Organ growth and fermentation profiles of broilers differing in body growth rate

Abstract

This study sought to determine the relationship among broiler performance, organ development, and indicators of microbiota colonization. A total of 1,200 two-day-old male Ross 308 broiler chicks, divided among 3 cohorts of equal size, were housed in battery cages, and allotted based on body weight. On study d 11, birds were weighed, and birds with BW gain within the 10th and 90th percentiles were assigned to the Slow and Fast groups, respectively. Birds (n = 30 for each group) selected on d 11 were provided water and a corn-soybean meal-based diet ad libitum while maintained individually through study d 25 (i.e., a 14-d growth period). Parameters regarding growth performance, organ and intestine weights and lengths, and intestinal volatile fatty acid concentrations were measured. All data were analyzed by one-way ANOVA using the Mixed procedure of SAS. Fast birds exhibited greater (P < 0.001) BW gain and feed intake than slow birds, but feed conversion ratio (FCR) did not differ (P = 0.19). Additionally, Slow birds had higher (P < 0.05) relative weights (% of BW) for nearly all organs on d 11 and 25, most notably the gizzard, proventriculus, pancreas, and liver. Conversely, intestinal sections were longer (P < 0.05) in the Fast birds. Measurement of gut histomorphology did not show any notable differences between growth rate groups in terms of villi height, crypt depth, or their ratio for either time-point (P > 0.05). In terms of volatile fatty acid concentrations of luminal contents, acetate concentrations were 10.2% higher (P < 0.001) in the ileum of the Slow birds compared with Fast birds on d 25. Overall, the findings suggest that total BW gain is influenced by the development of metabolically active organs, as supported by lower weight gain in Slow birds with relatively larger organ weights and shorter intestinal lengths than their Fast counterparts. The general lack of differences in fermentation end-product concentrations in luminal contents does not rule out influence of the microbiota on growth rate of broilers, which warrants further investigation.

INTRODUCTION

Global meat consumption is projected to increase by 14% and reach 374 metric tons by 2030 (OECD, 2021). Of this amount, 152 metric tons or 52%, is expected to be contributed by poultry. In the United States, 9.13 billion broilers were raised in 2021 alone, accounting for a market value of $31.5 billion (USDA-NASS, 2022). Reflecting the increased demand for chicken products, the industry has refined the broiler production process, and today's birds reach a market weight of 2.5 kg in 7 wk compared to 16 wk in the 1940′s (Havenstein et al., 2003). Much of this progress has been a result of applying continual genetic selection to greatly improve the growth rate and the feed conversion efficiency in broilers. As such, these dramatic improvements in nutrition, management, and efficiencies of vertical integration have allowed poultry to remain one of the cheapest sources of animal protein available today (Tallentire et al., 2016).

When considering input costs, feed constitutes approximately 70% of the total cost of production necessary to rear chickens, and any improvement in feed efficiency is beneficial to consumers and producers alike (Mallick et al., 2020). Broiler environmental management is highly refined, and its benefits are well documented, but there exist other areas with potential that are not as well understood and merit further examination and development. For example, flock uniformity is often assessed during production, which includes BW measurements and culling of underperforming or “runty” birds. These variations in flock uniformity typically result in profit reduction over the grow-out period (Gous, 2018). However, the underlying cause of variability amongst and within flocks is unknown, as production broilers have nearly identical genetics. It is thought that these variations may be the result of other factors such as parent flock characteristics and seasonal changes (Jespersen et al., 2024), digestive capacity, or the intestinal microbiota of individual birds (Lundberg et al., 2021). Notably, intestinal length is thought to be closely related to nutrient digestibility and absorption (Svihus and Itani, 2019), while diverse intestinal microbiome characteristics lead to growth differences and flock nonuniformity (Lundberg et al., 2021). However, little research has been conducted to explore these possibilities. Therefore, our objective was to evaluate tissue development and luminal profiles of fermentative end-products among a single population of broilers (i.e., biological variation within a group bred and raised under the same environmental conditions with limited genetic variability) that that differed in the rate of body weight gain.

MATERIALS AND METHODS

All animal care and experimental procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to initiation of the experiment.

Animal Husbandry

A total of 1,200 male Ross 308 broiler chicks across 3 cohorts (400 chicks per cohort) were received at 2 d posthatch from a commercial hatchery (Hoover's Hatchery, Rudd, IA) and transported to the University of Illinois Edward R. Madigan Laboratory animal care facility. Upon arrival of each cohort of birds, chicks were weighed and 300 chicks that gave the most uniform distribution of weight with the most minimal change in body weight were utilized. Average enrollment weights per cohort were as follows: cohort 1, 32 ± 2 g; cohort 2, 37 ± 2 g; cohort 3, 32 ± 2 g. Each cohort of birds was initially placed into battery cages according to initial body weight to the nearest gram, such that all birds in a single group cage (n = 30) had the same initial body weight. The 100 chicks excluded from the study were humanely euthanized by CO₂ asphyxiation, and 10 of these euthanized birds were randomly selected for the collection of baseline organ weights and lengths (Supplementary Table 1).

All chicks enrolled on study were housed in thermostatically controlled batteries (model SB5T; Alternative Design Manufacturing, Siloam Springs, AR) with raised wired floors in an isolated, environmentally controlled room with continuous lighting. All chicks received the same standard corn and soybean meal diet for the duration of the 25-d feeding study. Feed and water were provided ad libitum throughout the experiment, and the diet constituted a practical formulation that would be standard for the poultry industry (Table 1).

Table 1.

