ABSTRACT

Background: In modern industrial poultry farming practices, forced molting has not lost its importance and is widely employed in commercial poultry production. However, to effectively apply this technology, a more detailed understanding of the physiological processes that occur during molting is necessary. Moreover, new methods are needed to help the bird recover from molting, minimize stress, promote recovery, and ultimately increase productivity in the next cycle.

Aim: The objective of this study was to investigate the antioxidant status of laying hens during the molting period and to assess the effectiveness of combining probiotics with trace elements in alleviating postmolting syndrome in birds.

Methods: The study was conducted on a total of 250 Hisex-Brown laying hens at the age of 469 days, which were randomly divided into groups: a premolting group that received the basic diet without induced molting; a molting group that underwent a 10-day fasting period; a postmolting group that underwent molting with gradual feeding restoration; a control production group (n=50) that underwent molting with gradual feeding restoration; after the end of molting, the laying hens received the basic diet; an experimental production group that underwent molting with gradual feeding restoration and then received acidophilus, bifidobacteria, and chelated trace elements during 30 days. Blood samples were collected from the wing veins of laying hens in each group to assess the antioxidant status of the birds using colorimetric assays.

Results: Total superoxide dismutase activity increased by 33% (p=0.04), whereas catalase activity increased by 15% (p=0.02), concurrently with a 13% increase in malonic dialdehyde content, in laying hens undergoing forced molting (fasting period), in blood serum, compared with non-molted birds. By the end of the molting period, the activities of these enzymes had decreased, and the malonic dialdehyde content was 31% higher than the control values (p=0.002). Following molting, treatment with a probiotic mixture (Lactobacillus acidophilum and В. adolescentis) and trace elements (Cu, Mn, Fe, Se, and Zn) increased antioxidant capacity, accompanied by a 24% increase in superoxide dismutase activity (p=0.02), a 25% increase in glutathione peroxidase activity (p=0.05), and a 14% decrease in malonic dialdehyde levels (p=0.003).

Conclusion: The present study provides convincing evidence for the use of a combined supplement containing L. acidophilum and В. adolescentis with trace elements during the postmolting period to improve antioxidant status.

Keywords: Poultry, Forced molting, Stress, Antioxidant status, Probiotics, Trace elements.

Introduction

Modern technologies for laying hen husbandry have resulted from numerous scientific studies designed to optimize the physiological capabilities of birds to enhance their productivity (El-Sabrout et al., 2022; Korver, 2023). However, extending the productive lifespan of laying hens remains a significant challenge.

Numerous scientific studies have focused on extending the productive life of industrial laying hens (Bain et al., 2016; Arulnathan et al., 2024; Levkovich et al., 2024). According to a number of authors, enhancing the economic efficiency of the poultry industry, extending the production cycle of poultry, and enhancing egg quality can be achieved by forced molting (Lei et al., 2023; Wang et al., 2023). This technological method allows the natural molt to be forced in laying hens through stress, leading to a rapid recovery of high egg production and subsequently improving egg quality. The literature review showed that there are currently various methods of inducing forced molting (Wilson et al., 1967; Onbasilar and Erol, 2007; Sgavioli et al., 2011, 2013; Bozkurt et al., 2016; Abdulwahid et al., 2021). However, according to the analysis of the experimental studies, feeding and light conditions had the most significant influence on the effectiveness of the forced molting program.

According to a number of studies, forced molting is a specific stress for hens that contributes to the mobilization of internal reserves in the body and increases their oxidative, energy, and protein synthesis potential. As a result, it leads to a repeated increase in egg production (Berry, 2003; Mert and Yildirim, 2016). Exposure to stress factors leads to a chronic allostatic body load. This, in turn, can cause a breakdown in adaptation mechanisms and the development of serious disorders in multiple functional systems (Ruszler, 1998; Koelkebeck and Anderson, 2007). Studies have demonstrated that the periods of molting and subsequent recovery of the body are characterized by significant alterations in factors of local and general immune defense, vitamin and mineral metabolism, and oxidation-reduction processes (Alodan and Mashaly, 1999; Ricke, 2003). The data presented indicate that the effective implementation of this technological method requires a comprehensive examination of the physiological processes occurring in the avian body during the initiation of molting. Furthermore, the optimization of forced molting protocols and the development of strategies for the withdrawal of birds from this state are essential because they facilitate the recovery of the body from stressor effects and enhance productivity in the subsequent cycle.

