Effect of dietary supplementation with recombinant human lysozyme on growth performance, antioxidative characteristics, and intestinal health in broiler chickens

Abstract

Lysozyme is often used as a feed additive to act as an antibacterial protein that boosts the immune system of livestock and poultry while protecting against pathogens. To investigate the effects of recombinant human lysozyme (rhLYZ) from Pichia pastoris and chlortetracycline on broiler chicken’s production performance, antioxidant characteristics, and intestinal microbiota, a total of 200, 1-d-old male Arbor Acres broiler chickens (46.53 ± 0.42 g) were selected for a 42-d experiment. Dietary treatments included a basal diet of corn-soybean meal supplemented with either 0 mg/kg (CON), 50 mg/kg aureomycin (ANT), 20 mg/kg rhLYZ (LOW), 60 mg/kg rhLYZ (MEDIUM), or 180 mg/kg rhLYZ (HIGH). Compared with CON, MEDIUM diet increased (P < 0.05) average daily gain (67.40 g) of broilers from day 22 to 42. In the early (1.29) and overall phases (1.69), MEDIUM led to a reduction (P < 0.05) in the feed conversion ratio of broiler chickens. Furthermore, in comparison to the CON and ANT, MEDIUM exhibited reduced (P < 0.05) levels of INF-γ and tumor necrosis factor-α in the serum. In the cecum, the abundance of Monoglobus and Family_XIII_AD3011_group was lower (P < 0.05) in the MEDIUM treatment compared to CON. Overall, supplementation of 60 mg/kg of rhLYZ improved growth performance, nutrient utilization efficiency, and serum immune function, while also influencing the composition of intestinal microbiota. This suggests lysozyme’s potential to replace antibiotic additives in feed.

The study revealed that, through a 42-d experiment, the addition of 60 mg/kg of recombinant human lysozyme to broiler chickens’ feed led to significant improvements in average daily gain, feed conversion ratio, and serum immune markers. Furthermore, this supplementation influenced the composition of their intestinal microbiota, presenting a promising alternative to antibiotic use in poultry production.

Introduction

Antibiotic resistance represents a mounting challenge with significant ramifications for public health. This issue not only threatens the health of animals and humans but also has implications for environmental pollution (Martinez, 2009). For example, the prolonged and indiscriminate use of antibiotics in poultry farming, such as aureomycin, may contribute to the development of antibiotic-resistant strains, posing a potential risk to both poultry and public health. A systematic review report published in The Lancet Global Health found that antibiotic-resistant bacteria among these animals decreased by 39% by taking intervention measures to restrict the use of antibiotics in food animals. Many countries have taken actions to reduce the use of antibiotics in food animals. Therefore, developing strategies to replace antibiotics has become an important research topic in the field of animal husbandry.

In light of the current situation, the pursuit of viable alternatives to antibiotics has taken on utmost importance. These alternatives must enhance animal growth performance, preserve immune function, and yield positive effects on the environment and human health. Human lysozyme stands out as a promising candidate in this regard. Lysozyme, also known as muramidase, is a natural antibacterial protein that kills bacteria and viruses and promotes tissue repair (Jollès and Jollès, 2004). Lysozyme hydrolyzes the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in the peptidoglycan of bacterial cell walls (Weiser and Gotschlich, 1991), which causes bacterial death (Nielsen and McCammon, 2003). In addition to its antimicrobial properties, lysozyme exhibits various biological functions such as antiviral, antioxidant, anti-inflammatory, and gut microbiota modulation activities (Khorshidian et al., 2022; Kye et al., 2022). Some studies have investigated the potential of lysozyme to improve animal health and productivity. For example, lysozyme fed to weaned piglets mitigated the stress response induced by Escherichia coli. This benefit was achieved by reducing intestinal E. coli concentration, lowering white blood cell counts, decreasing circulating concentrations of stress hormone, and improving nutrient digestibility (Park et al., 2021). Therefore, lysozyme has the potential to modulate gut microbiota, improve gut health, and serve as an alternative to feed antibiotics. Due to the limited availability of natural lysozyme sources, using Pichia pastoris as an expression vector can achieve efficient and cost-effective production of lysozyme. Recombinant technology has been used widely to produce human lysozyme for food processing, pharmaceuticals, and animal feed (He et al., 2020).

The study investigated the impact of using recombinant human lysozyme (rhLYZ) from P. pastoris as a feed supplement to improve the growth performance, nutrient utilization efficiency, immune function, and intestinal health of broiler chickens. It evaluated the potential application of rhLYZ as a substitute for antibiotics in the rearing of broiler chickens.

Materials and Methods

The current investigation was conducted at China Agricultural University in an experimental poultry shed. The Institutional Animal Care and Use Committee of China Agricultural University approved the research protocol for this experiment (No.AW09089104-1, Beijing, China). All animal procedures followed the guidelines outlined in the National Research Council’s Handbook for the Welfare and Ethics of Laboratory Animals.

