Effects of 60-Week Feeding Diet Containing Bt Rice Expressing the

Subscriber access provided by READING UNIV

Article

Effects of 60-week feeding diet containing Bt rice expressing the Cry1Ab protein on the offspring of inbred Wuzhishan pigs fed the same diet Qiang Liu, Weigang Yang, Mingjie Li, Yi Wu, Yingzheng Wang, Shuaishuai Wu, Hui Gao, Ying Han, Feng Yang, Shutang Feng, and Shenming Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04067 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

Journal of Agricultural and Food Chemistry

1

Effects of 60-week feeding diet containing Bt rice expressing the Cry1Ab protein

2

on the offspring of inbred Wuzhishan pigs fed the same diet

3

Qiang Liu †, Weigang Yang †, Mingjie Li †, Yi Wu , Yingzheng Wang , Shuaishuai Wu , Hui Gao ,

a

a

a

a

a

a

a

c

a

a

a, b*

Ying Han , Feng Yang , Shutang Feng and Shenming Zeng

4 5 6

a

Laboratory of Animal Embryonic Biotechnology; National Engineering Laboratory for Animal

7

Breeding; Key Laboratory of Animal Genetics, Breeding, and Reproduction of the Ministry of

8

Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing,

9

China;

10 11

b

College of Animal Science and Technology, Yangzhou University, Yangzhou, China;

c

Institute of Animal Sciences, China Academy of Agricultural Sciences, Beijing, China.

12 13

*

14

186 1828 5928/010-62733744, China agricultural university. No. 2 Yuanmingyuan West Road,

15

Haidian District, Beijing, China; College of Animal Science and Technology, Yangzhou University,

16

No. 48 Wen hui East Road, Yangzhou, Jiangsu 225009, People's Republic of China (E-mail:

17

[email protected]).

To whom correspondence should be addressed. Shenming Zeng, Ph.D., Tel/Fax numbers: +86

18 19 20

†

The first three authors contribute equally to this work.

1

ACS Paragon Plus Environment


21

ABSTRACT

22

We evaluated the chronic effects of Bt rice carrying the Cry1Ab protein

23

(1.64mg/kg) on offspring of highly inbred WZSP, fed with Bt rice for 360 days, in a

24

60-week feeding study. The WZSP offspring (n=27) were assigned to two groups

25

(Minghui86 group, female, n=6; male, n=5; Bt group, female, n=11; male, n=5). The

26

average obtained Cry1Ab protein dosage for female and male pigs was 1.003 and

27

1.234 mg/kg body weight after 10 week feeding, respectively. The experimental feed

28

in the study was nutritionally matched in both groups. The average daily gain and

29

feed conversion ratio of the females in week 3 and males from weeks 1 to 10 were

30

different between the Bt and Minghui86 groups (P < 0.05), and the body weight of the

31

male pigs in week 2 was greater in the Minghui86 group than the Bt group (P < 0.05).

32

No other differences were observed, and there were no significant differences in the

33

serum sex steroid level, haematology parameters, relative organ weights or

34

histopathology. Although differences in some serum chemistry parameters (alanine

35

aminotransferase of female pigs and alkaline phosphatase of male pigs) were

36

observed, they were not considered treatment-related. Based on these results,

37

long-term intake of transgenic rice carrying Cry1Ab protein exerts no unintended

38

adverse effects on WZSP offspring.

39 40

Keywords: Bt rice, Cry1Ab protein, offspring, WZSP, food safety

2


Page 2 of 41

Page 3 of 41


41

INTRODUCTION

42

In crop production, conventional and modern breeding methods have long been

43

used by crossing one parental line with another to express a specific property (e.g.,

44

disease and insect resistance) 1. However, it takes a long time before the desired trait

45

is expressed. At present, the rate of increase in crop-yield (less than 1.7%) cannot

46

meet population growth demands 2. To control insect pests that are a significant

47

problem to crop production 3, broad spectrum chemical insecticides are used.

48

However, this approach repeatedly results in environmental contamination, ecosystem

49

deterioration, pest population outbreaks and pesticide resistance

50

biology, transgenic technology can provide crop lines containing desired traits more

51

rapidly and efficiently than traditional breeding methods.

4, 5

. Using molecular

52

Rice is cultivated and consumed worldwide as an important staple food.

53

According to statistics, global rice consumption in 2017/18 is projected at 480.5

54

million tons, and China’s rice consumption in 2017/18 is projected at 142.35 million

55

tons (data from USDA: Rice Outlook). Therefore, the application of transgenic

56

technology in rice production is also an inevitable trend. One of the most widely

57

grown transgenic rice varieties are those with an insertion of the cry1Ab gene from

58

Bacillus thuringiensis (Bt) whose spore and crystalline protein (Cry1Ab/Ac) have

59

been genetically engineered into many crops, such as maize, cotton and rice, to

60

control insect pests for almost 40 years

61

can only be found in the intestines of Lepidoptera insects. Therefore, mammals are

62

not theoretically affected by Bt 8, 9. However, there are still public concerns about the

6, 7

. Specific receptors for the crystal protein

3



Page 4 of 41

63

safety of Bt rice, primarily due to the three major health risks: toxicity, allergenicity

64

and genetic hazards

65

and food safety assessments for any novel GE plant before it is approved for

66

commercial cultivation 12.

. Therefore, it is necessary to undergo strict environmental

During last decade, although feeding studies in animals, such as rats 13-16, pigs 17,

67 68

10, 11

18

, cows 19, and broilers 20, have shown that the GM crops is no toxicity, there is still

69

no way to rule out doubts from public about the safety of GMs as a long-term food.

70

Because a few studies have shown that GM crops are toxic to animals. Séralini (2007)

71

re-analyzed the data of a 90-day rat-feeding with a transgenic corn MON863 and

72

showed that chemistry measurements revealed signs of hepatorenal toxicity

73

years later, De Vendômois (2009) suggested that three main commercialized

74

genetically modified (GM) maize (NK 603, MON 810, and MON 863) induced a state

75

of hepatorenal toxicity after 5 and 14 weeks of feeding on rats 22. A summary about

76

the impact of feeding Bt maize to pigs on health parameters was modified and showed

77

in Table 1

78

pigs is increasing, as the feeding period (from 4 to 28 weeks) is extended. In addition,

79

these studies were focused on assessing Bt maize, not Bt rice. The safety assessment

80

of Bt rice is mainly carried out in rats, not pigs. It can be seen that whether GM crops

81

are toxic or not is not conclusive. Apparently, it is insufficient to evaluate chronic

82

toxicity through short-term tests, and long-term or multigenerational studies ought to

83

be conducted.

84

21

. Two

23

. It shows that the adverse impact of Bt maize on health parameters of

On the other hand, those studies only revealed the effects of the GM rice on the 4


Page 5 of 41


85

first generation. Whether it is potentially toxic to the next generation of humans and

86

animals remains unknown. In general, if GM food is toxic to human or animal

87

reproduction after long-term consumption, it would likely appear in the next

88

generation. In this 60-week feeding experiment with a nutritionally matched

89

experimental feed, we used the offspring of the highly inbred line of WZSP, fed with

90

Bt rice for 360 days, to assess the safety of Bt rice containing the Cry1Ab protein on

91

growth performance, haematology and organ morphology.

92 93

MATERIALS AND METHODS

94

Rice and animals

95

The insect-resistant Bt rice line transformed by Fujian Academy of Agricultural

96

Sciences, mfb-Minghui86 [Environ Entomol. 2014 Apr, 43(2):528-36], and its

97

non-transgenic parental line, mfb-Minghui86, were cultivated simultaneously

98

side-by-side in an experimental field (Fuzhou, China). The Bt rice in the experiment

99

was not a commercial product and just for scientific research.

100

All pigs used in this experiment were the second generation of the high inbred

101

line of WZSP fed with Bt rice containing the Cry1Ab protein for 360 days and

102

cultivated by the Institute of Animal Sciences, China Academy of Agricultural

103

Sciences (Beijing, China). In this experiment, 17 females (Bt group, n=11; Minghui86

104

group, n=6), and 10 males (Bt group, n=5; Minghui86 group, n=5) with similar health

105

conditions were used. The average age of the second generation pigs used in the

106

experiment was about 3 months. The average initial weights of the males and females 5



Page 6 of 41

107

from the Bt and Minghui86 groups were 4.50 kg, 4.63 kg, 5.66 kg, and 4.96 kg,

108

respectively.

