Supply Organ Development in Young Broilers in Response to Changing Dietary Fat and Amino Acids in the Starter Period ^†

Abstract

Early growth in broilers depends on the rapid development of supply organs that enable nutrient use and support the growth of demand tissues such as muscle and bone. This study evaluated whether increasing dietary fat (and thereby AME) and amino acid concentration in starter diets enhances supply organ development and growth performance in Cobb male broilers. A 2 × 2 factorial design compared two fat levels, corresponding to two AME levels (F− 2750 vs. F+ 3050 kcal/kg), and two standardized ileal digestible lysine levels (AA− 1.0% vs. AA+ 1.2%) in an ideal ratio, to other essential AAs during days 0–11. Higher amino acid concentration consistently improved body weight gain, feed efficiency, and nutrient utilization throughout the trial, whereas the benefits of higher AME were mainly observed during the first 11 days. Diets high in both fat and amino acids reduced early feed intake, suggesting satiety effects. The effects on supply organ development were limited; only the pancreas and small intestine exhibited treatment-related differences in relative weight or allometric growth. In conclusion, increased amino acid concentration in starter diets improved overall broiler performance and nutrient efficiency, whereas dietary fat provided only short-term benefits. These improvements were not consistently associated with morphological changes in supply organs.

1. Introduction

Broiler chickens undergo dynamic shifts in nutrient requirements as their metabolic demands change across growth stages. These changes are influenced by dietary nutrient availability and the birds’ maintenance requirements. During the early stages of development, particularly from hatching until around day 7 or 8, broiler chickens show higher allometric growth in supply organs, such as the gastrointestinal tract and associated organs, as well as the respiratory and cardiovascular systems, while the maintenance needs for demand organs, such as the (breast) muscle, skeleton, and adipose tissue, remain relatively low [1,2]. From day 17 or 18 onward, growth shifts towards demand organs, resulting in increased maintenance requirements [1]. Inadequate development of supply organs at a young age may reduce nutrient efficiency and hinder the later growth of demand organs.

After hatching, energy utilization shifts from yolk lipids to dietary carbohydrates, altering endocrine and nutrient signaling and favoring starch digestion and glycolysis [3]. Lipase secretion peaks around day 18 of incubation and declines before hatching [4]. From hatching until day 7, lipase secretion remains lower than amylase or trypsin secretion, while bile secretion doubles by day 7 [4,5,6]. Insufficient bile salts in early life may compromise fat emulsification and, consequently, reduce lipase efficiency in hydrolyzing fat into fatty acids. Fat digestibility in young broilers is therefore relatively low, with reported values ranging from 42% to 69% [7,8,9]. Therefore, a high inclusion of dietary fat in starter phase diets is assumed to negatively impact performance parameters.

In contrast, increasing dietary fat together with overall nutrient concentration in a pre-starter diet increased the relative length and weight of the small intestine and cecum, but reduced relative pancreas and liver weights [10]. Similar effects on small intestinal weights were reported with dietary fat levels [11]. However, in both studies, these effects were confounded by variations in other nutrients. Previous work showed that increasing dietary energy through carbohydrates from day 0 to 11 reduced body weight gain (BWG) and feed intake (FI) at days 4 and 11, with these effects persist until day 35 [12]. The higher dietary energy from carbohydrates also resulted in higher allometric growth of the liver and ileum. Reported standardized ileal digestible lysine requirements in the first 11 days vary widely (≈0.98–1.44%) [13,14,15,16], reflecting differences in strain, sex, and amino-acid profiles. Consequently, the interactive effects of fat-derived AME and amino-acid concentration on early supply organ development and performance remain unclear.

Broiler chickens exhibit a highly dynamic metabolic state in early life. Although young broilers efficiently digest starch, its inclusion negatively impacts organ development and, consequently, growth performance [12]. Fat is less digestible than starch, especially at young age; however, because embryos rely on lipid-rich energy sources, fat may still be the preferred energy source to support organ development in newly hatched broilers. As broiler chickens transition from yolk-derived lipids to dietary carbohydrates after hatching, we hypothesize that increasing dietary fat (and thus AME) and amino acid concentration in the starter diet will better align with early digestive physiology, resulting in accelerated supply organ development, enhanced nutrient absorption, and improved growth efficiency.

2. Materials and Methods

All procedures were reviewed and approved by the Animal Welfare Committee of Schothorst Feed Research (AVD246002016450) on 1 of May 2019 and complied with Dutch laws and regulations on animal experiments. The experimental design complied with ARRIVE 2.0 guidelines [17].

2.1. Experimental Design and Diets

Four different starter phase diets were used in a 2 × 2 factorial design. Dietary fat was included at two levels to create an apparent metabolizable energy (AME) difference of 300 kcal (F−: <35 g/kg dietary fat with 2750 kcal/kg and F+: >75 g/kg dietary fat with 3050 kcal/kg; factor 1). Standardized ileal digestible (SID) lysine (Lys) was included at two levels (AA−: 1.00% and AA+: 1.20%; factor 2), with all balanced amino acids according to breed recommendations [18] (

Table 1. Ingredient and nutrient composition of the experimental starter phase diets (d0–11).

Dietary Fat/AME	F−	F−	F+	F+
Dietary Amino Acids	AA−	AA+	AA−	AA+
Ingredient composition (g/kg)
Corn	334.2	364.3	338.5	329.6
Wheat	250.0	250.0	250.0	250.0
Soybean meal > 48%CP	186.5	245.0	186.5	245.0
Wheat middlings	60.0	20.0	80.0	30.0
Peas	50.0	50.0	20.0	20.0
Potato protein	10.0	10.0	10.0	10.0
Corngluten meal 60%CP	5.0	5.0	7.5	7.5
Maize starch	5.0	5.0	0.0	0.0
Soybean oil	13.0	13.0	60.0	60.0
Limestone	17.7	17.7	17.7	17.7
Monocalciumphosphate	7.7	7.7	7.7	7.7
Salt	1.4	1.4	1.4	1.4
Sodiumbicarbonate	3.3	3.3	3.3	3.3
L-lysine HCl	2.7	3.2	2.7	2.5
DL-methionine	2.4	3.1	2.6	3.1
L-threonine	1.0	1.2	1.0	1.1
L-valine	0.1	0.1	0.1	0.1
L-arginine	0.1	0.1	0.1	0.1
L-isoleucine	0.1	0.1	0.1	0.1
Premix starter	5.0	5.0	5.0	5.0
Phytase	3.3	3.3	3.3	3.3
Glucanase-Xylanase	2.5	2.5	2.5	2.5
Total	1000.0	1000.0	1000.0	1000.0
Calculated chemical composition (g/kg)
Crude protein	185.0	215.0	185.0	215.0
Crude fat	34.1	34.9	82.6	79.4
Crude ash	56.5	56.9	56.2	56.7
Crude fiber	28.2	25.7	28.5	25.8
Dry matter	877.1	876.7	882.5	882.6
Starch (Ewers)	398.5	391.2	376.5	352.6
AME (kcal/kg) 1	2750	2750	3050	3050
Digestible lysine 1	10.0	12.0	10.0	12.0
Digestible methionine 1	4.8	5.9	4.8	5.9
Digestible methionine and cysteine 1	7.3	8.8	7.3	8.8
Digestible threonine 1	6.5	7.8	6.5	7.8
Digestible tryptophan 1	1.9	2.3	1.9	2.3
Digestible valine 1	8.0	9.6	8.0	9.6
Digestible isoleucine 1	6.6	8.0	6.6	8.0
Digestible arginine 1	10.5	12.6	10.5	12.6
Digestible glycine and serine 1	14.0	16.7	14.0	16.7
Digestible leucine 1	12.7	15.3	12.7	15.3
Calcium	9.0	9.0	9.0	9.0
Phosphorus	5.7	5.3	5.1	5.1
Analyzed chemical composition (g/kg)
Crude protein	184	213	182	216
Crude fat	34	35	83	80
Dry matter	883	881	885	887
Starch	378	383	372	348
Calcium	8.8	9.1	8.8	8.7
Phosphorus	5.5	5.4	4.8	4.8
Sodium	1.4	1.5	1.4	1.4
Manganese (mg)	91	118	111	98

F− = low dietary fat/AME; F+ = high dietary fat/AME; AA− = low amino acids; AA+ = high amino acids. ¹ Calculated according to CVB (2016). All vitamins and micro minerals were according to the Cobb breeder recommendations [19].