Experimental design and diets

The rhLYZ employed in this study is a powdered product obtained through centrifugation, purification, and lyophilization of the supernatant from a 30-L fermentation tank, where rhLYZ expressing strains were fermented for 192 h. The composition of the fermentation medium, as well as the procedure for purifying and lyophilizing the supernatant from the fermentation process, is consistent with the report by Wang et al. (2020a). The enzyme activity of the culture supernatant was 610,000 U/mL after 192 h of fermentation. The final lyophilized enzyme powder exhibits an activity of up to 12,960 U/mg. Two hundred 1-day-old male Arbor Acres broilers weighing 46.53 ± 0.42 g were tagged individually by attaching metal tags to their wings. Each cage (90 cm × 90 cm × 75 cm) was outfitted with an integrated trough feeder and an adjustable drinker, with the drinker connected to a water source featuring two nipples. Throughout the experimental period, the birds had unrestricted access to both feed and water. The broilers were administered Newcastle disease vaccine on days 7 and 28, and an inactivated infectious bursal disease vaccine on days 14 and 21. For the first 2 days preceding the placement of chicks into the poultry house, continuous illumination was provided. Subsequently, a lighting schedule of 23 h of light to 1 h of darkness was implemented daily. Broilers were raised under a constant lighting regimen in a temperature-controlled environment. The initial room temperature for 1-day-old chicks was approximately 33 °C. The room temperature decreased by 3 °C per week until reaching 24 °C, where it was maintained thereafter. Relative humidity was maintained between 45% and 55%. Broilers were allocated randomly to one of five dietary treatments. Each treatment was replicated in five cages with eight broilers per cage. Dietary treatments included: a basal diet of corn and soybean meal (CON) supplemented with 50 mg/kg aureomycin, a dosage commonly utilized in poultry studies (Liu et al., 2022b; Wang et al., 2022; Chia Tai Group, Henan, China; ANT), 20 mg/kg rhLYZ (LOW), 60 mg/kg rhLYZ (MEDIUM), or 180 mg/kg rhLYZ (HIGH). Broilers consumed an early-phase diet from day 0 to 21, and a late-phase diet from day 22 to 42 (Table 1). Experimental diets were formulated to satisfy dietary requirements specified by the NRC (Dale, 2014).

Table 1.

Composition and nutrient levels of basal diets (as-fed basis)

Item1	Diet
	Days 0 to 21	Days 22 to 42
Ingredient, %
Corn, 8.2% CP	58.17	64.31
Soybean meal, 46% CP	30.44	23.54
Corn gluten meal	2.00	3.02
Fish meal, 64.7% CP	2.00	2.06
Soybean oil	3.38	3.48
Dicalcium phosphate	1.50	1.04
Limestone	1.30	1.35
Salt	0.30	0.30
l-Lysine HCl	0.01	0.08
Methionine	0.14	0.04
Threonine	0.01	0.03
Vitamin–mineral premix2	0.50	0.50
Chromic oxide	0.25	0.25
Total	100.00	100.00
Nutritional levels3
Metabolizable energy, Kcal/kg	3,050	3,150
SID4 Lysine, %	0.86	0.72
SID Methionine, %	0.30	0.29
SID Threonine, %	0.63	0.74
Crude protein, %	21.00	19.14
Calcium, %	1.00	0.9
Available phosphorus, %	0.45	0.35
Analyzed composition
Crude protein, %	20.49	18.61
NDF, %	6.74	6.39
ADF, %	3.25	2.83
Ca, %	0.95	0.87
P, %	0.67	0.58

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH).

²The vitamin–mineral premix provided the following per kilogram of feed: vitamin A, 10,000 IU; vitamin D₃, 3,000 IU; vitamin E, 24 mg; vitamin K₃, 2.1 mg; vitamin B₁₂, 2 mg; riboflavin, 5.0 mg; pantothenic acid, 15 mg; niacin, 40 mg; choline chloride, 500 mg; folic acid, 0.9 mg; vitamin B₆, 3 mg; biotin, 0.05 mg; Mn (from MnSO₄·H₂O), 70 mg; Fe (from FeSO₄·H₂O), 80 mg; Zn (from ZnSO₄·H₂O), 100 mg; Cu (from CuSO₄·5 H₂O), 18.8 mg; I (from KI), 0.35 mg; Se (from Na₂SeO₃), 0.30 mg.

³Values were calculated according to Dale (2014).

⁴SID, standardized ileal digestible.

Sample collection and processing

Performance of broilers from day 1 to 42 was measured by calculating average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) based on body weight (BW) and feed intake. On days 21 and 42, one broiler chicken with uniform weight was selected and slaughtered from each cage. A blood sample (10 mL) was drawn from the jugular vein of each chicken and stored at room temperature for 30 min. Subsequently, the sample was centrifuged at 812 ×g  and 4 °C for 15 min. The serum was extracted and stored at −20 °C until further analysis of its biochemical immune parameters.

Birds were sacrificed by cervical dislocation and the posterior half of the duodenum, jejunum, and ileum were thoroughly flushed with phosphate-buffered saline. Gut tissues were fixed using a 4% paraformaldehyde solution, and subsequently subjected to meticulous morphological analysis. Concurrently, tissue samples were collected from the liver, spleen, thymus, and bursa of Fabricius to determine the visceral weight index, which was quantified using the following formula: viscera weight index (%) = (viscera weight/final BW) × 100%. Lastly, we procured digesta samples from the ileum and cecum for high-throughput sequencing of the 16S ribosomal RNA gene.

Serum immune and antioxidant parameters

Serum immunoglobulins (IgA, IgM, and IgG) were measured using commercially available ELISA kits from Nanjing Jiancheng Bioengineering Institute, China, following the manufacturer’s instructions. The antioxidant capacity, based on serum concentrations of total superoxide dismutase (T-SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC), was also evaluated using ELISA kits from the same manufacturer according to their instructions. Additionally, ELISA kits from Nanjing Jiancheng Bioengineering Institute were used to determine the concentrations of tumor necrosis factor-α (TNF-α), interferon-γ, interleukin-4 (IL-4), IL-6, and IL-8 in serum following the recommended protocols (Bao et al., 2020).

Microbiota analysis

Genomic DNA of the microbial community in ileal and cecal digesta was isolated using the E.Z.N.A. stool DNA kit (Omega Bio-tek, Norcross, GA, USA) in accordance with the manufacturer’s specifications. The V3–V4 regions of the bacterial 16S rRNA gene were amplified by PCR using universal primers 338F (5ʹ-ACTCCTACGGGAGGCAGCAG-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ) with the following protocol: initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, single extension at 72 °C for 10 min, and ending at 4 °C (Liu et al., 2018). Sequencing was performed using Illumina technology. Raw data underwent quality filtering with Trimmomatic, and merged using FLASH software, under the following criteria: 1) reads with an average quality score less than 20 were truncated; 2) sequences shorter than 50 bp or with ambiguous reads were discarded; and 3) samples were differentiated according to their barcode and primers, and reads with ambiguous bases were removed (Liu et al., 2018).