109

Experimental design

110

All pigs were housed in individual pens (1.2 m×1.2 m×1.0 m) with fully slatted

111

concrete floors at the Experimental Base of China Agriculture University (Hebei,

112

China) where the temperature ranged from 22°C-28°C, and relative humidity ranged

113

from 40 %-60 %. The pigs were confined to the same cage during the experiment.

114

Male pigs were housed together with a female pig from the same group for mating. A

115

floor heating system was used for maintaining the temperature in the winter. The pigs

116

in the Bt and Minghui86 groups were fed ad libitum with a diet containing Cry1Ab

117

protein rice and isogenic rice three times per day, respectively. An automatic drinking

118

system was used for water. A two-week acclimation period to the diets was used after

119

weaning, and the day that the new diets began marked the beginning of week 0. The

120

pigs were fed from October 30, 2014 to December 25, 2015 and observed closely at

121

least three times daily. All procedures involving experimental animals conformed to

122

the Guide for the Care and Use of Laboratory Animals (NIH publication No.86-23,

123

revised 1996) and were approved by the Animal Welfare Committee of China

124

Agricultural University.

125

Detection of the Cry1Ab protein and compositional analysis of the rice

126

During the experiment, ten random samples (five for each group) were analysed

127

for the Cry1Ab protein concentration with an antibody specific enzyme linked

128

immunosorbent assay kit (QualiPlate™ Kit; Envirologix, Portland, ME, USA) 6


24, 25

.

Page 7 of 41


129

Analysis of the nutrients (moisture, ash, crude fat, crude protein, NDF and starch) was

130

determined by Chinese standard methods (GB6435-86, GB/T6432-94, and

131

GB/T6433-94). Calcium and phosphorus were determined with EDTA titrimetric and

132

vanadium molybdenum acid colorimetric methods, respectively. Amino acids (except

133

tryptophan) were analysed with an automatic amino acid analyser (L-8800, Hitachi

134

Group, Tokyo, Japan), and tryptophan was measured by high performance liquid

135

chromatography (Agilent 1200, Agilent Technologies, Santa Clara, California) after

136

the rice was crushed with a mortar and pestle and hydrolysed for 22 h in 4 mol/L

137

sodium hydroxide solution at a temperature of 110oC. The comparison of the rice

138

compositions between groups is presented in Table 2. The content of the Cry1Ab

139

protein in the transgenic rice (Bt group) was 1.64 mg/kg, while no Cry1Ab protein

140

was detected in the control rice (Minghui86 group).

141

Diets and nutrient composition analysis

142

The diet formula was per the NRC standard 2012 (Nutrient Requirements of

143

Swine: Eleventh Revised Edition: NRC 2012 Models for Estimating Nutrient

144

Requirements of Pigs Case Studies). From the compositional analysis, flours of the Bt

145

and Minghui86 rice were formulated into diets at a concentration of 56-66 % by mass

146

based on the nutrient requirements at different growth stages. All diet ingredients

147

were identical except the rice. The diets were produced in the feed production

148

workshop of the National Feed Industry Centre (Beijing, China) and packed into

149

woven bags clearly labelled Bt or Minghui86. The composition lists and nutrient

150

compositions of the two diet formulations at five stages (piglet, fattening period, 7



151

earlier stage of gestation, latter period of gestation, and lactation) for the pigs are

152

summarized in Table 3. The diet formulation of the females in the fattening period

153

was the same as the males. The weaning time of the piglets was 42 days. Gestation

154

was divided into two periods, 1-80 days and 81-115 days. The total energy in the diet

155

formulation at the different stages met the nutrient requirements as stated in the 2012

156

Swine NRC standard, and the nutrient composition of the diets is shown in Table 3.

157

Effects of Bt rice on behaviour, average daily gain, feed intake, body weight and

158

feed conversion ratio

159

Throughout the study, signs of morbidity and other parameters that measure the

160

incidence of toxicity were observed and recorded daily. WZSP is a small breed with

161

early sexual maturity and tolerance to inbreeding

162

the WZSPs at an average age of 6 months were in oestrus and began their natural

163

mating behaviour. Therefore, the average daily gain (ADG), average daily feed intake

164

(ADFI), and feed conversion ratio (FCR), which measures an animal's efficiency in

165

converting feed mass to desired output and body weight (BW), were measured from

166

week 0 until week 10. The results are shown in Table 4 and Figure 1, respectively.

167

Birth/weaning weight, slaughter weight and serum sex steroid level

26

. At week 10 of the experiment,

168

The birth/weaning weight of the pigs was recorded after the natural production

169

and weaning date (42 days after birth). The slaughter weight was recorded on the last

170

day of the experiment. The results are shown in Figure 2.

171

Blood samples from the females for oestradiol analysis were collected in both

172

dioestrus and oestrus. Blood samples from the males for the testosterone test were 8


Page 8 of 41

Page 9 of 41


173

collected after slaughter on the last day of the study (December 25, 2015). The Bt

174

group pigs fed the experimental diet were approximately 3 to 7 months pre-pregnancy,

175

due to the different mating and pregnancy times. The gestation period was 114 to 116

176

days. The duration of the pigs fed with Bt rice after giving birth was approximately 42

177

days. The Bt group pigs were fed with Bt rice for the entire 60 weeks. The

178

concentration of testosterone and oestradiol in the serum was tested by radio

179

immunoassay (RIA) at the Northern Biotechnology Research Institute (Beijing,

180

China). The results are presented in Figure 1A and 1B, respectively.

181

Blood biochemistry and haematology analysis

182

The blood samples for the biochemical and haematological analyses were

183

collected from 27 pigs (Minghui86 group=10, Bt group=17) after slaughter into

184

non-anticoagulant and anticoagulant tubes, at volumes of 5 mL and 1 mL, respectively.

185

The blood samples for the haematology analysis were shaken after collection to

186

prevent agglutination, while the blood samples for the biochemistry analysis were

187

placed on a slant for at least ten minutes. Serum was collected after centrifugation at

188

room temperature at 3000 g for 15 minutes. The blood biochemical parameters were

189

analysed by the HITACHI4600 auto-analyser (Hitachi Group, Tokyo, Japan).

190

Haematological analysis was performed using an ABX pentra 80 (HORIBA ABX,

191

France) cell counter. The parameters are shown in Table 5 and Table 6, respectively.

192

Organ/body Weight Ratios and histopathology of the main tissues

193

To prevent contamination, the organs including heart, lung, liver, spleen, kidney,

194

testes and uterus were trimmed and weighed (paired organs were weighed together) 9



195

immediately after slaughter. The organ/body weight ratios were calculated and are

196

shown in Table 7. The tissues were placed in 4 % formalin for 24 h, embedded in

197

paraffin, sectioned at 5 µm and stained in haematoxylin and eosin for microscopic

198

observation.

199

Statistical analysis

200

All data are presented as the mean ± SD and were analysed using an independent

201

samples t-test and a one-way ANOVA with SPSS 17.0 for Windows. The ANOVA

202

followed by a post hoc Dunnett’s test was used to determine significant differences

203

between the Bt and Minghui86 groups. The differences were considered statistically

204

significant at P < 0.05. Data from the males and females were analysed separately.

205 206

RESULTS

207

Compositional analysis

208

The compositional analyses of the Bt and isogenic rice are shown in Table 2.

209

Most Bt rice compositions were similar to the Minghui86 rice except copper and iron.

210

The diet formulas and nutrient composition for the Bt and Minghui86 groups at

211

different stage are shown in Table 3.

212

Effects of feeding Bt rice diets to WZSP on growth performance

213

Throughout the feeding trial, the pigs were examined clinically every day. There

214

were no behavioural differences (morbidity, oestrous features, walking manners,

215

ingestion behaviour, mating mode), between the Bt and Minghui86 groups during the

216

experimental feeding period (from October 30, 2014 to December 25, 2015). The 10


Page 10 of 41

Page 11 of 41


217

ADG and FCR of the female pigs in week 3 and the male pigs in weeks 1 through 10

218

were different between the Bt and Minghui86 groups (P < 0.05), but no other

219

differences were observed (Table 4). There was no difference in the ADFI between the

220

Bt and Minghui86 groups from weeks 1 to 10 (Table 3). Except for the male pigs’ BW

221

in week 2 being greater in the Minghui86 group than in the Bt group (P < 0.05), no

222

differences were observed (Figure 1A, B). The daily intake of Cry1Ab protein for the

223

males and females from the Bt group were 1.234 and 1.003 mg/kg BW, respectively.

