| The investigations reported in this dissertation focused on pullets’nutrition.To obtain optimum production performance in laying hens,the birds must have proper development during the pullet phase,yet there is scarce information about this crucial phase in the modern strain of layer-type chicken after severe genetic selection for decades.Therefore,the effects of rearing dietary crude protein（CP）on the development of pullets,hypothalamus-pituitary-gonadal（HPG）axis,bone quality,and subsequent laying performance and egg quality were evaluated.Three experiments were conducted with the following aims:1)study the effects of pullets’dietary protein levels on the development of pullets and performance of laying hens;2)study the effect of protein regimen in the layer-pullet diet on the development of pullets and production performance of hens;3)study the interaction of pre-lay dietary protein and energy levels on the performance,egg quality,expression of genes in HPG axis,and bone parameters of laying hens.Experiment 1 was conducted to evaluate the effects of dietary CP levels on the growth performance and bone quality of pullets.The HPG axis at 18 wk was evaluated.384 Hy-Line Brown pullets at 8 wk of age were weighed and randomly assigned to one of the 3 dietary treatment groups（14%,16%,or 18%CP）.The energy level of all the pullet diets was the same（11.30 MJ/kg）.The pullets were offered with the grower diet for 10 wks（from 8 to 18 wk of age）and switched to the same pre-lay and laying diet till the end of the study（43 wk）.The birds were fed ad libitum throughout the study.Body weight（BW）was determined at 8,12,16,and 30 wk of age.Eight pullets per treatment were selected for sample collection at 12,15,and 18 wk.At 12wk of age,the BW of pullets in the 14%CP group was significantly higher（P<0.05）than in the other two groups,but BW at the end of the pullet phase（18 wk）was not affected（P>0.05）by the rearing CP levels.However,the BW of hens in the 14%CP group was lower（P<0.05）than in the 16%and 18%CP group at 30 wk of age.Tibia and femur length,width,index,and bending strength were not influenced by the dietary CP levels at 12,15,and 18 wk（P>0.05）.Similarly,the organ and muscle index of the birds at 12,15,and 18 wk of age were comparable（P>0.05）among the treatment groups.At 18 wk of age,dietary CP levels did not influence（P>0.05）the serum concentrations of calcium（Ca）,phosphorus（P）,calcitonin,estradiol（E2）,luteinizing hormone（LH）,while the parathyroid hormone and follicle stimulating hormone（FSH）decreased（P<0.05）in 14%CP group when compared to 18%CP group.Compared with the 16%CP group,the expression level of FSHR in the 14%CP group was down-regulated（P<0.05）.Low dietary CP during the pullet phase significantly increased the mortality rate in hens during the laying period（P<0.05）.The total number of reads generated from each hypothalamus sample using the Gallus gallus genome as a reference ranged from 43,918,252 to 55,030,150.A total of86 differentially expressed genes（DEGs）were identified in PB（16%）vs.PC（18%）,PB（16%）vs.PA（14%）,and PA（14%）vs.PC（18%）for the transcriptome study.Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis identified enriched GO terms and KEGG pathways,respectively;however,none was significantly enriched due to the few numbers of DEGs（P>0.05）.Laying performance was evaluated between 22 to 43 wk of age.At weeks 26,28,42,and 43,hens fed the 14%CP diet had significantly lower egg production than hens fed the 16%and 18%CP diets（P<0.05）.However,cumulative egg production was not influenced by the dietary CP levels（P>0.05）,suggesting that the low CP level during the pullet phase had a non-significant effect on laying performance.During wk 26,29,30,31,and 33 wk,compared to the other treatments,egg weight was lower for the 14%CP treatment（P<0.05）.In addition,the 14%CP had significantly lower egg mass than the other diet groups at weeks 33,42,and 43（P<0.05）.Dietary CP levels did not affect the Haugh unit at 28,34,and 41 wk of age（P>0.05）.Feed intake and feed conversion ratio（FCR）were comparable among the dietary treatment during the whole experimental period.In conclusion,these findings suggest that feeding a 14%CP diet during the rearing phase did not influence the growth performance and bone quality of pullets but had minimal effects on the HPG axis.Low dietary CP did not affect egg production and egg quality but increased the mortality of laying hens during the laying phase.Experiment 2 was designed to study the effects of pullets’dietary CP regimen on the performance,HPG axis,and bone quality of laying hens.A total of 384 9-wk-old Hy-Line Brown pullets were randomly distributed to one of the 3 dietary treatments（14-18%,16-16%,and 18-14%CP）with 8 replicates per treatment and 16 pullets per replicate.From 9 to 17 wk of age,the pullets in the 14-18%CP and 18-14%CP group were fed with 14%and 18%CP pullet diet,respectively,and from 18 to 21 wk,they were fed with 18%and 14%CP pre-lay diet,respectively.The pullets in the 16-16%CP group were fed with a 16%CP pullet diet from 9 to17 wk of age and a 16%CP pre-lay diet from 18 to 21 wk of age.The hens were fed with the same layer diet from 22 to 70 wk during the laying period.Performance was measured at 17 and21 wk.At 17 and 70 wk,24 birds（1 per replicate）were sampled for tibia and femur traits evaluation.At 21 wk,the development of the reproductive organ and HPG axis were evaluated.Blood samples were collected from randomly selected chickens（1 per replicate）for measurement of follicle FSH,LH,E2,and P4.Laying performance was evaluated till 70 wk of age.Eggs were collected at 28,32,36,and 70 wk of age for egg quality assessment.At the end of the 70 wk of age,8 laying hens per treatment were randomly selected for bone quality analysis.At 17 wk of age,the growth performance of the pullets was comparable among the dietary groups（P>0.05）.Compared with the 16%CP diet,the low CP（14%CP）diet significantly decreased the bone mineral density,bone strength and upregulated the m RNA expression of cathepsin K（P<0.05）.The serum concentration of ALP,osteocalcin（OC）,Ca,P,pyridinoline（PYD）,and