Zhu 2018

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Article
The deposition and elimination of glucosinolates metabolites
derived from rapeseed meal in eggs of laying hens
liping zhu, jianping wang, xuemei ding, shiping bai, qiufeng zeng, zhuowei su, yue xuan, and keying zhang
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 18 Jan 2018
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Page 1 of 35 Journal of Agricultural and Food Chemistry
The deposition and elimination of glucosinolates metabolites derived from
rapeseed meal in eggs of laying hens
L. P. Zhu1, J. P. Wang1, X. M. Ding1, S. P. Bai1, Q. F. Zeng1, Z. W. Su1, Y. Xuan1, K. Y.
Zhang1*
1
Institute of Animal Nutrition, Key Laboratory for Animal Disease-Resistance
Nutrition of China, Ministry of Education, Sichuan Agricultural University, Chengdu,
Sichuan, China, 611130
*Corresponding author: Telephone: 86-0835-2882232. Fax: 86-0835-2883153. E-mail:
zkeying@sicau.edu.cn
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry Page 2 of 35
1 ABSTRACT
2 This study was to investigate the deposition and elimination of glucosinolates
3 metabolites including 5-vinyl-1,3-oxazolidine-2-thione (5-VOT) and thiocyanate ion
4 (SCN-) derived from rapeseed meal (RSM) in hen eggs. During 12 weeks
5 accumulation phase, the serum triiodothyronine, thyronine, blood urea nitrogen,
6 kidney index and thyroid index linearly increased with the RSM at week 12 (P < 0.05).
7 The thyroid histopathology revealed a sign of hyperplastic goiter in hens fed with
8 17.64-29.40% RSM. The 5-VOT content of eggs (Y, ng/g) was correlated with 5-VOT
9 intake (X2, µg/d.bird) and 5-VOT feeding time (X1, week): Y = 54.94*X1 + 0.51*X2 -
10 430.34 (P < 0.01, R2 = 0.80). The SCN- content of eggs (Y, mg/kg) was correlated
11 with RSM intake (X2, µg/d.bird) and RSM feeding time (X1, week): Y = 0.095*X1 +
12 0.302*X2 - 0.4211 (P < 0.01, R2 = 0.70). After a 4-week withdrawal of RSM, the
13 5-VOT and SCN- didn’t show in eggs. Taken together, 5.88% RSM with dietary
14 supplements of 23.55 mg/kg 5-VOT and 10.76 mg/kg SCN- had no effects on hens
15 with regard to serum parameters, organ index and thyroid histopathology, and more
16 than 4 weeks withdrawal should be considered for human and hens health.
17 KEYWORDS: 5-vinyl-1,3-oxazolidine-2-thione, thiocyanate ion, laying hens, health,
18 deposition, elimination

19 INTRODUCTION
20 Rapeseed meal (RSM) is an important feedstuff for laying hens, but the use of
21 RSM is limited because of its low available energy and protein for animals as well as
22 the high contents presence of antinutritional factors such as glucosinolates (Gls),
23 erucic acid, sinapine, and crude fiber. Gls are the main antinutritional factors in RSM
24 which can be hydrolyzed by myrosinase enzyme (E.C.3.2.3.1) from either plant or gut
25 microbiota of animals.1,2 The Gls metabolites 5-vinyl-1,3-oxazolidine-2-thione
26 (5-VOT) and thiocyanate ion (SCN-) could cause strong metabolic disturbances.3,4 In
27 addition, 5-VOT and SCN- were recognized of as poisonous substances according to
28 FDA Poisonous Plant Database.5 After ingestion of 1 g 5-VOT, the synthesis of
29 thyroid hormone was inhibited in rats which showed a phenomenon of goiter after
30 ingestion of 5 g 5-VOT.6 A single oral dose of 25 mg of 5-VOT inhibited radioiodine
31 uptake in adult human (70 kg).7 30 µg potassium thiocyanate caused a significant
32 decline of the thyroid iodine concentration in rats.8 Feeding experiments in rats
33 indicated that consumption of erucic acid could cause cardiotoxicity and
34 hyperfunction of thyroid gland.9,10 5-VOT and SCN- could be transferred to serum,
35 milk, muscular tissues and to certain organs: liver, lung, kidney, and mainly thyroid.11
36 When RSM was added to layer diet, 5-VOT and SCN- would be transferred into eggs
37 which threatened human health. Although some researchers reported about the 5-VOT
38 and SCN- deposition in milk and tissues,12 there was no information about the 5-VOT
39 and SCN- deposition in eggs. Mabon et al. showed that the plasma SCN- and 5-VOT
40 contents recovered to the initial state after one to three weeks withdrawal of RSM

41 ingestion,12 but there was no information about the recovery of hens health condition
42 when the 5-VOT and SCN- were eliminated from layers diets.
43 Therefore, this present study was conducted to investigate the response of hens to
44 Gls from RSM and the recovery of hens after a 4-week withdrawal of RSM, and to
45 investigate the deposition and elimination of 5-VOT and SCN- in eggs.
46 MATERIALS AND METHODS
47 Chemicals.
48 5-VOT, with a purity of 98+%, was obtained from Alfa Aesar (China). NaSCN
49 (99 %) was obtained from Sigma-Aldrich (Shanghai, china). All chemicals and
50 solvents were HPLC-grade.
51 Birds, Diets and Management.
52 A total of 900 30-week-old Lohmann pink-shelled laying hens were randomly
53 assigned to 6 dietary treatments with 10 pens per treatment and 15 hens per pen (3
54 hens/cage). Six isoenergetic and isonitrogenous diets containing 0, 5.88, 11.76, 17.64,
55 23.52, and 29.40% RSM were formulated on a digestible amino acid basis. All hens
56 were fed mash form diets (Table 1) which were formulated to meet or exceed nutrient
57 requirements of laying hens according to National Research Council13 and Lohmann
58 management guide alternative systems. The ratio of digestible lysine to digestible
59 methionine, threonine, and tryptophan in all diets was 49, 73, and 20 according to
60 Coon and Zhang.14
61 The trial lasted for 16 weeks, and all hens were fed with the control diet after 12
62 12-week exposure of RSM. Hens were provided with free access to water and