224

The birth/weaning and slaughter weight of WZSP from the Bt and Minghui86

225

groups.

226

There were no significant differences in the birth/weaning and slaughter weights

227

among males and females of the Bt and Minghui86 groups (Figure 2A, B, C, P >

228

0.05).

229

Effects of feeding Bt rice diets to WZSP on the serum sex steroid levels

230

The testosterone concentration in the Minghui86 group male pigs (6.43 ± 2.21

231

ng/mL) was higher than that of the Bt group (4.61 ± 1.24 ng/mL), but there was no

232

significant difference (Figure 3A, P > 0.05). Similarly, the serum oestradiol

233

concentrations in the dioestrus and oestrus stages were not significantly different

234

between the Bt (8.69 ± 4.03 pg/mL, 28.53 ± 10.24 pg/mL) and Minghui86 groups

235

(10.42 ± 2.55 pg/mL, 31.88 ± 9.89 pg/mL) (Figure 3B, P > 0.05).

236

Effects of the Bt rice diets on WZSP haematological and serum chemistry

237

parameters

238

The haematological results (Table 5) demonstrated that no blood parameters 11



Page 12 of 41

239

significantly differed between the Bt and Minghui86 groups for either the male or

240

female pigs. The serum biochemical values are shown in Table 6. The ALP

241

concentration in the males was significantly higher in the Bt group (112.33 ± 16.62

242

U/L) than in the Minghui86 group (85.80 ± 9.44 U/L) (P < 0.05), but not in the

243

females. The ALT levels in the Minghui86 group blood samples (81.26 ± 23.29 U/L)

244

were higher than in the females of the Bt group (58.51 ± 9.69 U/L) (P < 0.05), but

245

there was no difference in those of the males (P > 0.05). There were no significant

246

differences in the other parameters.

247

Effects of feeding Bt rice diets to WZSP on organ/body weight ratios and

248

histopathology

249

No abnormal organs were observed in the Bt group pigs after slaughter. There

250

were no differences in the organ/body weight ratios between the Bt and Minghui86

251

groups (Table 7). Furthermore, no histopathological abnormal changes were found in

252

the liver, spleen, kidney, testis (male) and ovary (female) between the Bt and

253

Minghui86 groups (Figure 4).

254 255

DISCUSSION

256

The Wuzhishan miniature pig is an ideal animal model for safety assessments of

257

Bt rice expressing Cry1Ab because of its high inbreeding coefficient (0.968), stable

258

heredity and little variability between individual animals. It is also similar to humans

259

in haematology, organ histology, physiology, immunology and genetic level

260

this study, Wuzhishan miniature pig offspring, which have been inbred for more than 12


27-30

. In

Page 13 of 41


261

seventeen generations, were used to assess the risk of Bt rice as the main ingredient in

262

pig diets. To our knowledge, this is the first study that assesses the effects of parental

263

dietary exposure to Bt rice on the growth performance, serum biochemistry,

264

haematological parameters, organ weight, and histopathology of the main organs in

265

the Wuzhishan miniature pig offspring model at different growth stages over a

266

long-term (60-week) feeding period.

267

Although there was little difference in the ADG and FCR between the Bt and

268

Minghui86 groups, they were statistically, but not biologically, different. Moreover,

269

the differences did not occur in both sexes simultaneously, and they were only

270

distributed at the beginning of the experimental period. The ADG, ADFI and BW

271

measurements from weeks 0 to 10 of the study found no obvious differences in the

272

growth performance between the Bt and Minghui86 groups. This agrees with the

273

observation of a long-term (196 days) toxicity study on Bt corn (1.32 mg/kg BW) in a

274

WZSP model, which showed that the cry1Ac gene had no effect on the growth,

275

immune response or health indicators at any growth stage

276

birth/weaning and slaughter weights also showed that the Bt rice did not influence

277

growth performance. Similar to our findings, Walsh et al. reported that there were no

278

differences in feed consumption, average daily gain and feed conversion efficiency

279

between pigs fed with Bt MON810 maize and the control diet 18. Yuan et al. (2013)

280

measured parameters including microfloral composition, intestinal permeability,

281

epithelial structure and intestinal immunity and found no adverse effects on

282

gastrointestinal health from GM T2A-1 rice

32

31

. In addition, the

. Buzoianu et al. (2013) reported that

13



283

feeding Bt MON810 maize to sows during gestation and lactation and to their

284

offspring from weaning to 115 days post-weaning did not affect pig growth or health

285

33

. Brake et al. (2003) found no significant differences in survival rate, body weight,

286

feed efficiency, and carcass yield in a long-term chicken feeding study with BT11

287

maize and its non-GM control maize

288

(over 25 months) study with 36 cows fed with maize containing the Cry1Ab protein

289

or its isogenic counterpart, and no diet-related adverse effects were determined 19. Zhu

290

et al. (2015) assessed the effect of feeding GM rice expressing the Cry1Ab/Ac protein

291

for 90 days on the Xenopus laevis, and concluded that frog development was not

292

adversely affected by GM rice intake 35. This was similar to results reported by Chen

293

et al

294

unaffected by the Bt rice.

34

. Steinke et al. (2010) conducted a long-term

36

. Birth/weaning, slaughter weights and growth performance were also

295

Testosterone and oestradiol concentrations are the most important indicators of

296

reproductive performance. In this study, there were no significant differences in the

297

serum hormone, T and E2, levels between the Bt and Minghui86 groups. Similarly, a

298

90-day feeding study of transgenic maize, BT799, documented no differences in

299

serum sex hormone levels

300

adverse reproductive effects in animals fed MingHui63 or the control rice 38.

37

. Wang et al. (2013) also reported that TT51 caused no

301

This study demonstrated that no blood parameters differed between the Bt and

302

Minghui86 groups in either male or female pigs. Likewise, a 90-day safety study of

303

genetically modified rice expressing the Cry1Ab protein in Wistar rats conducted by

304

Schroder et al. found no adverse effects on animal behaviour, weight gain or standard 14


Page 14 of 41

Page 15 of 41


39

305

haematological and biochemical parameters

. Liang Chen et al. also reported that

306

haematological parameters did not differ in Wuzhishan miniature pigs that were fed

307

Bt corn expressing the Cry1Ac protein

308

biochemical values between the Bt and Minghui86 groups were noted. Increased liver

309

size and enzyme levels are indictors of liver dysfunction 40, 41. We found that the liver

310

enzyme, ALT, was lower in females fed the Bt rice than the isogenic rice. A long-term

311

(196 days) toxicity study on Bt corn (1.32 mg/kg BW) in a WZSP model found the

312

same results for males 31. In addition, these differences were not simultaneous in both

313

sexes and were within the normal reference intervals for Wuzhishan pigs 42. The liver

314

enzyme, AST, was lower in females fed Bt rice than in the control group, paralleling

315

the study of Buzoianu et al.

316

Minghui86 group in male pigs, but not females. This was not considered

317

treatment-related, as the ALP values for all groups were beyond the normal reference

318

intervals. In addition, a 10~70 fold increase in AST and a 5~10 fold concomitant

319

increase of ALP and GGT can be toxic to the liver

320

CREA is low compared to literature values. Plasma creatinine (CREA) concentration

321

is often used to assess the function of renal in both acute and chronic conditions, but

322

the CREA depends on the age and sex of the animals 45. So these differences were not

323

diet-related.

31

. Sporadic significant differences in serum

43

. ALP values were higher in the Bt group than the

41, 44

. Our results showed that the

324

Other parameters reflecting renal function such as urea, urea nitrogen, and uric

325

acid were similar between the Bt and Minghui86 groups. The liver and kidneys are

326

important in immune and toxin responses, and their changes suggest altered metabolic 15



327

processes. Our study revealed no histopathological abnormalities in the kidney or

328

liver between the Bt and MH86 groups. Therefore, any significant differences may be

329

associated with individual differences.

