Effects supplementation of novel multi-enzyme on laying performance, egg quality, and intestinal health and digestive function of laying hens

Abstract

This study investigated the effects of multi-enzyme supplementation on various aspects of laying hens, including laying performance, egg quality, intestinal health and digestive function. In total, 384 Jingfen No.6 laying hens at 65-week-age were randomly assigned to four distinct dietary treatments: a basal diet (CON), CON supplemented with 150 g/t multi-enzyme (T1), CON with 300 g/t multi-enzyme (T2), and 600 g/t multi-enzyme (T3). A significant linear (P = 0.044) and quadratic (P = 0.014) increase was observed in the laying rate, while the feed/egg ratio exhibited a linear (P = 0.001) and quadratic (P < 0.001) decrease with increasing multi-enzyme supplementation. Additionally, linear (P < 0.05) and quadratic (P < 0.05) increases were observed in yolk rate and haugh unit with increasing levels of multi-enzyme supplementation. The trypsin activity in the duodenum and crude protein digestibility showed linear (P < 0.05) and quadratic (P < 0.05) increase with the addition of multi-enzyme. Furthermore, lipase and amylase activities in the duodenum increased quadratically (P = 0.041) and linearly (P = 0.040), respectively. Both jejunal and ileal digesta viscosities showed linear (P < 0.05) and quadratic (P < 0.05) decrease with the increasing addition of multi-enzyme. Moreover, multi-enzyme supplementation significantly increased (P < 0.05) the number of goblet cells in the intestinal of the treatment groups. The mRNA expression of Occludin-1, mucin 2 (MUC-2), large neutral amino acids transporter small subunit 1 (LAT-1) in the jejunum were significantly increased (P < 0.05) in the treatment groups (T1, T2 and T3) compared to the CON group. Additionally, the mRNA expression of solute carrier family 6-member 19 (B⁰AT-1) and large neutral amino acids transporter small subunit 4 (LAT-4) were significantly evaluated (P < 0.05) in the T2 and T3 groups, respectively. In conclusion, multi-enzyme supplementation enhanced digestive enzyme activities and intestinal barrier function, reduced intestinal digesta viscosity, and regulated mRNA expression of intestinal amino acid and lipid transporter genes, thereby improving crude protein digestibility and positively affecting laying performance and egg quality in hens.

Introduction

Soybean meal is a vital protein source in the diets of laying hens. However, due to its high cost and inconsistent availability, researchers have been investigating alternative vegetable protein sources. Currently, cottonseed meal and rapeseed meal are gaining popularity as substitutes in the poultry feed industry. Nutritionally, cottonseed meal is recognized for its low-fat content and high levels of biologically valuable protein (Alford et al., 1996; Broderick et al., 2013). Rapeseed meal is notable for its elevated concentrations of sulfur-containing amino acids and essential minerals, such as calcium and phosphorus (Konkol et al., 2024). Nonetheless, both cottonseed meal and rapeseed meal contain high levels of crude fiber and anti-nutritional factors, including non-starch polysaccharides and gossypol, which can adversely impact growth performance and increase mortality rates among laying hens (He et al., 2017).

Non-starch polysaccharides can be categorized into water-soluble and insoluble types (Hosseini et al., 2018). The insoluble non-starch polysaccharides have a minimal impact on viscosity and do not adversely affect nutrient digestibility (Sarmiento-Franco et al., 2000). In contrast, the soluble non-starch polysaccharides can increase digesta viscosity, hindering the digestion and absorption of proteins, lipids, and starches (Knudsen, 2014). Free gossypol, found in cottonseed meal, is a toxic phenolic pigment harmful to animal health (Gadelha et al., 2014; Yang et al., 2021). Some studies have shown that free gossypol can lead not only to a decline in egg yolk quality but also to an imbalance in liver fat deposition, resulting in liver dysfunction (Blevins et al., 2010; Waldroup 1981).

Therefore, if anti-nutritional factors can be effectively removed, and digestion and absorption of crude fiber can be promoted, this can improve the health and production performance of poultry, reduce breeding cost, and increase overall benefits (Jazi et al., 2017). Several methods can address the negative effects of anti-nutritional factors, such as adding emulsifiers (Oketch et al., 2022; Wiśniewska et al., 2023), bile acids (Yang et al., 2022), and exogenous enzymes (Chen et al., 2021; Olgun et al., 2018; Wan et al., 2023) to the feed. Exogenous enzymes have been extensively used in poultry feed to mitigate the adverse effects of anti-nutritional factors and enhance production performance (Slominski, 2011). However, the efficacy of multi-enzyme combinations needs to be evaluated, as the effects of various enzyme combinations remain unclear. Currently, there is a scarcity of studies examining the impacts of different combinations of exogenous enzymes on laying hens fed vegetable protein diets supplemented with alternative soybean meal. An in-depth study of different enzyme combinations and related knowledge is crucial for advancing nutritional research in laying hens.

Thus, our study aims to evaluate the effects of a novel multi-enzyme combination (including alkaline protease, xylanase, glucanase, β-mannanase, cellulase, acid protease, glucamylase, and α-galactosidase) on laying performance, egg quality, apparent nutrient digestibility, digestive enzyme activities, intestinal morphology and nutrient transporter gene expression in laying hens, which fed diets containing dephenolized cottonseed and rapeseed meal as part of vegetable protein source.