In this context, the present study aimed to investigate the antioxidant status of laying hens during the molting period and to assess the efficacy of combining probiotic-containing preparations with trace elements in alleviating postmolting syndrome in birds.

Materials and Methods

Experimental design

The study was conducted on 250 Hisex-Brown laying hens (CJSC “Poultry Farm Orenburgskaya”) at an age of 448 days. Birds were allowed 3 weeks for acclimation during which they were fed a complete layer diet ad libitum and allowed full access to water and a lighting program of 16L:8D (469 days of age).

Then, they were randomly divided into groups: a premolting group (n=50, 469 days of age); a molting group (n=50) that underwent a 10-day fasting period; the birds received shell as feed at 50 g per head (479 days of age), lighting program of 12L:12D; and a postmolting group (n=50) that underwent molting according to the scheme adopted in poultry farms for 50 days. During the 10-day fasting period, feed was withheld from laying hens with free access to water and shell-based feed. Then, feeding was gradually restored, starting with daily feeding of 30 g of basic ration per head, increasing to 120 g by day 50 (519 days of age), lighting program of 14L:10D; a control production group (n=50) that underwent molting with gradual feeding restoration (549 days of age); an experimental production group (n=50) that underwent molting with gradual feeding restoration. After the end of molting, the probiotic preparation “Lactobifadol forte” (Component LLC BF, Russia) at a dosage of 1.5 g/kg of feed and mineral complex based on organic forms of trace elements, specifically: 2.5 mg Cu/kg (from copper glycinate), 100 mg Mn/kg (from manganese glycinate), 25 mg Fe/kg (from iron glycinate), 0.2 mg Se/kg (from selenium methionine), and 70 mg Zn/kg (from zinc glycinate) were introduced into the diet for 30 days (549 days of age), lighting program of 16L:8D. The selected doses meet the norms of trace element application in feed for laying hens of this age (Fisinin et al., 2011).

For clarity, Table 1 presents a schematic of forced molting.

“Lactobifadol-Forte” (Component LLC BF, Russia) is a probiotic preparation based on a mixture of live acidophilus and bifidobacteria. The preparation contains at a concentration of 1 x 10⁶ colony-forming units (CUs) and B. adolescentis at a concentration of 8.0 x 10⁷ CU. The bacteria were dried by the sorption method on a natural plant carrier.

The mineral complex contained essential trace elements in the chelate form. The feed additives used were: “B-Traxim 2C Cu-240” (Pancosma S.A., Switzerland), which contains at least 24% Cu; “B-Traxim 2C Mn-220” (Pancosma S.A., Switzerland), which contains at least 22% Mn; “B-Traxim 2C Fe-220” (Pancosma S.A., Switzerland), which contains at least 22% Fe; “Plexomin Zn 29” (Phytobiotics Futterzusatzstoffe GmbH, Germany), which contains 29% Zn.

Table 1. Schematic of the induced molting procedure.

Table 2 shows the nutritional value of the basic diet for laying hens aged 46 weeks or more. The temperature regime and relative humidity corresponded to the recommended norms for poultry. The photoperiod programme complied with European Social Security Regulation 43/2007 (Council Directive 2007/43/EU laying down the minimum rules for the protection of chickens kept for meat production).

