
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00703-X
10.1016/j.psj.2024.104124
104124
METABOLISM AND NUTRITION
The influence of breed, dietary energy and lysine on laying persistency and body composition of laying hens
van Eck Lieske lieske_van_eck@cargill.com
*†1
Chen Hsuan *
Carvalhido Ines *
Enting Henk *
Kwakkel Rene †
⁎ Cargill Animal Nutrition Innovation Center, NL-5334 LD, Velddriel, the Netherlands
† Department of Animal Sciences, Wageningen University, NL-6700 AH, Wageningen, The Netherlands
1 Corresponding author lieske_van_eck@cargill.com
07 8 2024
11 2024
07 8 2024
103 11 10412422 3 2024
24 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The effect of dietary energy and lysine levels on laying persistency and body composition in brown and white hens was studied. Dietary treatments with 2 Metabolizable Energy levels (ME lay; constant or reduction over time) and 2 apparent fecal digestible Lys levels (AFD Lys; constant or reduction over time), were fed to Lohmann white or brown hens, from 17 to 75 wk of age, in a 2 × 2 × 2 factorial design. Data were subjected to mixed model analyses. The egg production curve was modeled using a non-linear regression function. White hens showed an improved laying persistency and a higher number of total eggs per hen compared to brown hens, indicated by a shorter peak production phase but a significant lower slope of decline after peak (P < 0.05). Similarly, hensfed a reduced instead of a constant ME Lay diet had a better laying persistency, indicated by a shorter peak production phase and a significant smaller slope of decline after peak (P < 0.05). This improved laying persistency was probably related to a higher ADFI and nutrient intake of hens fed the reduced ME Lay diets (P < 0.05). A segmented regression analysis showed reasonable correlation between egg mass production and ME Lay intake, with an R2 adjusted of 0.78 for the overall model. The egg mass production was not increased for intakes above 330 kcal of ME Lay intake for white hens and 324 kcal for brown hens. Limited effects of dietary treatments on body crude fat and crude protein were found. Both energy and protein requirement seemed to increase over time, indicated by higher voluntary nutrient intake and a reducing body crude fat after wk 43 in all treatments. The egg mass production correlated poorly with the AFD Lys intake, with an R2 of 0.22. In conclusion, laying persistency was mostly influenced by breed and dietary ME Lay level, but not dietary AFD Lys level.

Key words

body composition
laying hen
laying persistency
nutrition
==== Body
pmcINTRODUCTION

During the domestication process of the red jungle fowl (Gallus gallus) over the last 6,000 to 8,000 years, selection has influenced hen egg production rhythm and potential. It shifted from seasonal breeding of 5 to 7 eggs per clutch to continuous production, with a current potential of 500 eggs at 100 wk of age (Anderson, 2019; Hata et al., 2021). During an initial peak production phase, laying hens produce almost 1 egg per day after which a period of egg production decline follows, including more days without egg laid and an increased length in the number of consecutive days without production (Lillpers and Wilhelmson, 1993). To support laying persistency, it could be reasoned that the peak of egg production should be prolonged (Grossman et al., 2000) and/or the slope of egg production decline should be reduced, but it has not been studied yet which scenario has a bigger impact or how to achieve this with current commercial breeds. The long-term egg production process is enabled by nutrient supply and influenced by the ovary and oviduct to produce the eggs, the liver to metabolize most nutrients and body fat reserves to supply part of the nutrients and potentially influence reproductive capacity (van Eck et al., 2023). Nutrient requirements change over time due to a reduction in laying rate, an increase in egg weight and an increase in maintenance requirements due to higher body weights, with potentially a difference in body composition as well. These changes require a shift in feeding strategies over time to support long-term egg production. We only found few recent, long-term studies (exceeding 40 wk) published investigating the nutritional requirements for laying persistency. In a 50 wk trial studying the effect of diet density (both energy and protein) on long-term egg production in Hyline W36 hens, it was found that higher dietary nutrient levels linearly increased egg production mostly during the second phase of lay after wk 50 (DePersio et al., 2015). The hens in this study were not able to maintain energy intake with lower density diets (2,711 kcal/kg), resulting in higher nutrient intake levels in the higher density diet treatments (2,977 kcal/kg). In another study with brown hens, a higher energy diet (2,680 kcal/kg vs. 2,780 kcal/kg) resulted in higher energy intake, resulting in higher egg weights but not egg production in wk 23 to 59 (Scappaticcio et al., 2021). Increasing digestible lysine from 0.68% to 0.80% also increased egg weights, but not egg production. Contrary, in a 94 wk trial with Lohmann LSL Lite hens, a higher dietary protein and digestible lysine level increased feed intake, egg production and egg weights from early lay and lasting until the end of the study (Da Nóbrega et al., 2022). So in general, long-term feed studies show improved egg weights or egg production when dietary energy, and possibly dietary amino acids, are increased.

None of the published long-term studies have included a model analysis on the egg production curve to understand in which phase of egg production nutrients can have the highest impact on laying persistency. This study therefore examined the effect of dietary energy and amino acid levels, supplied in constant levels or with a reduction of levels over time, on long-term egg production and body composition in a white and brown breed of laying hen. The energy and lysine levels in the constant treatments were chosen to meet minimum requirements during the peak production phase targeting 707 mg lysine /hen/day (Silva et al., 2015). The hens were fed ad libitum, so actual intake levels could vary. Egg production data was modeled to measure the influence of diet on the duration of peak production and the slope of egg production decline. Body composition was measured to study the energy and protein balance and examine the influence on laying persistency. We hypothesized that a reduction of dietary energy over time would increase feed intake and therefore amino acid intake, with a positive effect on the duration of peak production and persistency, resulting in hens with lower body fat levels, independent of the breed.

MATERIALS AND METHODS

All procedures were approved by the Animal Welfare Committee of the Cargill Innovation Center (Velddriel) in accordance with Dutch laws and regulations on the execution of animal experiments (AVD220002016765).

Animals and Housing

Dietary treatments with 2 Metabolizable Energy lay levels (ME; constant or reduction over time) and 2 apparent fecal digestible Lys levels (AFD; constant or reduction over time), were fed to Lohmann white and brown hens from 17 to 75 wk of age. A total of 576 Lohmann LSL-Classic white laying hens and 576 Lohmann Brown-Classic laying hens were obtained at 17 wk of age from a commercial breeder (Nieuwenhuizen Heesch, The Netherlands). All hens were housed in the same free-range rearing facility and received the same diet until the trial started. At 17 wk of age, the hens were randomly divided over 48 floor pens, separated per breed, with 23 hens per pen, at the Cargill Animal Nutrition Innovation Center (Velddriel, Netherlands). The pens were divided over 8 blocks with 6 pens per block; treatments were randomly allocated within block. Pens were 2.58 m2 with a deep littered floor covering 33% of the pen and an elevated floor with plastic slats covering 66% of the pen. Nest boxes of 120 cm in length and perches with at least 15 cm per hen were available per pen. Feed and water were provided ad libitum during the entire trial. Light and temperature schedules followed the Lohmann breeder recommendations for the white hens during the entire trial (Lohmann Tierzucht, 2018).

Diet Formulation

Breeder guidelines (Lohmann Tierzucht, 2018) were used for minimum levels of calcium, phosphorus, sodium, chloride and the ideal protein ratio considering methionine + cysteine, arginine, valine, tryptophane, threonine and isoleucine in ratio to lysine. Dietary ME Lay and AFD Lys followed the trial design (Table 1). Feed was provided in mash form in 4 phases including pre-lay (wk 17-20, one standard diet), pre-peak (wk 20–27), peak (wk 28–45) and post-peak (wk 45–75). Between treatments and feeding phases, major raw material shifts were avoided by assuring a minimum similar inclusion level of 80% of each raw material between treatments. Before diet formulation, batches of the main ingredients were reserved and analyzed in accordance with standard laboratory methods for: dry matter (DM; ISO 6496, 1999), crude protein (CP; ISO 16634-1, 2008), c rude fat (CF; ISO 6492, 1999), ash (ISO 5984, 2002), calcium (ISO 27085, 2009) and phosphorus (ISO 27085, 2009). Diet formulation was based on digestibility and nutrient calculations provided by CVB (2016), based on the analyzed nutrient levels of the raw materials. The diets were produced by Research Diet Services (Wijk bij Duurstede, The Netherlands). Diet composition and analysis are given in Table 2. The diet analysis was in reasonable accordance with formulated values, except for calcium and phosphorus in some phases. The analyzed calcium levels showed some differences with formulated levels, with a minimum of 95% and a maximum of 117% relative to the formulated values. Moreover, the P analysis showed a lower result than expected of 77% compared to formulated values in the final phase of the study. It is expected that these results did not influence any of the performance parameters, but they might have influenced eggshell quality. Eggshell quality was not assessed in this study.Table 1 Overview of the treatment structure and dietary ME Lay and AFD Lys levels in all periods.