Materials and methods

Animal ethics statement

The experimental procedures conducted in this study were ethically approved by the Institutional Animal Care and Use Committee of Zhejiang University (ZJU20240150). This approval ensures adherence to established standards for the ethical treatment and utilization of animals in research.

Enzyme preparation

The multi-enzyme used in this study was the commercial preparation from Wuhan Sunhy Biology Co., Ltd (Hubei, China), with eight enzyme activities (U/g): alkaline protease (produced by Bacillus subtilis), 129,600; xylanase (produced by Pichia pastoris), 30,000; glucanase (produced by Pichia pastoris), 3,500; β-mannanase (produced by Pichia pastoris), 1,000; cellulase (produced by Trichoderma reesei), 1,500; acid protease (produced by Bacillus subtilis), 1,000; glucamylase (produced by Trichoderma reesei), 3,000 and α-galactosidase (produced by Pichia pastoris), 200.

Experimental design, animals, diets and housing

The study involved a total of 384 65-week-old Jingfen No. 6 laying hens, which were randomly assigned to four dietary treatments, each with six replicates containing sixteen birds per replicate (four hens per cage) over an 8-week feeding period. Four dietary treatments were as follows: the basal diet (CON), the basal diet supplemented with 150 g/t multi-enzyme (T1), the basal diet supplemented with 300 g/t multi-enzyme (T2) and the basal diet supplemented with 600 g/t multi-enzyme (T3). The nutritional composition of the basal diet is shown in Table 1, and all diets were provided in mash form.

Table 1.

Ingredients and nutrient contents of the basal diet (as-fed basis)

Items	content
Ingredients, %
Corn (7.8 % crude protein)	65.50
Soybean meal (43 % crude protein)	11.50
Dephenolized cottonseed meal (60 % crude protein)	6.00
Rapeseed meal (38 % crude protein)	6.00
Limestone powder	7.00
Premix1	4.00
Total	100.00
Nutrient composition
Metabolism energy, Mcal/kg	2.62
Crude protein2 (%)	16.57
Crude fiber2 (%)	3.50
Crude ash2 (%)	9.70
Ether extract2 (%)	3.00
Methionine (%)	0.36
Lysine (%)	0.86
Calcium (%)	2.97
Total phosphorus (%)	0.46

¹Premix provided the following per kilogram of diet: vitamin A, 10000 IU; vitamin D3, 1800 IU; vitamin E, 10 IU; vitamin K, 10 mg; vitamin B12, 1.25 mg; thiamine, l mg; riboflavin, 4.5 mg; calcium pantothenate, 50 mg; NaCl, 3 g; niacin, 24.5 mg; pyridoxine, 5 mg; biotin, 1 mg; folic acid, 1 mg; choline, 500 mg; Mn, 60 mg; I, 0.4 mg; Fe, 80 mg; Cu, 8 mg; Se, 0.3 mg; Zn, 60 mg.

²Crude protein, crude fiber, crude ash, and ether extract were measured values; others were based on calculated values.

Every four hens were housed in 40 cm (width) × 45 cm (length) × 45 cm (height) stainless steel cages (4 cages per replicate) and the stocking density was 450 cm² per hen. During the experiment period, the hens were free access to food and water. Lighting was provided in a 16 h light and 8-hour dark cycle, using a combination of natural and artificial light to meet the recommended conditions. Room temperature was consistently maintained between 15 °C and 22 °C. The initial week served as an adaptation period, followed by seven weeks dedicated to the formal experiment.

Production performance and Egg quality

During the test period, the performance of hens, including egg production, egg weight, broken eggs, and mortality, was recorded daily for each replicate (with 6 replicates per treatment). Feed was weighed once a week. The laying rate, average egg weight, average daily feed intake (ADFI), and feed/egg ratio were calculated as follows:

$$\text{Laying}\text{rate}(\%)=\frac{\text{Total}\text{number}\text{of}\text{eggs}}{\text{The}\text{number}\text{of}\text{laying}\text{hens}\times \text{Days}}\times 100$$

$$\text{Average}\text{egg}\text{weight}\left(\mathrm{g}\right)=\frac{\text{Total}\text{eggs}\text{weight}}{\text{Total}\text{number}\text{of}\text{eggs}}$$

$$\text{AFDI}\left(\mathrm{g}/\text{hen}/\text{day}\right)=\frac{\text{Total}\text{final}\text{feed}\text{intake}-\text{Total}\text{initial}\text{feed}\text{intake}}{\text{The}\text{number}\text{of}\text{laying}\text{hens}\times \text{Days}}$$

$$\text{Feed}/\text{egg}\text{ratio}\left(\mathrm{g}/\mathrm{g}\right)=\frac{\text{Total}\text{feed}\text{consumption}\text{during}\text{the}\text{test}\text{period}}{\text{Total}\text{egg}\text{weight}\text{during}\text{the}\text{test}\text{period}}$$

A total of 144 eggs (6 eggs per replicate) were selected to evaluate egg quality at the end of the trial period. Egg weight, eggshell strength, haugh unit, and yolk color were measured by a digital egg tester (DET-6000, Nabel Co., Ltd., Kyoto, Japan). Eggshell thickness was measured (without shell membrane) with an egg shell thickness gauge (ESTG-1, Orka Food Technology Ltd., Ramat Hasharon, Israel). For the physical measurements, short axis and long axis of eggs were measured with 0.01 mm sensitive digital caliper, and the egg shape index is the ratio of the short axis to the long axis.

