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Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality
Theriogenology xxx (2016) 1–7
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Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality N. Iaffaldano a, *,1, G. Paventi b,1, R. Pizzuto b, M. Di Iorio a, J.L. Bailey c, A. Manchisi a, S. Passarella b a
Department of Agricultural, Environmental, and Food Sciences, University of Molise, Campobasso, Italy Department of Medicine and Health Sciences “Vincenzo Tiberio”, University of Molise, Campobasso, Italy c Département des Sciences Animales, Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2015 Received in revised form 3 February 2016 Accepted 28 February 2016
This study examines whether and how helium-neon laser irradiation (at fluences of 3.96– 9 J/cm2) of cryopreserved ram sperm helps improve semen quality. Pools (n ¼ 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm2, mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm2-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm2 (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Ram sperm Helium-neon laser Cytochrome c oxidase ATP Cryopreserved semen quality
1. Introduction The widespread application of artificial insemination depends largely on the use of cryopreserved sperm. However, the cryopreservation of sperm usually results in reduced fertility because of repercussions of this procedure on sperm quality [1]. In some mammalian species such as
* Corresponding author. Tel.: þ390874404697; fax: þ390874404855. E-mail address: [email protected] (N. Iaffaldano). 1 These authors contributed equally to this work. 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.02.031
sheep [2,3], cryopreserved semen usually gives rise to unacceptably low conception rates [4]. Freezing and/or thawing destabilize sperm membranes leading to mitochondrial damage, which impairs both sperm motility and their ability to survive in the female reproductive tract [5]. Consistently, the reduction in ram sperm motility produced in response to freezing/thawing has been linked to a sharp decrease in ATP contents [3,6]. Thus, to obtain acceptable fertility levels [7], techniques aimed at improving the quality of cryopreserved ram semen, including sperm motility, are required [8]. To date, research efforts have mainly focused mostly on trying to improve freezing
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procedures by finding suitable freezing extenders ([9] and references therein) and/or assessing the use of different additives ([3,10] and references therein) to protect sperm during the freezing-thawing process. However, recent findings indicate that certain cryoprotectants, such as the soy lecithin, could negatively affect sperm mitochondrial function [11], such that other strategies aimed at improving mitochondrial function must be considered. In this regard, photobiostimulation using a low-intensity helium-neon (He-Ne) laser has been shown to improve sperm motility (for a review see [12]). Indeed, photobiostimulation, firstly reported in 1969 [13], has been proven in mouse [14], human [15], sheep [16], dog [17,18], avian [19], and rabbit [20] sperm. This procedure applied to cryopreserved sperm has, nevertheless, only been investigated in cattle [21] and birds [22], thus, studies conducted in other species are needed. In addition, the mechanism whereby sperm photobiostimulation occurs remains to be clearly established. What has been established is that mitochondria are the main light targets in the cell, and photobiostimulation usually results in an increase in ATP synthesis (for references see [23]). It should also be mentioned that sperm motility is dependent on levels of cell ATP, which may be synthesized via both mitochondrial respiration and glycolysis [24–26]. Thus, to expand our preliminary work [27], this study was designed to determine whether mitochondrial stimulation through He-Ne laser irradiation could improve the quality of cryopreserved ram sperm. To this end, we assessed the effects of laser irradiation on certain qualitative parameters in thawed ram sperm, and in a parallel investigation, we also examined the effects on ATP contents and cytochrome c oxidase (COX) activity. 2. Materials and methods 2.1. Chemicals The LIVE/DEAD Sperm Viability Kit was purchased from Molecular Probes Inc. (Eugene, OR, USA). Other chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Semen treatments Cryopreserved semen from four mature Merinizzata Italiana rams was obtained from the Breeding Association APA (Potenza, Italy). The semen was frozen in straws (about 230 106 sperm/straw) according to FAO, 2012 guidelines. Seven semen pools were prepared each by thawing (in a water bath at 37 C for 30 seconds) and mixing 16 straws (four straws per ram). Thus, in seven different experiments, we used a total of 112 straws of cryopreserved semen. 2.3. Sperm irradiation Each semen pool was divided into four aliquots subjected to the treatments: no irradiation (control) or irradiation, as reported by Iaffaldano et al. [19], at room temperature, using a He-Ne laser (wavelength 632.8 nm; 6 mW, 1 cm2 light spot size) for 11 (t1), 17 (t2), or 25 (t3)
