Long term effect of dietary glycerol monolaurate on laying performance, internal [617675]

Title: Long term effect of dietary glycerol monolaurate on laying performance, internal
and external egg quality in Hy-line brown laying he ns
Type of Manuscript: Article
Abstract
Objective: As is known to us all that laying perfor mance and egg quality of commercial laying hens are rapidly
declined during the late laying period. But recent studies show that many supplements enable to extend production
cycle and improve egg production in aged hens. The aim of this experiment was to evaluate the effect o f glycerol
monolaurate (GML) supplementation on the laying per formance and egg quality in hens at 40–64 weeks of age.
Methods: Three hundred and seventy-eight 40-week-ol d Hy-Line Brown laying hens were randomly assigned into
three groups with six replicates each (n = 21). The control group received a basal diet, and the treat ed groups fed
basal diets supplemented with 150 and 300 mg/kg GML . Results: The results revealed that laying rate an d egg mass
were increased in 150 and 300 mg/kg GML supplementa tion groups during the late laying period, and the feed
conversion ratio was decreased compared to the CON group. Laying related hormones including serum foll icle
stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were dramatically increased in 300 mg/kg (p
< 0.001) GML supplementation groups. Moreover, sign ificant (p < 0.05) increased eggshell strength (the 4th
month), eggshell thickness (the 4th and 6th month) and flavor amino acids in albumen were observed in both 150
and 300 mg/kg GML supplementation groups. Conclusio n: These findings demonstrated that dietary GML has the
potential to improve egg production and feed effici ency during the late laying period, meanwhile, it c ould enhance
both internal and external egg quality.
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Long term effect of dietary glycerol monolaurate on laying performance, internal 1
and external egg quality in Hy -line brown laying hens 2
3
Tao Liu1, Chuang Li3, Fengqin Feng1,2* 4
*Corresponding Author: Fengqin Feng 5
Tel:+86 -571-88982192 , Fax: +86 -571-88982192, E -mail: [anonimizat] 6
1College of Biosystems Engineering and Food Science, Zhej iang University, Hangzhou 7
310058, China 8
2Ningbo Institute of Zhejiang University, Ningbo 315100, China 9
3School of Biological Science and Engineering, Hebei University of Science and 10
Technology, Shijiazhuang 050018, China 11
ORCID 12
Tao Liu 13
https://orcid.org/000 0-0001 -6709 -5110 14
Chuang Li 15
https://orcid.org/0000 -0002 -4039 -8610 16
Fengqin Feng 17
https://orcid.org/0000 -0002 -9770 -1721 18
19
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Title of the manuscript: Long-term effect of dietary glycerol monolaurate on Hy -line 20
brown laying hens. 21
22
Abstract 23
Objective: As is known to us all that laying performance and egg quality of 24
commercial laying hens are rapidly declined during the late laying period. But recent 25
studies show that many supplements enable to extend production cycle and improve 26
egg production in aged hens. The aim of this experiment was to evaluate the effect of 27
glycerol monolaurate (GML) supplementation on the laying performance and egg 28
quality in hens at 40 –64 weeks of age. 29
Methods: Three hundred and seventy -eight 40 -week -old Hy -Line Brown laying hens 30
were random ly assigned into three groups with six replicates each ( n = 21). The 31
control group received a basal diet, and the treated groups fed basal diets 32
supplemented with 150 and 300 mg/kg GML. 33
Results: The results revealed that laying rate and egg mass were incre ased in 150 and 34
300 mg/kg GML supplementation groups during the late laying period, and the feed 35
conversion ratio was decreased compared to the CON group. Laying related hormones 36
including serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and 37
estradiol (E2) were dramatically increased in 300 mg/kg ( p < 0.001) GML 38
supplementation groups. Moreover, significant ( p < 0.05) increased eggshell strength 39
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(the 4th month), eggshell thickness (the 4th and 6th month) and flavor amino acids in 40
albumen w ere observed in both 150 and 300 mg/kg GML supplementation groups. 41
Conclusion: These findings demonstrated that dietary GML has the potential to 42
improve egg production and feed efficiency during the late laying period, meanwhile, 43
it could enhance both inte rnal and external egg quality. 44
Keywords: glycerol monolaurate; late laying period hens; laying performance; egg 45
quality; albumen amino acids composition. 46
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INTRODUCTION 47
Eggs are rich in protein, fatty acids, vitamins, and minerals, etc. and are considered as 48