Operational taxonomic units (OTUs) were clustered with UPARSE (version 7.1, http://drive5.com/uparse/) using a 97% similarity cutoff. Chimeric sequences were filtered out. Each 16S rRNA representative gene sequence was categorized and analyzed using RDP Classifier (http://rdp.cme.msu.edu/) against the Silva (SSU128) 16S rRNA database at a confidence threshold of 70% (Wang et al., 2020b).

Statistical analysis

IBM SPSS software was used for data analysis (Chicago, IL, USA, version 2.0). The linear and quadratic responses of different doses of rhLYZ were evaluated using orthogonal polynomial contrast analysis. To separation of means was accomplished using Duncan’s test. Differences were deemed significant at P < 0.05. Bacterial communities in intestinal digesta were analyzed at the phylum, family, and genus levels using the Kruskal–Wallis method. The α-diversity, which represented the diversity of intestinal microorganisms, was analyzed using mothur (version 1.30.1), whereas β-diversity was evaluated through principal coordinate analysis (PCoA) using QIIME (University of California, San Diego, USA). Additionally, linear discriminant analysis (LDA) and LDA effect size (LEfSe) analyses were performed through the LEfSe tool (Shanghai Majorbio Bio-pharm Technology, China).

Results

Growth performance

Compared with CON and ANT, MEDIUM reduced the ADFI of birds from day 0 to 21 (Table 2). Compared with CON, MEDIUM increased (P < 0.05) ADG (67.40 g) of broilers from day 22 to 42. FCR of broilers was decreased (P < 0.05) in LOW (1.39) and MEDIUM (1.29) compared with CON (1.48) and ANT (1.43) from day 0 to 21. MEDIUM also decreased the FCR (1.69) of broilers over the entire experiment (P < 0.05). From day 0 to day 21, there was a quadratic effect (P < 0.05) on FCR and ADFI with increasing doses of rhLYZ. From day 22 to 42, there was a quadratic effect (P < 0.05) on ADG with increasing doses of rhLYZ. Throughout the experiment, there was a quadratic effect (P < 0.05) on FCR with increasing doses of rhLYZ.

Table 2.

Effects of antibiotic and rhLYZ by P. pastoris on growth performance of broilers¹

Item		ANT	rhLYZ				SEM	P-value
			CON	LOW	MEDIUM	HIGH		ANOVA2	Linear3	Quadratic4
0 to 21	Day 0 BW, g	46.82	47.17	48.35	47.29	46.69	0.33	0.455	0.439	0.293
	Day 21 BW, g	631.25	625.30	650.00	635.71	620.83	9.33	0.993	0.812	0.455
	ADFI, g	39.81a	40.81a	39.95a	36.01b	40.24a	0.58	0.029	0.217	0.027
	ADG, g	27.83	27.53	28.65	28.02	27.34	0.27	0.668	0.707	0.228
	FCR	1.43ab	1.48a	1.39b	1.29c	1.47a	0.02	0.001	0.257	0.001
22 to 42	Day 42 BW, g	2,040.48	1,967.46	2,054.76	2,051.59	1,997.62	18.10	0.954	0.534	0.047
	ADFI, g	120.55	115.04	120.48	120.05	118.21	0.91	0.274	0.346	0.111
	ADG, g	67.01ab	63.85b	67.01ab	67.40a	65.65ab	0.50	0.045	0.254	0.048
	FCR	1.80	1.80	1.80	1.78	1.80	0.01	0.934	0.922	0.611
0 to 42	ADFI, g	83.02	81.03	83.07	80.74	82.10	0.49	0.451	0.863	0.766
	ADG, g	47.46	45.72	47.76	47.73	46.45	0.40	0.435	0.629	0.120
	FCR	1.75a	1.77a	1.74a	1.69b	1.77a	0.01	0.028	0.397	0.014

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH); n = 5.

²Statistical significance was determined using one-way ANOVA among all groups.

^3,4Linear and quadratic effects of added rhLYZ levels, except for ANT group.

^abcWithin a row, values with different superscripts differ (P < 0.05).

Immune organ indices

No significant differences were observed among treatment groups for indices of the liver, spleen, thymus, or bursa of Fabricius (Table 3).

Table 3.

Effects of antibiotic and rhLYZ by P. pastoris on immune organ index of broilers¹

Item	ANT	rhLYZ				SEM	P-value
		CON	LOW	MEDIUM	HIGH		ANOVA2	Linear3	Quadratic4
Day 21
Liver, %	2.79	3.23	3.22	3.5	2.94	0.09	0.091	0.437	0.123
Spleen, %	0.04	0.03	0.03	0.04	0.03	0.00	0.507	0.620	0.580
Thymus, %	0.41	0.36	0.46	0.41	0.36	0.02	0.243	0.815	0.071
Bursa, %	0.17	0.15	0.15	0.15	0.13	0.01	0.940	0.706	0.865
Day 42
Liver, %	2.48	2.32	2.50	2.80	2.55	0.07	0.395	0.236	0.239
Spleen, %	0.11	0.11	0.14	0.12	0.13	0.01	0.401	0.521	0.580
Thymus, %	0.34	0.27	0.34	0.36	0.28	0.02	0.348	0.799	0.066
Bursa, %	0.05	0.05	0.06	0.05	0.05	0.00	0.934	0.890	0.540

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH); n = 5; (% of BW).

²Statistical significance was determined using one-way ANOVA among all groups.

^3,4Linear and quadratic effects of added rhLYZ levels, except for ANT group.

^abcWithin a row, values with different superscripts differ (P < 0.05).

