Effects of dietary Acremonium terricola culture on production performance, serum biochemical parameters, egg quality and yolk amino acid contents of Beijing You-chicken

Abstract

The effects of Acremonium terricola culture (ATC) on production performance, serum biochemical parameters, egg quality and amino acid contents in the yolk of eggs of Beijing You-chicken were conducted in the current study. A total of 216 Beijing You-chickens (330 days old) were randomly divided into 2 groups. The control group (CON) was fed a corn-soybean-based diet, and the experimental group was fed a basal diet supplemented with 0.20% ATC. The pretest period was 7 d, and the experiment period was 8 weeks. The production performance, serum biochemical parameters, egg quality, and the concentrations of amino acids in the yolk of eggs were measured at the 4 weeks (FW, the first stage) and the 8 weeks (EW, the second stage) of the experiment, respectively. Compared with the CON group, there were no significant differences (P > 0.05) in the production performance of the experimental group at the end of four- and eight-week periods of study. The concentration of serum LH, FSH and E2 increased significantly for the ATC group, at both time periods when compared to CON group, while the triglyceride (TG) content was only increased significantly (P < 0.05) in the first stage. The average egg weight, albumen height, and Haugh unit representing egg quality of Beijing You-chickens in the experimental group were increased significantly (P < 0.05) compared with the CON group at both time periods, while the egg shape index and yolk weight were only increased significantly (P < 0.05) in the second stage. The protein content in the yolk was increased significantly at both time periods (P < 0.05). Levels of lecithin and Vitamin A in yolks of the ATC supplemented group increased significantly (P < 0.05) compared to the CON group, at both FW and EW, respectively. The contents of aspartic acid, threonine, methionine, leucine, and arginine were increased significantly in the first stage. In addition, the contents of threonine, glutamine, and valine were increased significantly in the second stage (P < 0.05). Our results suggest that dietary supplementation with 0.20% ATC improves serum biochemical parameters and egg quality in Beijing You-chickens. Future studies should focus on optimizing ATC dosage and exploring its underlying mechanisms for enhanced poultry production.

Introduction

Antibiotics have been widely used in livestock and poultry farming since the 1950s because they can treat bacterial diseases, promote the growth of livestock and poultry, improve carcass quality and maintain the health of livestock and poultry [1]. However, antibiotic resistant of animal-derived bacteria and antibiotic residues in livestock and poultry products caused by long-term use of antibiotics may pose a serious threat to food security [2, 3]. With increasing concerns over the safety of animal-derived food products and the environmental impact of antibiotics, there is growing demand for alternatives to antibiotics in livestock and poultry farming. The global focus has shifted towards developing safe, sustainable, and effective substitutes for antibiotics to meet modern regulatory standards and address public concerns regarding antibiotic resistance and food safety [4]. The development of safe and effective antibiotic substitutes has become a hot spot in the current global research and also an important topic to explore in animal nutrition [2]. The abusive use of antibiotics in animal breeding is becoming increasingly prominent, and the voice of no-resistance animal breeding is rising daily. High feeding stocking density increases disease risk, as it may lead to higher stress levels and reduced immunity in poultry. Therefore, it is important to find antibiotic substitutes that can improve the growth performance and immune function of animals [2,3,4,5].

China’s poultry breeding industry developed rapidly in the past 30 years. The production of broiler chickens in China has been ranked among the top three in the world for many years [6, 7]. In China, chicken has become the second-largest livestock product [8]. Beijing You-chicken, local species domesticated in Beijing, China, is a kind of meat and egg type chicken, which is famous for its delicious meat and good egg quality. Its meat quality (e.g., breast muscle intramural fat, eggs containing fatty acids, and other nutrients) is high [9]. However, the average sexual maturity of Beijing You-chicken occurs approximately 16 weeks of age, and they typically begin laying at this time after hatching [10]. After the peak laying period, it is common for the laying rate of hens, including Beijing You-chickens, to gradually decline, which is a natural part of the reproductive cycle [11].

