Dietary resveratrol improved production performance, egg quality, and intestinal health of laying hens under oxidative stressRESVERATROL IN LAYING HENS

INTRODUCTION

Oxidative stress is a phenomenon caused by an imbalance between production and accumulation of oxygen reactive species (ROS) in cells and tissues. Oxidative stress has been proven to be linked to internal mechanism for many environmental stressors (i.e., UV, ionizing radiations, pollutants, and heat stress), xenobiotics (mycotoxins, heavy metals, pesticides, etc.), health disorders and aging (Pizzino et al., 2017; Liguori et al., 2018; Wang et al., 2019; Liu et al., 2021a; Liu et al., 2021b; Wang et al., 2021b). Moreover, it has been proved that oxidative stress plays a role in follicular atresia to lead to ovarian aging and reproductive disorders (Gupta et al., 2006; Devine et al., 2012; Shen et al., 2012). Serval previous studies has shown that oxidative stress could led to reduction in production performance and intestinal health (Mishra and Jha, 2019; Wang et al., 2019; Li et al., 2020; Wang et al., 2021b).

Resveratrol (trans-3, 5, 40-trihydroxytilbene; RV) is a naturally polyphenol compound found in berries, nuts, skins of grapes and thus in red wine (Jiang et al., 1997). RV has antioxidant, antitumor, antiviral, and free radical scavenging properties, that is thought to be an effective compound for the prevention aging and age-related diseases (Nunes et al., 2018; Li et al., 2018; Zhou et al., 2021). Also, RV is associated with protection against oxidative stress of ovary to increase follicle numbers and prolong the ovary life-extending effect in human and rat model (Liu et al., 2013; Li and Liu, 2018; Ochiai and Kuroda, 2019). Literature has shown that RV improved production performance, egg quality (extend egg shelf life, sensory score, egg quality) and antioxidant capacities of laying hens (Feng et al., 2017; Zhang et al., 2019). However, the response of layers to oxidative stress and protecting effect of RV on production performance, intestinal and ovarian function is not clear.

Therefore, the purpose of the current study was to verify the hypothesis that dietary resveratrol supplementation could attenuate the production performance, egg quality, intestinal health and ovarian function of laying hens under oxidative stress.

MATERIALS AND METHODS

The experiment was carried out according to the Chinese guidelines for animal Welfare and approved by the Animal Care and Use Committee of Sichuan Agricultural University.

RESULTS

Jejunal Inflammatory Cytokine and Barrier Integrity Related Gene Expression

Layers under tBHP challenge up-regulated mRNA expression of pro-inflammatory cytokine (including IL-1β, IL-6 and TNF-α) and its up-streaming nuclear factor NF-κB (Figure 1; P_(tBHP)<0.05). Dietary RV supplementation down-regulated mRNA gene expression of IL-1β, IL-6, TNF-α and NF-κB (P_(RV) ≤0.05).

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Figure 1. Effect of dietary resveratrol supplementation on inflammatory cytokine mRNA gene expression in jejunum. (A) IL-1β, (B) IL-6, (C) TNF-α, and (D) NF-κB mRNA expression. Abbreviations indicated: RV = 600 mg/kg resveratrol, tBHP = intraperitoneal injection of tert-butyl hydroperoxide (tBHP) (0 or 800 μmol/kg BW), IL-1β = interleukin-1 β, IL-6 = interleukin-1, TNF-α = tumor necrosis factor alpha, NF-κB = nuclear factor-kappa B.

Moreover, compared with the layers receive no challenge, layers under t-BHP challenge had lower mRNA expression of barrier-related proteins (claudin-1, claudin-2, Mucin-1, and Occludin) (Figure 2; P_(tBHP)<0.05) in jejunum mucosa. As expected, dietary RV up-regulated mRNA expression of intestinal barrier related protein (claudin-1, claudin-2, Mucin-1, and Occludin) (P_(RV) ≤0.05). Mucin-2 and ZO-1 were not affected by either tBHP or RV addition (P>0.05).

