Chlorella vulgaris or Spirulina platensis mitigate lead acetate-induced testicular oxidative stress and apoptosis with regard to androgen receptor expression in rats

The current research was constructed to throw the light on the protective possibility of Chlorella vulgaris (C. vulgaris) and Spirulina platensis (S. platensis) against lead acetate-promoted testicular dysfunction in male rats. Forty rats were classified into four groups: (i) control, (ii) rats received lead acetate (30 mg/kg bw), (iii) rats concomitantly received lead acetate and C. vulgaris (300 mg/kg bw), (vi) rats were simultaneously treated with lead acetate and S. platensis (300 mg/kg bw) via oral gavage for 8 weeks. Lead acetate promoted testicular injury as expressed with fall in reproductive organ weights and gonadosomatic index (GSI). Lead acetate disrupted spermatogenesis as indicated by sperm cell count reduction and increased sperm malformation percentage. Lead acetate-deteriorated steroidogenesis is evoked by minimized serum testosterone along with maximized follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels. Testicular oxidative, inflammatory, and apoptotic cascades are revealed by elevated acid phosphatase (ACP) and sorbitol dehydrogenase (SDH) serum leakage, declined testicular total antioxidative capacity (TAC) with elevated total oxidative capacity (TOC), tumor necrosis factor alpha (TNF-α), caspase-3 levels, lessened androgen receptor (AR) expression, and histopathological lesions against control. Our research highlights that C. vulgaris or S. platensis therapy can modulate lead acetate-promoted testicular dysfunction via their antioxidant activity as expressed by elevated TAC and reduced TOC, immunomodulatory effect as indicated by lessened TNF-α level, and anti-apoptotic potential that was revealed by minimized caspase-3 levels. As well as restoration of testicular histoarchitecture, androgen receptor, steroidogenesis, and spermatogenesis were detected with better impacts to S. platensis comparing with C. vulgaris. Therefore, further clinical trials are needed to test S. platensis and C. vulgaris as a promising candidate in treating male infertility.


Introduction
Male fertility could be seriously affected by environmental pollution (Sukhn et al. 2018). Lead (Pb) is one of the heavy metals and possesses non-biodegradable nature favoring its environmental accumulation with increasing hazards (Goto et al. 2020). Pb exposure is represented in food, air, water, paint, ceramics, cosmetics, and leaded gasoline as well as occupational sources (Adela et al. 2012;Ali et al. 2020).
Previous researches reported Pb-promoted reproductive system oxidative burden along with subsequently disrupted steroidogenesis and spermatogenesis in humans and animals (Haw et al. 2012;Udefa et al. 2020). Androgen receptor (AR) has fundamental roles in spermatogenesis and male fertility. Testosterone acts directly on both Sertoli cells and spermatogenic cells to enhance spermatogenesis via AR that requires normal spermatid differentiation and liberation from seminiferous epithelium (Hazra et al. 2013). Moreover, testosterone supports Leydig cell development and functions via AR and motivates growth and function of male reproductive system. Luteinizing hormone (LH) motivates steroidogenesis via LH receptor on the Leydig cells (Wang et al. 2009;Wang et al. 2018). However, follicle stimulating hormones (FSH) needed for normal functions and number of Sertoli cells that support germ cells' mitotic Responsible Editor: Mohamed M. Abdel-Daim activity through spermatogenesis stages (Oduwole et al. 2018). In this regard, Mokhtari and Zanboori (2011) validated that daily oral treating with lead acetate solution (50 and 100 mg/kg) for 28 days minimized sera testosterone and affected on sexual behavior, while the lower dose (25 mg/kg) did not exert any significant difference. Further, oral administration of lead acetate (60 mg/kg) for 28 days decreased weight of testis, sperm count, motility, and viability percentage (Offor et al. 2017). Many researches provoked the nutritional and therapeutic importance of microalgae (Camacho et al. 2019;Sajilata et al. 2008). Chlorella vulgaris and Spirulina platensis safety is well pronounced by the FDA comparing with other algae species (Bauer et al. 2017). Chlorella vulgaris (C. vulgaris) is a microalgae distributed in freshwater and rich in antioxidants, chlorophyll pigments, polysaccharides, amino acids, calcium, phosphorus, iron, iodine, manganese, omega-3, omega-6 polyunsaturated fatty acids, carotene, and vitamins C and E (Rahimnejad et al. 2017). Importantly, C. vulgaris reversed cancer in human lung cancer H1299 (Wang et al. 2010), mercury-induced renal toxicity (Blas-Valdivia et al. 2010), expression of brain c-fos in forced swimming stress (Souza Queiroz et al. 2016), cyclophosphamide-promoted testicular toxicity (Afkhami-Ardakani et al. 2018), diazinon-caused hepatic, splenic oxidative, and inflammatory burden (Abdelhamid et al. 2020).

