Effect of trolox added to freezing extenders over goat and ram spermatozoa

Semen cryopreservation is responsible for decrease the gamete fertility, due to structural and functional damages. Among the various causes, oxidative stress, resulting from the higher generation of reactive oxygen species (ROS), has been attributed to affect semen quality. Thus, it was objectified evaluate the effect of Trolox on ram and goat sperm, subjected to freezing. Semen pools of goat (n=5) and ram (n=6) were diluted in skimmed milk (7% glycerol) or Tris-egg yolk (5% glycerol) extender, respectively, added or not of Trolox (0, 20 or 40 μM/ml) and frozen. After thawing (37 °C/30 s), aliquots of semen were evaluated for lipid peroxidation by high performance liquid chromatography, coupled with a photodiode array detector (HPLC-DAD), and flow cytometry (C11BODIPY581/591), besides of plasma membrane and acrosome integrity by fluorescence microscopy, and sperm kinetics by computerized sperm analysis (CASA). The antioxidant treatment with Trolox did not determine significant effects (p>0.05) on lipid peroxidation, plasma membrane integrity, acrosomal integrity and on the kinetic parameters evaluated. Thus, it is concluded that Trolox (20 or 40 μM) did not have a protective or deleterious effect on goats and ram sperm, submitted to freezing.


Introduction
The semen cryopreservation process determines cellular changes that contribute to the fertility reduction, when compared to fresh semen (Bicudo et al., 2007), being the plasma membrane one of the most affected structures (Castro et al., 2016). These changes in sperm cells are associated with the biochemical, osmotic, thermal and mechanical stresses, which are seen at different stages of freezing process (Gangawar et al., 2016). Additionally, reactive oxygen species (ROS) can play a negative impact on sperm integrity during the cryopreservation, which have as main substrate the polyunsaturated fatty acids, mainly present in the cell membranes (Colagar et al., 2013;Castro et al., 2016).
The ROS reaction with cellular polyunsaturated fatty acids (PUFA), start a chain process known as lipid peroxidation (LPO) (Bollwein & Bittner, 2018). Because of the sperm damages, caused by ROS, occur a reduction in semen quality, that includes abnormalities in the intermediate piece, motility decrease, loss of the sperm capacity to perform the acrosome reaction, apoptosis acceleration and fertility impairment (Aitken et al., 1993). Thus, the evaluation and control of the oxidative status and antioxidant defenses system is important as an indicator of the male fertility, especially (Colagar et al., 2013).
Trying to minimize the sperm damage, caused by LPO, antioxidants have been tested (Solihati et al., 2018;Ugur et al., 2019). Among these, vitamin "E" and its hydro-soluble analog, Trolox, stands out. It is indicated as an excellent protector against lipid peroxidation and has been successfully tested in ram sperm (Maia et al., 2010;Sicherle et al., 2011;Silva et al, 2013). Sperm from humans (Taylor et al., 2009;Minaei et al., 2012), ram (Silva et al., 2013) and boar (Pena et al., 2003;Zanella et al., 2016), cryopreserved with Trolox supplementation, present better motility, that do of this agent an important ally to the semen conservation.
Based on the exposed above, was objected in this study evaluated the Trolox effect on goat and ram cryopreserved sperm, through the lipid peroxidation, membranes integrity and kinematics evaluation.