63 common trough feeders. The hens were housed in a windowless and environmentally
64 controlled room that was maintained at 24 °C and had a daily lighting schedule of
65 16-h light and 8-h dark. The Institutional Animal Care and Use Committee of Sichuan
66 Agricultural University approved all procedures used in the present study.
67 Sampling Procedure.
68 Glucosinolates, oxazolidine thione, isothiocyanates, and erucic acids contents of
69 RSM were analyzed by a commercial laboratory (Laboratory of Quality & Safety
70 Risk Assessment for Oilseed Products (Wuhan), MOA, PRC, Table 2) using the
71 national (PRC) standard methods SN/T 1868-2007, GB13089-91, NY/T 1596-2008,
72 and GB/T 23890-2009 respectively. At the end of 12th and 16th week, one hen was
73 chosen from each replicate and bled through a jugular vein (n = 10). The blood
74 samples were immediately placed on ice, transported to the laboratory within 3 h of
75 collection, and centrifuged at 2,000 × g for 15 min in a refrigerated centrifuge at about
76 4 °C. Serum was collected and stored at -20 °C until analyzed for the biochemical
77 parameters. Once blood was collected, birds were euthanized by cervical dislocation.
78 After that, the liver, thyroid, and kidney were weighed for calculating the relative
79 index (index (%) = organ fresh weight*100/each hen weight). The thyroid from hens
80 at the 12th week was put into fixative solution for histopathological lesions. To
81 evaluate the deposition of 5-VOT and SCN- in eggs, two egg samples were collected
82 from each replicate at week 4, 9, 12, and 16 after administration of RSM (n = 10).
83 Egg samples were stored at -20 °C before freeze drying.
84 Hormone Levels and Blood Parameters.

85 Serum glutamate pyruvate transaminase (GPT), glutamic-oxaloacetic
86 transaminase (GOT), triiodothyronine (T3), tetraiodothyronine (T4), and blood urea
87 nitrogen (BUN) were determined by colorimetric methods with a spectrophotometer
88 following the manufacturers’ protocols of the commercial kits (Nanjing Jiancheng
89 Institute of Bioengineering, Jiangsu, China).
90 Histopathology of Thyroid tissue.
91 The thyroid was dissected and fixed in 10% neutral formalin. The fixed tissues
92 were trimmed and embedded in paraffin. Thin sections (5 µm) were sliced and
93 mounted on a slide, and stained with hematoxylin and eosin for histopathological
94 examination by a pathologist. The stained slices were photographed using Motic
95 Microscope BA200 at 200 × magnification.
96 5-VOT and Thiocyanate Ions Analysis.
97 5-VOT was quantified by the high-performance liquid chromatography (HPLC)
98 analysis proposed by our previous study.15 The eggs were dried in a freezer dryer. The
99 dry samples were grinded by a pulverizer. 3.0 to 5.0 g samples and 3*15 mL
100 acetonitrile were added into a tube for extracting the 5-VOT. The organic solution was
101 transferred to a conic flask and then evaporated to dryness with a rotating evaporator
102 under vacuum (35 °C). The 5-VOT was recovered with 2 mL phosphate buffer, pH 7.0
103 and 2 mL n-hexane. The aqueous phosphate buffer solution was transferred to a 2 mL
104 tube and centrifuged for 5 min at 12000 r/min, then the aqueous phase was passed by
105 0.45 µm filter membrane for HPLC analysis.
106 A model Agilent 1200 HPLC system is equipped with a pump (model G1312A),

107 autosampler (Hip-Als, model G1367C), thermostat column compartment (TQ-SL,
108 model G1316B), UV diode array detector (model G1314F) and degasser (model
109 G1322A). The column of chromatogram is Alltima 5µm (250 mm*4.6 mm).
110 Thiocyanate ions were quantified using the international standard methods ISO
111 10304-3: 1997. 1.0 g dried egg powder and 4.0 g ultrapure water were added into a
112 tube which was shaken on vortex mixer for 2 minutes subsequently. And then it was
113 diluted to 10 mL with acetonitrile. After 20 minutes, it was centrifuged for 10 min at
114 6000 r/min. 1.0 mL supernatant liquid was diluted to 10 mL with ultrapure water and
115 filtered through a 0.22 µm filter membrane, then cleaned by Supelclean ENVI-18 SPE
116 tube (Part no. 57063, Supelco, Bellefont, PA) for HPIC analysis. A model Thermo
117 Dionex ICS-1100 system is equipped with a Dionex DS6 conductivity detection. The
118 column of chromatogram is DIONEX AS 19 (4 × 250 mm).
119 Method Validation.
120 The validation parameters were consisted of linearity range, precision,
121 repeatability, recovery, and limit of quantification (LOQ). Method validation of
122 5-VOT has been reported in our previous study.15 Quantification of SCN- has been
123 done using an external standard curve with seven points. The concentrations ranged
124 from 0.013 to 1.011 mg/L. The linearity range was evaluated by plotting the peak area
125 corresponding to analyte, as a function of the concentration introduced. The
126 intra-daily precision of the HPIC method was validated with a SCN- standard solution
127 under the selected optimal conditions 6 times a day. Six independently prepared
128 sample solutions of eggs from the same pen were analyzed and the variations within