330

In this 60-week study, we reported that different diets did not affect the relative

331

weights of the heart, liver, spleen, lung, kidney, testes and uterus. Similarly, no

332

changes in organ weight were found in rats fed GM rice containing the Cry1Ab

333

protein for 90 days 39 or in Wuzhishan pigs fed Bt corn containing the Cry1Ac protein

334

long-term (196 days) 31. In addition, there were no histopathological abnormalities in

335

the main tissues of the Bt and Minghui86 groups.

336

In summary, no adverse effects were observed in the present long-term (60-week)

337

feeding study, indicating the safety of feeding Bt rice containing the Cry1Ab protein

338

to offspring of WZSP that were fed Bt rice for 360 days. The results may provide a

339

scientific basis for evaluating the biosafety of GM rice containing Cry1Ab protein

340

after a long-term feeding.

341 342

AUTHOR CONTRIBUTIONS

343

Qiang Liu and Weigang Yang: Performed experiments, including fed and weighed the

344

animals; Shuaishuai Wu and Mingjie Li: analyzed nutrients of Bt and Minghui86 rice

345

and the diets for both groups; Yi Wu: responsible for observation of behaviour;

346

Yingzheng Wang: slaughter of the pigs; Hui Gao and Ying Han: determination of

347

indicators; Feng Yang: send samples; Shutang Feng: the pathologist, mainly

348

responsible for autopsy and pathological section analysis; Shenming Zeng: Designed 16


Page 16 of 41

Page 17 of 41


349

the study, supervised the project, wrote the paper.

350 351

ACKNOWLEDGMENTS

352

We express our gratitude to Professor Zhen Zhu from the Science Academy of

353

China, Professor Feng Wang from the Fujian Academy of Agricultural Sciences and

354

Professor Xue Wang from the National Centre for Drug Safety Evaluation for the

355

supply of transgenic rice and its near-isogenic control line. We also thank our

356

colleagues from Professor Defa Li’s team for their helpful advice on pig nutrition and

357

feeding.

358 359

ABBREVIATIONS:

360

WZSP, Wuzhishan pigs; Bt, Bacillus thuringiensis; GM, genetically modified; GE,

361

genetic engineering; RIA, radio immunoassay; FCR, feed conversion ratio; ADG,

362

average daily gain; ADFI, average daily feed intake; BW, body weight; SL,

363

subendothelial layer; Mc, myocardium; SM, smooth muscle; P, plica; AS, alveolar sac;

364

PA, pulmonary artery; HL, hepatic lobule; PA, portal area; CA, central artery; SN,

365

splenic nodule; PLS, periarterial lymphatic sheath; RP, red pulp; WP, white pulp; RC,

366

renal corpuscle; C, cortex; M, medulla; VP, vascular pole; UP, urinary pole; G,

367

glomus; PL, parietal layer; CS, capsular space; ST, seminiferous tubule; SC,

368

sustentacular cells; Sd, spermatid; Sz, spermatozoon; L, Leydigs cells of the testis; FC,

369

follicular cell; ZP, zona pellucida; WBC, white blood corpuscle; RBC, red blood

370

count; PLT, blood platelet count; HGB, haemoglobin; HCT, haematocrit; MPV, mean 17



371

platelet volume; MCV, erythrocyte mean corpuscular volume; MCH, mean

372

corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration;

373

RDW, red blood cell distribution width; TP, total protein; ALB, albumin; GLU,

374

Glucose; CREA, creatinine; CHOL, total cholesterol; TG, triglyceride; TB, total

375

bilirubin; ALT, the enzyme activities of alanine aminotransferase; AST, aspartate

376

aminotransferase; ALP, alkaline phosphatase; GGYP, gamma glutamyl transpeptidase

377

(GGTP); LDH, lactic dehydrogenase.

378 379

FUNDING SOURCES This research was funded by National Transgenic Major Projects (project no.

380 381

2012ZX08011001-006).

382 383

REFERENCES

384

1.

385

111, 493.

386

2.

387

to double global crop production by 2050. Plos One 2013, 8, e66428.

388

3.

389

review of entomology 2000, 45, 549-574.

390

4.

391

dynamics of insecticide resistance in field populations of Chilo suppressalis

392

(Lepidoptera: Crambidae) during 2002 – 2011 in China. Journal of economic

Oliver, M. J., Why we need GMO crops in agriculture. Missouri medicine 2014,

Ray, D. K.; Mueller, N. D.; West, P. C.; Foley, J. A., Yield trends are insufficient

Matteson, P. C., Insect pest management in tropical Asian irrigated rice. Annual

He, Y.; Zhang, J.; Gao, C.; Su, J.; Chen, J.; Shen, J., Regression analysis of

18


Page 18 of 41

Page 19 of 41


393

entomology 2012, 106, 1832-1837.

394

5.

395

Chinese rice varieties. Chinese Journal of Rice Science 2003, 17, 47-52.

396

6.

397

biotechnology 2003, 21, 1003-1009.

398

7.

399

Service of the Acquisition of Agri-biotech Applications (ISAAA). In Brief 2011.

400

8.

401

feed: the role of animal feeding trials. Food and chemical toxicology: an international

402

journal published for the British Industrial Biological Research Association 2008, 46,

403

S2.

404

9.

405

Zeigler, D.; Dean, D., Bacillus thuringiensis and its pesticidal crystal proteins.

406

Microbiology and molecular biology reviews 1998, 62, 775-806.

407

10. Zhang, C.; Wohlhueter, R.; Zhang, H., Genetically modified foods: A critical

408

review of their promise and problems. Food Science and Human Wellness 2016, 5,

409

116-123.

410

11. Seek Rhee, G.; Hyun Cho, D.; Hyuck Won, Y.; Hyun Seok, J.; Sun Kim, S.; Jun

411

Kwack, S.; Da Lee, R.; Yeong Chae, S.; Woo Kim, J.; Mu Lee, B., Multigeneration

412

reproductive and developmental toxicity study of bar gene inserted into genetically

413

modified potato on rats. Journal of Toxicology and Environmental Health, Part A

414

2005, 68, 2263-2276.

Sogawa, K.; Ge, Z.; Yang, X.; Liu, G., Whitebacked planthopper resistance in

Mendelsohn, M.; Kough, J.; Vaituzis, Z.; Matthews, K., Are Bt crops safe? Nature

James, C., Global status of commercialized biotech/GM crops. The International

EFSA, G., Safety and nutritional assessment of GM plants and derived food and

Schnepf, E.; Crickmore, N. V.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.;

19



415

12. Romeis, J.; Bartsch, D.; Bigler, F.; Candolfi, M. P.; Gielkens, M. M.; Hartley, S.

416

E.; Hellmich, R. L.; Huesing, J. E.; Jepson, P. C.; Layton, R., Assessment of risk of

417

insect-resistant transgenic crops to nontarget arthropods. Nature biotechnology 2008,

418

26, 203-208.

419

13. Cao, S.; He, X.; Xu, W.; Luo, Y.; Yuan, Y.; Liu, P.; Cao, B.; Shi, H.; Huang, K.,

420

Safety assessment of transgenic Bacillus thuringiensis rice T1c‐19 in Sprague–

421

Dawley rats from metabonomics and bacterial profile perspectives. Iubmb Life 2012,

422

64, 242-250.

423

14. Kroghsbo, S.; Madsen, C.; Poulsen, M.; Schrøder, M.; Kvist, P. H.; Taylor, M.;

424

Gatehouse, A.; Shu, Q.; Knudsen, I., Immunotoxicological studies of genetically

425

modified rice expressing PHA-E lectin or Bt toxin in Wistar rats. Toxicology 2008,

426

245, 24-34.

427

15. Tang, M.; Xie, T.; Cheng, W.; Qian, L.; Yang, S.; Yang, D.; Cui, W.; Li, K., A

428

90-day safety study of genetically modified rice expressing rhIGF-1 protein in

429

C57BL/6J rats. Transgenic Research 2012, 21, 499-510.

430

16. Wang, E. H.; Yu, Z.; Hu, J.; Jia, X. D.; Xu, H. B., A two-generation reproduction

431

study with transgenic Bt rice TT51 in Wistar rats. Food and chemical toxicology 2014,

432

65, 312-320.

