Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro

Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro Abdullateef Isiaka Alagbonsi, Luqman Aribidesi Olayaki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-34960/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2020 Read the published version in BMC Nutrition → Version 3 posted 4 You are reading this latest preprint version Show more versions Abstract Background : We investigated the in-vitro effects of vitamin C on delta-9-tetrahydrocannabinol (THC) -induced reduction in spermatozoa motility and kinematics. Methods : Six rats were used for the study. Semen from each of the 6 rats was randomly divided into 6 groups such that each rat’s semen was in all of the groups. Groups I-III received placebo, THC (1 mM), and vitamin C (5 mM) respectively. Group IV was pretreated with cannabinoid receptors’ blockers (CBs - ) 1 and 2, followed by THC. Groups V and VI received THC and vitamin C, but group VI was additionally pre-treated with CBs - . Results : The spermatozoa progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head (ALH) and beat cross frequency (BCF) were reduced by THC (6.08±1.16%; 5.64±0.82 µm/s; 6.96±0.74 µm/s; 2.75±0.23 µm/s; 0.31±0.02 µm; and 0.78±0.08 Hz respectively) but increased by vitamin C (51.20±1.32 %; 17.90±0.21 µm/s; 25.11±0.96 µm/s; 8.80±0.27 µm/s; 0.75±0.01 µm; and 3.15±0.03 Hz respectively) when compared to control (39.72±0.38 %; 13.70±0.29 µm/s; 18.04±0.58 µm/s; 7.54±0.34 µm/s; 0.65±0.02 µm; and 2.79±0.01 Hz respectively). Vitamin C inhibited the THC-induced reduction in these parameters (37.36±0.73 %; 10.98±0.45 µm/s; 13.58±0.30 µm/s; 7.11±0.22 µm/s; 0.58±0.01 µm; and 2.60±0.01 Hz respectively) in the absence of CBs - 1 and 2, and even caused additional increases in progressive motility (49.54±1.01 %), VAP (15.70±0.38 µm/s) and VCL (22.53±0.29 µm/s) above the control levels with CBs - . Conclusion : Vitamin C ameliorates the THC-induced reduction in spermatozoa motility in-vitro by modulation of their kinematics. Nutrition & Dietetics Kinematics Spermatozoa motility Spermatotoxicity Tetrahydrocannabinol Vitamin C Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1.0. Background Marijuana, preparation of the flower, leaves and seeds of the plant Cannabis sativa (CS), is the most widely abused illicit drug worldwide (1) and is becoming globally legalised due to its medicinal use by patients with pain (2), inflammation (3), epilepsy (4), cancer (5), etc. However, its detrimental effects on male reproductive functions have been well-established. For instance, it decreases germ cell proliferation, reproductive organ weight (6,7), and semen parameters (7–9) by eliciting endocrine disruption, hyperprolactinaemia (10), down-regulation of the hypothalamic-pituitary-gonadal axis (10,11), and oxidative stress (9). Delta-9-tetrahydrocannabinol (THC, an active psychoactive compound in CS), prescribed under the name Dronabinol (synthetic THC in sesame oil), is clinically being used for the treatment of acquired immunodeficiency syndrome (AIDS) -associated cachexia, multiple sclerosis, and cancer chemotherapy-associated nausea (12,13). However, it has been consistently shown to negatively alter spermatozoa motility, swimming behaviour, and acrosome reaction in sea urchin (14–16), rat (17), and human (18,19). The effects of vitamin C on spermatozoa parameters and fertility rate in subfertile males remain controversial. For instance, its protective effect against testicular damage has been well-reported in different conditions (20,21), while its toxic (20) and deoxyribonucleic acid (DNA)- damaging (22,23) potentials have also been shown. We have previously observed in our in-vivo studies that vitamin C exacerbates the CS-induced gonadotoxicity, oxidative stress and reproductive hormonal toxicities, but these CS-induced toxicities were ameliorated only when vitamin C was co-administered with melatonin (7,9,10). In our very recent in-vitro study, we observed that melatonin alone ameliorated THC-induced spermatotoxicity by attenuating the THC-induced reduction of hyper-activated motility (HAM) in the capacitated spermatozoa (17). The results of our recent in-vitro work (17) showed that melatonin alone attenuated, but did not abolish, the THC-induced reduction in spermatozoa motility and kinematics. This lack of abolishment is evident as the motility and kinematics were still significantly lower in spermatozoa that were treated with a combination of melatonin and THC than the control. Vitamin C has been reported to increase the motility and kinematics of caprine epididymal spermatozoa in-vitro (24). Since the combination of melatonin and vitamin C abolished the spermatotoxic effects of CS in-vivo, the lack of abolishment of the spermatotoxic effect of THC by melatonin alone in-vitro suggests that the effect of melatonin and vitamin C on the semen parameters might be additive or synergistic. This speculation led us to also investigate the effect of vitamin C on spermatozoa motility and kinematics in this study to clear our doubts whether vitamin C contributed to the combined effect of melatonin and vitamin C or whether the combined effect is a result of melatonin only. Following the procedures used in our previous in-vitro study, the present study investigated the effect of vitamin C on motility and kinematics of capacitated rat spermatozoa treated with cannabinoids using the computer-assisted sperm analyser (CASA). We hypothesised that vitamin C will ameliorate the THC-induced reduction in spermatozoa motility and kinematics. 2.0. Methods 2.1 Experimental protocol Six (6) male Wistar albino rats (200-210 g) sourced from the Department of Biochemistry’s Animal House, University of Ilorin, Nigeria were housed at room temperature under the daily light/dark cycle with free access to food and water ad libitum . In addition to the study approval by the Ethics Committee of the University of Ilorin, the “Principles of laboratory animal care (NIH publication No. 85-23, revised 1985)” were followed. Following the sacrifice of the rats by 0.8 ml kg -1 body weight of ketamine hydrochloride, the cauda epididymides were rapidly removed and minced in the modified Biggers–Whitten–Whittingham (BWW) capacitation medium, whose components have been previously described (17,25). The liberated spermatozoa were incubated in the medium. Dimethyl sulphoxide (DMSO) was used to prepare the stock solution of the drugs, which was then diluted to the required concentration before each experiment in a BWW capacitation medium such that the final DMSO concentration was 0.2 % (Vol/Vol) (25) that does not affect spermatozoa motility (17). Following the method of Charan and Kantharia (26) for the determination of animal sample size, semen from each rat (n= 6) was randomly divided into 6 treatment groups such that each rat’s semen was in all of the groups as follows: Groups I-III were respectively treated with placebo (control mixture), THC (1 mM), and vitamin C (5 mM). Group IV was pretreated with 1mM each of CBs - 1 (SR141716) and 2 (AM-630), followed by THC (1 mM). Groups V and VI were both treated with THC and vitamin C, but group VI was additionally pretreated with 1 mM each of CBs - 1 (SR141716) and 2 (AM-630). Experiments were carried out by incubating spermatozoa in the BWW capacitation medium for 30 minutes. We generated dose-response and time-course of modulation curves from our preliminary experiment to arrive at the doses of THC and vitamin C and the duration used for the study while the same dose of THC was used for the CBs - (17). 2.2 Determination of spermatozoa motility and kinematics Spermatozoa progressive motility and kinematics (average-path velocity [VAP], curvilinear velocity [VCL], straight-line velocity [VSL], the amplitude of lateral head displacement [ALH], beat cross frequency [BCF], wobble, straightness, and linearity) were recorded with CASA (JH-6004 Sperm Quality Analyser, Jiangsu Jiahua Electronic Instrument Co., Ltd, Jiangsu, China) as previously described (17). 2.3 Data analysis Data were blindly analysed using statistical software for the social sciences (SPSS) version 16.0 for Windows (IBM Corporation, Armonk, NY, USA). All values given were the Mean ± S.E.M of the variables. The normality of the data was assessed using the Shapiro-Wilk test. Significance was assessed by the one-way analysis of variance (ANOVA), followed by a posthoc Tukey test for multiple comparisons. The five experimental groups were compared with the control group that received placebo, while groups that received cannabinoids with(out) vitamin C were additionally compared with THC group. Receiver Operating Characteristic (ROC) curve was used to examine the predictive abilities of spermatozoa kinematics on their progressive motility. The p -values of 0.05 or less were considered statistically significant. 3.0. Results 3.1 Dose-response and time-course of modulation of spermatozoa motility by tetrahydrocannabinol and vitamin C Throughout the observation period, 10 µM, 100 µM, 1 mM and 10 mM but not 1 µM of THC reduced the rat spermatozoa motility when compared to the baseline (Figure 1A). On the contrary, 100 µM, 1 mM, 5 mM, and 10 mM but not 10 µM of vitamin C increased the rat spermatozoa motility when compared to the baseline (Figure 1B). 3.2 THC-induced reduction in progressive motility is attenuated by vitamin C THC reduced the spermatozoa progressive motility (6.08±1.16 %) when compared to the control (39.72±0.38 %) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (41.38±0.36 %). The progressive motility was increased by vitamin C (51.20±1.32 %) when compared to the control (39.72±0.38 %). Interestingly, vitamin C inhibited the THC-induced reduction in progressive motility (37.76±0.73 %), maintaining it at 94 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on the progressive motility and even caused its additional increase (49.54±1.01 %) by 20 % above the control level (Figure 2). 3.3 THC-induced reductions in some spermatozoa kinematics are attenuated by vitamin C THC reduced the spermatozoa VAP (5.64±0.82 µm/s) when compared to the control (13.70±0.29 µm/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (14.70±0.76 µm/s). The VAP was increased by vitamin C (17.90±0.21 µm/s) when compared to the control (13.70±0.29 µm/s). Moreover, vitamin C inhibited the THC-induced reduction in VAP (10.98±0.45 µm/s), maintaining it at 80% of the control. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VAP and even caused its additional increase (15.7±0.38 µm/s) by 13% above the control level (Figure 3). THC reduced the spermatozoa VCL (6.96±0.74 µm/s) when compared to the control (18.04±0.58 µm/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (21.39±0.73 µm/s). The VCL was increased by vitamin C (25.11±0.96 µm/s) when compared to the control (18.04±0.58 µm/s). Furthermore, vitamin C inhibited the THC-induced reduction in VCL (13.58±0.30 µm/s) by maintaining it at 75% of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VCL and even caused its additional increase (22.53±0.29 µm/s) by 20 % above the control level (Figure 4). THC reduced the spermatozoa VSL (2.75±0.23 µm/s) when compared to the control (7.54±0.34 µm/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (6.86±0.47 µm/s). The VSL was increased, albeit insignificant, by vitamin C (8.80±0.27 µm/s) when compared to the control (7.54±0.34 µm/s). Vitamin C inhibited the THC-induced reduction in VSL (7.11±0.22 µm/s) by maintaining it at 94 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VSL by maintaining it at a level (7.86±0.17 µm/s) comparable to that of the control (Figure 5). THC reduced the spermatozoa ALH (0.31±0.02 µm) when compared to the control (0.65±0.02 µm) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (0.69±0.00 µm). The VSL was increased by vitamin C (0.75±0.01 µm) when compared to the control (0.65±0.02 µm). Vitamin C inhibited the THC-induced reduction in ALH (0.58±0.01 µm) by maintaining it at 89 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on ALH by maintaining it at a level (0.67±0.01 µm) comparable to that of the control (Figure 6). THC reduced the spermatozoa BCF (0.78±0.08 Hz) when compared to the control (2.79±0.01 Hz) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (2.88±0.00 Hz). The BCF was increased by vitamin C (3.15±0.03 Hz) when compared to the control (2.79±0.01 Hz). Vitamin C inhibited the THC-induced reduction in BCF (2.60±0.01 Hz) by maintaining it at 93% of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on BCF by maintaining it at a level (2.77±0.03 Hz) comparable to that of control (Figure 7). 3.4 The effect of vitamin C on velocity ratios in THC-treated spermatozoa. THC and/or vitamin C caused no significant change in the spermatozoa wobble, straightness and linearity when compared to control. THC did not also have any effect on these velocity ratios when cannabinoid receptors 1 and 2 were blocked, either in the presence or absence of vitamin C (Table 1). 3.5 Prediction of progressive motility by the kinematics of spermatozoa Using the ROC curve, we determined if kinematics could predict the progressive motility of spermatozoa treated with THC and/or vitamin C. The curve showed that 100 % of progressively motile spermatozoa had 8.76 µm s -1 as VAP, 10.36 µm s -1 as VCL, 4.96 µm s -1 as VSL, 0.45 µm as ALH, 1.73 Hz as BCF, 66.49 % as wobble, and 32.46 % as linearity while 100 % of non-progressively motile spermatozoa had below these parameters’ cut-off values (except for wobble and linearity that the specificity is 33.30 %). The velocity ratios (wobble, straightness, and linearity) had lower confidence intervals and areas under the ROC curves than velocities, ALH, and BCF. Thus, there was a very low probability that the progressively motile and non-motile spermatozoa differ in their velocity ratios (wobble, straightness and linearity), while there was a very high probability that they differ in their velocities (VAP, VCL, and VSL), ALH and BCF (Table 2). 4.0. Discussion The reduction in spermatozoa motility by THC was initially reported in sea urchin by Schuel’s group (14–16). A series of studies later showed that cannabinoid receptors activation by anandamide (an endocannabinoid agonist) or THC inhibits spermatozoa functions like motility, velocities, capacitation and acrosome reaction in the frog, rat (6,17), boar (27), and human (19,28–31). Furthermore, the spermatotoxic potential and the consequent anti-fertility effect of marijuana (7,9,10) and its primary psychoactive cannabinoid (THC) (17) are well-known. Ejaculated spermatozoa capacitation process in the female reproductive tract involves membrane and metabolic modifications such as the generation of reactive oxygen species (ROS), an increase in intracellular ions and protein tyrosine phosphorylation, and changes in motility and plasma membrane fluidity (32,33). Events like spermatozoa binding to the zona pellucida (ZP), acrosome reaction, and oocyte fertilisation all depend on successful capacitation process (34), prevention of which will lead to fertilisation failure (35). During this process, spermatozoa acquire a motility pattern called hyper-activated motility (HAM) (36) that is characterised by asymmetrical flagellar beating (37), which is needed for fast swimming and generation of enough force necessary to penetrate cumulus cells and ZP during fertilisation (38). Because the HAM patterns of mammalian spermatozoa capacitated in-vivo and in-vitro have been reported to be similar (39,40), it has been very easy for researchers to study different aspects of spermatozoa physiology by in-vitro capacitation. Our data in this study showed that THC reduced the progressive motility of spermatozoa incubated in capacitation medium, which corroborates its spermatotoxic effect. Vitamin C has been previously reported to improve rat’s semen parameters in-vivo (9) and ameliorate the spermatotoxicity induced by cisplatin in rats (41,42) and p-dimethylaminoazobenzene in mice (43). In a randomised controlled clinical trial that included cases after varicocele surgery, vitamin C supplementation significantly increased motility and morphology but not count in infertile young adult males with low-quality spermatozoa, suggesting that it positively affects qualitative but not quantitative characteristics of semen analysis in this condition (44). Apart from the reported improvement in spermatozoa quality in smokers by increasing vitamin C dose from 200 to 1000 mg (45), another observational study on healthy, non-smoking Americans showed that vitamin C-containing diet or supplement increased spermatozoa count and motility (46). When combined with vitamin E, vitamin C has also been reported to enhance rabbit male fertility (47) by increasing concentration and total motile but decreasing abnormal and dead spermatozoa (47,48). A combination of vitamins C and E reportedly ameliorate the oxidative stress and spermatotoxicity induced by endosulfan in rats (49). Does vitamin C have spermatoprotective effect in-vitro ? Previous reports have shown that vitamin C is the most important seminal anti-oxidant, accounting for about 65% of the seminal anti-oxidant capacity, and is currently being used in-vitro to enhance spermatozoa quality in infertility clinics (50,51). An Egyptian study has linked smoking to decrease in seminal plasma vitamin C, which was shown to consequently reduce semen parameters and fertilisation potential (52). Our dose-response and time-course of modulation study showed that vitamin C increased the percentage motility of spermatozoa in a dose-dependent but not time-dependent manner. For instance, 100 µM, 1 mM, 5 mM, and 10 mM, but not 10 µM, significantly increased the spermatozoa motility throughout the observation period when compared to the baseline. Furthermore, incubation of spermatozoa in 5 mM of vitamin C solution also increased their motility by 22% when compared to control. The vitamin C-induced increase in spermatozoa motility was associated with an increase in VAP, VCL, VSL, ALH and BCF but decrease in straightness and linearity. These data provide pieces of evidence that vitamin C increases motility by enhancing the kinematics of spermatozoa. These data are consistent with the previous report of Babaie and Sivandi (24) that also reported an improvement in the motility and kinematics of caprine spermatozoa treated with vitamin C in-vitro . Similar to our study, Babaie and Sivandi (24) obtained epididymal spermatozoa from adult goat testes and incubated them in the capacitation medium (Ca 2+ -free Tyrode’s medium, spermatozoa TALP) supplemented with 50 µg/ml vitamin C, after which the motion parameters were assessed using the CASA at various intervals. They also observed that vitamin C increased total and progressive spermatozoa motility, VCL, VSL, linearity, ALH and BCF. Can vitamin C ameliorate the THC-induced spermatotoxicity? In our previous in-vivo studies, we reported that the CS-induced spermatotoxicities (and the associated endocrine disruption and oxidative stress) were exacerbated by either vitamin C or melatonin when administered separately, but ameliorated when vitamin C was co-administered with melatonin (7,9,10). However, our most recent in-vitro study showed that melatonin attenuated THC-induced reduction in spermatozoa motility and kinematics of rat. Based on our observation that the modulatory effects of melatonin on spermatotoxicity induced by CS or THC are different in-vivo and in-vitro , we were interested to know the effect of vitamin C on THC-induced spermatotoxicity in-vitro . In the present in-vitro study where spermatozoa were incubated with THC and vitamin C in the capacitation medium, we observed for the first time that vitamin C completely abolished the THC-induced reduction in progressive motility of spermatozoa by maintaining it at 94% of the control (which is not significantly different from the control level). We also observed that vitamin C attenuated the THC-induced reduction of VAP, VCL, ALH, and BCF but abolished the THC-induced reduction in VSL. These data provide pieces of evidence that vitamin C ameliorates the THC-induced spermatotoxicity, especially as regards motility and kinematics of spermatozoa. Taken together with our previous in-vivo study, it also suggests that vitamin C ameliorates cannabinoid-induced spermatotoxicity in-vitro but exacerbates it in-vivo. It is thus similar to our recent study where we reported that melatonin ameliorates cannabinoid-induced spermatotoxicity in-vitro but exacerbates it in-vivo (17). Is there a crosstalk between vitamin C and cannabinoid signalling? The existence of cannabinoid receptors (CbRs) and the endocannabinoid system in mammalian spermatozoa has been well-reported (31,53,54). We have reported that THC-induced reduction in the HAM of spermatozoa was abolished when both CbRs 1 and 2 were blocked, but was only attenuated when either CbR 1 or CbR 2 was selectively blocked. We also showed that CbR 1 contributed more than CbR 2 in THC-induced spermatotoxicity (17). Since the involvement of both CbRs in the regulation of spermatozoa motility has been established, the current study further investigated if the lack of complete abolishment of some kinematics by vitamin C is associated with CbRs activation by THC in spermatozoa incubated with a combination of THC and vitamin C. We blocked both CbRs (SR141716 as CB - 1 and AM-630 as CB - 2) and then added THC and vitamin C to the capacitation medium. We observed that prior blockade of CbRs cancelled the spermatotoxic effect of THC as vitamin C abolished the effect of THC on VSL, ALH and BCF, and even raised the progressive motility, VAP and VCL above the control value. This shows that vitamin C caused more inhibition of THC-induced spermatotoxicity when CbRs were blocked and is a pointer to the fact that there is a crosstalk between vitamin C and cannabinoid signalling. Is the ameliorative effect of vitamin C on motility related to the kinematics of spermatozoa? Across a wide range of animals like birds (55), fishes (56) and mammals (57), the competition of spermatozoa to fertilise the ovum is favoured by traits (e.g. kinematics) that promote the fertilising ability of male. To penetrate different barriers along its track, the spermatozoon requires a high flagellar BCF (58). The VCL and ALH signify the hyper-activated motility (HAM) of spermatozoa and the high-energy state required for successful fertilisation (59) while motility and velocities of spermatozoa reflect their mitochondrial function and energy status that have been