an excellent source of animal protein. With the improvement of laying hens’ production 49
performance, the laying age of commercial laying hens has been extended from th e 50
original 72 weeks to 80 weeks, and layer’s production cycle has been extended to 100 51
weeks in some breeding companies [1]. However, egg quality is rapidly declined during 52
the late laying period, along with the lower commercialization rate caused by enlar ged 53
egg size, thinner egg shell, higher cracked egg rate (% cracks), decreased albumen 54
height, shortened egg storage time and loss of flavor [2, 3] . Recently, improving the egg 55
quality as well as egg production performance of late laying period hens have a ttracted 56
continuous increasing concern in poultry feed nutrition and food science research. 57
Presently, various studies have revealed that some supplements such as rubber seed oil 58
[4], peppermint [5], tea polyphenol [6], Lonicera confusa and Astragali Radix extracts 59
[7], have positive impact on egg production, feed conversion ratio, egg sensory quality 60
and nutritional quality. These findings provide scientists and farmers an effective 61
approach to extend production cycle and enhance egg production in aged hen s. 62
63
Glycerol monolaurate (GML), a naturally occurring glycerol monoester of lauric acid 64
(C12:0), rich in coconut oil, is recognized as a safe food emulsifie approved by the US 65
Food and Drug Administration, and is nontoxic from 10 to 2000 mg/kg (21 CFR GRAS 66
182.4505) in food and health care products [8]. GML has strong antibacterial effects 67
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especially against gram positive bacteria by suppressing the growth and virulence of 68
numerous bacteria, fungi, and enveloped viruses in vitro [9]. Besides, it can be util ized 69
directly by the enterocytes for energy production and thereby helps to support the 70
integrity of the intestinal tissue in broilers and piglets [10]. Therefore, GML is widely 71
investigated as feed supplements or antibiotic substitute in farmed animals su ch as 72
piglets, broilers and calves in order to improve growth performance, increase feed 73
efficiency, promote health and enhance quality [11-14]. According to the results from 74
Fortuoso et al. [14], dietary glycerol monolaurate (GML) at 100 –300 mg/kg obvious ly 75
increases average body weight, feed consumption and carcass yield in male Cobb 500 76
broilers without toxic side effect. Likewise, Mustafa reports that inclusion of GML in 77
feed significantly improves body weight gain, reduces feed conversion ratio, and 78
greatly enhances the immunological and nutritional status in Ros s 308 broilers [15]. 79
Similarly, a previous study conducted by our lab proves that dietary GML at 300 and 80
450 mg/kg in layer’s diet improves functional properties of egg albumen protein such 81
as the hardness of egg white protein gels, foaming capacity and stability, and thermal 82
stability [16]. 83
84
In our previous study, we find that dietary GML have beneficial effect on productive 85
performance, egg quality, serum biochemical indices, and intestinal morphology of 86
laying hens during 44 -52 weeks of age [16, 17] . However, whether dietary GML 87
affect the production performance and egg quality of hens during the whole late 88
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laying period and in which time it begins to affect warrants further study . Thus, thi s 89
study was conducted to investigate the continuous effect of dietary GML on laying hens 90
from the end of the peak laying period to late laying period so as to evaluate its 91
long-term influence on laying performance and egg quality. Moreover, GML is a 92
typica l representative of medium chain fatty acids 1 -monoglycerides (MGs), t hese 93
findings in this study will comprehensively provide theoretical evidence for the 94
efficacy of dietary MGs on layers production and egg quality control. 95
96
MATERIALS AND METHODS 97
Experiment design 98
Three hundred seventy -eight 40 -week -old Hy -Line Brown laying hens were randomly 99
assigned into three groups with six replicates each ( n = 21). Birds were housed in 100
three -tier battery cages (40 × 35 × 60 cm, with a floor slope of 12˚) shari ng a room 101
maintained at 25 ± 2 ° C and 60% – 65% humidity with a 16 -h photoperiod, each cage 102
including 3 birds. Seven sequential cages fed with same diet trough were arranged as a 103
replicate, and all replicates were equally distributed in different spatial d irections. Feed 104
(115 g/hen/d) and water were provided ad libitum, and feeding and egg collection were 105
conducted daily. 106
The experimental diets were formulated according to NRC 1994 recommendations for 107
laying hens and nutrient levels were showed in Table 1 . Birds were fed with basal diets 108