Laboratory animals
This research was carried using forty male Wistar albino rats with 130-180 g, 4-5 weeks old. Rats were purchased from Lab. Animal House, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. Rats were acclimated for 2 weeks with food and drinking water supplied ad libitum. The diet composed of total protein (230 g/kg), corn starch (676 g/kg), soybean oil (50 g/kg), vitamin mix (4 g/kg), and mineral mix (40 g/kg) (da Ramos et al. 2010). Rats housed in stainless steel cages (545 × 395 × 200 mm 3 ) with sawdust bedding and maintained at healthy surroundings with 12h light-dark cycle, relative humidity (40%), and temperature (23 ± 2°C). Study protocol was complied with the ethical guidelines for laboratory animal use at the Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt (approval NO. 2021002).

Material list
Lead acetate trihydrate was obtained from Oxford Lab. Co., Mumbai, India (Catalog# 400002). C. vulgaris and S. platensis-lyophilized powders were obtained from national research center, Cairo, Egypt. The powder was dissolved in distilled water prior to rat's administration.

Study protocol
Rats were divided into four groups, 10 rats each and classified as follows: (i) Control: rats were administered only distilled water as vehicle. (ii) Lead: rats were given lead acetate (30 mg/kg b.w) of 1% solution per os (Elgawish and Abdelrazek 2014). (iii) Lead + C. vulgaris: rats concomitantly orally received lead acetate and C. vulgaris (300 mg/kg bw) by gavage tube (Zainul Azlan et al. 2020). (iv) Lead + S. platensis: rats were simultaneously orally treated with lead and S. platensis (300 mg/kg bw) by gavage tube (Bashandy et al. 2016). The experiment was continued for 8 weeks.

Dose justification
Several literatures demonstrated that lead acetate induced testicular toxicity in doses that are higher and lower than our used regimen. Oral administration of lead acetate at doses of 0.1% lead acetate in drinking water for 60 days (El-Sayed et al. 2010) and 0.05 and 0.15% for 55 days (Reshma Anjum and Sreenivasula Reddy 2015). A total of 0.1%, 0.25%, 0.5%, and 1.0% lead acetate (w/v) in double-distilled drinking water for 6 weeks (Li et al. 2018), 60 mg/kg for 28 days (Offor et al. 2019), and 100 mg/kg for 1 month (Abdrabou et al. 2019) induced testicular oxidative burden and lowered sperm quality.
The doses of C. vulgaris and S. platensis are safe and lower than LD50 which is higher than 5000 mg/kg for C. vulgaris (Himuro et al. 2014) and S. platensis (Ama Moor et al. 2017).

Blood and testicular tissue processing
After 8 weeks, each rat was weighted for determination of body weight gain. Then, rats were euthanized under xylazine effect. Blood samples were drawn from retro-orbital venous plexus, centrifuged at 1200×g for 20 min. Then, sera were harvested and stored at − 20°C for enzymes and hormonal analysis. After that, rats were decapitated and both testes, epididymis, seminal vesicles, and prostate glands were dissected and weighed. Gonadosomatic index (GSI) was calculated through dividing gonad weight by body weight and then multiplying by 100, where gonad weight = (weight of the right testis + weight of the left testis) / 2 (Latif et al. 2008). One testis was homogenized in phosphate-buffered saline (PBS), pH = 7.4 via a Teflon homogenizer (Glass Col homogenizer system, Vernon hills, USA), then harvested filtrate used for assessment of oxidative stress, inflammatory, and apoptosis markers. Additionally, another testis was prepared for histological and immunohistochemical screening.

