Introduction

Molybdenum (Mo) is a type of silver-white metal and exists in the form of molybdenite in nature. It is an essential trace element for animals which is nearly universally distributed in organisms [1]. Mo invades humans and animals through the digestive tract or respiratory tract and then accumulated in the liver and kidney [2]. It is a component of enzymes such as sulfite oxidase, xanthine oxidase (XOD), and aldehyde oxidase. These Mo enzymes are involved in intracellular metabolism [3]. Many studies have reported that improper mining and industrialization could lead to an increase in concentration of Mo in soil, water, and air and then it can be absorbed into by aquatic and terrestrial organisms causing chronic toxicity in fish and cattle [4, 5]. High dietary of Mo induced oxidative stress, as well as decreased antioxidant enzyme activities and increased free radical accumulation [6]. Adverse effects of Mo are observed on animals including reproductive impairments, growth depression, and anemia [7]. It can also impair the testicle which is associated with sterility in males [8, 9]. Exposure to HMo adversely affecting the reproductive system of male mice was approved in Wang HW’s study that administration of HMo not only decreased sperm density and motility but also increased the rate of teratosperm occurrence [10].

Cadmium (Cd) is a type of metal which mainly exists in greenockite. It is not an essential trace element for organisms and is harmful for humans and animals. It can be absorbed from water, food, and air contaminations and then accumulated in many tissues, such as kidney, liver, brain, muscle, testis, and so on, with long biological half-life in the body [11, 12]. This metal toxin is proved to damage various target organs. There are reports demonstrated that exposure to Cd caused pulmonary toxicity, hepatotoxicity, testicular injury, and lethality [13, 14]. In addition, this metal toxin can cause reproductive disturbance. Shuhua Yang et al. indicated that Cd exposure resulted in oxidative damage of hen’s ovary tissue by altering antioxidant defense enzyme systems, lipid peroxidation, apoptosis, and endocrine disturbance [15]. Li Jinlong et al. reported that Cd caused testicular toxicity in cocks [16]. The adverse effects include the rises of the percentage of apoptotic testicular cells and reduction of sperm concentration [17]. Many deleterious effects of Cd were reported including increasing generation of reactive oxygen species (ROS), destruction of proteins, inhibition of DNA repair enzymes and antioxidant enzyme activities, modulation of apoptosis, and regulation of many genes expression [18, 19]. There are reports indicating that Cd also declines sperm motility and damages the DNA of sperm [20, 21].

When exposed to the heavy metals, the body is protected from oxidative attacking through altering antioxidant enzyme activities [22]. Heavy metals can interact with cell membrane and alter the normal physiology, inducing oxidative stress, lipid peroxidase (LPO), and inhibit antioxidant enzyme activities [23]. It is reported that Mo and Cd have the ability to alter the expression levels of a variety of genes such as metal protein genes and apoptosis genes [24, 25]. In addition, Mo and Cd compete for the same transmembrane carriers with other essential nutrients and disturb the homeostasis of trace elements [26, 27].

More and more researchers have paid attention to investigating the effects and mechanisms of animal combined metal toxicity in recent years because of combined pollution of heavy metals. Our previous study showed that the damage on kidneys co-induced by Mo and Cd were more serious than separate Mo or Cd when investigating the effects and mechanism of Mo combined with Cd on duck kidneys [28]. Furthermore, there is a bare study about the prolonged toxicity of Mo combined with Cd on the testicle, especially on waterfowl. This study was carried out to examine the possible effects and the underlying injury mechanism of the co-induction of Mo and Cd on duck testicles. In this study, XOD, catalase (CAT) activities, the malonaldehyde (MDA) level, and the mRNA expression levels of metallothionein (MT) and ceruloplasmin (CP) were determined. In addition, we investigated the contents of Mo, Cd, iron (Fe), copper (Cu), Zinc (Zn), and selenium (Se) in testicle and pathological changes of testicle tissue. This research aimed at providing the toxic effects of Mo and Cd on the testicle of ducks and the underlying mechanisms of the injury.