China is the birthplace of traditional Chinese medicine. Chinese herbal medicine resources are very rich. Natural Chinese medicine feed additives have been widely studied and applied since the 1970s because they have no resistance, residual effects, and side effects. They can also promote animal growth, improve animal product quality, and enhance animal immunity and disease resistance [12, 13]. Chinese traditional medicines, such as Cordyceps militaris and Cordyceps guni, are rare Chinese herbal medicines with strong immune regulation functions [14]. Due to limited resources and high price, their popularization and application in livestock and poultry breeding are restricted. Acremonium terricola culture (ATC) is the first Cordyceps sinensis feed additive listed in China [15]. It is made from digarbospora isolated from C. sinensis through artificial fermentation. It has functional components similar to C. sinensis, such as cordycepin, cordyceps acid, cordyceps polysaccharide, sterol, and amino acids, which can be used as a substitute for natural C. sinensis [16]. ATC is a kind of inactivated fungal feed additive with high nutrition and good palatability, which can improve the growth performance, immune function, antioxidant capacity and intestinal morphology of ducks, geese, laying hens [17,18,19,20,21]. ATC contains C. sinensis and rich bioactive substances which are confirmed could regulate animal intestinal microbial flora, improve the body’s digestive function and promote animals’ growth development and has no residue and drug resistance [15, 22].

The application of ATC as a feed additive has been studied for its potential benefits in improving animal growth performance, immune function, and has no residue and drug resistance problem. However, while its use has been increasing in livestock such as pigs and ruminants, there is limited research on its application in poultry, particularly in Beijing You-chickens. This study aims to investigate the effects of dietary supplementation with ATC on production performance, serum biochemical parameters, egg quality, and yolk amino acid contents in Beijing You-chickens after their laying period. By examining these factors, the study to observe as a viable alternative to antibiotics and also for egg production and poultry health.

Materials and methods

Ethics approval and consent to participate

All animal-based experimental procedures conducted in the current study were approved by China Agricultural University and in accordance with the Guidelines of the Animal Ethical Committee (permit no. CAU20190425-2).

Dietary treatments and feeding

A total of 216 Beijing You-chickens (330 days old), specifically raised for egg production, were housed in an experimental poultry facility at China Agricultural University, Beijing, China. The chickens were procured from the College of Veterinary Medicine at China Agricultural University. All chickens were reared in 2-tier cages, with 6 chickens per cage. Each cage measured 50 cm × 40 cm × 40 cm, providing sufficient space for the chickens to move comfortably and exhibit natural behaviors. The chickens were exposed to artificial light for 14 to 16 h per day throughout the experimental period. They were kept in an air-conditioned house where the temperature was maintained at 22–25 °C and the humidity at 50–60%. Feed and clean drinking water were provided ad libitum. The chickens were randomly assigned to two treatments, each with six replicates (18 chickens per replicate). Each cage housed 6 chickens and three cages (18 chickens) represented one experimental replicate. The control group (CON) was fed a corn-soybean basal diet without antibiotics or growth promoters, while the experimental group received a corn-soybean basal diet supplemented with 0.20% ATC, following the manufacturer’s recommended dosage for poultry [20]. The pre-trial period lasted for 7 days, followed by a 56-day formal experimental period. The composition and nutrient levels of the basal diets were calculated to meet or exceed the requirements specified in the standard “Technical Code of Practice for Feeding and Management of Beijing-You Chicken (DB11/T1378-2023)” [23] for the first stage (4 weeks) and second stage (8 weeks) (Table 1).

The ATC used in this study was purchased from Hefei Micro Biological Engineering Co., Ltd., China.

Table 1 Composition and nutrient levels of the basal diets (air-dry basis)

Production performance

The egg production was recorded daily, and feed consumption was recorded weekly during the whole experimental period. Laying rate, average daily egg weight (ADEW), average daily feed intake (ADFI), and feed and egg ratio (F/E) were calculated.