Sample collection

At the end of the experiment, all laying hens were deprived of feed for 12 hours, but water was offered ad libitum. Then 48 laying hens (two bird from each replicate) were randomly selected and killed by cervical dislocation. Subsequently, blood samples (5 mL per bird) were collected from the brachial vein of each selected bird, and the resulting serum was obtained after centrifugation at 3,000 rpm for 15 minutes at 4°C, then stored at -20°C.

For intestinal tissue collection, the middle sections of the duodenum and ileum were preserved in 4 % paraformaldehyde. The middle portion of the jejunum was divided into two segments: one was fixed in 4 % paraformaldehyde, while the other was preserved in 2.5 % buffered glutaraldehyde. The remaining jejunum tissues were rinsed with cold sterile phosphate-buffered saline, and the intestinal mucosa was gently scraped using sterile slides. These samples were then stored in liquid nitrogen prior to final storage at -80°C.

Serum parameters analysis

The serum levels of various biomarkers, including total protein (TP), albumin (Alb), globulin (Glob), total bilirubin (TBil), serum creatinine (Crea), urea, aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransaminase (ALT), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), and total cholesterol (TChol) were quantified using standard laboratory techniques at the Second People's Hospital of Hangzhou (Hangzhou, China).

Hematoxylin and eosin (H&E) staining

The intestinal samples were prepared for histological analysis by initially dehydrating them in a graded series of alcohol solutions, followed by clearing with xylene, and embedding in paraffin. Next, the paraffin blocks were sliced into 5 μm sections and mounted onto glass slides. These sections were then stained with hematoxylin and eosin for histological examination. Subsequently, the tissue slices were examined under a microscope, and measurements of villi height (VH) and crypt depth (CD) were obtained using Image J software.

Transmission electron microscopy

The jejunum tissue was initially fixed in a 2.5 % glutaraldehyde buffer, followed by three washes in 0.1 mol/L cold phosphate buffer. Subsequently, the tissue was fixed in a 0.1 % osmium tetroxide buffer for two hours, followed by another wash with phosphate buffer. Rapid dehydration was performed using increasing concentrations of ethanol before embedding the tissue in a 1:1 mixture of epoxy propane and epoxy aldehyde resin. Ultrathin sections, measuring between 60 nm and 100 nm, were cut using an LKB Nova ultra-slicer (Leica Microsystems, Buffalo Grove, IL) and stained with uranyl acetate. The ultrastructure of the intestinal mucosal cells and microvilli was examined using transmission electron microscopy (JEOL, Tokyo, Japan) at 80 kV.

Scanning electron microscopy

Jejunum were initially fixed in 2.5 % glutaraldehyde at 4°C overnight, followed by washing with phosphate-buffered saline. Subsequently, the samples underwent dehydration, freeze-drying, and gold coating before observation and imaging with an EVO MA 15 scanning electron microscope (Carl Zeiss AG, Oberkochen, German).

Number of goblet cells

Segments approximately 1 cm in length from the middle duodenum, jejunum, and ileum of laying hens were collected and suspended in a 4 % paraformaldehyde solution. Tissue sections were then prepared and stained with periodic acid-Schiff (PAS) stain. Goblet cells stained with PAS were manually enumerated using the multipoint feature of ImageJ and expressed as goblet cells per 100 μm of villus. For each sample, six fields of view were selected to count goblet cells, and the average value was calculated for analysis.

Real-time quantitative PCR

Total RNA extraction from the jejunum was performed following established protocols outlined in previous studies (Liu et al., 2023; Xu et al., 2021). Trizol Reagent (Invitrogen, CA) was used for RNA isolation. The purity and concentration of the extracted RNA were assessed using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific, MA) at wavelengths of 260 and 280 nm. Only samples with absorbance ratios (260/280 nm) within the range of 1.8 to 2.0 were selected for subsequent analysis. For cDNA synthesis, the ReverAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA) and random primers were employed. Real-time quantitative PCR was conducted using a Roche LightCycler 480 PCR System with SYBR Green Master Mix. Specific primers for each gene are listed in Table S1. Relative gene expressions were calculated using the 2^−ΔΔCT method, with β-actin serving as the internal standard for normalization.

Digestive enzyme activity and viscosity parameters

To assess the activities of trypsin, lipase, and amylase, we followed the methods outlined in previous studies (Ding et al., 2021; Lu et al., 2023). Specifically, we utilized corresponding kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) in accordance with the manufacturer's guidelines to determine the activities of trypsin, lipase, and amylase in the duodenal mucosa.

At the conclusion of the trial, two hens from each replicate were selected for slaughter, and the viscosity of jejunal and ileal digesta was examined. Two grams of digesta from the middle of the jejunum and ileum were collected and mixed with 18 ml of HCl-KCl (0.2 mol/L, pH = 1.5). The mixture was then centrifuged at 10,000 rpm for 15 minutes, and the supernatants were collected for the determination of viscosity using an Ostwald viscosimeter (Huanguang, Taizhou, China).