129 six measurements were calculated for evaluation of repeatability. Recovery is reported
130 as the percent salvage in relation to the known three concentration levels of analyte
131 added to the sample (n = 4).
132 Statistical Analysis
133 The effects of RSM were assessed by one-way ANOVA followed by a linear and
134 quadratic contrast using the general linear model (GLM) procedure of SAS
135 software.16 LSD procedure was performed when ANOVA was significant at α = 0.05.
136 Data is expressed as means and standard error of the mean (SEM). Means were
137 considered significantly different when P < 0.05. A nonlinear regression model (Y =
138 a*X1 + b*X2 + c) was applied to predict nonlinear regression analysis of Gls
139 metabolites intake and deposition by SigmaPlot 12.5 (SyStat Software Inc.). In this
140 model, Y is 5-VOT (ng/g) or SCN- (mg/kg) content in eggs. X1 represented feeding
141 time (week). X2 represented 5-VOT (µg/d.bird) or RSM intake (g/d.bird).
142 RESULTS
143 Hormone Levels and Blood Parameters. In the present study, there was no
144 difference in serum GPT concentrate, GOT concentration, and GOT/GPT between
145 groups at week 12 (Table 3, P = 0.12; P = 0.18; P = 0.50). And supplementation of
146 29.4% RSM linearly increased serum T3 content of hens (P < 0.01), and feeding hens
147 with 23.52% RSM linearly increased serum T4 content at week 12 (P < 0.01). Serum
148 BUN content was linearly increased in hens fed with 17.64% RSM diet (P < 0.01).
149 After a 4-week withdrawal of RSM, there was no difference in serum GPT content,
150 GOT content, and GOT/GPT between groups at week 16 (Table 4, P = 0.63; P = 0.84;

151 P = 0.47). Although serum T3 and T4 content was linearly increased with dietary
152 RSM level (P < 0.05; P < 0.01) at week 16, there was no difference in serum T3 and
153 T4 content between groups at week 16. In addition, 17.64% RSM linearly increased
154 serum BUN content at week 16 (P < 0.01).
155 Organ Index and Organ Histopathology. In our study, RSM had no effect on liver
156 and kidney weight at week 12 (Table 5, P = 0.22; P = 0.94). 29.4% RSM linearly
157 increased thyroid weight at week 12 (P < 0.01). RSM exposure to hens had no effect
158 on liver index at week 12 (P = 0.39), but dietary supplementation with over 11.76%
159 RSM had a higher liver index value between groups. No difference in kidney index
160 between groups was observed (P = 0.47). 17.64% RSM linearly increased thyroid
161 index at week 12 (P < 0.01), and the thyroid histopathology revealed a sign of
162 hyperplastic goiter in hens fed with 17.64-29.40% RSM (Figure 1). Thyroid follicles
163 proliferation and epithelial cell proliferation was observed in hens that received over
164 17.64% RSM diet. The much severer thyroid follicles proliferation and epithelial cell
165 proliferation were occurred in hens when exposed to higher amount of RSM.
166 After 4 weeks withdrawal of RSM, the organs morphology recovered, and there
167 was no difference in liver, kidney, and thyroid weight and liver, kidney, and thyroid
168 index between groups at week 16 (Table 6, P =0.63; P = 0.88; P = 0.43; P = 0.79; P =
169 0.78; P = 0.32).
170 Method validation for quantitative determination of 5-VOT and SCN-.
171 Chromatograms of a calibration standard solution and cleaned-up ultrafiltrates of eggs
172 and diets for 5-VOT are displayed in Figure 2. The results of the validation

173 parameters for the 5-VOT were shown in previous study.15 Chromatograms of a
174 calibration standard solution and cleaned-up ultrafiltrates of eggs for SCN- are showed
175 in Figure 3. The results of the validation parameters for the SCN- are shown in Table
176 7. A good linearity range was found and the relative standard deviation (RSD) values
177 of precision is 0.81% (n = 6). The RSD value of repeatability is 1.90% (n = 6). The
178 recovery rates are between 89.03% and 99.59% at three concentration levels (n = 4),
179 and the RSD of recovery rate are between 0.035% and 1.74% (n = 4). The LOQ of
180 SCN- is 1.0 mg/kg.
181 Deposition and Elimination of 5-VOT in Eggs. The 5-VOT accumulation was
182 present in eggs was observed only after hens the ingested the RSM, and the dietary
183 5-VOT content in diet was increased with the increase of dietary RSM level (Table 8).
184 The 5-VOT content of eggs was linearly increased with the increase of dietary 5-VOT
185 level at week 4, 9, and 12 (P < 0.0001; P < 0.0001; P < 0.0001). Furthermore, the
186 5-VOT content in eggs was also linearly increased with increase of the feeding
187 duration of dietary RSM feeding time in each group (P = 0.001; P = 0.06; P = 0.017; P
188 < 0.0001; P = 0.06). The 5-VOT content of eggs (Y, ng/g) was significantly correlated
189 with dietary 5-VOT intake (X2, µg/d.bird) and dietary 5-VOT feeding time (X1, week):
190 Y = 54.94*X1 + 0.51*X2 - 430.34 (P < 0.01, R2 = 0.80). After 4 weeks withdrawal of
191 RSM, the 5-VOT couldn’t be detected in eggs. Thus, we speculated the 5-VOT
192 concentration in eggs came back to zero.
193 Deposition and Elimination of SCN- in Eggs. The SCN- content was 3.83, 10.76,
194 17.71, 27.56, 38.70, and 49.85 mg/kg diet in 0% to 29.40% RSM diet respectively.
10