minutes. The three irradiation treatments corresponded to energy doses of 3.96, 6.12, and 9 J/cm2, respectively. 2.4. Sperm quality assays In both control (nonirradiated) and irradiated ram sperm samples, the sperm variables motility, viability, osmotic resistance, acrosome integrity, and DNA integrity were assessed in duplicate as described in Rosato et al. [28], with minor adaptations as described below. Sperm motility was microscopically assessed 30 seconds after irradiation treatment. A drop of 10 mL of semen was deposited on a clean glass slide and covered with a coverslip. The preparation was then examined on a warmplate at 400 magnification using a phase-contrast microscope (Leica Aristoplan; Leitz Wetzlar, Heidelberg, Germany). Sperm mass motility and forward progressive motility were determined in five micrographs. Sperm mass motility was defined as the percentage of spermatozoa showing any form of sperm head movement. Forward progressive motility was recorded as the percentage of spermatozoa showing linear movement. The combination of stains, SYBR-14 and propidium iodide, in the LIVE/DEAD Sperm Viability Kit was used to assess sperm viability, as described in Rosato et al. [28]. In brief, sperm aliquots (5 mL) were added to 39-mL PBS solution containing 2 mL of SYBR-14 (diluted 1:100 in dimethyl sulfoxide) and incubated at 37 C for 10 minutes. Next, 5 mL of propidium iodide (diluted 1:100 in PBS) were added, and the mixture incubated at 37 C for a further 5 minutes. Then, 10 mL of the suspension were placed on microscopic slides, covered with coverslips and viable and/or nonviable spermatozoa detected by epifluorescence microscopy (Leica Aristoplan; Leitz Wetzlar, Heidelberg, Germany): blue excitation l ¼ 488 nm ( 1000 magnification using a 100 oil immersion objective, two slides/sample; 200 sperm/slide). A hypo-osmotic water test was used to assess sperm osmotic resistance. In this test, 10 mL of semen were mixed with 40 mL of distilled water in an 1.5-mL eppendorf tube and incubated for 5 minutes at 37 C. Ten microliters of the mixture were placed on a clean glass slide, covered with a thin cover slip and examined under a phase-contrast microscope. The typical “coiled tail” sperm osmotic reaction was easily detected. Hypo-osmotic water test–positive cells were identified in 200 cells counted in at least five fields at 800 total magnification [29]. Acrosome integrity was determined in duplicate airdried smears of a drop of semen from each treatment group. After fixation in methanol for 30 minutes, slides were washed with water and air-dried. Slides were incubated with fluorescein isothiocyanate–Pisum sativum agglutinin (FITC–PSA) for 30 minutes at room temperature, mounted with 50% glycerol (v:v) and coverslipped [30]. Acrosome intact and damaged cells were identified by counting 200 sperm in each sample at 1000 magnification using an oil immersion objective under epifluorescence illumination. Using this stain, acrosomeintact sperm show a uniform applegreen fluorescence, whereas acrosome-damaged sperm show little or no green fluorescence in the anterior head. The percentage of
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acrosome-intact sperm was calculated as a fraction of the total number of sperm. Sperm DNA integrity was evaluated using acridine orange (AO) according to the method described by Gandini et al. [31]. Acridine orange is a cationic fluorescent cytochemical stain that stains cell nuclei, specifically DNA. This stain fluoresces green when incorporated into native DNA (double-stranded and normal) as a monomer and orange-red when it binds to denatured (single-stranded DNA) as an aggregate. A drop of semen from each treatment group was smeared onto a slide, air-dried, fixed overnight in a 3:1 solution of methanol:glacial acetic acid and air-dried once more. Slides were rinsed several times with distilled water. Smears were then stained with an acridine orange solution (0.2 mg/mL in water) in the dark. After 5 minutes, each smear was washed with distilled water and protected with a coverslip. Slides were examined under a fluorescence microscope with a 490-nm excitation light and 530-nm barrier filter. In at least 200 sperm/slide, nuclei were examined and scored as green or orange fluorescing, and the percentage of normal and abnormal chromatin condensation calculated. Sperm displaying green fluorescence were taken to contain intact DNA, whereas those displaying a spectrum of yellow-orange to red fluorescence were recorded as having damaged DNA. 2.5. Cell ATP content Cell ATP contents were determined using an high performance liquid chromatography system (Kontron Instrument, Zurich, Switzerland) including a model 420 pump and a model 425 gradient former equipped with a data system 450 MT2. Adenylates were extracted by rapid centrifugation for 1 minute at 17,900 g in a refrigerated microcentrifuge (Eppendorf 5417R, Milan, Italy). Perchloric acid extracts were obtained, neutralized, and analyzed by high performance liquid chromatography as previously reported by de Bari et al. [32] with calibration in each experiment.