Chemical analysis

At the conclusion of the trial, two hens from each replicate underwent a digestive trial. The pre-trial phase lasted three days, followed by a four-day normal trial phase. During the normal trial, daily records were maintained for feed intake and fecal matter production. Upon completion of the normal trial, all fecal samples were collected and combined. Subsequently, both feed and fecal samples were analyzed for dry matter, crude protein (calculated as crude protein: N × 6.25), ether extract, crude ash, crude fiber and acid-insoluble ash, following the methods outlined by the AOAC (2023).

Statistical analysis

The experimental data were analyzed using one-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons, employing SPSS version 20.0 software (IBM-SPSS Inc, Chicago, IL). The results are presented as means ± standard error of the mean (SEM), with significance determined at a P-value of less than 0.05. Additionally, data visualization and further statistical analysis were conducted using GraphPad Prism version 8.0.2 software (Monrovia, CA).

Results

Laying performance

The effects of dietary multi-enzyme supplementation on laying performance are presented in Table 2. In this experiment, the laying rate exhibited a linear (P = 0.044) and a quadratic (P = 0.014) increase as the concentration of multi-enzyme increased. Compared to the CON group, the laying rates in the T1, T2 and T3 groups increased by 1.42 %, 4.11 % and 1.90 %, respectively. Additionally, the feed/egg ratio decreased linearly (P = 0.001) and quadratically (P < 0.001) with increasing levels of multi-enzyme in the diet. Compared to the CON group, the feed/egg ratio in the T1, T2 and T3 groups showed reductions of 6.44 %, 9.85 % and 7.58 %, respectively. However, no significant differences (P > 0.05) were observed in average egg weight, AFDI, broken eggs, or mortality among the groups throughout the study.

Table 2.

Effects of multi-enzyme supplementation on laying performance of laying hens

Items	CON	T1	T2	T3	SEM2	P-Value
						A	L	Q
Laying rate, %	80.45b	81.59ab	83.76a	81.98ab	0.379	0.010	0.044	0.014
Average egg weight, g	67.73	67.27	67.79	68.76	1.334	0.985	0.769	0.928
ADFI1, g/hen	132.52	129.03	135.67	132.90	1.179	0.272	0.473	0.769
Feed/egg ratio, g/g	2.64a	2.47b	2.38b	2.44b	0.025	<0.001	0.001	<0.001
Broken egg, %	0.54	0.32	0.50	0.80	0.108	0.501	0.326	0.315
Mortality, %	1.04	2.00	0.83	1.46	1.042	0.852	0.985	0.986

¹ADFI = average daily feed intake

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

²SEM, total standard error of means.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

Egg quality parameters

As shown in Table 3, the egg shape index increased quadratically (P = 0.001) with multi-enzyme supplementation in the diets. Compared to the CON group, the T1 group had the highest egg shape index, showing an increase of 3.1 %. Furthermore, significant linear (P = 0.044 and P = 0.002) and quadratic (P = 0.013 and P = 0.008) improvements were observed in the yolk rate and haugh unit with increasing levels of multi-enzyme supplementation. The T2 group exhibited the greatest increase in yolk rate, while the T3 group showed the highest improvement in the haugh unit, with increases of 5.14 % and 11.51 %, respectively, compared to the CON group. No significant differences (P > 0.05) were found in the egg weight, eggshell strength, eggshell thickness or egg yolk color across all groups.

Table 3.

Effects of multi-enzyme supplementation on egg quality of laying hens

Items	CON	T1	T2	T3	SEM1	P-Value
						A	L	Q
Egg weight, g	66.82	67.28	67.19	67.59	0.448	0.953	0.545	0.836
Egg shape index	1.30b	1.34a	1.33ab	1.32ab	0.004	0.012	0.434	0.001
Eggshell strength, N	34.98	37.33	38.48	37.54	0.950	0.537	0.310	0.419
Eggshell thickness, mm	0.34	0.33	0.34	0.33	0.003	0.681	0.482	0.736
Egg yolk color	10.46	10.46	10.21	10.38	0.074	0.619	0.461	0.659
Yolk rate, %	26.26b	27.55ab	27.61a	27.38ab	0.190	0.026	0.044	0.013
Haugh unit	79.66b	82.40ab	85.84ab	88.83a	1.191	0.025	0.002	0.008

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

¹SEM, total standard error of means.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

Apparent nutrient digestibility

Table 4 presents the results of the effects of multi-enzyme supplementation on apparent nutrient digestibility. As the levels of multi-enzyme supplementation increased, crude protein digestibility in hens increased both linearly (P = 0.003) and quadratically (P = 0.001), with the highest improvement observed in the T2 group. No significant differences (P > 0.05) in the digestibility of dry matter, ether extract, crude fiber, and crude ash were observed between the CON group and the treatment groups.

Table 4.