433

17. Cao, Z.; Wang, Z.; Gu, X., Residues and organ damage of exogenous gene and

434

protein from transgenic Bt brown rice in growing pigs. Chinese Journal of Animal

435

Nutrition 2014, 26, 1908-1915.

436

18. Walsh, M. C.; Buzoianu, S. G.; Gardiner, G. E.; Rea, M. C.; Ross, R. P.; Cassidy, 20


Page 20 of 41

Page 21 of 41


437

J. P.; Lawlor, P. G., Effects of short-term feeding of Bt MON810 maize on growth

438

performance, organ morphology and function in pigs. British Journal of Nutrition

439

2012, 107, 364-371.

440

19. Steinke, K.; Guertler, P.; Paul, V.; Wiedemann, S.; Ettle, T.; Albrecht, C.; Meyer,

441

H.; Spiekers, H.; Schwarz, F., Effects of long‐term feeding of genetically modified

442

corn (event MON810) on the performance of lactating dairy cows. Journal of Animal

443

Physiology and Animal Nutrition 2010, 94, e185-e193.

444

20. Qin, H., Safety assessment of rice genetically modified with Cry1Ac and sck by

445

feeding studies on broilers. Dissertation, Chinese Academy of Agriculture Science,

446

2012.

447

21. Séralini, G. E.; Cellier, D.; De Vendômois, J. S., New analysis of a rat feeding

448

study with a genetically modified maize reveals signs of hepatorenal toxicity.

449

Archives of environmental contamination and toxicology 2007, 52, 596-602.

450

22. De Vendômois, J. S.; Roullier, F.; Cellier, D.; Séralini, G. E., A comparison of the

451

effects of three GM corn varieties on mammalian health. International Journal of

452

Biological Sciences 2009, 5, 706-726.

453

23. Clazien, J. V.; Manon, S., Health effects of feeding genetically modified (GM)

454

crops to livestock animals: A review. Food and chemical toxicology 2017, 1-10.

455

24. Jin, L.; Wei, Y.; Zhang, L.; Yang, Y.; Tabashnik, B. E.; Wu, Y., Dominant

456

resistance to Bt cotton and minor cross‐resistance to Bt toxin Cry2Ab in cotton

457

bollworm from China. Evolutionary Applications 2013, 6, 1222-1235.

458

25. Mao, J.; Sun, X.; Cheng, J.H.; Shi, Y. J.; Wang, X. Z.; Qin, J. J.; Sang, Z. H.; He, 21



459

K.; Xia, Q., A 52-week safety study in cynomolgus macaques for genetically modified

460

rice expressing Cry1Ab/1Ac protein. Food and chemical toxicology 2016, 95, 1-11.

461

26. Feng, S.T., Chinese experimental miniature pig. Beijing: China Agriculture Press

462

2011, 408-418.

463

27. Huang, Q.; Xu, H.; Yu, Z.; Gao, P.; Liu, S., Inbred Chinese Wuzhishan (WZS)

464

minipig model for soybean glycinin and β -conglycinin allergy. Journal of

465

Agricultural and Food Chemistry 2010, 58, 5194-5198.

466

28. Fang, X.; Mou, Y.; Huang, Z.; Li, Y.; Han, L.; Zhang, Y.; Feng, Y.; Chen, Y.; Jiang,

467

X.; Zhao, W., The sequence and analysis of a Chinese pig genome. GigaScience 2012,

468

1, 16.

469

29. Jin, E.; Li, K.; Feng, S.; Mou, Y.; Wang, C.; Peng, K.; Yang, S., Histology of

470

immune organs in different-month-old WZS minipig inbred strain. Chinese Journal of

471

Comparative Medicine 2008, 18, 1-4.

472

30. Yang, S. L.; Ren, H. Y.; Wang, H.; Feng, S.T.; Gan, S.X.; Wang, A. D.; Li, K.,

473

Investigation on the hematology parameters of Chinese laboratory miniature pig

474

breeds. China Animal Husbandry & Veterinary Medicine 2007, 2, 38-41.

475

31. Chen, L.; Sun, Z.; Liu, Q.; Zhong, R.; Tan, S.; Yang, X.; Zhang, H., Long‐term

476

toxicity study on genetically modified corn with cry1Ac gene in a Wuzhishan

477

miniature pig model. Journal of the Science of Food and Agriculture 2016, 96,

478

4207-4214.

479

32. Yuan, Y.; Xu, W.; He, X.; Liu, H.; Cao, S.; Qi, X.; Huang, K.; Luo, Y., Effects of

480

genetically modified T2A-1 rice on the GI health of rats after 90-day supplement. 22


Page 22 of 41

Page 23 of 41


481

Scientific Reports-Uk 2013, 3.

482

33. Buzoianu, S.; Walsh, M.; Rea, M.; Cassidy, J.; Ryan, T.; Ross, R.; Gardiner, G.;

483

Lawlor, P., Transgenerational effects of feeding genetically modified maize to

484

nulliparous sows and offspring on offspring growth and health. Journal of Animal

485

Science 2013, 91, 318-330.

486

34. Brake, J.; Faust, M. A.; Stein, J., Evaluation of transgenic event Bt11 hybrid corn

487

in broiler chickens. Poultry Sci 2003, 82, 551-559.

488

35. Zhu, H. J.; Chen, Y.; Li, Y. H.; Wang, J. M.; Ding, J. T.; Chen, X. P.; Peng, Y. F.,

489

A 90 day safety assessment of genetically modified rice expressing Cry1Ab/1Ac

490

protein using an aquatic animal model. Journal of Agricultural and Food Chemistry

491

2015, 63, 3627-3633.

492

36. Chen, X.; Wang, J.; Zhu, H.; Li, Y.; Ding, J.; Peng, Y., Effects of Transgenic

493

cry1Ca Rice on the Development of Xenopus laevis. Plos One 2015, 10, e0145412.

494

37. Guo, Q.Y.; He, L.X.; Zhu, H.; Shang, J. L.; Zhu, L. Y.; Wang, J. B.; Li, Y., Effects

495

of 90-Day Feeding of Transgenic Maize BT799 on the Reproductive System in Male

496

Wistar Rats. International journal of environmental research and public health 2015,

497

12, 15309-15320.

498

38. Wang, E. H.; Yu, Z.; Hu, J.; Xu, H. B., Effects of 90-day feeding of transgenic Bt

499

rice TT51 on the reproductive system in male rats. Food and chemical toxicology

500

2013, 62, 390-396.

501

39. Schrøder, M.; Poulsen, M.; Wilcks, A.; Kroghsbo, S.; Miller, A.; Frenzel, T.;

502

Danier, J.; Rychlik, M.; Emami, K.; Gatehouse, A., A 90-day safety study of 23



503

genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in

504

Wistar rats. Food and chemical toxicology 2007, 45, 339-349.

505

40. Boone, L.; Meyer, D.; Cusick, P.; Ennulat, D.; Bolliger, A. P.; Everds, N.; Meador,

506

V.; Elliott, G.; Honor, D.; Bounous, D., Selection and interpretation of clinical

507

pathology indicators of hepatic injury in preclinical studies. Veterinary Clinical

508

Pathology 2005, 34, 182-188.

509

41. Kaneko, J. J.; Harvey, J. W.; Bruss, M. L., Clinical biochemistry of domestic

510

animals. Academic press 2008.

511

42. Min, F.; Wang, X.; Yuan, W.; ZHANG, Y.; PAN, J.C.; WANG, J., Determination

512

of some blood physiological and biochemical parameters in Wuzhishan Mini-Pigs of

513

closed colony. Acta Laboratorium Animalis Scientia Sinica 2008, 5, 373-379.

514

43. Buzoianu, S.; Walsh, M.; Rea, M.; Cassidy, J.; Ross, R.; Gardiner, G.; Lawlor, P.,

515

Effect of feeding genetically modified Bt MON810 maize to~ 40-day-old pigs for 110

516

days on growth and health indicators. Animal: an international journal of animal

517

bioscience 2012, 6, 1609.

518

44. Casteel, S.W.; Turk, J.R.; Cowart, R.P.; Rottinghaus, G.E., Chronic toxicity of

519

fumonisin in weanling pigs. Journal of Veterinary Diagnostic Investigation 1993

520

5,413–417.