Semen characteristics
The caudal part of epididymis was cut and squeezed in order to harvest the epididymal sperms. The concentration of sperms was carried post-dilution of epididymal content by sodium citrate dihydrate solution (2.9%) (Turk et al. 2007). The sperm cells count in 1 mm 3 of the suspension was detected by hemocytometer. Then, the total sperms/ epididymal tail were calculated = sperm number in 1 mm 3 of diluted suspension × dilution rate × 1 ml (total volume of the epididymal suspension) (Osama et al. 2019). Sperm abnormalities were assessed via slide smearing one drop of epididymal content, and then stained by Eosin Nigrosine. Then, a total number of 200 sperm were checked by a microscope for sperm abnormality such as bananashaped, straight, and other abnormalities (Okamura et al. 2005).

Testicular histopathology
Testicular specimens from study groups were immersed in 10% neutral buffered formalin, dehydrated in ascending concentrations of ethyl alcohol (70-100%), immersed in xylene, and processed formalin-fixed paraffin-embedded (FFPE) sections, and finally cut into 4 μm sections using a microtome. Further, one FFPE specimen was stained with hematoxylin and eosin (H&E) for microscopic examination and imaged each section using a light microscope (Axio Imager.A2, ZEISS).

Testicular immunohistochemistry
IHC protocol was implemented following the method of Williams et al. (2015). Formalin-fixed paraffin-embedded specimens were cut into 4-μm sections. After deparaffinization, sections were heated in Tris/HCL buffer (pH 9.0) for 20 min at room temperature for antigen retrieval. After that, sections were incubated with 0.3% H 2 O 2 in absolute methanol for 30 min. Then, incubation with primary antibody for androgen receptor (Thermo Fisher Scientific Co., USA, Catalog# MA1-150) and secondary antibodies as biotinylated polyvalent (Thermo Scientific Co., UK, Catalog# 32230) were performed. All slides are lightly counterstained with hematoxylin for 30 s prior to dehydration and mounting. Each section was captured using a light microscope (Axio Imager.A2, ZEISS). Testicular positively stained area percentage was quantified via ImageJ software per slide after light background subtraction.

Statistical analysis
Values were analyzed by the help of SPSS version 20. Results were expressed as mean ± SE. One-way analysis of variance (ANOVA) was conducted for comparison between study groups followed by Duncan multiple rang test (post hoc test). Significance was recorded at P < 0.01.

Results
Effect of C. vulgaris or S. platensis supplementation on body weight gain and accessory reproductive organ weights in lead acetate-exposed rats Lead acetate-intoxicated rats demonstrated significant (P < 0.01) reduction in body weight gain, weights of the testis, epididymis, prostate gland, seminal vesicle, and GSI against control. On contrary, co-therapy of C. vulgaris or S. platensis (300 mg/kg b.w) with lead acetate for 8 weeks increased weight gain and sex organ weights compared with lead acetate group. S. platensis could restore weight of prostate gland, seminal vesicle, and GSI to normal control values (Tables 1 and 2).
Effect of C. vulgaris or S. platensis supplementation on semen characteristics in lead acetate-exposed rats In comparison with control, lead acetate lowered sperm count 60.18 ± 0.61 vs. 81.62 ± 1.14 in line with elevation in percentage of total malformation sperms 40.05 ± 0.52 vs. 10 ± 0.3, while C. vulgaris or S. platensis supplementation with lead acetate increased sperm count and reduced total malformation sperm percentage comparing with lead acetate-exposed rats. Comparing S. platensis versus C. vulgaris-treated group, higher values of sperm count (77.91 ± 0.67 vs. 71.27 ± 0.68) and lower percentage of total malformation sperms (25.36 ± 0.39 vs. 30.98 ± 0.55) were observed (Table 3).