in mash form containing 0 mg/kg GML (CON), 150 mg/kg GML (GML150), 300 109
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mg/kg GML (GML300). GML with 95% purity acquired from Hangzhou Kangyuan 110
Food technology Co., Ltd (Hangzhou, China) and were included in the diet by 111
repla cing the same energy amount of oil. Birds were handled in accordance with the 112
guidelines of the Animal Care and Use Committee, Zhejiang University. 113
114
Data collection and sample preparation 115
During the experimental period, eggs laid were recorded daily on a r eplicate cage basis, 116
including eggs number, eggs weight, shell -less eggs, and cracked eggs. The laying rate, 117
average egg weight, and feed conversion ratio (FCR) were calculated per week, and the 118
statistical analysis was conducted every month (4 week). Bloo d samples were collected 119
from the wing vein. Serum samples were obtained by centrifugation (2000× g for 15 120
min at 4 ℃) and stored at −80 ℃ for further analysis. 121
At the end of 2nd, 4th and 6th month of the whole experimental period, thirty eggs per 122
treatmen t (5 eggs/replicate) were sampled, and were analyzed immediately for egg 123
quality, including haugh units (HU), albumen height, shell strength, shell thickness and 124
yolk percentage. After determining egg quality parameters, albumen and yolks of each 125
replicate (5 eggs) at the end of 6th month were collected and freeze -dried separately for 126
further analysis. 127
128
Egg quality determination 129
Shell thickness, shell strength, albumen height and haugh units were determined by a 130
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digital egg tester (DET 6000, NABEL Co., Ltd, Japan). After these measurements, the 131
yolk weights were weighed and the yolk percentage was calculated by the ration of yolk 132
weight to egg weight. 133
134
Serum parameters evaluation 135
Follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) were 136
measured using commercial ELISA kit (Catalog number: JYM0055Ch (FSH), 137
JYM0032Ch (LH) and JYM0049Ch (E2), Wuhan Colorful Gene biological technology 138
Co., Ltd. China). Total serum cholesterol (TC), triglycerides (TG), high -density 139
lipoprot ein cholesterol (HDL -C), low -density lipoprotein cholesterol (LDL -C), 140
glutamic oxalacetic transaminase (GOT), glutamic -pyruvic transaminase (GPT), 141
alkline phosphatase (AKP), calcium, total antioxigenic capacity (T -AOC), total 142
superoxide disumutase (T -SOD) and malondialdehyde (MDA) were determined using 143
kits from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China) by following the 144
manufacturer’s instructions. 145
146
Yolk fatty acids composition 147
Freeze -dried yolk samples were smashed and pass through a 40 -mesh sieve, then 0.10 g 148
dry mater (DM) was weighed out in duplicate into an ultra -sound cleaned culture tube. 149
To this 3.2 ml formyl chlorid (acetyl chloride : anhydrous methanol, 1:10; v/v) and 3.2 150
ml n -hexane containing 1 mg/ml C17:0, as an internal standa rd, were added prior to 151
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heating for 2 h at 75 ° C. Then 4 ml 7% potassium carbonate (v/v) was added and 152
centrifuged for 5 min at 3000× g. The organic solvent top layer was filtered through a 153
0.22-μm membrane filter prior to GC analysis [18]. 154
Fatty acid meth yl esters (FAME) were analyzed by a Shimadzu Gas chromatography 155
(GC-2014, Japan) with a FAME column (DB -23, 60 m×0.25 mm×0.25 μm, Agilent, 156
Santa Clara, CA, US) with split injection (30:1) and nitrogen at a constant flow of 0.65 157
ml/min as the carrier gas. F ID detector temperature and injector oven temperature was 158
set at 250 ° C and 230 ° C. The temperature program of the oven was: the initial oven 159
temperature was hold at 50 ° C for 1 min, then raised to 240 ° C at a rate of 1 ° C/min and 160
hold for 5 min. Identific ation of fatty acids and their response factors was aided with the 161
use of certified reference standards (37 component FAME Mix, Supelco 18919 -1AMP, 162
Sigma, USA) and quantified using the internal standard (C17:0). Fatty acids were 163
reported as mg/g dry materi al. 164
165
Albumen amino acids composition 166
Freeze -dried samples (20mg) were dissolved in 1ml 6M HCl in COD digestion tubes 167
under pure nitrogen atmosphere and heated for 2 h at 150 ° C in a digestion furnace. 168
Ten-microliter of digested samples or standard amino ac ids solution (Stock No. 169
AA-S-18, Sigma, USA) was pipetted into separated 1.5 -ml microcentrifuge tubes and 170
dried under vacuum. Then, a total of 20 μl derivatization reagent (ethanol: water: 171
triethylamine: phenyl -isothiocyanate, 7:1:1:1, v/v/v/v) was added a nd kept sealed for 172
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30 min at room temperature under nitrogen atmosphere [19]. The reagents were then 173
removed under vacuum. 174
