The ameliorative effect of Lactobacillus paracasei BEJ01 against FB1 induced spermatogenesis disturbance, testicular oxidative stress and histopathological damage

Abstract Fumonisin B1 (FB1) is a possible carcinogenic molecule for humans as classified by the International Agency for Research on Cancer (IARC) in 2B group. In livestock, it is responsible for several mycotoxicoses and economic losses. Lactobacillus strains, inhabitants of a wide range of foodstuffs and the gastrointestinal tract, are generally recognized as safe (GRAS). Thus, the aim of this work was to evaluate the protective effect of Lactobacillus paracasei (LP) against FB1-induced reprotoxicities including testicular histopathology, sperm quality disturbance, and testosterone level reduction. Pubescent mice were divided randomly into four groups and treated for 10 days. Group 1: Control; Group 2: FB1 (100 μg/kg b.w); Group 3: LP (2 × 109 CFU/kg b.w); Group 4: LP (2 × 109 CFU/kg b.w) and FB1 (100 μg/kg b.w). After the end of the treatment, animals were sacrificed. Plasma, epididymis, and testis were collected for reproductive system studies. Our results showed that FB1 altered epididymal sperm quality, generated oxidative stress, and induced histological alterations. Interestingly, these deleterious effects have been counteracted by the LP administration in mice. In conclusion, LP was able to prevent FB1-reproductive system damage in BALB/c mice and could be validated as an anti-caking agent in an animal FB1-contaminated diet.


Introduction
Fumonisin B1 (FB1) is one of the prevalent mycotoxins, produced by toxigenic fungi such as Fusarium verticilloides and Fusarium moniliforme. It is naturally present in a large variety of food and foodstuff with high incidence. In fact, FB1 is the most widely distributed Fusarium mycotoxin in North Africa (74%). In Tunisia, Jedidi et al. (2021) detected high fumonisin levels in wheat (20.83%), barley (40%), and maize (57.14%). These levels exceeded European limits and hence, suggested a risk for Tunisian cereals and consumers. This toxin was linked to human cancers (IARC 2002). Experimentally, FB1 toxicity can be traced to its analogy to sphingoid bases and the inhibition of ceramide synthase, a key enzyme in the biosynthesis of sphingolipids (Dellafiora et al. 2018).
Indeed, a large body of evidence has demonstrated that FB1 leads to several in vivo and in vitro biological alterations. In fact, Cao et al (2020) reported that FB1 affected the intestines, the first defense line of microorganisms against toxins, and caused homeostasis imbalance via the disturbance of Cyto P450 activity. Other studies demonstrated that liver and kidney, specific hosts for FB1 accumulation, could also be damaged (Rumora et al. 2007;Demirel et al. 2015;Hou et al. 2021). Moreover, FB1 was found to generate oxidative stress markers (Baldissera et al. 2020), neurochemistry toxicity (Gbore 2010), seizures, as well as hyperexcitability (Poersch et al. 2015) in the brain. Several studies reported also that FB1 induced immunotoxicity (Abb es et al. 2016), apoptosis (Kim et al. 2018;Zhang et al. 2018), and genotoxicity (M€ uller et al. 2012;Pinhão et al. 2020).
Furthermore, dietary exposure to FB1 may induce several physiological responses in the reproductive system. Regarding the female reproductive function, FB1 has been reported to affect granulosa cell proliferation and steroid production in swine and cattle species (Cortinovis et al 2014;Albonico et al 2016). In addition, FB1 has also been reported to have some adverse effects on male reproduction in rats (Voss et al. 1996) and pigs (Harrison et al. 1990). Moreover, many reports have investigated FB1's effects on sperm quality, mobility, and daily sperm production (Gbore and Egbunike 2008;Minervini et al. 2010;Egbunike 2010a, 2010b). In fact, it has been found that elevated ceramide levels were associated with apoptosis activation in Leydig cells and reduced testosterone production (Lu et al. 2003;Szab o-Fodor et al. 2015).
Despite the highly damaging effects of FB1 on various biological systems, only recently high concern has been attributed to mycotoxins bioavailability reduction in commodities using Lactobacillus strains in vitro and in vivo studies regarding their abilities to release antifungal substances, which could adsorb or degrade mycotoxins (Hassan and Bullerman 2008;Honor e et al. 2016). In the same way, a recent study from our laboratory showed that treatment with a select strain of Lactobacillus paracasei (LP), i.e. LPBEJ01, helped to protect BALB/c mice against hepatic and nephrotic damage that could be induced by FB1 (Ezdini et al. 2020). However, the underlying mechanisms of protection against FB1 nephrotoxicity are still none elucidated.
Therefore, the present study is a follow-up report to examine whether the use of this LP strain could impart protective effects against the adverse effects of FB1 on sperm parameters, daily sperm production, efficiency, testicular oxidative stress generation, and histopathology of the testis.

