Natural Astaxanthin From Haematococcus Pluvialis Enhanced Antioxidant Capacity And Improved Semen Quality In Aging Layer Breeder Roosters Through The Mitogen-Activated Protein Kinase/Nuclear Factor-Erythroid 2-Related Factor 2 (MAPK/Nrf2) Pathway

Shan Gao (  1197116620@qq.com ) Beijing University of Agriculture Nuo Heng Beijing University of Agriculture Fang Liu Beijing University of Agriculture Yong Guo Beijing University of Agriculture Yu Chen Beijing General Station of Animal Husbandry Liang Wang Beijing University of Agriculture Hemin Ni Beijing University of Agriculture Xihui Sheng Beijing University of Agriculture Xiangguo Wang Beijing University of Agriculture Kai Xing Beijing University of Agriculture Longfei Xiao Beijing University of Agriculture Xiaolong Qi Beijing University of Agriculture


Introduction
In poultry production, assisted reproductive technology such as arti cial insemination is widely used. A single male is responsible for fertilizing dozens of female birds [1]. Therefore, the reproductive performance of roosters is a key factor in production.
However, the sperm quality of roosters decreases gradually at approximately 50-55 weeks [2]. Several important reproductive traits change in aging roosters, including decreases in the sperm concentration, motility, testosterone level, and antioxidant capacity [3; 4]. Male fertility is one of the most important factors in determining hatchability. Therefore, it is necessary to nd a way to alleviate the in uence of age on the reproductive characteristics of breeding roosters. Recently, studies have shown that dietary supplementation with natural anti-oxidative compounds such as dietary linseed oil [5], lycopene[6] and curcumin [7] can alleviate the negative effects of age on roosters.
Astaxanthin (3,3-dihydroxy-β, β-carotene-4,4-dione) is a xanthophyll carotenoid, that is mainly divided into two forms: natural astaxanthin (ASTA) and synthetic astaxanthin. ASTA can be extracted from algae[8], yeast [9], sh, krill, and some microorganisms [10]. The microalgae Haematococcus pluvialis (a green microalga) is one of the major sources of ASTA, and can be extract a large amount of ASTA under stress conditions [12; 11]. Although synthetic astaxanthin has the same chemical formula as ASTA, the ability to scavenge singlet oxygen and free radical of ASTA is over 50 times and 20 times that of synthetic astaxanthin, respectively [13]. Meanwhile, the European Commission considers ASTA as a food dye [14]. Currently, ASTA has received much attention for its various bene cial characteristics, including its anti-oxidant [15], anti-in ammatory [16] and antiapoptotic properties [17]. However, there is little information on whether ASTA can improve the male reproductive performance.
The mitogen-activated protein kinase (MAPK) pathway, a family of serine/threonine protein kinases, is widely conserved in eukaryotes and is involved in antioxidant enzyme expression by modulating the Nrf2/ARE axis[18 ; 19]. Nuclear factor-erythroid 2related factor 2 (Nrf2) is an important regulator of antioxidant responsive element (ARE)-activated gene expression. Nrf2 can repair and degrade of damaged macromolecules caused by oxidative stress by upregulating the expression of antioxidant enzyme genes [20]. Studies have shown that the MAPK/Nrf2 pathway improves the antioxidant capacity of eggs by upregulating the mRNA expression of P38MAPK, Nrf2, and HO-1 [21]. Moreover, the Nrf2/ARE axis alleviates meat lipid peroxidation by regulating the expression of antioxidant enzymes and phase II enzymes in order to achieve long-term meat storage [22]. However, the potential effect of ASTA on sperm quality, antioxidant capacity, and the MAPK/Nrf2 antioxidant pathway in aging layer roosters was not clear.
Therefore, in the current study, we hypothesized that ASTA can improve semen quality by enhancing antioxidant capacity and activating the MAPK/Nrf2 pathway in aging roosters.

Materials And Methods
Animal care and use All experimental protocols were approved by the Animal Care and Use Committee of the Beijing University of Agriculture.