Serum biochemical parameters

Blood samples from twelve chickens per group were collected from the jugular vein on 28 d and 56 d and then centrifuged at 4,000×g for 5 min at 4 °C. The serum was separated and stored at -20 °C for subsequent analysis. The contents of serum total cholesterol (TC, Art. No. A111-1-1), triglyceride (TG, Art. No. A110-2-1), high-density lipoprotein cholesterol (HDL, Art. No. A112-1-1), low-density lipoprotein cholesterol (LDL, Art. No. A113-1-1), non-esterified fatty acid (NEFA, Art. No. A042-2-1), luteinizing hormone (LH, Art. No. H206-1-2), follicle stimulating hormone (FSH, Art. No. H101-1-2), and estradiol (E2, Art. No. H102-1) were determined using commercial kits (Nanjing Jiancheng Bioengineering Co., Ltd., Jiangsu, China) according to the manufacturer’s guidelines.

Egg quality

The egg weight was measured twice a week with the help of electronic balance. A total of sixty eggs without shell defects or cracks were randomly selected from each group for assessment of egg quality at 4 weeks and 8 weeks (at 28 d and 56 d, respectively). The egg albumen height and Haugh units were determined by a digital egg tester (Total Security Services, Bristol, England). Eggshell strength was determined and eggshell thickness was determined by the Hung-Ta (HT-8116) table-type compression strength tester. The egg shape index was assessed according to the following formula: vertical diameter/transect diameter. The egg yolks separated from the albumen were weighed by an electronic scale, and the weights were recorded. Yolk color was measured using a colorimeter (Minolta Chroma Meter, Tokyo, Japan).

Nutritional ingredients and amino acid contents in the yolk

The quantitation of nutritional ingredients such as protein, lecithin, cholesterol, vitamin A, vitamin D and vitamin E, and amino acids in egg yolk samples were analyzed by High-performance liquid chromatography (HPLC) (Agilent-1260; Agilent Technologies Inc., Santa Clara, CA, USA) [24]. Briefly, egg yolk extracts were prepared using ultrasound-assisted extraction. A total of 1.0 g of lyophilized egg yolk was subjected to extraction with 6 mL of various solvents: a 50/50 mixture of acetone and isopropanol, a 50/50 mixture of ethyl acetate and methanol, followed by ethyl acetate and methanol separately. Each extraction was performed for 30 min at a temperature range of 35–40 °C. After extraction, the samples were centrifuged at 1500 rpm for 10 min, and the resulting supernatants were collected. The extracts were then evaporated to dryness, and the yield percentage for each extract was determined. The absolute concentration of amino acids in yolk was calculated from the peak ratio between sample and standard.

Statistical analysis

Data were analyzed using SPSS statistical software (SPSS 22.0 Inc., Chicago, IL, USA). The differences among groups were analyzed by Student’s t-test of variance. P < 0.05 was considered as significant.

Results

Production performance

The effects of ATC on the production performance of Beijing You-chickens are as given in Table 2. Compared with the CON group, there were no significant differences (P > 0.05) in the laying rate, ADEW, ADFI, and F/E of the experimental group at both time periods.

Table 2 Effects of dietary Acremonium terricola culture treatment on production performance of Beijing You-chickens

Serum biochemical parameters

The effects of ATC on serum biochemical parameters of Beijing You-chicken are shown in Table 3. The concentration of serum LH, FSH and E2 increased significantly for the ATC group, at both time periods when compared to CON group. However, the triglyceride (TG) content was increased significantly (P < 0.05) only in the first stage.

Table 3 Effects of dietary Acremonium terricola culture treatment on serum biochemical parameters of Beijing You-chickens (n = 12)

Egg quality

The effects of ATC on the egg quality of Beijing You-chickens are shown in Table 4. The average egg weight, albumen height, yolk color, and Haugh unit of eggs of Beijing You-chickens in the experimental group were increased significantly (P < 0.05) compared with the CON group in the first stage, while average egg weight, egg shape index, albumen height, yolk weight, and Haugh unit of eggs of Beijing You-chickens were increased significantly (P < 0.05) in the second stage.

Table 4 Effects of dietary Acremonium terricola culture treatment on egg quality of Beijing You-chickens (n = 60)

Nutritional ingredients of yolk

The effects of ATC on nutritional ingredient contents in the yolk of eggs are shown in Table 5. The contents of protein and lecithin in yolk were increased significantly (P < 0.05) compared with the CON group in the first stage. The contents of lecithin and vitamin A were increased significantly (P < 0.05) compared with the CON group in the second stage, and the cholesterol content was decreased significantly (P < 0.05) compared with the CON group only in the second stage.