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2.7. Statistical analysis Sperm qualitative characteristics, ATP contents, and COX activity data were compared by analysis of variance followed by the Duncan multiple comparison test. Significance was set at P < 0.05. Correlations among qualitative sperm variables, COX activity, and ATP values recorded for sperm samples subjected to an irradiation dose of 6.12 J/cm2 were also assessed through Pearson’s correlation coefficients. Significance for these tests was set at both the P < 0.05 level (one-tailed) and P < 0.01 levels (two-tailed). All statistical tests were performed using the SPSS package (version 14.0 for Windows, 2005; SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of He-Ne laser irradiation on sperm qualitative parameters The data recorded on sperm motility, viability, osmotic resistance, and acrosome and DNA integrity in the control and irradiated thawed sperm samples are shown in Table 1. These data indicate that the energy dose of 6.12 J/cm2 significantly improved post-thaw semen quality giving rise to increases in sperm mass motility, progressive motility, and viability (P < 0.05). Contrarily, semen samples irradiated at fluences of 3.96 and 9 J/cm2 returned similar values of all the variables examined to control samples, with the exception of a lower DNA integrity recorded in the semen samples irradiated at 9 J/cm2 (P < 0.05). 3.2. Effect of He-Ne laser irradiation on sperm ATP contents The following ATP contents in nmol minute1 109 cells were recorded in the four treatment groups: 35.4 1.84 (control), 42.0 5.28 (irradiation at 3.96 J/cm2), 52.0 3.3 (irradiation at 6.12 J/cm2), and 41 2.9 (irradiation at 9 J/cm2). Thus, the sperm samples irradiated at 6.12 J/cm2 showed ATP contents that were around 125% of control values (P < 0.05) (Fig. 1).
2.6. Cytochrome c oxidase activity assay 3.3. Effect of He-Ne laser irradiation on sperm COX activity Cytochrome c oxidase reaction rates in lysated sperm were determined photometrically as described in [20,22] by monitoring the oxidation of externally added reduced cytochrome c as a decrease in absorbance at 550 nm (absorbance peak of the reduced form of cytochrome c) minus 540 nm (isosbestic point) by means of a JASCO V-560 and V-520 (Tokyo, Japan) dual-beam dualwavelength system (ε550540 ¼ 8 mM1 cm1). Briefly, sperm (about 250 106 cells) were completely lysated by the detergent Triton X-100 (0.1%) and 0.5 mg of lysate protein added to 1 mL of reaction medium consisting of Tris-HCl 50 mM, pH 7.2. The reaction was started by the addition of reduced cytochrome c. The COX reaction rate was obtained as the tangent at the initial part of experimental curve and expressed as nmol minute1 mg1 protein. Experimental data were plotted using the software package Grafit (Erithacus Software, East Grinstead UK).
Figure 2A shows a typical experiment in which the COX reaction was monitored by measuring the oxidation rate of externally added cytochrome c to lysates obtained from both control and irradiated sperm samples. As shown, rates of cytochrome c oxidation (measured as an absorbance decrease) were higher in the irradiated samples (t1 ¼ 3.96 J/cm2; t2 ¼ 6.12 J/cm2; and t3 ¼ 9 J/cm2). The addition of cyanide, which completely blocked the absorbance decrease, confirms that the oxidation of cytochrome c observed was entirely dependent on COX. The effect of He-Ne laser irradiation on COX was explored in more detail by checking the dependence of the COX reaction rate on increasing concentrations of externally added cytochrome c (Fig. 2B). In all the treatment groups, hyperbolic kinetics were found. Interestingly, laser irradiation led to a general decrease in Km values (Michaelis–Menten constant, measured as the
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Table 1 Sperm qualitative parameters (%) recorded in control (non irradiated) and He-Ne laser irradiated (3.96, 6.12 and 9 J/cm2) thawed ram sperm samples (n ¼ 7). Sperm treatment
Sperm parameter (%)
Control 3.96 J/cm2 6.12 J/cm2 9.00 J/cm2
48.78 49.07 57.28 51.60
Mass motility
1.30b 2.17b 1.76a 1.02b
Progressive motility 40.71 40.28 48.71 42.78
1.15b 1.62b 1.55a 1.16b
Viability 47.77 47.36 52.65 45.58
Osmotic resistance 1.63b 0.94b 1.59a 0.45b
39.56 40.02 43.62 38.53
1.57ab 1.76ab 1.79a 0.69b
Acrosome integrity 42.44 41.67 45.20 40.05
1.99a 2.09a 1.67a 0.63a
DNA integrity 98.53 98.08 98.58 97.82
0.15a 0.17ab 0.10a 0.27b
Sperm motility parameters were assessed in five micrographs using the methods: SYBR-14 and propidium iodide staining for sperm viability; hypo-osmotic water test (HOST) for osmotic resistance; FITC-PSA for acrosome integrity; and acridine orange test for DNA integrity (for further details see Methods Section 2.4). All qualitative parameters were determined in duplicate for each sample. a,b Different superscript letters within the same column indicate a significant difference (P < 0.05).