Chemicals and bacteria
Pure Fumonisin B1 (>98% by HPLC) was obtained from Sigma Aldrich and stored in DMSO at À80 C. The working solution of 100 lg/ml of FB1 in phosphate-buffered saline (PBS, pH 7) was prepared from the stock solution. The bacterial strain, Lactobacillus paracasei BEJ01 (LP BEJ01), was isolated from Tunisian artisanal butter (Abb es et al. 2013). All the other chemicals used in this study were of analytical grade.

Experimental animals
In this study, 40 male pubescent mice BALB/c (SEXUAL, St. Doulchard, France) were used (average body weight: 25 ± 0.3 g; age: 6 weeks old). These mice were given standard industrial granules mycotoxin free after check using HPLC and FB1 standard and drinking water ad libitum. They were divided into four treatment groups, of 10 mice each. Before treatment, the mice were acclimated for one week under the following conditions: temperature of 23-25 C, relative humidity (45%), and 12 h light/12 h dark cycle. The experimental protocols were approved with the guidelines of the Ethical Committee of the High Institute of Biotechnology of Monastir, Tunisia.
The specific doses of LP and FB1 used here were based on our previous report (Ezdini et al. 2020). In fact, FB1 (100 mg/kg b.w) showed a toxic effect on mice. However, no biological damage related to LP was shown. Each day, fresh LP and FB1 stock solutions were prepared to avoid any issue of LP non-viability or potential degradation of FB1. For the fourth group, mice were co-treated with FB1 and LP. The LP and FB1 mixture were prepared accordingly so that the final desired concentrations could be achieved in the fixed 200 ml volume. Dosing occurred at 8 AM each day for 10 days. 24 h after the final treatment, all mice were sacrificed and blood was collected by heart puncture. Plasma was obtained after cold centrifugation serving for the testosterone assay. The epididymis was freshly excised and used for sperm analysis. The right testis was stored at À80 C for the oxidative markers assay while the left testis was taken for the histological study.

Histology
Testes were immediately fixed in PFA 4%, followed by dehydration in increasing serial concentrations of ethanol then clarification in n-butanol. Samples were embedded in paraffin wax. Sections of 5 mm thick were made using a microtome. Ribbons containing serial sections of the organs were mounted on slides and stained with H&E before microscopic examination.

Sperm count and mobility
The epididymal sperm count was studied using the method of Linder et al (1986). The epididymis tail was excised and chopped into 1 ml of the pre-incubated RPMI medium at 32 C. The mixture was incubated for 2 min at 32 C and diluted to 1/10. A volume of 20 ml was placed on the hemocytometer cell. All spermatozoids (mobile and non-mobile) were counted in order to determine the percentage of mobility. A microscopic examination was done at a magnification of Â40. 3 replicas from each sample were carried out.

Daily sperm production
The daily sperm production (DSP) was assessed following the research protocol of Kyjovska et al. (2013) with some modifications. Indeed, the right testis was harvested after the sacrifice. Its half was homogenized for 60 s in a saline solution containing 0.9% NaCl and 0.05% Triton X-100. Only spermatids of the 14th and 16th stages were resistant to homogenization. To visualize them, staining with the Trypan blue 0.04% in PBS (1 M) was done for 30 min before counting. The spermatids were counted with a light microscope at Â400 magnification. The total elongated spermatids in the right testis were calculated by multiplication with the right testis weight. DSP was calculated by dividing the total of spermatids counted by 4.84 days that spermatids spent to develop into stages 14-16 in mouse species (Oakberg 1956).Then, DSP was divided by the weight of the testis to determine the number of sperm per gram of testis, which corresponds to the efficiency of sperm production.

Sperm viability
The viability was assessed using the Hypo-Osmotic Swelling (HOS) test described by Jeyendran et al (1984) as a test for evaluating the functional integrity of the sperm membrane. An aliquot of 0.1 ml of epididymal head suspension was added to 1 ml of hypo-osmotic solution and incubated for 1 hour at 37 C.
Microscopic observation was made at Â40 magnification and an enumeration of 200 spermatozoids was done per slide. 3 repetitions of each sample were made. The Viability was expressed as a percentage of viable spermatozoids of the total number counted.