The proposed solvent system consisted of two eluants. Solvent A was a solution of 20 175
mM sodium acetate containing 0.1% TEA (v/v) as m odifier. The pH was adjusted to 6.5 176
using glacial acetic acid, and the solution was filtered through a 0.22 -μm membrane 177
filter. Solvent B consisted of 20% water and 80% methanol. Before starting the gradient 178
for a certain run, the column was equilibrated for 30 min. Prior to HPLC (Waters 2695, 179
USA), the dried derivatized samples and standard amino acids were first dissolved in 180
50 μl solvent B and vortex mixed thoroughly. Then 450 μl solvent A was added and 181
vortex mixed thoroughly. The solution was filtered through a 0.22 -μm membrane filter 182
and ten -microliter was injected into the column (Ultimate AQ -C18, 4.6 mm × 250 mm 183
× 5 μm, Welch Co, Shanghai, China) by autosampler. Identification and quantification 184
of amino acids was aided with the certified reference standards (Amino Acids Mix 185
Solution, Supelco AA -S-18, Sigma, USA). Amino acids were reported as g/100g dry 186
material. 187
188
Statistical analysis 189
The data of results was collected individually, calculated for each treatment, checked 190
for normality test and express ed as mean ± standard deviation (SD). Statistical analysis 191
of the results was performed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). 192
Statistical differences between more than two groups were evaluated by one -way 193
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analysis of variance (ANOV A) wit h Tukey’s multiple comparison posttests. p-Value < 194
0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001) and 0.05 < 195
p-value < 0.10 was discussed as tendencies. 196
197
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RESULTS 198
Dynamic changes of egg production 199
As showed in Figure 1(a), the laying rate of CON group gradually decreased from 200
96.83% to 84.42% and the average egg weight ( Figure 1(b) ) rose gradually with 201
increasing weeks of age. The feed conversion rate (FCR) increased during 40 –52 weeks 202
of age, thereafter, the FCR tended to decreased t ill the end of the trail ( Figure 1(c)) . 203
Average egg broken rate showed continuous increase at the end of the trail ( Figure 1(d) ). 204
This plot visually displayed that dietary GML notably increased the laying rate and 205
reduced the feed conversion rate during 52 –64 weeks of age. Average egg weight and 206
broken rate were slightly improved by GML supplementation in the early experimental 207
period, but no significant differences were observed in the following experimental 208
period. 209
210
Laying performance 211
Laying performance f or all groups during the whole experiment was statistical 212
analyzed at an interval of four weeks and presented in Figure 2. Inclusion of 150 mg/kg 213
GML remarkably increased the laying rate at the 2nd (p < 0.05) 4th (p = 0.148), 5th (p = 214
0.219) and 6th (p < 0.05) month ( Figure 2(a) ), and the total egg mass for each month 215
increased by 2.42% ( p = 0.184), 3.97% ( p = 0.074), 2.48% and 4.34% ( p < 0.093) 216
(Figure 2(b) ), respectively. Moreover, the FCR of these periods sharply decreased by 217
5.12% ( p < 0.05), 2.89% ( p = 0.174), 2.07% ( p = 0.298) and 7.20% ( p = 0.063) ( Figure 218
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2(c)), separately. The changes of laying rate, total egg mass and FCR for 300 mg/kg 219
GML supplementation group were similar to that of 150 mg/kg GML group. The 220
broken rate at the first three months du ring this trail tended to increase in both 150 and 221
300 mg/kg GML supplementation group compared with the CON group, but no 222
differences were observed ( Figure 2(d) ). These findings indicated that dietary GML 223
started to affect the productive performance of laying hens at the 44 weeks of age and 224
lasted till the end of the experiment . 225
226
Egg quality 227
As showed in Figure 3 , the albumen height ( Figure 3(a) ) and eggshell thickness ( Figure 228
3(d)) of CON group were gradually reduced from the 2nd month to the 6th month, 229
indicating the decline of the egg quality in aged hens. In this trail, no significant 230
differences on albumen height, haugh units, and yolk percentage were observed, but the 231
eggshell strength (the 4th month, Figure 3(c) ) and thickness (the 4th and 6th mont h, 232
Figure 3(d) ) were significantly increased ( p < 0.05) in 150 and 300 mg/kg GML 233
supplementation group in relative to that of the CON group. 234
235
Serum parameters 236
Serum parameters of laying hens showed in Table 2 . Serum lipid profile including TG 237
(p < 0.01), TC ( p < 0.01), HDL -C and LDL -C (p < 0.05) in 300 mg/kg GML 238
supplementation group tended to increase in comparison with the CON group, whereas 239