Effects of multi-enzyme supplementation on apparent nutrient digestibility of laying hens

Items	CON	T1	T2	T3	SEM1	P-Value
						A	L	Q
Dry matter, %	59.63	59.83	61.57	62.74	0.836	0.567	0.147	0.354
Crude protein, %	45.40c	48.52bc	52.00a	50.30ab	0.797	0.001	0.003	0.001
Crude Fiber, %	16.38	17.33	19.10	21.90	1.197	0.426	0.086	0.228
Ether extract, %	89.45	89.37	88.62	89.72	1.005	0.988	0.995	0.965
Crude ash, %	19.68	20.71	19.00	21.05	0.483	0.466	0.601	0.779

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

¹SEM, total standard error of means.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

Digestive enzyme activity

The results from Table 5 indicate that the trypsin, lipase and amylase activities significantly changed (P < 0.05) following dietary supplementation with multi-enzyme. The trypsin activity in hens increased linearly (P = 0.043) and quadratically (P = 0.020) with the increasing levels of multi-enzyme. The lipase activity also increased quadratically (P = 0.041), while the amylase activity increased linearly (P = 0.040) as the level of multi-enzyme supplementation increased.

Table 5.

Effects of multi-enzyme supplementation on digestive enzymes in the duodenum of laying hens

Items	CON	T1	T2	T3	SEM1	P-Value
						A	L	Q
Trypsin, U/mg prot	273.02b	283.50b	322.54a	292.93ab	6.032	0.007	0.043	0.020
Lipase, U/g prot	27.64b	32.31a	30.77ab	30.32ab	0.600	0.036	0.233	0.041
Amylase, U/g prot	77.78b	81.12b	95.83a	87.35ab	2.030	0.003	0.040	0.102

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

¹SEM, total standard error of means.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

The viscosity of the intestinal content

As shown in Table 6, the viscosity of digesta in the jejunum and ileum decreased both linearly (P = 0.007 and P = 0.001) and quadratically (P = 0.019 and P = 0.003) with increasing concentrations of multi-enzyme. Specifically, the jejunal and ileal digesta viscosity in the T3 group was lower than that in the other groups.

Table 6.

Effects of multi-enzyme supplementation on digesta viscosity in the intestinal of laying hens

Items	CON	T1	T2	T3	SEM1	P-Value
						A	L	Q
Jejunal digesta viscosity (mPas)	1.56a	1.53ab	1.53ab	1.52b	0.006	0.034	0.007	0.019
Ileal digesta viscosity (mPas)	1.49a	1.46ab	1.43b	1.42b	0.008	0.012	0.001	0.003

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

¹SEM, total standard error of means.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

Serum biochemical parameters

Table 7 presents the effects of dietary multi-enzyme treatments on serum biochemical indexes in laying hens. There were no significant differences (P > 0.05) were observed in serum proteins (TP, Alb and Glob) between the CON group and treatment groups. Regarding kidney function parameters, significant linear (P = 0.002) and quadratic (P = 0.005) effects were observed for Crea content, while no significant differences were noted for TBil and Urea content with increasing levels of multi-enzyme in the diet. In liver functions parameters, the activity of AST decreased linearly (P = 0.006) and quadratically (P = 0.003) with increasing concentrations of multi-enzyme. For serum lipids parameters, the LDL-C and HDL-C levels decreased linearly (P = 0.003 and P = 0.016) and quadratically (P = 0.012 and P = 0.045) reduced due to the inclusion of multi-enzyme in the diet.

Table 7.

Effects of multi-enzyme supplementation on serum biochemical parameters of laying hens

Items1	CON	T1	T2	T3	SEM2	P-Value
						A	L	Q
Serum proteins
TP, g/L	45.24	46.09	43.98	45.17	0.574	0.650	0.657	0.898
Alb, g/L	15.75	15.27	15.24	15.33	0.175	0.551	0.178	0.374
Glob, g/L	31.63	29.53	28.10	28.80	0.627	0.221	0.077	0.112
Kidneys functions
TBil, umol/L	2.25	2.07	2.13	2.23	0.034	0.173	0.956	0.106
Crea, umol/L	4.75b	5.15ab	5.39a	5.45a	0.088	0.016	0.002	0.005
Urea, mmol/L	0.33	0.36	0.35	0.39	0.013	0.490	0.167	0.388
Liver functions
AST, U/L	244.80a	215.40b	219.10b	215.60b	3.499	0.003	0.006	0.003
ALP, U/L	444.83	376.75	623.17	354.67	40.772	0.078	0.948	0.479
ALT, U/L	<4	<4	<4	<4	NA3	NA3	NA3	NA3
Serum lipids
LDL-C, mmol/L	0.83a	0.81ab	0.76b	0.76b	0.011	0.025	0.003	0.012
HDL-C, mmol/L	0.87	0.82	0.72	0.74	0.023	0.081	0.016	0.045
TChol, mmol/L	2.50	2.37	2.25	2.47	0.106	0.849	0.814	0.709

¹TP, total protein; Alb, albumin; Glob, globulin; TBil, total bilirubin; Crea, creatinine; AST, aspartate aminotransferase; ALP, alkaline phosphatase; ALT, alanine aminotransferase; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TChol, total cholesterol.

^a−cMeans vary significantly within a row with different superscripts (P < 0.05).

²SEM, total standard error of means.

³NA is not statistically analyzed or not available.

CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme; A, ANOVA; L, Linear; Q, Quadratic.

Goblet cell density

The impact of multi-enzyme supplementation on the goblet cell density in intestines of laying hens was evaluated. Our results (Fig.1) indicated that multi-enzyme supplementation notably increased the number of goblet cells in the duodenum, jejunum and ileum of the treatment groups. Specifically, as the level of multi-enzyme supplementation in the diet increased, there was a corresponding rise in goblet cells density in the duodenum, with the T3 group exhibiting the highest density of goblet cells. In contrast, the hens in the T1 group achieved the highest number of goblet cells in both the jejunum and ileum across all treatment groups.