Serum immune and antioxidant parameters

At day 21, the broilers fed MEDIUM (0.95 g/L) exhibited increased (P < 0.05) IgA levels compared to CON (0.64 g/L); Table 4). Additionally, MEDIUM (1.10 g/L) broilers showed an increase (P < 0.05) in IgM levels compared to CON (0.78g/L) and ANT (0.81 g/L) birds. Similarly, at day 21, MEDIUM (6.83 g/L) birds had higher (P < 0.05) IgG levels than CON (4.96 g/L) and the ANT (5.42 g/L). With the increase in the dosage of rhLYZ, IgA exhibited linear and quadratic changes (P < 0.05), while IgM and IgG showed quadratic variations (P < 0.05). At day 42, there was an increase (P < 0.05) in serum IgG in MEDIUM compared to CON and ANT broilers. However, no significant differences were observed among the treatment groups (LOW, MEDIUM, and HIGH) for IgA and IgM levels at day 42.

Table 4.

Effects of antibiotic and rhLYZ by P. pastoris on serum immunoglobulin of broilers¹

Item	ANT	rhLYZ				SEM	P-value
		CON	LOW	MEDIUM	HIGH		ANOVA2	Linear3	Quadratic4
Day 21
IgA, g/L	0.93ab	0.64c	0.87ab	0.95a	0.79bc	0.03	0.003	0.006	<0.001
IgM, g/L	0.81b	0.78b	1.03a	1.10a	0.92ab	0.03	0.003	0.093	0.001
IgG, g/L	5.42b	4.96b	6.86a	6.83a	5.79ab	0.19	0.004	0.186	<0.001
Day 42
IgA, g/L	0.80	0.96	0.88	0.93	0.90	0.02	0.207	0.374	0.785
IgM, g/L	1.00	1.09	1.08	1.13	1.16	0.02	0.142	0.500	0.669
IgG, g/L	7.24b	7.53b	7.09b	8.78a	7.53b	0.16	0.003	0.931	0.522

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH); n = 5.

²Statistical significance was determined using one-way ANOVA among all groups.

^3,4Linear and quadratic effects of added rhLYZ levels, except for ANT group.

^abcWithin a row, values with different superscripts differ (P < 0.05).

At day 21, concentrations of IL-4 and IL-8 showed no significant differences among CON and the groups receiving different levels of rhLYZ (Table 5). In contrast, levels of IL-6, INF-γ, and TNF-α demonstrated significant variations among the groups. Notably, MEDIUM and HIGH showed lower (P < 0.05) levels of IL-6 and TNF-α compared to ANT group and CON groups. With the increase in the dosage of rhLYZ, IL-6 and TNF-α exhibited linear and quadratic changes (P < 0.05), while IL-8 and INF-γ showed linear variations (P < 0.05). At day 42, concentrations of IL-4, IL-6, IL-8, and TNF-α showed no significant differences. However, levels of INF-γ demonstrated significant variations. Birds fed LOW, MEDIUM, and HIGH exhibited lower (P < 0.05) levels of INF-γ compared to the CON group. INF-γ exhibited both linear and quadratic changes with the increase in the dosage of rhLYZ (P < 0.05).

Table 5.

Effects of antibiotic and rhLYZ by P. pastoris on serum immune factors of broilers¹

Item	ANT	rhLYZ				SEM	P-value
		CON	LOW	MEDIUM	HIGH		ANOVA2	Linear3	Quadratic4
Day 21
IL-4, pg/mL	62.88	62.23	61.04	53.14	62.99	2.75	0.72	0.813	0.897
IL-6, pg/mL	93.23a	82.51a	56.90b	58.65b	50.01b	3.06	<0.001	<0.001	0.005
IL-8, pg/mL	80.09	97.28	94.31	78.70	73.01	3.23	0.079	0.003	0.989
INF-γ, pg/mL	78.96ab	85.52a	73.11ab	63.63b	67.66ab	2.11	0.019	0.002	0.079
TNF-α, pg/mL	105.25a	101.74a	99.94ab	88.26bc	81.90c	2.30	0.002	0.001	0.041
Day 42
IL-4, pg/mL	47.90	48.28	41.19	40.71	48.82	1.42	0.161	0.377	0.121
IL-6, pg/mL	47.89	47.69	49.05	47.39	50.19	0.66	0.669	0.095	0.769
IL-8, pg/mL	60.99	58.03	59.83	58.56	57.69	0.77	0.641	0.982	0.813
INF-γ, pg/mL	61.58a	63.08a	53.51b	53.45b	53.51b	1.00	<0.001	<0.001	<0.001
TNF-α, pg/mL	68.823a	61.188b	67.96ab	65.415ab	66.091ab	0.89	0.033	0.895	0.489

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH); n = 5.

²Statistical significance was determined using one-way ANOVA among all groups.

^3,4Linear and quadratic effects of added rhLYZ levels, except for ANT group.

^abcWithin a row, values with different superscripts differ (P < 0.05).

At day 21, levels of T-SOD showed a slight increase in LOW, MEDIUM, and HIGH groups compared to CON, although the differences were not statistically significant (Table 6). In contrast, broilers fed MEDIUM (2.91 nmol/mL) showed lower (P < 0.01) levels of MDA compared to CON (4.01 nmol/mL). Levels of GSH-PX increase (P < 0.01) in LOW (203.37 U/mL) and MEDIUM (208.75 U/mL) compared to CON (164.84 U/mL) and ANT (173.31 U/mL). No significant difference was observed in T-AOC levels between MEDIUM-fed broilers and CON. With the increase in the dosage of rhLYZ, MDA exhibited quadratic changes (P < 0.05), while GSH-PX and T-AOC showed linear and quadratic variations (P < 0.05). At day 42, birds fed MEDIUM demonstrated higher (P < 0.01) levels of T-SOD (146.10 U/mL) and GSH-PX (251.62 U/mL) compared to birds fed CON. However, there were no significant differences in MDA and T-AOC levels between MEDIUM and CON. MDA and GSH-PX exhibited linear changes with the increase in the dosage of rhLYZ (P < 0.05).