Table 1Treatment description	Wk 17 to 20	Wk 20 to 28	Wk 28 to 45	Wk 45 to 75	
ME Lay, kcal	AFD Lys, %	ME Lay, kcal	AFD Lys, %	ME Lay, kcal	AFD Lys, %	ME Lay, kcal	AFD Lys, %	
Brown; constant ME Lay1; constant AFD Lys2	2800	0.72	2800	0.72	2800	0.72	2800	0.72	
Brown; constant ME Lay; reduction AFD Lys	2800	0.72	2800	0.66	2800	0.62	2800	0.58	
Brown; reduction ME Lay; constant AFD Lys	2800	0.72	2725	0.72	2675	0.72	2625	0.72	
Brown; reduction ME Lay; reduction AFD Lys	2800	0.72	2725	0.66	2675	0.62	2625	0.58	
White; constant ME Lay; constant AFD Lys	2800	0.72	2800	0.72	2800	0.72	2800	0.72	
White; constant ME Lay; reduction AFD Lys	2800	0.72	2800	0.66	2800	0.62	2800	0.58	
White; reduction ME Lay; constant AFD Lys	2800	0.72	2725	0.72	2675	0.72	2625	0.72	
White; reduction ME Lay; reduction AFD Lys	2800	0.72	2725	0.66	2675	0.62	2625	0.58	
1 Metabolizable energy, calculated according to CVB (2016).

2 Apparent fecal digestible, calculated according to CVB (2016). The ideal protein ratio was kept at minimum required levels in accordance with the breed guidelines (Lohmann Tierzucht, 2018).

Table 2 Ingredient and nutrient composition of the experimental diets.

Table 2Feeding phase	Wks 17 to 20	Feeding phase, wks 20 to 28	Feeding phase, wks 28 to 45	Feeding phase, wks 45 to end	
ME1 Lay	All	Constant	Reduction	Constant	Reduction	Constant	Reduction	
AFD2 Lysine		Con stant	Reduction	Con stant	Reduction	Con stant	Reduction	Con stant	Reduction	Con stant	Reduction	Con stant	Reduction	
Ingredients composition, g/kg	
 Corn	381	365	379	403	418	350	372	399	424	304	330	382	416	
 Wheat	200	200	200	200	200	200	200	200	200	200	200	200	200	
 Soybean meal	192	202	176	215	188	202	159	218	172	202	139	225	159	
 Sunflowermeal	80	80	80	35	46	80	80	35	56	80	80	35	66	
 Wheat middlings	61	20	31	10	10	37	58	10	10	82	120	10	10	
 Soybean oil	17	19	19	18	18	18	17	21	20	15	13	21	21	
 Limestone	46	92	92	94	94	90	90	92	92	95	95	102	102	
 Monocalcium-phosphate	7.3	7.6	7.7	8.3	8.4	7.6	7.7	8.4	8.6	6.1	6.3	7.4	7.7	
 Sodiumbicarbonate	2.5	2.1	2.1	2.0	2.0	2.1	2.2	2.0	2.1	2.0	2.0	1.9	2.0	
 Salt	1.9	2.0	2.0	2.0	2.0	2.0	1.9	2.0	2.0	1.8	1.7	2.0	2.0	
 Potassium carbonate 52%	0.0	0.0	0.7	0.5	1.3	0.0	1.1	0.9	2.1	0.0	1.9	1.6	3.6	
 DL-Methionine	0.6	1.6	1.3	1.8	1.4	1.6	1.0	1.8	1.2	1.5	0.6	1.7	0.8	
 NSP enzymes3	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	
 Phytase4	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	
 Premix5	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	10.0	
Calculated chemical composition	
 Dry matter	%	87.82	88.49	88.43	88.46	88.44	88.43	88.37	88.46	88.48	89.45	89.41	89.58	89.63	
 Crude protein	%	17.90	17.59	16.64	17.07	16.18	17.61	16.02	17.05	15.59	17.65	15.45	16.87	14.85	
 Crude fat	%	4.00	4.00	4.00	4.00	4.00	3.70	3.70	4.00	4.00	3.50	3.50	4.00	4.00	
 Ash	%	8.40	12.76	12.76	12.92	12.95	12.74	12.74	12.90	12.93	13.30	13.35	13.90	13.97	
 Calcium	%	2.00	3.73	3.73	3.83	3.83	3.73	3.73	3.83	3.83	3.85	3.85	4.11	4.11	
 Phosphorous	%	0.57	0.53	0.53	0.51	0.51	0.55	0.55	0.51	0.51	0.58	0.58	0.55	0.55	
 Available Phosphorous	%	0.38	0.38	0.38	0.39	0.39	0.38	0.38	0.39	0.39	0.36	0.36	0.38	0.38	
 Sodium	%	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.15	0.15	0.16	0.16	
 Potassium	%	0.83	0.79	0.79	0.79	0.79	0.80	0.80	0.80	0.80	0.92	0.92	0.92	0.92	
 Chloride	%	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.16	0.15	0.15	0.16	0.16	
 ME lay	kcal	2800	2725	2725	2800	2800	2675	2675	2800	2800	2625	2625	2800	2800	
 AFD Lysine	%	0.720	0.720	0.660	0.720	0.660	0.720	0.620	0.720	0.620	0.720	0.580	0.720	0.580	
 AFD Methionine	%	0.319	0.408	0.369	0.416	0.376	0.408	0.334	0.416	0.342	0.397	0.290	0.410	0.302	
 AFD Meth.+Cyst.	%	0.580	0.660	0.610	0.660	0.610	0.660	0.570	0.660	0.570	0.650	0.520	0.650	0.520	
 AFD Threonine	%	0.540	0.534	0.500	0.520	0.490	0.533	0.476	0.520	0.470	0.530	0.451	0.516	0.447	
 AFD Tryptophan	%	0.185	0.181	0.170	0.176	0.165	0.182	0.163	0.176	0.157	0.184	0.158	0.175	0.149	
 AFD Isoleucine	%	0.645	0.641	0.599	0.624	0.586	0.638	0.568	0.624	0.561	0.634	0.535	0.620	0.534	
 AFD Valine	%	0.728	0.715	0.674	0.692	0.656	0.715	0.647	0.692	0.632	0.714	0.620	0.686	0.603	
 AFD Arginine	%	1.060	1.044	0.976	0.993	0.935	1.043	0.932	0.992	0.896	1.054	0.900	0.989	0.858	
Analyzed chemical composition	
 DM	%	87.77	88.37	88.33	88.33	88.10	88.63	88.81	88.92	88.89	89.44	89.34	89.55	89.43	
 Crude protein	%	17.19	17.90	16.87	17.12	15.17	18.86	15.76	17.62	16.69	18.60	16.05	16.73	15.22	
 Crude fat	%	3.92	3.70	3.70	3.80	3.30	3.80	3.30	3.90	4.00	3.50	3.20	4.20	4.00	
 Calcium	%	2.30	4.01	3.61	3.65	3.73	3.98	3.74	3.65	4.35	3.81	4.25	4.36	4.21	
 Phosphorous	%	0.60	0.40	0.51	0.51	0.51	0.60	0.58	0.47	0.55	0.55	0.53	0.42	0.43	
1 Metabolizable energy, calculated according to CVB (2016).

2 Apparent fecal digestible, calculated according to CVB (2016).

3 Hostazym X 15000, Huvepharma.

4 Phyzyme XP 10000 TPT - 500 FTU, Danisco.

5 Supplied per kg diet rearing: Vitamin A (retinyl-acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3,500 IU; vitamin E (DL-α-tocopherol), 100 mg; vitamin K3 (menadione), 3.0 mg; vitamin B1 (thiamine), 3.0 mg; vitamin B2 (riboflavin), 6.0 mg; vitamin B6 (pyridoxine-HCL) 3.0 mg; vitamin B12 (cyanocobalamine), 20 μg; niacine, 35 mg; D-pantothenic acid, 15 mg; choline chloride, 600 mg; folic acid, 1.5 mg; biotin, 150 μg; Fe, 40 mg; Cu, 16 mg; Mn, 120 mg; Zn, 110 mg; I, 0.8 mg; Se, 0.3 mg.

Supplied per kg diet lay: Vitamin A (retinyl-acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,000 IU; vitamin E (DL-α-tocopherol), 25 mg; vitamin K3 (menadione), 1.5 mg; vitamin B1 (thiamine), 1.0 mg; vitamin B2 (riboflavin), 3.5 mg; vitamin B6 (pyridoxine-HCL) 1.0 mg; vitamin B12 (cyanocobalamine), 15 μg; niacine, 30 mg; D-pantothenic acid, 12 mg; choline chloride, 350 mg; folic acid, 0.8 mg; biotin, 100 μg; Fe, 50 mg; Cu, 10 mg; Mn, 60 mg; Zn, 54 mg; KI, 0.7 mg; Se, 0.1 mg.

Data Collection

Individual weights of all hens were measured and based on BW, ADG was calculated as follows: (BW at the end of a period – BW at the start of a period) / total days in the period. On the same day as the BW measurements, total feed intake per pen was measured to calculate ADFI per hen: total feed intake / number of pullets per pen corrected for mortality Eggs were collected per pen and classified according to first class eggs or second class eggs (including broken eggs, dirty eggs, shell less eggs, double yolk eggs, floor eggs or other). Each wk all eggs laid on 1 d were collected and weighed. Laying rate was calculated as total eggs produced per pen / the number of hens corrected for mortality. Egg mass was calculated as laying rate multiplied by average egg weight (EW) of first class eggs. Feed conversion ratio (FCR) for egg mass was calculated as: ADFI/egg mass.

In wk 66, 4 eggs per experimental unit were randomly selected and egg quality was determined. All eggs were weighed individually, broken and the albumen and yolk were separated and weighed. Haugh Units were determined using a Digital Egg Tester 6500 (Nabel Co.,Ltd. Kyoto, Japan), by measuring the height of the thick albumen and the egg weight. Lastly, albumen pH was measured.