Experimental design
In the present study, a total of ninety-six 53-week old Jinghong No.1layer breeder roosters with a similar weight and genetic background were supplied by Beijing Huadu Group Co., Ltd. (Beijing, China), and the birds were randomly distributed in four treatments, following a completely randomized design. Each treatment had six replicates, and there were four birds per replicate with one bird per cage (70 cm × 60 cm × 75 cm). All birds were fed a basal diet for 1week and then assigned to a corn-soybean meal-based diet containing 0, 25, 50, and 100 mg/kg ASTA for 6 weeks. Haematococcus pluvialis was purchased from Jingzhou Natural Astaxanthin Inc. (Jingzhou, China), and the ASTA content was 1.54%. The composition and nutrient levels of the cornsoybean meal-based diet are shown in Table 1. During the experimental period, the birds had 16-h light cycles and were allowed ad libitum access to water and the treatment diets. Sperm viability was evaluated using the eosin-nigrosin stain method. Brie y, a 5 µL sperm sample was mixed with 25 µL of stain on a pre-warmed slide before the test. Then, the stain was spread with another slide to make a sperm suspension smear. An Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) was used at a nal magni cation of 400 to analyze the sperm viability. For each sperm sample, ve microscopic elds were observed subjectively, and 200 sperms were counted to evaluate viability. Sperm with unstained heads of spermatozoa were considered viable, while sperm displaying partial or complete purple staining were considered nonviable.

Antioxidant enzymes and oxygen free radicals analysis
After analyzing the semen quality, the semen sample was centrifuged at 4000 × g for 10 min at 4 ℃, and then the seminal plasma was separated and stored at -20 ℃. At the end of the 6-week feeding trial, one bird from each replicate was randomly selected and sacri ced by cervical dislocation. Immediately after euthanasia, blood samples were collected from the left jugular vein of one bird per replicate. Plasma was separated by centrifugation at 3000 × g for 10 min and stored at -20 ℃ until analysis. The testis, liver, and kidney were removed immediately and quickly frozen at -80 ℃ for later analysis. These samples were used to analyze the activities of antioxidant enzymes and the ability to scavenge oxygen free radicals. Brie y, antioxidant capacity included the glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC), while oxygen free radical scavenging abilities included hydroxyl radical scavenging ability and superoxide anion scavenging ability. Meanwhile, malondialdehyde (MDA), as the product of lipid metabolism, was analyzed using a spectrophotometric method. These assays were performed using a commercial kit (Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China). The method and principle used to determine the antioxidant indicators with these kits were previouly described elsewhere [23].

Quantitative PCR Analysis
The mRNA expression levels of p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase 1 (JNK1), c-Jun Nterminal kinase 2 (JNK2), c-Jun N-terminal kinase 3 (JNK3), Nrf2, Cu-Zn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), CAT, glutathione peroxidase 1 (GPX1), and peroxidase 4 (GPX4) were determined via real-time quantitative PCR (RT-qPCR) with β-actin (ACTB) as an internal control in the testis tissues. Total RNA was isolated from 0.1 g of the testis sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Then, 1% agarose gel stained with ethidium bromide was used to con rm the 18S and 28S bands. Finally, the total RNA concentration was measured using a spectrophotometer at 260 nm. Total RNA was reverse transcribed into cDNA using a Thermo Fisher First cDNA Synthesis Kit (#K1621; Thermo Fisher Scienti c, Waltham, MA, USA). A Step One Plus Real-time PCR system (Applied Biosystems, Foster City, CA, USA) was used for the quantitative PCR analyses. The primers for the selected genes are listed in Table 2. After initial denaturation at 95 ℃ for 10 min, 40 cycles of ampli cation were performed (95 ℃ for 10 s and 58 ℃ for 30 s), generating melt curves to verify ampli cation speci city. The relative gene expression levels were calculated using the 2 −ΔΔCt method [24], with ACTB as the reference gene. The protein expression of Nrf2, p38, p-p38, ERK, p-ERK, JNK, and p-JNK was evaluated by western blotting in the testis tissues, and the antibodies are shown in Table 3. Total proteins were extracted from testis tissues using RIPA Buffer (W1001; SinoGene, Beijing, China) according to the manufacturer's instructions. The protein concentration of the extracts was determined using the Bradford method (W1014; SinoGene). Samples containing 30 µg of protein and protein markers (SM26616; Thermo Fisher Scienti c) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 4% stacking gel, 12% separating gel) at 120 V for 2 h. Proteins were transferred to polyvinylidene di uoride (PVDF) membranes using a Bio-Rad mini transfer system (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with Fast Protein-free Block Buffer (#W1028; SinoGene) for 5 min and then incubated with primary antibody (1:1000 dilution) overnight at 4 ℃. After rinsing four times with TBST for 10 min per rinse, HRP-conjugated secondary antibody (SA00001-2, ptg) was applied to the membranes at a dilution of 1:3000 for 1 h. Then, after rinsing three times with Tris-buffered saline with 0.1% Tween ® 20 Detergent (TBST) for 10 min per rinse, immunological signals were detected using a chemiluminescent (ECL) kit (29050; Engreen) and exposed to X-ray lms in the dark. Protein bands were quanti ed by densitometric analysis using Image J analysis software (National Institutes of Health, Bethesda, MD, USA).