Table 6.

Effects of antibiotic and rhLYZ by P. pastoris on serum antioxidant indexes of broilers¹

Item	ANT	rhLYZ				SEM	P-value
		CON	LOW	MEDIUM	HIGH		ANOVA2	Linear3	Quadratic4
Day 21
T-SOD, U/mL	131.04	128.72	138.00	133.07	131.21	1.15	0.139	0.845	0.181
MDA, nmol/mL	4.01a	3.78ab	3.35bc	2.91c	3.58ab	0.09	0.001	0.581	0.012
GSH-PX, U/mL	173.31bc	164.84c	203.37a	208.75a	189.22ab	4.03	0.002	0.031	0.02
T-AOC, mmol/mL	0.48b	0.64a	0.63a	0.67a	0.49b	0.02	<0.001	0.006	0.011
Day 42
T-SOD, U/mL	144.61a	137.08bc	140.96ab	146.1a	133.91c	1.01	<0.001	0.968	0.179
MDA, nmol/mL	4.42a	3.31b	3.58b	3.34b	3.12b	0.11	<0.001	0.032	0.455
GSH-PX, U/mL	224.59b	201.85c	229.01ab	251.62a	249.84a	4.16	<0.001	<0.001	0.187
T-AOC, mmol/mL	0.48	0.49	0.46	0.46	0.42	0.01	0.080	0.111	0.953

¹Experimental diets were control diet (CON), CON + 50 mg/kg aureomycin (ANT), CON + 20 mg/kg rhLYZ (LOW), CON + 60 mg/kg rhLYZ (MEDIUM), CON + 180 mg/kg rhLYZ (HIGH); n = 5.

²Statistical significance was determined using one-way ANOVA among all groups.

^3,4Linear and quadratic effects of added rhLYZ levels, except for ANT group.

^abcWithin a row, values with different superscripts differ (P < 0.05).

Intestinal microbial diversity

Classification of bacterial communities based on available sequences was conducted using a 97% similarity threshold for operational taxonomic units (OTUs). In the ileum on day 42, a total of 67 OTUs were shared among the five treatment groups, with 31, 12, 94, 13, and 29 OTUs specifically associated with the CON, ANT, LOW, MEDIUM, and HIGH groups, respectively (Figure 1). In the cecum on day 42, 522 OTUs were shared among the five treatment groups, while the CON, ANT, LOW, MEDIUM, and HIGH groups exhibited 19, 17, 5, 15, and 13 unique OTUs, respectively.

Figure 1.

The bacterial operational taxonomic unit community composition of the ileum and cecum in broiler chickens. Venn diagrams of the bacterial operational taxonomic unit community among five treatment groups: control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 20 mg/kg rhLYZ (LOW), con + 60 mg/kg rhLYZ (MEDIUM), con + 180 mg/kg rhLYZ (HIGH).

The α-diversity of microbial communities in each sample was evaluated using Shannon (Figure 2) indices. Regarding the microbial diversity and abundance, MEDIUM and HIGH birds exhibited higher values compared to the CON and the ANT in the ileum at day 21 and the cecum at day 21. At day 42 in the ileum, the Shannon index of MEDIUM was higher than the CON and the ANT, although the difference was not significant.

Figure 2.

The α-diversity of microbial communities was evaluated using Shannon indices. Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 20 mg/kg rhLYZ (LOW), con + 60 mg/kg rhLYZ (MEDIUM), con + 180 mg/kg rhLYZ (HIGH). A) The α-diversity for day 21 ileal bacterial communities. B) The α-diversity for day 42 ileal bacterial communities. C) The α-diversity for day 21 cecal bacterial communities. D) The α-diversity for day 42 cecal bacterial communities.

PCoA was conducted using the Weighted UniFrac distance metric to assess the β-diversity of microbial communities among different groups (Figure 3). Although the addition of rhLYZ to the basal diet improved the growth performance of broilers, no significant clustering of bacterial communities was found due to its supplementation.

Figure 3.

PCoA of bacterial community. Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 20 mg/kg rhLYZ (LOW), con + 60 mg/kg rhLYZ (MEDIUM), con + 180 mg/kg rhLYZ (HIGH). A) The PCoA for day 21 ileal bacterial communities. B) The PCoA for day 42 ileal bacterial communities. C) The PCoA for day 21 cecal bacterial communities. D) The PCoA for day 42 cecal bacterial communities.

At the phylum level on day 21, the dominant phylum in both the ileal and cecal microbiota was Firmicutes (Firmic.), accounting for over 96% in all treatment groups (Figure 4A and B). At the family level on day 21, the predominant families in the ileal microbiota were Lactobacillaceae (Lb.), Clostridiaceae (Clostrid.), Lachnospiraceae (Lachnosp.), Ruminococcaceae (Ruminococc.), and Peptostreptococcaceae (Peptostreptococc.; Figure 5A and B). In the cecal microbiota, the major families were Lb., Lachnosp., norank_o_Clostridia_UCG-014, and Ruminococc.

Figure 4.

Characterization of communities on the phylum level. Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 20 mg/kg rhLYZ (LOW), con + 60 mg/kg rhLYZ (MEDIUM), con + 180 mg/kg rhLYZ (HIGH). A) Day 21 ileal bacterial communities. B) Day 42 ileual bacterial communities. C) Day 21 cecal bacterial communities. D) Day 42 cecal bacterial communities.

Figure 5.

Characterization of communities on the family level. Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 20 mg/kg rhLYZ (LOW), con + 60 mg/kg rhLYZ (MEDIUM), con + 180 mg/kg rhLYZ (HIGH). A) Day 21 ileal bacterial communities. B) Day 42 ileal bacterial communities. C) Day 21 cecal bacterial communities. D) Day 42 cecal bacterial communities.