Every 4 wk on the days that BW was measured, 1 hen per pen was randomly selected for dissection. Hens were euthanized by using CO2 and weighed. The whole hens, including feathers, were individually stored in a freezer and cut into pieces with a saw and ground. A subsample of approximately 100g of the ground material were taken and freeze dried. Freeze dried material was analyzed for a full body composition analysis for DM (ISO 6496, 1999), CF (ISO 6496, 1999), CP (ISO 6496, 1999) and ash (ISO 6496, 1999). The weight of each body component was calculated based on the analyzed results and the dissected BW, and expressed as percentage of total body.

Statistical analysis

Data were analyzed using pen as the experimental unit. Model assumptions for normality and equal variance of the error terms were checked by inspection of the residual plots. Data were subjected to mixed model analyses, using R version 4.1.1 (R Core Team, 2013). A completely randomized block design with 8 treatments arranged as a 2 × 2 × 2 factorial design was used:(1) Yijk=μ+αi+βj+γk+αβij+αγik+βγjk+αβγijk+Bl+εijkl

where Yij = dependent variable, μ = overall mean, αi = ME Lay effect (i = constant or reduction), βj = AFD Lys effect (j = constant or reduction), γk = breed effect (k = white or brown), αβij = interaction effect between ME Lay and AFD Lys, αγik = interaction effect between ME Lay and breed, βγjk = interaction effect between AFD Lys and breed, αβγijk = interaction effect between ME Lay, AFD Lys and breed, Bl = random block effect (I = 1 - 8) and εijkl = residual error. Data were expressed as least square (LS) means and effects were considered to be significant when P≤0.05.

Model for Laying Persistency

To be able to quantify laying persistency, the egg production curve was modeled using the following function (Grossman et al., 2000):yt=r*(ypt2−t1)*[Ln(et/r+et1/r1+et1/r)−Ln(et/r+et2/r1+et2/r)]+rb4Ln(et/r+e(t2+P)/r1+e(t2+P)/r)

Where yt = egg production in percentage at time t, t1 and t2 = times at transition in wk, r = duration of transition constant = 0.3, yP = level of constant peak production in percentage, b4 = rate of decline in production in percentage, and P = persistency of constant production in wk. The curves were fitted using the nls function in the stats package of R. Model parameter estimates were evaluated using goodness of fit criteria. These criteria included: Pearson correlation coefficient, Durbin-Watson statistic and the Bayesian information criterion (BIC). The model parameters and goodness of fit were determined for all data combined and for the main effects separately. To compare the model parameters between treatments, a one sample t-test was used.

Segmented Regression Model

To determine the relationship between egg mass production and ME Lay intake and AFD Lys intake, inflection points were determined as described by van Eck et al. (2024):(2) yx=α0+βixi+∑j=1n−1βj+1(xi−γj)*I(xi>γj)

Where yx = egg mass production (g), xi = nutrient intake (ME Lay, kcal and AFD Lys, mg), a0 = intercept, ßi and ßj = slope at phase i or j, respectively, ɣj = the estimated inflection point between phase 1 and 1+1, I=1 when xi > ɣj and 0 otherwise, and n = number of phases. Curves were fitted using a segmented regression procedure in R (Muggeo, 2008). Model parameter estimates and number of inflection points were evaluated using goodness of fit criteria as described above, and an F-test comparing the number of inflection points ranging from 0 to 2. Initially, the model parameters and goodness of fit were determined for all data without treatment effect and in a next step, the model parameters were determined for each main effect separately. To compare the model parameters between main effects, a one sample t-test was used.

RESULTS

Intake and Growth

Brown hens were significantly heavier at the start of the study in wk 17 (P < 0.05; BW brown hens = 1.47 ± 0.09 kg and BW white hens = 1.19 ± 0.06 kg) and this difference remained relatively constant throughout the entire trial. There were no differences in ADFI in wk 17 to 20.

No 3-way interaction effects and only one 2-way interaction effect on ADFI, ME Lay intake and AFD Lys intake were found, so only main effects will be discussed (Table 3). In wk 27 to 35, brown hens had a higher ADFI than white hens, resulting in a higher ME Lay and AFD Lys intake (P < 0.05). After wk 35 until the end of the study, white hens had a higher ADFI (P < 0.05) than brown hens, with the difference increasing over time adding up to a difference of 18.7 grams in wk 71 to 75. As a result, the ME Lay and AFD Lys intake were also higher in the white hens from wk 35 onwards (P < 0.05). Apart from breed, also dietary ME Lay influenced ADFI, with significantly lower ADFI when hens were fed the constant ME Lay diets in all periods. This resulted in similar ME Lay intake levels between treatments in all periods, but significantly lower AFD Lys intake of hens fed the constant ME Lay diets. Dietary AFD Lys did not impact ADFI, and therefore resulted in higher AFD Lys intake of hens fed the constant AFD Lys treatments compared to hens fed the reduced AFD Lys treatments (P < 0.05). As the difference in AFD Lys between treatments became larger towards the end of the study, the AFD Lys intake increased relatively more in the hens fed the constant AFD Lys diets compared to the reduced AFD Lys diets.Table 3 Effects of breed, dietary ME Lay and AFD Lys on average daily feed intake, ME Lay intake and AFD LYS intake (g), expressed as least squares means.

Table 3			Average daily feed intake, g	ME lay intake, kcal	
			20 to 27	27 to 35	35 to 45	45 to 55	55 to 63	63 to 71	71 to 75	20 to 27	27 to 35	35 to 45	47 to 55	55 to 63	63 to 71	71 to 75	
Breed	ME Lay1	AFD Lys2															
Brown	Con	Con	105.3	117.8	119.0	117.7	117.9	118.3	120.0	294.9	329.9	333.1	329.5	330.2	328.8	336.1	
Brown	Con	Red	104.6	118.3	122.4	122.8	123.6	123.5	127.5	293.0	331.2	342.6	343.7	346.2	340.2	339.3	
Brown	Red	Con	107.6	123.4	125.6	126.7	132.2	139.2	144.7	295.2	332.0	336.1	332.6	346.9	358.2	373.1	
Brown	Red	Red	109.5	122.7	127.3	127.2	133.5	135.4	137.9	298.3	330.0	340.6	333.9	350.4	352.1	362.0	
White	Con	Con	104.4	113.9	121.9	123.6	130.6	141.2	152.2	293.0	322.6	341.4	341.5	365.6	380.0	421.7	
White	Con	Red	104.8	116.3	126.6	127.3	139.0	142.4	144.6	293.3	321.8	354.4	353.4	371.5	385.0	394.5	
White	Red	Con	110.4	120.5	133.9	134.4	145.7	152.5	152.9	301.0	319.6	352.2	341.7	368.9	396.3	401.4	
White	Red	Red	110.7	122.0	128.4	130.4	138.5	150.4	155.2	299.4	327.7	343.3	342.4	363.5	388.5	407.4	
SEM (n = 6)			1.3	1.5	1.9	2.9	4.4	4.6	5.9	3.3	4.0	4.7	6.1	11.9	14.0	16.7	
Breed effect	
Brown	106.8	120.5	123.6	123.6	126.8	129.1	132.5	295.4	330.8	338.1	334.9	343.4	344.8	352.6	
White	107.6	118.2	127.7	128.9	138.4	146.6	151.2	296.7	322.9	347.8	344.8	367.4	387.4	406.2	
SEM (n = 24)	0.7	1.0	1.1	1.6	2.2	2.4	2.9	1.6	2.4	2.6	3.0	5.5	7.6	8.6	
ME Lay effect	
Constant	104.8	116.6	122.5	122.8	127.8	131.4	136.1	293.6	326.4	342.9	342.0	353.4	358.5	372.9	
Reduction	109.6	122.1	128.8	129.7	137.5	144.4	147.7	298.5	327.3	343.0	337.7	357.4	373.8	386.0	
SEM (n = 24)	0.7	1.0	1.1	1.6	2.2	2.4	2.9	1.6	2.4	2.6	3.0	5.5	7.6	8.7	
AFD Lys effect	
Constant	106.9	118.9	125.1	125.6	131.6	137.8	142.5	296.0	326.0	340.7	336.4	352.9	365.8	383.1	
Reduction	107.4	119.8	126.2	126.9	133.6	137.9	141.3	296.0	327.7	345.2	343.4	357.9	366.4	375.8	
SEM (n = 24)	0.7	1.0	1.1	1.6	2.2	2.4	2.9	1.6	2.4	2.6	3.0	5.5	7.6	8.5	
P-values	
Breed x ME Lay x AFD Lys	N.S.3	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
Breed x ME Lay	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
Breed x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
ME Lay x AFD Lys	N.S.	N.S.	0.031	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	0.047	N.S.	N.S.	N.S.	N.S.	
Breed	N.S.	0.019	0.004	0.010	0.001	<.0001	<.0001	N.S.	0.004	0.005	0.028	0.004	<.0001	<.0001	
ME Lay	<.0001	<.0001	<.0001	0.001	0.004	0.000	0.008	0.040	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
	