Results And Discussion
Semen quality Semen quality is considered to be a crucial factor in roosters' fertility because the fertility rate is positively correlated with sperm motility, sperm concentration, and sperm motility parameters in roosters [25]. Few reports have described the effects of ASTA on semen quality in aging roosters. We evaluated the effects of dietary ASTA on semen quality in aging roosters (Table 4). Brie y, there were no differences in semen quality between the control group and the ASTA groups at week 0. The semen volume was signi cantly higher in the 25 mg/kg ASTA group than that in the other groups at week 6 (P < 0.05). Meanwhile, with an increase in dietary ASTA, sperm viability and sperm concentration signi cantly increased compared to those in the control group (P < 0.05).
In particular, the sperm concentration was linearly and quadratically affected (P < 0.05) by the dietary ASTA levels. In addition, the sperm motility parameters of aging layer roosters in the 50 mg/kg ASTA group were signi cantly linearly increased relative to those in the control group (P < 0.05). Moreover, sperm motility increased from 64.395-76.234% in the 0-100 mg/kg ASTA-treated group (P < 0.05). It has been previously demonstrated that sperm viability, total motility, and sperm kinematic parameters increase after ASTA treatment in mice [26]. A recent study also showed similar results: ASTA can improve post-thawed rooster sperm motility and kinetic parameters, including total motility, progressive motility, VAP, VSL, and LIN. However, there was no signi cant difference in LIN, STR, ALH, and BCF in the ASTA treatment groups compared to those in the control [27]. Interestingly, the opposite result was observed in the current study that the motility parameters including STR, LIN, WOB, VSL, VAP, BCF, and ALH in the ASTA groups were signi cantly higher than those in the control group (P < 0.05). The discrepant results may be partly related to the level of ASTA addition, the duration of supplementation, and the test subjects. Taken together, these ndings indicated that dietary ASTA supplementation signi cantly increased the semen quality during the test period (0-6 weeks). However, the possible mechanisms by which dietary ASTA affects semen quality are not well understood, but may be related to antioxidant defense system. Other studies have reported that senescence occurs due to the damage caused by free radicals and reactive oxygen species (ROS) to DNA, lipids, and proteins [29; 30]. Sophisticated enzymatic (SOD, CAT and GSH-Px) and non-enzymatic (vitamins A, C, E, and carotenoids) antioxidants constitute an antioxidant defense system that can regulate overall ROS levels to maintain physiological homeostasis [31]. ASTA, as a natural carotenoid antioxidant, can be transferred to the right place in the tissues and exert antioxidant effects at an appropriate concentration [32]. Previous studies haves demonstrated that ASTA has a higher antioxidant activity relative to α-carotene, lycopene, lutein, and β-carotene [33], and the capacity of scavenging singlet oxygen is about 550 times more than that of vitamin E [34]. Thus, in this study, the effects of dietary ASTA addition on antioxidant enzyme activity and free radical scavenging ability in the plasma, seminal plasma, testes, liver, and kidney of aging layer breeder roosters are shown in Figures 1 to 5. Brie y, the 25 mg/kg ASTA level linearly and quadratically increased the GSH-Px activity in the plasma, testis, and liver of aging layer roosters compared with that in the control group (P < 0.05). The activities of SOD and CAT also increased gradually with increasing dietary ASTA supplementation from 50 to 100 mg/kg (P < 0.05). Furthermore, MDA concentrations decreased linearly with increasing dietary ASTA levels (P < 0.05) in birds. With an increase in dietary ASTA, the levels of T-AOC increased linearly (P < 0.05). This observation is consistent with a previous report that in general, the activities of SOD, CAT, and GSH-Px in the brain and liver of rats increased dramatically at treatment with ASTA, respectively[36 ; 35]. In addition, we found that dietary ASTA supplementation linearly and quadratically increased the abilities of scavenging hydroxyl radicals and superoxide anions compared with those in the control group, which was consistent with a previous study [37]. In particular, the abilities of scavenging hydroxyl radical and superoxide anion of aging roosters were signi cantly increased in the 50 mg/kg ASTA group (P < 0.05).
Relationship between semen quality and antioxidant enzyme activity When the age of male poultry is over 45 weeks, semen volume, sperm concentration, viability, and forward motility decrease, whereas lipid peroxidation in seminal plasma increases [38]. Therefore, in the present study, we aimed to improve the quality of semen by improving the antioxidant capacity of aging roosters using dietary ASTA. Moreover, it has been demonstrated that antioxidant capacity plays an important role in maintaining sperm viability because it can protect against lipid peroxidation in semen [39]. MDA is an important indicator of lipid peroxidation. A previous study showed that sperm motility was negatively correlated with MDA content in spermatozoa [40]. In the current study, the MDA content in the semen plasma, plasma, and tissues showed a dose-dependent decrease with changes in dietary ASTA. Similar ndings were obtained in rats in which ASTA group showed a signi cant increase in SOD and CAT activities, as well as a signi cant decrease in MDA levels and repaired the oxidative stress-induced fertility disorder [41]. Therefore, the improvement of semen quality in aging roosters may be related to two factors. First, antioxidant enzyme activity remarkably improved, and MDA concentration decreased linearly with increasing dietary ASTA concentration, which plays an important role in the prevention of oxidative damage in the sperm plasma membrane. Second, dietary ASTA may improve the ability to neutralize singlet oxygen and scavenge radicals to inhibit lipid peroxidation, protect sperm from free radicals, and improve semen quality.