cytochrome c concentration giving half the maximum rate): 14.3 2.0 (control), 6.5 0.7 (irradiation at 3.96 J/cm2, t1), 9.4 0.9 (irradiation at 6.12 J/cm2, t2), and 10.8 1.7 mM (irradiation at 9 J/cm2, t3), indicating increased affinity between COX and its substrate. Irradiation also gave rise to increased Vmax values expressed as nmol minute1 mg1 protein of: 60.9 5.2 (control), 63.5 1.9 (irradiation at 3.96 J/cm2), 71.8 1.9 (irradiation at 6.12 J/cm2), and 66.3 3.2 (irradiation at 9 J/cm2). Thus, with respect to controls, significant (P < 0.05) increases in COX activity were recorded for semen samples irradiated at fluences of 6.12 J/cm2 (118 4.4%) and 9 J/cm2 (112 4.0%; Fig. 2C). To check whether this mitochondrial “stimulation” was somehow related to the improved quality of the cryopreserved sperm, we examined correlations between sperm bioenergetic and quality parameters for the samples in the 6.12 J/cm2 irradiation group. The results of this analysis revealed significant positive correlation between COX activity and ATP contents (P < 0.05), and more importantly, positive correlation was also detected between these variables and the sperm motility variables examined. Thus, ATP contents showed correlation with progressive motility (P < 0.05), whereas COX activity was correlated with both sperm mass and progressive motility (P < 0.01; Table 2).
Fig. 1. ATP contents recorded in control and Helium-neon laser-irradiated thawed ram sperm. ATP contents, expressed in nmol/109 sperm, are reported as the mean values standard error of the mean (7 experiments) recorded in control or irradiated sperm samples. a,bDifferent letters indicate significant differences (P < 0.05).
4. Discussion In this article, we describe how the post-thaw quality of cryopreserved ram sperm can be improved by He-Ne laser irradiation. According to our data, sperm photobiostimulation by He-Ne laser irradiation (at a fluence of 6.12 J/cm2) can improve the motility and viability of cryopreserved sperm in the sheep, an animal species that is highly susceptible to freezing and/or thawing damage [2,3,5,6]. Improved sperm quality in response to photobiomodulation has already been shown in cow [21], goat [33], dog [17], tilapia [16], turkey [19], rabbit [20], and buffalo [34] sperm. The main contribution of the present study is the finding that laser irradiation improves the quality of frozen-thawed ram sperm. In this regard, an increase in both sperm motility and acrosome integrity has been reported in cattle as the result of low-level laser irradiation before semen cryopreservation [35]. It has been well-established that in sperm cells, ATP generated by the mitochondria is mainly required for motility [17] and that a major effect of He-Ne laser irradiation is to increase ATP synthesis in both cells and isolated mitochondria (for a review see [23]). Hence, we hypothesize that the increased ATP production reported here (Fig. 1) could play a key role in the sperm quality improvement observed. Also, given that most cellular ATP is synthesized via oxidative phosphorylation, we investigated in some detail a key component of the respiratory chain, the COX [36], to determine whether and how sperm photobiostimulation could be related to mitochondrial activity. Helium-neon laser irradiation gave rise to reduced Km values, indicating increased substrateenzyme affinity and confirming the findings of a study in which a decrease in Km values for purified COX was observed in response to He-Ne laser treatment [37]. In the present study, COX activity was also enhanced as a result of He-Ne laser irradiation. Increased COX activity promotes COX proton pumping, which generates the electrochemical proton gradient that drives ATP synthesis. This notion is substantiated by the positive correlation detected here between COX activity and ATP content. Given that positive correlation was also observed between COX activity or ATP content and motility parameters, it would appear that the sperm motility increase observed here was the outcome of mitochondrial stimulation due to laser treatment.
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Fig. 2. Cytochrome c oxidase (COX) activity assay in control and Helium-neon (He-Ne) laser–irradiated thawed ram sperm. (A) Lysated sperm (0.5 mg protein) from samples not irradiated (ctrl B) or irradiated with He-Ne laser for different time periods with resultant energy doses of 3.96 (t1 C), 6.12 (t2 :), and 9 (t3 -) J/cm2 were incubated at 25 C in 1 mL of standard medium consisting of Tris-HCl 50 mM, pH 7.2. Absorbance (l ¼ 550–540 nm) was continuously monitored (for details see Methods Section). Arrows indicate the following additions: reduced cytochrome c (cyt c, 0.05 mM), potassium cyanide (KCN, 1 mM). (B) Dependence of the cyt c oxidation rate on increasing concentration of externally added cyt c. The reaction rate was measured as tangent to the initial part of the progress curve obtained as in (A), and expressed as nmol cyt c oxidized minute1 mg1 protein. (C) COX activity values (mean standard error of the mean; seven experiments), expressed as percentages of control values (nonirradiated sperm) recorded in thawed sperm samples subjected to three different energy doses of He-Ne laser irradiation. a,bDifferent letters indicate significant differences (P < 0.05).