195 The SCN- couldn’t be detected in eggs of hens fed with control diet during
196 accumulation phase. After a 4-week administration of RSM, the SCN- content
197 increased to 1.87, 2.28, 5.02, 6.99, and 8.32 mg/kg egg of hens fed with 5.88% to
198 29.40% RSM diet respectively, and the SCN- concentration in eggs was linearly
199 increased with the increasing dietary RSM at week 4, 9, and 12 (Table 9, P < 0.0001,
200 P = 0.004, P = 0.0003). But the SCN- content of eggs didn’t increase progressively
201 and remained almost constant at different time points in 5.88% to 29.40% RSM diet
202 group (P = 0.05, P = 0.22, P = 0.69, P = 0.38, P = 0.35). The SCN- content of eggs (Y,
203 mg/kg) was significantly correlated with dietary RSM intake (X2, g/d.bird) and dietary
204 RSM feeding time (X1, week): Y = 0.095*X1 + 0.302*X2 - 0.4211 (P < 0.01, R2 =
205 0.70). After a 4-week withdrawal of RSM, the SCN- came back to control level and
206 couldn’t be detected in eggs.
207 DISCUSSION
208 The supplementation of RSM in animal diets is limited because of the presence
209 of antinutritional factors.17 It was recommended that high Gls RSM inclusion should
210 be restricted to a maximum of 5% in hens diet, while the recommended maximum
211 inclusion level of low Gls RSM was increased to 10% as their detrimental effects are
212 lighter.18 Gls are precursors of compounds which exert goitrogenic activity in birds
213 and induced metabolic disorders,19 and it might also cause abnormalities in internal
214 organs containing liver, kidney, and mainly in thyroid gland.20
215 In our research, RSM didn’t affect serum GPT, GOT, and GOT/GPT which was
216 consistent with Ahmed21 and Busato22 who reported that RSM replacing 100% of the
11

217 soybean meal didn’t affect serum GPT concentration on chickens.23 But RSM linearly
218 increased serum T3 and T4 content in the present study. And similar research reported
219 that 30% RSM linearly increased serum T3 and T4 concentration on broilers.24 In
220 addition, 17.64% RSM increased serum BUN content in the present study. However,
221 there was little information about the effect of RSM on serum blood in poultry. And
222 previous study showed that 32% RSM increased the serum BUN content in fish diet25,
223 and 16% RSM didn’t affect serum BUN content. 30-40% canola meal had no
224 influence on serum BUN in weanling pigs.26 Different levels of antinutritional factors
225 in RSM and different experimental animals used in these studies might cause the
226 different results, and more researches need to conduct to investigate the effect of RSM
227 on serum BUN content in poultry. And RSM had no effect on liver index and kidney
228 index. This was in line with Marangos et al.27 who reported that 12% RSM diet with
229 324 mg/kg Oxazolidine thiones and 480 mg/kg isothiocyanates had no effect on layers
230 liver index during a 12-week experiment. Similarly, 3.84 µmol Gls/g diet had no
231 effect on liver index in hens,28 and 2.08 µmol Gls/g diet increased hens thyroid index.
232 17.64% RSM linearly increased thyroid index at week 12 in the present study which
233 was consistent with Rotkiewicz who reported that long-term ingestion of 10% RSM
234 could enlarge thyroid follicle size and weight.29 Gls metabolites impaired the thyroid
235 uptake of iodide, its oxidation, the iodine binding to thyroglobulin, synthesis and
236 release of hormone, and then increased the release of the thyroid-stimulating hormone
237 (TSH).30 TSH promoted thyroid follicle proliferation and enlargement,31 and thus
238 thyroid weight increased. And erucic acid might be another reason for thyroid
12

239 follicular epithelium proliferation.9
240 The 5-VOT accumulation was present in eggs only after the ingestion of
241 rapeseed meal, and the 5-VOT content in eggs was linearly increased with the dietary
242 5-VOT level at week 4, 9, and 12. While 5-VOT content in eggs was also linearly
243 increased with increase of dietary RSM feeding time in each group. This was in line
244 with Mabon who reported that the 5-VOT was present exclusively with the ingestion
245 of rapeseed meal in lamb, and the more RSM lamb ingested, the more 5-VOT the
246 thyroid accumulated.32 The SCN- couldn’t be detected in eggs of hens fed with control
247 diet during accumulation phase. However, Shuaib et al.33 showed control group had
248 an average of 0.5 mg/kg SCN- detected by gas chromatography. The limit of
249 determination is 1.0 mg/kg in present study, thus the difference of analysis method
250 might cause the different results. And the SCN- content of eggs didn’t increase
251 progressively and remained almost constant after a 4-week administration of RSM,
252 this was in lined with Shuaib et al. who reported that the levels of SCN- reached 1.6
253 mg/kg in the experimental eggs and then remained almost constant after 4 weeks of
254 administration of 10% RSM diet.33 Moreover, Mabon et al. showed the SCN-
255 concentration in the plasma increased during the first three weeks and then stay
256 relatively constant until the end of the experiment.32
257 Although 5-VOT showed in FDA Poisonous Plant Database5, there was no limit
258 of 5-VOT in food. Langer reported 25 mg of 5-VOT inhibited radioiodine uptake in
259 adult human (70 kg).7 Extrapolation from the results obtained for rats indicated that
260 thyroid enlargement is possible at a 5-VOT level of 2 µg/L of milk for an infant of 6
13