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Figure 2. Effect of dietary resveratrol supplementation on intestinal barrier-related mRNA gene expression in jejunum. (A) Claudin-1 (B) Claudin-2, (C) Mucin-1, (D) Mucin-2, (E) ZO-1, and (F) Occludin mRNA expression. Abbreviations indicated: RV = 600 mg/kg resveratrol, tBHP = intraperitoneal injection of tert-butyl hydroperoxide (tBHP) (0 or 800 μmol/kg BW); ZO-1 = zonula occluden-1.

Ovarian Reproductive Receptor Gene Expression

As shown in Figure 3, dietary supplementation with RV increased reproductive receptor mRNA expression, including AR, ESR1, ACVR1 and StAR compared with no addition (P_(RV) ≤0.01). No effects of tBHP were found on reproductive receptor gene expression (P_(tBHP)>0.05).

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Figure 3. Effect of dietary resveratrol supplementation on ovarian reproduction related hormone receptor mRNA gene expression. (A) Androgen receptor, (B) Estrogen receptor 1, (C) Activin a receptor type 1, and (D) Steroidogenic acute regulatory protein mRNA expression. Abbreviations indicated: RV = 600 mg/kg resveratrol, tBHP = intraperitoneal injection of tert-butyl hydroperoxide (tBHP) (0 or 800 μmol/kg BW); ACVR1 = activin a receptor type 1.

DISCUSSION

Oxidative stress induces apoptosis and affects cellular homeostasis, which was demonstrated to decrease ovarian function by inducing follicle atresia (Shen et al., 2017; Cao et al., 2018). In this study, we confirmed that the oxidative stress induced by injection of tBHP significantly reduced egg production rate and feed intake of laying hens; moreover, the administration of RV diet improved the egg production performance of laying hens in present study. Oxidative stress resulted from environmental toxicant and other stressors were reported to damage the health and production performance of layers (Devine et al., 2012; Wang et al., 2020b; Abbs et al., 2020). Tert-butyl hydroperoxide (t-BHP) known to cause peroxidation of membrane lipids and deplete cellular GSH and is commonly used as a model substance for evaluation of mechanisms of cellular alterations resulting from oxidative stress in cells and tissues (Kučera et al., 2014). The tBHP were found to successfully induce oxidative damage and decreased laying performance in our previous study (Wang et al., 2021b). In accordance with our current study, oxidative stress induced by high levels of molybdenum and vanadium were also led to egg production reduction and the addition of antioxidants (tea polyphenols) was able to reverse this effect by improving the antioxidant capacity and intestinal health in layers (Yuan et al., 2017; Wang et al., 2018; Wang et al., 2021b). Studies in mammals have found that oxidative stress can reduce the number of follicles in each stage of the ovarian cycle and impair ovarian function (Miyamoto et al., 2010; Abou-Seif et al., 2001; Brannian and Hansen, 2002). Additionally, previous studies have also reported that oxidative stress decreases hormone secretion (including lower estradiol, FSH, LH and IGF-1) and impairs the glutathione redox cycle (Miyamoto et al., 2010; Yu et al., 2019). Leptin were reported to exert an important role in the regulation of ovarian folliculogenesis indirectly by controlling of luteinizing hormone and FSH secretion (Brannian and Hansen, 2002). Similarly, the OS decreased estradiol, FSH, AMH and leptin levels in the current study, which explain the reduction in egg laying rate. However, the literature about oxidative stress on the ovary function of poultry is not well elucidated, which needs to be explored in future studies. Plant polyphenols were chosen in laying hens’ diet to increase production performance recently due to its antioxidative, antiaging, reducing antiinflammation, and antimicrobial properties. Zhang et al. (2019) found that 200 mg/kg RV supplementation improved feed intake, egg laying rate and feed efficiency of layers. Liu et al. (2014) also reported that RV improves growth performance and reduces oxidative stress in heat-stressed blacked-boned chickens (Liu et al., 2014). This may because the RV can increase the health status of laying hens by improving the health status of intestine, ovary and oviduct (enhance antioxidant capacities, decreasing inflammation) (Reis et al., 2019).