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the changes of these indexes in 150 g/kg GML supplementation group were 240
insignificant, indicating that high levels of GML affected layer’s blood lipid 241
metabolism. Serum Ca in 150 and 300 mg/kg GML supplementation was 2.27% and 242
6.82% higher than that of the CON group. Total antioxidant capacity in 150 ( p = 0.206) 243
and 300 ( p < 0.05) mg/kg GML supplementation groups was enhanced. Serum FSH, 244
LH and E2 content in 150 mg/kg GML supplementation group increased by 25.77% 245
(32.32 vs 40.65 ng/ml), 27.23% (487.95 vs 620.84 pg/ml) and 18.13% (161.62 vs 246
190.92 pg/ml), separately, but no significant differences were observed in relat ive to 247
that of the CON group. However, FSH ( p < 0.001), LH ( p < 0.001) and E2 ( p < 0.001) 248
in 300 mg/kg GML supplementation group were 1.82, 1.57 and 2.12 times higher than 249
that of the CON group, respectively. The changes of s erum parameters in GML treated 250
hens indicated that GML supplementation affected the metabolic status of laying hens. 251
252
Yolk fatty acids composition 253
Fatty acids profile of yolk at 64 weeks of age showed in Table 3 . The yolk fatty acids 254
composition in CON group was mostly dominated by unsaturated fatty acids (UFA), 255
which accounted for more than 63.56% of total fatty acids with C18:1n9c (67.83%), 256
being the most representative. Palmitic acid (C16:0, 70.40%) and stearic acid (C18:0, 257
27.63%) fatty acids were the most abundant saturated fatty acids (SFA). No significant 258
differences were observed in all groups. 259
260
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Albumen amino acids composition 261
Protein amino acids composition and content of albumen at 64 weeks of age showed in 262
Table 4 . Compared to the CON group, the Asp, Glu, Ala, Arg and Val content in 150 263
mg/kg GML supplementation group increased by 42.48% ( p < 0.05), 21.33% ( p = 264
0.216), 12.23% ( p = 0.128), 18.65% ( p = 0.214) and 14.33% ( p = 0.202), respectively, 265
and the ch anges of these amino acids in 300 mg/kg GML supplementation group 266
showed the same tendency. Total amino acids content in 150 mg/kg and 300 mg/kg 267
GML supplementation group increased by 11.68% ( p = 0.197) and 5.27% compared to 268
the CON group, respectively, bu t no significant differences were observed. However, 269
the total flavor amino acids content including Asp, Glu, Ser, Gly, Ala and Thr in 150 270
(20.80 vs 26.18 g/100g) and 300 (20.80 vs 27.00 g/100g) mg/kg GML supplementation 271
groups increased significantly. These results suggested that dietary GML may change 272
the taste of eggs by affecting the composition of a lbumen . 273
274
DISCUSSION 275
Trails conducted by our previous study have demonstrated that dietary GML has the 276
potential to improve the productive performance, egg quality and serum biochemical 277
indices in laying hens during 44 -52 weeks of age [16, 17] . However, the present study 278
indicated that dietary GM L has long -term and consistent beneficial effect on laying 279
performance and egg external quality during 40 -64 weeks of age, and these 280
improvements were notable in 52 -64 weeks of age. Moreover, dietary GML was 281
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reported to affect the amino acids composition o f albumen, especially in the flavor 282
amino acids, suggesting that improving egg internal quality via optimal feed 283
supplements was feasible . 284
285
The dynamic changes of laying performance including l aying rate, egg weight, FCR 286
and broken rate (Figure 1) during 40 -64 weeks of age were in consistent with the 287
performance description of layers after peak egg -laying period , which was defined as 288
the late laying period. Hy-line brown laying hens commonly begin producing eggs at 289
18 weeks of age and continue f or over a year. After the onset of lay, egg production 290
generally increases for about 6 weeks to reach a maximum and maintains for around 20 291
weeks, which is described as peak egg -laying period [20]. Thereafter, the laying rate 292
gradually decreases with the i ncreasing weeks of age and the eggs tend to increase in 293
size along with thinner egg shell and increased egg broken rate. However, in the current 294
study, it's worth noting that obvious increases of laying rate after 52 weeks of age were 295
observed, which was l ikely to be in contradiction with common laying laws. This could 296
be explained by the extreme weather outside the farmhouse at this experimental time. It 297
was summertime of 44 -52 weeks of ag e, and the inside farmhouse temperature was a 298
little bit higher than designed , thus, t he continuous high temperature at this time 299
accelerated the reduction of laying rate. However, t he environment al temperature 300
gradually fell after 52 weeks of age as the autumn coming, which resulted in slightly 301