261 kg ingesting 1 L of milk per day (2 µg 5-VOT/per day).34 Based on our findings, we
262 thought that eggs of hens which ingested 5.88% RSM containing 23.55 mg/kg 5-VOT
263 with 4 weeks might be safe for infant. According to the CAC (Codex Alimentarius
264 Commission), 14 mg/L NaSCN is allowed to add in milk, and the 10.02 mg/kg SCN-
265 is allowed to add in milk accordingly.35 Previous study reported that the serum
266 thyroxine, triiodothyronine and TSH levels were in the normal range for all man both
267 at the beginning and end of the experimental period after 4 weeks daily intake of 4.75
268 mg of thiocyanate by way of milk (19 mg/L), therefore a thiocyanate concentration of
269 19 mg/L didn’t have a negative effect on the thyroid function.36 In present study, the
270 maximum deposition of SCN- was lower than 10.02 mg/kg in eggs, thus it might be
271 safe for human.
272 After a 4-week withdrawal of RSM, the 5-VOT and SCN- concentration in eggs
273 couldn’t be detected. And there was no difference in serum T4 and T3 content
274 between groups. Akiba et al.37 found that blood T4 content had rebound after 14 days
275 withdrawal of 0.05% PTU (propylthiouracil; goitrogen), but blood T3 content had no
276 change. Likewise, blood T4 content obviously rebounded after a 10-day withdrawal of
277 0.05% goitrin (5-vinyl-2-oxazolidinethione) prepared from rapeseed, but T3 content
278 had no distinct changes.38 The reason for different results among distinction between
279 previous study studies and the present study may be owed to the different withdrawal
280 duration. While the serum BUN content had no rebound phenomena, so hens might
281 need longer time to restore kidney function. The organ morphology had recovered
282 after 4 weeks withdrawal of RSM at week 16, and this was in line with Lipner who
14

283 reported that thyroid weight approached the control base-line level after 8 days
284 withdrawal of 0.025% PTU,39 and thyroid weight decreased to twice that of control
285 group after 14 days withdrawal of 0.05% PTU and 10 days withdrawal of 0.05%
286 goitrin prepared from rapeseed.37,38 It seemed that the more goitrogens animals
287 ingested, the longer time thyroid need to recover.
288 In conclusion, Gls metabolites had a negative effect on laying hens. 5.88% RSM
289 with dietary supplements of 23.55 mg/kg 5-VOT and 10.76 mg/kg SCN- had no
290 effects on hens with regard to serum parameters, organ index and thyroid
291 histopathology, and eggs of hens fed with 23.55 mg 5-VOT/kg diet in 4 weeks might
292 be safe for infants. And 4 weeks withdrawal of diets containing 5-VOT and SCN-
293 should be considered for human and hens health.
294 CONFLICTS OF INTEREST
295 The authors declare that there are no known conflicts of interest have been
296 associated with this publication and there has been no significant financial support for
297 this work that could have influenced its outcome.
298 FUNDING SOURCES
299 This work was financially supported by national science and technology project
300 (2014BAD13B04, 201203015) and Sichuan Provincial science and technology project
301 (2014NZ0043, 2014NZ0002).
302
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347 Nahrung. 1995, 39, 21-31.
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18

369 25. Tan, Q.; Liu, Q.; Chen, X.; Wang, M.; Wu, Z. Growth performance, biochemical
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375 27. Marangos, A.; Hill, R.; Laws, B.; Muschamp, D. The influence of three rapeseed
376 meals and a mustard seed meal on egg and broiler production. Brit Poultry Sci. 1974,
377 15, 405-414.
378 28. Campbell, L.; Slominski, B. In Nutritive quality of low-glucosinolate canola meal
379 for laying hens, Proc. 8th Int. Rapeseed Congress, Saskatoon, SK, 1991, 442-447.
380 29. Rotkiewicz, T.; Bomba, G.; Falkowski, J.; Glogowski, J.; Kozera, W.; Kozłowski,
381 M. Studies on a long-term use of rapeseed products in diets for boars.
382 Pathomorphological changes in the reproductive system, liver and thyroid gland.
383 Reprod Nutr Dev. 1997, 37, 675-690.
384 30. Schone, F.; Jahreis, G.; Richter, G.; Lange, R. Evalulation of rapeseed meals in
385 broiler chicks: effect of iodine supply and glucosinolate dgradation by myrosinase or
386 copper. J Sci Food Agr. 1993, 61, 245-252.
387 31. Wallig, M. A.; Belyea, R. L.; Tumbleson, M. E. Effect of pelleting on
388 glucosinolate content of Crambe meal. Anim Feed Sci Tech. 2002, 99, 205-214.
389 32. Mabon, N.; Wathelet, J.-P.; Derycke, G.; Mandik, N.; Bister, J.-L.; Marlier, M.;
390 Paquay, R. In Correlation between breakdown products of glucosinolates and the
19

391 amount of rapeseed meal introduced in the diets of 80 lambs and 32 bulls. Evolution
392 of 5-vinyl-1, 3-oxcazolidine-2-thione (5-VOT) and thiocyanate ions in biological
393 fluids and organs, Proc. 10th Int. Rapeseed Congr., Canberra, 1999.
394 33. Shuaib, A.; Beswick, G.; Tomlins, R. I. The thiocyanate ion (SCN-) content of
395 eggs from hens (Gallus domesticus) fed on a diet containing rapeseed meal. J Sci
396 Food Agr. 1981, 32, 347-352.
397 34. Benns, G.; L'Abbé, M. R.; Lawrence, J. F. High-pressure liquid chromatography
398 detection of the antithyroid compound 5-vinyloxazolidine-2-thione in milk. J Agr
399 Food Chem. 1979, 27, 426-8.
400 35. Commission, C. A. Guidelines for the preservation of raw milk by use of the
401 lactoperoxidase system In Codex Alimentarius Commission: 1991; Vol. CAC/GL
402 13-1991.
403 36. Dahlberg, P. A.; Bergmark, A.; Eltom, M.; Björck, L.; Claesson, O. Effect of
404 thiocyanate levels in milk on thyroid function in iodine deficient subjects. Am J Clin
405 Nutr. 1985, 41, 1010-4.
406 37. Akiba, Y.; Itoh, H.; Matsumoto, T. Rebound phenomena in thyroid function after
407 withdrawal of propylthiouracil in growing chicks. Gen Comp Endocr. 1971, 17,
408 444-450.
409 38. Akiba, Y.; Matsumoto, T. Thyroid Function of Chicks after Withdrawal of
410 (-)-5-Vinyl-2-oxazolidinethione, a Goitrogen in Rapeseed. Poultry Sci. 1973, 52,
411 562-567.
412 39. Lipner, H.; Wagner, B. P.; Morris, H. P. Augmentation of mouse thyroid iodide
20