Materials and Methods

Animals and Treatments

One hundred twenty healthy 11-day-old male ducks were randomly divided into six groups with 20 ducks in each group. Duck model of excessive exposure of Mo and Cd was developed as described in our previous publication [28]. Briefly, ducks in each group were fed with basal diet with different concentrations of Mo and Cd: control group (0 mg/kg Mo, 0 mg/kg Cd), low dietary of Mo group (LMo group, 15 mg/kg Mo), high dietary of Mo group (HMo group, 100 mg/kg Mo), Cd group (4 mg/kg Cd), LMo + Cd group (15 mg/kg Mo, 4 mg/kg Cd), and HMo + Cd group (100 mg/kg Mo, 4 mg/kg Cd). The basal diet was formulated according to the National Research Council (NRC) (1994). Ammonium heptamolybdate ([(NH4)6Mo7O24·4H2O]) and cadmium sulfate (3CdSO4·8H2O) were added into the basal diet as the sources of Mo and Cd, respectively. Ducklings were fed with duckling basal diet and duck basal diet before and after 21 days, respectively. All ducks were maintained in isolation cages at a constant temperature with good ventilation and light and were given free access to water and feed. The composition of basal diet for ducks and the contents of Mo, Cd, Cu, Zn, Fe, and Se in the basal diet and water are shown in Tables 1 and 2. The experiment lasted for 120 days. The ducks used in this study were handled and treated in accordance with the strict guiding principles of the National Institution of Health for experimental care and use of animals.

Table 1 Contents of molybdenum, cadmium, copper, zinc, iron, and selenium in the basal diet and water
Full size table
Table 2 Composition and nutrient levels in the basal diet for the ducks
Full size table

Sample Collection

After being treated for 60 and 120 days, the testicles of ten ducks from each group were removed immediately after euthanasia. Then, testicle tissues were placed into sampling tubes which were transferred to liquid nitrogen immediately. The rest of the testicles were stored at −20 °C for the production of tissue homogenate and determination of the contents of trace elements. In addition, 10 g of testicle tissue specimen on the 120th day was fixed in formalin for the production of pathological sections. All other samples were stored at −80 °C until analyzed.

Production Method of the Testicle Tissue Homogenate

One gram of testicle tissue was weighed accurately and placed in glass homogenizer (Bo Tai, Haimen, China) containing 10 mL of cold saline and then the tissue was grinded until it was completely crushed. The obtained tissue homogenate was transferred into centrifuge tubes and centrifuged at 1100×g for 10 min using refrigerated centrifuge (Heal Force, Hong Kong, China). The supernatant was transferred into other new tubes and then were stored at −20 °C.

Determination of Antioxidant Enzyme Activities

The MDA level, XOD, and CAT activities in the testicle tissue homogenate were determined according to the manufacturer’s instructions. The kits for these assays were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Determination of Trace Elements

The trace elements including Mo, Cd, Cu, Fe, Zn, and Se in the testicle were analyzed using a Shimadzu AA 680 flame atomic absorption spectrophotometer (Shimadzu, Japan) according to the method described by Xiaofei Liu [29]. All analyses were carried out according to the manufacturer’s instruction by a trained technician.

Histopathological Examination

The testicle tissue specimens at 120 days were fixed in formalin and routinely processed in paraffin. Thin sections (5 μm) of each tissue were sliced from each block and mounted on glass. Slides were stained with hematoxylin and eosin (H&E). Afterward, pathological sections were observed using an optical microscope and photographs were taken.

RNA Isolation and Primer Designing

Total RNA was isolated from testicle samples using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions and was then reverse transcribed. The resultant cDNA was synthesized using a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). The reverse transcription reaction (20 μL) was conducted in a mixture containing 2 μL of 5× DNA Eraser Buffer, 1 μL of gDNA Eraser, 1 μL of total RNA, and 6 μL of RNase-free double distilled water (ddH2O) and was then incubated for 2 min in a 42 °C environment. Next, 4 μL of 5× Prime Script Buffer 2, 1 μL of Prime Script RT Enzyme Mix I, 1 μL of RT Primer Mix, and 5 μL of RNase-free dH2O were added to the reaction solution, and the reaction was run at 37 °C for 15 min, 85 °C for 5 s, and 4 °C for 10 min. The reverse transcription products (cDNA) were stored at −20 °C for TaqMan real-time PCR. Gene-specific primers and probes of MT and CP were designed using Primer Premier software (PREMIER Biosoft International, CA, USA). The GAPDH housekeeping gene was used as an internal reference. The primer sequences and probes are shown in Table 3.