). Higher fat and amino acid concentrations were achieved by adjusting ingredient inclusion levels (soybean meal, soybean oil, wheat middlings, and lysine-HCl). Diets differed not only in AME and SID Lys, but also in fiber content and other nutrients. Although other nutrients may have contributed to confounding effects, these compositional changes reflect practical diet formulations, rather than isolated effects of these nutrients. To balance digestible amino acids relative to SID Lys, feed-grade amino acids were used. The starter phase diets (2.3 mm pellet) were fed from day 0 to 11, followed by a standard commercial grower (3.0 mm pellet; day 12 to 28; 3000 kcal; 1.10% SID Lys) and finisher phase diets (3.0 mm pellet; d 28 to d 35; 3100 kcal; 0.99% SID Lys). All treatments had similar raw materials, according to breeder recommendations [18]. All diets were formulated based on digestibility, energy calculation, and nutrient data from the Centraal Veevoerderbureau (2016). The diets were balanced using ingredients such as maize, soybean meal, wheat middlings, and peas. The composition of the starter phase diets, including all amino acids balanced to SID Lys, are presented in Table 1. Diets were manufactured by ABZ Diervoeding (Leusden, The Netherlands).

All experimental diets were analyzed for dry matter (ISO 6496:1999) [20], ash (ISO 5984:2002) [21], crude protein (ISO 16634-1:2008) [22], crude fat by acid hydrolysis (ISO 6492:2002) [23], starch (ISO 6493:2000) [24], and calcium, sodium, and manganese content by ICP-AES (ISO 27085:2009) [25].

2.2. Animals and Housing

In total, 3200 eggs were selected within a weight range of 62 to 68 g from a single-origin commercial Cobb 500 fast-feathering flock. Incubation was performed at a commercial hatchery (Lagerwey, Lunteren, The Netherlands) according to breed standards [19]. The first 600–620 chicks hatched at approximately 12 h before the estimated hatch time and were removed from the hatching baskets. About 8 h later, approximately 75% of the total hatched chicks were sexed and selected. Only male chickens were selected for the experiment to improve homogeneity, as males typically have greater growth potential and nutrient requirements. The 820 selected chickens were transported to the research facility (Schothorst Feed Research, Lelystad, The Netherlands). Upon arrival, average chick weight was determined, and 20 chickens per pen were randomly selected for placement. After placement, initial pen weight was confirmed to fall within ±1 sd of the average estimated pen weight. Pens (1.00 × 2.00 m) were arranged in 10 blocks, with four treatments randomly assigned within each block (10 pen replicates/treatment). From the time of placement, feed and water were supplied ad libitum. Temperature and lighting were managed according to breed recommendations [19]. Birds were checked twice daily by caretakers, and mortality was recorded daily.

2.3. Data Collection

Pen body weight (BW) and FI were measured on days 0, 4, 11, 28, and 35 of age. For each period, BWG and feed-to-gain ratio (F:G) were calculated. Nutrient intake (AME, Lys, starch, and fat) and nutrient-to-gain ratios were subsequently calculated from the analyzed diet composition and FI.

For organ measurement, 20 broiler chickens were randomly selected at day 0, and one representative broiler chick per pen was randomly selected on days 4 and 11. Euthanasia was performed using CO₂. Carcasses were weighed before and after bleeding. The following supply organs were dissected and weighed: heart, liver, pancreas, proventriculus, gizzard, duodenum, jejunum, ileum, colon, and ceca. The remainder of the carcass (including head, feathers, and all other parts not dissected) was also weighed. The proventriculus and gizzard were cut open and rinsed to remove feed residuals before weighing. Intestinal segments were emptied by gentle squeezing. The lengths of the duodenum, jejunum (the end of duodenum till the Meckel’s diverticulum), ileum (Meckel’s diverticulum till the ileal–cecal junction), ceca, and colon were measured.

2.4. Histology

During dissection on days 4 and 11, a tissue segment from the midpoint of the duodenum, jejunum, and ileum was collected from each selected bird. Approximately 1 mL 10% formaldehyde solution was injected 5 cm from the midpoint to flush the intestinal content. The corresponding section was excised and fixed in 10% formaldehyde solution at room temperature.

The fixed samples were embedded in paraffin, sectioned at 5–6 µm using a microtome (Microm HM355S; Thermo Fisher Scientific, Waltham, MA, USA) and mounted on glass slides. Slides were stained with hematoxylin and eosin (HE) to measure villus length, crypt depth, and goblet cell count, using cellSens Dimensions (Olympus; Tokyo, Japan). Cells expressing mucin glycoprotein were identified by combined Alcian Blue (AB) and periodic acid Schiff staining, as described by [26].

2.5. Allometric Growth Modeling

Allometric relationships were modeled based on Huxley’s allometric growth model [27]. Growth curves were estimated for each treatment over the period’s days 0–4, 4–11, 0–11, and 0–4–11 to predict the weights of the heart, liver, proventriculus, gizzard, pancreas, duodenum, jejunum, ileum, and ceca, relative to yolk-free BW, using the following equation:

$$y=a{x}^b$$

where y is the organ weight (g), x is yolk-free BW (g), and a and b are coefficients estimated using the NLIN procedure in SAS (Version 9.3, 2011, SAS Institute Inc., Cary, NC, USA). The coefficient a represents the scale variable or intercept. The coefficient b indicates the organ growth rate relative to overall body growth, =1 is an equal growth rate (isometry), <1 is a slower growth rate, and >1 is a faster growth rate. It should be noted that the coefficients a and b are not fully independent, as a higher intercept (a) can be offset by a lower slope (b), or vice versa, leading to similar fitted values.

2.6. Statistical Analysis

Data were checked for outliers; however, no data points were excluded. The normality of residuals and the homogeneity of variances were verified prior to analysis. Data were analyzed using a two-way ANOVA, via the PROC GLM procedure in SAS. Mortality data were analyzed assuming a binomial distribution, using the same statistical model. Results are presented as least square (LS) means ± pooled SEM. Statistical significance was declared at p ≤ 0.05.

The fit of the allometric growth curves was first evaluated using pseudo-R² and normalized root mean square error (nRMSE). All models produced pseudo-R² values above 0.75, and nRMSE% values ranged from 3.8% to 11.6% relative to the mean observed organ weights across all ages and treatments. These metrics confirmed an adequate model fit. Allometric growth curves were compared between treatments using non-linear analysis of covariance (ANCOVA). The null hypothesis of curve equality was tested using a paired F-test procedure described by [28]. Pairs of treatments (trt) were compared by fitting separate allometric curves for each treatment using the NLIN procedure in SAS. From these analyses, the overall sum of squares (SS_sep = SS_trt1 + SS_trt2) and their corresponding degrees of freedom (df) (df_sep = df_trt1 + df_trt2) were obtained. Subsequently, data from the two paired treatments were pooled, and a single allometric curve was estimated to obtain the total sum of squares (SS_pool), and degrees of freedom (df_pool) were determined. Next, the F ratio was calculated:

$$\mathrm{F}={\displaystyle \frac{({\mathrm{S}\mathrm{S}}_{\mathrm{p}\mathrm{o}\mathrm{o}\mathrm{l}}-{\mathrm{S}\mathrm{S}}_{\mathrm{s}\mathrm{e}\mathrm{p}})/({\mathrm{d}\mathrm{f}}_{\mathrm{p}\mathrm{o}\mathrm{o}\mathrm{l}}-{\mathrm{d}\mathrm{f}}_{\mathrm{s}\mathrm{e}\mathrm{p}})}{{\mathrm{S}\mathrm{S}}_{\mathrm{s}\mathrm{e}\mathrm{p}}-{\mathrm{d}\mathrm{f}}_{\mathrm{s}\mathrm{e}\mathrm{p}}}}$$

A large F value (corresponding to a low p-value) indicated that the variation was better explained in separate fits than by the pooled fit. When p ≤ 0.05, the null hypothesis was rejected and the allometric curves were considered different. To control for type I error inflation resulting from multiple pairwise F-tests, p-values were adjusted using the Bonferroni correction. For each period, separate fits between two treatments were tested, resulting in 18 comparisons for each measured organ.