Gene expression of SOD1, SOD2, CAT, GPX1, and GPX4 The activity of an antioxidant enzyme is closely related to its gene expression [42]. The mRNA levels of SOD1, SOD2, CAT, GPX1, and GPX4 were determined to evaluate the effects of ASTA on the gene expression of antioxidant enzymes in aging rooster testis tissues ( Figure 6). In the present study, the SOD1 and SOD2 mRNA levels in the 50 mg/kg ASTA groups were higher than those in the other groups (P < 0.05). SOD1 is mainly soluble in the cytoplasm and SOD2 is mitochondrial, and ASTA has both lipophilic and hydrophilic properties, which can directly produce effects in or outside the cell membrane [43]. This may be the reason why the gene expression of SOD1 and SOD2 was improved by adding dietary ASTA. Additionally, in the dietary ASTA supplementation group, the GPX1 and GPX4 mRNA levels were signi cantly increased relative to those in the control group (P < 0.05). However, there were no differences in the GPX1 mRNA levels among the 50 and 100 mg/kg ASTA-treated groups. Furthermore, an increase in dietary ASTA supplementation from 0 to 100 mg/kg linearly increased the mRNA expression of CAT (P < 0.05). Similarly, in a previous study, dietary ASTA increased the oxidative damage repair potential of mice and upregulated the mRNA levels of GPX1, SOD1, SOD2, and CAT in the liver and kidney [44]. Therefore, it would be interesting to further study the mechanism of how dietary ASTA affects semen quality in aging roosters by improving the antioxidant defense system.
Gene and protein expression in the MAPK/Nrf2 pathway Nrf2 plays an important role in the antioxidant response, and it has been reported that the MAPK kinase pathway regulates the Nrf2 action. When the nuclear transcription factor Nrf2 is phosphorylated and activated by MAPKs (such as ERK and p38), it can be translocated to the nucleus, leading to the upregulation of antioxidant enzyme expression [22]. Therefore, in order to clarify whether ASTA could activate this pathway, the gene and protein expression in the MAPK/Nrf2 pathway in testis tissues were measured in the four groups, and the results are presented in Figures 7 and 8. In this study, signi cant differences in the mRNA levels of Nrf2, ERK, P38, and JNK were observed among all of the ASTA-added groups in the testis (Figure 7). Brie y, when the concentration of dietary ASTA reached 50 mg/kg, the mRNA levels of Nrf2, ERK, P38, and JNK2 were signi cantly higher than those in the control group (P < 0.05) and reached the maximum. A signi cant increase in JNK1 mRNA expression was observed in the 100 mg/kg ASTA group (P < 0.05). Moreover, an increase in dietary ASTA from 0 to 100 mg/kg linearly enhanced the mRNA expression of JNK3 (P < 0.05). This nding indicated that dietary ASTA increased the gene expression of Nrf2 and upregulated the expression of upstream signals (MAPKs). Western blot analysis revealed that dietary ASTA effectively elevated (P < 0.05) the ratio of the phosphorylated MAPKs (p38, ERK, and JNK) to total MAPKs (p38, ERK, and JNK) compared to that in the control ( Figure 8). Brie y, the results showed that ASTA treatment remarkably upregulated the p-p38, p-ERK, and p-JNK expression in the testis (P < 0.05). In particular, the p-p38, p-JNK, and Nrf2 protein levels in the 25 mg/kg ASTA group were higher than those in the other groups. In addition, when the concentration of dietary ASTA reached 50 mg/kg, p-ERK protein levels were signi cantly higher than those in the control group (P < 0.05) and reached the maximum. However, Niu et al., 2018 [45] observed an opposite result in that dietary ASTA supplementation did not affect the p-38 and p-JNK levels but signi cantly increased the p-ERK levels.
Differences in test subjects, and test conditions may explain these discrepant results.
Relationships among the MAPK/Nrf2 pathway and antioxidant enzymes and semen quality Aging is a complex phenomenon that is associated with an irreversible and progressive decline in body function due to biochemical and morphological changes [46]. In the process of aging, there are obvious characteristics such as increase in oxidative stress, a decline in cell function, and defects in tissues and organs [47]. In aging male animals, morphological changes in the testis include a reduction in the volume and quantity of germ cells, which leads to a decrease in sperm quality and leading to the upregulation of the gene expression of antioxidant enzymes [50]. There is a high proportion of polyunsaturated fatty acids in the plasma membrane of bird sperm, and with an increase in age, the antioxidant capacity of sperm decreases.
Once the plasma membrane of bird sperm is exposed to ROS, it is prone to lipid peroxidation [51]. Thus, the decrease in total antioxidant capacity of semen is considered to be one of the reasons for the decline of fertilization ability in aging roosters [52]. In the current study, our results revealed that dietary ASTA elevated the MAPK phosphorylation (p38, ERK, JNK), and the mRNA and protein expression of Nrf2 were remarkably enhanced by adding 50 mg/kg ASTA in the diets, which was in agreement with a previous study [53]. In addition, the SOD1, SOD2, CAT, GPX1, and GPX4 mRNA levels were higher in the dietary ASTA group than those in the control group, which led to the enhancement of antioxidant capacity and semen quality in aging roosters. These results indicate that dietary ASTA can activate the MAPK/Nrf2 pathway, upregulate Nrf2 transcription and translation, and promote the expression of downstream antioxidant enzyme genes, enhancing the antioxidant capacity and improving the semen quality in aging roosters.