Composition of the experimental diet.¹

Ingredient	Concentration, g/kg
Corn	525.8
Soybean meal	390.0
Soy oil	35.0
Sodium chloride	4.0
Limestone	12.0
Dicalcium phosphate	21.0
Vitamin premix2	2.0
Mineral premix3	1.5
Choline chloride	3.2
L-Lys HCl	1.4
DL-Met	3.2
L-Thr	0.9
Calculated Composition
Protein, g/kg	229.3
ME, kcal/kg	3046
Ca, g/kg	10.6
Total P, g/kg	8.0
nPP, g/kg	5.3
Ca:tP	1.3
Ca:nPP	2.0
Na, g/kg	2.0
Total AA, g/kg
Arg	14.3
His	5.8
Ile	8.8
Leu	17.3
Lys	12.7
Met	6.4
Met + Cys	9.5
Phe	10.3
Phe + Tyr	16.3
Thr	8.3
Trp	2.5
Val	9.5

¹Abbreviations: nPP: nonphytate phosphorous; tP: total phosphorous; ME: metabolizable energy.

²Provided per kilogram of complete diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 μg; dl-α-tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin, 4.41 mg; d-Ca-pantothenate, 10 mg; niacin, 22 mg; and menadione sodium bisulfite complex, 2.33 mg.

³Provided per kilogram of complete diet: Mn, 75 mg from MnO; Fe, 75 mg from FeSO₄ ·7H₂O; Zn, 75 mg from ZnO; Cu, 5 mg from CuSO₄ ·5H₂O; I, 0.75 mg from ethylenediamine dihydroiodide; and Se, 0.1 mg from Na₂SeO₃.

On study d 11, all birds were weighed, and individual BW gain was calculated for the starter phase (Supplementary Table 1). Afterwards, the 20 birds closest to the 10th and 90th BW gain percentiles were identified and selected for further evaluation (40 birds per cohort). The 10th percentile BW group (Slow) was characterized with initial average BW as follows: cohort 1, 214 ± 2 g; cohort 2, 240 ± 1 g; cohort 3, 203 ± 2 g. Similarly, the 90th percentile BW group (Fast) was characterized with initial average BW as follows: cohort 1, 310 ± 1 g; cohort 2, 324 ± 1 g; cohort 3, 286 ± 1 g. Of these selected birds, the 10 birds closest to each of the 10th and 90th BW percentiles (n = 20 birds total) were continued on to study d 25, and the remaining birds (n = 20 birds total) were euthanized to permit d 11 sample collection. The 20 birds advancing to study d 25 were re-identified as either Slow or Fast. Each of these birds was moved to clean cages, randomly assigned to cage location, and individually housed on a half battery deck such that each level of the battery had a pair of one Fast and one Slow bird (i.e., F1 with S1). Birds were housed individually and isolated to prevent the consumption of other birds' excreta to prevent undue influence on outcomes involving luminal contents.

Growth Performance

Individual BW, BW gain, feed intake, and feed efficiency (gain: feed ratio and feed conversion ratio) were calculated over the grower phase from study d 11 to 25, at which point all birds were humanely euthanized to permit collection of tissue and luminal content samples. Growth performance is reported for the Slow and Fast groups as an overall average across cohorts (treatment n=60) and separated by cohort (treatment n=20).

Organ Weights and Lengths

On study d 11 and 25, absolute weights of the following organs were recorded following euthanasia of birds: crop (flushed), gizzard (flushed), proventriculus (flushed), duodenum (from gizzard to end of pancreatic ducts), jejunum (from end of pancreatic ducts to Meckel's diverticulum), ileum (from Meckel's diverticulum to ileocecal junction), paired ceca, colon, liver, spleen, bursa, cecal tonsils, and pancreas. To minimize variability, the crop, gizzard and proventriculus were all flushed prior to weighing due to variable weights based on luminal fill with contents. Small intestinal and cecal tissues were not flushed before weighing to permit whole tissue sub-samples to be sent for histomorphology analysis and to allow for digesta collection for microbiota-related outcomes. Organs devoid of luminal contents were flushed with filtered 1 × phosphate buffered saline prior to being weighed, and all other organs were weighed without flushing shortly after dissection. Total organ weight was calculated by summing the absolute weight (g) of all organs listed above. Subsequently, this total organ weight was subtracted from each bird's live BW to generate empty BW.

Additionally, on study d 11 and 25, lengths of the following organs were recorded: duodenum, jejunum, ileum, the total length of both ceca, and colon. Total small intestinal length was calculated by compositing lengths of the duodenum, jejunum, and ileum. All organ lengths and weights, except for the ceca, were characterized both on an absolute and relative basis. Relative organ weight (% of BW) was calculated by taking the weight of the respective organ (g), dividing it by the BW of the individual bird (g), then multiplying by 100. Relative total organ weight (% of BW) was calculated by taking the cumulative weight of all collected organs (g), then multiplying by 100. Relative empty bird weight (% of BW without organs) was calculated by taking the empty BW of the bird (g), dividing it by the live body weight of the bird (g), then multiplying by 100. Relative organ length (% of total small intestinal length) was calculated by taking the length of the respective organ (cm), dividing it by the total length for each bird (cm), and then multiplying by 100.