521

45. George, J. S.; Luc, P. B.; Adrian, S., The use of plasma creatinine concentration

522

for estimating glomerular filtration rate in inants, children, and adolescents. Pediatric

523

Nephrology 1987, 34, 571-591.

524 24


Page 24 of 41

Page 25 of 41


525

Figure captions

526

Figure 1 Mean body weights of male and female WZSP from the Bt and

527

Minghui86 groups.

528

*: indicates significant differences (P < 0.05). The results are presented as the mean ±

529

SD, n=7, for females in the Bt group, n=5, for males in the Bt group, n=5, for both

530

males and females in the Minghui86 group.

531 532

Figure 2 Birth/weaning and slaughter weight of male and female WZSP from the

533

Bt and Minghui86 groups. No significant differences were observed in the above

534

criteria (P > 0.05). The results are presented as the mean ± SD, n=7, for females of the

535

Bt group, n=5, for males of the Bt group, n=5, for both males and females of the

536

Minghui86 group.

537 538

Figure 3 Serum sex steroid levels in the Bt and Minghui86 groups. No significant

539

differences were observed in the above criteria. The results are presented as the mean

540

± SD; n=11, for females of the Bt group, n=5, for males of the Bt group, n=5, for both

541

males and females of the Minghui86 group.

542 543

Figure 4 Histopathological images of tissues from the Bt and Minghui86 group

544

pigs. Heart：a, b, c, d; Lung: e, f, g, h; Liver: i, j, k, l; Spleen: m, n, o, p; Kidney: q, r,

545

s, t; Testis: u, v; Ovary: w, x. The letters indicate: (a, b, c, d) E: endothelium SL:

546

subendothelial layer Mc: myocardium; (e, f, g, h) SM: smooth muscle P: plica AS: 25



547

alveolar sac PA: pulmonary artery (i, j, k, l) HL: hepatic lobule PA: portal area; (m, n,

548

o, p) SN: splenic nodule PLS: periarterial lymphatic sheath RP: red pulp WP: white

549

pulp; (q, r, s, t) RC: renal corpuscle C: cortex M: medulla VP: vascular pole UP:

550

urinary pole G: glomus * podocyte PL: parietal layer CS: capsular space; (u, v) ST:

551

seminiferous tubule SC: sustentacular cell Sd: spermatid Sz: spermatozoon L: leydigs

552

cells of testis; (w, x) FC: follicular cell ZP: zona pellucida. Bar: 100 μm

553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 26


Page 26 of 41

Page 27 of 41


569

Tables

570

Table 1 Impact of feeding Bt maize to pigs on health parameters

GM crop

Experimental

Duration of

animal

experiment

Bt maize

Pigs

28 days

(MON810)

Age 35 d

(4 weeks)

Studied healthy parameters

Results (GM fed_vs_non-GM fed)

Immune response

Higher IL-4 and IL-6 level in splenocytes; less

CD4+ T cells in spleen; more CD4+ T-cells and

less B cells and macrophages in ileum;

Bt maize

Male pigs

31 days

Immune response,

Lower IFNα production from PBMC in GM-fed

(MON810)

Age 35 d

(4.5 weeks)

histopathology, serum

animals

biochemistry, organ weight

Bt maize

Male pigs

110 days

Haematology, immune

Differences observed in leukocyte, lymphocyte,

(MON810)

Age 40 d

(15.5 weeks)

response, histopathology,

monocyte couts, and serum urine biochemistry

serum biochemistry, organ

weight, gastrointestinal

microbiota

Bt maize

Piglets

115 days

Serum biochemistry,

Lower spleen weight, greater duodenal crypt

(MON810)

Age 28 d

(16.5 weeks)

histopathology,

depths, lower villus height/crypt depth ratios,

organ weight,

higher urea concentrations on day 0; lower

gastrointestinal microbiota

creatinine concentrations on day 30; higher AST

concentrations on day 115, higher faecal

Enterobacteriaceae and faecal total anaerobe

counts; higher ileal total anaerobe counts; 27



Page 28 of 41

differences in relative abundance of faecal

microbiota.

Bt maize

Nulliparous

143 days

Haematology

Higher blood monocyte count and percentage;

(MON810)

sows/ offspring

(20.5 weeks)

Immune response

lower granulocyte percentage on day 110 of

serum biochemistry

gestation. Lower leukocyte and granulocyte count

gastrointestinal microbiota

and percentage; Higher lymphocyte percentage in

organ weight (offspring only)

offspring of Bt maize-fed sows. Lower percentage of monocytes, ,CD4+,CD8+

lymphocytes on day 28 of lactation, day 110 of

gestation.

Bt maize (NK603,

Weaned piglets

MON863,

160 days

Serum biochemistry

Higher uterus weight and higher rate of severe

(22.7 weeks)

histopathology

stomach inflammation in stomach

MON810) and RR

organ weight

soybean

Bt corn (Cry1Ac

Wuzhishan

196 days

Growth performance

Higher IFN-γ concentration at day 134; lower

protein)

miniature pig

(28 weeks)

Blood biochemistry

level of ALT at day 196; less lymphocyte counts

haematology

at day 134;lower kidney weight and some

Isolation of peripheral blood

immune response

Age 40 d

lymphocytes and proliferation

assay

571

Note： Modified from 23

572 28


Page 29 of 41


573

Table 2. Nutritional ingredient comparison between the Bt and Minghui86 rice in diet

574

formulation at different stages (as-is basis).

Different

Piglet

Fattening period

stage

Group

Earlier period of

gestation（80 days）

Bt

Minghui86

Bt

Minghui86

Bt

Ingredients

Minghui86

Latter period

Lactation

of gestation (35 days)

Bt

Minghui86

Bt

Minghui86

kg/100kg

Moisture

7.6164

7.7748

7.1548

7.3036

6.4624

6.5968

6.5778

6.7146

6.9240

7.0682

Crude protein

4.2504

4.9104

3.9928

4.6128

3.6064

4.1664

3.6708

4.2408

3.8640

4.4640

Crude Fat

0.3960

0.5280

0.3720

0.4960

0.3360

0.4480

0.3420

0.4560

0.3600

0.4800

NDF

6.0918

7.1082

5.7226

6.6774

5.1688

6.0312

5.2611

6.1389

5.5380

6.4620

Starch

48.9192

48.2658

45.9544

45.3406

41.5072

40.9528

42.2484

41.6841

44.4720

43.8780

Crude Ash

0.2640

0.2640

0.2480

0.2480

0.2240

0.2240

0.2280

0.2280

0.2400

0.2400

Calcium

0.0066

0.0066

0.0062

0.0062

0.0056

0.0056

0.0057

0.0057

0.0060

0.0060

Phosphorus

0.0594

0.0726

0.0558

0.0682

0.0504

0.0616

0.0513

0.0627

0.0540

0.0660

Zinc

1.0560

1.1120

0.9920

1.0540

0.8960

0.9520

0.9120

0.9690

0.9600

0.1020

Manganese

0.5940

0.6138

0.5580

0.5766

0.5040

0.5208

0.5130

0.5301

0.5400

0.5580

Copper

0.1452

0.3168

0.1364

0.2976

0.1232

0.2688

0.1254

0.2736

0.1320

0.2880

Iron

0.6600

0.3036

0.6200

0.2852

0.5600

0.2576

0.5700

0.2622

0.6000

0.2760

575 576 577

29



Page 30 of 41

578

Table 3. Diet formulas and nutrient composition for the Bt and Minghui86 groups at

579

different stage per the NRC（2012）(as-is basis, kg 100 kg-1).