At the phylum level on day 42, both the ileal and cecal microbiota remained predominantly composed of the phylum Firmic., accounting for over 90% in all treatment groups (Figure 4C and D). At the family level on day 42, the dominant family in the ileal microbiota was primarily Lb. (Figure 5C). In the cecal microbiota, the major families were Lachnosp., Ruminococc., norank_o_Clostridia_UCG-014, Oscillospiraceae, and Lb. (Figure 5D).

To assess significant differences in families among treatments, Welch’s t-test was performed to investigate the effect of rhLYZ on gut microbiota of broiler chickens. Changes in the abundance of families between CON and MEDIUM birds were examined (Figure 6). At day 21, abundance of norank_f_Lachnospiraceae in MEDIUM was higher (P < 0.05) than CON in the ileum. However, no significant differences in microbial communities were observed between MEDIUM and CON in the cecum at day 21. At day 42 in the ileum, abundance of Escherichia-Shigella (E.-Sh.) in birds fed MEDIUM was lower (P < 0.05) than CON. In the cecum, abundance of Monoglobus and Family_XIII_AD3011_group was lower (P < 0.05) in MEDIUM compared to CON. Conversely, abundance of Eubacterium_nodatum_group in MEDIUM broilers was higher (P < 0.05) than CON.

Figure 6.

Bacterial genera with significantly different abundances between CON and MEDIUM. Experimental diets were control (CON), con + 60 mg/kg rhLYZ (MEDIUM). A) Day 21 ileal bacterial communities. B) Day 42 ileal bacterial communities. C) Day 42 cecal bacterial communities.

In this study, a LEfSe was employed with a LDA score threshold of 2.0 to assess the differential impact of rhLYZ on the composition of intestinal microbiota in broiler chickens across CON, ANT, and MEDIUM treatments. On day 21 in the ileum (Figure 7A and B), CON exhibited a higher relative abundance of the phylum Firmic., while the ANT showed a significant increase in abundance of the class Actinobacteriota (Actinobact.) and the family Streptomycetaceae (Streptomycet.). Additionally, MEDIUM led to enrichment of Lachnosp., Anaerofustaceae, Staphylococcaceae (Staphylococc.), and the genus Ruminococcus (P < 0.05). On day 21 in the cecum (Figure 8A and B), ANT demonstrated higher relative abundances of the phylum Cyanobacteria, the class Vampirivibrionia, the order Gastranaerophilales, and the genus Tyzzerella (P < 0.05), while MEDIUM showed a higher relative abundance of the genus unclassified_o_Erysipelotrichales (P < 0.05). On day 42 in the ileum (Figure 7C and D), CON birds exhibited enrichments of the phylum, Proteobacteria (Proteobact.), the class Gammaproteobacteria (Gammaproteobact.), and the order Enterobacterales (Enterobact.; P < 0.05). Additionally, MEDIUM broilers displayed higher relative abundances of the family, Peptostreptococc., as well as the genera, Streptococcus (Strep.), Roseburia, Bacillus (Bacill.), Leucobacter (Leucobact.), and Paracoccus (Paracocc; P < 0.05). On day 42 in the cecum (Figure 8C and D), ANT broilers showed an enrichment of the genus, Eubacterium_nodatum_group (P < 0.05), while MEDIUM broilers demonstrated enrichments of the class norank_c__Clostridia and the family Hungateiclostridiaceae (P < 0.05).

Figure 7.

Different structures of ileal bacterial communities from phylum to genus level among three groups (CON, ANT and MEDIUM). Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 60 mg/kg rhLYZ (MEDIUM). A) Taxonomic representation of day 21 distinct bacteria with statistically significant higher abundances. B) Histogram of day 21 LDA plots indicating scores for differentially abundant genera. C) Taxonomic representation of day 42 distinct bacteria with statistically significant higher abundances. D) Histogram of day 42 LDA plots indicating scores for differentially abundant genera.

Figure 8.

Different structures of cecal bacterial communities from phylum to genus level among three groups (CON, ANT and MEDIUM). Experimental diets were control diet (CON), con + 50 mg/kg aureomycin (ANT), con + 60 mg/kg rhLYZ (MEDIUM). A) Taxonomic representation of day 21 distinct bacteria with statistically significant higher abundances. B) Histogram of day 21 LDA plots indicating scores for differentially abundant genera. C) Taxonomic representation of day 42 distinct bacteria with statistically significant higher abundances. D) Histogram of day 42 LDA plots indicating scores for differentially abundant genera.

Discussion

Global concerns regarding the reduction or replacement of antibiotic dietary supplementation in poultry have spurred expanded research efforts to explore new alternatives. Feeding nigella seed oil as an alternative to avilamycin antibiotic resulted in improved growth performance and modified the fatty acid profile in muscles (Saleh, 2014). Adding L. acidophilus (0.1 g/kg) to the broiler diet positively impacted broilers’ growth, feed intake, feed efficiency, digestibility, and plasma lipid levels (Saleh et al., 2020). Supplementing broiler diets with BG or lincomycin enhanced their growth performance and immune response (Saleh et al., 2024). Results of this study indicate that the addition of rhLYZ to the diet of broilers has a significant impact on their growth and feed efficiency. Specifically, the MEDIUM level reduced ADFI from day 0 to 21 but increased ADG from day 22 to 42. Additionally, FCR improved in LOW and MEDIUM-fed broilers compared to CON and ANT-fed broilers, both in the early paction and the entire experiment. The improvement in broiler growth performance and nutrient utilization observed in the present study is consistent with previous studies. Abdel-Latif et al. (2017) demonstrated that the addition of 90 g/t lysozyme 10% (Nan Chang Lifeng Industry and Trading Co., Ltd., Jiangxi, China) to broiler feed without antibiotics significantly improved BW gain and FCRs. The improvement in growth performance observed in the present experiment highlights the potential role of rhLYZ in bolstering avian productivity. Importantly rhLYZ is not an antibiotic and may therefore provide a more sustainable alternative to traditional methods of improving broiler performance.

