 2002; Flachowsky &

Aulrich, 2003; Williams 2003; El Sanhoty et al. 2004; Weil 2005).

However, it has to be emphasized that the occurrence of effects due to

changes of nutrient composition is not specific for genome modification

through recombinant DNA technology – it also occurs frequently in

conventional breeding (e. g., Jousse et al. 1998; Tovar-Palacio et al. 1998;

Fafournoux et al. 2000; Saggau et al. 2000; Allred et al. 2001).

The present study was conducted to compare the composition and the

nutritional value of the two genetically modified potato lines, Desiree

35Svp60SEK-6 (Des-VP60) and Albatross GBSSctxBvp60SEK (Alb-VP60),

expressing the VP60 viral antigen as the ‘novel’ protein (Broer, Rostock

University, Faculty of Agro- and Environmental Sciences, Rostock, Germany),

and their conventional parental counterparts (NORIKA Ltd., Groß Luesewitz,

Germany). VP60 is a major structural protein of the Calicivirus causing rabbit

hemorrhagic disease (RHDV) (Ohlinger et al. 1990, Parra & Prieto, 1990) and

can induce immunoprotection of RHDV when expressed in baculovirus

(Laurent et al. 1994; Nagesha et al. 1995; Plana-Duran et al. 1996) or vaccinia

virus (Bertagnoli et al. 1996). Successful parenteral immunization with VP60

produced in transgenic potatoes has been described (Castanon et al. 1999;

2002). To study potential effects of potatoes expressing VP60 on rats’

metabolism, hematological and biochemical indices were measured in animals

fed wild type and modified potatoes. A nitrogen balance study investigating the

nutritional value of semi-synthetic diets containing casein protein supplemented

with 15 % N of the corresponding potatoes was undertaken.

Janczick et al.14

MATERIAL AND METHODS

Animals, diets and experimental design

The effects of two transgenic potatoes and their two parental counterparts

on metabolism was studied in an experiment with 32 young male Wistar rats

(Charles River Laboratories, Germany) of the weight of 99.8 ± 9.02 g at the

beginning of feeding experiment. Animals were housed individually under

standardized humidity (60 ± 5 %) and temperature (21.5 ± 1 °C) in metabolism

cages that allowed separate and quantitative collection of uneaten food, urine

and feces, with a 12/12 hrs light regime. Animals were allocated to four groups

and fed a semi-synthetic basis diet where the sole protein source was casein

supplemented with either lyophilised Albatros (Alb) or Desirée (Des) parental

potatoes (NORIKA Ltd, Gross Luesewitz, Germany) or transgenic Albatross

GBSSctxBvp60SEK (Alb-VP60) and Desiree 35Svp60SEK-6 potatoes (Des-

VP60) (Broer, Rostock University, Faculty of Agro- and Environmental

Sciences, Rostock, Germany, personal communication). A control group was

fed a casein diet only. The amount of protein in the diet was calculated on N

basis. The amount of nitrogen offered to rats was 0.150 g/100g body weight

(BW) what was equal to 0.94 g protein/100 g BW. To investigate the different

potato lines, and in order to be able to feed the required amount of protein in the

diets, casein was chosen as the main protein source to become 85% of protein,

15% of protein (on N basis) came from the investigated potato line (Table 1).

Table 1. Semi-synthetic diets composition fed to rats in a nitrogen balance study (g kg

-

1

DM)

Potato

Desiree

(wild type)

Desiree

35Svp60SEK-6

Albatros

(wild type)

Albatros

GBSSctxBvp60SEK

Cas Des Des-VP60 Alb Alb-VP60

Corn

starch

200 69 127 79 68

N-free

mixture*

700 700 700 700 700

Casein** 100 85 85 85 85

Potato 0 146 88 136 147

*N-free mixture consisted of 18.5% cellulose, 37.1% sugar, 14.8% oil, 7.4% vitamins (Vitamin A – 750 IE,

B1- 1 mg, B

2– 1 mg, B

6– 0.5 mg, B

12– 0.5 mg, C – 1 mg, D

3– 25 IE. E – 2.5 mg, K

3– 0.1 mg, Panthotenic

acid – 1 mg, Nicotine acid amide – 2.5 mg, Choline hydrochloride – 100 mg, Folic acid – 0.1 mg, Biotin –

