*6.4. Natural Compounds—Traditional Medicine*

Natural compounds have been used traditionally to treat diseases. For instance, beneficial e ffects on reproductive outcome have been reported using products derived from tea (*Camelia sinensis* (L.)), which is the second most consumed beverage after water [63]. For example, an in vitro experiment using green tea extract or epigallocatechin-3-gallate (EGCG) added to human spermatozoa media improved sperm capacitation hallmarks, such as tyrosine phosphorylation and cholesterol e fflux, through the estrogen receptor pathway [64]. EGCG has been shown to have beneficial e ffects when extreme stresses are applied to male mice [65,66]. Interestingly, adverse e ffects induced by artificial testicular hyperthermia were ameliorated by oral administration of green tea extract [65]. Positive effects were visible after 28 days of heat stress induction, improving sperm concentration, percentage of motile and progressive spermatozoa, and sperm membrane integrity [65]. Another example of the beneficial e ffects of EGCG were described when intraperitoneal administration (50 mg/kg) protected against testicular injury induced by ionizing radiation in rats [66]. Thus, treated animals restored testicular function with an improvement in the number of pups by littler reducing LPO (TBARs) and protein carbonyl levels [66]. EGCG's mechanism of action is via the mitogen-activated protein kinase/BCL2 family/caspase 3 pathway [66]. In another work, the combination of two di fferent tea extracts, white and green, where evaluated as additives to improve ART sperm of rats stored at room temperature. The authors found doubled levels of epigallocatechin (EGC) and EGCG in white tea in comparison with green tea [67], highlighting the variability associated with the type of tea extract used. Moreover, although both extracts had positive e ffects, the white tea extract had better ferric reducing antioxidant power than the green tea extract and the control. The beneficial effects were proportional to the concentration used, with 1 mg/mL of white tea extract being the best concentration tested for improving sperm survival and decreasing LPO over 72 hours of storage at room temperature [67]. Encouraged by the antioxidant e ffects on sperm parameters of white tea, the same group explored the oral administration potential of the extract to improve prediabetic type II (PreDM) male reproduction features known to be decreased due to oxidative stress [68]. PreDM is characterized by mild hyperglycemia, glucose intolerance, and insulin resistance and has been related with infertility or subfertility problems in males [69]. Consequently, using rat as an animal model, drinking white tea counteracted the negative e ffects of PreDM on the male reproductive tract. For example, white tea consumption improved testicular antioxidant power and decreased lipid peroxidation and protein oxidation [68]. Ingestion of white tea also restored sperm motility and restored sperm showing morpho-anomalies to normal levels [68].

### **7. Antioxidants as a Tool to Improve Male ART Outcomes**

Human infertility already a ffects one of six couples worldwide [70] and male factors contribute to 20–50% of infertility [71]. Infertile men tend to have higher ROS levels than fertile men. To counteract fertility problems, di fferent ART have been developed, mainly IVF and ICSI. In both cases, gametes are extracted from the body and incubated in in vitro conditions and, after a while, an embryo is transferred into the uterus. It should be noted that due to legislation and ethical issues, it is easier to perform experiments in animal models than in humans to test antioxidant e ffects on di fferent ART. The interest in the use of antioxidants to improve sperm parameters is not new. As early as 1943, in a study focused on sperm metabolism and oxygen consumption, MacLeod showed that sperm produce hydrogen peroxide, which has a deleterious e ffect on sperm motility, and it can be counterbalanced by addition of catalase to the media [72]. Later, some authors followed the same rationale and tried to adapt MacLeod's hypothesis to di fferent ART, such as cryopreservation, IVF, and ICSI.

Sperm conservation for long periods of time in liquid nitrogen (cryopreservation) is designed to keep sperm viable. From a practical point of view, cryopreservation is a tool to enable male fertility before, for example, chemotherapy, radiotherapy, vasectomy, or exposure to toxicants, or just to have time to screen donors for infectious agents, such as the human immunodeficiency or hepatitis B viruses [73]. On the other hand, from the animal industry point of view, the use of cryopreservation aims to maximize the number of services (inseminations) that can be performed from a simple ejaculation, ensuring the quality of genetical material preserved, or allowing the transportation of this genetical material to distant places. Cryopreservation is also of special interest to preserve endangered species. However, cryopreservation is not a harmless technique, inducing DNA and LPO damage and other adverse e ffects [74]. Moreover, cryopreservation, like ART, involves centrifugation, which is associated with production of ROS [13] and removal of seminal plasma which contains the main sperm antioxidant scavenger systems.