Testicular histopathological findings
H&E testicular sections of control group delineated seminiferous tubules (ST) with well-defined regular basement membrane and germinal epithelium. In addition to presence of regularly arranged spermatogenic cells and uniform maturation of spermatozoa formation in ST, normally organized Leydig cells are seen (Fig. 1a, b). In lead acetate, intoxicated testis revealed that ST suffered from thinned irregular basement membrane, apparent disarrangement, and damage, in addition to spermatogenesis distribution with no evidence of spermatozoa formation and hyperplasia of Leydig cells (Fig. 1c, d).
On the opposite, testicular section from C. vulgaris-treated rats exhibited that ST are regular in shape and outline and closely backed. Spermatogenesis is maintained in some tubules (50% of tubules), while others show vacuolization with evidence of disturbed spermatogenesis in line with absence of spermatozoa formation. Moreover, interstitium shows Leydig cell hyperplasia (Fig. 1e, f).
Regarding S. platensis-supplemented rats, seminiferous tubules are evenly spaced, uniform in shape, with regular outlines. Uniform spermatogenesis and spermatozoa formation are evident in most of tubules, in addition to diminution of Leydig cell hyperplasia (Fig. 1g, h). Collectively, supplementation of S. platensis significantly reversed the normal testicular histology with dynamic spermatogenesis implying the antioxidant, anti-inflammatory, and antiapoptotic potency

Testicular immunohistochemistry
Nuclear AR expression pattern was strong in spermatogenic cells and Leydig cells in control group (Fig. 2a, b). However, lead acetate group showed weak staining intensity of the expression area of AR in nuclei of spermatogenic cells and moderate staining intensity in Leydig cells (Fig. 2c, d).
Combined treating of C. vulgaris with lead acetate was able to moderately restore AR testicular loss as outlined by moderate expression of AR in spermatogenic cells in uniform tubule, while in injured tubules, there is weak expression of AR in nuclei of remaining spermatogenic cells. There is strong AR expression in Leydig cells (Fig. 2e, f).
Furthermore, co-therapy of S. platensis with lead acetate can restore normal AR testicular expression as pronounced by moderate to strong nuclear expression of AR in nuclei of spermatogenic cell. As well as, there is strong AR expression in Leydig cells (Fig. 2g, h).
Quantification of these findings pronounced that administration of lead acetate significantly downregulated AR expression against control rats. However, combined therapy of S. platensis or C. vulgaris with lead acetate significantly upregulated AR expression comparing with lead acetate-treated rats with the best ameliorative effect for S. platensis (Fig. 3).