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Table 2 Pearson correlation coefficients recorded for sperm qualitative versus bioenergetic parameters in thawed ram sperm subjected to Helium-neon laser irradiation at a fluence of 6.12 J/cm2. Parameter
Mass motility
Progressive motility
Viability
Osmotic resistance
Acrosome integrity
DNA integrity
ATP
ATP COX
0.488 0.689**
0.549* 0.736**
0.307 ns 0.352 ns
0.155 ns 0.375 ns
0.096 ns 0.338 ns
0.002 ns 0.242 ns
1 0.545*
Pearson correlation coefficients were calculated for the sperm qualitative parameters (Table 1) versus ATP contents (Fig. 1) or COX activity values (Fig. 2C) provided for sperm samples in the 6.12 J/cm2 irradiation group in the indicated table and figures (for further details see Methods Section 2.6). Values in bold represent significant correlation detected: *at the 0.05 level; **at the 0.01 level. Abbreviations: COX, Cytochrome c oxidase; ns, not significant.
Despite these observations, further aspects related to laser irradiation warrant consideration: (1) a cellular stress response [38] induced by laser irradiation; (2) Ca2þ transport across both the mitochondrial and plasma membrane, which plays a major role in controlling sperm motility and proved to be stimulated in irradiated sperm [14]; and (3) the formation of reactive oxygen species [16], known to mediate the effects of light [23]. Thus, besides ATP synthesis, COX could play also a role in ATP-independent Ca2þ transport, as reported in ram sperm [39] and could also act as a potent antioxidant-scavenging reactive oxygen species in the mitochondria, as suggested in [20]. The positive effects of He-Ne laser irradiation observed were dependent on the energy dose used. Effectively, both the lower (3.96 J/cm2) and higher (9 J/cm2) doses used in this study were ineffective at improving post-thaw sperm quality. Moreover, DNA integrity was impaired by the 9 J/cm2 treatment. We propose that this higher energy dose adds to the DNA damage incurred by the ram sperm during cryopreservation [2]. In contrast, other authors have noted a progressive decline in the mortality of bull sperm subjected to increasing doses of He-Ne laser irradiation (fluence 2–16 J/cm2) although sperm motility was not investigated [21]. In stored turkey semen, as in the present study, we observed that while lower energy doses were ineffective, higher doses of He-Ne laser irradiation could even be damaging [19]. Whether sperm photobiostimulation via laser light is a new method to be used in artificial insemination remains a matter of debate. Further work is needed to confirm in vivo that sperm quality is effectively improved by laser irradiation. Moreover, because laser irradiation could affect numerous biomolecules thus modifying a variety of biochemical processes (for references see [23]), we need to take a cautious approach to avoid, e.g., possible modification of the mammalian genome. Our findings nevertheless indicate that He-Ne laser irradiation is an innovative tool to be considered in future investigations aimed at improving the quality of cryopreserved mammalian sperm. Acknowledgments The work was supported by the Marie Curie Actions for International Research Staff Exchange (FP7-PEOPLE-2011IRSES, project no. 295137). The authors thank APA, Potenza for kindly providing the cryopreserved ram semen, the PhD student Martina Rocco for help with the laboratory analyses, and Ana Burton for editorial assistance. The authors N.I., G.P., designed the experiments reported in Tables 1 and 2 and Figs. 1 and 2, respectively; M.D.I., R.P., and G.P.
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N. Iaffaldano et al. / Theriogenology xxx (2016) 1–7 [19] Iaffaldano N, Meluzzi A, Manchisi A, Passarella S. Improvement of stored turkey semen quality as a result of He–Ne laser irradiation. Anim Reprod Sci 2005;854:317–25. [20] Iaffaldano N, Rosato MP, Paventi G, Pizzuto R, Gambacorta M, Manchisi A, et al. The irradiation of rabbit sperm cells with He-Ne laser prevents their in vitro liquid storage dependent damage. Anim Reprod Sci 2010;119:123–9. [21] Ocaña-Quero JM, Gomez-Villamandos R, Moreno-Millan M, Santisteban-Valenzuela JM. Biological effects of helium–neon (He–Ne) laser irradiation on acrosome reaction in bull sperm cells. J Photochem Photobiol B 1997;40:294–8. [22] Iaffaldano N, Paventi G, Pizzuto R, Passarella S, Cerolini S, Zaniboni L, et al. The post-thaw irradiation of avian spermatozoa with He–Ne laser differently affects chicken, pheasant and turkey sperm quality. Anim Reprod Sci 2013;142:168–72. [23] Passarella S, Karu T. Absorption of monochromatic and narrow band radiation in the visible and near IR by both mitochondrial and nonmitochondrial photoacceptors results in photobiomodulation. This paper is devoted to the memory of Prof. Lorenzo Bolognani who was one of the pioneers in the field of photobiomodulation. J Photochem Photobiol B 2014;140:344–58. [24] Bartoov B, Bar-Sagie D, Mayevsky A. The effect of pH on ram sperm collective motility driven by mitochondrial respiration. Int J Androl 1980;3:602–12. [25] Breitbart H, Nass-Arden L. Relationship between intracellular calcium, energy metabolism, and motility of ram sperm. Arch Androl 1995;35:83–92. [26] Storey BT. Mammalian sperm metabolism: oxygen and sugar, friend and foe. Int J Dev Biol 2008;52:427–37. [27] Dobrin N, Zamfirescu S, Anghel AH, Topoleanu I, Iaffaldano N, Paventi G, et al. Study of the effects of exposure to different doses of energy generated by a He-Ne laser on the quality of frozen-thawed semen of ram. Rom Biotech Lett 2015;20:10381–7. [28] Rosato MP, Iaffaldano N. Effect of chilling temperature on the longterm survival of rabbit spermatozoa held either in a tris-based or a jellified extender. Reprod Dom Anim 2011;46:301–8.