Table 3 Primers and probes used in this study
Full size table

Real-Time Quantitative Polymerase Chain Reaction with TaqMan Probe

Gene expression levels were assessed by real-time quantitative polymerase chain reaction (RT-qPCR) with TaqMan probe using ABI 7900HT (Applied Biosystems, USA) as the following profiles: stage 1: 1 cycle at 50.0 °C 2 min; stage 2: 1 cycle at 95.0 °C 5 min; stage 3: 40 cycles at 95.0 °C 15 s, 60.0 °C 1 min. At the end of PCR reactions, melt curve analyses were performed for all genes. Relative gene expression level was normalized according to the expression of GAPDH gene. Results (fold changes) were expressed as \( {2}^{-\varDelta \varDelta Ct} \) in which

$$ \varDelta \varDelta C\mathrm{t}=\left({Ct}_{\mathrm{target}}\right.-{\left.{Ct}_{\mathrm{GAPDH}}\right)}_t-\left({Ct}_{\mathrm{target}}-{\left.{Ct}_{\mathrm{GAPDH}}\right)}_C\right. $$

Cttarget and CtGAPDH are the cycle thresholds for target gene and GAPDH genes in the experimental groups, respectively, t is the treatment group, and C is the control group.

Statistical Analysis

One-way analysis of variance was used to find out the differences between groups using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Differences between the data were assessed using Tukey’s honestly significant difference (HSD) test for post hoc multiple comparisons. All statements of significance are based on the 0.05 level of probability and P < 0.01 was considered a highly significant difference. All data were presented as means ± standard deviation.

Results

Results of Antioxidant Enzyme Activities

The antioxidant enzyme activities and MDA level in the testicles of ducks were presented in Fig. 1a, b, c. At 60 days, the activities of XOD in HMo + Cd group and CAT in groups of LMo + Cd and HMo + Cd decreased significantly (P < 0.01) while the XOD activity in LMo group increased significantly (P < 0.01) compared with the control group. However, there was no significant difference (P > 0.05) in the XOD activity between groups of HMo, Cd, LMo + Cd, and control group (Fig. 1a). CAT activity in Cd group, LMo + Cd group, and HMo + Cd group decreased highly significantly (P < 0.01) compared with control group while it decreased not significantly (P > 0.05) in LMo group (Fig. 1b). Furthermore, the difference between LMo + Cd group, HMo + Cd group, and Cd group was highly significant (P < 0.01). The MDA levels in experimental groups at 60 days were not significantly changed (P > 0.05) in comparison with the control group (Fig. 1c). At 120 days, the MDA level in LMo group, HMo group, LMo + Cd group, and HMo + Cd group increased significantly (P < 0.05) compared with the control group and Cd group. However, there was no significant difference (P > 0.05) between the Cd group and control group, LMo group, and HMo group. At 120 days, the XOD activity decreased highly significantly (P < 0.01) except that of LMo group in comparison with that of the control group. High dietary of Mo combined with Cd reduced the XOD activity significantly (P < 0.05). The CAT activity in groups of Cd, LMo + Cd, and HMo + Cd was significantly lower than that of other groups (P < 0.05) while in HMo + Cd group, it decreased significantly (P < 0.05) than that in LMo + Cd group. There was no significant difference between LMo, HMo groups, and control group (P > 0.05) (Fig. 1b).

Fig. 1
figure 1

Effects of co-induction of Mo and Cd on the antioxidant enzyme activities in testicles. a, b, c The results of XOD activity, CAT activity, and MDA level, respectively. Each value presented the means ± SD. Different capital letters mean highly significant difference (P < 0.01) between groups; different lowercase letters mean significant difference (P < 0.05) between groups; the same small letters mean that there is no significant difference (P > 0.05) between groups. These designations of letters are the same for Figs. 2 and 3

Full size image

Effects of Mo, Cd, and Their Combination on the mRNA Expression Levels of MT and CP