Discussion
Lead is one of the male reproductive toxicants in humans and animals leading to testicular dysfunction and infertility (Al-Megrin et al. 2020). In the running study, lead acetate promoted reduction in body weight gain comparing with control that may be due to lead acetate-prompted reactive oxygen species (ROS) generation along with antioxidant downregulation favoring oxidative stress and inflammatory response (Albarakati et al. 2020;Oyagbemi et al. 2015). Proinflammatory cytokine expression such as TNF-α enhanced apoptosis, muscle wasting, and weight gain loss ( . Thereby, lessened GSI may imply loss of germ cell. We herein explored that lead acetate significantly (p < 0.01) elevated ACP and SDH activity in sera against control. ACP is secreted by Sertoli cells and detected in lysosomes of Leydig cellmediated lysis of abnormal sperms (Olayinka and Ore 2015), while SDH is distributed in spermatogenic cells of seminiferous tubules and correlated with germ cell maturation via energy supply by conversion of sorbitol to fructose (Tripathi et al. 2016). Remarkably, ROS-induced cellular injury could be associated with enzyme liberation into the circulation (Hassan et al. 2013). Hence, elevated serum ACP and SDH can imply the testicular oxidative burden promoted by lead acetate. Concerning hormonal profile, lead acetate disrupted steroidogenesis that confirmed by minimized serum testosterone levels along with maximized FSH, LH levels, and downregulated nuclear AR expression in spermatogenic cells and Leydig cells against control. Fall in testosterone level can attribute to lead-promoted testicular oxidative and immune response via macrophage-expressed TNF-α consistently with Allouche et al. (2009) and Udefa et al. (2020). These are similar to our findings that revealed decline in testicular TAC with elevation in TOC and TNF-α levels. These may account to lead acetate counteracted antioxidant enzyme activities through targeting SH groups and/or metal cofactors of antioxidants (Patra et al. 2011). As well as, lead acetate downregulated Nfe212 gene expression-promoted downregulation of antioxidant enzyme expression (Kabel and Elkhoely 2017). These oxidative and inflammatory responses mediated steroidogenesis inhibition in Leydig cells whereby TNF-α lessened testicular 3b-, 17b-17ß-hydroxysteroid dehydrogenase activity and inhibited testosterone synthesis (Hales 2002;Hong et al. 2004). Consequently, AR damage occurred in response to testicular oxidative burden (Chang et al. 2019;Yao et al. 2007). AR possessed a fundamental role in testosterone feedback regulation via autocrine action on Leydig cells, and gonadotropin-releasing hormone (GnRH) reduction resulted in pituitary LH inhibition (Amory and Bremner Within the same column, means ± SE with different lowercase letters are significantly (P < 0.01) differed Environ Sci Pollut Res Fig. 1 Graphical photomicrographs of testicular sections from rats treated with C. vulgaris or S. platensis against lead acetate revealing (H&E) between the experimental groups (scale bar: 50 μm, 100 μm); a, b control group delineated seminiferous tubules (ST) with well-defined regular basement membrane and germinal epithelium (black arrows, a; red arrows, b). In addition to presence of regularly arranged spermatogenic cells and uniform maturation of spermatozoa formation in ST (red arrow, a; arrow head, b). As well as, normally organized Leydig cells are seen (arrow heads, a; blue arrows, b). c, d Lead group outlined ST suffered from thinned irregular basement membrane, apparent disarrangement, and damage (black arrows, c; red arrows, d).
In addition to spermatogenesis distribution with no evidence of spermatozoa formation (red arrow, c; arrow head, d) and hyperplasia of Leydig cells (arrow heads, c; blue arrows, d). e, f Lead + C. vulgaris group exhibited ST with regular shape and outline, and closely backed (black arrows, e). Spermatogenesis is maintained in some tubules (50% of tubules) (blue arrows, e), while others show vacuolization (arrow heads, e) with evidence of disturbed spermatogenesis in line with absence of spermatozoa formation (red arrow, e; black arrow, f). Moreover, interstitium shows Leydig cell hyperplasia (green arrows, e; blue arrows, f). g, h Lead + S. platensis group showed that seminiferous tubules are evenly spaced, uniform in shape, with regular outlines (black arrows, g; red arrows, h). Spermatogenic cells (arrow head, h) revealed that uniform spermatogenesis and spermatozoa formation are evident in most of tubules (red arrow, g; black arrows, h). In addition, diminution of Leydig cell hyperplasia (arrow heads, g; blue arrows, h) 2001). Further, downregulation of AR expression in highfat diet-supplemented mice was associated with minimization in testosterone level and infertility (Fan et al. 2015). Therefore, regressed