0.01 mg, Inositol – 12.5 mg, p-Aminobenzoic acid – 5 mg, fulfilled ad. 1 g with wheat starch). 14.8%

minerals (CaCO3

- 6.86%, Ca-citrate – 30.83%, CaHPO4⋅H

20 – 11.28, K

2HPO

4– 21.88, KCl – 12.47%, NaCl

– 7.71%, MgSO4

– 3.83%, MgCO3 – 3.52%, Fe-Ill ammoncitrate – 1.53%, MnSO

4⋅H

2O – 0.02%, CnS0

4⋅ H

20

– 0.0078%, KJ - 0.0041%, NaF – 0.051%, AlNH4(S0

4)

2⋅12 H

20 – 0.009%, ZnCO

3– 0.006%) and 7.4% wheat

starch; corn starch was added to obtain 100%.

**Methionine was added to casein in the amount of 3% casein.

All four diets were isoenergetic and isonitrogenous, contained 150 mg

nitrogen per 10 g dry matter (DM), and were fed for 14 days. Food for each

Archiva Zootechnica vol. 10, 2007 15

animal was adjusted weekly according to the body weight (10 g DM per 100 g

BW) and fed once a day (at 8.00 a.m.). Water was administered in nipple bottles

ad libitum, and the access to the food was not limited. BW was recorded on day

1, 8 and day 14 of the feeding experiment, before feeding. From day 8 to day 14

feed intake was recorded and urine and feces were collected daily. HCl was

added to urine and feces samples for storing at 4ºC until analyzes were done.

Nitrogen balance

Samples were weighed and the N-content, as well as DM was analyzed.

DM content in pure potato lyophilisates, feed, uneaten feed and fecal samples

was determinedusing the Weender standard procedure (Naumann & Bassler,

1993). The nitrogen content of all samples was analyzed using an elementary

analyzer (LECO CNS 2000, LECO Instrumente GmbH, Moenchengladbach,

Germany) applying the Dumas combustion technique (Hauck, 1982; Kirsten,

1983) and was used for evaluation of N balance. Data obtained in the N-balance

study was used for calculation of total tract apparent crude protein and amino

acids digestibility, net protein utilization (NPU) and protein efficiency ratio.

Endogenous and metabolic nitrogen were calculated using regression formulas

based on rats’ BW and DM intake, which were obtained in previous nitrogen-

free experiments on rats performed at this Institute (Janczyk, 2005).

Amino acid composition of the potato protein

Amino acids (AAs) were determined in lyophilized potatoes using

automated ion exchange chromatography (Biochrom 20 Plus analyzer,

Biochrom Ltd, Cambridge, UK) according to the methods of Kreienbring &

Wuensche (1974) and Kreienbring (1983).

Blood and serum indices

To follow possible adverse acute effect of feeding the transgenic potatoes,

blood parameters were investigated. On day 14 rats were exsanguinated under

inhalant isofluran (IsoFlo

®

) anesthesia. Whole blood was collected (in EDTA

tubes) for hematological examination. Serum was prepared for estimation of

alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and

glutamate dehydrogenase activities, as well as for determination of the

concentrations of bilirubin, total protein, urea, creatinine. These analyses were

done at the Office for Veterinary and Foodstuff Examination of the

Mecklenburg-Vorpommern (LVL), Rostock, Germany, using internal protocols.

All procedures involving animal handling and treatment were approved by

the Committee for Animal Use and Care of the Agricultural Department of

Mecklenburg-Western Pommerania, Germany, according to the German Law

for Animal Protection.

Janczick et al.16

PCR-DGGE

Bacterial composition of the caecal digesta was investigated. After the rats

had been killed by the exsanguination, caecal digesta was collected and stored

at 4ºC until DNA isolation. The DNA was isolated from 250 mg of undiluted

chyme using DNA Spin Kit for Soil

®

(MP Biomedicals, Heidelberg, Germany)