correlated with their fertility (60). Moreover, the progressive motility of spermatozoa has been positively correlated with the VAP, VCL, VSL, ALH, BCF, straightness, and linearity but negatively correlated with wobble in bulls (57,61,62). In our recent study, velocities (VAP, VCL and VSL), ALH and BCF showed strong predictive power on HAM, suggesting that these kinematics determine the response of spermatozoa capacitation-induced HAM to cannabinoids and melatonin (17). In the present study, there was a very low probability that the progressively motile and non-motile spermatozoa differ in their velocity ratios (wobble, straightness and linearity), while there was a very high probability that they differ in their velocities (VAP, VCL, and VSL), ALH and BCF. These suggest that velocities, ALH and BCF determine the modulation of spermatozoa capacitation-induced HAM by cannabinoids and vitamin C. Even though hydrogen peroxide is the major ROS produced in spermatozoa, a previous study has shown that 87% of infertile patients have superoxide anion (63). Spermatozoa are sensitive to assault from free radicals due to the existence of high membrane polyunsaturated fatty acids and lack of capacity for DNA repair (64). Increased ROS level has also been shown to correlate with decreased motility (65) and many structural and functional abnormalities of spermatozoa. Meanwhile, non-enzymatic antioxidants like vitamin C, pyruvate, vitamin E, taurine, vitamin A, hypotaurine, albumin, ubiquitol, urate, and enzymatic antioxidants like catalase, superoxide dismutase, and glutathione peroxidase/reductase have been reported to be contained in the seminal fluid (66), even though vitamin C is considered as the major antioxidant in the testis (67). Furthermore, the ROS-induced damage in spermatozoa has been shown to reduce with supplementation of the latter’s clinical media with antioxidant (64). Despite the physiological significance of vitamin C in the body, especially on spermatozoa, humans (unlike most animals) are unable to synthesise vitamin C, making its inclusion in diet or supplement a necessity (68). Vitamin C transporters have been reported to be expressed in the Sertoli cells, spermatocytes, spermatids, spermatozoa and the testis (69,70). However, our present study is limited due to our inability to establish the relationship between vitamin C transporters and cannabinoid receptors as regards HAM of capacitated spermatozoa, which is worthy of consideration in a future study. Another limitation of our study is our inability to estimate the seminal ROS and antioxidant levels in all the groups, which would have enabled us to understand the relationship between spermatozoa kinematics and redox system and their modulation by vitamin C and/or cannabinoid system. Notwithstanding the limitations, our study is the first to provide information on the in-vitro effect of vitamin C on hyperactivated motility and kinematics of capacitated spermatozoa treated with cannabinoids. Conclusion In conclusion, our present acute study shows that vitamin C ameliorates THC-induced reduction in spermatozoa motility in-vitro by modulation of their kinematics. Since the success rate of in-vitro fertilisation among users of CS is known to be low (71), the outcome of our present acute study is of clinical importance as it suggests that the success rate of in - vitro fertilisation among CS users could be improved by in-vitro supplementation of their spermatozoa with vitamin C. We recommend that a similar study should be conducted on human spermatozoa. Abbreviations THC, tetrahydrocannabinol; CBs - , cannabinoid receptors’ blockers; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity; ALH, the amplitude of lateral head; BCF, beat cross frequency; CS, Cannabis sativa; AIDS, acquired immunodeficiency syndrome; DNA, deoxyribonucleic acid; HAM, hyper-activated motility; CASA, computer-assisted sperm analyser; BWW, Biggers–Whitten–Whittingham; BSA, bovine serum albumin; DMSO, dimethyl sulphoxide; ANOVA, one-way analysis of variance; ROC, Receiver Operating Characteristic; ROS, reactive oxygen species; ZP, zona pellucida; CbRs, cannabinoid receptors Declarations Ethics approval and consent to participate All the animals were well-catered according to the criteria outlined in the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the National Academy of Science and approved by the Ethical Research Committee of the University of Ilorin, Nigeria. The consent to participate in the study is not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contributions IAA and LAO both conceived and designed the study. IAA carried out the study, interpreted the data, and drafted the manuscript. LAO supervised the study. Both authors read and approved the final manuscript to be published. Acknowledgement The authors acknowledge Oyeyemi Abdulwahab of Igbinedion University, Okada, who was a Ph.D. candidate in the Department of Physiology, University of Ibadan, Nigeria for permitting us to use his computer-assisted semen analyser. 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In Testicular Development, Structure and Function. A. Steinberger ES, editor. New York: Raven Press; 1980. 481–489 p. Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. Reproduction. 1985 Mar;73(2):441–9. Farrell PB, Foote RH, McArdle MM, Trouern-Trend VL, Tardif AL. Media and dilution procedures tested to minimize handling effects on human, rabbit, and bull sperm for computer-assisted sperm analysis (CASA). J Androl. 1996;17(3):293–300. Gasparini C, Simmons LW, Beveridge M, Evans JP. Sperm Swimming Velocity Predicts Competitive Fertilization Success in the Green Swordtail Xiphophorus helleri. Halsey LG, editor. PLoS One. 2010 Aug;5(8):e12146. Perumal P, Srivastava SK, Ghosh SK, Baruah KK. Computer-Assisted Sperm Analysis of Freezable and Nonfreezable Mithun ( Bos frontalis ) Semen. J Anim. 2014;2014:1–6. Mazzilli F, Rossi T, Marchesini M, Ronconi C, Dondero F. Superoxide anion in human semen related to seminal parameters and clinical aspects. Fertil Steril [Internet]. 1994 Oct;62(4):862–8. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0015028216570174 Donnelly ET, O’Connell M, McClure N, Lewis SEM. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod [Internet]. 2000 Jul;15(7):1552–61. Available from: https://academic.oup.com/humrep/article-lookup/doi/10.1093/humrep/15.7.1552 Jedlinska-Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A. Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J Reprod Dev [Internet]. 2006 Apr;52(2):203–9. Available from: http://joi.jlc.jst.go.jp/JST.JSTAGE/jrd/17028?from=CrossRef Kim JG, Parthasarathy S. Oxidation and the spermatozoa. Semin Reprod Endocrinol [Internet]. 1998 Dec 15;16(4):235–9. Available from: http://www.thieme-connect.de/DOI/DOI?10.1055/s-2007-1016283 Augustine LM, Markelewicz RJ, Boekelheide K, Cherrington NJ. Xenobiotic and endobiotic transporter mRNA expression in the blood-testis barrier. Drug Metab Dispos [Internet]. 2005 Jan;33(1):182–9. Available from: http://dmd.aspetjournals.org/lookup/doi/10.1124/dmd.104.001024 Subramanian VS, Marchant JS, Reidling JC SH. N-Glycosylation is required for Na+-dependent vitamin C transporter functionality. Biochem Biophys Res Commun. 2008;374:123–7. Angulo C, Castro MA, Rivas CI, Segretain D, Maldonado R, Yañez AJ, et al. Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells. J Cell Physiol [Internet]. 2008 Dec;217(3):708–16. Available from: http://doi.wiley.com/10.1002/jcp.21545 Angulo C, Maldonado R, Pulgar E, Mancilla H, Córdova A, Villarroel F, et al. Vitamin C and oxidative stress in the seminiferous epithelium. Biol Res [Internet]. 2011;44(2):169–80. Available from: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602011000200009&lng=en&nrm=iso&tlng=en Klonoff-Cohen HS, Natarajan L, Victoria Chen R. A prospective study of the effects of female and male marijuana use on in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) outcomes. Am J Obstet Gynecol. 2006 Feb;194(2):369–76. Tables Table 1: Effect of vitamin C on velocity ratios in tetrahydrocannabinol-treated spermatozoa. Groups Wobble (VAP/VCL, %) Straightness (VSL/VAP, %) Linearity (VSL/VCL, %) Control 76.08 ± 2.91 55.18 ± 3.27 42.01 ± 3.13 THC 81.45 ± 9.23 49.81 ± 4.49 40.52 ± 5.70 CBs - + THC 68.64 ± 1.43 46.89 ± 3.74 32.16 ± 2.55 Vitamin C 71.44 ± 1.98 49.14 ± 0.91 35.07 ± 0.39 Vitamin C+THC 80.96 ± 4.01 64.83 ± 1.96 # 52.35 ± 1.36 # CBs - + THC + Vitamin C 69.74 ± 2.59 50.15 ± 2.04 34.88 ± 0.63 THC = tetrahydrocannabinol, CBs - = cannabinoid receptors’ blockers; VSL = straight line velocity; VCL = curvilinear velocity; VAP = average-path velocity; # p <0.05 vs. THC Table 2: Prediction of hyper-activated motility by the kinematics of capacitated spermatozoa treated with tetrahydrocannabinol with(out) vitamin C Outcomes Predictors Sensitivity (%) Specificity (%) Cutoff value AUC 95% CI p -values Progressive motility VAP (µm/s) 100.00 100.00 8.76 1.000 1.000 to 1.000 0.000 VCL (µm/s) 100.00 100.00 10.36 1.000 1.000 to 1.000 0.000 VSL (µm/s) 100.00 100.00 4.96 1.000 1.000 to 1.000 0.000 ALH (µm) 100.00 100.00 0.45 1.000 1.000 to 1.000 0.000 BCF (Hz) 100.00 100.00 1.73 1.000 1.000 to 1.000 0.000 Wobble (%) 100.00 33.30 66.49 0.333 -0.200 to 0.867 0.405 Straightness (%) 88.90 66.70 48.43 0.778 0.455 to 1.101 0.166 Linearity (%) 100.00 33.30 32.46 0.593 0.181 to 1.004 0.644 VAP= Average-path velocity; VCL= Curvilinear velocity; VSL= Straight-line velocity; ALH= Amplitude of lateral head displacement; BCF= Beat/cross frequency; AUC= Area under the receiver operating characteristic curve; CI= Confidence interval. Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2020 Read the published version in BMC Nutrition → Version 3 posted Editorial decision: Accept 30 Sep, 2020 Editor assigned by journal 28 Sep, 2020 Submission checks completed at journal 27 Sep, 2020 Editor invited by journal 27 Sep, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-34960","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":2863803,"identity":"4cc72b57-04c5-47b2-b137-6e0c49a2e8af","order_by":0,"name":"Abdullateef Isiaka Alagbonsi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYLCCBwwMciD6wAOitSQwGBiDtSSQoiWxAcIgAvCzH38mkVDzJ31+2OGHQFvs5HQbCGiR7Mkxk0g4ZpC78XaaAVBLsrHZAQJaDA7ksEkksAG1zE4AaTmQuI2QFvvzz4EO+2eQbjg7/QNxWgwkEswkEtsMEuSlc4i0ReLGG2OLxD5jww3SOQUHEgyI8At/f/rDGx++ycnLz07f/OFDhZ0cQS1AwCIBdiFYpQFh5SDA/AFEyjcQp3oUjIJRMApGIAAAnzNGNZva1NIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5462-9950","institution":"University of Rwanda","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Abdullateef","middleName":"Isiaka","lastName":"Alagbonsi","suffix":""},{"id":2863804,"identity":"4c23ca21-b109-411f-b164-e865d9f04575","order_by":1,"name":"Luqman Aribidesi Olayaki","email":"","orcid":"","institution":"University of Ilorin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Luqman","middleName":"Aribidesi","lastName":"Olayaki","suffix":""}],"badges":[],"createdAt":"2020-06-12 04:24:39","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.3.rs-34960/v3","doiUrl":"https://doi.org/10.21203/rs.3.rs-34960/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40795-020-00387-y","type":"published","date":"2020-11-24T15:02:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":2749273,"identity":"4e6cfc8d-cba2-4c74-a879-5c88c9d3d108","added_by":"auto","created_at":"2020-10-02 17:04:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22613,"visible":true,"origin":"","legend":"Dose-response and time-course of modulation of the percentage motility (progressive + non-progressive) of spermatozoa after incubation in tetrahydrocannabinol (A) or vitamin C (B).