The mechanisms underlying the observed effects of rhLYZ on broiler growth and feed efficiency are not entirely clear. One possibility is that rhLYZ directly affects the gut microbiota of broilers, which can in turn influence nutrient absorption and utilization. Alternatively, rhLYZ may have a more indirect effect by enhancing the immune function of broilers, which could ultimately improve their overall health and performance. Overall, the application potential of rhLYZ as a feed additive in poultry production is noteworthy. As the pursuit of alternatives to antibiotics gains momentum, human lysozyme emerges as a promising candidate. The observed enhancement in growth performance attributed to human lysozyme aligns with the industry’s pursuit of sustainable practices aimed at improving productivity while addressing concerns regarding antibiotic resistance.

Lysozyme plays a significant role in defense against bacterial pathogens and in regulating interactions between gut microbiota and host immune systems (Xiong et al., 2019). As the largest immune organ in the body, the gut is constantly exposed to attacks from exogenous and commensal bacteria and antigens (Mowat and Agace, 2014). Immunoglobulins and cytokines serve as crucial indicators of immune function in the gut. In this study, under the same vaccination conditions, where broiler chickens were immunized with Newcastle disease vaccine and inactivated infectious bursal disease vaccine, we observed relatively higher levels of IgA, IgM, and IgG in the MEDIUM compared to CON. This preliminary finding suggests that the addition of rhLYZ at an appropriate dosage may have a beneficial effect on the immune status of broiler chickens.

Our study findings indicate that rhLYZ supplementation has a notable impact on serum immune factors of broilers, particularly in reducing concentrations of IL-6, IL-8, INF-γ, and TNF-α. These results suggest that the P. pastoris expression of human lysozyme may contribute to immune regulation and modulation of inflammation in broilers. With the expansion of poultry farming, birds face various threats, including heat stress, fungal toxins, and pathogens. These factors can disrupt the redox status of the organism (Mishra and Jha, 2019), leading to reduced growth performance and compromised economic efficiency. To address this issue, nutritional regulation of the redox status has become a prominent topic in poultry nutrition research (Surai and Kochish, 2019).

In our study, rhLYZ supplementation positively influenced serum antioxidant indexes in broilers, as evidenced by increased T-SOD and GSH-PX levels, along with decreased MDA concentrations. These results underscore the potential of rhLYZ to enhance the antioxidant capacity of broilers. Further research is needed to elucidate the underlying mechanisms and explore practical applications of rhLYZ in promoting a beneficial antioxidant status in broilers.

The apparent lack of a beneficial effect, and in some instances, the inferior performance of the antibiotic group compared to the control group may be attributed to several factors below. Antibiotics, despite their historical use in promoting growth and health in poultry, can exhibit varying efficacy depending on factors such as dosage, administration duration, and the specific antibiotic agent used (Polycarpo et al., 2017; Oso et al., 2019). Additionally, the complex interplay between the gut microbiota, immune system, and dietary interventions could influence the observed outcomes. That might be the reason why the supplementation of antibiotic destroyed the normal morphology and structural integrity of the intestine in this study (Liu et al., 2022a).

The intimate connection between intestinal microbiota composition and host health is well-established (Ley et al., 2006). Observed alterations in microbial diversity and relative abundance in response to human lysozyme supplementation underline its potential to bolster growth performance and the overall well-being of broiler chickens. (Huang et al., 2018) conducted a study examining the microbial communities present in both the foregut and hindgut of chicken, and found that Firmic., Proteobact., Bacteroidetes, and Actinobacteria were the dominant phyla. Additionally, they observed significant temporal changes in these populations over time. Our findings align with theirs, as we also identified the dominance of the Firmic. phylum in both the ileum and cecum among the different treatment groups at day 21. In the ileum, Lb., Clostrid., Lachnosp., Ruminococc., and Peptostreptococc were prominent, while in the cecum, Lb., Lachnosp., norank_o_Clostridia_UCG-014, and Ruminococc. were more prevalent. Recombinant rhLYZ’s inherent ability to modulate both detrimental and beneficial microbial populations could foster a favorable environment for nutrient absorption, immune function, and metabolic activities. By examining these patterns over time, we can better understand the factors that influence microbial composition and develop strategies to promote gut health.

At day 21, MEDIUM-fed birds exhibited a greater abundance of norank_f_Lachnospiraceae in the ileum compared to CON-fed birds. At day 42, MEDIUM broilers showed a lower abundance of E.-Sh. in the ileum and lower abundances of Monoglobus and Family_XIII_AD3011_group in the cecum compared to CON broilers. Additionally, abundance of Eubacterium_nodatum_group was higher in MEDIUM compared to CON broilers in the cecum. These results are consistent with some previous studies. For example, a meta-analysis of changes in gut microbiota showed that abundance of Lachnosp. family is relatively low in patients with inflammatory bowel disease (IBD), while it is high in healthy individuals (Sokol et al., 2008). Other researchers found that Escherichia coli significantly increased in IBD patients’ intestines and was related to severity of the disease (Baumgart et al., 2007). A marked increase in abundance of the E.-Sh. genus is a characteristic of dysbiosis in the intestinal flora and has emerged as a distinctive feature among patients with IgA nephropathy (Zhao et al., 2022).