3. Results

3.1. Growth Performance and Feed Intake

The effects of the starter diet on performance from the time of placement to d 35 are shown in

Table 2. Effects of dietary fat/AME and amino acid concentration in the starter phase on body weight, body weight gain, feed intake, and feed-to-gain ratio ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	20	20	20	20	10	10	10	10
BW (g/bird)
0 d	45.85	45.9	45.75	46.0	45.7	46.0	45.8	46.0	0.1	0.521	0.214	0.787
4 d	107.6	108.7	107.2	108.9	105.3 c	109.6 a	109.5 a	108.2 b	0.2	0.364	0.011	0.027
11 d	347.3	356.7	339.5	364.6	329.5 c	365.1 a	349.4 b	364.0 a	3.0	0.030	<0.001	0.010
28 d	1839.3	1856.6	1813.5	1882.4	1801.5	1876.1	1823.5	1888.6	11.3	0.291	<0.001	0.178
35 d	2659.2	2653.4	2616.3	2695.2	2616.1	2701.2	2616.7	2689.0	17.1	0.998	0.032	0.395
BW gain (g/bird)
0–4 d	61.6	62.9	61.6	62.9	59.6 c	63.6 a	63.7 a	62.2 b	0.2	0.442	0.437	0.027
4–11 d	240	248	232	256	224 c	255 a	240 b	256 a	2.5	0.031	<0.001	0.010
0–11 d	301	311	293	319	284 c	319 a	304 b	318 a	2.7	0.036	<0.001	0.042
11–28 d	1512	1499	1473	1538	1472	1551	1474	1525	9.2	0.293	<0.001	0.579
28–35 d	820	796	804	813	814	825	793	800	7.2	0.093	0.112	0.396
0–35 d	2613	2607	2571	2649	2570	2655	2571	2643	16.8	0.442	0.020	0.437
FI (g/bird)
0–4 d	57.4	55.5	56.7	56.2	56.7 b	58.2 a	56.8 b	54.2 c	0.2	0.163	0.930	0.042
4–11 d	288	281	285	284	286 ab	291 a	284 b	278 c	1.8	0.020	0.656	0.049
0–11 d	346	337	342	341	342 b	349 a	341 b	332 c	2.1	0.009	0.747	0.048
11–28 d	2086	2088	2062	2112	2053	2119	2071	2104	10.8	0.702	0.012	0.185
28–35 d	1286	1267	1255	1298	1282	1290	1227	1307	9.6	0.636	0.023	0.147
0–35 d	3718	3692	3659	3751	3677	3758	3639	3744	18.2	0.163	0.043	0.052
F:G (g:g/bird)
0–4 d	0.932	0.880	0.918	0.893	0.945 c	0.918 b	0.889 ab	0.870 a	0.008	<0.001	<0.001	<0.001
4–11 d	1.206	1.136	1.230	1.113	1.274 d	1.138 b	1.185 c	1.087 a	0.009	<0.001	<0.001	<0.001
0–11 d	1.148	1.083	1.168	1.068	1.203 d	1.093 b	1.122 c	1.044 a	0.006	<0.001	<0.001	<0.001
11–28 d	1.398	1.392	1.400	1.390	1.394 ab	1.403 b	1.404 b	1.378 a	0.004	0.064	0.051	<0.001
28–35 d	1.568	1.580	1.561	1.587	1.574 ab	1.562 ab	1.548 a	1.612 b	0.005	0.429	0.237	0.037
0–35 d	1.424	1.415	1.424	1.414	1.431 a	1.414 b	1.415 b	1.416 b	0.003	0.051	0.051	<0.001

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a–d values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

. Interactions between dietary fat (F; thus a 300 kcal AME difference) and amino acid concentration (AA) were found for BW on days 4 and 11 and for BW gain from day 0 to 4 and 4 to 11. The BW gain from day 0 to 4 was lower in the F−AA− group compared to the other groups. From day 4 to 11, BW gain remained the lowest in the F−AA− group and increased with AA+ and F+. From day 11 onwards, BW was consistently higher in the AA+ groups, resulting in the highest BW on day 35, compared to the AA− groups. The initial positive effect of additional fat/AME on BW gain diminished over time.

For FI, F × AA interactions were found in the periods from day 0 to 4, 4 to 11, and 0 to 11. The highest FI was found in F−AA+, whereas the lowest FI was in F+AA+. After d 11, FI was higher in the AA+ groups compared to the AA− groups, resulting in the highest overall FI.

For the F:G ratio, F × AA interactions were found in each period of measurement. The F:G ratio was highest in the F−AA− group from day 0 to 4 and 4 to 11, with F+AA+ showing the lowest F:G ratio. From day 11 to 28, the F:G ratio was highest in the F−AA+ and F+AA− groups, while still lowest in the F+AA+ group. In the final period (day 28 to 35), the F:G ratio was highest in the F+AA+ group and lowest in the F+AA− group. Overall, the F−AA− group had the highest F:G ratio, while the other groups had similar, lower F:G ratios.

3.2. Nutrient Intake and Efficiency

Nutrient intake and gain per nutrient for day 0 to 4 and 4 to 11 are shown in

Table 3. Effects of dietary fat/AME and amino acid concentration in the starter phase on the nutrient intake and nutrient-to-gain ratio ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	20	20	20	20	10	10	10	10
Nutrient intake
AME (kcal/bird)
0–4 d	165.4	169.5	167.8	167.1	162.2 c	168.5 ab	173.4 a	165.6 bc	2.2	0.083	0.873	0.040
4–11 d	827.1	893.4	865.6	854.8	823.0 c	831.1 c	908.2 a	878.5 b	2.9	<0.001	<0.001	<0.001
Fat (g/bird)
0–4 d	1.98	4.75	3.47	3.26	1.94 d	2.03 c	5.00 a	4.50 b	0.01	<0.001	<0.001	0.031
4–11 d	9.91	22.70	16.45	16.16	9.67 d	10.15 c	23.23 a	22.17 b	0.08	<0.001	0.643	0.048
Lys (g)
0–4 d	0.64	0.61	0.57	0.68	0.57 c	0.71 a	0.57 c	0.65 b	0.02	0.147	<0.001	0.022
4–11 d	3.18	3.10	2.86	3.41	2.87 c	3.48 a	2.85 c	3.33 b	0.05	0.315	<0.001	0.001
Starch (g/bird)
0–4 d	22.7	18.7	21.1	22.7	22.6 a	22.8 a	19.6 b	17.9 c	1.3	<0.001	0.245	0.044
4–11 d	113.8	105.2	112.0	113.8	113.8 a	113.8 a	110.1 ab	100.4 b	1.5	<0.001	0.167	<0.001
Nutrient-to-gain ratio
AME:g (Kcal:g/bird)
0–4 d	2.70	2.76	2.77	2.69	2.73	2.68	2.79	2.71	0.03	0.394	0.069	0.061
4–11 d	3.40	3.58	3.70	3.25	3.62	3.16	3.84	3.32	0.05	0.061	0.023	0.056
Fat:g (g:g/bird)
0–4 d	0.044	0.076	0.064	0.055	0.049 b	0.037 c	0.078 a	0.073 a	0.001	<0.001	<0.001	<0.001
4–11 d	0.056	0.092	0.082	0.066	0.066 c	0.045 d	0.097 a	0.087 b	0.001	<0.001	<0.001	<0.001
Lys:g (mg:g/bird)
0–4 d	1.024	0.975	0.929	1.085	0.938 c	1.112 a	0.923 d	1.057 b	0.004	0.057	<0.001	0.003
4–11 d	1.295	1.252	1.215	1.334	1.236 c	1.355 a	1.193 d	1.311 b	0.005	<0.001	<0.001	<0.001
Starch:g (g:g/bird)
0–4 d	0.36	0.30	0.33	0.32	0.36 a	0.35 ab	0.31 b	0.29 b	0.01	0.017	0.201	0.048
4–11 d	0.45	0.42	0.48	0.41	0.49 a	0.43 bc	0.46 ab	0.39 c	0.01	0.053	<0.001	0.001

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a–d values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

. There were F × AA interactions for AME, lysine, starch, and fat intake, showing the same effects for both periods (day 0 to 4 and 4 to 11). The AME intake was highest in the F+AA− group and lowest in the F−AA− group. Lysine intake was highest in F−AA+ and lowest in F−AA− and F+AA−. Starch intake was highest in the F−AA− and F−AA+ and lowest in F+AA+ group. Fat intake was highest in the F+AA− group and lowest in the F−AA− group. There were F × AA interactions for the nutrient-to-gain ratio of AME, lysine, starch, and fat. From day 4 to 11, the AME:gain ratio was higher in AA− groups, compared to the AA+ groups. The Lys:gain ratio was highest in F−AA+ and lowest in F+AA− in all periods, whereas the starch:gain ratio was highest in the F−AA− group and lowest in the F+AA+ group. The fat:gain ratio was highest in the F+AA− group and lowest in the F−AA+ group in all periods.