according to manufacture’s protocol. V6 – V8 fragments of bacterial rRNA

gene present in isolated DNA were amplified by means of polymerase chain

reaction (PCR) using forward primer S-D-Bact-0968-a-S-GC and reverse

primer S-D-Bact-1401-a-A-17 (Konstantinov et al, 2004), synthesized by

Invitrogen (UK). PCR reactions and amplifications were done according to

Konstantinov et al. (2004) in a Biometra model (Biometra, Goettingen,

Germany) thermocycler. Denaturing gradient gel electrophoresis (DGGE) was

performed to separate the amplicons. The DGGE protocol was adapted from

Konstantinov et al. (2004) and the electrophoresis was done in Mutation

Detecting System (BioRad

®

, Munich, Germany). Completed DGGE gels were

stained with SYBR

®

Gold Nucleic Acid Gel Stain (Molecular Probes, Eugene,

OR, USA) (stock solution diluted 10.000-fold in 0.5 x TAE buffer of pH 7.5)

for 15-30 minutes. Stained gels were illuminated with UV light

(transilluminator Biometra TI 1, Germany) and photographed with a digital

camera (Power Shot G3, Canon). The DGGE images (400 dpi) were analyzed

with Bionumerics 2.1 software, and number of bands, diversity (Shannon) index

and similarity (Dice) coefficient were calculated essentially as described by

Konstantinov et al. (2004) and Kluess (2004).

Statistics

All results were subjected to one factor analysis of variance outlined in the

software Statistica Version 6.0. All differences between means were tested by

P<0.05, using the post-hoc HSD Tukey test included in the same software.

RESULTS

Nutrient composition of the potatoes

To study nutrient composition as a possible reason of possible differences

in nutritional value between the conventionally bred Des and Alb potato lines

and the parental Des, Alb and the transgenic Des-VP60 and Alb-VP60 potato

lines, respectively, both the parental and transgenic potato lines were analyzed

for crude nutrients and AAs content (Table 2). The content of DM was

approximately the same in all four potato lines (Des: 95.4, Des-VP60: 96.2,

Alb: 93.0, Alb-VP60: 93.2 %). However, crude nutrient content differed both

between conventional bred parental lines Des and Alb and the GM Des-VP60

line and its parental counterpart (Table 2). Des potatoes exhibited increased

content of crude fiber (cF) and crude fat (ether extract, EE) in comparison with

Alb potatoes. The modified Des-VP60 potato had more protein, more cF and

less nitrogen-free extracts compared to the Des parental line. The starch content

Archiva Zootechnica vol. 10, 2007 17

of Alb potatoes was higher than in Des. Unfortunately, the amount of harvested

Des-VP60 was insufficient to conduct all analyses so the starch content of this

potato line has not been established.

Table 2. Composition of lyophilized wild type and isogenic potato lines.

Parameter Des1

Des-VP60 Alb Alb-VP60

DM (%) 95.36 96.18 93.02 93.20

cA (%DM) 4.20 4.86 3.74 3.57

cP (%DM) 9.15 15.38 9.62 9.60

cF (%DM) 2.98 9.20 2.23 2.26

EE (%DM) 0.41 0.36 0.28 0.26

NfE (%DM) 83.15 69.65 85.63 86.33

Glucose (%) 1.66 0.61 0.01 0.01

Fructose (%) 2.89 0.79 0.01 0.01

Saccharose (%) 1.15 1.29 1.11 0.74

Starch (%) 67.12 ne** 77.40 77.51

AA (% cP)

ASP 18.19 17.47 7.13 15.71

THR* 2.83 2.58 4.35 3.84

SER 2.60 2.31 4.67 3.57

GLU 11.28 14.10 16.14 10.39

GLY 2.19 1.63 1.79 3.08

ALA 2.37 1.75 3.31 3.23

VAL 3.73 4.11 4.84 4.14

ILE* 2.67 2.76 4.24 3.18

LEU* 3.84 3.20 8.06 5.79

TYR 1.90 1.78 3.03 1.77

PHE 3.19 2.67 3.91 3.73

HIS 1.47 1.51 2.21 1.53

LYS* 4.57 4.87 7.36 5.12

ARG 3.73 6.11 2.92 4.05

PRO 2.77 2.63 7.06 3.95

CYS 1.09 0.87 1.56 1.58

MET* 0.83 0.83 1.18 1.22

TRP* 1.09 0.89 0.83 0.91

All AAs 70.33 72.06 84.57 76.19

*essential amino acids

**ne – not possible to establish as too few material

1

– see Table 1

DM- dry matter; cA – crude ash; cP – crude protein; cF – crude fiber; EE – ether extract; crude fat; NfE –

nitrogen free extract

In addition, the amounts of several AAs were quite different, both between

the conventionally bred potato lines and between the parental and transgenic

lines. Glutamate and arginine contents were higher in the Des-VP60 in

comparison to the Des, but the modified Alb-VP60 had less Glu and more Arg

than the parental Alb potato. Lysine and aspartate content did not change

Janczick et al.18

between Des lines, but the modified Alb line had less Lys and twice the amount

of Asp when compared to the parental plant.