\n*p\u003c0.01; #p\u003c0.001 vs. Baseline (0 min)","description":"","filename":"fig1.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig1.PNG"},{"id":2749274,"identity":"042e2622-8077-4c54-b323-dc55e318eacd","added_by":"auto","created_at":"2020-10-02 17:04:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40554,"visible":true,"origin":"","legend":"Vitamin C abolished the tetrahydrocannabinol-induced reduction in the progressive motility of rat spermatozoa.\nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig2.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig2.PNG"},{"id":2749275,"identity":"6c9ee65b-f58b-4189-8312-0d9cbf9cd514","added_by":"auto","created_at":"2020-10-02 17:04:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46799,"visible":true,"origin":"","legend":"Vitamin C attenuated the tetrahydrocannabinol-induced reduction in the average path velocity of rat spermatozoa.\nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig3.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig3.PNG"},{"id":2749276,"identity":"ac5c77aa-7944-49da-be02-c5a85533f350","added_by":"auto","created_at":"2020-10-02 17:04:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43090,"visible":true,"origin":"","legend":"VVitamin C attenuated the tetrahydrocannabinol-induced reduction in the curvilinear velocity of rat spermatozoa.\nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig4.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig4.PNG"},{"id":2749277,"identity":"85a6ea7c-fb19-4c0d-8d94-b0c782aa1844","added_by":"auto","created_at":"2020-10-02 17:04:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":47481,"visible":true,"origin":"","legend":"Vitamin C abolished the tetrahydrocannabinol-induced reduction in the straight line velocity of rat spermatozoa.\nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig5.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig5.PNG"},{"id":2749278,"identity":"b353ccec-78c6-4f54-9a09-59b6ce371691","added_by":"auto","created_at":"2020-10-02 17:04:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50042,"visible":true,"origin":"","legend":"Vitamin C attenuated the tetrahydrocannabinol-induced reduction in the amplitude of lateral head of rat spermatozoa. \nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig6.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig6.PNG"},{"id":2749279,"identity":"a0964cd4-6b0f-4e45-aea7-7b9d810b2c8f","added_by":"auto","created_at":"2020-10-02 17:04:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":46980,"visible":true,"origin":"","legend":"Vitamin C attenuated the tetrahydrocannabinol-induced reduction in the beat cross frequency of rat spermatozoa. \nTHC, tetrahydrocannabinol; CBs-, cannabinoid receptors’ blockers; *p\u003c0.05 vs. Control; #p\u003c0.05 vs. THC","description":"","filename":"fig7.PNG","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/fig7.PNG"},{"id":13597251,"identity":"01146612-12d4-4139-8864-653f5be5ac72","added_by":"auto","created_at":"2021-09-17 05:31:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":593914,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-34960/v3/a9c67543-fbe8-4aa9-beef-10d925eff4fc.pdf"}],"financialInterests":"","formattedTitle":"Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro","fulltext":[{"header":"1.0. Background","content":"\u003cp\u003eMarijuana, preparation of the flower, leaves and seeds of the plant \u003cem\u003eCannabis sativa \u003c/em\u003e(CS), is the most widely abused illicit drug worldwide (1) and is becoming globally legalised due to its medicinal use by patients with pain (2), inflammation (3), epilepsy (4), cancer (5), etc. However, its detrimental effects on male reproductive functions have been well-established. For instance, it decreases germ cell proliferation, reproductive organ weight (6,7), and semen parameters (7\u0026ndash;9) by eliciting endocrine disruption, hyperprolactinaemia (10), down-regulation of the hypothalamic-pituitary-gonadal axis (10,11), and oxidative stress (9).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eDelta-9-tetrahydrocannabinol (THC, an active psychoactive compound in CS), prescribed under the name Dronabinol (synthetic THC in sesame oil), is clinically being used for the treatment of acquired immunodeficiency syndrome (AIDS) -associated cachexia, multiple sclerosis, and cancer chemotherapy-associated nausea (12,13). However, it has been consistently shown to negatively alter spermatozoa motility, swimming behaviour, and acrosome reaction in sea urchin (14\u0026ndash;16), rat (17), and human (18,19).\u003c/p\u003e\n\u003cp\u003eThe effects of vitamin C on spermatozoa parameters and fertility rate in subfertile males remain controversial. For instance, its protective effect against testicular damage has been well-reported in different conditions (20,21), while its toxic (20) and deoxyribonucleic acid (DNA)- damaging (22,23) potentials have also been shown.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; We have previously observed in our \u003cem\u003ein-vivo \u003c/em\u003estudies that vitamin C exacerbates the CS-induced gonadotoxicity, oxidative stress and reproductive hormonal toxicities, but these CS-induced toxicities were ameliorated only when vitamin C was co-administered with melatonin (7,9,10). In our very recent \u003cem\u003ein-vitro \u003c/em\u003estudy, we observed that melatonin alone ameliorated THC-induced spermatotoxicity by attenuating the THC-induced reduction of hyper-activated motility (HAM) in the capacitated spermatozoa (17). \u0026nbsp;The results of our recent \u003cem\u003ein-vitro\u003c/em\u003e work (17) showed that melatonin alone attenuated, but did not abolish, the THC-induced reduction in spermatozoa motility and kinematics. This lack of abolishment is evident as the motility and kinematics were still significantly lower in spermatozoa that were treated with a combination of melatonin and THC than the control.\u003c/p\u003e\n\u003cp\u003eVitamin C has been reported to increase the motility and kinematics of caprine epididymal spermatozoa \u003cem\u003ein-vitro\u003c/em\u003e (24). Since the combination of melatonin and vitamin C abolished the spermatotoxic effects of CS\u003cem\u003e in-vivo,\u003c/em\u003e the lack of abolishment of the spermatotoxic effect of THC by melatonin alone \u003cem\u003ein-vitro\u003c/em\u003e suggests that the effect of melatonin and vitamin C on the semen parameters might be additive or synergistic. This speculation led us to also investigate the effect of vitamin C on spermatozoa motility and kinematics in this study to clear our doubts whether vitamin C contributed to the combined effect of melatonin and vitamin C or whether the combined effect is a result of melatonin only.\u003c/p\u003e\n\u003cp\u003eFollowing the procedures used in our previous \u003cem\u003ein-vitro\u003c/em\u003e study, the present study investigated the effect of vitamin C on motility and kinematics of capacitated rat spermatozoa treated with cannabinoids using the computer-assisted sperm analyser (CASA). We hypothesised that vitamin C will ameliorate the THC-induced reduction in spermatozoa motility and kinematics.\u003c/p\u003e"},{"header":"2.0. Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix (6) male Wistar albino rats (200-210 g) sourced from the Department of Biochemistry\u0026rsquo;s Animal House, University of Ilorin, Nigeria were housed at room temperature under the daily light/dark cycle with free access to food and water \u003cem\u003ead libitum\u003c/em\u003e. In addition to the study approval by the Ethics Committee of the University of Ilorin, the \u0026ldquo;Principles of laboratory animal care (NIH publication No. 85-23, revised 1985)\u0026rdquo; were followed.\u003c/p\u003e\n\u003cp\u003eFollowing the sacrifice of the rats by 0.8 ml kg\u003csup\u003e-1\u003c/sup\u003e body weight of ketamine hydrochloride, the cauda epididymides were rapidly removed and minced in the modified Biggers\u0026ndash;Whitten\u0026ndash;Whittingham (BWW) capacitation medium, whose components have been previously described (17,25). The liberated spermatozoa were incubated in the medium. Dimethyl sulphoxide (DMSO) was used to prepare the stock solution of the drugs, which was then diluted to the required concentration before each experiment in a BWW capacitation medium such that the final DMSO concentration was 0.2 % (Vol/Vol) (25) that does not affect spermatozoa motility (17).\u003c/p\u003e\n\u003cp\u003eFollowing the method of Charan and Kantharia (26) for the determination of animal sample size, semen from each rat (n= 6) was randomly divided into 6 treatment groups such that each rat\u0026rsquo;s semen was in all of the groups as follows: Groups I-III were respectively treated with placebo (control mixture), THC (1 mM), and vitamin C (5 mM). Group IV was pretreated with 1mM each of CBs\u003csup\u003e-\u003c/sup\u003e 1 (SR141716) and 2 (AM-630), followed by THC (1 mM). Groups V and VI were both treated with THC and vitamin C, but group VI was additionally pretreated with 1 mM each of CBs\u003csup\u003e-\u003c/sup\u003e 1 (SR141716) and 2 (AM-630). Experiments were carried out by incubating spermatozoa in the BWW capacitation medium for 30 minutes. We generated dose-response and time-course of modulation curves from our preliminary experiment to arrive at the doses of THC and vitamin C and the duration used for the study while the same dose of THC was used for the CBs\u003csup\u003e- \u003c/sup\u003e(17).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Determination of spermatozoa motility and kinematics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpermatozoa progressive motility and kinematics (average-path velocity [VAP], curvilinear velocity [VCL], straight-line velocity [VSL], the amplitude of lateral head displacement [ALH], beat cross frequency [BCF], wobble, straightness, and linearity) were recorded with CASA (JH-6004 Sperm Quality Analyser, Jiangsu Jiahua Electronic Instrument Co., Ltd, Jiangsu, China) as previously described (17).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were blindly analysed using statistical software for the social sciences (SPSS) version 16.0 for Windows (IBM Corporation, Armonk, NY, USA). All values given were the Mean \u0026plusmn; S.E.M of the variables. The normality of the data was assessed using the Shapiro-Wilk test. Significance was assessed by the one-way analysis of variance (ANOVA), followed by a posthoc Tukey test for multiple comparisons. The five experimental groups were compared with the control group that received placebo, while groups that received cannabinoids with(out) vitamin C were additionally compared with THC group. Receiver Operating Characteristic (ROC) curve was used to examine the predictive abilities of spermatozoa kinematics on their progressive motility. The \u003cem\u003ep\u003c/em\u003e-values of 0.05 or less were considered statistically significant.