			AFD Lys intake, mg								
			20 to 27	27 to 35	35 to 45	47 to 55	55 to 63	63 to 71	71 to 75								
Breed	ME Lay1	AFD Lys2															
Brown	Con	Con	758.4	848.3	856.6	847.4	849.0	845.4	864.2								
Brown	Con	Red	690.6	733.4	758.7	712.0	717.1	704.6	700.5								
Brown	Red	Con	780.1	888.2	904.6	912.3	951.6	982.5	1022.1								
Brown	Red	Red	722.5	760.5	789.4	737.8	774.2	777.9	799.9								
White	Con	Con	753.5	829.5	877.8	878.2	940.1	977.2	1084.0								
White	Con	Red	691.4	712.6	784.6	732.0	769.6	797.4	816.5								
White	Red	Con	795.2	853.8	947.8	937.4	1011.9	1087.1	1100.9								
White	Red	Red	725.2	755.8	795.8	756.5	803.1	858.4	900.2								
SEM (n=6)	8.1	9.6	11.7	13.9	28.5	32.8	39.6								
Breed effect	
Brown	737.9	807.6	827.3	802.4	823.0	827.6	846.7								
White	741.3	787.9	851.5	826.0	881.2	930.0	975.4								
SEM (n = 24)	4.1	5.8	6.4	7.0	13.2	18.1	21.1								
ME Lay effect	
Constant	723.5	780.9	819.4	792.4	818.9	831.2	866.3								
Reduction	755.8	814.6	859.4	836.0	885.2	926.5	955.8								
SEM (n = 24)	4.1	5.8	6.4	7.0	13.2	18.1	21.4								
AFD Lys effect	
Constant	771.8	855.0	896.7	893.8	938.2	973.0	1017.8								
Reduction	707.4	740.5	782.1	734.6	766.0	784.6	804.3								
SEM (n = 24)	4.1	5.8	6.4	7.0	13.2	18.1	20.9								
P-values	
Breed x ME Lay x AFD Lys	N.S.3	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.								
Breed x ME Lay	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.								
Breed x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.								
ME Lay x AFD Lys	N.S.	N.S.	0.023	0.068	N.S.	N.S.	N.S.								
N.S.	0.003	0.005	N.S.	0.003	0.005	0.021	0.003	<.0001	<.0001								
N.S.	0.003	0.005	<.0001	<.0001	<.0001	<.0001	0.001	0.000	0.002								
N.S.	0.003	0.005	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001	<.0001								
1 Metabolizable energy, calculated according to CVB (2016); Con = Constant level over time and Red = Reduced levels over time.

2 Apparent fecal digestible, calculated according to CVB (2016).

3 N.S. indicates non-significant results with P>0.10.

The difference in ME Lay and AFD Lys intake only resulted in minor differences in ADG (Table 4). At the start of the study in wk 20 to 27, white hens had a lower ADG than brown hens (P < 0.05). In wk 45 to 55, white hens had a more severe ADG loss than brown hens (P < 0.05; -0.61 g vs. -0.20 g) but in wk 71 to 75, white hens showed a higher ADG than brown hens (P < 0.05). Only in the final period in wk 70 to 75, hens fed the constant dietary ME Lay showed significantly higher ADG (P<0.05). No other effects of dietary treatment on ADG were found.Table 4 Effects of breed, dietary ME Lay and AFD Lys on average daily gain (g), expressed as least squares means.

Table 4			Average Daily Gain	
			20 to 27	27 to 35	35 to 45	45 to 55	55 to 63	63 to 71	71 to 75	
Breed	ME Lay1	AFD Lys2								
Brown	Con	Con	6.97	1.60	−0.10	−0.01	−0.43	−0.16	0.35	
Brown	Con	Red	6.71	1.75	−0.04	−0.14	−0.61	0.17	−0.31	
Brown	Red	Con	6.83	1.45	0.14	−0.19	−0.93	0.06	−0.18	
Brown	Red	Red	6.71	1.44	−0.05	−0.45	−0.47	−0.01	0.28	
White	Con	Con	4.93	1.91	0.11	−0.50	−0.08	0.25	0.90	
White	Con	Red	4.48	1.73	−0.18	−0.86	−0.27	0.00	1.30	
White	Red	Con	5.15	1.51	0.02	−0.56	−0.62	−0.11	−0.45	
White	Red	Red	4.70	1.72	0.25	−0.55	−0.52	0.07	0.31	
SEM (n = 6)			0.36	0.19	0.25	0.19	0.26	0.19	0.41	
Breed effect									
 Brown	6.81	1.56	−0.01	−0.20	−0.61	0.01	0.04	
 White	4.81	1.72	0.05	−0.61	−0.37	0.05	0.51	
SEM (n = 24)			0.23	0.13	0.17	0.10	0.18	0.14	0.19	
ME Lay effect									
 Constant	5.77	1.75	−0.05	−0.38	−0.35	0.06	0.56	
 Reduction	5.85	1.53	0.09	−0.44	−0.63	0.00	−0.01	
 SEM (n = 24)	0.23	0.13	0.17	0.10	0.18	0.14	0.20	
AFD Lys effect									
 Constant	5.97	1.62	0.04	−0.31	−0.51	0.01	0.16	
 Reduction	5.65	1.66	−0.01	−0.50	−0.47	0.06	0.39	
 SEM (n = 24)	0.23	0.13	0.17	0.10	0.18	0.14	0.20	
P-values										
 Breed x ME Lay x AFD Lys	N.S.3	N.S.	N.S.	N.S.	N.S.	0.053	N.S.	
 Breed x ME Lay	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	0.015	
 Breed x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
 ME Lay x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
 Breed	<.0001	N.S.	N.S.	0.003	N.S.	N.S.	0.048	
 ME Lay	N.S.	0.077	N.S.	N.S.	0.079	N.S.	0.021	
 AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
1 Metabolizable energy, calculated according to CVB (2016); Con = Constant level over time and Red = Reduced levels over time.

2 Apparent fecal digestible, calculated according to CVB (2016).

3 N.S. indicates non-significant results with P>0.10.

Egg Production and Quality

The shape of the egg production curve was significantly impacted by breed and dietary ME Lay, and only to a small extent by dietary AFD Lys (Table 5; Figure 1). The time of transition to start laying (t1) and reaching the peak (t2) was significantly later in brown hens than white hens (t1: difference = 0.77 wks; t2: difference = 1.43 wks). The height of peak production was significantly lower in brown hens than in white hens (yp; difference =2.5%). At the same time, the duration of the peak production phase was significantly longer in brown hens (P; difference = 3.4 wks). Finally, the slope of decline was significantly higher in brown hens than in white hens (b4; difference = 0.24%/wk).Table 5 Estimates of model parameters, SE of parameter estimates, total egg production yt, adjusted R2, RSE and BIC separated by breed (white or brown), dietary ME Lay treatment (constant or reduction) and dietary AFD Lys treatment (constant or reduction).

Table 5	Parameter	Goodness-of-fit	
	t1 (wk)	t2 (wk)	yp (%)	P (wk)	b4 (%/wk)	R2	RSE b	BIC c	
Overall model	20.48 (0.05)	24.26 (0.05)	96.11 (0.18)	31.88 (0.87)	−0.58 (0.04)	0.81	6.71	17006	
Breed									
 Brown	21.20b (0.06)	24.72b (0.06)	94.87b (0.20)	33.61b (0.85)	−0.72b (0.06)	0.88	5.89	8023	
 White	20.43a (0.06)	23.29a (0.06)	97.37a (0.21)	30.21a (1.13)	−0.48a (0.04)	0.85	4.97	7910	
P-valuedtreatment effect P < 0.01	
 ME Laye									
 Constant	20.54 (0.07)	24.19 (0.07)	95.62a (0.24)	33.58a (0.78)	−0.91a (0.06)	0.85	7.22	8628	
 Reduction	20.43 (0.07)	24.34 (0.07)	96.76b (0.27)	25.66b (2.37)	−0.26b (0.04)	0.79	5.68	8167	
P-valuedtreatment effect P < 0.01	
 AFD Lysf									
 Constant	20.55 (0.07)	24.20 (0.07)	96.03 (0.25)	32.56 (1.42)	−0.50 (0.06)	0.82	6.35	8303	
 Reduction	20.42 (0.07)	24.33 (0.07)	96.23 (0.25)	30.98 (1.08)	−0.65 (0.05)	0.80	7.00	8704	
P-valuedtreatment effect P < 0.01	
a yt=r*(ypt2−t1)*[Ln(et/r+et1/r1+et1/r)- Ln(et/r+et2/r1+et2/r)]+rb4Ln(et/r+e(t2+P)/r1+e(t2+P)/r).

b Residual Standard Error.

c Bayesian information criterion, lower values indicate a better model fit.

d The P-value obtained with an F-test. P-values compare the overall model with the added treatment factor. If the F-test was significant for treatment effect (P < 0.05), the model parameters between treatments were compared using a one-sample t-test. Values with unique superscripts in a column for breed, ME Lay or AFD LYS were significantly different (P < 0.05).

e Metabolizable energy, calculated according to CVB (2016).

f Apparent fecal digestible, calculated according to CVB (2016).

Figure 1 The egg production performance in percentage (y-axis) from wk 17 to 75 (x-axis) showing the modeled data, with in blue the description of the model parameters of yp = level of constant peak production, t1 and t2 = transition in wk, P = persistency of constant production in wk and b4 = rate of decline in production in percentage for (A) the overall model, (B) the effect of breed with brown hens (dotted line) and white hens (continuous line), (C) dietary ME Lay effect with a constant ME Lay (dotted line) and a reduced ME Lay over time (continuous line) and (D) dietary AFD Lys effect with a constant AFD Lys (dotted line) and reduced AFD Lys (continuous line).