Volatile Fatty Acids and Dry Matter Analysis

Fresh luminal contents from the ileum and ceca were collected on study d 11 and 25 for determination of DM content and volatile fatty acid (VFA) concentrations [i.e., short-chain fatty acids (SCFA) plus branched-chain fatty acids (BCFA)]. DM was analyzed after drying digesta samples in a 105°C oven for a minimum of 48 h. Fresh samples for VFA analysis were weighed and preserved at the time of collection using a 1:1 ratio of 2 N HCl before placement in a –20°C freezer for long term storage. Absolute VFA concentrations (μmol/g DM) were quantified using gas chromatography (Hewlett-Packard 5890A series 99, Hewlett-Packard, Palo Alto, CA) with a custom packed column (1.83 m × 6.35 mm i.d.) packed with 10% SP-1200/1% phosphoric acid on 80/100 Chrom-WAW (Supelco, Bellefonte, PA). Temperature parameters for the gas chromatography equipment were as follows: injector, 175°C; FID detect, 180°C; and the oven at 125°C. Relative molar ratio values were calculated by dividing absolute concentrations of each analyte (μmol/g) by the total concentration (μmol/g; within either SCFA or BCFA category) and multiplying by 100.

Histomorphology and Histopathology

Directly after euthanasia, tissue samples (approximately 1 cm in length) were obtained to determine histomorphology outcomes in the ileum and histopathology outcomes in both the ileum and ceca. Samples were excised and placed in 10% neutral buffer formalin before shipment to an external laboratory (Veterinary Diagnostic Pathology LLC; Fort Valley, VA) for histomorphology and histopathology analyses by a certified poultry histopathologist. Prior to analysis, tissues were embedded in paraffin to permit preparation of two 5-µm sections per tissue block for each individual bird, and subsequent staining with hematoxylin and eosin. The histopathologist remained blinded to treatment identities during all phases of tissue evaluation and lesion scoring. Each section of the ileum (i.e., 2 independent sections per bird) had 10 representative and well-oriented villi and associated crypts evaluated, with villi height and crypt depth measured, and the villi height-to-crypt depth ratio was calculated per individual bird.

Separate from these structural outcomes, the ileal and cecal tissues underwent histopathological lesion scoring using 2.5 cm rings of tissue. Tissues were embedded in paraffin, then 5-µm sections were cut and stained with hematoxylin and eosin. A certified histopathologist who remained blinded to treatment identity, examined, and reported tissue scores. Scoring was on a 0-5 scale with 0 being no lesions, 1 = rare or slight, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Overall averages were taken for each measure per chicken to allow one value for each experimental unit for a given outcome. For histopathology, the scoring system and lesions investigated in the data analysis were only included where at least 1 bird exhibited a score of 1 or more.

Statistical Analysis

Each individual bird was considered the experimental unit for statistical analyses of all measured outcomes. Data were subjected to a 1-way ANOVA using the MIXED procedure of SAS (version 9.4; SAS Institute; Cary, NC), except for the starter phase data (not statistically analyzed). A random effect of cohort was included in the statistical model. The level of significance was set at P < 0.05. Results are presented as least squares means with their respective SEM. Outliers were identified as having an absolute Studentized residual value of 3 or greater and were removed from the final dataset. Histomorphology data were subjected to an ANOVA using the MIXED procedure of SAS (version 9.4; SAS Institute; Cary, NC). A 1-way ANOVA was used to determine whether the overall model was significant, and in those instances, mean separation was conducted assuming an alpha level of 0.05.

Due to lesion scores being discrete values using a 6-point scale (0–5, inclusive), cumulative probabilities were produced via the post-fitting linear model (PLM) procedure of SAS (version 9.4; SAS Institute; Cary, NC), specifically a Tukey-Kramer test. Probabilities of a specific score within a treatment were calculated, with the sum of all equal to 1 within a treatment. Afterwards, weighted average scores were calculated to allow comparison across treatments.

RESULTS

Growth Performance

Growth performance results during the grower phase are presented in Table 2. On both d 11 and 25, BW was higher (P < 0.001) in Fast birds compared with Slow birds. BW gain and feed intake were also higher (P < 0.05) over the 14-d growth period in Fast versus Slow birds. However, no differences (P > 0.05) were observed in feed conversion ratio (FCR) between the 2 groups.

Table 2.

Growth performance of fast vs. slow growing chicks during the grower phase.¹

	Treatment				SEM	P-value
	Slow		Fast
Item	n	Mean	n	Mean
BW d 11, g	60	219	60	306	2.19	< 0.001
BW d 25, g	30	897	30	1,145	18.28	< 0.001
BW gain d 11-25, g/chick	30	678	30	838	17.98	< 0.001
Feed intake d 11-25, g/chick	30	1,123	30	1,266	32.7	0.002
Gain:feed, g/kg	29	631	30	671	18.8	0.14
FCR, g/g	28	1.60	30	1.52	0.05	0.19

¹n_: number of birds; BW: body weight; FCR: feed conversion ratio; SEM: standard error of the mean.

Organ Weights and Lengths

On d 11 and 25, all measured organs had higher (P < 0.05) absolute weights for Fast birds compared with the Slow group (Table 3), except for the crop on d 11 (P > 0.05). When organs were compared on a relative basis, some differences between the 2 groups were lost. Specifically, on d 11, significance of the ileum, ceca, colon, spleen, and bursa were lost (P > 0.05) when compared on a relative weight basis. Similarly, on d 25, no differences (P > 0.05) in relative organ weights were observed for the jejunum, ceca, colon, spleen, bursa, cecal tonsils, or crop. On both d 11 and 25, the total sum weight of organs was greater (P < 0.001) on an absolute basis in Fast birds. When the same outcome was calculated on a relative basis, however, the Slow birds had a greater (P < 0.001) total weight. Empty body weight was also higher (P < 0.001) in the Fast group on d 11 and 25 and remained higher in Fast birds (P < 0.001) when calculated on a relative basis.

Table 3.