Sperm morphology
For the sperm morphology assessment, a fragment of the epididymis was ground in NaCl saline solution (0.9%) and formalin (10%). The previous suspension was diluted with distilled water for a final volume of 10 ml. Then, 1 ml of eosin 1% was supplemented to the above suspension followed by incubation at room temperature between 40 and 120 min. A drop of the mixture was placed afterward, spread between slide and coverslip serving for microscopic examination, which allowed us to detect morphological abnormalities. The main focus was on the anomalies of the head and tail, the principal parts of the spermatozoids. 300 spermatozoids were counted per slide (3 replicates/sample). Each anomaly was expressed as a percentage of the total number counted.

Testosterone assay
The plasmatic testosterone concentration was determined by the competitive ELISA technique, according to the Demeditec Testosterone ELISA kit (Demeditec Diagnostics GmbH, REF: DE1559) instructions. The Optical Density (OD) was read at 450 nm and the results were obtained automatically using a 4 Parameter Logistics curve fit by an ELISA reader (øayto, RT-2100C).

Oxidative stress markers
Testis samples from each group were homogenized into ice and centrifuged on cold for 15 min at 12 000 rpm. The supernatant was stored at À80 C until use, while the cellular debris was thrown. The total protein content was quantified using the Bradford method while we used the bovine serum albumin (BSA) to prepare the standard curve.

Malondialdehyde
Malondialdehyde (MDA) was measured following the method described previously by Yoshioka et al (1979). A mixture of 100 ml of the testis homogenate, 2 ml of TBA (0.67%), and 800 ml of TCA (20%) was heated in a boiling bath of water for 30 min. The heated samples were cooled and centrifuged at 4000 rpm for 10 min. Then, 3 ml of n-butanol was added to obtain a pink colorant extract. The absorbance of the supernatant was measured at 532 nm in parallel with a blank that contained the entire reagents except for the homogenate. The MDA concentration was expressed as the OD (532 nm) of the molar coefficient of extinction of the TBARS (1.56 Â 105).

Conjugated dienes
Conjugated Dienes (CD) as one of the major lipid oxidations was assessed by the method of Esterbauer et al. (1989). In fact, a mixture of 100 ml homogenate, 1 ml of NaCl (0.9%), and 3 ml of chloroform-methanol (2v/1v) was vortexed for 2 min and then centrifuged (4000 rpm, 10 min). The lower phase was transformed into a sterile tube to be evaporated into the oven (70 C). Finally, 1 ml of hexane was added and the OD at 243 nm was measured in parallel with a blank (hexane). The CD quantity was expressed in mmoles/mg of proteins.

Catalase (CAT)
The catalase (CAT) activity was carried out by measuring the decrease in absorbance at 240 nm of the sample for 1 min. The Enzyme activity was expressed in mmoles of H 2 O 2 /min/ mg of proteins, following the protocol of Aebi (1974).

Protein sulfhydryl groups (PSH)
Protein sulfhydryl groups (PSH) activity was assessed as described by Sedlak and Lindsay, (1968). A mixture containing 50 ml of homogenate, 150 ll of 0.2 M Tris (pH ¼ 8.2), 40 ll of 0.02 M EDTA, 790 ll of methanol, and 10 ll of 0.01 M DTNB was incubated for 15 min to be then centrifuged for 14 500 rpm at 4 C for 10 min. After a first record of the absorbance OD1 at 412 nm, samples were supplemented with TCA (5%) in order to precipitate the sulfhydryl proteins. Afterward, 300 ml of the supernatant was incubated for 3 min with the 0.4 M Tris and 0.01 M DTNB. A second absorbance OD2 was done at the same wavelength as OD1. In fact, the PSH content corresponds to subtracting the OD2 (Containing the non-sulfhydryl proteins) from OD1 (the total proteins). The reduced glutathione (GSH) was used to prepare a calibration curve. Finally, the PSH activity was expressed as nmol/mg of protein.