At the end of the experiment, blood was drawn from the wing veins of the laying hens. Antioxidant defense (activity of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and peroxidation (malonic dialdehyde) were assessed using a specialized T-SOD Activity Assay Kit (Elabscience, China), CAT Activity Assay Kit (Elabscience, China), GSH-Px Activity Assay Kit (Elabscience, China), and MDA Assay Kit (Elabscience, China) by colorimetric method using an INNO Spectrophotometer (LTek, South Korea).

Statistical analysis

We analysed all data using Statistica version 10 (StatSoft Inc., USA). The normality of the obtained data was assessed using the Shapiro–Wilk test. This test has the best power for a given significance. The hypothesis that the data belonged to a normal distribution was rejected in all cases with a probability of 95%, which justified the use of nonparametric procedures for downstream statistical analyses; we, therefore, examined differences among group means using Mann–Whitney U-tests. This nonparametric statistics method is convenient for comparing small samples and is used by many authors when describing data with non normal distribution. The data were presented as median (Me) and as 25th–75th centiles (Q₂₅-Q₇₅). Differences were considered statistically significant when p < 0.05.

Ethical approval

The experimental studies were conducted in accordance with the instructions and recommendations of the Russian regulations (Order of the Ministry of Health of the USSR No.755 of August 12, 1977 “On measures to further improve the organizational forms of work using experimental animals”), the protocols of the Geneva Convention, and the principles of good laboratory practice (National Standard of the Russian Federation GOST R 53434-2009). All animal procedures were performed in accordance with the rules of the Animal Ethics Committee of the FSSI FRC BST RAS. The design of the experiment was approved by the local ethics committee of the FSSI FRC BST RAS (No. 7 dated 06/04/2023).

Results

The analysis revealed that livestock safety remained high throughout the experiment.

Blood analysis plays a crucial role during forced molting, enabling timely adjustments to the process and effective monitoring of the bird’s condition. The results indicate variations in antioxidant content in the blood serum of laying hens during forced molting (Table 3).

Table 2. Basic diet nutrient levels in laying hens aged 46 weeks or more.

It was found that in laying hens of the molting group, the activity of total superoxide dismutase increased significantly by 33 % (p=0.04) compared with the birds of the premolting group. Notably, upon resumption of feeding, a decrease in enzymatic activity was observed, with a 14 % reduction.

Catalase activity was found to be significantly higher in laying hens of the molting group, increasing by 15% (p=0.02) compared with the premolting group. Notably, by the end of molting, the catalase activity decreased, similar to the decrease observed in the superoxide dismutase enzyme activity.

It should be noted that glutathione peroxidase activity did not undergo any significant changes.

The highest level of malonic dialdehyde was found in laying hens of the postmolting group, with a 31% increase in blood serum content compared with the premolting group (p=0.002), indicating the presence of oxidative stress in the studied birds.

During the study, we found that a complex supplement containing a mixture of live acidophilus and bifidobacteria, combined with essential trace elements, was effective in increasing the activity of antioxidant enzymes and reducing the levels of malonic dialdehyde in the serum of laying hens (Table 4).

Table 3. Indicators of antioxidant defense and peroxidation in laying hens during the induced molting period.

Table 4. Indicators of antioxidant defense and peroxidation in laying hens following the introduction of probiotics and trace elements into their diets.

The introduction of a probiotic preparation in combination with trace elements (Cu, Mn, Fe, Se, and Zn) was found to have a positive impact on the antioxidant status of laying hens in the experimental group.

The activity of total superoxide dismutase was significantly higher in the experimental group than in the control group, with a 24% increase (p=0.02). In contrast to superoxide dismutase, the catalase activity did not differ significantly from that of the control. In contrast, glutathione peroxidase activity was significantly increased by 25% (p=0.05). It should be noted that against the background of increased antioxidant enzyme activity in the blood serum of birds, the content of malonic dialdehyde decreased by 14% (p=0.003).

The obtained data indicate that laying hens in the control group were in a state of redox imbalance, whereas the introduction of a complex supplement based on a mixture of live acidophilus and bifidobacteria, combined with essential trace elements, enhanced the birds’ antioxidant functions.