Organ weights of fast vs. slow growing chicks during the grower phase.¹

	Absolute weight, g				Relative weight2, % of BW
Item	Slow	Fast	SEM	P-value	Slow	Fast	SEM	P-value
D 11
Empty body weight	168.36	244.41	3.22	< 0.001	76.64	79.78	0.51	< 0.001
Total Organs3	51.01	61.72	1.26	< 0.001	23.36	20.22	0.51	< 0.001
Crop	1.54	1.70	0.10	0.19	0.71	0.56	0.04	0.006
Proventriculus	2.08	2.67	0.06	< 0.001	0.94	0.88	0.02	0.018
Gizzard	10.25	11.90	0.48	< 0.001	4.72	3.93	0.21	< 0.001
Pancreas	1.29	1.58	0.05	< 0.001	0.59	0.52	0.02	0.013
Duodenum	4.62	5.54	0.13	< 0.001	2.12	1.83	0.05	< 0.001
Jejunum	9.14	11.62	0.32	< 0.001	4.18	3.77	0.12	0.013
Ileum	7.26	9.13	0.33	< 0.001	3.30	3.02	0.12	0.12
Ceca	3.08	4.01	0.23	< 0.001	1.40	1.33	0.09	0.52
Colon	0.76	1.01	0.03	< 0.001	0.35	0.33	0.01	0.45
Liver	9.92	11.44	0.26	< 0.001	4.57	3.79	0.13	< 0.001
Spleen	0.28	0.35	0.02	0.002	0.13	0.12	0.01	0.11
Bursa	0.39	0.61	0.03	< 0.001	0.18	0.21	0.01	0.07
Cecal tonsils	0.17	0.20	0.01	0.013	0.08	0.06	0.004	0.018
D 25
Empty body weight	776.81	1003.52	16.11	< 0.001	86.75	87.68	0.20	< 0.001
Total organs3	119.99	141.01	3.25	< 0.001	13.25	12.32	0.20	< 0.001
Crop	2.39	2.88	0.18	0.003	0.27	0.25	0.02	0.28
Proventriculus	4.31	4.95	0.12	0.001	0.48	0.44	0.01	0.002
Gizzard	21.92	24.62	1.23	< 0.001	2.43	2.19	0.12	0.001
Pancreas	3.14	3.43	0.09	0.027	0.35	0.30	0.01	< 0.001
Duodenum	8.86	10.13	0.31	0.002	0.99	0.89	0.02	0.002
Jejunum	22.60	27.84	0.82	< 0.001	2.51	2.42	0.06	0.30
Ileum	18.44	21.31	0.68	0.006	2.05	1.86	0.05	0.015
Ceca	9.50	12.11	0.64	0.007	1.04	1.06	0.05	0.83
Colon	1.99	2.43	0.13	0.001	0.22	0.21	0.01	0.34
Liver	23.23	26.86	0.58	< 0.001	2.57	2.36	0.06	0.002
Spleen	0.84	1.06	0.05	0.002	0.09	0.10	0.005	0.81
Bursa	1.67	2.15	0.10	0.002	0.19	0.19	0.01	0.84
Cecal tonsils	0.60	0.67	0.05	0.014	0.07	0.06	0.005	0.09

¹All means represent 30 birds unless a value was removed from the final data set. The following organs were not flushed prior to weighing: duodenum, jejunum, ileum, and ceca. The following organs were flushed prior to weighing: colon, crop, gizzard, proventriculus.

²Calculated as: (Absolute organ weight / live BW) × 100.

³Sum of all organs listed. Empty body weight calculated as: total organ weight – live BW.

When comparing absolute intestinal lengths on d 11 and 25, Fast birds had longer (P < 0.05) individual intestinal sections and total small intestinal length, except for the ileum on d 25 and colon at both collections (P > 0.05) (Table 4). However, these differences were not observed (P > 0.05) when small intestinal sections were expressed as a percentage of total small intestinal length.

Table 4.

Organ lengths of fast vs. slow growing chicks during the grower phase.¹

	Absolute Length2				Relative Length3
Item	Slow	Fast	SEM	P-value	Slow	Fast	SEM	P-value
D 11
Duodenum	19.04	20.19	0.32	0.004	21.19	20.37	0.43	0.16
Jejunum	37.02	41.06	1.01	0.006	40.31	41.33	0.50	0.16
Ileum	35.23	38.65	0.86	0.006	35.51	38.63	0.52	0.87
Ceca4	14.03	16.36	0.30	< 0.001	.6	.	.	.
Colon	4.36	4.58	0.10	0.07	.	.	.	.
Total small intestinal length5	91.55	99.90	1.75	< 0.001	.	.	.	.
D 25
Duodenum	23.65	25.51	0.43	0.003	18.81	18.83	0.37	0.98
Jejunum	54.24	57.11	0.95	0.014	42.05	41.83	0.53	0.74
Ileum	50.53	53.34	1.14	0.09	39.61	39.00	0.58	0.46
Ceca4	23.11	25.74	0.51	< 0.001	.	.	.	.
Colon	5.78	6.11	0.12	0.06	.	.	.	.
Total small intestinal length5	127.78	136.63	1.91	< 0.001	.	.	.	.

¹All means represent a total of 30 birds unless a value was removed from the final data set.

²Absolute length was measured in centimeters.

³Relative length (%) was calculated by taking the length of the organ (cm) and dividing it by the total small intestinal length of the bird (cm), then multiplying by 100.

⁴Ceca length was calculated as the combined length of both ceca.

⁵Total small intestinal length was calculated as the combined length of duodenum, jejunum, and ileum.

⁶Periods denote values that were unable to be calculated.