3.3. Supply Organ Weight and Intestinal Histo-Morphological Parameters

The effects of the starter diet on relative organ weights for days 4 and 11 are shown in

Table 4. Effects of dietary fat/AME and amino acid concentration in the starter phase on supply organ weight as percentage of body weight at day 4 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	20	20	20	20	10	10	10	10
BW (g)	116.5	118.4	116.9	118.0	113.9 b	119.2 a	119.9 a	116.9 ab	1.8	0.352	0.516	0.035
Supply organ weight d 4, % of BW
Heart	0.96	0.95	0.94	0.97	0.94	0.97	0.94	0.97	0.11	0.931	0.473	0.975
Liver	4.92	4.36	4.49	4.79	4.60 b	5.24 a	4.39 c	4.34 c	0.07	0.003	0.098	0.044
Proventriculus	1.14	1.11	1.11	1.14	1.13	1.14	1.08	1.15	0.01	0.761	0.411	0.527
Gizzard	4.35	4.54	4.47	4.42	4.56	4.14	4.39	4.69	0.17	0.441	0.947	0.084
Pancreas	0.42	0.45	0.41	0.45	0.40	0.43	0.43	0.47	0.02	0.161	0.099	0.118
Duodenum	1.79	1.96	1.86	1.89	1.80	1.77	1.91	2.02	0.08	0.120	0.562	0.627
Jejunum	3.47	3.76	3.63	3.61	3.40	3.56	3.86	3.66	0.12	0.123	0.964	0.244
Ileum	2.59	2.76	2.70	2.65	2.63	2.54	2.77	2.75	0.09	0.204	0.638	0.720
Ceca	0.75	0.85	0.75	0.83	0.72	0.77	0.78	0.90	0.07	0.433	0.106	0.956

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a–c values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

and

Table 5. Effects of dietary fat/AME and amino acid concentration in the starter phase on supply organ weight as percentage of body weight at day 11 ¹.

	F		A		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	20	20	20	20	10	10	10	10
BW (g)	363.2	368.1	354.8	376.4	341.7 c	384.7 a	368.0 b	368.1 b	3.7	0.599	0.025	0.025
Supply organ weight d 11, % of BW
Heart	0.75	0.76	0.77	0.74	0.77	0.73	0.78	0.76	0.09	0.624	0.495	0.700
Liver	4.08	3.95	3.98	4.05	4.11	4.06	3.85	4.05	0.12	0.213	0.517	0.059
Proventriculus	0.76	0.77	0.79	0.73	0.79	0.72	0.79	0.75	0.01	0.520	0.005	0.507
Gizzard	2.97	3.05	3.05	2.97	3.11	2.82	2.99	3.12	0.08	0.406	0.472	0.064
Pancreas	0.43	0.41	0.45	0.40	0.46 a	0.41 bc	0.42 ab	0.39 c	0.01	0.116	0.040	0.046
Duodenum	1.41	1.54	1.48	1.48	1.44	1.39	1.51	1.57	0.11	0.078	0.901	0.388
Jejunum	2.61	2.66	2.67	2.61	2.65	2.57	2.69	2.64	0.09	0.648	0.555	0.890
Ileum	1.95	1.95	2.00	1.91	2.03	1.88	1.98	1.93	0.06	0.959	0.223	0.551
Ceca	0.60	0.68	0.63	0.65	0.61	0.59	0.64	0.72	0.06	0.129	0.409	0.218

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a–c values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

, respectively, and the length and relative weights for the small intestine for both days are shown in

Table 6. Effects of dietary fat/AME and amino acid concentration in the starter phase on the length and weight per cm of the intestine on day 4 and 11 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n					10	10	10	10
Day 4
Duodenum
Length (cm)	14.4	15.8	15.0	15.2	14.5	14.2	15.5	16.2	0.2	<0.001	0.288	0.491
Weight (g/cm)	0.147	0.146	0.146	0.147	0.145	0.149	0.147	0.145	0.003	0.306	0.797	0.671
Jejunum
Length (cm)	32.6	33.8	33.9	32.4	32.9	32.3	35.1	32.7	0.4	0.234	0.298	0.098
Weight (g/cm)	0.124	0.100	0.111	0.114	0.120	0.129	0.101	0.099	0.002	<0.001	0.914	0.268
Ileum
Length (cm)	33.0	32.5	32.6	33.0	32.6	33.4	32.5	32.6	0.5	0.602	0.450	0.486
Weight (g/cm)	0.091	0.133	0.112	0.111	0.093	0.088	0.132	0.133	0.003	<0.001	0.845	0.657
Day 11
Duodenum
Length (cm)	10.8	11.4	11.3	10.9	11.1	10.4	11.4	11.3	0.2	0.079	0.322	0.387
Weight (g/cm)	0.485	0.494	0.463	0.516	0.451	0.519	0.476	0.512	0.008	0.416	<0.001	0.284
Jejunum
Length (cm)	47.6	48.5	47.70	48.45	45.3 b	50.0 a	50.1 a	46.9 b	0.9	0.567	0.611	0.026
Weight (g/cm)	0.203	0.202	0.201	0.203	0.207	0.198	0.195	0.208	0.006	0.857	0.723	0.088
Ileum
Length (cm)	48.3	48.1	48.9	47.4	48.7	47.8	49.2	47.0	0.4	0.996	0.522	0.716
Weight (g/cm)	0.151	0.152	0.149	0.154	0.149	0.153	0.150	0.154	0.004	0.714	0.101	0.285

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a,b values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

. The BW of the dissected birds is also reported herein. On day 4, the BW of randomly selected birds was higher overall in all treatments compared to the average weights. While the BW of randomly selected birds on day 11 was higher for the F−AA+ group compared to the average weights, the other groups were similar in BW to the treatment average. There were F × AA interactions found for relative liver weight on day 4 and relative pancreas weight on day 11. Relative liver weight was highest in F−AA+ on day 4, while it was lowest in F+AA− and F+AA+. On day 11, relative pancreas weight was highest in F−AA−, while it was lowest in F+AA+. Relative proventriculus weight was higher in the AA− groups on d 11. In the relative weights of all other organs, no differences were observed among treatments.

On day 4, the duodenum was longer in F+ than F−, without observed differences in weight per cm. Jejunum weight per cm was higher in F− than F+, without differences in length. Ileum weight per cm was higher in F+ than F−, without observed differences in length. There was an F × AA interaction on day 11 for jejunal length, where F−AA+ and F+AA− resulted in the longest jejunal length and F−AA− and F+AA+ resulted in the shortest jejunal length. Duodenal weight per cm was higher in the AA+ groups than the AA− groups on day 11.

The results of villus length, crypt depth, and villus-to-crypt ratio (V:C) for days 4 and 11 are shown in

Table 7. Effects of dietary fat/AME and amino acid concentration in the starter phase on villus length and crypt depth at day 4 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	20	20	20	20	10	10	10	10
Duodenum
Villus length (µm)	1053	999	1025	1027	1048	1058	1001	997	17	<0.001	0.328	0.191
Crypt depth (µm)	166	181	169	177	169 b	162 b	169 b	192 a	5.7	0.196	0.487	0.047
V:C ratio	6.6	6.1	6.5	6.1	6.4 a	6.7 a	6.6 a	5.5 b	0.2	0.218	0.240	0.032
Jejunum
Villus length (µm)	518	560	540	536	530 ab	503 b	549 ab	570 a	13.9	<0.001	0.078	<0.001
Crypt depth (µm)	138	157	146	148	144 bc	132 c	148 b	165 a	4.0	0.010	0.472	0.021
V:C ratio	3.9	3.6	3.8	3.7	3.8 a	3.9 a	3.8 a	3.5 b	0.1	0.174	0.458	0.002
Ileum
Villus length (µm)	401	404	402	404	406	396	398	411	10	0.853	0.921	0.697
Crypt depth (µm)	127	140	125	149	123 b	129 b	128 b	152 a	4.3	0.103	0.070	<0.001
V:C ratio	3.3	3.1	3.3	3.0	3.4	3.1	3.2	2.9	0.1	0.202	0.089	0.097