Antioxidant supplementation to cryopreservation media has been proposed as a way to overcome ROS production and OS status in spermatozoa (summarized in Table 2). For example, supplementation with a synthetic phenolic antioxidant, butylated hydroxytoluene (BHT), during boar sperm cryopreservation improved post-thawing sperm survival, decreased MDA levels at the concentration of 0.4 mM BHT, and embryo development was improved (28.8% vs. 15.8%) without modification of embryo cleave percentage in comparison to the control [75]. Later, it was described that 1 mM BHT improved antioxidant sperm activity, pregnancy rate (86.7 vs. 63.6%), the number of gilts farrowing (86.7 vs. 45.4%), and the number of piglets born (10.8 ± 1.6 vs. 8.2 ± 2.2) after performing intrauterine artificial insemination (IUI) using cryopreserved sperm versus control [76]. Subsequently, in a multitest in which four di fferent compounds with antioxidant activity (BHT 2 mM, ascorbic acid 8.5 mg/mL, hypotaurine 10 mM, and cysteine 5 mM) were added during goa<sup>t</sup> sperm cryopreservation, LPO was decreased but only ascorbic acid and BHT significantly improved fertility in comparison with control after performing artificial insemination (AI) [77].





The importance and the use of the amino acid cysteine in the fight against ROS impacts on the cell is due to the fact it is a limiting substrate for glutathione synthesis [90]. Cysteine (2 mM) and taurine (2 mM) (a cysteine derived) antioxidant properties were controversial when they were used during the cryopreservation procedure of bull spermatozoa [80]. Taurine decreased GSH and SOD levels, while CAT levels were five times higher than control, but MDA levels were also higher. However, cysteine increased SOD and CAT levels without an effect on MDA levels [80]. The nonreturn rate was not modified when IUI were performed by neither of the compounds; however, a nonsignificant (*p* > 0.05) tendency of improvement was observed in cysteine-treated straws 74.54% (41/55) in comparison to control 57.14% (28/49) [80]. Similar results were obtained when a higher concentration of cysteine (5 mM) and trehalose (25 mM) were added again to bull cryopreservation media. Thus, the antioxidant features of these compounds were not proved; neither MDA nor GPx levels were enhanced [82]. Furthermore, no improvement on the nonreturn rate was found after IUI [82]. Similarly, using cysteamine (5 μM), a decarboxylated derivative of cysteine and lycopene (500 μg/mL) during bull sperm cryopreservation, no differences were found in the nonreturn rate [83]. In other study, the authors used N-acetyl-l-cysteine (NAC), an acetylated cysteine residue which has been shown to effectively reduce ROS formation when H2O2 stress were used in thawed bull spermatozoa [87]. However, neither sperm DNA, nor the number of blastocysts were not improved after performing ICSI using spermatozoa cryopreserved in the presence of NAC [87]. Nevertheless, in an IVF study on mice using fresh spermatozoa, where gametes and embryos were stressed by incubation under 20% oxygen atmosphere (over physiological levels on oviduct and uterine from 2–8% [91]), a combination of substances with antioxidant activity were tested (LAC 10 μM, NAC 10 μM, α-Lipoic Acid 5 μM) in either IVF media, embryo culture media, or both. Treated samples had lower intracellular levels of H2O2, accelerated embryo development, and significantly increased trophectoderm (TE) cell numbers, inner cell mass (ICM), and total cell numbers [88]. All these effects were exacerbated when the antioxidant combo were added during the whole process [88].

Positive effects were also described when thawed bull spermatozoa were supplemented with an antioxidant combination (zinc chloride 10 μg/mL, D-aspartic acid 500 μg/mL, and coenzyme-Q10 40 μg/mL), obtaining a better percentage of total sperm motile and progressive motility and a decrease of DNA fragmentation through sperm incubation [89]. Moreover, antioxidant supplementation improved embryo development. Although no differences were found in the cleave percentage, the number of blastocysts that reached the eight-cell stage was 37.1% in the control versus 51.7% in the treated group [89].

Following the rationale of MacLeod [72], adding antioxidant enzymes to counteract the adverse effects of ROS on spermatozoa was used to improve sperm cryopreservation. Enzymes with antioxidant properties were added to bull cryopreservation media—0.5 and 1.0 mM of reduced glutathione (GSH) or a combination of 0.5 mM of GSH and 100 U/mL of SOD— but did not modify the nonreturn rates [84]. In another study, the use of CAT (200 IU/mL) was used to cryopreserve ram (*Capra pyrenaica*) epidydimal spermatozoa obtained postmortem [79]. At this concentration, no differences were found in sperm parameters but negative effects were described on fertility: fewer pronucleus zygotes (25.5% control vs. 13.2% treated) and cleaved embryos were obtained from treated samples after IVF (16.7% control vs. 7.6% treated) [79].

Natural compounds with antioxidant activity have also been tested in ART. Metformin, a biguanide isolated from *Galea o*ffi*cialis* used worldwide as a treatment for diabetes type II [92], was recently added to the cryopreservation sperm media of chicken due to its antioxidant properties, among other properties [93]. Cryopreserved mouse spermatozoa treated with metformin displayed better motility, sperm viability, doubled fertilization rate and embryo development, and halved DNA fragmentation rate [86]. These promising results of supplementation of cryopreservation media with metformin appeared to be related to the activation of 5'AMP-activated protein kinase (AMPK). However, recently, negative results have been described when metformin (1 and 10 mM) was used to improve boar sperm preservation at 17 ◦C, decreasing sperm motility and mitochondria potential [94]. In an in vitro study performed in human spermatozoa kept at physiological temperature, metformin (10 mM) induced a reduction of sperm motility, where the mechanism of action was associated with PKA pathway inhibition [95]. Boar spermatozoa were coincubated during cryopreservation with rosemary extract (*Rosmarinus o*ffi*cinalis*) or cysteine (10 mM) or a combination of both [81]. Although both compounds enhance some sperm properties, the most noticeable e ffects were found by rosemary compound, enhancing total sperm motility, progressive motility, and preventing acrosome membrane damage three hours post-thawing in comparison to control [81]. Rosemary-treated spermatozoa yielded better cleave percentages without a ffecting blastocyst formation rate after performing IVF [81].