Carbonyl proteins
As previously described by Colombo et al (2016), the carbonyl proteins (CP) were dosed as follows: A mixture tube containing 200 ml of the homogenate, and 800 ml of DNPH dissolved into HCL (2.5 N), was incubated at room temperature in the dark for 1 h. A vortex every quarter of an hour was made. Then, 1 ml of TCA (20%) was added and the tube on ice was re-incubated for 10 min, after which short centrifugation was done. The supernatant was discarded and the pellet was washed by TCA (10%). New centrifugation was made and the pellet was washed with ethanol/ ethyl acetate (1v/1v). The final pellet was dissolved into 500 ml of guanidine hydrochloride (6 M). The absorbance was recorded at 370 nm after incubating the mixture for 10 min at 37 C.

Statistical analysis
The results of the in vivo study were statistically analyzed with SPSS, IBM 23 using the one-way ANOVA test followed by Tukey as a Post Hoc test. All values were expressed as the mean ± SD and the difference is significant at p 0.05.

Histology
Histological examination indicated a normal testicular structure in control mice, as well as in that of the LP-treated group, proved by normal spermatogenesis, and well-organized distribution of cells in the Seminiferous Epithelium (Figure 1). FB1 exposure clearly altered the testicular structure of adult mice, highlighted by the presence of several altered seminiferous tubules, showing hemorrhage, dilation of blood vessels, and infiltration of mononuclear cells into the interstitial space compared to the control animals ( Figure 1). No difference in the histological structure was observed in the testis of FB1 þ LP treated animals, compared to those of the control, with active spermatogenesis in almost all seminiferous tubules.
In addition, in the testis of control and LP treated mice, very few positive-TUNEL cells could be seen, like spermatogonia (SPG; arrow, Figure 1), spermatocytes (SPC; arrowhead, Figure 1), and spermatids (SPT; Figure 1). On the contrary, FB1 exposure produced a conspicuous increase in the number of TUNEL positive cells in all testicular cells, particularly in the Leydig cells (LC; asterisk, Figure 1). LP treatment partially decreased the FB1-induced apoptosis, since the number of interstitial and germinal apoptotic cells diminished (Figure 1).
The spermatozoid viability was also studied ( Table 1). The present data showed a slight decrease in the vitality (nonsignificant) of the treated mice's sperm. The administration of LP alone or with the FB1 showed 95% of viability as indicated in the control group.
For the motility, which is an important parameter in masculine fertility, our findings showed a decrease in the percentage of sperm motility in the treated mice's Table 1. The co-treated mice demonstrated similar motility as the control. LP alone revealed a preventive effect on sperm motility affected by FB1 treatment.
The morphological study of the epididymal spermatozoa (Figures 2-4) illustrated that FB1 affected the morphology of the spermatozoa with a high percentage of 85% compared to the other treated and control groups. In particular, a percentage of 60% was recorded as tail abnormalities in parallel with 25% of the head abnormalities (Figures 3 and 4). Interestingly, the administration of LP in combination with FB1 displayed restoration of these malformations caused by FB1-mycotoxin.  Data are expressed as mean ± SD from 6 mice per group. a Significantly different from the control (p < 0.05). b Not Significantly different from the control (p < 0.05)

DSP and DSP efficiency
The DSP and DSP efficiency values are mentioned in Table 2. Results revealed that there is no difference between the control (25 ± 2, p 0.05) and LP groups (26 ± 2, p 0.05). Mice exposed to FB1 induced a significant decrease (8 ± 2, p 0.05) in the DSP and DSP efficiency (106 ± 10, p 0.05) compared to the control group (359 ± 39, p 0.05). The cotreatment of LP with FB1 justified a powerful effect to restore these altered parameters in the treated group.

Testosterone level
Testosterone quantification by Elisa kit in the control, FB1, LP, and LP combined with FB1 groups is demonstrated in Figure 5. The plasmatic concentration observed in the LP group was similar to the control one. The exposure of animals to FB1 caused a notable decline in testosterone levels. Combined with the FB1, the LP strain showed a potential effect to reverse the FB1 disturbance.

Oxidative stress
The results of the oxidative stress biomarkers Table 3 revealed that treatment of mice with LP alone did not alter the balance of the oxidative stress markers as compared with the control. The FB1 administration generated oxidative stress via the increase of MDA, CD, and PC accompanied by a decline in CAT and PSH activities. Inversely, the companied treatment with FB1 plus LP improved the failure of the oxidant system caused by FB1.