\u003c/p\u003e"},{"header":"3.0. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Dose-response and time-course of modulation of spermatozoa motility by tetrahydrocannabinol and vitamin C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThroughout the observation period, 10 \u0026micro;M, 100 \u0026micro;M, 1 mM and 10 mM but not 1 \u0026micro;M of THC reduced the rat spermatozoa motility when compared to the baseline (Figure 1A). On the contrary, 100 \u0026micro;M, 1 mM, 5 mM, and 10 mM but not 10 \u0026micro;M of vitamin C increased the rat spermatozoa motility when compared to the baseline (Figure 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 THC-induced reduction in progressive motility is attenuated by vitamin C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa progressive motility (6.08\u0026plusmn;1.16 %) when compared to the control (39.72\u0026plusmn;0.38 %) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (41.38\u0026plusmn;0.36 %). The progressive motility was increased by vitamin C (51.20\u0026plusmn;1.32 %) when compared to the control (39.72\u0026plusmn;0.38 %). Interestingly, vitamin C inhibited the THC-induced reduction in progressive motility (37.76\u0026plusmn;0.73 %), maintaining it at 94 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on the progressive motility and even caused its additional increase (49.54\u0026plusmn;1.01 %) by 20 % above the control level (Figure 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 THC-induced reductions in some spermatozoa kinematics are attenuated by vitamin C\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa VAP (5.64\u0026plusmn;0.82 \u0026micro;m/s) when compared to the control (13.70\u0026plusmn;0.29 \u0026micro;m/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (14.70\u0026plusmn;0.76 \u0026micro;m/s). The VAP was increased by vitamin C (17.90\u0026plusmn;0.21 \u0026micro;m/s) when compared to the control (13.70\u0026plusmn;0.29 \u0026micro;m/s). Moreover, vitamin C inhibited the THC-induced reduction in VAP (10.98\u0026plusmn;0.45 \u0026micro;m/s), maintaining it at 80% of the control. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VAP and even caused its additional increase (15.7\u0026plusmn;0.38 \u0026micro;m/s) by 13% above the control level (Figure 3).\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa VCL (6.96\u0026plusmn;0.74 \u0026micro;m/s) when compared to the control (18.04\u0026plusmn;0.58 \u0026micro;m/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (21.39\u0026plusmn;0.73 \u0026micro;m/s). The VCL was increased by vitamin C (25.11\u0026plusmn;0.96 \u0026micro;m/s) when compared to the control (18.04\u0026plusmn;0.58 \u0026micro;m/s). Furthermore, vitamin C inhibited the THC-induced reduction in VCL (13.58\u0026plusmn;0.30 \u0026micro;m/s) by maintaining it at 75% of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VCL and even caused its additional increase (22.53\u0026plusmn;0.29 \u0026micro;m/s) by 20 % above the control level (Figure 4).\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa VSL (2.75\u0026plusmn;0.23 \u0026micro;m/s) when compared to the control (7.54\u0026plusmn;0.34 \u0026micro;m/s) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (6.86\u0026plusmn;0.47 \u0026micro;m/s). The VSL was increased, albeit insignificant, by vitamin C (8.80\u0026plusmn;0.27 \u0026micro;m/s) when compared to the control (7.54\u0026plusmn;0.34 \u0026micro;m/s). Vitamin C inhibited the THC-induced reduction in VSL (7.11\u0026plusmn;0.22 \u0026micro;m/s) by maintaining it at 94 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on VSL by maintaining it at a level (7.86\u0026plusmn;0.17 \u0026micro;m/s) comparable to that of the control (Figure 5).\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa ALH (0.31\u0026plusmn;0.02 \u0026micro;m) when compared to the control (0.65\u0026plusmn;0.02 \u0026micro;m) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (0.69\u0026plusmn;0.00 \u0026micro;m). The VSL was increased by vitamin C (0.75\u0026plusmn;0.01 \u0026micro;m) when compared to the control (0.65\u0026plusmn;0.02 \u0026micro;m). Vitamin C inhibited the THC-induced reduction in ALH (0.58\u0026plusmn;0.01 \u0026micro;m) by maintaining it at 89 % of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on ALH by maintaining it at a level (0.67\u0026plusmn;0.01 \u0026micro;m) comparable to that of the control (Figure 6).\u003c/p\u003e\n\u003cp\u003eTHC reduced the spermatozoa BCF (0.78\u0026plusmn;0.08 Hz) when compared to the control (2.79\u0026plusmn;0.01 Hz) but this reduction was inhibited by cannabinoid receptors 1 and 2 blockers (2.88\u0026plusmn;0.00 Hz). The BCF was increased by vitamin C (3.15\u0026plusmn;0.03 Hz) when compared to the control (2.79\u0026plusmn;0.01 Hz). Vitamin C inhibited the THC-induced reduction in BCF (2.60\u0026plusmn;0.01 Hz) by maintaining it at 93% of the control value. When cannabinoid receptors 1 and 2 were blocked, vitamin C completely abolished the effect of THC on BCF by maintaining it at a level (2.77\u0026plusmn;0.03 Hz) comparable to that of control (Figure 7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 The effect of vitamin C on velocity ratios in THC-treated spermatozoa.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHC and/or vitamin C caused no significant change in the spermatozoa wobble, straightness and linearity when compared to control. THC did not also have any effect on these velocity ratios when cannabinoid receptors 1 and 2 were blocked, either in the presence or absence of vitamin C (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Prediction of progressive motility by the kinematics of spermatozoa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the ROC curve, we determined if kinematics could predict the progressive motility of spermatozoa treated with THC and/or vitamin C. The curve showed that 100 % of progressively motile spermatozoa had 8.76 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e as VAP, 10.36 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e as VCL, 4.96 \u0026micro;m s\u003csup\u003e-1\u003c/sup\u003e as VSL, 0.45 \u0026micro;m as ALH, 1.73 Hz as BCF, 66.49 % as wobble, and 32.46 % as linearity while 100 % of non-progressively motile spermatozoa had below these parameters\u0026rsquo; cut-off values (except for wobble and linearity that the specificity is 33.30 %). The velocity ratios (wobble, straightness, and linearity) had lower confidence intervals and areas under the ROC curves than velocities, ALH, and BCF. Thus, there was a very low probability that the progressively motile and non-motile spermatozoa differ in their velocity ratios (wobble, straightness and linearity), while there was a very high probability that they differ in their velocities (VAP, VCL, and VSL), ALH and BCF (Table 2).\u003c/p\u003e"},{"header":"4.0. Discussion","content":"\u003cp\u003eThe reduction in spermatozoa motility by THC was initially reported in sea urchin by Schuel\u0026rsquo;s group (14\u0026ndash;16). A series of studies later showed that cannabinoid receptors activation by anandamide (an endocannabinoid agonist) or THC inhibits spermatozoa functions like motility, velocities, capacitation and acrosome reaction in the frog, rat (6,17), boar (27), and human (19,28\u0026ndash;31). Furthermore, the spermatotoxic potential and the consequent anti-fertility effect of marijuana (7,9,10) and its primary psychoactive cannabinoid (THC) (17) are well-known.\u003c/p\u003e\n\u003cp\u003eEjaculated spermatozoa capacitation process in the female reproductive tract involves membrane and metabolic modifications such as the generation of reactive oxygen species (ROS), an increase in intracellular ions and protein tyrosine phosphorylation, and changes in motility and plasma membrane fluidity (32,33). Events like spermatozoa binding to the zona pellucida (ZP), acrosome reaction, and oocyte fertilisation all depend on successful capacitation process (34), prevention of which will lead to fertilisation failure (35). During this process, spermatozoa acquire a motility pattern called hyper-activated motility (HAM) (36) that is characterised by asymmetrical flagellar beating (37), which is needed for fast swimming and generation of enough force necessary to penetrate cumulus cells and ZP during fertilisation (38). Because the HAM patterns of mammalian spermatozoa capacitated \u003cem\u003ein-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e have been reported to be similar (39,40), it has been very easy for researchers to study different aspects of spermatozoa physiology by \u003cem\u003ein-vitro\u003c/em\u003e capacitation. Our data in this study showed that THC reduced the progressive motility of spermatozoa incubated in capacitation medium, which corroborates its spermatotoxic effect.\u003c/p\u003e\n\u003cp\u003eVitamin C has been previously reported to improve rat\u0026rsquo;s semen parameters \u003cem\u003ein-vivo\u003c/em\u003e (9) and ameliorate the spermatotoxicity induced by cisplatin in rats (41,42) and p-dimethylaminoazobenzene in mice (43). In a randomised controlled clinical trial that included cases after varicocele surgery, vitamin C supplementation significantly increased motility and morphology but not count in infertile young adult males with low-quality spermatozoa, suggesting that it positively affects qualitative but not quantitative characteristics of semen analysis in this condition (44). Apart from the reported improvement in spermatozoa quality in smokers by increasing vitamin C dose from 200 to 1000 mg (45), another observational study on healthy, non-smoking Americans showed that vitamin C-containing diet or supplement increased spermatozoa count and motility (46). When combined with vitamin E, vitamin C has also been reported to enhance rabbit male fertility (47) by increasing concentration and total motile but decreasing abnormal and dead spermatozoa (47,48). A combination of vitamins C and E reportedly ameliorate the oxidative stress and spermatotoxicity induced by endosulfan in rats (49).\u003c/p\u003e\n\u003cp\u003eDoes vitamin C have spermatoprotective effect \u003cem\u003ein-vitro\u003c/em\u003e? Previous reports have shown that vitamin C is the most important seminal anti-oxidant, accounting for about 65% of the seminal anti-oxidant capacity, and is currently being used \u003cem\u003ein-vitro\u003c/em\u003e to enhance spermatozoa quality in infertility clinics (50,51). An Egyptian study has linked smoking to decrease in seminal plasma vitamin C, which was shown to consequently reduce semen parameters and fertilisation potential (52). Our dose-response and time-course of modulation study showed that vitamin C increased the percentage motility of spermatozoa in a dose-dependent but not time-dependent manner. For instance, 100 \u0026micro;M, 1 mM, 5 mM, and 10 mM, but not 10 \u0026micro;M, significantly increased the spermatozoa motility throughout the observation period when compared to the baseline. Furthermore, incubation of spermatozoa in 5 mM of vitamin C solution also increased their motility by 22% when compared to control. The vitamin C-induced increase in spermatozoa motility was associated with an increase in VAP, VCL, VSL, ALH and BCF but decrease in straightness and linearity. These data provide pieces of evidence that vitamin C increases motility by enhancing the kinematics of spermatozoa. These data are consistent with the previous report of Babaie and Sivandi (24) that also reported an improvement in the motility and kinematics of caprine spermatozoa treated with vitamin C \u003cem\u003ein-vitro\u003c/em\u003e. Similar to our study, Babaie and Sivandi (24) obtained epididymal spermatozoa from adult goat testes and incubated them in the capacitation medium (Ca\u003csup\u003e2+\u003c/sup\u003e-free Tyrode\u0026rsquo;s medium, spermatozoa TALP) supplemented with 50 \u0026micro;g/ml vitamin C, after which the motion parameters were assessed using the CASA at various intervals. They also observed that vitamin C increased total and progressive spermatozoa motility, VCL, VSL, linearity, ALH and BCF.