Volatile Fatty Acids in Luminal Contents

On d 11, no differences (P > 0.05) between Fast and Slow groups were observed in the luminal concentrations of VFA sampled from the ileum or ceca (Table 5). On d 25, absolute and relative ileal concentrations of acetate were higher (P < 0.001 and P = 0.027, respectively) in Slow birds, and Slow birds also had a higher (P = 0.05) absolute concentration of propionate in the cecum. No other individual VFA concentrations differed (P > 0.05) on d 25 (Table 6).

Table 5.

Concentration of volatile fatty acids in luminal contents of the ileum and ceca on d 11.¹

	Treatment				SEM	P-value
	Slow		Fast
Item	n	Mean	n	Mean
Ileum
DM, %	27	31.34	30	29.67	3.11	0.25
SCFA absolute, μmol/g DM
Acetate	28	137.05	29	137.07	11.61	1.00
Propionate	18	0.52	18	0.52	0.05	0.98
Butyrate	8	0.34	3	0.40	0.07	0.31
Total SCFA2	28	137.51	29	137.50	11.62	1.00
SCFA relative, % of total3
Acetate	26	99.65	29	99.69	0.07	0.51
Propionate	17	0.44	20	0.45	0.05	0.90
Butyrate	8	0.51	3	0.61	0.13	0.44
Cecum
DM, %	30	37.69	28	35.24	3.47	0.38
SCFA absolute, μmol/g DM
Acetate	29	229.73	28	218.75	20.56	0.62
Propionate	29	5.42	28	6.18	0.67	0.20
Butyrate	28	28.29	28	29.03	2.32	0.81
Total SCFA	29	265.9	28	254.45	23.40	0.65
SCFA relative, % of total3
Acetate	29	85.95	27	85.90	0.58	0.94
Propionate	29	2.23	28	2.57	0.23	0.21
Butyrate	29	11.81	27	11.50	0.56	0.64
BCFA absolute, μmol/g DM
Isovalerate	18	1.30	17	1.38	0.20	0.77
Valerate	17	2.06	18	2.24	0.25	0.62
Isobutyrate	28	0.85	27	0.70	0.09	0.20
Total BCFA	28	2.94	27	2.90	0.46	0.95
BCFA relative, % of total
Isovalerate	18	32.37	18	32.64	2.85	0.94
Valerate	17	47.51	18	49.65	3.32	0.62
Isobutyrate	28	49.97	28	47.94	7.01	0.75

¹n: number of birds; BCFA: branched-chain fatty acids; DM: dry matter; SCFA: short-chain fatty acids; SEM: standard error of the mean.

²Total SCFA calculated as the sum of all absolute concentrations (μmol/g) for acetate, propionate, and butyrate.

³Relative values calculated by dividing absolute concentrations of each analyte (μmol/g) by the total concentration (μmol/g; within either SCFA or BCFA category) and multiplying by 100.

Table 6.

Concentration of volatile fatty acids in ileal and cecal contents on study d 25.¹

	Treatment
	Slow		Fast
Item	n	Mean	n	Mean	SEM	P-value
Ileum
DM, %	30	17.28	30	17.66	0.27	0.27
SCFA absolute, μmol/g DM
Acetate	28	217.85	29	196.71	7.29	< 0.001
Propionate	19	0.66	30	0.67	0.07	0.93
Butyrate	5	0.43	8	0.43	0.15	1.00
Total SCFA2	28	218.58	29	197.49	7.30	< 0.001
SCFA relative, % of total3
Acetate	28	99.75	29	99.64	0.04	0.027
Propionate	21	0.35	30	0.34	0.04	0.89
Butyrate	5	0.20	7	0.17	0.04	0.45
Cecum
DM, %	30	16.92	30	18.17	0.55	0.12
SCFA absolute, μmol/g DM
Acetate	30	477.99	29	442.38	22.32	0.26
Propionate	29	17.54	28	13.48	1.41	0.050
Butyrate	30	108.53	29	96.11	5.72	0.12
Total SCFA	30	604.98	29	552.91	27.85	0.19
SCFA relative, % of total3
Acetate	30	79.12	29	79.98	0.56	0.27
Propionate	30	3.01	29	2.56	0.21	0.14
Butyrate	30	17.87	29	17.47	0.58	0.58
BCFA absolute, μmol/g DM
Isovalerate	28	2.94	28	2.63	0.21	0.27
Valerate	30	4.90	29	4.70	0.41	0.67
Isobutyrate	29	1.94	29	1.84	0.21	0.61
Total BCFA	30	10.28	29	9.08	0.81	0.25
BCFA relative, % of total3
Isovalerate	27	29.15	28	29.35	1.49	0.90
Valerate	29	50.13	29	52.68	2.04	0.23
Isobutyrate	30	20.34	29	18.98	1.18	0.24

¹n: number of birds; BCFA: branched-chain fatty acids; DM: dry matter; SCFA: short-chain fatty acids; SEM: standard error of the mean.

²Total SCFA calculated as the sum of all absolute concentrations (μmol/g) for acetate, propionate, and butyrate.

³Relative values calculated by dividing absolute concentrations of each analyte (μmol/g) by the total concentration (μmol/g; within either SCFA or BCFA category) and multiplying by 100.

Intestinal Histology and Histopathology

No significant differences (P > 0.05) were observed between Fast and Slow groups in terms of villi height, crypt depth, or the villi height-to-crypt depth ratio in the ileum on d 11 or 25 (Table 7). However, there appeared to be a trend on d 25 where the villi height-to-crypt depth ratio was numerically higher (P = 0.054) in the Fast group, mainly attributed to the numerically higher (P = 0.09) villi height in the Fast birds. Additionally, no apparent differences (P > 0.05) in expected probabilities of expressing histopathology scores were evident between Fast and Slow groups, and scores for nearly all outcomes yielded weighted averages below 1 (Table 8).