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study. ^a–c values within rows with different superscripts differ significantly at p ≤ 0.05 on the interaction between F and AA.

and

Table 8. Effects of dietary fat/AME and amino acid concentration in the starter phase on villus length and crypt depth at day 11 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n					10	10	10	10
Duodenum
Villus length (µm)	1523.1	1600.6	1557.8	1565.9	1534.2	1512	1581.4	1619.7	27.9	0.080	0.888	0.097
Crypt depth (µm)	276.1	282.1	279.6	278.6	270.3	281.9	288.9	275.2	5.0	0.567	0.919	0.227
V:C ratio	5.6	5.8	5.7	5.8	5.8	5.5	5.6	6.0	0.1	0.547	0.878	0.109
Jejunum
Villus length (µm)	875.5	907.3	871.7	911.1	861.6	889.4	881.8	932.8	22.5	0.493	0.398	0.802
Crypt depth (µm)	227.1	242	241.7	227.4	239.5	214.6	243.9	240.1	5.8	0.205	0.205	0.368
V:C ratio	4.0	3.9	3.8	4.1	3.8	4.2	3.7	4.0	0.1	0.379	0.075	0.662
Ileum
Villus length (µm)	565.4	520.85	545.2	541.05	566.3	564.5	524.1	517.6	15.6	0.068	0.896	0.941
Crypt depth (µm)	189.0	183.1	178.2	193.9	178.3	199.7	178.1	188.1	4.1	0.073	0.040	0.483
V:C ratio	3.1	2.9	3.1	2.9	3.2	2.9	3.0	2.8	0.1	0.122	0.042	0.527

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study.

, respectively. There were F × AA interactions for duodenal crypt depth, duodenal V:C ratio, jejunal villus length, jejunal crypt depth, jejunal V:C ratio, and ileal crypt depth on day 4. Duodenal crypts were shallowest in F−AA+ and deepest in F+AA+. The F+ had the lowest duodenal villus length, leading to the highest V:C ratio in the F−AA+ group and the lowest V:C ratio in the F+AA+ group. In the jejunum, villi were longest and crypts deepest in F+AA+, while F–AA+ had the shortest villi and shallowest crypts, leading to the highest V:C ratio in F–AA+ and the lowest in F+AA+. Ileal crypts were deepest in the F+AA+ group, whereas crypt depth was smallest in the F−AA− group. On day 11, ileal crypts were deepest in the AA+ groups, leading to the lowest V:C ratio.

There were no differences observed in the total number of goblet cells, goblet cell size, area of the goblet cells per villus, or number of goblet cells with acidic mucus (Appendix A,

Table A1. Effects of dietary fat/AME and amino acid concentration in the starter phase on goblet cell development on day 4 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	16	16	16	16	8	8	8	8
Duodenum
Total goblet cells per villus	80	71	80	72	82	77	78	66	4.8	0.184	0.196	0.051
Acidic goblet cells per villus	62	59	64	59	65	61	62	57	4.7	0.638	0.715	0.203
Goblet cells surface per 1 mm of villus	1921	1700	1952	1671	2029	1817	1875	1525	121	0.397	0.466	0.101
Goblet cell size (µm)	24	23	24	23	24	23	24	22	0.6	0.869	0.843	0.461
Goblet cell surface as % of villus	1.34	1.32	1.45	1.21	1.45	1.24	1.46	1.19	0.10	0.930	0.127	0.116
Jejunum
Total goblet cells per villus	55	62	57	60	54	56	61	65	2.9	0.217	0.604	0.192
Acidic goblet cells per villus	44	59	50	53	42	45	59	60	3.0	0.213	0.897	0.188
Goblet cells surface per 1 mm of villus	1347	1410	1345	1412	1355	1338	1334	1487	89	0.618	0.575	0.206
Goblet cell size (µm)	24	22	23	23	24	24	22	23	0.7	0.719	0.973	0.651
Goblet cell surface as % of villus	2.08	2.23	2.17	2.14	2.13	2.02	2.21	2.26	0.12	0.382	0.604	0.237
Ileum
Total goblet cells per villus	60	60	59	61	61	58	56	64	3.5	0.969	0.902	0.210
Acidic goblet cells per villus	47	45	45	46	50	43	41	49	3.3	0.756	0.843	0.428
Goblet cells surface per 1 mm of villus	1251	1177	1133	1294	1167	1334	1100	1254	66.9	0.644	0.717	0.619
Goblet cell size (µm)	23	20	20	22	21	24	20	20	0.9	0.489	0.508	0.337
Goblet cell surface as % of villus	2.87	3.09	2.82	3.14	2.71	3.02	2.93	3.25	0.17	0.552	0.427	0.199

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study.

and

Table A2. Effects of dietary fat/AME and amino acid concentration in the starter phase on goblet cell development on day 11 ¹.

	F		AA		F × AA					p-Values
	F−	F+	AA−	AA+	F−AA−	F−AA+	F+AA−	F+AA+	SEM	F	AA	F × AA
n	16	16	16	16	8	8	8	8
Duodenum
Total goblet cells per villus	174	197	179	193	160	188	198	198	8.1	0.117	0.312	0.104
Acidic goblet cells per villus	125	135	126	134	116	134	136	135	7.7	0.764	0.801	0.225
Goblet cells surface per 1 mm of villus	4159	4823	4314	4667	3665	4652	4964	4682	224	0.163	0.356	0.082
Goblet cell size (µm)	23	24	23	24	22	24	24	23	0.7	0.874	0.791	0.814
Goblet cell surface as % of villus	1.60	1.69	1.55	1.74	1.42	1.77	1.68	1.70	0.08	0.653	0.469	0.181
Jejunum
Total goblet cells per villus	139	139	129	149	130	148	127	151	8.9	0.956	0.679	0.548
Acidic goblet cells per villus	105	103	95	113	96	114	94	112	7.6	0.971	0.365	0.296
Goblet cells surface per 1 mm of villus	2988	3456	2947	3497	2655	3321	3239	3673	243	0.348	0.384	0.165
Goblet cell size (µm)	22	24	23	23	21	22	24	23	0.6	0.785	0.917	0.696
Goblet cell surface as % of villus	2.16	2.31	2.09	2.39	1.95	2.39	2.23	2.40	0.14	0.761	0.517	0.498
Ileum
Total goblet cells per villus	115	112	126	102	132	98	119	106	7.3	0.951	0.296	0.117
Acidic goblet cells per villus	86	87	94	78	99	75	93	81	7.6	0.916	0.137	0.192
Goblet cells surface per 1 mm of villus	1708	1395	1567	1535	1852	1564	1282	1507	109	0.145	0.864	0.286
Goblet cell size (µm)	16	13	13	16	15	17	11	14	0.8	0.349	0.371	0.318
Goblet cell surface as % of villus	2.29	2.25	2.46	2.08	2.40	2.17	2.52	1.99	0.11	0.865	0.349	0.318

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Values represent pen means; each treatment consists of 10 replicate pens with 20 male broiler chickens at the start of the study.

).

3.4. Allometric Growth

Allometric growth coefficients as indicators for supply organs development from day 0 to 11 are shown in

Table 9. Allometric growth coefficients ¹ for effects of dietary fat/AME and amino acid concentration in the starter phase on supply organs from day 0 to 11.