Effects of Mo, Cd, and their co-induced effects on the mRNA levels of MT and CP were presented in Fig. 2a, b. The mRNA expression level of CP was reduced significantly (P < 0.01) in LMo + Cd group and HMo + Cd group in comparison with control group and other experimental groups at 60 and 120 days. In addition, it was significantly lower (P < 0.01) in HMo + Cd group than in LMo + Cd group at 120 days (Fig. 2a). The mRNA expression level of CP in groups of LMo, HMo, and Cd decreased significantly (P < 0.05) compared with that in control group, and it was significantly lower (P < 0.05) in Cd group than that in LMo and HMo groups at 60 days. However, the difference between the LMo, HMo groups, and control group was significant (P < 0.01) while it was not significant (P > 0.05) between Cd group and control. At 60 and 120 days, the mRNA expression level of MT in Cd group, LMo + Cd group, and HMo + Cd group showed a highly significant increase (P < 0.01) compared with that in control group and Mo-treated groups (Fig. 2b). Notably, the mRNA level of MT in Cd group was highly significantly higher than that in LMo + Cd group and HMo + Cd group (P < 0.01). There was no significant difference in the mRNA level of MT between control group and Mo groups (P > 0.05).

Fig. 2
figure 2

The mRNA expression levels of CP and MT at 60 and 120 days. a CP; b MT

Full size image

Contents of Mo, Cd, Cu, Fe, Zn, and Se in Testicles

Results of contents of Mo, Cd, Cu, Fe, Zn, and Se in testicle tissue of experimental ducks were shown in Fig. 3a–f. Less Mo was accumulated in the testicle of Cd group compared with the HMo group (P < 0.05) at 60 and 120 days (Fig. 3a). Meanwhile, the content of Mo in LMo + Cd group and HMo + Cd group showed a downtrend compared with the HMo group at 60 and 120 days. At 60 days, content of Cd in Cd group, LMo + Cd group, and HMo + Cd group was highly significantly (P < 0.01) higher than that in control group and single treated by Mo and Cd group (Fig. 3b). Content of Cu in the testicle of HMo group and HMo + Cd group was significantly lower than that of the control group at 60 and 120 days (P < 0.01 and P < 0.05, respectively) (Fig. 3c). Accumulation of Cu in the testicle of HMo + Cd group had a downtrend compared with that of HMo group and Cd group. At 60 days, the content of Fe in HMo group and LMo + Cd group was significantly (P < 0.05) lower than that in the control group while it was highly significantly (P < 0.01) decreased in HMo + Cd group (Fig. 3d). There was no significant difference (P < 0.05) between experimental groups except for HMo + Cd group and the control group at 120 days. Zn concentration decreased significantly (P < 0.05) in HMo + Cd group compared with the groups of control, LMo, and Cd at 60 days while there was no significant difference (P > 0.05) between those groups at 120 days (Fig. 3e). Deposition of Se in testicles of experimental groups had no significant change (P > 0.05) in comparison with control groups at 60 and 120 days (Fig. 3f).

Fig. 3
figure 3

Contents of Mo, Cd, Cu, Fe, Zn, and Se in testicles at 60 and 120 days

Full size image

Pathological Observation

No abnormal findings were observed in control and low dietary of Mo-treated duck testicle tissues. Congestion of blood vessel (black arrow) in the interstitial tissue, reduction of the number of spermatocyte and mature sperm in the seminiferous tubule, and sperm delay (red arrow) were observed in both HMo group (Fig. 4c) and Cd group (Fig. 4d). In addition, deciduous spermatocytes (green arrow) were also observed in HMo group and HMo + Cd group. A severe congestion in interstitial tissue was observed in the testicle tissue of LMo + Cd group. The number of spermatocyte increased with a low sperm count and sperm malformation. A part of sperms were short of tail (yellow arrow) (Fig. 4e). In HMo + Cd group, morphology of the testicles showed a sever congestion in interstitial tissue and degeneration of spermatocyte. The number of spermatocyte and sperm decreased obviously. Moreover, almost all mature sperms had abnormal form (Fig. 4f).