Figure 1

A reduction in dietary ME Lay resulted in a significantly higher height of peak production (yp; difference =1.1%). The duration of the peak production phase was significantly shorter when hens were fed the reduced ME Lay diets (P; difference = 7.9 wks) but the slope of decline was significantly lower (b4; difference = 0.65%/wk).

For dietary AFD Lys, the F-test showed a significant difference between the constant and reduced AFD Lys model, but the pairwise t-test was only able to detect a trend in a larger slope of decline for the hens fed the reduced AFD Lys diets (P=0.08; b4; difference = 0.15%/wk).

The EW showed inconsistent results in different periods and most effects were limited to the earlier production phase before wk 55. At start of lay in wk 20 to 35, EW of the brown hens was significantly higher (Table 6), but after this period the breed no longer significantly impacted EW. In wk 27 to 35, a short-term breed x ME Lay interaction was found (P < 0.05), showing that the EW increased in brown hens fed constant vs. reduced ME Lay diets, whereas in white hens the EW decreased when fed constant versus reduced ME Lay diets. In general, ME Lay did not influence EW, except in wk 55 to 63, in which EW were significantly higher when hens were fed a constant ME Lay diet. Hens fed a constant AFD Lys had higher EW in wk 27 to 35 and wk 45 to 55 (P < 0.05), with a similar trend in wk 35 to 45 (P = 0.88).Table 6 Effects of breed, dietary ME Lay and AFD Lys on egg weight (g) and egg mass (g), expressed as least squares means.

Table 6			Egg weight, g	Egg mass, g	
			20 to 27	27 to 35	35 to 45	45 to 55	55 to 63	63 to 71	71 to 75	20 to 27	27 to 35	35 to 45	45 to 55	55 to 63	63 to 71	71 to 75	
Breed	ME Lay1	AFD Lys2															
 Brown	Con	Con	53.92	62.98	64.56	65.88	66.87	66.24	65.88	35.58	58.94	60.17	61.37	62.48	55.96	52.98	
 Brown	Con	Red	54.56	61.73	63.43	64.09	65.16	64.75	64.58	34.02	58.71	60.11	59.48	60.64	52.57	47.56	
 Brown	Red	Con	53.50	61.70	63.52	64.66	65.09	65.62	65.51	34.11	58.58	60.27	60.44	60.42	60.10	59.54	
 Brown	Red	Red	53.12	61.72	63.12	64.06	64.71	65.08	64.88	35.33	60.02	61.05	60.48	60.29	59.37	57.26	
 White	Con	Con	53.02	61.16	64.14	65.25	65.35	64.69	65.26	42.14	59.69	61.25	63.56	62.91	55.88	57.30	
 White	Con	Red	52.00	60.30	63.90	64.73	66.00	66.26	65.60	43.17	58.37	62.03	62.59	61.32	53.70	56.53	
 White	Red	Con	53.05	61.68	64.40	65.18	65.37	65.16	65.05	43.21	60.33	62.07	63.36	62.27	59.87	59.14	
 White	Red	Red	52.73	61.43	64.31	65.30	65.24	64.98	64.68	43.14	60.58	63.24	62.99	61.52	60.10	58.16	
 SEM (n = 6)			0.50	0.39	0.37	0.41	0.47	0.65	0.77	0.79	0.78	0.88	0.89	0.98	1.45	1.76	
Breed effect																
Brown	53.78	62.03	63.66	64.67	65.46	65.42	65.21	34.76	59.07	60.40	60.44	60.96	57.00	54.34	
 White	52.70	61.14	64.19	65.12	65.49	65.27	65.15	42.92	59.74	62.15	63.13	62.00	57.39	57.78	
 SEM (n = 24)			0.25	0.19	0.19	0.20	0.23	0.33	0.39	0.40	0.39	0.44	0.45	0.59	0.85	1.01	
ME Lay effect																
 Constant	53.38	61.54	64.01	64.99	65.84	65.48	65.33	38.73	58.93	60.89	61.75	61.84	54.53	53.59	
 Reduction	53.10	61.63	63.84	64.80	65.10	65.21	65.03	38.95	59.88	61.66	61.82	61.12	59.86	58.53	
 SEM (n = 24)	0.25	0.19	0.19	0.20	0.23	0.33	0.39	0.40	0.39	0.44	0.45	0.59	0.85	1.01	
AFD Lys effect																
 Constant	53.38	61.88	64.15	65.24	65.67	65.43	65.42	38.76	59.39	60.94	62.18	62.02	57.95	57.24	
 Reduction	53.10	61.29	63.69	64.54	65.28	65.27	64.93	38.92	59.42	61.61	61.38	60.94	56.44	54.88	
 SEM (n = 24)	0.25	0.19	0.19	0.20	0.23	0.33	0.39	0.40	0.39	0.44	0.45	0.59	0.85	1.01	
 P-values																	
 Breed x ME Lay x AFD Lys	N.S.3	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	0.092	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
 Breed x ME Lay	0.073	0.011	0.066	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	0.005	
 Breed x AFD Lys	N.S.	N.S.	N.S.	0.093	0.054	0.071	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
 ME Lay x AFD Lys	N.S.	0.097	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	
 Breed	0.004	0.002	0.053	N.S.	N.S.	N.S.	N.S.	<.0001	N.S.	0.008	<.001	N.S.	N.S.	0.002	
 ME Lay	N.S.	N.S.	N.S.	N.S.	0.031	N.S.	N.S.	N.S.	0.092	N.S.	N.S.	N.S.	<.0001	<.0001	
 AFD Lys	N.S.	0.039	0.088	0.021	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	N.S.	0.032	
1 Metabolizable energy, calculated according to CVB (2016); Con = Constant level over time and Red = Reduced levels over time.

2 Apparent fecal digestible, calculated according to CVB (2016).

3 N.S. indicates nonsignificant results with P > 0.10.

Egg mass production of the white hens was significantly higher in wk 20 to 27, wk 35 to 55 and wk 71 to 75 (P < 0.05; Table 6). This effect was more heavily influenced by number of eggs than egg weights. Similarly, in the final production period in wk 63 to 75, egg mass of hens fed the reduced ME Lay diets was significantly higher (P < 0.05). Lastly, the constant dietary AFD Lys treatment only resulted in higher egg mass in wk 71 to 75.

Egg quality was measured toward the end of the trial in wk 66 and only breed showed an impact on albumen pH, albumen percentage and yolk percentage. Eggs collected from brown hens had relatively more albumen with a lower pH than white hens (P<0.05; Table 7). At the same time, the relative yolk weight of white hens was higher (P<0.05).Table 7 Effects of breed, dietary ME Lay and AFD Lys on egg quality including sample egg weight, Haugh Unit, albumen pH, albumen weight as percentage of egg weight and yolk weight as percentage of egg weight, expressed as least squares means.

Table 7			Egg weight, g	HU	Albumen pH	Albumen, %	Yolk, %	
Breed	ME Lay1	AFD Lys2						
 Brown	Con	Con	64.52	87.35	7.76	0.56	0.25	
 Brown	Con	Red	64.68	87.58	7.72	0.56	0.25	
 Brown	Red	Con	66.88	85.35	7.77	0.55	0.25	
 Brown	Red	Red	64.71	84.03	7.77	0.55	0.25	
 White	Con	Con	64.46	88.73	7.80	0.51	0.29	
 White	Con	Red	65.62	88.68	7.80	0.52	0.28	
 White	Red	Con	64.37	90.27	7.82	0.52	0.28	
 White	Red	Red	65.36	88.67	7.82	0.52	0.28	
 SEM (n = 6)			1.03	2.53	0.04	0.01	0.00	
Breed effect							
 Brown	65.20	86.08	7.75	0.56	0.25	
 White	64.95	89.09	7.81	0.52	0.28	
 SEM (n = 24)			0.56	1.19	0.02	0.00	0.00	
ME Lay effect							
 Constant	64.82	88.08	7.77	0.54	0.27	
 Reduction	65.33	87.08	7.80	0.54	0.27	
 SEM (n = 24)	0.56	1.18	0.02	0.00	0.00	
AFD Lys effect							
 Constant	65.06	87.92	7.79	0.54	0.27	
 Reduction	65.09	87.24	7.78	0.54	0.27	
 SEM (n = 24)	0.56	1.18	0.02	0.00	0.00	
P-values								
 Breed x ME Lay x AFD Lys	N.S.3	N.S.	N.S.	N.S.	N.S.	
 Breed x ME Lay	N.S.	N.S.	N.S.	N.S.	N.S.	
 Breed x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	
 ME Lay x AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	
 Breed	N.S.	0.069	0.014	<.0001	<.0001	
 ME Lay	N.S.	N.S.	N.S.	N.S.	N.S.	
 AFD Lys	N.S.	N.S.	N.S.	N.S.	N.S.	
1 Metabolizable energy, calculated according to CVB (2016); Con = Constant level over time and Red = Reduced levels over time.

2 Apparent fecal digestible, calculated according to CVB (2016).

3 N.S. indicates non-significant results with P > 0.10.

Body composition

In general, no interaction effects were found so only main effects will be discussed (P>0.05; Table 8). Body CF levels were only influenced by breed in wk 17, with higher body CF percentages in brown hens. Regarding body CP, breed showed consistently higher body CP percentage in brown hens than in white hens (significant wk 31 and 59, tendencies wk 35, 39, and 43). Reducing ME Lay over time resulted in significantly higher body CP in wk 20 and 84, with a similar trend in wk 47 and 75. In wk 39 and 55, the result was reversed with lower body CP levels in hens fed the reducing ME Lay diets.Table 8 Effects of breed, dietary ME Lay and AFD Lys on body crude fat and body crude protein in several weeks.