In recent years, a limited number of studies have indicated an association of Family_XIII_AD3011_group with inflammation. Zhong et al. (2020) identified Family_XIII_AD3011_group, Prevotella, and Ruminococcus as potential microbial markers for diagnosing bovine mastitis. Xiao et al. (2021) demonstrated that the abundance of Family_XIII_AD3011_group and Fusobacterium, both associated with inflammation, was elevated in highly fatigued participants, suggesting that inflammation may be a key driving factor in fatigue-related cancer. This evidence suggests that an increase in abundance of Family_XIII_AD3011_group may trigger inflammation. Decreased abundance of E.-Sh., Monoglobus, and Family_XIII_AD3011_group in the ileum of MEDIUM birds can potentially be attributed to the antimicrobial properties of rhLYZ. The microbiota influences the immune status of the host, and inflammation associated with dysbiosis enhances insulin resistance (Cavallari and Schertzer, 2017). Our previous results showed that rhLYZ supplementation reduced serum levels of immune factors, IL-6, IL-8, INF-γ, and TNF-α in broilers. The primary function of lysozyme is to disrupt the bacterial cell wall, resulting in the elimination of bacteria. The addition of human lysozyme to broiler chicken feed effectively regulates the intestinal microbiota, reducing the population of harmful microorganisms such as Clostridia, thereby maintaining intestinal microecological balance and enhancing the digestive and absorptive capabilities of broiler chickens, which ultimately promotes growth. However, excessively high dosages may further eliminate beneficial bacteria, disrupting the intestinal microbial balance and compromising intestinal health, ultimately exerting a negative impact on growth performance. In the present study, based on the growth performance exhibited by the three groups with different supplementation dosages, we can preliminarily infer that the MEDIUM (60 mg/kg) dosage of rhLYZ is optimal. This dosage range effectively suppresses the growth of harmful bacteria while maintaining the balance of intestinal flora, and it also demonstrates favorable serum biochemical indicators and antioxidant capacity.

The LDA-LEfSe plot provides valuable insights into the relationship between taxon abundance and the observed differential effects. Notably, a higher LDA score corresponds to a stronger effect. The present study investigated the differential impact of rhLYZ on the composition of the intestinal microbiota in broiler chickens across the CON, ANT, and MEDIUM. On day 21, antibiotic-fed broilers showed a significant increase in abundance of the class Actinobact. and the family Streptomycet. compared with CON broilers. Furthermore, the MEDIUM led to enrichment of Lachnosp., Anaerofustaceae, Staphylococc., and the genus Ruminococcus. Lachnosp., a bacterial genus frequently found in the gut of healthy individuals, is considered a potentially beneficial microorganism due to its significant involvement in carbohydrate metabolism. Fermentation conducted by Lachnosp. contributes to the production of acetate and butyrate, which serve as essential energy sources for the host organism. Under anaerobic conditions, specific strains of Anaerofustaceae exhibit the ability to metabolize inositol, resulting in the production of short-chain fatty acids (SCFAs) such as propionate and butyrate. Notably, certain strains within Anaerofustaceae possess capacity to convert dietary inositol into propionate and butyrate salts, implying a potential role for inositol in regulating intestinal homeostasis and metabolic pathways within the host organism (Bui et al., 2021). These findings also highlight the potential of Anaerofustaceae as a probiotic for promoting and maintaining the overall health of the host.

Moving to day 42, CON birds exhibited significant enrichments of the phylum, Proteobact., the class, Gammaproteobact., and the order, Enterobact., compared to the other groups. Additionally, MEDIUM broilers displayed higher relative abundances of the family, Peptostreptococc., as well as the genera, Strep., Roseburia., Bacill., Leucobact., and Paracocc. These findings suggest that feeding moderate levels of rhLYZ may have a positive effect on gut microbiota composition of broilers. In the cecum on day 42, the antibiotic group showed a significant enrichment of the genus, Eubacterium_nodatum_group, while MEDIUM broilers demonstrated significant enrichments of the class, norank_c__Clostridia, and the family, Hungateiclostridiaceae. These findings are consistent with a previous study that reported changes in the gut microbiota of chickens due to antibiotic treatment (Stanley et al., 2014) and the potential prebiotic effects of lysozyme on gut microbiota composition (Savijoki et al., 2006). These intestinal microorganisms contribute to the metabolism of less digestible carbohydrates into different SCFA molecules, such as acetate, butyrate, and propionate. SCFAs possess favorable chemical properties, and their impact on health has been extensively documented (Mansuy-Aubert and Ravussin, 2023). These compounds have the capacity to regulate the gut environment and various metabolic pathways (de Vos et al., 2022), potentially influencing the efficiency of nutrient utilization and consequently improving the growth rate of broiler chickens. The observed improvement in growth performance attributed to human lysozyme could be attributed, at least in part, to beneficial modifications in the intestinal microbiota structure. Therefore, human lysozyme may potentially enhance broiler growth rate by improving microbiota structure, optimizing digestion, and enhancing nutrient absorption.

Conclusion

The present study evaluated the effects of dietary supplementation with rhLYZ from P. pastoris on growth performance, antioxidative characteristics, and intestinal health in broiler chickens. Recombinant rhLYZ supplementation at 60 mg/kg to the diet improved broiler growth performance, nutrient utilization, and serum biochemical indicators. Moreover, recombinant rhLYZ supplementation at 60 mg/kg to the diet altered the intestinal microbial community. The practical implications of these findings extend to the realm of sustainable poultry production. Integrating recombinant rhLYZ as a feed additive holds the potential to enhance broiler health and performance while circumventing concerns associated with antibiotic resistance.

Glossary

Abbreviations

ADFI

average daily feed intake

ADG

average daily gain

BW

body weight

FCR

feed conversion ratio

GSH-Px

glutathione peroxidase;

Ig

immunoglobulin

IL

interleukin

LDA

linear discriminant analysis

LEfSe

linear discriminant analysis effect size

MDA

malondialdehyde

PCoA

principal coordinate analysis; rhLYZ, recombinant human lysozyme

T-AOC

total antioxidant capacity; TNF-α, tumor necrosis factor-α

T-SOD

total superoxide dismutase

Conflict of interest statement

The authors state that they do not have any known competing financial interests or personal ties that could to have influenced the work disclosed in this study.