413 pump activity after withdrawal of propylthiouracil. American Journal of
414 Physiology--Legacy Content. 1959, 196, 981-982.
415
21

416 FIGURE CAPTIONS
417 Figure 1. H&E 200×. Photomicrographs of histologic preparations of thyroid from
418 hens fed different levels of RSM for 12 weeks (n = 5). (A) Portion of thyroid from the
419 control group. No thyroid follicles proliferation. (B) Portion of thyroid from hens that
420 received RSM at 5.88%. No thyroid follicles proliferation but mild epithelial cell
421 proliferation was evident, and this appearance was similar to that of thyroid from the
422 control group. (C) Portion of thyroid from hens that received RSM at 11.76%. No
423 thyroid follicles proliferation but moderate epithelial cell proliferation was evident. (D)
424 Portion of thyroid from hens that received RSM at 17.64%. Mild thyroid follicles
425 proliferation and moderate epithelial cell proliferation were evident. (E) Portion of
426 thyroid from hens that received RSM at 23.52%. Moderate thyroid follicles
427 proliferation and moderate epithelial cell proliferation were evident. (F) Portion of
428 thyroid from hens that received RSM at 29.4%. Moderate thyroid follicles
429 proliferation and severe epithelial cell proliferation were evident.
430 Figure 2. HPLC-UV chromatogram relative to the (A) 5-VOT standard at a
431 concentration of 71.4 µg/mL, (B) 5-VOT from the egg sample at a concentration of
432 614.23 ng/g, (C) 5-VOT from the 23.52% RSM diet sample at a concentration of
433 88.49 mg/kg.
434 Figure 3. Ion chromatography relative to the (A) SCN- standard at a concentration of
435 0.744 µg/mL, (B) SCN- from the egg sample at a concentration of 0.04 µg/mL.
22

Table 1. Ingredients and Nutrient Composition of Experimental Diets (g/kg, as fed basis)
RSM
Items
0.00 58.80 117.60 176.40 235.20 294.00
Ingredient
Corn 625.80 612.44 599.08 585.72 572.36 559.00
Soybean meal 264.50 211.60 158.70 105.80 52.90 0.00
Rapeseed meal 0.00 58.80 117.60 176.40 235.20 294.00
Rapeseed oil 6.00 13.02 20.04 27.06 34.08 41.10
Calcium carbonate 78.00 77.80 77.60 77.40 77.20 77.00
Dicalcium phosphate 11.90 11.34 10.78 10.22 9.66 9.10
NaCl 4.00 4.00 4.00 4.00 4.00 4.00
a
Mineral premix 5.00 5.00 5.00 5.00 5.00 5.00
Vitamin premixb 0.30 0.30 0.30 0.30 0.30 0.30
L-lysine.HCl 0.90 1.86 2.82 3.78 4.74 5.70
DL-methionine 1.90 1.92 1.94 1.96 1.98 2.00
L-theronine 0.70 0.86 1.02 1.18 1.34 1.50
L-tryptophan 0.00 0.06 0.12 0.18 0.24 0.30
Choline chloride 1.00 1.00 1.00 1.00 1.00 1.00
Total 1000.00 1000.00 1000.00 1000.00 1000.00 1000.00
Calculated Nutrient levels
(%)
AMEn (kcal/Kg) 2700 2700 2700 2700 2700 2700
Crude protein 16.5 16.5 16.5 16.5 16.5 16.5
Ca 3.50 3.50 3.50 3.50 3.50 3.50
Available P 0.32 0.32 0.32 0.32 0.32 0.32
Digestible Lys 0.85 0.85 0.85 0.85 0.85 0.85
Digestible Met 0.42 0.42 0.42 0.42 0.42 0.42
Digestible Thr 0.62 0.62 0.62 0.62 0.62 0.62
Digestible Trp 0.17 0.17 0.17 0.17 0.17 0.17
Dietary Gls (µmol/g) 0 0.92 1.84 2.76 3.67 4.59
5-VOT (mg/kg)c 0 23.55 68.71 83.16 88.49 94.25
SCN- (mg/kg)c 3.83 10.76 17.71 27.56 38.7 49.85
c
Crude fiber 3.93 3.85 4.14 5.16 5.45 6.22
a
Provided per kilogram of diet: 60 mg Fe (FeSO4·7H2O), 8 mg Cu (CuSO4·5H2O), 60 mg
Mn (MnSO4·H2O), 80 mg Zn (ZnSO4·7H2O), 0.3 mg Se (NaSeO3) and 0.35mg I (KI).
b
Provided per kilogram of diet: 8000 IU vitamin A, 1600 IU vitamin D3, 5 IU vitamin E,
0.8 mg vitamin B1, 2.5 mg vitamin B2, 1.5 mg vitamin B6, 0.004 mg vitamin B12, 2.2
mg D-pantothenic acid, 0.25 mg folic acid, 20 mg nicotinic acid and 0.1 mg biotin.
c
Value is analyzed.
23

Table 2. Antinutritional Factors Composition of Rapeseed Meal (as fed basis)

Items Rapeseed meal
Glucosinolates (µmol/g) 15.62
Oxazolidine thione (mg/g) 1.16
Isothiocyanates (mg/g) 1.32
Erucic acids (%) 8.40
24