Discussion
Scientific research about mycotoxins and particularly in FB1 has received greater attention because of the significant economic losses that crops would face by a fungal infestation. It is therefore important to develop several strategies to reduce its spread in the matrix used in the alimentary chain for both human food and animal feed. In fact, numerous harmful effects related to FB1-mycotoxin have been discovered. They have been found to be carcinogenic for humans (IARC 2002), immunotoxic (Stoev et al. 2012), neurotoxic (Suarez et al. 2012), and genotoxic (Chuturgoon et al. 2014). In contrast, only a few reports about its infestation in the male reproductive system are available. Consequently, the purpose of this study was to evaluate the effect of FB1 on BALB/c mice sperm quality, testosterone level, oxidative stress induction as well as histological alterations in the reproductive organs. Furthermore, based on the consumer demand to benefit from safe food devoid of mycotoxins, we evaluated the Lactobacillus paracasei BEJ01, to mitigate FB1-reprotoxicities. The mentioned bacteria were already used in our previous study and by itself was safe and disclosed potential protective effects against general toxicities (Abb es et al. 2016;Ezdini et al. 2020).
In this study, mice exposed to FB1 for10 days through an oral route showed a positive correlation between FB1 treatment and the reproductive system disturbance. Our results revealed testicular alterations marked by the presence of such debris and vacuolization in the lamina of the seminiferous tubules. A wide interstitial space was also observed.
These findings were in accordance with those of Abdel-Waheb et al (2018) who reported that rats treated with FB1 (100 mg/kg b.w) showed vacuolation (V) in seminiferous tubules lumen, distorted spermatocytes, and the interstitium containing vacuoles and edematous spaces. These structural abnormalities may be attributed not only due to the toxin accumulation in the testis but also due to the indirect effect of FB1 on the disruption of the cell membrane as a sphingolipids inhibitor.
Furthermore, the present data highlights the effect of this toxin on sperm parameters. First, the concentration of sperm was linked to spermatogenesis occurrence in the seminiferous tubule and associated with the maturation of germ cells. As a consequence of the abnormalities located in the testis as described by the histological micrographs (Figure 1), sperm concentration, daily sperm production, and efficiency significantly declined. The suggested mechanism of FB1 toward the spermatogenesis process was probably through its potential to elevate the sphingolipids contents, which was associated with the apoptosis event. Here, we hypothesized that a germ cell apoptosis event occurs in the testicular epithelium as an effect of FB1 treatment leading to a reduction of the germ cell population, and hence, a reduction in the studied parameters of the sperm quantity in both testis and epididymis. These findings could support the results suggested by Szab o-Fodor et al. (2015) in which the disturbance of meiosis and mitosis of the germinal epithelial cells induced by FB1 alone or combined with ZEN and DON in rabbit buck was documented. Table 2. Testicular daily sperm production and efficiency on male balb/ c mice.
Moreover, a slight reduction in sperm vitality was recorded in the treated mice. In addition, a sharp decline in motility was observed. In the main context, our results were supported by several studies. In fact, Minervini et al. (2010) reported that FB1 reduced the total and progressive motility of equine spermatozoa. A similar phenomenon that affected spermatogenesis and sperm parameters were reported as a result of a diet containing more than 5 mg/kg of FB1 in wild boar (Gbore and Egbunike 2008). Likewise, livestock was affected as shown by Ewuola and Egbunike (2010a). As a matter of fact, Spermatic mass, motility, and viability of the rabbit's semen declined in correlation with an increase in the dietary of FB1.
The mobility was sensitive to the morphology of the spermatozoa. Actually, the mice's exposure to FB1 revealed a deleterious effect on sperm morphology; in particular, 60% of tail abnormalities and 25% of the head were recorded. The dominance of the tail abnormalities revealed the decline in sperm motility mentioned earlier. How could FB1 significantly disrupt spermatogenesis and spermatozoa morphology? Two mechanisms were suggested: the first one was the reduction of the hormonal level, especially the testosterone quantity. Similarly, Abdel-Waheb et al. (2018) stated the sensitivity of the testosterone level in the FB1 treatment alone or combined with AFB1.This hormonal disruption may be described by the capability of FB1 to adjust cholesterol and lipid homeostasis in the absence of LXR in the liver (R egnier et al. 2019). An involvement of LPCAT3, a sphingolipid enzyme, was suggested in FB1 cholesterol modulation (Rong et al. 2013;Wang et al. 2016). In the same vein, FB1 decreased LDLR and consequently inhibited the cholesterol influx (Abdul and Chuturgoon 2021).