Melatonin (MLT) is a hormone endogenously synthesized mainly by the pineal gland. It has been detected in human seminal fluid [96] and melatonin receptors have been described in sperm of several species [97]. MLT's antioxidant property was tested in cryopreserved human spermatozoa [98]. MLT increased the expression of the antioxidant-related gene Nrf2 as well as its downstream genes SOD2, CAT, HO-1, and GSTM1, leading to lower ROS levels and LPO [98]. On the other hand, MLT (1 μM) used during boar semen preservation at 17 ◦C only showed a modest membrane protective e ffect [99]. By contrast, cryopreserved ram sperm supplemented with MLT achieved higher viability rates, higher percentages of total motile and progressive motile spermatozoa, and higher DNA integrity [85]. However, after IVF, only faster first embryonic division without any other embryo output di fference was observed in those samples supplemented with MLT [85].

Yamaguchi, et al. [100] showed that thawed boar spermatozoa supplemented with ca ffeine improved fertility [100]. Later, the same authors tested a combination of ca ffeine (1.15 mM) with the antioxidant compound β-mercaptoethanol (50 μM) but pregnancy rate was not modified (20 vs. 21% control and treatment respectively) after AI. However, litter size (10.0 ±1.0) almost doubled the data from control samples (5.7 ± 1.5) (*p* < 0.07) [78].

### **8. Antioxidants as a Therapy to Improve Reproduction Outcome**

Sperm produce ROS as consequence of high aerobicmetabolism. ROS production at nonphysiological levels overwhelm cellular scavenger systems and result in deleterious effects, such as lipid and protein peroxidation and DNA damage. Infertile men are known to possess pathological ROS levels, leading to sperm DNA fragmentation and lower ART outcome [29]. Thus, to deal with ROS overproduction and their deleterious effects at cellular levels in the male reproductive system, different strategies have been tested: (i) antioxidant oral consumption and (ii) antioxidants used as additives to media during ART.

Literature concerning the use of compounds with antioxidant activity and the improvement of sperm function is extensive. Nevertheless, others have found negative results [101,102], questioning the beneficial impact of antioxidant prescription and arguing that there is not clear evidence supporting prescription of antioxidants [103] or even that the over exposure to antioxidants can lead to other pathologies [104]. Others have found that administration of high doses of antioxidants have harmful effects on health [105,106]. Most trials have the handicap of using a lower number of men or are not double-blind or placebo-controlled. Moreover, the heterogeneity of the treatments and concentrations used as well as the experimental design make it hard to establish solid conclusions. Studies with greater numbers of patients should be performed, including large control groups to address the e ffects of oral antioxidant consumption on reproductive outcome. Moreover, arbitrary formulations of antioxidants should be avoided and classical pharmacological concentration-dependent experiments should be performed in order to find e ffective concentrations of antioxidants. Rather than by oral consumption, better reproductive outcome results are described when antioxidants were implemented in ART, especially during cryopreservation-thawing procedures. Antioxidant supplementation decreased LPO and improved reproductive outcome. Antioxidant concentration should be adapted to each form of ART. The future of antioxidant therapy to improve ART involves the development of nonintrusive technologies that can discern between sperm with or without lipid peroxidation or DNA damage, allowing physicians to inject healthy sperm into the oocyte by ICSI.

**Author Contributions:** Conceptualization, D.M.-H.; M.G.A. and P.F.O.; Writing-Original Draft Preparation, D.M.-H and M.G.A. Writing-Review & Editing, D.M.-H.; M.J.B.; A.R.B.; M.G.A. and P.F.O. Supervision, M.G.A. Funding Acquisition, M.G.A and P.F.O.

**Funding:** This work was supported by the Portuguese "Fundação para a Ciência e a Tecnologia"—FCT: M.G. Alves (IFCT2015, PTDC/BIM-MET/4712/2014 and PTDC/MEC-AND/28691/2017); P.F. Oliveira (IFCT2015 and PTDC/BBB-BQB/1368/2014); UMIB (Pest-OE/SAU/UI0215/2014) and co-funded by FEDER via Programa Operacional Fatores de Competitividade-COMPETE/QREN & FSE and POPH funds.

**Acknowledgments:** David Martin Hidalgo is recipient of a post-doctoral fellowship from Junta de Extremadura and Fondo Social Europeo (PO17020).

**Conflicts of Interest:** The authors declare no conflict of interest.