sustained increased laying rate till the end of the experiment. 302
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303
Statistical analysis revealed that dietary GML supplementation led to an sustained 304
increase in laying rate and egg mass, and a decrease in FCR during 40 -64 weeks of 305
age, which were similar to our previous reports of im provements in productive 306
performance by GML supplementation during 44 -52 weeks of age [17]. These results 307
were also in consistent with a previous study, where dietary MCFA improved 308
production performance and egg quality in laying hens [21]. Additionally, e gg quality 309
also showed significant improvement after GML supplementation . The eggshell 310
strength (the 4th month, Figure 3(c) ) and thickness (the 4th and 6th month, Figure 3(d) ) 311
were increased in 150 and 300 mg/kg GML supplementation group in relative to tha t of 312
the CON group. The Eggshell strength is an essential attribute for consumable eggs, 313
which is closely associated with high fragility and decreased feasibility for carriage and 314
storage. Moreover, fragile eggs cause economic damage and food safety concer ns 315
because even the occurrence of hair cracks raises the risk for bacterial contamination of 316
the broken egg and of other eggs when leaking, creating problems with internal and 317
external quality and food safety [22]. 318
319
Laying performance and egg quality are profoundly reflected by the serum parameters 320
of laying hens. The level of serum hormones including FSH, LH and E2 has been 321
considered as a sensitive indicator of laying performance. FSH is the main hormone 322
responsible for the development and maturation of small follicles, while LH mainly 323
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promotes the secretion of progesterone. E2 can promote follicular development via 324
feedback effects on the hypothalamus and pituitary [23]. Moreover, the main reason for 325
the decline in the egg production of laying hens in th e late laying period was the yolk 326
synthesis and accumulation decrease caused by decreased hormone concentrations [7, 327
23]. Therefore, the increased serum FSH, LH and E2 level resulted from dietary GML 328
may be the main reason for the significant increased lay ing rate in late laying period 329
hens (see Figure 1 and Figure 2 ). Similar results were reported by our previous study, 330
where dietary GML improved the laying rate via inducing the secretion of 331
gonadotropins including FSH and LH [17]. As demonstrated by Zhang et al., dietary 332
γ-aminobutyric acid significantly increased laying performance, with an increasing 333
contents of serum FSH, LH, and other growth hormones [24]. On the other hand , the 334
increased serum calcium content in 150 (1.32 vs 1.35 mM) and 300 (1.32 vs 1.41 mM, 335
p < 0.05) mg/kg GML supplementation group was associated with an improvement in 336
the eggshell thickness and strength of laying hens. As Świątkiewicz found that dietar y 337
MCFAs notably enhanced both eggshell density and eggshell breaking strength [25]. 338
339
The improvement s of laying performance and egg quality in GML supplementation 340
group s may be related to its unique physiological and biological properties. Compared 341
to long chain fatty acids (LCFAs), medium chain fatty acid s (MCFAs) are more easily 342
absorbed and metabolized due to their carnitine -independent transport into hepatic 343
mitochondria, which could provide rapid energy supply for extrahepatic organs in the 344
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body. As GML provided an immediate, extra source of energy sup ply, the expenditure 345
of protein as a source of energy was decreased and the protein synthesis of lying hens 346
was consequently increased [26]. This hypothesis was consistent with a previous study 347
where dietary MCT significantly increased the protein retentio n and the efficiency of 348
protein utilization [27]. Moreover, MCFAs were beneficial for intestinal health and 349
balance due to its strong antibacterial effects especially against gram positive bacteria 350
[10], which were also good for animal performance. In addition, animal growth was 351
also related to the gut microbiota alteration modulated by GML supplementation. 352
MCFAs supplementation had positive effects on fecal microbiota content and they had 353
more selective effects in the upper digestive tract and changed the composition of gut 354
microbiota . Thus, t he effects of GML on gut microbiota might play a key role in the 355
modulation of the metabolic status of laying hens. However, this needed further 356
analysis. 357
358
Egg quality is a comprehensive concept that encompasses both internal and external 359
quality. External egg quality consists of egg shape index, shell color, shell thickness and 360