In the current study, tBHP challenge were found to result in reduction in eggshell quality (including pale shell color, lower thickness and strength) and albumen quality. Moreover, the RV treatment resulted in a higher shell color in OS-challenged birds, which is in agreement with previous observation where the decrease in oxidative stress-induced shell color was reversed by antioxidant supplementation studies (Yuan et al., 2016; Wang et al., 2016).The eggshell pigmentation has been widely used as a potential indicator for stress and disease condition in commercial laying hens (Samiullah et al., 2015). It is evident that stress stimuli, including environmental stressors (heat stress, crowding, fear, etc.) and dietary hazards (heavy metals, mycotoxins, toxicants, etc.) led to pale coloration eggshell eggs (Yuan et al., 2016; Wang et al., 2016; Wang et al., 2018; Wang et al., 2020b). Ma et al. (2021) also reported that dietary arsenic supplementation (40.67 mg/kg) would lead to oxidative stress to reduce egg laying rate, albumen height, Haugh unit, and eggshell strength of laying hens. The green tea polyphenol epigallocatechin-3-gallate were found to increase eggshell color by increasing morphology and antioxidant capacity of uterus in layers exposed to oxidative stress (induced by 5 or 10 mg/kg vanadium) in previous studies (Yuan et al., 2016; Wang et al., 2018). Resveratrol also can affect calcium metabolism (Sahin et al., 2010). Liu et al. (2005) showed that resveratrol (0.7 mg/kg) supplementation increased bone mineral density and inhibited femur calcium loss associated with estrogen deficiency in ovariectomized rats. Zhang et al. (2019) found that 400 mg/kg RV extent the egg shelf lives (increase albumen quality) during storage. Moreover, we found that egg yolk color was decreased by tBHP challenge and recovered by RV supplementation. Egg yolk color is produced by oxycarotenoids, commonly known as xanthophyll pigments, derived from the hen’s feed. It has been reported that the oxycarotenoids can be oxidized by agents such as peroxides and trace minerals. Previous studies have suggested that antioxidants, such as vitamin E, quercetin, selenium were found to increase the magnitude of the yolk color and antioxidant capacities of eggs (Surai et al., 2000; Amevor et al., 2021). However, Sahin et al. (2010) didn’t observe any effect of RV (200 or 400 mg/kg) on feed intake, egg production, inner and outer egg quality indicators in quails. The discrepancy may rely on the difference poultry strains, physiology stage and the stress model they are exposed to.