Table 5 Effects of dietary Acremonium terricola culture treatment on nutritional ingredient in yolk of eggs (n = 60)

Amino acid contents in the yolk

The effects of ATC on amino acid contents in the yolk of eggs are shown in Table 6. The contents of aspartic acid, threonine, methionine, leucine, and arginine increased significantly (P < 0.05) in the first stage. In addition, the contents of threonine, glutamine, valine and total amino acids were increased significantly (P < 0.05) in the second stage.

Table 6 Effects of dietary Acremonium terricola culture treatment on amino acid contents in the yolk of eggs (n = 60)

Discussion

Production performance is a key indicator for assessing animal health. Dietary ATC supplementation in dairy cows, sheep, piglets, ducks, geese, laying hens and broilers can improve production performance [15,16,17,18,19,20,21,22]. Dietary ATC supplementation can increase the laying rate and egg weight of laying hens, and decrease the egg breaking rate and F/E, but the changes in these indexes did not reach a significant level [25]. Sun found that the laying rate and egg weight of laying ducks were increased after supplementing C. sinensis feed additive in the diet for 28 d [26]. Feed intake and F/E had a decreasing trend, but the differences were not significant. In this study, we found that although dietary supplementation with 0.20% ATC improved the production performance of Beijing You-chickens, the differences were not statistically significant. This result is consistent with findings from previous studies on poultry, where the effects of ATC on performance were less pronounced during the later stages of laying. The selected Beijing You-chickens were after the time of laying stage, and the effect of ATC on the performance might be better in the early laying stage. Under normal feeding conditions in this study, the addition of ATC to the diet did not significantly affect the performance of Beijing You-chicken after the best time of laying stage.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are the main hormones affecting laying rate [27]. The main physiological function of FSH is to stimulate the growth and development of follicles, while the main physiological function of LH is to play a synergistic role with FSH to promote the maturation of follicles and the hyperplasia of granulosa membrane, so that the granulosa layer can produce estrogen. When the ratio of LH to FSH reaches a certain level, ovulation can be induced, which plays a major regulatory role in ovulation [28]. Progesterone plays a crucial role in ovulation laying hens, while estrogen is involved in the regulation of follicular development. The observed increase in serum levels of LH, FSH, and E2 suggests that ATC supplementation may enhance hormone regulation, potentially improving ovulation and egg production.

The evaluation indexes of egg quality are divided into external indexes (egg shape index, eggshell strength, and eggshell thickness, etc.) and internal indexes (yolk weight, yolk color, albumen height, and Haugh unit, etc.) [29]. The strength and thickness of the eggshell are the key parameters to measure the quality of the eggshell. The good quality of eggshells can reduce the rate of deformity, the occurrence of soft and deformed eggs, and the damage of eggs during transportation. Eggshell strength refers to the anti-damage strength of the eggshell, indicating the pressure per square centimeter. Eggshell thickness and egg shape index are closely related to each other. Shell thickness ranges from 0.3 to 0.4 mm, and even small changes can have a significant impact on the rate of shell breakage. The Egg shape index ranges from 1.32 to 1.39, which is a classification index of chicken species and a key factor affecting the hatching rate [30, 31]. Yolk color is determined by the type and number of fat-soluble pigments in the diet. Oxygen-containing carotenoid (lutein) plays a key role in the deposition of yolk pigments, which cannot be synthesized by poultry itself and must be obtained from the diet. Egg white height and Haugh unit are the key parameters to measure protein quality. Within a certain range, the higher the value is, the greater the viscosity and the higher the quality of the egg. In the later stage of egg-laying, eggshell strength and thickness gradually decrease with the increase in age and egg weight resulting in a decrease in the overall quality of the eggshell [32, 33]. Our results align with previous studies, indicating that ATC supplementation can improve protein quality and egg freshness without adversely affecting eggshell strength and thickness. This suggests that ATC supplementation has a positive impact on egg quality, which is particularly important for the commercial production of eggs. The yolk color of the experimental group was increased significantly, indicating that the supplementation of 0.20%ATC in the diet could promote the absorption of yolk pigment in the intestinal tract. ATC (0.20%) has a variety of antioxidant components, which can enhance the activity of antioxidant enzymes in the body, prevent the oxidation of pigment, accelerate the deposition of pigment, and deepen the color of egg yolk. Wang et al. found that the discarded medium of C. militaris could significantly increase the protein height of laying hens [25]. The diet supplemented with 0.20% ATC significantly increased protein height and Haugh unit of Beijing You-chicken at the late laying stage, which is consistent with the above results [34]. The Haugh unit of the experimental group was higher than 78 in both stages, indicating that feeding a 0.20% ATC diet could significantly improve the freshness of eggs and the quality of Beijing You-chicken eggs at the later stage of laying.