Fig. 1.

The results of the number of goblet cells in the duodenum, jejunum and ileum (magnification, 100 ×). ^{a – b} Within a row, values with no common superscripts differ significantly. The red arrow means the goblet cell in intestinal villi. One-way ANOVA was used to analyze the data (means ± SEM). CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme.

Intestinal morphology and physical barrier function

The influence of multi-enzyme supplementation on the intestinal morphology of laying hens was assessed using anatomical sectioning and microscopic observation (Fig. 2). Light microscopy indicated that the VH of the jejunum in the T2 group were significantly higher than in the CON group, with no significant changes observed in CD and VH/CD (Fig. 2A, C). Scanning electron microscopy revealed that the microvilli in the T2 group was more densely arranged and more neatly ordered compared to those in the CON group (Fig. 2B). Furthermore, transmission electron microscopy revealed that the morphology and arrangement of microvilli in the T2 and T3 group were more neatly, and the tight junctions were more closely aligned compared to those in the CON group (Fig. 3A). Moreover, the results of the mRNA expression of tight junction proteins in the jejunum are presented in Fig. 3B. It was found that the mRNA expression of Occludin-1 and mucin 2 (MUC-2) significantly increased (P < 0.05) in the treatment groups (T1, T2 and T3) compared to the CON group. The highest mRNA expression of Occludin-1 and MUC-2 were observed in the T2 group and T1 group, respectively. Additionally, the mRNA expression of zonula occluden 1 (ZO-1) in the T2 and T3 groups also notably increased (P < 0.05) compared to the CON group. However, there was no significant difference in the mRNA expression of Claudin-1.

Fig. 2.

Effects of multi-enzyme supplementation on intestinal morphology of laying hens. (A) Histomorphology of the jejunum (magnification, 40 ×). (B) Scanning electron micrographs of the jejunum microvilli (magnification, 20,000 ×). (C) Villus height, crypt depth and villus height/crypt depth of the jejunum. ^{a – b} Within a row, values with no common superscripts differ significantly. One-way ANOVA was used to analyze the data (means ± SEM). CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme.

Fig. 3.

Effects of multi-enzyme supplementation on intestinal physical barrier function of laying hens. (A) Transmission electron micrographs of the jejunum microvilli in laying hens (magnification, 60,000 ×). (B) The relative mRNA expressions of ZO-1, Claudin-1, Occludin-1 and MUC-2 were analyzed by real-time qPCR. ^{a – b} Within a row, values with no common superscripts differ significantly. One-way ANOVA was used to analyze the data (means ± SEM). ZO-1, Zonula occluden 1; MUC-2, Mucin 2. CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme.

Effects on the gene expression of nutrient transporter genes in the intestinal

To further evaluate the effects of multi-enzyme supplementation on nutrient digestion and absorption in laying hens, we assessed the mRNA expression of genes related to amino acid, lipid, and glucose transporters in the jejunum by using RT-qPCR (Fig. 4). The results indicated that the mRNA expression of amino acid transporters in the jejunum showed a notable increase in large neutral amino acids transporter small subunit 1 (LAT-1) in the treatment groups (T1, T2 and T3). Additionally, solute carrier family 6-member 19 (B⁰AT-1) and large neutral amino acids transporter small subunit 4 (LAT-4) were significantly enhanced in the T2 group and T3 group, respectively. Furthermore, the mRNA expression of lipid transporters in the jejunum revealed that fatty acid-binding protein 2 (FABP-2) had the highest expression in the T2 group, followed by the T3 and CON groups. However, the mRNA expression levels of glucose transporters (sodium glucose cotransporter 1 (SGLT-1) and glucose transporter 5 (GLUT-5)), excitatory amino acid transporter 3 (EAAT-3), and fatty acid translocase (CD36) were no discernible changes.

Fig. 4.

Effects of multi-enzyme supplementation on the relative mRNA expression of intestinal nutrient transporters. ^{a – b} Within a row, values with no common superscripts differ significantly. One-way ANOVA was used to analyze the data (means ± SEM). B⁰AT-1, Solute carrier family 6-member 19; EAAT-3, Excitatory amino acid transporter 3; LAT-1, Large neutral amino acids transporter small subunit 1; LAT-4, Large neutral amino acids transporter small subunit 4; CD36, Fatty acid translocase; FABP-2, Fatty acid-binding protein 2; SGLT-1, Sodium glucose cotransporter 1; GLUT-5, Glucose transporter 5. CON, the basal diet; T1, the basal diet supplemented with 150 g/t multi-enzyme; T2, the basal diet supplemented with 300 g/t multi-enzyme; T3, the basal diet supplemented with 600 g/t multi-enzyme.

Discussion

The beneficial effects of exogenous enzymes on poultry performance and nutrition have already been well documented. These enzymes can enhance the nutritional value of diets, reduce feed costs, maintain digestive health, promote growth, and minimize environmental pollution (Musigwa et al., 2021; Poudel et al., 2023). The objective of this study was to assess the impact of a novel multi-enzyme combination on laying performance, egg quality, intestinal health and nutrient digestibility in laying hens fed diets containing dephenolized cottonseed and rapeseed meal as part of vegetable protein source.