Growth and nutritional value

Daily DM intake did not show any differences between all feeding groups.

However, rats fed Des and Des-VP60 potatoes exhibited a significantly

increased growth in comparison to rats fed Alb and Alb-VP60 potatoes (Table

3). The daily weight gain of rats fed potatoes of both Des lines was higher by

about 20 % compared to rats fed potatoes of both Alb lines; however this

difference was not significant. There were no obvious differences among the

corresponding GM potatoes and their conventional counterparts.

Table 3. Weight gain and parameters of nutritional value of protein consisted of 15% N

from potato and 85% N from casein (n=8/group, mean ± SD)*

Group1

dWG (g) dDMI (g)

N-balance

(mg/d)

TTaPD

(%)

TTtPD

(%) NPU (%) PPV (%) PER

Cas

2.8 ± 0.22

a

10.7 ±

0.08 a

119.0 ±

4.20 a

83.4 ±

1.10 a

88.7 ±

1.22 a

89.4 ±

2.16 a

62.7 ±

2.19 ac

2.3 ± 0.18

Des 2.3 ± 0.30

11.5 ±

0.12

94.2 ± 6.22

b

73.9 ±

1.50 b

79.3 ±

1.50 b

76.6 ±

2.72 bde

50.3 ±

3.57 a

2.0 ± 0.26

Des-

VP60

2.4 ± 0.25

11.5 ±

0.23

108.1 ±

3.78 ab

79.8 ±

0.82 a

85.2 ±

0.88 a

82.7 ±

2.35 ad

56.8 ±

1.33 ac

2.0 ± 0.22

Alb

2.0 ± 0.61

b

11.1 ±

0.44 b

77.3 ± 6.11

c

68.5 ±

3.95 c

73.9 ±

3.80 c

69.8 ±

2.84 ce

42.9 ±

3.13 b

1.7 ± 0.54

Alb-

VP60

1.9 ± 0.81

b

11.4 ±

0.59

72.4 ±

18.08 c

66.2 ±

4.43 c

71.8 ±

4.30 c

68.1 ±

7.80 c

40.6 ±

9.01 b

1.7 ± 0.69

*values in columns marked with different letters differ significantly, P<0.05

1

– see Table 1

dWG – daily weight gain; dDMI – daily dry matter intake; TTaPD – total tract apparent crude protein

digestibility; TTtPD – total tract true crude protein digestibility; NPU – net protein utilization; BV –

biological value; PPV – productive protein value, PER – protein efficiency ratio

These observations are in correspondence with other results of the nitrogen

balance study (Table 3). Rats fed potatoes of both Des lines exhibited

significantly increased N-balance of 20 – 50 % and increased total tract

apparent crude protein digestibility in comparison to rats fed potatoes of both

Alb lines (P < 0.05). In correspondence to these findings, other nutritional

characteristics, based on nitrogen retention, as NPU and productive protein

value showed similar results due to their calculation on the basis of the N-

balance values (Table 3).

The genetic modification of the two parental potato lines has affected

differentially nutritive value of the transgenic potatoes. The genetic

modification of the Desiree potato led to significant improvement of N-balance

and the total tract apparent crude protein digestibility, whereas N-balance and

Archiva Zootechnica vol. 10, 2007 19

the other nutritional characteristics remained unchanged in the Alb-VP60 potato

in comparison with the parental Alb potato.

Bacterial DGGE profiles in gut

The DGGE profile of V6-V8 fragments of bacterial 16S rDNA isolated

from caecal digesta showed a high individual diversity of the gut microbial

community (Fig. 1). Similarity (Dice) coefficient within groups was 60% in

Des, 59% in Des-VP60, 41% in Alb, and 52% in Alb-VP60. In addition,

obtained number of bands in the universal DGGE-profile (Des-groups: app. 18,

Alb-groups: app. 13) and calculated diversity (Shannon) index (Des-groups:

approx. 1.2, Alb-groups: approx. 0.9) was obviously higher in both Des-groups

in comparison to both Alb-groups.