Results of our study indicate that ATC supplementation significantly increased essential amino acids such as threonine, leucine, and arginine, improving the nutritional profile of eggs. These findings suggest that ATC can improve the nutritional quality of eggs, making them more desirable for consumption. Eggs have high nutritional value and are rich in high-quality protein, fat, vitamins and minerals, and other nutrients. Eggs also become one of the essential animal foods in people’s daily life. Amino acids are the basic components of protein, and their content often reflects the quality of eggs [35]. Egg and human body amino acid composition and proportion are similar and very conducive to digestion and absorption of the body. The utilization rate can reach more than 94%. It is an ideal protein source in natural food [36]. Lecithin, is recognized as the third nutrient alongside protein vitamins, and its content accounts for about 10% of egg yolk, which is much higher than other natural food sources [37]. Compared with other natural foods, egg cholesterol content is slightly higher. Also, frequent intake of eggs causes hypertension, hyperlipidemia, atherosclerosis, and coronary heart disease, and the consumption level of eggs is limited [38]. Lecithin, a blood vessel scavenger, plays an important role in lipid metabolism. It can neutralize excessive cholesterol intake and restore the blood cholesterol level to normal [39]. In this study, in the first stage, the lecithin content of the experimental group was increased significantly. The levels of yolk cholesterol decreased, but the difference was not significant. In the second stage, cholesterol levels dropped significantly, which is consistent with a previous study in which waste C. militaris medium significantly reduced cholesterol content in eggs, indicating that the supplementation of 0.20% ATC in the diet could make eggs meet the needs of consumers.

Amino acids are the basic components of protein and the basic substances needed for life activities. Essential amino acids (EAA) are an important index to evaluate egg quality and are closely related to the body’s immune function. Free amino acids include alanine, glycine, glutamate, arginine, aspartic acid, etc [40]. When heated with reducing sugars/carbonyl compounds, it triggers the Maillard reaction and produces extremely important volatile flavor substances. The latter has a positive effect on egg flavor. It is also known as flavor amino acids [41]. Threonine aspartic acid and glutamic acid are the flavoring substances responsible for the sweet and umami taste of eggs [42]. In this study, a total of 15 kinds of amino acids were detected in Beijing You-chicken eggs, and the contents of essential amino acids, such as threonine, valine, and leucine in eggs of the experimental group were significantly increased. The levels of the free amino acids such as aspartate, threonine, glutamate, and arginine were also significantly increased. Egg protein content was increased significantly in both phases. The results indicated that dietary 0.20% ATC supplementation can promote the digestion and absorption capacity of dietary amino acids of Beijing You-chickens, significantly improving the contents of amino acids and protein in eggs and improving the nutritional value and deliciousness of eggs.

However, further studies with longer experimental durations and varying dosages of ATC may help to clarify its long-term effects and optimal dosages for improving both production performance and egg quality.

Conclusions

In conclusion, our study suggests that dietary supplementation with 0.20% ATC can significantly enhance serum biochemical parameters, improve egg quality, and increase the nutritional content and amino acid composition in the yolk of eggss during the late laying period. These findings indicate that ATC could be a promising dietary supplement to improve poultry health and product quality, offering an alternative to conventional antibiotics in poultry farming. Future research should focus on optimizing the dosage of ATC and exploring its long-term effects on poultry performance and health, as well as investigating the underlying mechanisms through which ATC mediates these improvements.