Organ	Diet Treatment	a	a (95% CI)	b	b (95% CI)	Pseudo-R2
Heart	F−AA−	0.013	0.011–0.014	0.903	0.861–0.958	0.90
	F−AA+	0.014	0.012–0.016	0.896	0.868–0.912	0.91
	F+AA−	0.012	0.010–0.013	0.919	0.891–0.942	0.94
	F+AA+	0.014	0.011–0.015	0.895	0.879–0.914	0.93
Liver	F−AA−	0.031	0.027–0.036	1.048	1.007–1.102	0.89
	F−AA+	0.037	0.034–0.041	1.019	0.992–1.056	0.90
	F+AA−	0.033	0.030–0.038	1.025	1.001–1.048	0.93
	F+AA+	0.027	0.026–0.029	1.069	1.038–1.103	0.97
Proventriculus	F−AA−	0.074	0.067–0.077	0.601	0.581–0.624	0.90
	F−AA+	0.077	0.071–0.082	0.602	0.574–0.636	0.86
	F+AA−	0.064	0.051–0.079	0.644	0.592–0.721	0.76
	F+AA+	0.074	0.068–0.076	0.612	0.595–0.631	0.94
Gizzard	F−AA−	0.051	0.036–0.056	0.919	0.881–0.953	0.79
	F−AA+	0.059	0.052–0.063	0.879	0.853–0.907	0.87
	F+AA−	0.063	0.039–0.076	0.863	0.817–0.961	0.77
	F+AA+	0.056	0.051–0.059	0.907	0.880–0.936	0.89
Pancreas	F−AA−	0.002	0.002–0.002	1.122	1.008–1.168	0.88
	F−AA+	0.003	0.002–0.003	1.069	1.014–1.153	0.82
	F+AA−	0.003	0.003–0.004	1.051	1.009–1.114	0.89
	F+AA+	0.005	0.005–0.005	0.971	0.939–0.994	0.92
Duodenum	F−AA−	0.020	0.017–0.021	0.949	0.901–0.980	0.82
	F−AA+	0.020	0.017–0.021	0.941	0.913–0.991	0.86
	F+AA−	0.021	0.017–0.025	0.935	0.889–0.975	0.79
	F+AA+	0.019	0.016–0.022	0.970	0.941–1.001	0.82
Jejunum	F−AA−	0.027	0.021–0.035	1.007	0.967–1.089	0.75
	F−AA+	0.035	0.031–0.037	0.951	0.920–0.982	0.81
	F+AA−	0.039	0.032–0.044	0.937	0.883–0.967	0.80
	F+AA+	0.034	0.026–0.039	0.962	0.878–0.993	0.79
Ileum	F−AA−	0.033	0.027–0.036	0.927	0.869–0.997	0.76
	F−AA+	0.037	0.033–0.039	0.888	0.857–0.927	0.83
	F+AA−	0.039	0.035–0.042	0.889	0.889–0.928	0.88
	F+AA+	0.042	0.039–0.044	0.869	0.853–0.901	0.93
Ceca	F−AA−	0.005	0.004–0.006	1.054	1.002–1.188	0.77
	F−AA+	0.005	0.004–0.006	1.038	1.001–1.183	0.78
	F+AA−	0.004	0.004–0.005	1.059	0.997–1.123	0.75
	F+AA+	0.005	0.004–0.006	1.065	1.003–1.145	0.75

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Coefficients for the allometric equation y = ax^b; y = supply organ weight in g; x = yolk-free body weight in g; a and b estimated coefficients with confidence interval (CI). Values represent model-derived parameters describing the relationship between organ weight and body weight. Goodness-of-fit was evaluated using pseudo-R² and root mean square error (RMSE). No statistical significance is reported for these parameters, as between-treatment comparisons are presented separately in Table 10.

; coefficients for other calculated periods can be found in the Appendix B 

Table A3. Allometric growth coefficients ¹ for effects of dietary fat/AME and amino acid concentration in the starter phase on supply organs from day 0 to 4.

Organ	Diet Treatment	a	a (95% CI)	b	b (95% CI)	Pseudo-R2
Heart	F−AA−	0.005	0.004–0.006	1.129	1.099–1.166	0.91
	F−AA+	0.005	0.004–0.006	1.121	1.087–1.154	0.90
	F+AA−	0.006	0.005–0.007	1.095	1.063–1.124	0.93
	F+AA+	0.005	0.004–0.006	1.152	1.118–1.189	0.92
Liver	F−AA−	0.003	0.002–0.004	1.580	1.525–1.633	0.88
	F−AA+	0.003	0.002–0.004	1.594	1.546–1.639	0.87
	F+AA−	0.005	0.004–0.006	1.450	1.411–1.481	0.91
	F+AA+	0.005	0.004–0.006	1.443	1.402–1.482	0.92
Proventriculus	F−AA−	0.090	0.082–0.097	0.561	0.533–0.590	0.83
	F−AA+	0.079	0.072–0.085	0.596	0.568–0.628	0.85
	F+AA−	0.096	0.087–0.103	0.544	0.515–0.575	0.81
	F+AA+	0.079	0.072–0.086	0.594	0.563–0.623	0.87
Gizzard	F−AA−	0.007	0.006–0.008	1.379	1.328–1.422	0.86
	F−AA+	0.008	0.007–0.009	1.341	1.297–1.385	0.88
	F+AA−	0.007	0.006–0.008	1.370	1.323–1.418	0.85
	F+AA+	0.005	0.004–0.006	1.451	1.405–1.492	0.89
Pancreas	F−AA−	0.001	0.001–0.002	1.241	1.189–1.291	0.80
	F−AA+	0.001	0.001–0.002	1.268	1.215–1.322	0.79
	F+AA−	0.001	0.000–0.002	1.264	1.219–1.315	0.81
	F+AA+	0.001	0.000–0.002	1.351	1.301–1.398	0.85
Duodenum	F−AA−	0.004	0.003–0.004	1.321	1.278–1.365	0.84
	F−AA+	0.005	0.004–0.006	1.281	1.242–1.322	0.88
	F+AA−	0.004	0.003–0.005	1.316	1.272–1.359	0.85
	F+AA+	0.003	0.002–0.004	1.395	1.341–1.441	0.80
Jejunum	F−AA−	0.003	0.002–0.004	1.488	1.432–1.539	0.82
	F−AA+	0.004	0.003–0.005	1.482	1.427–1.534	0.83
	F+AA−	0.003	0.002–0.004	1.569	1.503–1.628	0.81
	F+AA+	0.003	0.002–0.004	1.515	1.457–1.567	0.82
Ileum	F−AA−	0.010	0.009–0.011	1.196	1.159–1.237	0.83
	F−AA+	0.013	0.012–0.015	1.142	1.111–1.178	0.81
	F+AA−	0.009	0.008–0.010	1.220	1.179–1.257	0.82
	F+AA+	0.008	0.007–0.009	1.251	1.208–1.292	0.82
Ceca	F−AA−	0.000	0.000–0.001	1.706	1.623–1.783	0.78
	F−AA+	0.000	0.000–0.001	1.766	1.681–1.844	0.79
	F+AA−	0.000	0.000–0.001	1.697	1.624–1.772	0.77
	F+AA+	0.000	0.000–0.001	1.923	1.849–2.001	0.76

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Coefficients for the allometric equation y = ax^b; y = supply organ weight in g; x = yolk-free body weight in g; a and b estimated coefficients with confidence interval (CI).

and

Table A4. Allometric growth coefficients ¹ for effects of dietary fat/AME and amino acid concentration in the starter phase on supply organs from day 4 to 11.