Fig. 4
figure 4

Histological results of the testicles in ducks. Original magnification, ×400. a control group; b LMo group; c HMo group; d Cd group; e LMo + Cd group; f HMo + Cd group. Congestion of blood vessel in the interstitial tissue (black arrow), sperm delay (red arrow), deciduous spermatocytes (green arrow), and a part of sperms which were short of tail (yellow arrow)

Full size image

Discussion

It is a widely recognized that both Mo and Cd exposure could induce oxidative stress and inhibit antioxidant enzyme activities in the body [30]. Free radical can cause DNA damage and lipid peroxidation because of its strong oxidizing power. Lipid peroxidation is an important indicator of oxidative damage induced by ROS. Numerous studies have demonstrated that MDA concentration in vivo is an indicator of lipid peroxidation level and an indirect biomarker of free radical generation [31, 32]. XOD is an enzyme containing Mo which is involved in intracellular metabolism and its activity is associated with free radical generation [3]. Raisbeck MF et al. stated high dietary of Mo-induced oxidative stress and resulted in a depression of antioxidant enzymes [6]. In this study, co-exposure to Mo and Cd reduced the XOD and CAT activities in testicle compared with single-treated groups at the end of experiment. The MDA level increased significantly except for Cd group at 120 days. Several studies have suggested that Cd causes oxidative stress and induces oxidative damage by disturbing the antioxidant defense systems [15, 33]. The increase in MDA level and inhibition of XOD activity in co-treated groups indicated that co-induction of Mo and Cd generated more free radicals, induced higher lipid peroxidation, and disturbed intracellular metabolism. Meanwhile, the change of CAT activity in combination groups suggested that co-induction of Mo and Cd disturbed antioxidant defense systems and inhibited antioxidant enzyme. In general, changes of the XOD activity, CAT activity, and MDA level in Mo + Cd groups showed that the activities of XOD in HMo+Cd group and CAT in groups of LMo+Cd, HMo+Cd decreased significantly indicated that the interaction of Mo and Cd may have by seriously inhibiting antioxidant enzyme and lipid peroxidation level in vivo. Ming Zhang et al. revealed that cadmium reduced the antioxidative stress enzyme activity that may be related to DNA damage and/or cell apoptosis of testis in human which may adversely affect spermatogenesis and male reproduction [34]. In this study, pathological sections of testicle tissue in combination-treated groups showed severe congestion in interstitial tissue, degeneration of spermatocyte, and sperm malformation. It has been described that Cd could result in cell death of male germ cells, damage Sertoli cells, and induce sperm abnormality in rodents [35–37] which is in agreement with the results of this study. The present study indicated that co-exposure to Mo and Cd might induce testicular toxicity via the production of more reactive oxygen species and the interference with antioxidant defense mechanisms more seriously. The damage of Mo combined with Cd on testicle was more serious than Mo or Cd single-treated groups. The result of histomorphology also reflected this condition similarly. The increase of XOD activity in LMo group at 60 days may be explained that XOD is a Mo enzyme; low dosage of Mo added into diet may increase XOD activity.

MT is a cysteine-rich, low molecular weight, metal-binding protein with high affinity for metal ions in biological systems which was first discovered in horse kidney [38, 39]. MT gene is one of the many metal-responsive genes. It has been proposed that Cd, Zn, and Cu induces the expression and synthesis of metallothionein, which is then stored in many organs as a Cd-MT, Zn-MT, and Cu-MT complex which are non-toxic within many organs and play the protective role against metal toxicity [40]. Furthermore, Cd has the ability to alter the mRNA expression levels of a variety of genes and their corresponding proteins possibly via linking to specific transcriptional activators and respressors [13]. Several studies demonstrated that exposure to low dosage of Cd significantly enhanced MT expression level of various species [41, 42]. Masters BA and Satarug S reported that Cd exposure could cause transcriptional activation of the MT genes [13, 43]. In this study, expression level of MT was up-regulated in combined groups but it was lower in Cd group. MT functions in Cd detoxication primarily through the high affinity binding of the metal to MT, thus sequesters Cd away from critical macromolecules [40]. In addition, MT also maintains metal homeostasis, scavenges reactive oxygen species, and regulates the expression levels of genes and tissue regeneration. Those functions maybe contribute to MT protection against metal toxicity which induced oxidative stress [44]. In the present study, lower level of up-regulation of MT in combination groups compared with Cd group was observed. Cu is an essential element and cofactor of several enzymes in organisms. Changes in Zn and Cu homeostasis have been found to be closely associated with metallothionein levels [45]. In the present study, that content of Cu decreased in combined groups might suggest that Mo + Cd treatment intensified the disturbance of Cu absorption and excretion. The deposition of Cd increased the synthesis of MT in testicle and caused the alteration in redox state and cellular essential metal homeostasis. It suggested that Mo might be involved in inhibition of MT gene transcription which regulates MT protein synthesis when ducks were exposed together to Mo and Cd.