\u003c/p\u003e\n\u003cp\u003eCan vitamin C ameliorate the THC-induced spermatotoxicity? In our previous \u003cem\u003ein-vivo\u003c/em\u003e studies, we reported that the CS-induced spermatotoxicities (and the associated endocrine disruption and oxidative stress) were exacerbated by either vitamin C or melatonin when administered separately, but ameliorated when vitamin C was co-administered with melatonin (7,9,10). However, our most recent \u003cem\u003ein-vitro\u003c/em\u003e study showed that melatonin attenuated THC-induced reduction in spermatozoa motility and kinematics of rat. Based on our observation that the modulatory effects of melatonin on spermatotoxicity induced by CS or THC are different \u003cem\u003ein-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e, we were interested to know the effect of vitamin C on THC-induced spermatotoxicity\u003cem\u003e in-vitro\u003c/em\u003e. In the present \u003cem\u003ein-vitro\u003c/em\u003e study where spermatozoa were incubated with THC and vitamin C in the capacitation medium, we observed for the first time that vitamin C completely abolished the THC-induced reduction in progressive motility of spermatozoa by maintaining it at 94% of the control (which is not significantly different from the control level). We also observed that vitamin C attenuated the THC-induced reduction of VAP, VCL, ALH, and BCF but abolished the THC-induced reduction in VSL. These data provide pieces of evidence that vitamin C ameliorates the THC-induced spermatotoxicity, especially as regards motility and kinematics of spermatozoa. Taken together with our previous \u003cem\u003ein-vivo \u003c/em\u003estudy, it also suggests that vitamin C ameliorates cannabinoid-induced spermatotoxicity \u003cem\u003ein-vitro\u003c/em\u003e but exacerbates it \u003cem\u003ein-vivo. \u003c/em\u003eIt is thus similar to our recent study where we reported that melatonin ameliorates cannabinoid-induced spermatotoxicity \u003cem\u003ein-vitro \u003c/em\u003ebut exacerbates it \u003cem\u003ein-vivo\u003c/em\u003e (17).\u003c/p\u003e\n\u003cp\u003eIs there a crosstalk between vitamin C and cannabinoid signalling? The existence of cannabinoid receptors (CbRs) and the endocannabinoid system in mammalian spermatozoa has been well-reported (31,53,54). We have reported that THC-induced reduction in the HAM of spermatozoa was abolished when both CbRs 1 and 2 were blocked, but was only attenuated when either CbR 1 or CbR 2 was selectively blocked. We also showed that CbR 1 contributed more than CbR 2 in THC-induced spermatotoxicity (17). Since the involvement of both CbRs in the regulation of spermatozoa motility has been established, the current study further investigated if the lack of complete abolishment of some kinematics by vitamin C is associated with CbRs activation by THC in spermatozoa incubated with a combination of THC and vitamin C. We blocked both CbRs (SR141716 as CB\u003csup\u003e-\u003c/sup\u003e 1 and AM-630 as CB\u003csup\u003e-\u003c/sup\u003e 2) and then added THC and vitamin C to the capacitation medium. We observed that prior blockade of CbRs cancelled the spermatotoxic effect of THC as vitamin C abolished the effect of THC on VSL, ALH and BCF, and even raised the progressive motility, VAP and VCL above the control value. This shows that vitamin C caused more inhibition of THC-induced spermatotoxicity when CbRs were blocked and is a pointer to the fact that there is a crosstalk between vitamin C and cannabinoid signalling.\u003c/p\u003e\n\u003cp\u003eIs the ameliorative effect of vitamin C on motility related to the kinematics of spermatozoa? Across a wide range of animals like birds (55), fishes (56) and mammals (57), the competition of spermatozoa to fertilise the ovum is favoured by traits (e.g. kinematics) that promote the fertilising ability of male. To penetrate different barriers along its track, the spermatozoon requires a high flagellar BCF (58). The VCL and ALH signify the hyper-activated motility (HAM) of spermatozoa and the high-energy state required for successful fertilisation (59) while motility and velocities of spermatozoa reflect their mitochondrial function and energy status that have been correlated with their fertility (60). Moreover, the progressive motility of spermatozoa has been positively correlated with the VAP, VCL, VSL, ALH, BCF, straightness, and linearity but negatively correlated with wobble in bulls (57,61,62). In our recent study, velocities (VAP, VCL and VSL), ALH and BCF showed strong predictive power on HAM, suggesting that these kinematics determine the response of spermatozoa capacitation-induced HAM to cannabinoids and melatonin (17). In the present study, there was a very low probability that the progressively motile and non-motile spermatozoa differ in their velocity ratios (wobble, straightness and linearity), while there was a very high probability that they differ in their velocities (VAP, VCL, and VSL), ALH and BCF. These suggest that velocities, ALH and BCF determine the modulation of spermatozoa capacitation-induced HAM by cannabinoids and vitamin C.\u003c/p\u003e\n\u003cp\u003eEven though hydrogen peroxide is the major ROS produced in spermatozoa, a previous study has shown that 87% of infertile patients have superoxide anion (63). Spermatozoa are sensitive to assault from free radicals due to the existence of high membrane polyunsaturated fatty acids and lack of capacity for DNA repair (64). Increased ROS level has also been shown to correlate with decreased motility (65) and many structural and functional abnormalities of spermatozoa. Meanwhile, non-enzymatic antioxidants like vitamin C, pyruvate, vitamin E, taurine, vitamin A, hypotaurine, albumin, ubiquitol, urate, and enzymatic antioxidants like catalase, superoxide dismutase, and glutathione peroxidase/reductase have been reported to be contained in the seminal fluid (66), even though vitamin C is considered as the major antioxidant in the testis (67). Furthermore, the ROS-induced damage in spermatozoa has been shown to reduce with supplementation of the latter\u0026rsquo;s clinical media with antioxidant (64). Despite the physiological significance of vitamin C in the body, especially on spermatozoa, humans (unlike most animals) are unable to synthesise vitamin C, making its inclusion in diet or supplement a necessity (68). Vitamin C transporters have been reported to be expressed in the Sertoli cells, spermatocytes, spermatids, spermatozoa and the testis (69,70). However, our present study is limited due to our inability to establish the relationship between vitamin C transporters and cannabinoid receptors as regards HAM of capacitated spermatozoa, which is worthy of consideration in a future study. Another limitation of our study is our inability to estimate the seminal ROS and antioxidant levels in all the groups, which would have enabled us to understand the relationship between spermatozoa kinematics and redox system and their modulation by vitamin C and/or cannabinoid system. Notwithstanding the limitations, our study is the first to provide information on the \u003cem\u003ein-vitro\u003c/em\u003e effect of vitamin C on hyperactivated motility and kinematics of capacitated spermatozoa treated with cannabinoids.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our present acute study shows that vitamin C ameliorates THC-induced reduction in spermatozoa motility \u003cem\u003ein-vitro\u003c/em\u003e by modulation of their kinematics. Since the success rate of \u003cem\u003ein-vitro\u003c/em\u003e fertilisation among users of CS is known to be low (71), the outcome of our present acute study is of clinical importance as it suggests that the success rate of \u003cem\u003ein\u003c/em\u003e-\u003cem\u003evitro\u003c/em\u003e fertilisation among CS users could be improved by \u003cem\u003ein-vitro\u003c/em\u003e supplementation of their spermatozoa with vitamin C. We recommend that a similar study should be conducted on human spermatozoa.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTHC, tetrahydrocannabinol; CBs\u003csup\u003e-\u003c/sup\u003e, cannabinoid receptors\u0026rsquo; blockers; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity; ALH, the amplitude of lateral head; BCF, beat cross frequency; CS, \u003cem\u003eCannabis sativa; \u003c/em\u003eAIDS, acquired immunodeficiency syndrome; DNA, deoxyribonucleic acid; HAM, hyper-activated motility; CASA, computer-assisted sperm analyser; BWW, Biggers\u0026ndash;Whitten\u0026ndash;Whittingham; BSA, bovine serum albumin; DMSO, dimethyl sulphoxide; ANOVA, one-way analysis of variance; ROC, Receiver Operating Characteristic; ROS, reactive oxygen species; ZP, zona pellucida; CbRs, cannabinoid receptors\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animals were well-catered according to the criteria outlined in the \u0026lsquo;Guide for the Care and Use of Laboratory Animals\u0026rsquo; prepared by the National Academy of Science and approved by the Ethical Research Committee of the University of Ilorin, Nigeria.\u003c/p\u003e\n\u003cp\u003eThe consent to participate in the study is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIAA and LAO both conceived and designed the study. IAA carried out the study, interpreted the data, and drafted the manuscript. LAO supervised the study. Both authors read and approved the final manuscript to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Oyeyemi Abdulwahab of Igbinedion University, Okada, who was a Ph.D. candidate in the Department of Physiology, University of Ibadan, Nigeria for permitting us to use his computer-assisted semen analyser. We also appreciate Mr. Adebowale Olabanji of Bridge Scientifik Enterprises Ilorin for his technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Salam O. Gastric acid inhibitory and gastric protective effects of Cannabis and cannabinoids. Asian Pac J Trop Med. 2016 May;9(5):413\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eLynch ME. Cannabinoids in the management of chronic pain: a front line clinical perspective. 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J Reprod Immunol. 2002 Jan;53(1\u0026ndash;2):133\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eDe Jonge C. Biological basis for human capacitation. Hum Reprod Update. 2005 May;11(3):205\u0026ndash;14.\u003c/li\u003e\n\u003cli\u003eYanagimachi R. Fertility of mammalian spermatozoa: its development and relativity. Zygote. 1994 Nov;2(4):371\u0026ndash;2.