Effects of Low-Energy Laser Irradiation on Sperm Cells Dynamics of Rabbit ( Oryctolagus Cuniculus )

Infertility is a world disease in which a couple is unable to achieve pregnancy. There are numerous parameters to determinate fertility; nevertheless, sperm motility is by consensus one of the most important attributes to evaluate male fertility. Contributions to a better understanding of this crucial parameter are imperative; hence, the aim of this investigation was to assess the effect of low-energy laser irradiation on sperm cell dynamics in thawed samples that were cryopreserved. We used a 405 nm blue laser beam to irradiate spermatic cells from rabbit inside a temperature-controlled dispersion chamber at 37 °C; then, we applied an image recognizing system to calculate individual sperm trajectories and velocities. We found that sperms raise its motility after irradiation suggesting that λ=405 nm is an optimal wavelength for spermatic photo-stimulation.


INTRODUCTION
Infertility is considered a global public health problem in which an active sexual couple is incapable of achieving pregnancy [28]; between 10% and 15% of coupleshavedifficultiesingettingpregnant [27].Tahmasbpourandcoworkers de Jesús-Miranda, JM Mandujano, LA Méndez, F Castillo, YJ Mulia, J García, C Felipe, YE Osorio-González, D (2014) affirm that about75% of infertility problems are due to disorders in sperm quality like oligospermia (low sperm count) and asthenospermia (poor sperm motility); this last term could compromise fertility [5].Sperm count, motility, normal forms, pH, and viscosity are a few out of many factors to evaluate sperm fertility [17]; nevertheless, motility is an inherent characteristic of a good sperm since abnormal sperms cells never show good motility.As a general rule, a spermatozoon is naturally motile, although not all motile spermatozoa are fertile [14].Sperm motility then is a vital factor to evaluate male fertility [8,19].
Sperm motility is based on metabolic and regulatory mechanisms [23]: concentration of reactive oxygen species [2] and intracellular calcium [11], energy ATP, supply products of glycolysis [21] and mitochondria as well as in flagellamotilesystem [16].
Laser beams were used in assisted human reproduction for a first time in the 70s [25]; since then, its use has been expanded for the treatment of sperm and fertility problems [7,20].Despite the numerous publications about sperm stimulation using lasers to improve fertility, it has been suggested that more research is needed to understand the mechanisms better, as well as the interactions and effects in biological and cryopreserved samples photobiostimulated [14,21].Abdel-Salam and Harith (2015) suggested that short wavelengths as λ= 532 nm and λ=405 nm could be better tools in photobiostimulating procedures since it is easier to absorb short wavelengths than longer ones.
A good sperm quality is a good biomarker of health in general, and it is related to survival expectancy; mortality decreases as the level of normal sperms and good sperm motility rises [13].Asthenospermia could be related to defects in mitochondrial function, as mitochondria are the main source of energy supply for spermatic movement [9].A bigger mitochondrial load, as well as a greater sperm motility, would imply that the sperm capability of fertilizing is increased [3].
Motility, vitality, and morphology of spermatic cells are important attributes for a good spermatic function [28].However, most of the procedures and techniques to evaluate motility are subjective estimations (Schirren, 1982a, cited by Push, 1987).Measurements of semen quality must be precise [6,12]; as mentioned above, spermatic motility is one the most important criteria to determine male fertility [8,19,24,28].Thus, the accurate description of the distinctive features of sperm motility becomes necessary.The aim of this work was to evaluate the effect of low energy radiation on rabbit sperm cell dynamics of thawed cryopreserved samples using a 405 nm blue beam laser.

Spermatic samples
Several straws containing 0.25 mL of cryopreserved sperm cells from rabbit (Oryctogalus cuniculus) were thawed in a warm water bath at 37 °C.Samples were obtained from the semen bank of the Laboratorio de Biología de la Reproducción, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México.The spermatic pool was obtained using the artificialvaginamethodinmalerabbitsabout18monthsage;allmaleswere sexually active with more than 80% of motility and viability, and less than 10%ofmorpho-abnormalities.Eachsampleconsistedof15μLofthesperm pool on a slide inside a temperature-controlled dispersion chamber at 37 °C.Each sample was disposed to be irradiated using an experimental set as shown infigure1.

Spermatic stimulation
A total of 27 samples from the spermatic pool were divided into three groups of nine samples each; a) control group (non-irradiated), b) group irradiated with 4 J energy (samples irradiated for 60 s) and c) group irradiated with 6 J energy (samples irradiated for 90 s.).Irradiation was performed using a solid-state 405nmbluebeam(CW,Fermion1series)withanopticalfibercoupledand operating at 70 mW.A lens arrangement was used to optimize the beam power density at the focal point; for this purpose, a laser beam expander was used to increase the circular diameter which was later incised in a plane-convex lens.This beam expander was adjusted with micrometric displacement to correct the divergence laser beam.The focus of the beam was made so that the beam waist hit a 40.5 mm 2 area.Videos of 30 s from each sample were recorded.

Determination of dynamic parameters
We used a Computer Assisted Sperm Analyses (CASA) to analyze sperm motility.Such system is based on the reconstruction of the individual trajectory of each sperm cell from all videotaped samples.The images of the videos were captured using a camera adapted to a microscope and stored in a computer.The calculated parameters (Fig. 2) were straight-line velocity (VSL, micrometers/s), defined as the distance a sperm travels from the first to the  end point of its travel by time; average path velocity (VAP, micrometers/s), defined as the distance traveled long its average trajectory per unit time; curvilinear velocity (VCL, micrometers/s), which description is in terms of its real trajectory; linearity (LIN = VSL/VCL), percentage ratio between straightline and curvilinear velocities, and wobbling (WOB = VAP/VCL).