Our findings revealed that exhibited both linear and quadratic increases with increasing levels of multi-enzyme in the feed during the trial, suggesting that the inclusion of multi-enzyme in the diet could promote poultry production. This result is consistent with the previous study by Sun and Kim (2019), which indicated that increasing dietary levels of non-starch polysaccharide-degrading multi-enzymes positively affect egg production. Khan et al. (2011) reported that dietary supplementation with multi-enzyme (xylanase and β-glucanase) significantly increased egg production in hens at 40 weeks of age when fed with a corn-based diet. Moreover, in this study, the feed/egg ratio decreased both linearly and quadratically with the inclusion of increasing levels of multi-enzyme in the diet. Similarly, Chen et al. (2021) indicated that supplementation with alkaline protease significantly decreased the feed/egg ratio in hens. A lower feed/egg ratio corresponds to a higher feed conversion ratio. Khempaka et al. (2018) reported that mixed enzymes added to the feed improved the degradation of cell wall polysaccharides from plant feed ingredients, thereby enhancing nutrient utilization in chickens. Animal feed contains a substantial amount of non-starch polysaccharides, which limit nutrient absorption and contribute to low feed digestibility, directly affecting nutrient utilization and production performance (Morgan et al., 2022). However, the addition of enzymes to feed has demonstrated positive effects on the digestion and utilization of protein, starch and other major nutrients, thus promoting animal growth (Park and Kim, 2018). The studies above indicate that dietary supplementation with multi-enzyme can enhance nutrient digestibility, which may improve the feed conversion ratio and laying rate.

Our results demonstrated that the egg shape index, yolk rate, and haugh unit in the treatment group were remarkably increased compared to the CON group. These findings are consistent with a previous study by Deniz et al. (2013), which showed that hens fed an enzyme complex containing β-glucanases and β-xylanases in a corn-soybean meal diet had improved outcomes. Seyedoshohadaei et al. (2023) indicated that laying hens fed wheat-based diets supplemented with enzymes could increase the egg shape index, suggesting a positive impact of enzymes on egg morphology. Cai et al. (2024) found that adding protease to feed tended to increase the haugh unit and decrease water content in yolk, which may be related to improved protein utilization efficiency in the diet. Akbar et al. (1983) noted that the yolk percentage increased as dietary protein level rose from 15 % to 19 % in large-graded eggs, indicating elevated protein intake could enhance yolk ratio. Adding protease to feed improves the digestibility of crude protein and amino acids in the ration, promoting nutrient deposition in laying hens and enhancing their production performance (Lahaye et al., 2018), which likely contributes to the increased yolk rate. Egg quality serves as both an indicator of the production performance of laying hens and a factor influencing the nutritional and commercial value of eggs. The morphology of an egg is commonly expressed in terms of the shape index, representing the ratio of the short diameter to the long diameter (Kayadan and Uzun, 2023). The standard egg shape index is approximately 74 %, indicating an oval shape, with the usual index ranging from 72 % to 76 %. Eggs with shape indices below 72 % are considered too long, while those above 76 % are considered too round (Oguz et al., 2017). In our study, all eggs fell within the typical index range, and the shape index in the T2 and T3 groups were closer to the standard egg shape index. The haugh unit, a measure of egg protein quality, is determined by the height of the thick albumen and egg weight, serving as an indicator of egg freshness (Sun and Kim, 2019). With extension of storage time, water in the egg evaporates through the pores, leading to protein hydrolysis and a decrease in albumen height, resulting in a lower haugh unit (Jiang et al., 2022; Liu et al., 2016). Egg yolk, being the most nutritious part of the egg, provides essential nutrients for embryo development in chicks and contains bioactive lipids essential for human nutrition and health (Xiao et al., 2020). Compared to the CON group, the yolk rate in all treatment groups notably increased, especially in the T2 group, indicating that the addition of the novel multi-enzyme combination is significant for improving the nutritional value of eggs.

Blood biochemical indexes are important indicators of health status, nutritional and physiological status in animals, including birds. The level of LDL-C in serum is recognized as a biomarker for blood lipid metabolism (Wu et al., 2022). Dai et al. (2021) reported that t dietary supplementation with flavonoids led to a decline in serum LDL-C levels, which improved reproductive performance by regulating liver lipid metabolism in aged breeder hens. In the current study, the LDL-C content in serum showed both linear and quadratic reductions due to the inclusion of multi-enzyme in the diet. Additionally, the activity of AST in serum is commonly used as an indicator of liver damage (Lin et al., 2023). Our study found that the activity of AST decreased linearly and quadratically with increasing concentrations of multi-enzyme, suggesting that the laying hens in the treatment groups experienced less hepatocyte damage.