Different stage

Piglet

Fattening period

Early gestation

period（80 days）

Latter period

Lactation

of gestation (35

days)

Group

MH86

Bt

MH86

Bt

Ingredients

MH86

Bt

MH86

Bt

MH86

Bt

%

Rice

66

66

62

62

56

56

57

57

60

60

Soybean meal

22

22

15

15

20

20

20

20

24

24

Wheat bran

5

5

17

17

20

20

18

18

9

9

Fish Flour

2

2

2

2

\

\

\

\

2

2

Soybean oil

\

\

\

\

\

\

1

1

1

1

Granulated sugar

1

1

\

\

\

\

\

\

\

\

Vitamin-mineral premix*

4

4

4

4

4

4

4

4

4

4

DE（Kcal/kg）1

3544

3542

3513

3511

3387

3385

3459

3457

3589

3587

CP2

21.75

21.43

20.89

20.60

20.45

20.23

22.32

22.25

23.25

23.18

Ca3

0.94

0.93

0.86

0.83

0.79

0.75

0.85

0.82

0.93

0.92

P4

0.95

0.92

0.63

0.63

0.56

0.55

0.78

0.74

0.83

0.80

STTD P5

0.27

0.27

0.35

0.34

0.31

0.31

0.29

0.28

0.37

0.35

Lys

1.90

1.84

1.65

1.57

1.63

1.61

1.76

1.75

1.83

1.81

Nutrient composition/%

30


Page 31 of 41


SID Lys6

0.98

0.95

0.78

0.73

0.82

0.80

0.89

0.82

0.92

0.91

Met

0.66

0.64

0.58

0.53

0.64

0.62

0.60

0.56

0.70

0.61

dig Met7

0.40

0.40

0.45

0.45

0.51

0.51

0.49

0.43

0.53

0.49

Thr

1.15

1.13

1.05

1.02

1.10

1.08

1.18

1.15

1.23

1.19

dig Thr8

0.63

0.61

0.64

0.63

0.59

0.52

0.66

0.65

0.70

0.62

Trp

0.35

0.35

0.28

0.25

0.33

0.32

0.43

0.36

0.49

0.40

dig Trp9

0.26

0.26

0.27

0.24

0.28

0.25

0.26

0.26

0.33

0.32

580

* In mg/kg diet: Vit.A: 10,000(IU); Vit.D3: 2,000(IU); Vit.E:24(IU); Vit.K: 2; Vit.B2: 6; Vit.B6: 4;

581

Vit.B12:0.024; Panthothenate: 20; Niacin: 30; Biotin: 0.4: Folic acid: 3.6; Zn: 120; Fe: 96; Mn: 40;

582

Cu: 8; I: 0.56; Se: 0.24; Acetylcholine: 2.0. 1DE: Digestible energy (kcal kg−1); 2CP: Crude

583

protein; 3Ca: Calcium; 4P: Phosphorus; 5STTD P: Standardized total tract digestive phosphorus;

584

6

585

threonine; 9dig Trp: Digestive tryptophan.

SID lys: Standard ileum digestive lysine; 7dig Met: Digestive methionine; 8dig Thr: Digestive

586 587 588 589 590 591 592 593 594 31



Page 32 of 41

595

Table 4. Average daily gain, average daily feed intake and feed conversion ratio

596

between the Bt and Minghui86 groups. Females

Items

Males

Bt

Minghui86

P

Bt

Minghui86

P

week 1

0.19 ± 0.04

0.23 ± 0.07

0.36

0.21 ± 0.05

0.27 ± 0.08

0.08

week 2

0.19 ± 0.10

0.24 ± 0.08

0.39

0.22 ± 0.07

0.29 ± 0.10

0.21

week 3

0.24 ± 0.11

0.38 ± 0.10

0.04*

0.27 ± 0.16

0.29 ± 0.13

0.73

week 4

0.36 ± 0.13

0.32 ± 0.11

0.63

0.30 ± 0.14

0.23 ± 0.04

0.37

week 5

0.23 ± 0.09

0.29 ± 0.09

0.33

0.19 ± 0.10

0.30 ± 0.10

0.16

week 6

0.34 ± 0.09

0.28 ± 0.04

0.17

0.19 ± 0.06

0.17 ± 0.07

0.57

week 7

0.22 ± 0.08

0.21 ± 0.06

0.69

0.16 ± 0.02

0.19 ± 0.06

0.22

week 8

0.19 ± 0.07

0.19 ± 0.04

0.94

0.15 ± 0.05

0.15 ± 0.04

0.86

week 9

0.16 ± 0.02

0.17 ± 0.06

0.68

0.17 ± 0.05

0.18 ± 0.05

0.75

week 10

0.21 ± 0.08

0.21 ± 0.06

0.88

0.20 ± 0.03

0.17 ± 0.05

0.27

week 1-week 10

0.23 ± 0.02

0.25 ± 0.01

0.21

0.25 ± 0.01

0.22 ± 0.02

0.03*

week 1

0.39 ± 0.05

0.41 ± 0.09

0.61

0.42 ± 0.07

0.49 ± 0.08

0.07

week 2

0.38 ± 0.11

0.42 ± 0.11

0.53

0.41 ± 0.11

0.53 ± 0.16

0.20

week 3

0.43 ± 0.18

0.56 ± 0.12

0.20

0.50 ± 0.25

0.52 ± 0.18

0.70

week 4

0.62 ± 0.19

0.55 ± 0.12

0.46

0.55 ± 0.18

0.44 ± 0.02

0.28

week 5

0.43 ± 0.12

0.49 ± 0.16

0.47

0.41 ± 0.21

0.57 ± 0.14

0.25

ADG (kg/d)

ADFI (kg/d)

32


Page 33 of 41


week 6

0.60 ± 0.14

0.48 ± 0.08

0.11

0.45 ± 0.18

0.35 ± 0.13

0.29

week 7

0.42 ± 0.14

0.41 ± 0.07

0.91

0.35 ± 0.04

0.39 ±0.14

0.56

week 8

0.37 ± 0.10

0.44 ± 0.11

0.27

0.36 ± 0.14

0.34 ± 0.09

0.80

week 9

0.33 ± 0.06

0.38 ± 0.10

0.29

0.39 ± 0.12

0.37 ± 0.09

0.78

week 10

0.41 ± 0.13

0.44 ±0.09

0.65

0.49 ± 0.07

0.35 ± 0.10

0.09

week 1-week 10

0.44 ± 0.05

0.46 ± 0.01

0.38

0.43 ± 0.04

0.43 ± 0.02

0.92

week 1

2.07 ± 0.33

1.91 ± 0.50

0.52

2.02 ± 0.14

1.87 ± 0.27

0.23

week 2

2.13 ± 0.33

1.84 ± 0.25

0.14

1.88 ± 0.20

1.86 ± 0.18

0.89

week 3

1.87 ± 0.31

1.49 ± 0.19

0.04

2.06 ± 0.57

1.89 ± 0.21

0.38

week 4

1.78 ± 0.21

1.79 ± 0.27

0.96

1.82 ± 0.23

1.95 ± 0.39

0.60

week 5

1.92 ± 0.26

1.71 ± 0.23

0.18

2.18 ± 0.18

1.93 ± 0.27

0.23

week 6

1.77 ± 0.12

1.70 ± 0.09

0.34

2.37 ± 0.20

2.18 ± 0.23

0.26

week 7

1.90 ± 0.21

2.02 ± 0.22

0.37

2.22 ± 0.25

2.06 ± 0.12

0.37

week 8

1.99 ± 0.38

2.26 ± 0.22

0.19

2.46 ± 0.46

2.24 ± 0.14

0.43

week 9

2.06 ± 0.25

2.25 ± 0.28

0.24

2.35 ± 0.30

2.11 ± 0.26

0.34

week 10

2.05 ± 0.48

2.14 ± 0.35

0.74

2.42 ± 0.30

2.14 ± 0.22

0.28

week 1-week 10

1.88 ± 0.08

1.82 ± 0.04

0.15

1.72 ± 0.15

1.96 ± 0.11

0.02*

FCR

*

597

ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio was

598

calculated as ADFI divided by the ADG; *: indicate significant differences (P < 0.05). The results

599

are presented as the mean ± SD, n=7, for females in the Bt group, n=5, for males in the Bt group,

600

n=5, for both males and females in the Minghui86 group. 33



601

Page 34 of 41

Table 5. Haematological parameters in the Bt and Minghui86 group pigs. Males

Items

Females

Literature

Bt

Minghui86

P

Bt

Minghui86

P

NR

WBC (10^9/L)

16.63 ± 4.19

16.48 ± 6.13

0.96

17.67 ± 5.91

15.23 ± 3.74

0.46

15.81 ± 3.97

RBC (10^12/L)