Table 8			Body crude fat, %	
			17	20	24	27	31	35	39	43	47	51	55	59	63	67	75	
Breed	ME Lay1	AFD Lys2																
 Brown	Con	Con	13.13	11.97	14.25	14.98	15.88	17.44	18.76	19.23	17.52	13.52	17.31	14.33	17.38	17.74	15.67	
 Brown	Con	Red	10.47	13.73	14.86	13.53	17.01	18.82	20.44	19.11	17.64	12.77	17.35	16.20	14.42	14.45	14.68	
 Brown	Red	Con	10.21	12.28	14.47	12.70	15.81	16.61	19.93	17.72	18.05	13.23	16.20	15.69	16.56	13.57	13.32	
 Brown	Red	Red	12.27	12.04	15.06	14.25	17.05	18.94	19.63	19.16	16.02	13.82	18.14	16.90	17.35	15.77	14.48	
 White	Con	Con	9.11	13.23	13.82	14.88	15.64	18.39	20.47	17.78	16.19	13.94	15.41	16.17	16.19	15.90	16.23	
 White	Con	Red	9.48	12.94	14.14	15.80	16.91	17.45	19.25	17.27	18.52	15.02	16.22	15.52	12.59	15.00	11.94	
 White	Red	Con	9.20	11.89	13.19	15.27	17.67	17.91	19.93	19.09	15.78	12.97	16.93	16.35	13.01	16.25	15.02	
 White	Red	Red	9.09	11.98	16.38	14.08	16.47	18.44	19.32	18.30	16.47	9.95	16.34	15.74	15.75	13.76	12.90	
 SEM (n = 6)			1.12	0.88	1.13	1.03	0.91	1.24	0.79	1.61	1.37	1.81	1.35	1.54	2.13	1.48	1.34	
Breed effect																	
Brown	11.52	12.51	14.66	13.86	16.44	17.95	19.69	18.81	17.31	13.33	17.25	15.78	16.43	15.39	14.54	
White	9.22	12.51	14.38	15.01	16.67	18.05	19.74	18.11	16.74	12.97	16.22	15.95	14.39	15.23	14.02	
 SEM (n = 24)			0.52	0.44	0.60	0.52	0.46	0.58	0.35	0.69	0.64	1.39	0.63	0.74	0.83	0.63	0.84	
ME Lay effect																	
 Constant	10.55	12.97	14.27	14.80	16.36	18.02	19.73	18.35	17.47	13.81	16.57	15.56	15.15	15.77	14.63	
 Reduction	10.19	12.05	14.78	14.07	16.75	17.97	19.70	18.57	16.58	12.49	16.90	16.17	15.67	14.84	13.93	
 SEM (n = 24)	0.52	0.44	0.60	0.52	0.46	0.58	0.36	0.69	0.64	1.39	0.63	0.74	0.83	0.62	0.84	
AFD Lys effect																	
 Constant	10.41	12.35	13.93	14.46	16.25	17.59	19.77	18.45	16.89	13.41	16.46	15.64	15.79	15.87	15.06	
 Reduction	10.33	12.67	15.11	14.42	16.86	18.41	19.66	18.46	17.16	12.89	17.01	16.09	15.03	14.75	13.50	
 SEM (n = 24)	0.52	0.44	0.60	0.52	0.47	0.58	0.36	0.69	0.64	1.39	0.63	0.72	0.83	0.63	0.84	
 P-values																		
 Breed x ME Lay x AFD Lys	0.081	N.S.3	N.S	0.088	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.028	N.S	
 Breed x ME Lay	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.056	N.S	N.S	N.S	N.S	N.S	
 Breed x AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.061	
 ME Lay x AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.027	N.S	N.S	
 Breed	0.003	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.071	N.S	N.S	
 ME Lay	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	
 AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.075	
	
			Body Crude Protein, %	
			17	20	24	27	31	35	39	43	47	51	55	59	63	67	75	
	
 Breed	ME Lay1	AFD Lys2																
 Brown	Con	Con	21.96	22.26	22.62	20.84	20.87	19.54	20.08	19.64	16.91	18.30	19.61	19.84	18.16	18.28	18.85	
 Brown	Con	Red	22.40	22.80	22.14	20.22	20.99	19.38	20.19	20.40	17.93	19.13	19.12	19.60	17.29	17.86	18.69	
 Brown	Red	Con	22.63	22.59	22.58	21.17	20.75	19.41	19.68	20.02	18.61	18.95	18.88	20.16	18.77	18.45	19.03	
 Brown	Red	Red	22.25	23.10	22.63	20.22	20.89	19.35	19.03	20.14	18.54	17.84	18.93	19.44	18.26	18.17	19.27	
 White	Con	Con	22.75	22.30	21.81	20.38	19.72	18.84	19.10	19.64	17.90	17.86	18.84	18.83	16.60	17.15	18.18	
 White	Con	Red	23.36	21.95	22.38	20.14	20.07	19.05	19.66	19.70	17.31	17.84	18.35	18.01	16.51	17.43	17.63	
 White	Red	Con	21.83	23.10	22.17	19.84	20.28	18.61	19.46	19.52	17.81	18.45	17.95	19.16	17.28	17.03	19.15	
 White	Red	Red	21.40	23.19	21.02	20.02	20.82	19.25	19.26	19.82	17.80	18.13	18.43	18.59	17.60	17.52	18.81	
 SEM (n = 6)			0.58	0.44	0.64	0.55	0.45	0.40	0.29	0.42	0.53	0.45	0.34	0.39	0.61	0.62	0.51	
Breed effect																	
 Brown	22.31	22.69	22.49	20.61	20.88	19.42	19.75	20.05	18.00	18.56	19.13	19.76	18.12	18.19	18.96	
 White	22.34	22.63	21.84	20.09	20.22	18.94	19.37	19.67	17.70	18.07	18.39	18.65	17.00	17.28	18.45	
 SEM (n = 24)			0.27	0.25	0.36	0.25	0.21	0.20	0.13	0.17	0.25	0.23	0.16	0.18	0.24	0.27	0.26	
ME Lay effect																	
 Constant	22.62	22.33	22.24	20.39	20.41	19.20	19.76	19.84	17.51	18.28	18.98	19.07	17.14	17.68	18.34	
 Reduction	22.03	23.00	22.10	20.31	20.68	19.16	19.36	19.87	18.19	18.34	18.55	19.34	17.98	17.79	19.07	
 SEM (n = 24)	0.28	0.25	0.36	0.25	0.21	0.20	0.14	0.17	0.25	0.22	0.16	0.18	0.24	0.26	0.26	
AFD Lys effect																	
 Constant	22.29	22.56	22.29	20.55	20.41	19.10	19.58	19.70	17.81	18.39	18.82	19.50	17.70	17.73	18.80	
 Reduction	22.35	22.76	22.04	20.15	20.69	19.26	19.54	20.02	17.89	18.24	18.71	18.91	17.41	17.74	18.60	
 SEM (n = 24)	0.27	0.25	0.36	0.25	0.21	0.20	0.13	0.17	0.25	0.22	0.16	0.18	0.24	0.27	0.26	
 P-values																		
 Breed x ME Lay x AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	
 Breed x ME Lay	0.024	N.S	N.S	N.S	N.S	N.S	0.052	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	
 Breed x AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	
 ME Lay x AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	0.054	N.S	N.S	0.061	0.093	N.S	N.S	N.S	N.S	
 Breed	N.S	N.S	N.S	N.S	0.030	0.093	0.055	0.093	N.S	0.099	0.002	<.001	0.001	0.008	N.S	
 ME Lay	N.S	0.029	N.S	N.S	N.S	N.S	0.041	N.S	0.055	N.S	0.057	N.S	0.011	N.S	0.052	
 AFD Lys	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	N.S	0.024	N.S	N.S	N.S	
1 Metabolizable energy, calculated according to CVB (2016); Con = Constant level over time and Red = Reduced levels over time.

2 Apparent fecal digestible, calculated according to CVB (2016).

3 N.S. indicates non-significant results with P > 0.10.

Nutrient Intake vs. Egg Mass Production

The segmented regression model shows the relationship between ME Lay intake and egg mass production. A linear model was compared with a model with 1 inflection point (or breakpoint), and tested for the best fit. When the model with 1 inflection point showed the best fit, it indicated that the effect of ME Lay intake on egg mass production was not linear, and changed after a specific amount of ME Lay intake (inflection point).

The statistical analysis showed a significantly better fit when 1 inflection point was added versus the linear response for ME Lay intake vs. egg mass production (P < 0.05; Table 9). The egg mass production increased significantly more with higher ME Lay intake in white hens (P < 0.05; slope βi), with a higher inflection point (P < 0.05; γiof 330 kcal intake), compared to the brown hens (Table 9, Figure 2). Similarly, egg mass production increased significantly more with higher ME Lay intake, when hens were fed a reduced ME Lay diet over time (P < 0.05), but the inflection point was reached earlier at 322 kcal intake vs. 331 kcal for hens fed the constant ME Lay diets (P < 0.05). Lastly, egg mass production increased significantly with higher ME Lay intake when hens were fed a constant AFD Lys diet over time (P < 0.05), and reached the inflection point earlier at 322 kcal vs. 330 kcal intake for hens fed the constant AFD Lys diets (P<0.05). The egg mass production kept increasing slightly, but significantly, with higher ME Lay intake beyond this inflection point (P<0.05; slope βj) for the hens fed the constant AFD Lys diets, but not for the hens fed the reduced AFD Lys diets over time.Table 9 Functional specifications, coefficients, and goodness-of-fit criteria of the segmented regression models describing the ME Lay intake (kcal/d) x Egg mass production (g/d) of laying hens, separated by breed (white or brown), dietary ME Lay treatment (constant or reduction) and dietary AFD Lys treatment (constant or reduction).