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[29] Lomeo AM, Giambersio AM. ‘‘Water test’’: a simple method to assess sperm-membrane integrity. Int J Androl 1991;14: 278–82. [30] Mendoza C, Correras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one–step staining method using Pisum sativum agglutinin. J Reprod Fertil 1992;95:755–63. [31] Gandini L, Lombardo F, Lenzi A, Spanò M, Dondero F. Cryopreservation and sperm DNA integrity. Cell and Tissue Banking 2006;7: 91–8. [32] de Bari L, Valenti D, Pizzuto R, Atlante A, Passarella S. Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria. Biochim Biophys Acta Bioenerg 2007;1767:281–94. [33] Wenbin Y, Wenzhong L, Mengzhao L, Baotian Z, Laizeng AI, Tongya L. Effects of laser radiation on Saanen buck’s sperm energy metabolism. In: Proceedings of the Sixth International Conference on goats, Beijing, China; 1996. [34] Abdel-Salam Z, Dessouki SH, Abdel-Salam SA, Ibrahim MA, Harith MA. Green laser irradiation effects on buffalo semen. Theriogenology 2011;75:988–94. [35] Fernandes GHC, De Tarso Camillo De Carvalho P, Serra AJ, Crespilho AM, Schatzman Peron JP, Rossato C, et al. The effect of low-level laser irradiation on sperm motility, and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm. PLoS One 2015;10:e0121487. [36] Arnold S. The power of life-cytochrome c oxidase takes center stage in metabolic control, cell signalling and survival. Mitochondrion 2012;12:46–56. [37] Pastore D, Greco M, Passarella S. Specific helium-neon laser sensitivity of the purified cytochrome c oxidase. Int J Radiat Biol 2000;76: 863–70. [38] Fulda S, Gorman AM, Hori O, Samali A. Cellular stress responses: cell survival and cell death. Int J Cell Biol 2010;2010:214074. [39] Zarca A, Rubinstein S, Breitbart H. Transport mechanism for calcium and phosphate in ram spermatozoa. Biochim Biophys Acta 1988; 944:351–8.
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Helium-neon laser irradiation of cryopreserved ram sperm enhances cytochrome c oxidase activity and ATP levels improving semen quality N. Iaffaldano a, *,1, G. Paventi b,1, R. Pizzuto b, M. Di Iorio a, J.L. Bailey c, A. Manchisi a, S. Passarella b a
Department of Agricultural, Environmental, and Food Sciences, University of Molise, Campobasso, Italy Department of Medicine and Health Sciences “Vincenzo Tiberio”, University of Molise, Campobasso, Italy c Département des Sciences Animales, Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 August 2015 Received in revised form 3 February 2016 Accepted 28 February 2016
This study examines whether and how helium-neon laser irradiation (at fluences of 3.96– 9 J/cm2) of cryopreserved ram sperm helps improve semen quality. Pools (n ¼ 7) of cryopreserved ram sperm were divided into four aliquots and subjected to the treatments: no irradiation (control) or irradiation with three different energy doses. After treatment, the thawed sperm samples were compared in terms of viability, mass and progressive sperm motility, osmotic resistance, as well as DNA and acrosome integrity. In response to irradiation at 6.12 J/cm2, mass sperm motility, progressive motility and viability increased (P < 0.05), with no significant changes observed in the other investigated properties. In parallel, an increase (P < 0.05) in ATP content was detected in the 6.12 J/cm2-irradiated semen samples. Because mitochondria are the main cell photoreceptors with a major role played by cytochrome c oxidase (COX), the COX reaction was monitored using cytochrome c as a substrate in both control and irradiated samples. Laser treatment resulted in a general increase in COX affinity for its substrate as well as an increase in COX activity (Vmax values), the highest activity obtained for sperm samples irradiated at 6.12 J/cm2 (P < 0.05). Interestingly, in these irradiated sperm samples, COX activity and ATP contents were positively correlated, and, more importantly, they also showed positive correlation with motility, suggesting that the improved sperm quality observed was related to mitochondria-laser light interactions. Ó 2016 Elsevier Inc. All rights reserved.