9.22 ± 0.89

8.74 ± 0.31

0.29

9.13 ± 0.89

9.35 ± 0.64

0.68

8.22 ± 0.79

239.5 ± 67.71

241.2 ± 96.82

0.97

252.9 ± 76.69

229.25 ± 112.94

0.65

236.92 ± 54.56

Lymphocyte (10^9/L)

7.57 ± 2.04

8.52 ± 3.72

0.60

10.93 ± 3.75

11.23 ± 2.00

0.88

8.61 ± 3.02

Neutrophil (10^9/L)

6.78 ± 1.2

6.68 ± 3.36

0.95

5.03 ± 1.51

4.87 ± 0.65

0.80

5.23 ± 1.54

Eosinophils (10^9/L)

1.9 ± 2.81

0. 84 ± 0. 49

0.43

1.17 ± 0.77

0. 68 ± 0. 43

0.25

——

Basophilic (10^9/L)

0.07 ± 0.03

0.08 ± 0.04

0.69

0.11 ± 0.11

0.06 ± 0.03

0.42

——

Monocyte (10^9/L)

0.26 ± 0.08

0.36 ± 0.25

0.41

0.56 ± 0.28

0.57 ± 0.33

0.95

1.10 ± 0.35

174.00 ± 13.40

170.00 ± 13.10

0.63

182.09 ± 13.94

188.50 ± 14.82

0.45

166.97 ± 16.25

HCT (%)

59.05 ± 6.29

57.58 ± 5.28

0.69

56.41 ± 5.23

50.17 ± 5.02

0.24

52.60 ± 5.69

MPV (fl)

8.65 ± 0.33

8.58 ± 0.33

0.78

8.21 ± 0.60

8.78 ± 0.64

0.14

10.57 ± 1.68

MCV (fl)

64.17 ± 4.82

65.78 ± 4.14

0.57

63.97 ± 2.82

64.45 ± 4.50

0.83

62.09 ± 4.13

MCH (pg)

18.95 ± 1.27

19.42 ± 1.05

0.53

20.24 ± 0.88

20.15 ± 0.20

0.85

19.50 ± 1.38

MCHC (g/L)

295.5 ± 13.28

295.6 ± 14.58

0.99

316.75±12.90

313.75±20.10

0.76

314.13 ± 7.55

11.6 ± 1.27

11.48 ± 0.88

0.86

12.26 ± 1.26

11.95 ± 1.07

0.68

16.77 ± 0.88

Groups

PLT (10^9/L)

HGB (g/L)

RDW (%)

602

Values are means ± SD (n=7, for females in the Bt group, n=5, for males in the Bt group, n=5, for

603

both males and females in the Minghui86 group.). P > 0.05 (ANOVA) means no significant

604

difference. white blood cell (WBC), red blood cell (RBC), blood platelet count (PLT),

605

haemoglobin (HGB), haematocrit (HCT), mean platelet volume (MPV), erythrocyte mean 34


Page 35 of 41


606

corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular

607

haemoglobin concentration (MCHC), red blood cell distribution width (RDW).

608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 35



628

Page 36 of 41

Table 6. Blood biochemical parameters in the Bt and Minghui86 group pigs Males

Items

Females

Literature

Groups

Bt

Minghui86

P

Bt

Minghui86

P

NR

Tp (g/L)

87.25 ± 3.78

85.70 ± 6.05

0.62

84.79 ± 10.07

82.04 ± 4.28

0.57

84.55 ± 4.56

ALB (g/L)

44.05 ± 3.41

44.24 ± 2.33

0.92

45.65 ± 2.92

47.88 ± 2.31

0.17

44.23 ± 3.40

IgA (mg/dL)

13.52 ± 2.78

11.02 ± 3.33

0.21

18.57 ± 3.90

18.52 ± 4.84

0.98

——

IgG (mg/dL)

543.98 ± 34.20

526.24 ± 34.20

0.41

578.82 ± 76.02

557.32 ± 90.88

0.63

——

IgM (mg/dL)

185.95 ± 27.27

201.92 ± 25.28

0.34

232.41 ± 43.45

214.86 ± 45.57

0.47

——

Glu (mmol/L)

5.33 ± 1.90

4.50 ± 1.52

0.46

7.29 ± 1.79

8.06 ± 2.10

0.49

6.03 ± 1.32

CREA (umol/L)

90.83 ± 21.39

85.80 ± 14.72

0.67

91.00 ± 26.55

96.80 ± 23.39

0.68

157.12 ± 28.26

UREA (mmol/L)

4.82 ± 0.89

4.38 ± 1.50

0.57

4.65 ± 0.83

4.50 ± 0.85

0.74

5.26 ± 0.89

CHOL (mmol/L)

1.63 ± 0.46

1.75 ± 0.38

0.66

2.72 ± 0.24

2.65 ± 0.34

0.71

1.66 ± 0.45

TG (mmol/L)

0.35 ± 0.14

0.29 ± 0.07

0.43

0.67 ± 0.32

0.71 ± 0.24

0.83

0.85 ± 0.25

TB (umol/L)

2.91 ± 0.55

2.82 ± 0.47

0.76

2.45 ± 0.72

2.64 ± 0.50

0.62

——

A/G

1.03 ± 0.13

1.07 ± 0.06

0.52

1.22 ± 0.24

1.41 ± 0.15

0.13

1.20 ± 0.18

ALT (U/L)

62.75 ± 6.46

56.64 ± 10.55

0.27

58.51 ± 9.69

81.26 ± 23.29

0.01*

75.04 ± 7.56

AST (U/L)

106.38 ± 26.63

90.62 ± 29.44

0.38

128.52 ± 76.56

260.08 ± 191.15

0.06

——

ALP (U/L)

112.33 ± 16.62

85.80 ± 9.44

0.01*

82.55 ± 19.27

97.20 ± 14.14

0.15

53.29 ± 10.4

GGTP (U/L)

86.17 ± 17.46

77.40 ± 15.48

0.41

112.27 ± 33.93

103.60 ± 40.41

0.66

76.32 ± 6.25

LDH (U/L)

916.33 ± 131.45

847.20 ± 134.04

0.41

895.72 ± 247.18

797.6 ± 138.62

0.43

430.97 ± 78.46

629

Values are means ± SD (n=7, for females in the Bt group, n=5, for males in the Bt group, n=5, for

630

both males and females in the Minghui86 group.) *: P < 0.05 (ANOVA) means significant 36


Page 37 of 41


631

difference; P> 0.05 (ANOVA) means no significant difference. total protein (TP), albumin (ALB),

632

glucose (GLU), creatinine (CREA), total cholesterol (CHOL), triglyceride (TG), total bilirubin

633

(TB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase

634

(ALP), gamma glutamyl transpeptidase (GGTP), lactic dehydrogenase (LDH).

635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 37



653

Table 7. Ratios of organs to body weight in the Bt and Minghui86 group pigs. Items

Bt (%)

Minghui86 (%)

P

Males

Heart

0.34 ± 0.05

0.37 ± 0.09

0.35

Lung

0.53 ± 0.08

0.60 ± 0.12

0.26

Liver

1.70 ± 0.13

1.90 ± 0.22

0.09

Spleen

0.19 ± 0.02

0.21 ± 0.03

0.24

Kidney

0.44 ± 0.08

0.50 ± 0.03

0.13

Testis

0.38 ± 0.07

0.38 ± 0.09

0.96

Heart

0.30 ± 0.05

0.32 ± 0.08

0.67

Lung

0.39 ± 0.14

0.45 ± 0.11

0.46

Liver

1.39 ± 0.35

1.44 ± 0.16

0.77

Spleen

0.19 ± 0.04

0.19 ± 0.01

0.96

Kidney

0.31 ± 0.05

0.30 ± 0.07

0.69

Uterus

0.77 ± 0.33

0.84 ± 0.25

0.69

Females

654 655 656 657 658 38


Page 38 of 41

Page 39 of 41


659

Figure graphics

660 661

Figure 1

662 663

Figure 2

664

39



665

Figure 3

666

Figure 4

667 668 669 670 671 672 673 674 675 40


Page 40 of 41

Page 41 of 41


676

TOC Graphic：：

677

41


Effects of 60-Week Feeding Diet Containing Bt Rice Expressing the

Recommend Documents