The intestinal epithelium, composed of single layer of cells, sits at the interface between an organism and its luminal environment, and as such is prone to oxidative stress (Circu et al., 2012). Intestinal morphology determines the nutrient absorption capacity and villus height is generally recognized as a good indicator for intestinal health. An increase in villus height indicates an increased absorption surface and higher capability of absorption available nutrients, whereas deeper crypt depth indicates that the villi in the small intestinal mucosa are atrophied (Celi et al., 2019). ROS, such as hydrogen peroxide and nitric oxide, disrupt tight junctions (TJs) and elevate paracellular permeability in vitro. TJs (such as zonula occludens-1 (ZO-1), occludin, and claudin-1 and -4) regulate the paracellular transport of ions, molecules, and water, and it determined the integrity of epithelia cell and mucus gel layer (Moretó and Pérez-Bosque, 2009; Ulluwishewa et al., 2011). In this study, we found that OS led to down-regulation of intestinal barrier associated protein (claudin-1, claudin-2, mucin-1, occludin). Also, OS induced inflammation (indicated by higher mRNA expression of pro-inflammatory cytokine, such as IL-1β, IL-6 and TNF-α) and its up-streaming nuclear factor NF-κB), decreased the villus height and antioxidant capacities of small intestine, as demonstrated by lower activities of antioxidant enzymes, SOD, GSH-Px and T-AOC, and higher corresponding levels of MDA. Wang et al. (2020) also reported that layers exposed to high stocking density stress also led to deceases in villus height of small intestine. RV has been evident to restrain oxidative stress and alleviate inflammatory responses in a rat model of polycystic ovary syndrome (Sadi et al., 2015). It also plays several roles in antioxidative and anti-inflammatory pathways and ameliorates metabolic syndrome (Javkhedkar et al., 2015). Xing et al. (2019) and Wang et al. (2021a) reported that RV (400 mg/kg) supplementation decreased oxidative stress in fatty liver hemorrhagic syndrome by enhancing antioxidant enzymes (Nrf2, SOD-1 and HO-1) and decreasing inflammatory cytokines (NF-κB, IL-1β, IL-6 and TNF-α) mRNA expression in ovary. Intestinal oxidative stress produces pro-inflammatory cytokines, which increase mucus layer permeability, leading to intestinal and systemic inflammation. Mucosal oxidative stress would result from the disruption of these unique redox control nodes, and subsequent contribute to the development of inflammation and other intestinal pathology. Yang et al. (2021) also reported that dietary supplementation with 400 mg/kg RV protect against oxidative damage and inflammation injury (induced by heat stress) indicated by improving tight junction protein (claudin 1, occludin) mRNA expression, antioxidant enzyme activities (SOD and CAT) and reducing the secretion of IL-1β in duck jejunum.

In the current study, we found that dietary supplementation with RV increased mRNA expression of AR, ESR1, ACVR1 and StAR. Androgen is an important regulator of prostate gland development and function, including proliferation, differentiation, maintenance (Chang et al., 1988). The androgen receptor (AR) is a crucial mediator of androgen action and a ligand-dependent transcription factor that belongs to the nuclear steroid hormone receptor super-family. Resveratrol has been reported to exert estrogenic effects, increasing uterine and ovarian wet weight (Henry and Witt, 2002; Wang et al., 2021). Han et al. (2021) reported that RV increased estrogen receptor 2 (ESR2) expression. This may also explain the increasing effect of RV on egg production performance. Also, Morita et al. (2012) found that resveratrol increased the expression of LH receptor and StAR mRNA expression in rat GCs, suggesting a possibility that resveratrol may promote steroidogenesis and luteinization (a process of terminal differentiation of granulosa cells) in the ovary of rat. StAR is known to govern the rate-limiting step in steroidogenesis, which is the translocation of cholesterol from the outer to the inner mitochondrial membrane (Kiriakidou et al., 1996; Devoto et al., 2002). However, other studies demonstrated that RV depressed the AR transcriptional activity in cancer cells and in ovarian follicle (Bowers et al., 2000; Ozatik et al., 2020). The discrepancies may due to the different animal type, stress model and RV levels used the study. As the oxidative stress is the general mechanism for aging and hazardous toxicant in animals, further study is needed to elucidate the mechanism within the response of RV in oxidative stress.

CONCLUSION

These results indicated that tBHP (intraperitoneal injection of 800 μmol/kg BW of tBHP) induced oxidative stress in layers, which may lead to reduction in production performance, egg quality, intestinal health and induced ovarian inflammation. Dietary supplementation with 600 mg/kg RV was able to maintain intestinal and ovarian function and mitigate the negative impact of tBHP challenge on production performance and egg quality.

Conflict of interest

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have been approved the manuscript that is enclosed.

Uncited References

Abbas et al., 2020, Jang et al., 1997, Abou-Seif and Youssef, 2001, Muhammad et al., 2021, Surai, 2000, Wang et al., 2018, Wang et al., 2020a, Circu and Aw, 2012