Conclusions
In summary, the results of this study con rmed our hypothesis that dietary ASTA supplementation improves the semen quality of aging roosters, as re ected by the upregulation of the antioxidant system (Fig. 9). Therefore, these ndings suggest that dietary ASTA could attenuate age-related sub-fertility in aging layer breeder roosters. However, the possible mechanisms by which ASTA ameliorates sperm quality are not well understood. Therefore, it is necessary to evaluate the effect of dietary ASTA on semen quality by the MAPK/Nrf2 signaling pathway in vitro.   capacity (T-AOC) in the testis. The data represent the mean ± standard deviation; n=6 in each group. a-d Means within a row with no common superscripts differ signi cantly (P < 0.05).   Effects of dietary natural astaxanthin (ASTA) on the expression of proteins related to the mitogen-activated protein kinase/nuclear factor-erythroid 2-related factor 2 (MAPK/Nrf2) pathway in the testis tissues of aging layer breeder roosters. The experimental groups included the control group (0 mg/kg) and ASTA groups (25-100 mg/kg). The results are expressed as the means ± standard deviation (n = 3). a-c Means within a row with no common superscripts differ signi cantly (P < 0.05).

Figure 9
Schematic diagram summarizing the mechanisms by which natural astaxanthin (ASTA) promotes the antioxidant defense system in aging roosters. The antioxidant defense system is down-regulated in the natural aging process in roosters. ASTA attenuated the oxidative stress in the testis via the activation of the mitogen-activated protein kinase/nuclear factor-erythroid 2related factor 2 (MAPK/Nrf2) pathway to attenuate the age-related sub-fertility in aging layer breeder roosters. ARE, antioxidant responsive element; CAT, catalase; GSH-Px, glutathione peroxidase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; SOD, superoxide dismutase.