Figure 1. DGGE banding profile of PCR products of V6-V8 regions of 16S rRNA gene

sequences retrieved from bacterial DNA isolated from rats’ caecal content. Primers used

for PCR-DGGE were S-D-Bact-968-a-S-GC (forward) and S-D-Bact-1401-a-A-17

(reversed) (32). Rats were fed protein consisted of 85% N from casein and 15% N from

isogenic or transgenic Desiree or Albatros potatoes (Des, Des-VP60, Alb, Alb-VP60,

respectively). There are fewer bands seen in the profiles in Alb and Alb-VP60 in

compare to Des and Des-VP60 what shows a less complex caecal microbial community

in Alb and Alb-VP60. Each lane is the result from a single rat. M lane - electrophoresis

marker lane.

Blood and serum indices

No significant differences were seen in hematological and biochemical

indices (Table 4 and Table 5), suggesting that there is no acute effect of VP60

on rat metabolism.

Janczick et al.20

DISCUSSION

Genetically modified organisms (GMO) have to be tested for safety before

they can be introduced into the marketplace. The decision on what

characteristics should be investigated to assess the risk of feeding GMOs has

not been made clearly to date (Flachowsky et al. 2005). A part of the risk

assessment is “determination of substantial equivalence”, meaning, in the case

of new component, that there should be no significant differences compare to

the parental (isogenic) plant (Flachowsky et al. 2005; OECD, 1993; EC, 1997).

This is based mostly on the chemical composition analysis (OECD, 2003). In

the case where modification might substantially change the bioavailability of a

component, animal feeding studies are of prime importance (OECD, 2003).

Beside the possible influence of the ‘novel’ protein in the digestive tract and

tissues of food-producing animals, insertion of the ‘foreign’ DNA sequences

into the plant genome may modify the nutritional value of feedstuffs (e. g., El

Sanhoty et al. 2004; Weil, 2005; Liu-Stratton et al. 2004). However, we have to

consider that the occurrence of effects due to changes of nutrient composition is

not specific for genome modification through recombinant DNA technology,

but also occurs frequently in conventional breeding.

Table 4. Hematological parameters of blood from rats fed protein consisted of 15% N

from potato and 85% N from casein (mean ± SD)

Group1

Ht (%)

RBC

(106/µL)

Hb

(g/dL)*

Thr

(103/µL)

WBC

(103/µL)

Lymph

(%)

Non-

segmented

neutrophils

(%)

Segmented

neutrophil

s (%)

Des 39 ± 3.5 6.3 ± 0.22

14.6 ±

0.83

1143 ±

110.6

3.1 ± 2.16 80 ± 7.9 2 ± 1.7 18 ± 7.0

Des-

VP60

40 ± 3.6 6.2 ± 0.50

15.0 ±

0.98

1233 ±

227.4

3.2 ± 2.11 83 ± 7.7 2 ± 1.8 15 ± 7.5

Alb 41 ± 2.8 6.5 ± 0.45

15.5 ±

0.99

1459 ±

200.2

5.2 ± 2.23 79 ± 14.2 2 ± 2.0 17 ± 12.1

Alb-

VP60

39 ± 2.1 5.9 ± 0.34

14.3 ±

0.84

1385 ±

167.1

2.9 ± 2.30 85 ± 7.9 1 ± 1.0 12 ± 6.3

1

– see Table 1

Ht – hematocrite, RBC – red blood cells, erythrocytes; Hb – haemoglobin; Thr – thrombocytes; WBC – white

blood cells, leucocytes; Lymph - lymphocytes

g/dL x 0.6206=mmol/L

Therefore, the present investigation was conducted to compare the

nutritional value of two genetically modified potato lines, and their

conventional parental counterparts in rats. The protein content of potatoes is too

low to cover the maintenance protein requirement of rats alone. Moreover,

lyophilised potatoes are not found tasty by animals, and so to ensure the rats

take all offered feed, a semi-synthetic diet containing 85% N from casein as

basal protein source and 15% N from the investigated potato lines was prepared.

Differences in protein digestibility between groups can therefore be only

induced by the potato protein. The nutritional value of the diets was investigated

Archiva Zootechnica vol. 10, 2007 21

based on weight gain, nitrogen balance, total tract apparent crude protein

digestibility and net protein utilization. The feed intake and growth of rats fed a

semi-synthetic diet containing 100% N from casein served as a reference

control diet.