strength, while internal egg quality is composed of yolk and albumen quality, suc h as 361
albumen height, yolk color and haugh unit, and determined by the composition of such 362
components [28]. In order to confirm whether GML supplementation changed the 363
internal egg quality, we further analyze the main composition of eggs. Dietary GML 364
had no effect on yolk fatty acid composition, though the blood lipid metabolism of 365
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laying hens was affected in laying hens. However, the amino acids composition of 366
albumen was notably changed in GML -treated g roups. Protein is made of amino acids, 367
and the amounts of protein and content and composition of amino acids could influence 368
the nutritive values and flavor of the eggs. Essential amino acid (EAA) content is an 369
important nutritive value index for eggs, while flavor amino acids (FAA) content play 370
an important role on the taste of eggs. In the current study, no significant differences of 371
EAA were observed in all groups , but the FAA content in the GML supplementation 372
groups increased significantly . In comparison with the CON group, the FAA content in 373
150 mg/kg an d 300 mg/kg GML supplementation groups increased by 25.87% and 374
29.81%, respectively. Moreover, the relative contents of total flavor amino acids 375
detected in the present study accounted for approximate 32.37% (in 150 mg/kg GML 376
supplementation group) in the total amino acids content . Furthermore , the relative 377
quantity of desirable amino acids possessing an umami (Glu and Asp), or sweet (Ser, 378
Gly, Ala and Thr) taste [29], and t he concentration of Asp, Glu and Ala in GML 379
supplementation group s increased notably . Therefore, the remarkable changes on these 380
amino acids could inevitably lead to the taste alteration of the eggs produced during late 381
laying period , especially in the umami and sweet taste of the albumen . In addition, the 382
total amino acids of 150 and 300 mg/kg GML supplementation group increased by 383
11.68% and 5.27% compared to the CON group, indicating that dietary also increased 384
the nutritive values of eggs. With the increasing of laying hen’s age, the albumen 385
height and HU decreased due to the decline of protein utilization efficiency in feed, and 386
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the sensory properties of eggs were also decreased [30]. Therefore, eggs produced by 387
conventional cages (modern p roduction system) in high rearing density , limited space 388
and extended egg production cycle are generally with poor taste [31]. The improvement 389
of nutritive values and taste of eggs derived from GML supplementation was of great 390
importance. 391
392
In this study, w e observed a long -term beneficial effect of dietary GML on laying rate, 393
egg mass and feed efficiency in laying hens during 40 -64 weeks of age. T his 394
improvement was related to the increase of laying related hormones including FSH, LH 395
and E2. Moreover, dieta ry GML had positive impact on both external egg quality 396
(eggshell thickness and strength) and internal egg quality (albumen amino acids 397
composition). These results provided us a new approach to increase egg production in 398
aged hens as well as to improve egg quality. Considering the laying performance, egg 399
quality and serum parameters , a supplementation level of 150 mg/kg GML addition is 400
recommended in the diet of late laying period Hy -Line Brown laying hens. 401
402
CONFLICT OF INTEREST 403
The authors declare that the y have no conflict of interest. 404
405
Acknowledgements 406
This work was supported by the Technology and Achievement Transformation Project 407
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of Hangzhou, China (Grant NO.20161631E01), Zhejiang University New Rural 408
Development Research Institute Agricultural Technology Promotion Fund (Grant NO. 409
2017ZDNT006), Key Pro ject of Natural Science Foundation of Zhejiang Province 410
(Grant No. LD19C200001). We're especially grateful for the Tingting Bu, Yanyun Ying 411
Jianli Wang, and all people participating in the preparations and analyses of this study. 412
413
Compliance with ethics requirements 414
All applicable international, national, and/or institutional guidelines for handling of the 415
animals were followed. 416
417
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Table 1 . Composition and nutrient level of the basal diet for 2 sub -trials (as fed -basis). 519
Ingredients, % 40–58 week of age 58–64 week of age
Corn 62.90 64.90
Soybean meal 23.55 21.48
Limestone 7.93 7.93
Salt 0.30 0.30
Fish oil 0.03 0.03
Rapeseed oil 0.59 0.66
1Premix 4.70 4.70
Total 100 100
Calculated values
2Metabolizable energy
(MJ/kg) 11.85 11.98
3Crude protein, % 16.10 15.80
