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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