Methodology
All reagents used to the scientific research were purchased from Sigma-Aldrich (St. Louis, USA), with the exception of 2-tiobarbituric acid (MERCK, Darmstadt, Germany), potassium hydroxide (VETEC, Rio de Janeiro, Brazil), sodium hydroxide (NUCLEAR, São Paulo, Brazil) and methanol (CARLOS ERBA, Val de Reuil, France). Moreover, national and institutional guidelines for the care and use of animals were followed, having been the animal procedures approved by the Ethics Committee on the Use of Animals (UFRPE Process Number CEUA / UFRPE 014/2012).
To perform the study around the Trolox effect on goat and ram cryopreserved sperm, were used five goats and six ram sexually mature, with a fertility history and handled intensively. The animals were feed with Tifton hay and 400 g/day of commercial concentrated, besides water and mineral salt ad libitum. From these, ejaculated were obtained by artificial vagina, in presence of a female as a dummy (Oliveira et al., 2013), in alternate days, totaling five ejaculated per goats and six per ram (25 and 36 ejaculated, respectively). The semen samples were subjectively evaluated macroscopic and microscopically, under a phase contrast microscope (Olympus, Japan; 100x), and only ejaculated with motility ≥ 70% (CBRA, 2013) were approved and destined to form the semen pools.
Each experimental repetition corresponded to a semen pool, formed by 5 goats or 6 ram ejaculated, totaling 5 and 6 pools per experiment, respectively. It was done to eliminate the individual variation (Bucak et al., 2008). The minimum requirements to freeze the seminal pool were: volume ≥ 3.0 mL, motility ≥ 70% and sperm concentration ≥ 2x10 9 sperm/mL. Spermatic motility was subjectively analyzed using a phase contrast microscope (Olympus, Japan; 100x) and spermatic concentration in a Neubauer chamber (400x), after dilution of semen aliquots in saline formalin solution (1:400) (CBRA, 2013).
The diluted semen was packaged in straws (0.25 mL) and frozen in an automated system (TK 3000® -TK Tecnologia em Congelação Ltda., Brazil). The cooling curve used showed a drop of -0.25 °C/min in temperature until reaching 5 °C, at which the material remained for 120 min (stabilization time). Then the freezing curve was initiated, with a drop of -20 °C/min, until reaching -120 °C, when the straws were immersed and stored in liquid nitrogen (-196 °C).
Regarding the ram semen pools, they were diluted in Tris-egg yolk extender (Silva et al., 2012), for the final concentration of 200 x10 6 sperm/mL, fractionated and treated according to the experimental groups (G1=control, G2=20 µM and G3=40 µM of Trolox). Subsequently, the samples were stored in straws (0.25 mL) and frozen in an automated system, as previously described for goat semen.
The evaluation of the lipid peroxidation, normally associated with sperm damages during cryopreservation (Aitken et al., 1993;Bollwein & Bittner, 2018) and acting as an indicator of the male fertility (Colagar et al., 2013), was did dosing MDA (malondialdehyde) by HPLC according Candan & Tuzmen (2008), with modifications. For this, work solutions were prepared and, previously to the lipoperoxidation analysis in semen samples, a standard curve was prepared with TEP. The TEP working solution (40 nmol/mL; 2 mL TEP stock solution, 50 mL sulfuric acid) was diluted in series using water to standard concentrations of 10, 5, 2.5, 1.25 and 0.625 nmol/mL, and incubated for 2 h at room temperature.
The chromatographic separations were performed using Rexchorm ODS column (150 mm x 4.6 mm x 5 µm) and Regarding the study of plasma membrane integrity (PMi), parameter positively correlated with fertility (Lee et al., 2009) and most affected by semen cryopreservation (Castro et al., 2016), it was performed by double staining with carboxifluorescein diacetate (CFD) and propidium iodide (PI) (Silva et al., 2019). A total of 200 spermatozoa were evaluated under an epifluorescence microscope (Carl Zeiss, Germany; 400x) and classified as carriers of intact membrane, when stained in green, or damaged membrane, when stained in red.
The acrosome integrity, essential to the fertility (Fernandes & Pimentel, 1997), was evaluated through fluoresceine isothiocyanate-conjugated peanut agglutinin (FITC-PNA) (Silva et al., 2019). A total of 200 spermatozoa per slide were analyzed under a fluorescence microscope (Carl Zeiss, Germany) and classified as having intact acrosome (Aci), when stained in green, or reacted acrosome (Acr), when stained in mixed green, without staining or stained only in the equatorial region of the sperm head. The statistical analyses were performed using GraphPad InStat (version 3.10, 2009). The data obtained to lipid peroxidation, plasma membrane integrity, acrosome integrity and sperm kinematic were tested for normality and homogeneity of variance by Kolmogorov-Smirnov method and Bartlett´s test. Subsequently, these were submitted to analysis of variance (ANOVA), according to each species and experimental group, to determine the effects of the treatments and their interactions, considering significance of 5%. When presented significance, Tukey-Kramer multiple comparison test was realized for mean comparisons. The results were expressed as means and standard deviation (means ± SD).