Table 7.

Ileal tissue histomorphology in birds differing in growth rate.¹

	Treatment
Outcome	Slow	Fast	SEM	P-value
D 11
Villi height, µm	356.5	330.2	15.06	0.22
Crypt depth, µm	113.7	107.9	3.38	0.20
V:C2 ratio	3.19	3.10	0.097	0.52
D 25
Villi height, µm	376.2	409.8	14.04	0.09
Crypt depth, µm	126.8	126.5	4.08	0.96
V:C ratio	3.03	3.25	0.091	0.054

¹All means represent a total of 30 birds unless a value was removed from the final data set.

²V:C ratio is the villi height-to-crypt ratio.

Table 8.

Expected probabilities and weighted averages of histopathology scores in the ileum and cecum on study d 11 and 25.¹

	Score
Item	0	1	2	3	4	5	Average score2
Ileum, d 11
LP GALT presence
Slow	0.97	0.03	0.01	0.00	0.00	0.00	0.05
Fast	0.94	0.05	0.01	0.00	0.00	0.00	0.07
Heterophils LP
Slow	0.97	0.02	0.01	0.00	0.00	0.00	0.04
Fast	0.99	0.01	0.00	0.00	0.00	0.00	0.01
Bacteria/dysbacteria
Slow	0.99	0.01	0.00	0.00	0.00	0.00	0.01
Fast	100.00	0.00	0.00	0.00	0.00	0.00	0.00
Intraepithelial leukocytes
Slow	0.14	0.76	0.10	0.00	0.00	0.00	0.96
Fast	0.82	0.18	0.00	0.00	0.00	0.00	0.18
Mucus/goblet cells
Slow	0.91	0.08	0.01	0.00	0.00	0.00	0.10
Fast	0.89	0.10	0.01	0.00	0.00	0.00	0.12
Cecum, d 11
LP GALT presence
Slow	0.07	0.41	0.50	0.02	0.00	0.00	1.47
Fast	0.10	0.47	0.43	0.02	0.00	0.00	1.39
Heterophils LP
Slow	0.93	0.03	0.02	0.01	0.00	0.00	0.10
Fast	0.99	0.00	0.01	0.00	0.00	0.00	0.02
Ileum, d 25
LP GALT presence
Slow	100.00	0.00	0.00	0.00	0.00	0.00	0.00
Fast	100.00	0.00	0.00	0.00	0.00	0.00	0.00
Heterophils LP
Slow	0.94	0.04	0.01	0.00	0.01	0.00	0.10
Fast	0.35	0.32	0.07	0.11	0.15	0.00	1.39
Bacteria/dysbacteria
Slow	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Fast	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Intraepithelial leukocytes
Slow	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Fast	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Mucus/goblet cells
Slow	0.81	0.14	0.05	0.00	0.00	0.00	0.24
Fast	0.93	0.05	0.02	0.00	0.00	0.00	0.09
Cecum, d 25
LP GALT presence
Slow	0.92	0.07	0.01	0.00	0.00	0.00	0.09
Fast	0.58	0.36	0.05	0.01	0.00	0.00	0.49
Heterophils LP
Slow	1.00	0.00	0.00	0.00	0.00	0.00	0.00
Fast	1.00	0.00	0.00	0.00	0.00	0.00	0.00

Abbreviations: LP, lamina propria; GALT, gut-associated lymphoid tissues.

¹Broilers were reared from 0 to 27 d of age with sample collection occurring on either d 13 or 27.

²Weighted average score was calculated to account for the probability of each score occurring. Specifically, the value was multiplied by the proportion and then the sum calculated within treatment.

DISCUSSION

The objective of this study was to identify whether organ development or microbiota-derived fermentation end-products may be an influential factor for explaining differences in the rate of BW gain in broilers. Although the current study demonstrated clear BW differences between Fast and Slow groups, there were variable differences in organ weights and lengths, VFA concentrations in luminal contents, and intestinal histology.

Comparison of Growth Performance

When selecting birds on d 11 for Fast and Slow groups, the 10th and 90th percentiles were chosen to create disparate populations, and by d 25, the BW difference between treatment groups remained clear. As all birds within each cohort were obtained from the same hatchery source, these populations successfully simulated variations often observed in commercial flocks. Interestingly, during the 14-d study period, the Fast group gained more weight and ate more feed than the Slow group, but no differences in efficiency of gain were observed. This may be due to the fact that all birds were of the same genetic background as selected from within a single hatch per cohort. In the industry, faster growing birds have the potential for a higher ultimate carcass yield than slower growing birds when grown to the same market age. This was portrayed in a study by Narinc et al. (2015), where fast- and slow-growing broilers were reared to specific slaughter weights, which reported that the fast-growing breed exhibited higher carcass and breast yields and smaller wing and fat pad weights compared with the slow-growing breed. A similar study conducted by Canoğulları Doğan et al. (2019) reported that the fast-growing breed attained market weight 34 d earlier while also consuming less feed and producing higher breast weights, whereas the slow-growing breed had a higher percentage of abdominal fat and leg weights. Whether or not the same conclusions could be drawn from our study cannot fully be determined, as both groups in the current study originated from the same genetic line (i.e., were genetically similar) and carcass outcomes were also not measured. However, it is possible that the distinct groups in our study may have yielded similar carcass outcomes as the different breeds used by the other researchers, based on the differences we did observe for BW gain, feed intake, and organ measurements.