Discussion

In modern industrial poultry farming, forced molting remains important and is widely used in egg production. Scientists from various countries are developing forced molting schemes that can have a complex impact on the bird’s body, enabling the rapid restoration of high productivity and extending the bird’s use into a second cycle of egg-laying. As previously mentioned, a common zootechnical method for inducing molting is to significantly decrease the birds’ nutritional intake and daily light exposure. However, our study revealed that this process is accompanied by significant oxidative stress, as indicated by changes in oxidative stress markers, the 13% increase in malonic dialdehyde content, alongside elevated activity of superoxide dismutase (33% higher) and catalase (15% higher) in the blood serum of laying hens during starvation. The changes in antioxidant enzyme activity in the blood serum of laying hens during starvation appear to constitute a compensatory mechanism that protects the birds’ body from oxidative stress, which arises in response to stressor exposure and represents a self-regulated, adaptive response. Starvation is regarded as a type of stress, as evidenced by the metabolic changes occurring in the body, which are typical of a state of functional stress induced by food deprivation (Bulent and Niyazi, 2018). Several experimental studies have demonstrated that fasting increases malonic dialdehyde levels, a marker of lipid peroxidation, thereby indicating elevated oxidative stress (Omidi et al., 2016; Barim-Oz, 2018; Zengin, 2021). Scientists have also demonstrated that these conditions lead to increased activity of various antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (Barim-Oz and Şahin, 2016; Houmani et al., 2022). Consequently, the stress factor inducing forced molting in this case triggers the mobilization of the birds’ internal reserves and enhances their redox potential.

In contrast, at the end of the 50-day experiment, when the birds had completed molting, a marked decline in antioxidant enzyme activity was observed, implying that long-term dietary restrictions can lead to a substantial impairment of the body’s antioxidant capacity. Prolonged starvation not only deprives the body of macronutrients (carbohydrates, fats, and proteins) but also leads to a deficiency in various micronutrients, notably vitamins and trace elements, which serve as cofactors for multiple antioxidant enzymes (Lebedev et al., 2023). It is well established that dietary deficiencies in certain trace elements have a profound impact on antioxidant enzyme activity. Specifically, selenium deficiency leads to a decrease in glutathione peroxidase activity, copper deficiency suppresses Cu and Zn-superoxide dismutase activity, and manganese deficiency reduces Mn-superoxide dismutase activity (de Rosa et al., 1980; Takahashi et al., 1986; Gomi and Matsuo, 1995). Furthermore, it is possible that increased free radical formation may have both direct and indirect impacts on the activity of antioxidant enzymes, which cannot be ruled out. Free radicals can induce structural modifications in these molecules, thereby altering their catalytic activity. It should be noted that the results obtained are consistent with those of other authors. One study observed a decline in the activity of antioxidant enzymes, such as catalase and glutathione peroxidase, during the postmolt period in birds (Mert and Yildirim, 2016). Despite an initial increase in antioxidant enzyme activity, oxidative damage ultimately surpasses the body’s defense mechanisms, leading to elevated oxidative stress. The induction of forced molting results in the depletion of antioxidant stores in laying hens, accompanied by an increase in free radical release.

Refeeding after a period of fasting can reverse oxidative damage and restore antioxidant enzyme activity (Wang et al., 2017; Musazadeh et al., 2023). The findings indicate that the antioxidant status of laying hens can be improved by providing supplemental nutrients in their diets after the molting period.