AR expression may raise LH and FSH release to compensate the testicular disruptions and testosterone depletion. Furthermore, TNF-αmediated disruption of mitochondrial membrane that accompanied by cytochrome c release, caspase-9, and caspase-3 activation resulted in apoptosis (Zhou et al. 2019). Moreover, lead acetate in spermatogenic cells upregulated caspase-3 and Bax in line with downregulated Bcl2 (Hassan et al. 2019). Consequently, further germ cell apoptosis, sperm membrane perturbation, and DNA fragmentation are detected (Henkel et al. 2010). Currently, these findings were confirmed by the testicular architectural deteriorations that revealed irregular shape of seminiferous tubules with no evidence of spermatozoa formation and Leydig cell hyperplasia. Totally, lower testosterone level, AR expression, and higher sperm abnormalities revealed the oxidative, inflammatory, and apoptosis cascade in lead acetateintoxicated rats. Also, sperm count and sex organ weights were indicative for testosterone role in differentiation of spermatogenic cell mass and sperm production. On the contrary, co-therapy of lead acetate with C. vulgaris or S. platensis significantly (p < 0.01) attenuated lead acetatepromoted testicular dysfunction. Concerning the reproductive defensive mechanism of C. vulgaris was supported by our data such as restoration of weight gain, sex organ weight, sperm cell count, sperm malformation percentage, serum Fig. 2 Graphical photomicrographs of testicular sections from rats treated with C. vulgaris or S. platensis against lead acetate revealing androgen receptor immunostaining area % between the experimental groups (scale bar: 50 μm, 100 μm); a, b control group outlined that nuclear AR expression pattern was strong in spermatogenic cells (black arrows, b) and Leydig cells (red arrow, b). c, d Lead group exhibited weak staining intensity of the expression area of AR in nuclei of spermatogenic cells (black arrow) and moderate staining intensity in Leydig cells (red arrow, d). e, f Lead + C. vulgaris group revealed moderate expression of AR in spermatogenic cells in uniform tubule (upper right; black arrow, f), while in injured tubules, there is weak expression of AR in nuclei of remaining spermatogenic cells (red arrow, f). There is strong AR expression in Leydig cells (arrow heads, f). g, h Lead + S. platensis group delineated moderate to strong nuclear expression of AR in nuclei of spermatogenic cell (black arrow, h). As well as, there is strong AR expression in Leydig cells (arrow heads, h) ACP, SDH activity, testosterone, FSH, LH levels, and upregulation of testicular AR expression. AS well as, upregulation in testicular TAC, downregulation in TOC, TNF-α, and caspase-3 expression were recorded agreeing with Abdelhamid et al. (2020), Mustafa (2015), Raj et al. (2013), andVijayavel et al. (2007). These findings emphasized many previously published researches. Feeding of C. vulgaris enriched diet 0, 3, or 5% upregulated liver and plasma antioxidant enzymes in cadmium-exposed rats for 10 weeks (Son et al. 2009). Moreover, C. vulgaris (150 mg/kg b.w) ameliorated lipid peroxidation, DNA damage, TNF-α expression, as well as restored antioxidant enzymes in STZ diabetic rats (Aizzat et al. 2010). Furthermore, C. vulgaris supplementation (50 mg/kg bw) counteracted aging-promoted oxidative stress in C57BL/6 mice for 8 weeks (Aliahmat et al. 2012). Similarly, Mustafa (2015) proposed that co-treating of C. vulgaris (50 mg/kg bw) with lead acetate 200 mg/l for 12 weeks was able to restore thickness of germinal epithelium diameter of seminiferous tubules and sperm structure that is due to its chelating and antioxidant potential. Recently, C. vulgaris dietary supplementation upregulated hepatic and ovarian antioxidant gene expression, as well as body performance enhancement in New Zealand White rabbits (Sikiru et al. 2019). These curative effects of C. vulgaris may attribute to its antioxidant content such as lycopene, eicosatetraenoic, docosahexaenoic acid, astaxanthin, polysaccharides, polyphenols, lutein and fucoxanthin, omega 3, omega 6, reduced glutathione, chlorophyll, and vitamin C, E (Rahimnejad et al. 2017;Renju et al. 2014;Vijayavel et al. 2007). Accordingly, lycopene upregulated the epididymal AR expression in polychlorinated biphenyl-intoxicated rats (Raj et al. 2013). Eicosapentaenoic and docosahexaenoic acid counteracted cisplatin-promoted testicular and seminal toxicity in rats (Ciftci et al. 2014). Further, astaxanthin ameliorated testicular and sex hormone alterations in high fructose-supplemented rats (Dokumacioglu et al. 2018). In vitro and in vivo, C. vulgaris polysaccharides evoked antioxidant possibilities via scavenging superoxide, DPPH, and hydroxyl radical-scavenging (Yu et al. 2019). Furthermore, omega-3 fatty acids pronounced antioxidant and anti-inflammatory activities in a rat model of stress-induced liver injury (Ali and Rifaai 2019). Collectively, C. vulgaris can restore steroidogenesis in Leydig cells and testosterone-mediated spermatogenesis in seminiferous tubules via AR that was indicated by increased sperm count. As well as, restoration of testosterone enhanced sex organ growth that was revealed by their weight elevation in our study.