\u003c/li\u003e\n\u003cli\u003eAMIEUX PS, MCKNIGHT GS. The Essential Role of RI\u0026alpha; in the Maintenance of Regulated PKA Activity. Ann N Y Acad Sci. 2002 Jun;968(1):75\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eGoodson SG, Zhang Z, Tsuruta JK, Wang W, O\u0026rsquo;Brien DA. Classification of Mouse Sperm Motility Patterns Using an Automated Multiclass Support Vector Machines Model1. Biol Reprod. 2011 Jun;84(6):1207\u0026ndash;15.\u003c/li\u003e\n\u003cli\u003eDemott RP, Suarez SS. Hyperactivated Sperm Progress in the Mouse Oviduct1. 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Andrologia. 2006 Dec;38(6):221\u0026ndash;4.\u003c/li\u003e\n\u003cli\u003eAquila S, Guido C, Santoro A, Perrotta I, Laezza C, Bifulco M, et al. Human Sperm Anatomy: Ultrastructural Localization of the Cannabinoid1 Receptor and a Potential Role of Anandamide in Sperm Survival and Acrosome Reaction. Anat Rec Adv Integr Anat Evol Biol. 2010 Feb;293(2):298\u0026ndash;309.\u003c/li\u003e\n\u003cli\u003eAmoako AA, Gebeh AK, Marczylo EL, Willets JM, Elson J, Marczylo TH, et al. Impact of reference gene selection for type 2 cannabinoid receptor gene expression studies in human spermatozoa. Andrologia. 2013 Aug;45(4):278\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eBirkhead TR, Martinez JG, Burke T, Froman DP. Sperm mobility determines the outcome of sperm competition in the domestic fowl. Proc R Soc London Ser B Biol Sci. 1999 Sep;266(1430):1759\u0026ndash;64.\u003c/li\u003e\n\u003cli\u003eGage MJG, Macfariane CP, Yeates S, Ward RG, Searle JB PG. 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J Anim. 2014;2014:1\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eMazzilli F, Rossi T, Marchesini M, Ronconi C, Dondero F. Superoxide anion in human semen related to seminal parameters and clinical aspects. Fertil Steril [Internet]. 1994 Oct;62(4):862\u0026ndash;8. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0015028216570174\u003c/li\u003e\n\u003cli\u003eDonnelly ET, O\u0026rsquo;Connell M, McClure N, Lewis SEM. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum Reprod [Internet]. 2000 Jul;15(7):1552\u0026ndash;61. Available from: https://academic.oup.com/humrep/article-lookup/doi/10.1093/humrep/15.7.1552\u003c/li\u003e\n\u003cli\u003eJedlinska-Krakowska M, Bomba G, Jakubowski K, Rotkiewicz T, Jana B, Penkowski A. Impact of oxidative stress and supplementation with vitamins E and C on testes morphology in rats. J Reprod Dev [Internet]. 2006 Apr;52(2):203\u0026ndash;9. 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Molecular identification and functional characterization of the vitamin C transporters expressed by Sertoli cells. J Cell Physiol [Internet]. 2008 Dec;217(3):708\u0026ndash;16. Available from: http://doi.wiley.com/10.1002/jcp.21545\u003c/li\u003e\n\u003cli\u003eAngulo C, Maldonado R, Pulgar E, Mancilla H, C\u0026oacute;rdova A, Villarroel F, et al. Vitamin C and oxidative stress in the seminiferous epithelium. Biol Res [Internet]. 2011;44(2):169\u0026ndash;80. Available from: http://www.scielo.cl/scielo.php?script=sci_arttext\u0026amp;pid=S0716-97602011000200009\u0026amp;lng=en\u0026amp;nrm=iso\u0026amp;tlng=en\u003c/li\u003e\n\u003cli\u003eKlonoff-Cohen HS, Natarajan L, Victoria Chen R. A prospective study of the effects of female and male marijuana use on in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) outcomes. Am J Obstet Gynecol. 2006 Feb;194(2):369\u0026ndash;76.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003eTable 1: Effect of vitamin C on velocity ratios in tetrahydrocannabinol-treated spermatozoa.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width:6.75in;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 31pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eGroups\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 31pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eWobble\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e(VAP/VCL, %)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 31pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eStraightness\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e(VSL/VAP, %)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 31pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eLinearity\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e(VSL/VCL, %)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eControl\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e76.08 \u0026plusmn; 2.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e55.18 \u0026plusmn; 3.27\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e42.01 \u0026plusmn; 3.13\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eTHC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e81.45 \u0026plusmn; 9.23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e49.81 \u0026plusmn; 4.49\u003c/span\u003e\u003csup\u003e\u003cspan style='font-size:16px;font-family: \"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e40.52 \u0026plusmn; 5.70\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCBs\u003csup\u003e-\u003c/sup\u003e + THC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e68.64\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026plusmn; 1.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e46.89\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026plusmn; 3.74\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e32.16\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026plusmn; 2.55\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eVitamin C\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e71.44 \u0026plusmn; 1.98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e49.14 \u0026plusmn; 0.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e35.07 \u0026plusmn; 0.39\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eVitamin C+THC\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e80.96 \u0026plusmn; 4.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e64.83 \u0026plusmn; 1.96\u003csup\u003e\u0026nbsp;#\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e52.35 \u0026plusmn; 1.36\u003csup\u003e\u0026nbsp;#\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 149.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCBs\u003csup\u003e-\u003c/sup\u003e + THC + Vitamin C\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 121.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e69.74 \u0026plusmn; 2.59\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107.1pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e50.15 \u0026plusmn; 2.04\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1.5in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e34.88 \u0026plusmn; 0.63\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:5px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size: 16px; line-height: 115%; font-family: \"Times New Roman\", serif;'\u003eTHC = tetrahydrocannabinol,\u003c/span\u003e\u003cspan style='font-size: 16px; line-height: 115%; font-family: \"Times New Roman\", serif;'\u003e\u0026nbsp;CBs\u003csup\u003e-\u003c/sup\u003e = cannabinoid receptors\u0026rsquo; blockers;\u003c/span\u003e\u003cspan style='font-size: 16px; line-height: 115%; font-family: \"Times New Roman\", serif;'\u003e\u0026nbsp;VSL = straight line velocity; VCL = curvilinear velocity; VAP = average-path velocity;\u003csup\u003e\u0026nbsp;#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 vs. THC\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;line-height:115%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:115%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-family: \"Times New Roman\", serif; font-size: 16px; text-align: justify;'\u003eTable 2: Prediction of hyper-activated motility by the kinematics of capacitated spermatozoa treated with tetrahydrocannabinol with(out) vitamin C\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 5.2e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 1in;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eOutcomes\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90.9pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ePredictors\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eSensitivity (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eSpecificity (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCutoff value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eAUC\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e95% CI\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border-top: 1pt solid black;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;font-family: \"Times New Roman\",serif;'\u003ep\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-values\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"8\" style=\"width: 1in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eProgressive motility\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90.9pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eVAP (\u0026micro;m/s)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e8.76\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000 to 1.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.9pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eVCL (\u0026micro;m/s)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e10.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000 to 1.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.9pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eVSL (\u0026micro;m/s)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e4.96\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1.000 to 1.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.9pt;border: none;padding: 0in 5.4pt;height: 27.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eALH (\u0026micro;m)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;height: 27.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;height: 27.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border: none;padding: 0in 5.4pt;height: 27.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;height: 27.6pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan 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90.9pt;border: none;padding: 0in 5.4pt;height: 18.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eWobble (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;height: 18.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;height: 18.15pt;vertical-align: top;\"\u003e\n \u003cp 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style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.333\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border: none;padding: 0in 5.4pt;height: 18.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-0.200 to 0.867\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border: none;padding: 0in 5.4pt;height: 18.15pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.405\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.9pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eStraightness (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e88.90\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e66.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e48.