Crude fat, % 2.86 2.91
3Calcium, % 3.73 3.78
Total phosphorus, % 0.60 0.57
Lysine, % 0.81 0.75
Methionine + Cysteine, % 0.65 0.60
Methionine, % 0.35 0.32
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1Provided the following per kilogram of diet: vitamin A, 10,000IU; vitamin D 3, 4000 IU, 520
vitamin E, 200 IU, vitamin B 1, 4 mg, vitamin B 2, 6 mg, vitamin B 6, 5 mg, vitamin B 12, 521
1 mg, vitamin K 3, 3 mg, biotin 0.5 mg, folic acid, 3.0 mg; D-pantothenic acid, 20mg; 522
nicotinic acid, 20 mg; Cu, 10mg; Fe, 100 mg; Mn, 100 mg; Zn, 100 mg Se, 0.40 mg. 523
2Values were calculated according to metabolizable energy of feedstuffs for poultry 524
provided by NRC 1994. 525
3The numbers were analyzed values 526
527
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Table 2 Effect of dietary GML on serum parameters in Hy -line brown laying hens. 528
Parameters CON GML150 GML300 SEM P-value
TG (mM) 10.57 13.88 17.86** 0.77 0.001
TC (mM) 4.96 5.194 6.78** 0.22 0.001
HDL -C (mM) 2.69 2.74 3.23 0.12 0.122
LDL -C (mM) 0.74 0.78 0.89* 0.08 0.038
Ca (mM) 1.32 1.35 1.41** 0.01 0.001
AKP (mM) 26.20 27.61 26.58 1.57 0.821
Glucose (mM) 16.55 15.47 18.43 0.54 0.074
Total protein (g protein/L) 28.64 29.86 29.21 0.46 0.562
T-AOC (U/ml) 364.83 435.56 457.92* 19.01 0.008
FSH1 (ng/ml) 32.32 40.65 91.30*** 12.34 0.001
LH2 (pg/ml) 487.95 620.84 1254.40*** 8.34 0.001
E23 (pg/ml) 120.39 147.58 404.29** 40.68 0.001
1Follicle Stimulating Hormone. 529
2Luteinizing Hormone. 530
3Estradiol. All data were expressed as means ± SD ( n = 18). Asterisk indicates differences 531
(*p < 0.05, ** p < 0.01, *** p < 0.001). 532
533
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Table 3 Effect of dietary GML on yolk fatty acids composition of Hy -line brown laying 534
hens. 535
mg/g dry material CON GML150 GML300 SEM P-value
C14:0 3.41 3.39 4.81 0.37 0.197
C16:0 122.12 122.40 123.59 1.89 0.952
C16:1 15.62 15.19 15.59 0.35 0.142
C18:0 47.93 43.93 43.90 1.03 0.194
C18: ln9c 205.24 202.77 206.01 2.96 0.907
C18: 2n6c 74.18 70.22 67.14 1.44 0.135
C24:1 7.91 5.94 7.24 0.56 0.363
SFA1 173.47 169.72 172.30 2.58 0.849
UFA2 302.57 294.13 295.98 4.34 0.730
MUFA3 228.39 223.90 228.84 3.57 0.840
PUFA4 74.18 70.22 67.14 1.44 0.135
SFA: UFA 0.57 0.58 0.59 0.01 0.108
Total FA5 476.04 463.85 468.27 6.79 0.781
1Saturated fatty acid (C14:0 + C16:0 + C18:0). 536
2Unsaturated fatty acid (C18: ln9c + C18:2n6c). 537
3Monounsaturated fatty acid (C18: ln9c). 538
4Polyunsaturated fatty acid (C18:2n6c); 5Total fatty acid. 539
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All data were expressed as means ± SD ( n = 6). Asterisk indicates differences (* p < 540
0.05, ** p < 0.01, *** p < 0.001). 541
542
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Table 4 Effect of dietary GML on albumen amino acids composition of Hy -line brown 543
laying hens at 64 weeks of age. 544
g/100g dry material CON GML150 GML300 SEM P-value
Asp1 5.32 7.58* 7.44* 0.41 0.018
Glu1 4.83 5.86 5.45 0.27 0.286
Ser1 3.50 3.88 3.29 0.14 0.247
Gly1 2.80 3.04 2.87 0.07 0.344
Ala1 5.23 5.87 5.69 0.15 0.166
Arg 3.86 4.58 4.37 0.19 0.270
His 1.58 1.67 1.60 0.05 0.784
Pro 5.33 5.50 5.02 0.14 0.460
Tyr 2.90 3.07 2.79 0.11 0.680
Ile2 5.16 5.53 5.35 0.13 0.514
Thr1,2 2.72 2.80 2.72 0.13 0.955
Val2 5.86 6.70 6.70 0.22 0.190
Met2 3.22 3.39 3.22 0.08 0.615
Leu2 8.29 8.83 8.50 0.17 0.441
Phe2 5.30 5.58 5.44 0.12 0.646
Lys2 5.44 6.08 4.89 0.25 0.198
Cys 0.71 1.99 1.40 0.39 0.476
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FAA1 20.80 26.18* 27.00** 0.99 0.050
EAA2 35.99 38.92 37.19 0.95 0.453
TAA3 72.42 80.88 76.24 2.18 0.271
1FAA, flavor amino acid (Asp, Glu, Ser, Gly, Ala, Thr). 545
2EAA, essential amino acid (Ile, Thr, Val, Met, Leu, Phe, Lys). 546
3TAA, total amino acid. 547
All data were expressed as means ± SD ( n = 18). Asterisk indicates differences (* p < 548
0.05, ** p < 0.01, *** p < 0.001). 549
550
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Figure captions 551
Figure 1. Dynamic changes of laying performance for Hy -line brown laying hens 552
during the whole experimental period. (a) Laying rate; ( b) Egg weight; (c) FCR; (d) 553
Broken rate. 554
555
Figure 2. Effect of dietary GML on laying performance in Hy -line brown laying hens 556
during 25 weeks of feeding. (a) Laying rate; (b) Egg mass; (c) FCR; (d) Broken rate. All 557
data were expressed as means ± SD ( n = 6). Asterisk indicates differences (* p < 0.05, 558
**p < 0.01, *** p < 0.001). 559
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560
Figure 3. Effect of dietary GML on egg quality of Hy -line brown laying hens. (a) 561
Albumen height; (b) Haugh units; (c) Shell strength; (d) Shell thickness. All data were 562
expressed a s means ± SD ( n = 6). Asterisk indicates differences (* p < 0.05, ** p < 0.01, 563
***p < 0.001). 564
565
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