The integrity of the intestinal epithelium serves as a crucial physical barrier against the invasion of intestinal pathogens, while also facilitating nutrient absorption and waste secretion. This barrier primarily consists of intestinal epithelial cells and structural complexes, such as tight junctions and adherens junctions (Mowat and Agace, 2014; Otani and Furuse, 2020). Our study revealed that the tight junctions of microvilli in the treatment groups were both denser and longer. To further explore the underlying mechanisms, we found that the mRNA expressions of Occludin and MUC-2 were significantly increased in the treatment groups, with ZO-1 notably elevated in the T2 and T3 groups. Occludin and ZO-1 are essential tight junction proteins that play a critical role in regulating intestinal permeability and integrity (Yan et al., 2024). Thus, our results indicated that the addition of multi-enzyme could stimulate the production of goblet cells and mucin-2, consequently enhancing the defensive abilities of gut in hens. MUC-2, secreted by goblet cells, constitutes the first physical barrier of the intestine, preventing the invasion of pathogenic bacteria (Li et al., 2023; Yang et al., 2019). In our study, the mRNA expression level of MUC-2 and the number of goblet cells in the jejunum were increased in the treatment groups. These findings suggest that dietary supplementation with multi-enzyme is beneficial for intestinal mucosal development and enhances intestinal health in laying hens.

High levels of insoluble non-starch polysaccharides have been reported to affect intestinal mobility and digesta transit time by forming a barrier to endogenous digestive enzymes, such as amylases and proteases (Moita et al., 2022). Studies have shown that the addition of exogenous enzymes effectively increases the activities of digestive enzymes in the intestine and promotes the degradation of non-starch polysaccharides, thereby improving the digestion and absorption of nutrients in the feed (Hosseini et al., 2018; Yi et al., 2024b). Our study's results demonstrated that the activities of trypsin, lipase, and amylase in the duodenum were notably increased, while the digesta viscosity of the jejunum and ileum was significantly reduced with the addition of multi-enzyme in laying hens. These results suggested that the addition of multi-enzyme may degrade indigestible viscous substances in the intestine, thereby facilitating nutrient digestion and absorption.

Intestinal villi are crucial for poultry's ability to absorb nutrients from feed. Mehri et al. (2010) suggested that a decrease in the viscosity of digesta in the intestine could lead to increased villi length and width, thereby increasing their surface area. In our study, laying hens fed with multi-enzyme exhibited longer villi in the jejunum, along with a greater number of microvilli, which collectively improved the absorptive surface area of their mucosa. Similar findings were reported by Yi et al. (2024b), who observed that protease supplementation increased villi height and villi height to crypt depth ratios in broilers. Kim et al. (2021) indicated that dietary supplemental exogenous multi-enzyme containing phytase showed a significant improvement in VH, CD, and VH/CD, compared to basal diets.

The addition of enzymes to the diet can promote the digestion of nutrients by reducing the viscosity of digesta and enhancing the activity of endogenous digestive enzymes in the intestine. Furthermore, the reduction in digesta viscosity resulting from enzyme supplementation contributes to improved intestinal villi structure and morphology and promotes the division and renewal of intestinal cells, potentially affecting the expression of nutrient transporters in intestinal epithelial cells (Iji et al., 2001). Nutrients in the feed are degraded in the intestine into various small molecules that are transported through different nutrient transporters, whose expression affects the availability of nutrients and energy for animal growth (Mott et al., 2008). Therefore, to evaluate the effects of multi-enzyme supplementation on nutrient transporters, we examined the gene expression of nutrient transporters in the jejunum tissues. The amino acid transporter LAT-4 is involved in the transport of phenylalanine, leucine, isoleucine, and methionine (Guetg et al., 2015). LAT-1 is a system L, Na⁺-independent amino acid transporter responsible for transport of large neutral amino acids such as leucine, valine, phenylalanine, tyrosine, Tryptophan, and methionine (Chrostowski et al., 2010; Kaira et al., 2013). B⁰AT-1, is a neutral amino acid transporter that has the properties of system B⁰, mediating the Na⁺-dependent transport of neutral amino acids (Zhang et al., 2022). In this study, the mRNA expression level of B⁰AT-1, LAT-1, LAT-4 were upregulated in the multi-enzyme supplemented groups compared to the CON group, suggesting that the multi-enzyme may enhance the uptake of corresponding amino acids by increasing the expression of their transporters. Additionally, the mRNA expression level of FABP-2 was notably upregulated in the T2 group compared to the CON group. FABP-2 is involved in lipid metabolism in the intestine (Meena et al., 2023), indicating that multi-enzyme supplementation may effectively enhance the intestinal absorption of long-chain fatty acids in laying hens. These results align with research conducted by Yi et al. (2024a), which demonstrated that the inclusion of multiple enzymes in the diet led to a significant increase in the mRNA expression of peptide transporter 1, B⁰AT, and FABP-1, resulting in enhanced nutrient absorption within the broiler's intestinal. Based on these findings, it can be suggested that dietary multi-enzyme supplementation positively affects intestinal digestion and absorption, thereby improving feed digestion efficiency. As anticipated, the crude protein digestibility in this study increased linearly and quadratically with increasing multi-enzyme supplementation. Similarly, previous studies have shown that protease supplementation can increase crude protein digestibility (Ding et al., 2016; Erdaw et al., 2017).

Our study demonstrates that dietary supplementation with multi-enzyme improves laying performance and egg quality in laying hens. This enhancement is achieved by enhancing digestive enzyme activities and apparent nutrient digestibility, reducing jejunal and ileal digesta viscosity, and modulating the mRNA expression of intestinal amino acid and lipid transporter genes.

Declaration of competing interest

The authors declare no conflict of interest.