Results
The results demonstrated that the Trolox addition, at 20 or 40 µM concentrations, to the goat and ram semen cryopreservation extender does not determine significant differences (p>0.05) in the levels of MDA or in the percentage of cells marked with C11-BODIPY 581/591 , when compared to the control group (Table 1). Similarly, to the above exposed, no differences (p>0.05) were observed between the experimental groups for sperm kinematics, plasma membrane integrity, and acrosome integrity of goat and ram semen, after freezing-thawing, in presence or absence of Trolox (Table 2).

Discussion
High ROS levels in semen lead to lipid peroxidation, that is a process harmful to spermatozoa, which may contribute to low motility, morphology and sperm count (Colagar et al., 2013). In this sense, a negative correlation between oxidative stress and sperm motility has been described in ram (Peris et al., 2007). However, in the present study, there was no decline in motility, as well as in any other kinetic parameter, plasma membrane and acrosome integrity after cryopreservation, regardless of antioxidant addition (0, 20 or 40 μM of Trolox), being the total motility higher than 70% for goats and 60% for ram.
Based on the above exposed, is evident the absence of a protective effect of Trolox, in the concentrations studied, on the sperm of goats and ram. These findings corroborate with Sicherle et al. (2011) that did not observe a positive action of this agent at 100 µM, on ram semen, after thawing, except when lipid peroxidation was induced. Similarly, Cabrita et al. (2011) when using 0.1 and 0.5 mM of α-tocopherol for freezing fish sperm, did not find a better preservation of gametes kinetics, viability or DNA integrity.
In opposition to the previous reports, Silva et al. (2013) showed that the Trolox addition at 60 or 120 µM to the egg yolk medium improved the parameters of PM, VSL, VAP, LIN, WOB, STR and PMi. Similarly, Azawi & Hussein, (2013) also saw improvement to plasma membrane integrity, but not to acrosome, for up to 120 h of refrigeration (5 °C) of ram sperm, added with 1 mg/mL of vitamin E.
The absence of protective effect in relation to the antioxidant studied can be attributed to factors such as the dosedependent action (Aitken, 1995) once that, despite Silva et al. (2013) having used the same cryopreservation methodology of the present report, the antioxidant concentrations applied were higher. In addition, it is worth highlighting the specie-specific variation, and the composition of the medium to which the antioxidant is added (Guthrie & Welch, 2007;Lecewicz et al., 2018;Silva et al., 2019).
The use of 20 and 40 µM of Trolox did not reduce the concentrations of malonaldehyde present in cryopreserved semen of goats and ram, when compared to the control group. This fact also was observed by Sicherle et al. (2011) after the use of Trolox (100 µM/10 8 spermatozoa), during the process of ram semen frozen and thawed. On the other hand, inducing the lipid peroxidation with 0.24 nmol FeSO4, these authors verified that the antioxidant became efficient in reduce this harmful process (Sicherle et al., 2011). The same was observed by Maia et al. (2010), when using Trolox (50 µM Trolox/10 8 spermatozoa) for ram semen freezing, as well as by Cerolini et al. (2000), when added α-tocopherol for boar semen refrigeration.
According to the above exposed, it is possible that the protective antioxidant effect of Trolox, beyond to be a dosedependent way, is evidenced only in situations of extreme stress, as is the case of lipoperoxidation induction (Partyka et al., 2011). The variability of results obtained after Trolox therapy may be justified by factors as the influence caused by the iron concentration in semen samples over the ROS production (Peris et al., 2007), the quantity and associations of antioxidants added and present in the semen, as well as the variations in the sperm membrane lipid composition (Guthrie & Welch, 2007;Lecewicz et al., 2018).

Conclusion
It is concluded that Trolox (20 or 40 μM) does not favor the preservation of kinematic parameters, plasma membrane and acrosome integrity, as well as the reduction of lipid peroxidation degree in goat and ram sperm submitted to freezing.
However, is important realize new investigations that consider a greater variety of antioxidant concentrations and correlated it with the specie and extender employed, as well as the induction or not of the lipid peroxidation.