Table 9	Parametera	Goodness-of-fit	
	α0	ßi	ßj	ɣi	
	Estimate	SEM	Estimate	SEM	Estimate	SEM	Estimate	SEM	R2	R2 adj	DWb	BIC c	
Overall model													
 Linear	−3.255	4.416	0.177	0.013					0.40	0.39	−	1922	
 1 inflection point	−160.342	10.976	0.681	0.036	0.011	0.038	324.198	1.393	0.78	0.78	1.46	1643	
P-value for number of inflection points P < 0.001d	
 Breed													
 Brown	−192.526a	13.880	0.777a	0.045	0.035	0.052	324.032a	1.610	0.56	0.56	1.90	949	
 White	−102.450b	11.003	0.499b	0.036	−0.025	0.038	330.777b	2.101	0.78	0.77	1.11	778	
 ME Laye													
 Constant	−114.295a	10.713	0.530a	0.034	−0.016	0.040	331.088a	2.064	0.76	0.76	1.39	838	
 Reduction	−200.838b	15.987	0.810b	0.052	0.017	0.054	322.529b	1.491	0.82	0.82	1.50	804	
 AFD Lysf													
 Constant	−181.799a	15.596	0.752a	0.051	0.011a	0.053	322.483a	1.673	0.81	0.81	1.55	816	
 Reduction	−124.138b	12.653	0.560b	0.041	0.000b	0.045	330.050b	2.240	0.76	0.76	1.59	837	
a Yik is the inflection pointin phase i ro j, a0 is the intercept, Bijk is the allometric slope at phase i, j, or k.

b Durbin-Watson statistic; values range from 0 to 4, values of 2 indicate no autocorrelation, value less than 2 indicates a positive autocorrelation, a value greater than 2 a negative autocorrelation.

c Bayesian information criterion, lower values indicate a better model fit.

d The P-value obtained with an F-test to compare the linear model with a one-inflection point model. The model parameters between treatments were compared using a pairwise t-test. Values with unique superscripts in a column for breed, ME Lay or AFD LYS were significantly different (P < 0.05).

e Metabolizable energy, calculated according to CVB (2016).

f Apparent fecal digestible, calculated according to CVB (2016).

Figure 2 The egg mass production in g/d (y-axis) plotted against the ME Lay intake in kcal/day (x-axis) showing the segmented regression with the linear model (continuous line) and the 1-inflection point model (dotted lines) for (A) the overall model, (B) the effect of breed with white hens (black line) and brown hens (red line), (C) dietary ME Lay effect with a constant ME Lay (black line) and a reduced ME Lay over time (red line) and (D) dietary AFD Lys effect with a constant AFD Lys (black line) and reduced AFD Lys (red line).

Figure 2

The segmented regression model for AFD Lys intake versus egg mass production showed a significantly better fit when 1 inflection point was added versus the linear response, but in general the model did not have a strong correlation (P<0.05; R2 adj = 0.22 for the 2 phase model; Table 10). Only dietary AFD Lys significantly influenced the response, with higher egg mass production when hens were fed a constant AFD Lys diet over time, and reached the breakpoint at 850 mg AFD Lys intake versus 775 mg AFD Lys intake for hens fed the reduced AFD Lys diets (P < 0.05; Figure 3).Table 10 Functional specifications, coefficients, and goodness-of-fit criteria of the segmented regression models describing the AFD Lys intake (mg/d) x Egg mass production (g/d) of laying hens, separated by breed (white or brown), dietary ME Lay treatment (constant or reduction) and dietary AFD LYS treatment (constant or reduction).

Table 10	Parametera		Goodness-of-fit	
	α0	ßi	ßj	ɣi	
	Estimate	SEM	Estimate	SEM	Estimate	SEM	Estimate	SEM	R2	R2 adj	DWb	BIC c	
Overall model													
 Linear	26.92	3.84	0.04	0.00					0.18	0.17	−	2010	
 1 inflection point	8.21	6.89	0.06	0.01	−0.002	0.02	882.87	27.42	0.22	0.22	0.63	2005	
P-value for number of inflection points P <0.001	
 Breed													
 Brown	14.73	7.87	0.05	0.01	−0.007	0.02	893.22	35.43	0.25	0.23	0.61	952	
 White	1.18	12.50	0.07	0.02	0.005	0.03	858.19	41.96	0.18	0.17	0.55	1042	
 ME Laye													
 Constant	6.76	8.17	0.06	0.01	−0.020	0.03	884.94	33.58	0.25	0.24	0.68	1001	
 Reduction	0.72	17.75	0.07	0.02	0.007	0.03	852.47	42.08	0.20	0.18	0.70	1023	
 AFD Lysf													
 Constant	−149.07a	13.43	0.25a	0.02	0.002a	0.02	849.72a	4.85	0.78	0.77	1.64	841	
 Reduction	−43.84b	17.14	0.14b	0.02	−0.001b	0.04	775.06b	15.81	0.30	0.29	0.53	989	
a Yik is the inflection point in phase i ro j, a0 is the intercept, Bijk is the allometric slope at phase i, j or k.

b Durbin-Watson statistic; values range from 0 to 4, values of 2 indicate no autocorrelation, value less than 2 indicates a positive autocorrelation, a value greater than 2 a negative autocorrelation.

c Bayesian information criterion, lower values indicate a better model fit

dThe P-value obtained with an F-test to compare the linear model with a one-inflection point model. The model parameters between treatments were compared using a pairwise t-test. Values with unique superscripts in a column for breed, ME Lay or AFD LYS were significantly different (P < 0.05).

e Metabolizable energy, calculated according to CVB (2016).

f Apparent fecal digestible, calculated according to CVB (2016).

Figure 3 The egg mass production in g/d (y-axis) plotted against the AFD Lys intake in mg/day (x-axis) showing the segmented regression with the linear model (continuous line) and the 1-inflection point model (dotted lines) for (A) the overall model, (B) the effect of breed with white hens (black line) and brown hens (red line), (C) dietary ME Lay effect with a constant ME Lay (black line) and a reduced ME Lay over time (red line) and (D) dietary AFD Lys effect with a constant AFD Lys (black line) and reduced AFD Lys (red line).

Figure 3

DISCUSSION

Nutrients and Feed Intake

The energy intake of the hens was remarkably constant between treatments, and only influenced by breed (all periods), and dietary ME Lay levels in the first period (wk 20–27). The maximum difference in energy intake between treatments was 27 kcal per day for the white hens and 86 kcal per day for the brown hens, but neither difference was significant (Table 3). The AFD Lys intake fluctuated much more between treatments, with a significant impact of breed in all periods except wk 20 to 27, a significant higher AFD Lys intake when hens were fed a reduced ME Lay diet and significantly higher AFD Lys intake when hens were fed the constant AFD Lys diets. This indicates that the hens standardized their feed intake based on dietary ME Lay but not AFD Lys.

The breed guide indicates that the energy intake should be higher in brown hens than in white hens throughout lay (Lohmann Breeders GmbH, 2021). The current trial shows the opposite with a higher voluntary ADFI and energy intake of the white hens than the brown hens. The total egg mass production of the white hens was higher than the brown hens (Table 6) which could have driven a higher feed and energy requirement. No interaction effect between any of the treatments in egg mass and egg weight production was found. In general, diets did not influence egg weights (Table 6). This is contradictory to the results by Da Nóbrega et al. (2022) and Scappaticcio et al. (2021), who found increased egg weights with higher dietary lysine intake. In both studies, the higher lysine diets were confounded with higher soybean oil inclusion. Higher dietary oil levels may increase egg weights (Whitehead et al., 1991). Moreover, in the study by Da Nóbrega et al. (2022), the lowest daily lysine intake was probably limiting egg mass production, with intake levels of only 336 mg per hen per day at the start, and 510 mg per hen per day at the end of the study. These factors could have accounted for egg weight differences found in these studies, which were not found in to the current study.

Laying Persistency

The egg production curve was significantly impacted by breed and dietary energy, but not dietary lysine. The white hens started to produce eggs earlier (t1), reached the peak earlier (t2), had a higher peak production (yp), a shorter peak production phase (P) and a smaller slope of decline (b4; Table 5, Figure 1) compared to the brown hens. In the end, this summed up to a higher total number of eggs (white hens 356.6 ±8.3; brown hens 339.8±10.5). Similarly, hens fed a reduced ME Lay diet had a higher peak production (yp), a shorter peak production phase (P) and a smaller slope of decline (b4; Table 5, Figure 1) compared to the hens fed a constant ME Lay diet. In the end, feeding a reduced energy diet during lay, resulted in a higher total number of eggs (constant energy 343.6 ± 12.7; reduced energy 352.4 ± 7.4). Both of these results show that for laying persistency, a shorter peak production period with a smaller slope of decline was beneficial. Grossman et al. (2000) hypothesized that for laying persistency, the duration of the peak of egg production (P) should be prolonged, but the current study shows that this is not the case.