Connecting Organ Development With Growth Performance

On an absolute basis, birds in the Fast group exhibited longer organ lengths than their Slow counterparts; however, significance was lost when expressing organ lengths on a relative basis. This corroborates research by Lumpkins et al. (2010), which observed that different genetic lines can have different rates of intestinal development, with relatively longer and heavier duodenum, jejunum, and ileum segments in a heritage breed when compared with modern high-yield broilers. Conversely, Schmidt et al. (2009) reported that modern broilers exhibited jejunal and ileal sections that were 20% longer compared with a heritage line representing genetics from the 1950′s. In our current study, however, these differences between the Fast and Slow groups persisted when all birds were from the same genetic line, hatch date (within cohort), and raised within the same environment. Therefore, the differences observed indicate that variability in intestinal length still exists within a commercial genetic line reared in a common environmental context. These differences might partially explain the BW differences between groups by that the longer absolute length of the intestine in Fast birds, together with lower total SCFA concentrations in the ileum and the tendency of taller villi on d 25, contributes to a better nutrient digestion and absorption and thus higher BW.

Previous research indicates a longer absolute length of the small intestine may translate to a higher capacity for digestibility, nutrient absorption, and potentially higher nutrient utilization, thereby leading to improved growth performance in broilers (Konarzewski et al., 2000; Svihus and Itani, 2019). This conclusion coincides with our results, where birds in the Fast group had longer absolute intestinal lengths and better growth performance, except for feed conversion ratio. Nevertheless, these statistical differences were lost when intestinal section lengths were placed on a relative basis, which indicates that allometric growth of the organs was unchanged between groups (Alshamy et al., 2018). More research in this is warranted to better understand this phenomenon.

Although relative organ length did not differ between the 2 treatment groups, relative organ weight differences were consistently observed. Many factors have been reported to influence organ growth in broilers, with selection for body growth rate chief among them (Nir et al., 1993). As such, selection for higher growth rate in broilers has resulted in decreasing the average age of broilers at slaughter weight. For example, rearing a slow-growing heritage breed (e.g., Delaware) to market weight took 2.5-times longer, and cost nearly twice as much as it did for modern broilers (McCrea et al., 2014). The same researchers also noted that, although processed at the same live market weight as commercial broilers, the Delaware birds had significantly lower carcass dressing weights, suggesting they were less efficient at developing muscle, and more efficient at building bones and internal organs. Our data supports these conclusions, where Slow birds had higher total relative organ weights than the Fast birds. Additionally, when empty carcass weight was calculated on a relative basis, which was the weight of the bird after most organs were removed as a proportion of live BW, the Fast group exhibited a higher percentage of BW not attributable to those organs. In this way, empty carcass weight, although not perfect, yields an outcome that is comparable to the traditional calculation of hot carcass weight, and expressing the empty carcass weight as a proportion of live BW approximates the calculation of dressing percentage (Jespersen et al., 2024). Genotype effects on carcass yield are well documented, where decreased carcass yield occurs in Slow-growing broiler breeds (Tang et al., 2009; Devatkal et al., 2019; Singh et al., 2021). However, birds in the current study were assumed to have the same genotype, which complicates these results. Nevertheless, based on our results and the literature, it is plausible that our Slow group of broilers would have exhibited a reduced carcass yield at market weight, although more research is needed to confirm this speculation.

Organ Maturation

Multiple organs mature earlier than others due to genetic disposition, including multiple digestive organs such as the proventriculus, gizzard, and small intestine (Katanbaf et al., 1988; Mitchell and Smith, 1991; Nir et al., 1993). Other researchers reported the highest relative weight of small intestinal segments at 5-to-8 d of age before declining thereafter (Nitsan et al., 1991; Nir et al., 1993; Iji et al., 2001; Ravindran et al., 2006), which suggests that accelerated development of digestive organs may be important to sustain post-hatch muscle growth in fast-growing broilers. The digestive system also appears to mature earlier in modern breeds when compared with older genetic lines. In the current study, fast-growing birds exhibited notable reductions in relative organ weights and mass of segments in the small intestine at the same age of slow-growing birds. Mitchell and Smith (1991) reported an increase in the absolute weight and length of the small intestine, but a notable decrease in relative intestinal weight and length in broiler lines selected for a rapid growth rate. Findings from our study demonstrate the same principle; Fast birds had higher absolute organ weights compared with Slow birds, but the effect was reversed when placed on a relative weight basis. Although it has been reported that the relative weight of the small intestine decreases as birds mature, Ravindran et al. (2006) observed a steady increase in intestinal mass from hatch to study conclusion. This indicates that while the small intestine relative mass appears to decrease as birds mature, any decrease may be compensated by increased intestinal mass to help support nutrient supply throughout the body.

Measuring Digestive Capacity

Proventriculus. The proventriculus, or the glandular stomach in chickens, is where the release of digestive acids and enzymes occurs prior to passage into the gizzard. Singh et al. (2021) observed that a fast-growing broiler breed had a higher relative weight of the proventriculus than the comparator slow-growing breed. These results contradict our own results, where despite the Fast group demonstrating higher absolute weights for both time-points, the proventriculus was significantly heavier in the Slow group on a relative weight basis. However, the 2 groups in our study came from a single genetic line and from a single hatch per cohort, which might partially contribute to the contradictory findings here. Havenstein et al. (2003) reported that a reduction in visceral organ weight relative to body weight was associated with higher growth rates in modern breeds. This reduction in relative organ size is tied to a decrease in overall maintenance energy required, and results in an overall increase in energy efficiency and utilization (Tallentire et al., 2016). The relative proventriculus weight increase observed in the Slow group in our study aligns with these findings.