Keywords: Ram sperm Helium-neon laser Cytochrome c oxidase ATP Cryopreserved semen quality
1. Introduction The widespread application of artificial insemination depends largely on the use of cryopreserved sperm. However, the cryopreservation of sperm usually results in reduced fertility because of repercussions of this procedure on sperm quality [1]. In some mammalian species such as
* Corresponding author. Tel.: þ390874404697; fax: þ390874404855. E-mail address: [email protected] (N. Iaffaldano). 1 These authors contributed equally to this work. 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.02.031
sheep [2,3], cryopreserved semen usually gives rise to unacceptably low conception rates [4]. Freezing and/or thawing destabilize sperm membranes leading to mitochondrial damage, which impairs both sperm motility and their ability to survive in the female reproductive tract [5]. Consistently, the reduction in ram sperm motility produced in response to freezing/thawing has been linked to a sharp decrease in ATP contents [3,6]. Thus, to obtain acceptable fertility levels [7], techniques aimed at improving the quality of cryopreserved ram semen, including sperm motility, are required [8]. To date, research efforts have mainly focused mostly on trying to improve freezing
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procedures by finding suitable freezing extenders ([9] and references therein) and/or assessing the use of different additives ([3,10] and references therein) to protect sperm during the freezing-thawing process. However, recent findings indicate that certain cryoprotectants, such as the soy lecithin, could negatively affect sperm mitochondrial function [11], such that other strategies aimed at improving mitochondrial function must be considered. In this regard, photobiostimulation using a low-intensity helium-neon (He-Ne) laser has been shown to improve sperm motility (for a review see [12]). Indeed, photobiostimulation, firstly reported in 1969 [13], has been proven in mouse [14], human [15], sheep [16], dog [17,18], avian [19], and rabbit [20] sperm. This procedure applied to cryopreserved sperm has, nevertheless, only been investigated in cattle [21] and birds [22], thus, studies conducted in other species are needed. In addition, the mechanism whereby sperm photobiostimulation occurs remains to be clearly established. What has been established is that mitochondria are the main light targets in the cell, and photobiostimulation usually results in an increase in ATP synthesis (for references see [23]). It should also be mentioned that sperm motility is dependent on levels of cell ATP, which may be synthesized via both mitochondrial respiration and glycolysis [24–26]. Thus, to expand our preliminary work [27], this study was designed to determine whether mitochondrial stimulation through He-Ne laser irradiation could improve the quality of cryopreserved ram sperm. To this end, we assessed the effects of laser irradiation on certain qualitative parameters in thawed ram sperm, and in a parallel investigation, we also examined the effects on ATP contents and cytochrome c oxidase (COX) activity. 2. Materials and methods 2.1. Chemicals The LIVE/DEAD Sperm Viability Kit was purchased from Molecular Probes Inc. (Eugene, OR, USA). Other chemicals used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Semen treatments Cryopreserved semen from four mature Merinizzata Italiana rams was obtained from the Breeding Association APA (Potenza, Italy). The semen was frozen in straws (about 230 106 sperm/straw) according to FAO, 2012 guidelines. Seven semen pools were prepared each by thawing (in a water bath at 37 C for 30 seconds) and mixing 16 straws (four straws per ram). Thus, in seven different experiments, we used a total of 112 straws of cryopreserved semen. 2.3. Sperm irradiation Each semen pool was divided into four aliquots subjected to the treatments: no irradiation (control) or irradiation, as reported by Iaffaldano et al. [19], at room temperature, using a He-Ne laser (wavelength 632.8 nm; 6 mW, 1 cm2 light spot size) for 11 (t1), 17 (t2), or 25 (t3)
minutes. The three irradiation treatments corresponded to energy doses of 3.96, 6.12, and 9 J/cm2, respectively. 2.4. Sperm quality assays In both control (nonirradiated) and irradiated ram sperm samples, the sperm variables motility, viability, osmotic resistance, acrosome integrity, and DNA integrity were assessed in duplicate as described in Rosato et al. [28], with minor adaptations as described below. Sperm motility was microscopically assessed 30 seconds after irradiation treatment. A drop of 10 mL of semen was deposited on a clean glass slide and covered with a coverslip. The preparation was then examined on a warmplate at 400 magnification using a phase-contrast microscope (Leica Aristoplan; Leitz Wetzlar, Heidelberg, Germany). Sperm mass motility and forward progressive motility were determined in five micrographs. Sperm mass motility was defined as the percentage of spermatozoa showing any form of sperm head movement. Forward progressive motility was recorded as the