Here, we suggest as a hypothesis that the established mechanism of FB1 affecting the cholesterol pathway in the liver could be adopted as the same in the Leydig cells via the absence of reports studying this detailed mechanism in the testis.
Secondly, we hypothesize that the ROS levels and their antioxidant defense imbalance led to oxidative damage in the spermatozoa (Fraczek and Kurpisz 2005). Consequently, we focused on measuring the oxidative stress occurring in the testis upon FB1 exposure. Our data revealed that the FB1 treatment elevated the MDA, CD, and protein carbonyl levels, while, catalase and PSH activities decreased.
The oxidative stress involved through the free radicals and peroxides resulted in the damage of the sperm membrane, and decreased sperm motility. Thus, the displayed abnormalities and impaired motility of sperm could be a result of the abnormal functionality of the mitochondria (Chai et al. 2017).
Several pathways may be involved in the effect of FB1 on the mitochondria generating ROS. Due to its analogy to sphingolipids, FB1 was able to increase the polyunsaturated fatty acids content, trigger the mitochondrial permeability, and then the induction of apoptosis.
These key mechanisms proposed were previously indicated by (Aitken et al. 2012) who reported that ROS generation drives the spermatozoa along the intrinsic apoptotic cascade by the loss of Mitochondrial Membrane Potential, leading to DNA adduct formation and fragmentation, and ultimately cell death. In the main content, Nowicka-Bauer and Nixon (2020) observed that ROS deregulated sperm bioenergetic pathways, along with the structural and signaling machinery of the sperm tail confirming 60% of tail abnormalities also found in this study.
The supplementation of Lactobacillus strain alone did not cause any sign of toxicity and all the studied parameters showed normal spermatogenesis as well as good sperm quality in comparison with the normal group. Thus, several reports proved that Lactobacillus strains benefit from a wide range of advantageous effects. In fact, in previous studies, Lactobacillus was demonstrated to have a regulatory activity of cholesterol (Huang et al. 2020), and a promoter effect to treat allergic rhinitis (G€ uvenc¸et al. 2016). Also, lactic acid bacteria have also shown anti-inflammatory (Abb es et al. 2016), antifungal (Mundula et al. 2019), antimicrobial (Pattani et al. 2013), and antiviral (Lehtoranta et al. 2014) effects.
Supplemented with FB1, our strain showed powerful protective effects against FB1 toxicities on the mouse reproductive system. In fact, the preventive effect of Lactobacillus on FB1 spermatogenesis toxicities was due to its antioxidant activities playing a key role in improving sperm motility via its ion pumps present in the flagella. Sertoli cells benefit also from the powerful antioxidant activities of probiotics boosting the survival and maturation of spermatozoa (Inatomi and Otomaru 2018). Experimentally, several in vitro and in vivo studies showed that probiotics, in particular, Lactobacillus strain enhanced antioxidant enzymes via chelating Fe 2þ and Cu 2þ (Lee et al. 2005), reduced ROS imbalance, and by consequence counteracted apoptosis pathways (Yan and Polk 2002;Wu et al. 2019). Likewise, probiotics effectively protect spermatogenic cells, sperm quality,and subsequently protect the reproductive system (Chen et al. 2013;Valcarce et al. 2019;Abasi and Keshtmand 2020). Another study has demonstrated a positive correlation between the regulation of cholesterol by probiotics and the plasmatic level of testosterone (Dardmeh et al. 2017).
Finally, another mechanism for the protective role of LP may be involved suggested in probiotics counteracted mycotoxins by sequestration and/or degradation in the gastrointestinal tract (Zhao et al. 2016;Vanhoutte et al. 2017;Ben Salah Abb es et al. 2020).

Conclusion
In summary, our findings showed that the male reproductive system was menaced by FB1, leading to enhance oxidative stress damage, deleterious effects on sperm parameters, reduced testosterone levels, and testicular histological alterations.
LP showed a potential protective effects against the toxic damage of FB1 via several mechanisms, including (i) the antioxidant power of FB1, and (ii) the ability of LP to adsorb/or degrade FB1 in the digestive system and then decrease their bioavailability.
Consequently, it would be interesting to investigate whether Lactobacillus metabolites were involved in detoxification processes.

Ethics approval and consent to participate
Experimental protocols were approved with the guidelines of the Ethical Committee of the High Institute of Biotechnology of Monastir, Tunisia.

Animal welfare statement
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this is a meta-analysis article with no original research data.

Disclosure statement
No potential conflict of interest was reported by the author(s).  Lactobacillus strains for their ability to remove fumonisins B1 and B2. Food Chem Toxicol. 97:40-46.