Organ	Diet Treatment	a	a (95% CI)	b	b (95% CI)	Pseudo-R2
Heart	F−AA−	0.026	0.023–0.029	0.790	0.752–0.828	0.87
	F−AA+	0.024	0.021–0.027	0.800	0.764–0.838	0.88
	F+AA−	0.020	0.019–0.022	0.838	0.803–0.874	0.89
	F+AA+	0.029	0.026–0.032	0.771	0.737–0.806	0.86
Liver	F−AA−	0.096	0.087–0.105	0.854	0.819–0.890	0.90
	F−AA+	0.123	0.111–0.134	0.814	0.781–0.846	0.88
	F+AA−	0.087	0.079–0.096	0.858	0.822–0.894	0.91
	F+AA+	0.061	0.056–0.067	0.930	0.892–0.969	0.94
Proventriculus	F−AA−	0.057	0.051–0.063	0.636	0.606–0.668	0.82
	F−AA+	0.073	0.067–0.080	0.611	0.581–0.644	0.83
	F+AA−	0.046	0.040–0.051	0.701	0.663–0.743	0.80
	F+AA+	0.067	0.060–0.073	0.629	0.598–0.661	0.85
Gizzard	F−AA−	0.195	0.179–0.210	0.687	0.657–0.719	0.81
	F−AA+	0.197	0.183–0.210	0.673	0.643–0.704	0.82
	F+AA−	0.267	0.244–0.291	0.615	0.582–0.650	0.78
	F+AA+	0.230	0.213–0.248	0.663	0.633–0.694	0.83
Pancreas	F−AA−	0.003	0.002–0.003	1.072	1.024–1.121	0.84
	F−AA+	0.004	0.003–0.005	1.000	0.957–1.046	0.83
	F+AA−	0.005	0.004–0.006	0.973	0.932–1.015	0.85
	F+AA+	0.012	0.010–0.013	0.799	0.764–0.837	0.81
Duodenum	F−AA−	0.057	0.050–0.063	0.765	0.728–0.804	0.86
	F−AA+	0.050	0.044–0.056	0.785	0.746–0.823	0.87
	F+AA−	0.068	0.061–0.075	0.737	0.696–0.779	0.85
	F+AA+	0.058	0.052–0.064	0.779	0.742–0.819	0.88
Jejunum	F−AA−	0.084	0.077–0.093	0.809	0.772–0.848	0.80
	F−AA+	0.127	0.115–0.139	0.731	0.692–0.772	0.79
	F+AA−	0.196	0.179–0.212	0.660	0.621–0.703	0.77
	F+AA+	0.134	0.121–0.147	0.726	0.686–0.766	0.80
Ileum	F−AA−	0.077	0.070–0.084	0.779	0.742–0.820	0.85
	F−AA+	0.077	0.070–0.085	0.762	0.726–0.802	0.84
	F+AA−	0.110	0.100–0.120	0.709	0.669–0.751	0.82
	F+AA+	0.128	0.116–0.140	0.679	0.641–0.718	0.81
Ceca	F−AA−	0.021	0.018–0.024	0.788	0.741–0.839	0.78
	F−AA+	0.024	0.021–0.027	0.746	0.699–0.793	0.77
	F+AA−	0.020	0.018–0.023	0.799	0.754–0.845	0.79
	F+AA+	0.027	0.024–0.030	0.773	0.729–0.816	0.76

F = dietary fat/AME; F− = low dietary fat/AME; F+ = high dietary fat/AME; AA = dietary amino acid; AA− = low amino acids; AA+ = high amino acids. ¹ Coefficients for the allometric equation y = ax^b; y = supply organ weight in g; x = yolk-free body weight in g; a and b estimated coefficients with confidence interval (CI).

. The results from the equality of the null hypothesis are presented in

Table 10. Probabilities comparing the separate and pooled fits for each treatment combination of the allometric growth curves.

		Day 0–4			Day 0–11			Day 4–11			Day 0–4–11
		F−AA−	F−AA+	F+AA−	F−AA−	F−AA+	F+AA−	F−AA−	F−AA+	F+AA−	F−AA−	F−AA+	F+AA−
Heart	F−AA+	0.960			0.547			0.311			0.398
	F+AA−	0.743	0.577		0.616	0.834		0.296	0.365		0.396	0.341
	F+AA+	0.817	0.859	0.362	0.760	0.618	0.453	0.404	0.230	0.172	0.382	0.617	0.288
Liver	F−AA+	0.274			0.177			0.003			0.054
	F+AA−	<0.001	<0.001		<0.001	<0.001		0.653	0.007		0.104	0.347
	F+AA+	<0.001	<0.001	0.486	0.159	0.171	<0.001	<0.001	<0.001	0.011	0.096	0.051	0.110
Proventriculus	F−AA+	0.147			0.682			0.193			0.511
	F+AA−	0.763	0.188		0.713	0.836		0.198	0.058		<0.001	<0.001
	F+AA+	0.134	0.692	0.234	0.698	0.765	0.814	0.395	0.237	0.066	0.337	0.418	<0.001
Gizzard	F−AA+	0.674			0.059			0.633			0.487
	F+AA−	0.837	0.332		0.561	0.382		0.136	0.179		0.364	0.587
	F+AA+	0.086	0.051	0.091	0.864	0.035	0.453	0.682	0.694	0.201	0.381	0.607	0.696
Pancreas	F−AA+	0.109			0.114			<0.001			<0.001
	F+AA−	0.428	0.790		0.067	0.340		0.108	<0.001		0.264	<0.001
	F+AA+	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	0.003	<0.001	<0.001
Duodenum	F−AA+	0.989			0.886			0.745			0.637
	F+AA−	<0.001	0.001		<0.001	0.748		0.236	0.295		0.536	0.549
	F+AA+	<0.001	<0.001	0.014	<0.001	<0.001	0.037	0.742	0.639	0.377	<0.001	<0.001	<0.001
Jejunum	F−AA+	0.324			0.623			<0.001			<0.001
	F+AA−	0.067	0.072		0.617	0.708		<0.001	0.082		<0.001	0.496
	F+AA+	0.071	0.069	0.142	0.529	0.606	0.731	<0.001	0.851	0.095	<0.001	0.374	0.467
Ileum	F−AA+	<0.001			<0.001			0.163			<0.001
	F+AA−	0.143	<0.001		0.094	<0.001		0.006	0.042		<0.001	<0.001
	F+AA+	<0.001	<0.001	<0.001	<0.001	0.164	<0.001	<0.001	<0.001	0.087	<0.001	0.432	<0.001

F− = low dietary fat/AME; F+ = high dietary fat/AME; AA− = low amino acids; AA+ = high amino acids. p-values based on overall sum of squares fits, using F = ((SS_pooA − (SS_trt1 + SS_trt2))/(df_pooA − (df_trt1 + df_trt2))/((SS_trt1 + SS_trt2) − (df_trt1 + df_trt2)). p ≤ 0.05 indicates that the separate fit between any two treatments explains the variation better than the pooled fit. Grey-shaded cells indicate treatment comparisons with identical F-test results; values are shown once to avoid duplication.

. Allometric growth relationships between organ weight and yolk-free body weight were fitted accurately (pseudo-R² > 0.75; nRMSE < 12%). Liver allometric growth was higher in the F− groups during days 0 to 4 and higher in the F+AA− group during d 0 to 11. During days 4 to 11, the highest allometric growth rate occurred in the F+AA+ group, indicating an effect of increasing dietary fat, and thus AME, and amino acid concentration. When considering all data points (days 0, 4, and 11), there was no difference in allometric growth observed among treatments.

Proventriculus allometric growth was higher in the F+AA− group compared to the other groups across the overall period. The pancreas’ allometric growth was higher in F+AA+ group during days 0 to 4 and lower during days 0 to 11. From day 4 to 11, the highest allometric growth was in the F−AA− group and the lowest was in the F+AA+ group. When including all data points, the allometric growth rate of the pancreas was higher in the AA− groups (Figure 1).

The duodenal allometric growth was higher in the F+AA+ group during days 0 to 4, days 0 to 11, and the overall period (Figure 2). In contrast, the jejunal allometric growth was highest in the F−AA− group during days 4 to 11 and the overall period (Figure 3). For the ileum, allometric growth was highest in the F+AA+ group and lower in the F−AA+ group during days 0 to 4. F− resulted in higher allometric growth during days 4 to 11, whereas AA− resulted in higher allometric growth during days 0 to 11. Moreover, when considering all the measurement points, allometric growth of the ileum was highest in the F−AA− group, with an effect of amino acid concentration (Figure 4). For the other organs, no differences were observed in allometric growth among treatments.

4. Discussion

The purpose of this study was to assess whether increasing dietary fat (resulting in a 300 kcal AME difference) or higher amino acid concentration, or both, in starter diets could support the early development of the supply organs.

4.1. Growth Performance and Nutrient Intake

Dietary amino acid concentration had a stronger and more consistent effect on early growth than dietary fat, suggesting that amino acid availability, rather than energy density, is more limiting in early life. This agrees with earlier research [11]. The transient effect of higher dietary fat (and thus AME) on body weight gain likely reflects an improved early energy supply, coupled with limited fat digestibility before day 10 [29]. However, the results of the present study diverge from those reported by Diehl et al., 2024 [12], where increasing 300 kcal AME, through starch, resulted in a decrease in BW gain in the first week of age. These discrepancies likely reflect differences in how fat and carbohydrates are metabolic utilization. The transient performance response to the high-fat (F+) diet likely reflects the limited lipid digestion capacity of chicks during the first week post-hatch [29], when bile salt secretion and lipase activity are still developing [4,5,6]. Consequently, the additional dietary energy from fat may not have been fully utilized, leading to a short-lived effect. In contrast, the consistent improvement in the AA+ group suggests that amino acid supply more directly supports metabolic and structural growth processes during early development. The treatments in this study may resemble an imbalanced titration of the AME-to-lysine ratio; however, it is important to note that diets were not intentionally formulated as a titration series. Body weight gain did not follow the response pattern that would be expected from such titration. This aligns with findings by Plumstead et al., 2007 [30], who reported that, in young broilers, the AME-to-lysine ratio only partially accounts for variations in body weight gain. Variability among studies likely reflects how young broilers prioritize nutrient partitioning between maintenance and growth when dietary energy and amino acids are not balanced. This highlights that nutrient interactions, rather than single nutrients, determine early growth efficiency.