CP is a Cu-containing protein and is an oxidase with high antioxidation capacity which is involved in scavenging oxygen free radical and protecting various organs from lipid peroxidation and other oxidative attack in extracellular [22]. The status of CP in vivo is an indicator of Cu level and distribution of free radical generation in extracellular matrix. In this study, co-induction of Mo and Cd triggered a repression of the CP expression. Content of Cu in HMo + Cd group had a downtrend compared with HMo group and Cd group, which might suggest that Mo + Cd treatment intensified the disturbance of Cu absorption and excretion of Cu. In a word, diet added with Mo indirectly increased oxidative stress to exacerbate Cd toxicity through reducing the expression of MT and CP which protected testicles from lipoperoxidation and other oxidative stress.

Toxic concentrations of Cd and Mo are known to disturb the metabolism of zinc, copper, and iron [26, 27, 46]. The homeostasis of trace elements is very necessary for the body to maintain protein structures and catalyze enzymatic reactions [47]. Nemmiche S et al. have investigated that transporters for Fe, calcium (Ca), Zn, manganese (Mn), and magnesium (Mg) have been proposed to be involved in Cd uptake in mammalian cells [48]. The results about the contents of Mo and Cd illustrated that Mo and Cd probably crossed the blood-testis barrier, and persistent low dosage of Mo and Cd led to different degrees of the accumulation of Mo and Cd in testicles of ducks that resulted in a balance disturbance of trace elements such as Cu, Fe, and Zn in testicles. In this study, Fe deposition in testicle of HMo + Cd group presented a decreasing tendency along with the whole experiment process. Chronic Mo aggravates Cd toxicity possibly by hindering influx of Fe from the systemic circulation to blood-testis barrier to alter Fe homeostasis besides disturbing the Fe absorption.

Zn is an essential micronutrient and component of more than 300 enzymes involved in cellular metabolism and expression of genes. Bersényi reported that Mo can reduce the contents of Zn in the liver and muscle [7]. Cd can replace Cu and Zn to bind with MT which leads to Zn being redistributed in plasma/serum [49]. The result of the present study showed a significant decrease in the content of Zn in Mo + Cd groups, which indicated that combined action of Mo and Cd may reduce the content of Zn in duck testicles.

Se is also an essential trace element and component of several antioxidant enzymes such as glutathione peroxidases (GPx) and thioredoxin reductase [48]. Se deficiency-induced oxidative stress and antioxidant protection of selenoproteins such as GPx are perceived to be involved in the pathogenesis of Se deficiency-related diseases [50]. Cardin et al. demonstrated that interactions between SeO4 2− and MoO4 2− were competitive for a common intestinal transport mechanism in chick small intestine [51]. It has been investigated that metabolism of Se was deranged to against cadmium toxicity. In Chen X’s study, the toxic effect of cadmium can be decreased because of the protection of Se [52]. Ellingsen et al. and Wasowicz W et al. demonstrated that Se concentration and Cd concentration was inversed in whole blood and plasma [53, 54]. But no obvious changes were observed in the deposition of Se in testicles in our study. This result agreed with those obtained by White CL and Abdel Rahim AG, who demonstrated that dietary with Cd (1–5 mg/kg diet) and Mo (0.3–50 mg/kg diet) had no significant effects on Se [55, 56]. The dosage of Mo and Cd implemented in the experiment was possibly not enough to affect the metabolism of Se.

Conclusion

In conclusion, our results suggested that Mo might exacerbate Cd toxic by increasing lipid peroxidation, inhibiting enzyme activities, synergistic aggravating testicular degeneration, repressing of metal, and disturbing trace element homeostasis of testis in vivo. Mo-Cd interaction may have a synergistic effect on testicular toxicity.