In this context, oral S. platensis administration reserved weight gain and reproductive organ weights that may relate to direct lead adsorption by S. platensis favoring rapid body lead elimination (Banji et al. 2013). As well as, S. platensis nutritional ingredient such as protein, essential fatty acids, vitamins, iron, zinc, polysaccharides, phenols, carotenoids, chlorophyll, C-phycocyanin pigment enriched the body (Hosseini et al. 2013). More confirmation in our work, S. platensis increased sperm cell count, lessened sperm malformation percentage, decreased testicular ACP, SDH leakage, and restored testosterone, FSH, LH alterations. Also, S. platensis upregulated testicular AR, TAC along with downregulated TOC, TNF-α, caspase-3 expression agreeing with many previous researches (Abdel -Daim et al. 2019;Bashandy et al. 2016;Esener et al. 2017;Sadek et al. 2017). Oral pretreatment with S. platensis (500 and 1000 mg/kg bw) 1 h prior to deltamethrin exposure for 5 days improved hepatic and renal antioxidant prospect (Abdel- Daim et al. 2013). Moreover, twice weekly oral supplementation of S. platensis (500 mg/kg b.w) for 8 weeks exhibited hepatic and pancreatic β cell antioxidant, anti-inflammatory, and anti-apoptotic potentials in STZ-diabetic rats (Sadek et al. 2017). Furthermore, administration of S. platensis (300 mg/kg bw) for 4 weeks could alleviate sperm quality and sex hormone alterations in furan-treated rats (Abd El-Hakim et al. 2018). Similarly, another strain, S. maxima, was able to up-reregulate testicular antioxidant enzymes and downregulate caspase-3 in lead acetate-exposed rats (Abdrabou et al. 2019). These ameliorative events may attribute to antioxidant active ingredients of S. platensis. Phenols scavenged ROS and improved testicular antioxidant status in alloxan-diabetic rats (Roy et al. 2015). Specifically, C-phycocyanin is detected in Spirulina only and revealed antioxidant and antiapoptotic potentials in cardiomyocytes of doxorubicin-treated rats (Khan et al. 2006) and in paraquat-promoted acute lung injury in rats (Sun et al. 2011). Chlorophyll counteracted sodium nitrateinduced renal and hepatic oxidative burden in rats (Suparmi et al. 2016). Moreover, vitamin C, E influenced testicular antioxidant attributes against lead in rats (Ayinde et al. 2012).These are consolidated by architectural restoration as evident by presence of uniform seminiferous tubules and Fig. 3 Column chart revealing the androgen receptor immunostaining area % in rats treated with C. vulgaris or S. platensis against lead acetate. Data are expressed as mean ± SE. Means with different superscripts are significantly (P < 0.01) different spermatozoa. Therefore, the antioxidant and antiinflammatory potency of S. platensis can reverse lead acetate-fostered oxidants, TNF-α, and caspase 3 expressionmediated testicular apoptosis, AR damage, and steroidogenesis inhibition in Leydig cells. Hence, testosterone and AR restoration boosted spermatogenesis that are expressed by remarkably raised sperm count and sex organ weight.

Conclusion
The above-mentioned data can consolidate the protective potential of oral supplementation of C. vulgaris or S. platensis (300 mg/kg bw) for 8 weeks in modulating lead acetateboosted testicular dysfunction via antioxidant, immunomodulatory, anti-apoptotic potentials enhancing testicular cells, and androgen receptor restoration with superior impacts to S. platensis comparing with C. vulgaris. Totally, regenerated testicular cells can restore steroidogenesis producing testosterone that mediated spermatogenesis in seminiferous tubules with respect to AR.
Abbreviations GSI, gonadosomatic index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; ACP, acid phosphatase; SDH, sorbitol dehydrogenase; TAC, total antioxidative capacity; TOC, total oxidative capacity; TNF-α, tumor necrosis factor alpha; AR, androgen receptor Data availability All required data will be available with the corresponding author upon request.

Declarations
Ethics approval Study protocol was complied with the ethical guidelines for laboratory animal use at the Faculty of Veterinary Medicine, Suez Canal University, Egypt (approval NO. 2021002).
Consent to participate Not applicable as the study did not include human subject.

Consent for publication All authors approve this submission.
Conflict of interest The authors declare that they have no conflict of interest.