Statistical analysis
Kruskall-Wallis one-way ANOVA tests were performed using generalized linearmethodsata0.05significancelevelforassessingtheeffectofradiation on VLS, VAP, VCL, LIN and WOB variables among groups (control, 4 J and 6 J groups).Only variables with significant differences among groups were further analyzed performing pairwise comparisons with p-value adjustment according to the number of tests carried out.

RESULTS AND DISCUSSION
One of the most frequent problems in assisted reproduction is to improve sperm viability; currently, there are several pharmacological treatments available, but energy stimulation using a low energy laser represents an alternative solution to the use of drugs.Although some studies have shown contradictory results because an inappropriate dose can affect the integrity of the plasma membrane ortheacrosome,andevencauseirreversibleDNAdamage,thefirstpropertyto be tested in a sample that was stimulated should be the motility.The rheometry tests showed that the shear stress of the fresh samples is in the range 0.5-0.16Pa and for samples that were thawed they were in the range 0.18-0.48Pa as shown in Figure 3.
The shear stress values were used to calculate the viscosities which were 7.5 cP and 9.7 cP for fresh and thawed semen samples, respectively.These values were incorporated into CASA to estimate the velocities and motility parameters, which are shown in Table 1.
Motility variables VLS, VAP and VCL, were significantly different among groups; for VLS, H(2)=15.884,forVAP,H(2)=16.427,andforVCL, H(2)Table 1. Mean ± standard deviation of motility variables from each of the three sample groups.Significant differences in a motility variable among the groups are indicated with an asterisk; Kruskall Wallis H and its associated p-values are shown.Pairwise comparisons: equal alphanumeric characters represent non-significant differences (p>0.05),different alphanumeric characters correspond to significant differences with adjusted p-value shown (according to the number of tests performed).17.471; p<0.001 for these three test statistic values.Pairwise comparisons with adjusted p-values showed that therearesignificantdifferencesinthesethreevariablesbetweenthecontrol group and 4J group, and between control group and 6J group (see p-values in Table 1).These differences corroborate that laser irradiation indeed has an effect on different variables evaluating sperm motility.To evaluate the size of such an effect in each variable, we used the effect size estimate, r, as explained in Fritz et al.,2012.ThesignificantdifferencesinVLSbetweencontrolgroup and4Jgroup(p<0.001)havealargeeffectsize(r=-0.92);however,inthesignificant differences between control group and 6J group (p=0.029), the effectsizeissmaller(r=-0.61).VAPwerealsosignificantlydifferentbetween controland4Jgroups(p<0.001,r=-0.91),andbetweencontroland6Jgroups(p=0.008,r=-0.71).Finally,therewerestatisticallysignificantdifferencesin VCLbetweencontroland4Jgroups(p<0.001,r=-0.94),andbetweencontroland 6J groups (p=0.007,r = -0.71).It is remarkable that all the calculated effect sizes are considered large in the context of this analysis.
As it was assessed, all the laser stimulated samples increased their spermatic velocities with respect to the control group; the spermatic trajectories are not predominantly linear, and they have moderate oscillations; these observations corroborate that irradiated groups have a high probability of possessing spermatic viability.
Increasing spermatic motility is imperative for assisted reproduction specialists as to achieve fecundation of the ovum, spermatozoa must undergo a process named sperm capacitation, which takes place in the female tract [29].When capacitation is achieved, spermatozoa acquire an increase in motility, which is called hyperactivation [30].When spermatozoa come into contact withfollicularfluidacrosomereactiontakesplace,whichistheruptureofthe acrosome membrane followed by the exposition of substances in the sperm head.
Increased motility in sperm samples is reported to be due to stimulation of mitochondrial ATP production by the blue laser beam; mitochondrial ATP is responsible for responding to light stimulation and propitiating the generation of reactive oxygen species (ROS).Nonetheless, this interpretation must be carefully assessed as the blue light stimulation (405nm) effects has also been reported in cell types different from sperm, where intracellular ROS production has been observed [31].Although ROS have an important function in sperm capacitation, hyperactivation, and acrosome reaction, a correlation between high ROS concentrations and decrease in motility has been found [32].

Figure 1 :
Figure 1: Experimental set.Sperm samples inside a temperature-controlled dispersion chamber (center) were irradiated with a 405 nm blue laser (left); videos were captured using a camera adapted to a microscope (right) and stored in a computer.Adapted from Beltrán et al., 2016.

Figure 2 .
Figure 2. Schematic representation of dynamic parameters.Curvilinear (green), average (red) and straight velocities.Green frames represent images captured in videos.

Figure 3 :
Figure 3: The graph compares the shear stress between a sample of fresh sperm (black squares) and one that was previously cryopreserved (red squares).

Table 1 :
Mean ± standard deviation of motility variables from each of the three sample groups.Significantdifferencesinamotilityvariableamongthegroupsareindicated with an asterisk; Kruskall Wallis H and its associated p-values are shown.Pairwise comparisons: equal alphanumeric characters represent non-significant differences (p>0.05),differentalphanumericcharacterscorrespondtosignificantdifferenceswithadjusted p-value shown (according to the number of tests performed).