Table 5. Biochemical parameters of blood from rats fed protein consisted of 15% N

from potato and 85% N from casein (mean ± SD)

Group1

ALAT

(U/L)

ASAT

(U/L)

ALP

(U/L)

GLDH

(U/L)

Bilirubine

(µM/L)

Total

protein

(g/L)

Urea

(mM/L)

Creatinine

(µM/L)

Des 37 ± 5.0

107 ±

11.4

611 ±

41.5

18 ± 5.5 3 ± 0.5 44 ± 1.2 1 ± 0.6 26 ± 1.6

Des-

VP60

36 ± 4.8

107 ±

26.1

545 ±

140.7

18 ± 5.7 2 ± 0.3 46 ± 2.1 1 ± 0.9 27 ± 2.4

Alb 41 ± 8.6

110 ±

25.0

631 ±

106.0

14 ± 2.5 2 ± 0.3 43 ± 1.8 1 ± 0.6 26 ± 2.0

Alb-

VP60

37 ± 4.8

108 ±

16.4

647 ±

58.7

15 ± 2.4 2 ± 0.7 43 ± 1.5 2 ± 1.9 26 ± 1.9

ALAT – alanine aminotransferase, ASAT – aspartate aminotransferase, ALP – alkaline aminotransferase,

GLDH – glutamate dehydrogenase

1 – see Table 1

The results of nitrogen balance and protein digestibility studies revealed

strong differences of nutritive value between the two parental potato lines Des

and Alb, these differences were also seen in the genetically modified derivative

strains. The higher nutritive value of the Desiree potatoes, when compared to

the Albatros potatoes, is probably due to differences in chemical composition.

We could determine a different amino acid composition of the protein from the

different potato lines. We did not study the rate of hydrolysis of the protein in

the digestive tract, but it cannot be excluded, the potato protein differed also in

tertiary and quaternary structures.

The Albatros potato starch content was higher then in Desiree line. As the

protein content of Albatros was lower, more potato was in the diet to ensure the

same amount of protein as in Desiree groups. The higher amount of potato

starch could therefore influence the bacterial composition in the caecum, where

the most fermentation processes in the rat gut occur.. The changes in the

composition of the bacterial population were recorded using a molecular

approach of 16S rRNA gene amplification and denaturing gradient gel

electrophoresis (Muyzer, 1999). The DGGE profiles showed a high degree of

animal-specific diversity in the gut microbial community that is in accordance

with the results of other studies (Simpson et al. 2003). Nevertheless, it is

obvious that both Alb groups had less diverse bacterial communities in the

caecum than the Des groups, possibly due to the higher concentration of starch

in the Albatros potatoes and better growth of bacteria fermenting the best this

kind of starch.

The genetic modification of the two parental potato lines has differentially

affected the nutritive value of the transgenic potatoes when fed as 15% N

Janczick et al.22

supplementation to casein. The genetic modification of the Desiree potato led to

a significant improvement of N-balance and total tract apparent crude protein

digestibility, whereas N-balance and the other nutritional characteristics

remained unchanged in the Alb-VP60 potato in comparison with the Alb

isogenic potato. The increased nutritional value of the GM Des-VP60 potato, in

comparison with the Des parental potato, is probably due to changes in

chemical composition (e.g. increased protein, increased crude fiber content,

decreased nitrogen free extract content, changed amino acids composition,

decreased glucose and fructose content). The present paper does not describe

molecular reason of these changes. However the significant differences between

both parental potato lines indicate strong effects due to somaclonal variation.

Differences between parental and corresponding transgenic potato lines, in

terms of their nutritive qualities, could be due to somaclonal variations,

pleiotropic effects (of gene expression changes within the potato or of the

expressed transgenic protein in the rat) or genetic modification. Pleiotropic

effects of the expressed viral protein can be probably excluded since

hematological and biochemical indices in all four potato lines support the

assumption that VP60 lacks any systemic affects in rats.

Acknowledgement

The authors thank Ms. Ingetraud Prignitz, Aenne Koepnick, Sonja Bruesch

and Brigitte Wuerfel for their excellent technical assistance. The study was

supported by the German Federal Ministry of Education and Research (No. of

grant 0312744).

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