The hens fed the reduced ME Lay diets had a similar energy intake, but a higher AFD Lys intake, compared to the hens fed the constant ME Lay diet. This indicates that the higher egg production persistency that was found for the hens fed the ME Lay reduced diets, was due to a higher AFD Lys intake. Yet, the laying persistency of the hens fed the constant AFD Lys diets, resulting in a higher AFD Lys intake, was not improved compared to the hens fed the reduced AFD Lys diet. There was a larger difference in AFD Lys intake between the dietary AFD Lys treatment groups than between the dietary ME Lay treatment groups. So, if the improved laying persistency in the reduced ME Lay treatment was only caused by a higher AFD Lys intake, it would be expected that a similar effect was found in the constant AFD Lys group compared to the reduced AFD Lys group, but that was not the case. The total ADFI of the hens fed the reduced ME Lay was the highest between treatments, so a higher intake of other nutrients or ingredients in the diet could have also contributed to the improved laying persistency.

An ingredient that could have influenced laying persistency is oil. Dietary oil can be used for lipoprotein production, which is especially important for yolk formation and thus egg production (van Eck et al., 2023). Even though the dietary oil inclusion in the reduced ME Lay diets was slightly lower compared to the constant ME Lay diets, due to a higher ADFI of these hens, the oil intake of the hens fed the reduced ME Lay diets was higher. Especially from wk 45 onwards, the oil intake of the hens fed the reduced ME Lay diets was 0.7 grams higher per day than for the hens fed the constant ME Lay diets (data not shown). The difference between the constant and reduced AFD Lys group was much smaller with only 0.1 gram per day, and in-between the intake levels of the hens fed the constant and reduced ME Lay. These values might seem low, but with an average fat content of 4.7 grams in yolk (Réhault-Godbert et al., 2019), a 0.7 gram increase per day could be a significant contribution to yolk formation. This might indicate that oil intake, rather than dietary energy level, facilitates long-term egg production. Kleyn et al. (2022) found that a higher lipid intake improves energy utilization in laying hens, but it did not increase egg production. However, they did not study long-term effects and included hens of different age periods in their study.

Body Composition

Limited effects of dietary treatments on body CF and CP were found. Unfortunately, the variation in the data of CF analysis in all periods was larger than the least significant difference that could be detected. This might have contributed the lack of results. In general, the body CF levels increased until wk 39, after which they slowly decreased and reached similar levels in wk 75 as at the start of production in wk 20. This pattern of body compositional change is similar to broiler breeders (Salas et al., 2019). The hens continued to increase energy intake over time (Table 3) and ME Lay intake seemed higher than required for egg production after wk 55. Hence, the catabolism of body CF was not needed to account for low ME Lay intake, and was therefore potentially related to other processes. At the end of a laying cycle, for example, hens naturally start to molt (Bennion and Warren, 1933), resulting in protein and fat catabolism (Webster, 2003). Perhaps this natural process influences metabolism and the body composition over time, already starting during the end of a laying cycle. Additionally, nutrient digestibility reduces from wk 48 to 75 by 15% for CP and 17% for CF (Gu et al., 2021b). So, even though nutrient intake was increasing over time, due to a reduction in nutrient digestibility, the hens potentially still needed to deplete their reserves.

Energy Utilization

Considering the influence of ME Lay intake on egg mass production, the segmented regression analysis showed reasonable correlation between egg mass and ME Lay intake with an R2 adjusted of 0.78 for the overall model (Table 9; Figure 2). The egg mass production was not increased above 330 kcal of ME Lay intake for white hens and 324 kcal of ME Lay intake for brown hens (Table 9; Figure 2). The reduced ME Lay diets resulted in a higher slope and earlier inflection point for egg mass, compared to the constant ME Lay diets. So the hens reached their maximum egg mass production with slightly lower ME Lay intake, when fed a reduced ME Lay diet. Similarly, the constant AFD Lys diets resulted in a higher slope and earlier inflection point for egg mass, compared to the reduced AFD Lys diets. So the hens reached their maximum egg mass production with slightly lower AFD Lys intake, when fed a reduced AFD Lys diet. For all of these effects, the inflection point for egg mass production was constant around 324 kcal of ME Lay intake. In other words, below an intake of 324 kcal of ME Lay, energy intake increased egg production. Above an intake of 324 kcal of ME Lay, egg mass production was no longer increased by higher ME Lay intake levels. The hens consumed this amount of energy around wk 35, but kept increasing their voluntary ME Lay intake beyond this point. Since egg mass was no longer affected by higher ME Lay intake levels, other processes related to maintenance or growth probably influenced the increasing ME Lay intake over time.

There are several factors to consider which could have influenced the increasing ME Lay intake over time. First, as discussed previously, the nutrient digestibility reduces over time (Gu et al., 2021b). Second, the metabolic rate was considered constant throughout the entire laying cycle. In humans, the basal metabolic rate can increase over time due to chronic inflammation, potentially as a result of high abdominal adipose tissue accumulation and non-alcoholic fatty liver disease (Bartke et al., 2021; Reddavide et al., 2019). The fat level of the hens in the current study increased over time reaching a maximum at 39 wk and then slowly reduced again (Table 8). No inflammation levels were tested. In hens, also liver functionality is decreased in older laying hens with reduced antioxidant capacity and altered lipid metabolism (Gu et al., 2021a). This might explain the seemingly increasing energy requirement over time, which was unrelated to egg mass production, but future studies should include liver functionality and inflammation parameters. Lastly, feather coverage can change over time and in general reduces as hens age. We did not measure the feather coverage differences in time and between treatments, but in general hens were fully covered when they arrived and still had some feather coverage at the end of the study. With a temperature around 21°C, the difference in expected increased energy requirement for maintenance is around 17 kcal per kg BW per day from 100 to 50% feather coverage and 21 kcal per kg BW per day from 50 to 0% coverage (Peguri and Coon, 1993). If we assume the hens reduced feather coverage by 50% from the beginning of the study towards the end, this could sum up to a maximum of 33 kcal per day increase in maintenance energy requirement for white hens and 39 kcal per day increase in maintenance requirement for brown hens. This increase in energy requirement could have accounted for the increase in voluntary ME Lay intake over time . We recommend for future studies to include feather coverage measurements and for energy requirement equations to be updated to include different maintenance requirements based on feather coverage.

Lysine Utilization

The AFD Lys intake in this study should be sufficient to maximize egg mass production (Silva et al., 2015). The egg weights did not significantly differ between treatments and was in general slightly lower than the breed average (lower weights varying 1 to 2 grams per egg at the end of production). If AFD Lys intake was driving egg mass production, it would be expected that the egg mass production was also higher in the treatments with the highest AFD Lys intake. However, in general, the egg mass production correlated poorly with the AFD Lys intake, with an R2 adj of 0.22 (Table 10). So in the current trial, the AFD Lys intake did not seem to correlate to egg mass production as much as ME Lay did. The egg mass production only increased with higher AFD Lys intake up to 775 mg lysine intake for the hens fed the reduced AFD Lys diets, and 850 mg lysine intake for the hens fed the constant AFD Lys diets (Table 10, Figure 3). Interestingly, the maximum egg mass of the hens fed the reduced AFD Lys was not higher than the maximum egg mass of the hens fed the constant AFD Lys. This indicates that the hens fed the reduced AFD Lys diets were more efficient in converting the AFD Lys into egg mass.

Practical Implications

The results of the current trial can help poultry producers and formulators to optimize laying hen diets. Reducing ME Lay over time can support long-term egg production both in white and brown hens. Moreover, the model of the egg production curve showed that a lower slope of decline after peak production is more important for laying persistency than a longer peak production phase. The results also indicate that white hens need 324 kcal of daily energy intake and brown hens need 331 kcal of daily energy intake for optimal egg mass production.

CONCLUSIONS

Laying persistency was influenced by breed and dietary ME Lay level, but not dietary AFD Lys level. White hens showed an improved laying persistency compared to brown hens, with a shorter peak production phase but a significant smaller slope of decline after peak, resulting in a higher total number of eggs per hen. Similarly, hens fed a reduced instead of constant ME Lay diet over time improved their laying persistency, with a shorter peak production phase and a significant smaller slope of decline after peak. Hens adapted their feed intake to have similar ME Lay intake levels between treatments, within breed. This resulted in higher ADFI (and thus nutrient intake) for hens fed the reduced ME Lay levels. So the improved laying persistency of hens fed the reduced ME Lay levels was probably related to a higher nutrient intake in general, although the impact of AFD Lys intake seemed limited in this study. Both energy and protein requirement seemed to increase over time, indicated by higher voluntary nutrient intake and reducing body CF after wk 43.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This study was financially supported by Cargill. The funding agency had no role in the study design, data collection, and analysis, but was involved in the preparations of this article. The authors would like to thank Eveline Spek, Esther Quick-Vos, Anke van Bergen, Maud Brugman, Marijn Derksen – Erinkveld, Jennifer de Jong as well as the rest of the staff of the Cargill Animal Nutrition Innovation Center Velddriel, for their assistance and care of the animals during this study.

DISCLOSURES

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Lieske van Eck reports financial support was provided by Cargill Animal Nutrition. Hsuan Chen reports financial support was provided by Cargill Animal Nutrition. Ines Carvalhido reports financial support was provided by Cargill Animal Nutrition . Henk Enting reports financial support was provided by Cargill Animal Nutrition.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104124.
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