The introduction of probiotics and essential trace elements into the diet of laying hens after molting accelerates recovery and improves overall bird health. The experimental data suggest that the introduction of a complex additive into the diet of laying hens modulates their enzymatic defense system. The study found that receiving probiotics based on a microbial mixture of Lactobacillus acidophilum and Bifidobacterium adolescentis, along with trace elements Cu, Mn, Fe, Se, and Zn in chelate form, after molting increased antioxidant capacity (by 24% and 25% for superoxide dismutase and glutathione peroxidase activities, respectively) and decreased oxidative status (by 14% for malonic dialdehyde levels), which may lead to improved health, increased egg production, and reduced mortality. Probiotics, known for their diverse mechanisms of action and supported by numerous scientific papers demonstrating their effectiveness in improving gut function through competition with pathogenic and opportunistic microbiota, strengthening of the protective intestinal barrier, and immunomodulation, have also been explored by researchers for their potential antioxidant properties (Krysiak et al., 2021; Latif et al., 2023). Over the past decades, researchers have demonstrated that different strains of probiotic bacteria can exhibit antioxidant capacity, which may contribute to their ability to mitigate oxidative stress (Feng and Wang, 2020). However, data concerning the antioxidant mechanisms of action of probiotics are insufficient. Scientists have suggested that probiotic bacteria can chelate metal ions, thereby inhibiting oxidative processes. Additionally, probiotics exhibit their own antioxidant enzymatic systems and are able to produce various metabolites with antioxidant activity. Furthermore, they can stimulate the host antioxidant system, increase the activity of antioxidases, and regulate signaling pathways that produce reactive oxygen species (Kullisaar et al., 2002; Lee et al., 2005; LeBlanc et al., 2011; Ahire et al., 2013; Wang et al., 2016). Scientists have experimentally in vitro and in vivo found that bacterial culture supernatant, intact cells, and intracellular cell-free extracts scavenge hydroxyl radicals and superoxide, reducing malonic dialdehyde levels and enhancing antioxidant activity (Shen et al., 2011).

Currently, there is a growing interest in researching the efficacy of supplementing bird diets with various microelements during the postmolting period, which is a critical phase in the life cycle (Naseem et al., 2021). Although there is consensus on the priority and promise of probiotics in organic agriculture, research on the combined use of probiotic-containing preparations with trace elements during the postshedding period remains understudied. Our study found that combining probiotics with essential trace elements has a synergistic positive effect on the antioxidant status of laying hens. It is well known that trace elements such as Cu, Mn, Fe, Se, and Zn play a key role in the body’s antioxidant defense mechanisms, being cofactors for various antioxidant enzymes and helping to neutralize reactive oxygen species and maintain oxidative balance (Zidenberg-Cherr and Keen, 1991; Wołonciej et al., 2016). Supplements with these trace elements boost glutathione peroxidase and superoxide dismutase activity, stimulate protective cell signaling, and enhance antioxidant defenses by reducing oxidation susceptibility (Khan et al., 2018; Hübner and Haase, 2021; Lv et al., 2023).

Conclusion

The use of feed enriched with biologically active feed additives has been widely used in practice for a long time due to its proven effectiveness in improving animal health and productivity. Various feed additives in the form of vitamins, minerals, pre- and probiotics are widely used in the poultry industry to promote healthy growth and development in birds, as well as to improve their immune systems and reduce the risk of disease (Bhagwat et al., 2021; Shojadoost et al., 2021; Yaqoob et al., 2022; Yousaf et al., 2023). However, the use of these additives in poultry diets during the postmolt period is a fairly new practice that can yield promising results in improving bird health and productivity during this critical phase.

The study provides convincing evidence in favor of using a combined supplement based on acidophilus and bifidobacteria with trace elements in the period after molting to restore the antioxidant status of the body. Using different feeding strategies can help birds recover from molt and maintain productivity during the second feeding cycle.

Acknowledgments

None.

Conflict of interest

The authors declare no conflict of interest.

Funding

This research was supported by grants for major scientific projects on priority directions of scientific and technical development (No. 075-15-2024-550).

Authors’ contributions

All authors contributed to the study conception and design. SL: The study was supervised and the manuscript. TK: Data and sample collection, laboratory tests, data analysis and interpretation, critical review, and manuscript drafting. OM: Conceptualization, laboratory tests, data analysis and interpretation, manuscript drafting, editing, and revision. All authors have read and approved the final manuscript.

Data availability

All data were provided in the manuscript.

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