bone ash were not influenced by the CP levels（P>0.05）.During the pre-lay period,the rearing CP regimen did not influence the growth performance,body composition（lean and fat percentage）,and ovarian development at the end of 21 wk of age（P>0.05）.The dietary treatments had no effect on the serum concentration of FSH and E2 of laying hens at 21 wk of age（P>0.05）.The serum concentration of LH was significantly higher in the 18-14%CP group compared to the other groups（P<0.05）,and the concentration of P4 in the 14-18%CP group was significantly higher compared to the other groups（P<0.05）.In the 18-14%CP group,ESR-1 was significantly downregulated in the hypothalamus and large white follicles.However,Gn RH,Gn IH（hypothalamus）,LH,FSH,Gn RH-R1,Gn IHR（pituitary）,LHR,and FSHR（large white follicle）,LHR,ESR-1 and FSHR（small white follicles）,were not differentially expressed.The dietary treatments significantly influenced the laying performance during some period of the study,such that the egg production and egg mass in the 18-14%CP group was significantly lower compared to the other two groups（P<0.05）.However,there was no significant difference in the egg weight among the treatments groups（P>0.05）.Also,compared to the other treatment groups,the total egg production tends to be lower（P=0.056）in the 18-14%CP group,and hens took a longer time to reach 50%egg production（P<0.05）,suggesting that laying hens are sensitive to CP level switch during the pre-lay stage.The egg quality analyses showed that the Haugh unit was significantly lower in the 14-18%CP group at 28 and34 wk of age（P<0.05）.However,there was no significant difference in the Haugh unit at subsequent times（P>0.05）.At 70 wk of age,the 14-18%CP group tends to have better shell quality,but the bone quality was similar among the treatment groups.Also,serum concentrations of ALP,OC,Ca,P,PYD,and bone ash were not affected by the CP regimen（P>0.05）.Feed intake and FCR were comparable during most of the period of the experiment（P>0.05）,and cumulative mortality was not influenced by the dietary CP regimen（P>0.05）.In conclusion,the results of this study indicated that a high protein diet tends to improve the bone quality of laying hens pullets.However,the slight improvement in bone quality during the pullet phase was annulled at the end of the laying cycle after a switch of diet during the pre-lay phase.The dietary CP regimen did not affect the performance of the pullets but had minimal effects on the HPG axis at 21 wk of age,which tended to adversely affect the subsequent laying performance in hens fed with the low CP diet during the pre-lay phase（18-14%CP group）.In experiment 3,the interaction of pre-lay dietary protein and energy levels on the performance during the laying period,egg quality,bone quality,and expression of genes in the HPG axis of laying hens at the end of the laying cycle was investigated.1,856 Hy-Line Brown pullets at 15 weeks of age were distributed randomly to 4 dietary treatment groups using a 2x2factorial arrangement with 2 metabolizable energy（11.30 and 11.72 MJ/kg,respectively）and 2CP levels（15%and 16.5%CP,respectively）.There were 4 replicates per treatment with 116birds each.The experimental diet was fed from 15 to 20 wk of age,and the same laying diet was fed afterward till the end of the experiment（72 wk）.At the end of the 72 wk of age,eggs were randomly collected for egg quality analysis,and 2 laying hens per replicate were randomly selected for evaluating the expression of genes in the HPG axis and bone quality.The laying performance,BW,muscle weight（leg and breast）,and organ index of the aged laying hens were not significantly influenced（P>0.05）by the dietary CP,ME,and their interaction.Egg shape-index and eggshell thickness were increased with the high pre-lay CP diet,while the high pre-lay energy diet increased the yolk color at 72 wk of age（P<0.05）.However,the Haugh unit and other egg quality traits were not influenced by the pre-lay protein and energy levels（P>0.05）.The diet with 11.30 MJ/kg-15%CP had a higher expression level of ESR-1 in the hypothalamus,ovary,large white follicles,and dominant follicles compared with the other groups.However,the circulating FSH,LH,P4,and E2 concentrations in the aged laying hens were not affected by the pre-lay diet（P>0.05）.The femur quality traits were comparable among the dietary treatment groups（P>0.05）.The tibia of hens fed with the 15%CP during the pre-lay phase was significantly longer than hens fed with the 16.5%CP（P<0.05）.Also,the tibia length increased in hens fed with the high pre-lay energy diet（P<0.05）;however,other tibia quality traits were not influenced by the dietary treatments（P>0.05）.These results suggest that pre-lay protein and energy levels had minimal effect on egg quality characteristics and tibia parameters but had no effect on the laying performance,BW,muscle weight,and organ index at the end of lay.These results showed that the pre-lay protein and energy levels minimally altered the HPG axis genes at the end of the laying cycle,but the concentration of the circulating sex hormone was unchanged.Conclusively,the findings of this study suggest that some slight experiment to experiment variation exists on some parameters in studies with dietary CP levels based on the timing,duration,and time of sampling in the experiments.Pullets fed with 14%CP during the rearing phase had a higher mortality rate during the laying phase;however,in the dietary CP regimen study,switching to 18%CP at the pre-lay phase in the 14-18%CP group prevented this.The dietary CP levels ranging from 14%to 18%had no detectable influence on the bone quality in laying hens.Dietary CP level during the rearing phase had no unbeneficial effect on the development of the HPG axis.It is recommended to feed pullets during the grower and pre-lay phase with 15%CP to 16%CP and 11.30 MJ/kg ME diet without a further increase to 16.5%or18%since there was no difference in the production performance and bone quality when fed with higher energy and protein level. |
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