Table 3. Effects of RSM on Serum Parameters at 12 wka (a,b,cMeans in the Same Row
with Different Superscripts Differ, P < 0.05)
Itemsb RSM ratio (%) p-value
0 5.88 11.76 17.64 23.52 29.40 SEM ANOVA Linear
GPT 5.0 3.7 4.1 4.1 3.8 4.1 0.34 0.12 0.16
GOT 187.6 178.6 166.9 166.9 165 181 7.40 0.18 0.25
GOT/GPT 40.88 51.94 38.93 45.83 46.39 46.07 4.91 0.5 0.7
T3 1.26b 1.29b 1.36ab 1.31ab 1.44ab 1.61a 0.06 <0.01 <0.01
T4 12.22c 13.92c 14.57bc 15.1bc 19.05ab 20.92a 0.99 <0.01 <0.01
BUN 1.41c 1.20c 1.24c 2.23b 2.59a 2.37ab 0.07 <0.01 <0.01
a
Data are presented as mean values with SEM. Each mean represents 10 hens;
b
GPT = glutamate pyruvate transaminase, U/L; GOT = glutamic-oxaloacetic transaminase,
U/L; T3 = triiodothyronine, ng/mL; T4 = tetraiodothyronine, ng/mL; BUN = blood urea
nitrogen, mmol/L.
25

Table 4. Effects of RSM on Serum Parameters at 16 wka (a,b,cMeans in the Same Row
with Different Superscripts Differ, P < 0.05)
Itemsb RSM ratio (%) p-value

0 5.88 11.76 17.64 23.52 29.40 SEM ANOVA Linear
GPT 4 4.1 4.6 4.7 4.4 4.3 0.33 0.63 0.37
GOT 184.9 179.5 187.1 191 197.3 189.4 9.38 0.84 0.31
GOT/GPT 48.83 45.11 42.25 40.52 47.14 46.52 3.25 0.47 0.79
T3 0.68ab 0.58b 0.60b 0.65ab 0.93a 0.72ab 0.07 0.02 <0.05
T4 6.68 7.07 6.96 6.76 8.55 8.12 0.82 0.48 <0.01
BUN 2.11c 2.16bc 2.20bc 2.45ab 2.66a 2.63a 0.10 <0.01 <0.01
a
Data are presented as mean values with SEM. Each mean represents 10 hens.
b
GPT = glutamate pyruvate transaminase, U/L; GOT = glutamic-oxaloacetic
transaminase, U/L; T3 = triiodothyronine, ng/mL; T4 = tetraiodothyronine, ng/mL; BUN
= blood urea nitrogen, mmol/L.
26

Table 5. Effects of RSM on Organ Weight and Organ Index at 12 wka (a,b,cMeans in
the Same Row with Different Superscripts Differ, P < 0.05)
Item RSM ratio (%) p-value

0 5.88 11.76 17.64 23.52 29.40 SEM ANOVA linear
Liver weight (g) 29.20 27.20 31.16 28.60 26.79 28.42 1.29 0.22 0.48
Kidney weight (g) 10.51 11.02 11.36 11.12 11.59 11.23 0.72 0.93 0.41
Thyroid weight (g) 0.189bc 0.182c 0.235abc 0.244ab 0.248ab 0.254a 0.012 <0.001 <0.001
Liver index (%) 1.69 1.62 1.87 1.75 1.72 1.79 0.089 0.46 0.39
Kidney index (%) 0.607 0.653 0.683 0.681 0.740 0.710 0.045 0.46 0.04
Thyroid index (%) 0.0109b 0.0108b 0.0141ab 0.0149a 0.0159a 0.0160a 0.0008 <0.0001 <0.0001
a
27

Table 6. Effects of RSM on Organ Weight and Organ Index at 16 wka (a,b,cMeans in
the Same Row with Different Superscripts Differ, P < 0.05)
Item RSM ratio (%) p-value

0 5.88 11.76 17.64 23.52 29.40 SEM ANOVA linear
Liver weight (g) 29.59 31.98 30.53 29.21 30.69 28.94 1.36 0.63 0.46
Kidney weight (g) 10.60 11.10 10.80 10.24 10.40 10.56 0.51 0.88 0.51
Thyroid weight (g) 0.165 0.174 0.192 0.187 0.192 0.182 0.011 0.43 0.14
Liver index (%) 1.861 2.013 1.847 1.878 1.947 1.861 0.094 0.79 0.83
Kidney index (%) 0.651 0.697 0.650 0.653 0.661 0.608 0.041 0.78 0.35
Thyroid index (%) 0.0102 0.0109 0.0115 0.0119 0.0121 0.0117 0.0006 0.32 0.04
a
28

Table 7. Validation Parameters for The Ion Chromatography Method.

Linearity
Item (R2) Precision Repeatability Recovery
Contents Contents RSD Added Recovery RSD
(mg/L) RSD (%) (mg/kg) (%) amount (µg) rates (%) (%)
-
SCN 1.0 0.09548 0.81 10.07 1.9 4.0 97.74 0.035
10.0 99.59 0.035
80.0 89.03 1.741
29

Table 8. 5-VOT Concentration in Eggs of Laying Hensa (dry matter basis, ng/g)
p-valuec
Items 4wk 9wk 12wk 16wk
SEM ANOVA Linear Quadratic
RSM ratio (%)
0 N.Db N.D N.D N.D
5.88 82.62dB 113.64eB 176.22cA N.D 11.09 <0.01 <0.01 0.09
11.76 399.04cA 422.57dA 551.78bcA N.D 44.58 0.10 0.06 0.24
17.64 571.01bcB 614.23cB 792.87bA N.D 45.13 0.03 0.02 0.14
23.52 669.86bB 748.52bB 1456.98aA N.D 47.72 <0.01 <0.01 <0.01
29.40 1075.69aB 1190.86aAB 1837.45aA N.D 217.47 0.09 0.06 0.23
SEM 58.62 28.97 167.2
ANOVA <0.01 <0.01 <0.01
d
p-value Linear <0.01 <0.01 <0.01
Quadratic <0.01 <0.01 <0.01
a
b
N.D means the 5-VOT is lower than determination limit;
c
The capital letters in the same row are significantly different, P < 0.05;
d
The lowercases in the same column are significantly different, P < 0.05.
30