Differences in FI could partly explain the contrasting performance results. FI regulation in broilers is complex and has been associated with several nutrient-based feedback mechanisms, including glucostatic, lipostatic, ionostatic, and aminostatic theories [31,32]. The reduction in FI observed with higher dietary fat and amino acid levels are in line with observations by Lamot et al., 2019 [10]. This indicates that a synergistic interaction between dietary fat and amino acids can trigger satiety. Interestingly, when dietary fat was increased without additional amino acids (F+AA−), AME and fat intake increased while FI remained unchanged. According to the lipostatic theory, elevated plasma free fatty acids would be expected to activate satiety signals in the hypothalamus [31,33]. Likewise, raising amino acid concentration alone (F−AA+) increased lysine intake but did not affect FI. This suggests that circulating amino acids in the blood plasma alone may not be a dominant driver of early satiety in broilers. Alternatively, the threshold required to trigger an aminostatic response was not reached under the present experimental conditions.

Feed conversion ratio (F:G) and nutrient-to-gain ratios help interpret the BW and FI results in the context of nutrient requirements and the energy cost of protein deposition. The most consistent reduction in F:G was observed in the AA+ groups and persisted throughout the entire study. Dietary fat also reduced F:G, suggesting that the AME-to-lysine ratio plays an important role in determining efficiency. Increasing amino acid concentration lowered the AME:gain ratio (day 4 to 11), whereas increasing AME alone had no effect, consistent with the findings of Plumstead et al., 2007 [30]. Likewise, the Lys:gain ratio was reduced when digestible lysine was lowered and AME increased, underscoring the importance of the energy-to-lysine balance for protein deposition. Although the combination of higher AME and high amino acid concentration did not result in the highest BW at day 11, it resulted in the lowest F:G, indicating improved efficiency. This partly agrees with Hirai et al., 2020 [34], who found that increasing amino acid concentration reduced F:G, although they reported no effect of AME. The discrepancy may be explained by differences in diet formulation, as the lowest AME level in the present study was substantially lower than in Hirai et al., 2020 [34]. Finally, the higher fat:gain ratio observed may be partly related to a reduced fat digestibility in (very) young broilers [29], although this was not measured in the current study.

4.2. Supply Organ Weights, Intestinal Histo-Morphological Parameters, and Allometric Growth

Relative liver weight was lower in the F+ groups on d 4 but showed higher allometric growth from d 4–11, resulting in similar overall weight. These findings are partly consistent with Lamot et al., 2019 [10], though diet concentrations in that study were higher. The maturation rate (b) of the liver was above 1 for all treatment groups, particularly in the first period, indicating that liver growth outpaced body growth. This may be linked to genetic selection for reduced residual feed intake and lower body fatness [35]. Such selection has been associated with declining serum VLDL concentrations over time [36,37,38,39]. As VLDL is the principal transporter of triglycerides from the liver [40,41], reduced secretion could predispose modern broilers to hepatic lipid accumulation, especially under high-AME diets. Collectively, they emphasized the liver’s central role in energy metabolism, though the developmental response appears to be biphasic across timepoints.

Relative proventriculus weight was higher in the AA− groups on d 11, and the proventriculus showed a higher allometric growth in F+AA− compared with the other groups. This contrasts with Mansilla et al., 2022 [42], who found no effect of AME or amino acids on proventriculus weight, suggesting responses may depend on diet formulation or experimental conditions.

Relative pancreas weight was lower in the F+AA+ group on day 11, consistent with the observed allometric growth patterns. This finding is in contrast with Swatson et al., 2002 [43], who reported increased relative pancreas weight in response to higher dietary amino acid levels. However, their study was performed in older birds (from d 10 to 24), which may explain the discrepancy, as pancreatic development during this later stage may follow different dynamics compared to the early post-hatch period. The higher relative pancreas weight in the AA− groups aligns with the literature suggesting that low-protein diets can stimulate pancreatic hypertrophy as a compensatory mechanism to increase enzyme production under reduced amino acid availability [44]. Protease secretion, particularly trypsin and chymotrypsin, peaks early (d 4–7), whereas lipase activity increases more gradually, plateauing between d 10 and 16 [45,46]. These discrepancies suggest that the effect of amino acid level on pancreas development is context-dependent, influenced by amino acid balance, bird age, and other dietary components. Although an enlarged pancreas in early life could theoretically enhance digestive capacity and thereby improve BW gain or feed efficiency later, this was not supported in the current study. Despite having heavier pancreases, birds on AA− diets did not show superior growth performance or efficiency at later stages.

Analyses of the small intestinal sections showed different responses because of dietary fat (and thus AME) and amino acid concentration, complicating the interpretation of the results. The greater duodenal allometric growth rate in F+ and a trend in duodenum length may be linked to increased cholecystokinin production in response to high-fat diets [47]. Further research is needed to support this suggestion, as the literature on the topic is scarce. Digestion of dietary lipids is around 15–25% in the duodenum, but reaches about 75% in the jejunum [9]. Despite comparable jejunal weights among treatments, the allometric growth coefficient was higher in F−AA−, possibly reflecting minimum growth development. Additionally, at d 4, F+AA+ birds had longer villi with deeper crypts in the jejunum, which may reflect higher nutrient availability stimulating mucosal development. Overall, lower AME and amino acid concentrations may stimulate compensatory allometric growth, whereas longer villi and deeper crypts in high-nutrient diets may reflect improved absorptive efficiency driven by greater nutrient availability. Ileal allometric growth showed different developmental patterns across age periods. However, when considering all measurement points, the allometric growth of the ileum was fastest in the F−AA− group, decreasing with dietary fat, amino acid concentration, or both. These findings indicate that duodenal, jejunal, and ileal development did not uniformly respond to dietary energy and amino acids but may have adapted differentially to optimize early nutrient absorption.

Several studies have shown differences in the goblet cell development with age or dietary changes [48,49,50,51,52,53,54]. In the present study, no effects were observed on goblet cell development, indicating that neither increased amino acid concentration nor additional energy from fat (nor their combination) altered goblet cell development under the current experimental conditions and diet formulations.

The formulation of the experimental diets, as described in Materials and Methods, inherently altered the proportions of soybean meal and wheat middlings when adjusting amino acid concentration. It could therefore be hypothesized that the observed variations in supply organ weights were influenced not only by fat/AME or lysine content but also by these associated changes in soybean meal and fiber levels. Soybean meal provides, in addition to its crude protein contribution, various bioactive and anti-nutritional factors that may affect gastrointestinal development in young chicks [52,53]. However, potential interactions related to soybean meal composition were beyond the scope of the present study and cannot be evaluated here. Similarly, the inclusion of wheat middlings as a fiber source could have stimulated gastrointestinal tract (GIT) development, yet no differences in gizzard weight or allometric growth were observed, suggesting that the effect of fiber was negligible [48]. While excessive fiber may also increase endogenous protein losses, further research is warranted to clarify these potential effects.

5. Conclusions

This study provides valuable insights into the interactions between dietary fat (and thus AME) and amino acid concentration, and their effects on early supply organ development, general growth performance, and nutrient intake in broiler chickens. Elevated amino acid levels (AA+) improved BW gain, FCR, and nutrient use efficiency, suggesting an enhanced metabolic utilization during early development. Although dietary fat supports early performance, the effects diminished with age. These improvements in performance were not always reflected in morphological changes in supply organs; however, the observed effects on pancreas weight and intestinal development suggest that AA+ supported digestive capacity and nutrient absorption efficiency. In contrast, additional dietary fat (F+) primarily enhanced early growth in the first days post-hatch, with effects on organ development being inconsistent and diminishing over time. Taken together, these results demonstrate that amino acid density in starter diets plays a more critical role than added fat for promoting the functional development of supply organs and ensuring efficient nutrient use in early broiler growth. Practically, young broilers can be offered higher-fat starter diets, as long as amino acid supply is optimized to match their rapid early growth demands.