Gizzard. The absolute weight of the gizzard was higher in the Fast treatment throughout the study. This corroborates evidence from Maisonnier et al. (2001), which reported that the gizzard tended to increase in absolute size when birds were selected for high digestive efficiency, suggesting that a larger gizzard allowed for longer luminal residency time. Additionally, an indigenous slow-growing breed (Venda) had higher gizzard and crop weights than Ross 308 broilers at the same age (Mabelebele et al., 2013). In the current study, when gizzard weight was expressed on a relative basis, the difference between the groups reversed with the Slow birds expressing an increased relative gizzard weight at both time-points. Singh et al. (2021) observed a higher relative gizzard weight in a slow-growing broiler breed than in Cobb 500 broilers reared on the same diet, suggesting that slower growing birds have relatively heavier gizzards. The same authors concluded that the gizzard is an organ particularly susceptible to genetic changes, especially when selecting for high digestive efficiency (Singh et al., 2021). Additionally, Rougière et al. (2009) found the digestion efficiency positively correlated with gizzard weight. Our results showed that Fast birds had higher absolute gizzard weight but lower relative weight, while the opposite was found in Slow birds. This probably explained that the numerically lower FCR in Fast birds was positively related to the birds’ higher absolute gizzard weight, despite their lower relative gizzard weight compared with Slow counterparts.

Pancreas. A crucial organ responsible for secretion of digestive enzymes is the pancreas. Again, the current study observed a smaller relative weight of the pancreas in Fast group birds. Péron et al. (2006) reported that relative pancreas weight was reduced when genetically selecting for improved feed efficiency, although feed efficiency did not differ in the current study. Other previous research reported that supplementing broiler diets with pancreatic enzymes reduced the relative size of the pancreas (Gracia et al., 2003) and endogenous pancreatic enzyme secretions (Mahagna et al., 1995). Therefore, we attribute the reduction of relative pancreas weight in the Fast birds to sufficient or ‘better’ digestive function compared with the Slow birds.

Small Intestinal Histomorphology. The small intestine is lined with villi that increase the overall surface area of the small intestine, thereby permitting more nutrients to be absorbed by the animal. Measuring villi height is a practical outcome that may indicate absorptive capacity (Prakatur et al., 2019), and could provide insight into possible variations in absorption capabilities. In the current study, no statistical differences were observed in villi height; however, a greater numerical difference was noted between groups on d 25, where the Fast birds had increased villi height and villus height-to-crypt depth ratio. Both outcomes, which were numerically higher the Fast birds, may suggest greater intestinal activity, with evidence that crypt depth increases linearly with age with a subsequent increase in villus height (Poole et al., 2003). It is also possible that these differences would have become statistically significant if the groups of birds had been able to mature further.

Volatile Fatty Acids and the Microbiome. The intestinal tract of the broiler chicken, although relatively short, is inhabited by diverse microbial communities. Chicks hatch with an undeveloped microbiome that does not become established until they are roughly 2 to 3 wk old (Oakley et al., 2014; Shang et al., 2018; Jurburg et al., 2019), and the cecal microbial community can take up to 30 d to fully develop (Amit-Romach et al., 2004). These microbial communities play a key role in the growth and gut health of animals through VFA production (Dunkley et al., 2007), but can also modulate intestinal morphology and influence nutrient absorption and digestion throughout the tract (Shakouri et al., 2006; Yang et al., 2009).

Butyrate is considered essential for villi development (Panda et al., 2009), and in mice, butyrate has been established as the primary source of energy for colonocytes (Donohoe et al., 2011). It is also known to act in regulating gene expression as a histone deacetylase inhibitor suppressing colonic tumor cell lines, promoting osteoblast formation, and preventing and treating diet-induced obesity among other effects (Steliou et al., 2012; Berni Canani et al., 2012). In our experimental context, butyrate did not differ between groups, whereas some differences in acetate and propionate were observed. Although not statistically compared, total VFA concentrations appeared to be lower on d 11 than d 25. Additionally, on d 25, higher absolute concentrations of acetate and propionate were measured in the ileum and ceca of Slow birds, respectively. In the current study, despite the SCFA differences observed in the slow treatment, it is unlikely that these elevations significantly impacted bird performance. This is due to the estimation that broilers, like humans and pigs, attain only approx. 5 to 15% of their total caloric dietary energy from SCFA (Bergman, 1990; Rinttilä and Apajalahti, 2013). Thus, based on the small differences in VFA concentrations between birds selected for divergent growth rate, we conclude that intestinal luminal profiles are not a likely source of growth variability in broilers. However, we cannot rule out the possibility that Fast birds had better digestion and absorption efficiency than Slow birds, as the former had a lower propionate level and a tendency of lower total SCFA and butyrate levels in the cecum, indicating more nutrient absorption in the small intestine and less substances for cecal microbial fermentation. Moreover, directed studies focused specifically on microbiota composition may offer additional value in this context.

CONCLUSIONS

To summarize, birds from the Fast group had a higher BW gain than the Slow group and were able to maintain this difference throughout the study. Despite having lighter BW, birds in the Slow group exhibited notably higher relative organ percentages. However, this difference in BW gain was unable to be fully explained through the characterization of differences in organ growth, intestinal histomorphology and histopathology, or through concentrations of VFA in luminal contents as indicators of microbial fermentation.

DISCLOSURES

A. H. Smith is an employee of Arm & Hammer Animal and Food Production, Church & Dwight Co., Inc. (Waukesha, WI, USA). No other authors have any conflict of interest to report.