Table 9. SCN- Concentration in Eggs of Laying Hensa (dry matter basis, mg/kg)
p-valuec
Items 4wk 9wk 12wk 16wk
SEM ANOVA Linear Quadratic
RSM ratio (%)
0 N.Db N.D N.D N.D
cA eA bA
5.88 1.87 1.89 2.62 N.D 0.24 0.05 0.35 0.47
cB dA bAB
11.76 2.82 4.64 3.49 N.D 0.46 0.22 0.18 0.04
17.64 5.02bA 5.99cA 5.27abA N.D 1.07 0.69 0.81 0.56
aA bA aA
23.52 6.99 6.31 6.88 N.D 1.07 0.38 0.91 0.72
aA aA aA
29.40 8.32 8.71 8.27 N.D 0.91 0.35 0.99 0.78
SEM 0.59 1.47 0.95
ANOVA <0.01 <0.01 0.04
d
p-value Linear <0.01 <0.01 <0.01
Quadratic 0.80 0.73 0.79
a
b
N.D means the 5-VOT is lower than determination limit;
c
The capital letters in the same row are significantly different, P < 0.05;
d
The lowercases in the same column are significantly different, P < 0.05.
31

Figure 1
32

Figure 2
33

Figure 3
34

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Journal of Agricultural and Food Chemistry is published by the American Chemical

The deposition and elimination of glucosinolates metabolites derived from

rapeseed meal in eggs of laying hens

L. P. Zhu1, J. P. Wang1, X. M. Ding1, S. P. Bai1, Q. F. Zeng1, Z. W. Su1, Y. Xuan1, K. Y.

Nutrition of China, Ministry of Education, Sichuan Agricultural University, Chengdu,

Sichuan, China, 611130

*Corresponding author: Telephone: 86-0835-2882232. Fax: 86-0835-2883153. E-mail:

ACS Paragon Plus Environment

2 This study was to investigate the deposition and elimination of glucosinolates

3 metabolites including 5-vinyl-1,3-oxazolidine-2-thione (5-VOT) and thiocyanate ion

5 accumulation phase, the serum triiodothyronine, thyronine, blood urea nitrogen,

17 KEYWORDS: 5-vinyl-1,3-oxazolidine-2-thione, thiocyanate ion, laying hens, health,

ACS Paragon Plus Environment

22 the high contents presence of antinutritional factors such as glucosinolates (Gls),

25 microbiota of animals.1,2 The Gls metabolites 5-vinyl-1,3-oxazolidine-2-thione

27 addition, 5-VOT and SCN- were recognized of as poisonous substances according to

28 FDA Poisonous Plant Database.5 After ingestion of 1 g 5-VOT, the synthesis of

30 ingestion of 5 g 5-VOT.6 A single oral dose of 25 mg of 5-VOT inhibited radioiodine

31 uptake in adult human (70 kg).7 30 µg potassium thiocyanate caused a significant

32 decline of the thyroid iodine concentration in rats.8 Feeding experiments in rats

33 indicated that consumption of erucic acid could cause cardiotoxicity and

34 hyperfunction of thyroid gland.9,10 5-VOT and SCN- could be transferred to serum,

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45 investigate the deposition and elimination of 5-VOT and SCN- in eggs.

46 MATERIALS AND METHODS

50 solvents were HPLC-grade.

51 Birds, Diets and Management.

52 A total of 900 30-week-old Lohmann pink-shelled laying hens were randomly

57 requirements of laying hens according to National Research Council13 and Lohmann

58 management guide alternative systems. The ratio of digestible lysine to digestible

60 Coon and Zhang.14

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66 Agricultural University approved all procedures used in the present study.

68 Glucosinolates, oxazolidine thione, isothiocyanates, and erucic acids contents of

69 RSM were analyzed by a commercial laboratory (Laboratory of Quality & Safety

71 national (PRC) standard methods SN/T 1868-2007, GB13089-91, NY/T 1596-2008,

74 samples were immediately placed on ice, transported to the laboratory within 3 h of

75 collection, and centrifuged at 2,000 × g for 15 min in a refrigerated centrifuge at about

83 Egg samples were stored at -20 °C before freeze drying.

84 Hormone Levels and Blood Parameters.

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85 Serum glutamate pyruvate transaminase (GPT), glutamic-oxaloacetic

86 transaminase (GOT), triiodothyronine (T3), tetraiodothyronine (T4), and blood urea

87 nitrogen (BUN) were determined by colorimetric methods with a spectrophotometer

88 following the manufacturers’ protocols of the commercial kits (Nanjing Jiancheng

89 Institute of Bioengineering, Jiangsu, China).

90 Histopathology of Thyroid tissue.

94 examination by a pathologist. The stained slices were photographed using Motic

95 Microscope BA200 at 200 × magnification.

96 5-VOT and Thiocyanate Ions Analysis.

97 5-VOT was quantified by the high-performance liquid chromatography (HPLC)

105 0.45 µm filter membrane for HPLC analysis.

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107 autosampler (Hip-Als, model G1367C), thermostat column compartment (TQ-SL,

118 column of chromatogram is DIONEX AS 19 (4 × 250 mm).

119 Method Validation.

120 The validation parameters were consisted of linearity range, precision,

121 repeatability, recovery, and limit of quantification (LOQ). Method validation of

125 corresponding to analyte, as a function of the concentration introduced. The

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131 added to the sample (n = 4).

132 Statistical Analysis

141 time (week). X2 represented 5-VOT (µg/d.bird) or RSM intake (g/d.bird).