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.778\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.455 to 1.101\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.166\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 90.9pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eLinearity (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e100.00\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e33.30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e32.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.593\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.181 to 1.004\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0.75in;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid black;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e0.644\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family: \"Times New Roman\", serif; font-size: 16px;'\u003eVAP= Average-path velocity; VCL= Curvilinear velocity; VSL= Straight-line velocity; ALH= Amplitude of lateral head displacement; BCF= Beat/cross frequency; AUC= Area under the receiver operating characteristic curve; CI= Confidence interval.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:10.0pt;margin-left:0in;line-height:200%;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nutn","sideBox":"Learn more about [BMC Nutrition](http://bmcnutr.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nutn/default.aspx","title":"BMC Nutrition","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Kinematics, Spermatozoa motility, Spermatotoxicity, Tetrahydrocannabinol, Vitamin C","lastPublishedDoi":"10.21203/rs.3.rs-34960/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-34960/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: We investigated the \u003cem\u003ein-vitro\u003c/em\u003e effects of vitamin C on delta-9-tetrahydrocannabinol (THC) -induced reduction in spermatozoa motility and kinematics. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Six rats were used for the study. Semen from each of the 6 rats was randomly divided into 6 groups such that each rat’s semen was in all of the groups. Groups I-III received placebo, THC (1 mM), and vitamin C (5 mM) respectively. Group IV was pretreated with cannabinoid receptors’ blockers (CBs\u003csup\u003e-\u003c/sup\u003e) 1 and 2, followed by THC. Groups V and VI received THC and vitamin C, but group VI was additionally pre-treated with CBs\u003csup\u003e-\u003c/sup\u003e. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The spermatozoa progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head (ALH) and beat cross frequency (BCF) were reduced by THC (6.08±1.16%; 5.64±0.82 µm/s; 6.96±0.74 µm/s; 2.75±0.23 µm/s; 0.31±0.02 µm; and 0.78±0.08 Hz respectively) but increased by vitamin C (51.20±1.32 %; 17.90±0.21 µm/s; 25.11±0.96 µm/s; 8.80±0.27 µm/s; 0.75±0.01 µm; and 3.15±0.03 Hz respectively) when compared to control (39.72±0.38 %; 13.70±0.29 µm/s; 18.04±0.58 µm/s; 7.54±0.34 µm/s; 0.65±0.02 µm; and 2.79±0.01 Hz respectively). Vitamin C inhibited the THC-induced reduction in these parameters (37.36±0.73 %; 10.98±0.45 µm/s; 13.58±0.30 µm/s; 7.11±0.22 µm/s; 0.58±0.01 µm; and 2.60±0.01 Hz respectively) in the absence of CBs\u003csup\u003e-\u003c/sup\u003e 1 and 2, and even caused additional increases in progressive motility (49.54±1.01 %), VAP (15.70±0.38 µm/s) and VCL (22.53±0.29 µm/s) above the control levels with CBs\u003csup\u003e-\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: \u0026nbsp;Vitamin C ameliorates the THC-induced reduction in spermatozoa motility \u003cem\u003ein-vitro\u003c/em\u003e by modulation of their kinematics.\u003c/p\u003e","manuscriptTitle":"Vitamin C ameliorates tetrahydrocannabinol-induced spermatotoxicity in-vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2020-10-02 17:04:42","doi":"10.21203/rs.3.rs-34960/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-09-30T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-09-28T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-27T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-27T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nutn","sideBox":"Learn more about [BMC Nutrition](http://bmcnutr.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nutn/default.aspx","title":"BMC Nutrition","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-09-24 18:17:56","doi":"10.21203/rs.3.rs-34960/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2020-09-24T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-23T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-23T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nutn","sideBox":"Learn more about [BMC Nutrition](http://bmcnutr.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nutn/default.aspx","title":"BMC Nutrition","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-06-16 15:38:27","doi":"10.21203/rs.3.rs-34960/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-07-27T12:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nThe authors have conducted a well-designed animal study on the effect of vitamin C on in rats treated with.\n- Please provide more details on study methods and results in the abstract (e.g. the number of rats in each group should be mentioned...).\n- The introduction too short. It is better to include the main objectives and the rational of the study.\n- How did the researchers determine the dosages of applied supplements? More details on preliminary experiment are needed.\n- Although there is not any detailed information on duration of the study, it seems only acute effects of vitamin C administration were compared with other groups. Thereby, the current conclusion which definitely confirm positive outcomes of this vitamin should be revised accordingly.\n- How did the researchers calculate the current sample size?\n- How did the authors check for normality of data?\n- Consider ANCOVA statistical analyses (adjusted for baseline values) as the statistical test of choice for analyzing the results of trials.\n- Table 1. Please add the exact P-values.\n- Table 2. How did the authors calculate sensitivity, specificity, Cut-off values and 95% CI? Also, the findings presented in this table needs to be more described in the manuscript..\n\n- In the discussion, it is needed to include more updated studies to compare their findings with the current results. Also, the illustrations on vitamin C mechanisms of action is limited.\n- At the last paragraph of discussion, please describe your study's strengths and weaknesses.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please publish my name with my report.**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **No**\n* Is the use of statistics and treatment of uncertainties appropriate?: **No**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"decision","content":"Major revision","date":"2020-07-27T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-08T12:00:00+00:00","index":4,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-07T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-07T12:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-06-26T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThe authors presented a series of analysis to test the effect of Vitamin C on a number of different sperm motility and kinematics parameters after exposure to THC. The authors show that Vitamin C can reduce the spermatotoxic effects of THC in vitro, leading to an even greater recovery in presence of cannabinoid receptors' blockers.\nThe work presented here is rigorous and of interest for the field, especially if considered in light of previous literature produced by these and other authors. However, in my opinion, there are a few points that need to be addressed before the paper is published.\n\n\n1. In the Background section, the authors report that in their previous works (Alagbonsi et al, 2016; Gundersen et al, 2015; Alagbonsi et al, 2017) vitamin C ameliorated spermatotoxicity parameters only when in combination with melatonin. They then point to another work (Alagbonsi et al, 2018) where they showed that melatonin alone was able to ameliorate the spermatotoxic effects of THC, and state that therefore they would like to test whether vitamin C alone can show similar results. Did the authors use different techniques wrt their previous works, or why do they think that they could get different results? Why did they not think that it was melatonin alone responsible for the vitamin C + melatonin effect on THC-induced spermatotoxicity, in light of their second study (Alagbonsi et al, 2018)? Please clarify this point in the manuscript for the reader to understand the rationale behind this choice.\n2. Did the authors consider also the effect on the number/ratio of normal and abnormal forms? If yes, was it not reported because there was no significant difference between treatment and control? If no, why did the authors choose not to consider this parameter? It is known that THC can have an effect on sperm morphology (see https://doi.org/10.1097/JU.0000000000000248), which is an important indicator in human fertility. Moreover, as reported by the authors in lines 243-244, vitamin C supplements showed positive effects on sperm morphology in infertile humans, therefore it would have been of key relevance to have these data. Please, comment on this.\n3. In Lines 297-299, the authors speculate about the cross-talk between vitamin C and cannabinoid signalling. However, to better understand the effect of vitamin C in presence of cannabinoid receptors' blockers, it would have been useful to have data also on the same parameters for THC + CB- alone, without the vitamin C addition. We do not know which would have been the effect of this treatment alone, therefore it is difficult to draw conclusions. For instance, vitamin C might have caused a worsening of the phenotype, not necessarily an amelioration, when compared with THC + CB- alone, possibly exposing variable effects of vitamin C in presence and absence of CB activation. Maybe the authors could explain why they chose not to test this combination.\n4. In Lines 216-219 the sentence is a bit convoluted, please try to clarify the meaning by expanding a bit.\n\nFigures:\n- for all figures in which significance is reported, please do not use the letters because it is extremely difficult to understand which comparisons were statistically significant and which were not. This is particularly true for the bar plots in figures 2 to 7, in which using a line with asterisks on top (the usual way to report significance) would definitely help the reader\n- Figure 1. Since the symbols for the different concentrations are very small and the lines in the graph are really close together, it is difficult to understand which line links to which concentration. Please, either enlarge the symbols or use other ways to differentiate the lines (colors, dashed lines, etc)\n\n\nTypos:\nline 55: pShychoactive compounds\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Are the methods appropriate and well described?: **Yes**\n* Does the work include the necessary controls?: **Yes**\n* Are the conclusions drawn adequately supported by the data shown?: **Yes**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I am able to assess the statistics**\n* Quality of written English: **Acceptable**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. 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