percentage of spermatozoa showing linear movement. The combination of stains, SYBR-14 and propidium iodide, in the LIVE/DEAD Sperm Viability Kit was used to assess sperm viability, as described in Rosato et al. [28]. In brief, sperm aliquots (5 mL) were added to 39-mL PBS solution containing 2 mL of SYBR-14 (diluted 1:100 in dimethyl sulfoxide) and incubated at 37 C for 10 minutes. Next, 5 mL of propidium iodide (diluted 1:100 in PBS) were added, and the mixture incubated at 37 C for a further 5 minutes. Then, 10 mL of the suspension were placed on microscopic slides, covered with coverslips and viable and/or nonviable spermatozoa detected by epifluorescence microscopy (Leica Aristoplan; Leitz Wetzlar, Heidelberg, Germany): blue excitation l ¼ 488 nm ( 1000 magnification using a 100 oil immersion objective, two slides/sample; 200 sperm/slide). A hypo-osmotic water test was used to assess sperm osmotic resistance. In this test, 10 mL of semen were mixed with 40 mL of distilled water in an 1.5-mL eppendorf tube and incubated for 5 minutes at 37 C. Ten microliters of the mixture were placed on a clean glass slide, covered with a thin cover slip and examined under a phase-contrast microscope. The typical “coiled tail” sperm osmotic reaction was easily detected. Hypo-osmotic water test–positive cells were identified in 200 cells counted in at least five fields at 800 total magnification [29]. Acrosome integrity was determined in duplicate airdried smears of a drop of semen from each treatment group. After fixation in methanol for 30 minutes, slides were washed with water and air-dried. Slides were incubated with fluorescein isothiocyanate–Pisum sativum agglutinin (FITC–PSA) for 30 minutes at room temperature, mounted with 50% glycerol (v:v) and coverslipped [30]. Acrosome intact and damaged cells were identified by counting 200 sperm in each sample at 1000 magnification using an oil immersion objective under epifluorescence illumination. Using this stain, acrosomeintact sperm show a uniform applegreen fluorescence, whereas acrosome-damaged sperm show little or no green fluorescence in the anterior head. The percentage of
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acrosome-intact sperm was calculated as a fraction of the total number of sperm. Sperm DNA integrity was evaluated using acridine orange (AO) according to the method described by Gandini et al. [31]. Acridine orange is a cationic fluorescent cytochemical stain that stains cell nuclei, specifically DNA. This stain fluoresces green when incorporated into native DNA (double-stranded and normal) as a monomer and orange-red when it binds to denatured (single-stranded DNA) as an aggregate. A drop of semen from each treatment group was smeared onto a slide, air-dried, fixed overnight in a 3:1 solution of methanol:glacial acetic acid and air-dried once more. Slides were rinsed several times with distilled water. Smears were then stained with an acridine orange solution (0.2 mg/mL in water) in the dark. After 5 minutes, each smear was washed with distilled water and protected with a coverslip. Slides were examined under a fluorescence microscope with a 490-nm excitation light and 530-nm barrier filter. In at least 200 sperm/slide, nuclei were examined and scored as green or orange fluorescing, and the percentage of normal and abnormal chromatin condensation calculated. Sperm displaying green fluorescence were taken to contain intact DNA, whereas those displaying a spectrum of yellow-orange to red fluorescence were recorded as having damaged DNA. 2.5. Cell ATP content Cell ATP contents were determined using an high performance liquid chromatography system (Kontron Instrument, Zurich, Switzerland) including a model 420 pump and a model 425 gradient former equipped with a data system 450 MT2. Adenylates were extracted by rapid centrifugation for 1 minute at 17,900 g in a refrigerated microcentrifuge (Eppendorf 5417R, Milan, Italy). Perchloric acid extracts were obtained, neutralized, and analyzed by high performance liquid chromatography as previously reported by de Bari et al. [32] with calibration in each experiment.
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2.7. Statistical analysis Sperm qualitative characteristics, ATP contents, and COX activity data were compared by analysis of variance followed by the Duncan multiple comparison test. Significance was set at P < 0.05. Correlations among qualitative sperm variables, COX activity, and ATP values recorded for sperm samples subjected to an irradiation dose of 6.12 J/cm2 were also assessed through Pearson’s correlation coefficients. Significance for these tests was set at both the P < 0.05 level (one-tailed) and P < 0.01 levels (two-tailed). All statistical tests were performed using the SPSS package (version 14.0 for Windows, 2005; SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Effect of He-Ne laser irradiation on sperm qualitative parameters The data recorded on sperm motility, viability, osmotic resistance, and acrosome and DNA integrity in the control and irradiated thawed sperm samples are shown in Table 1. These data indicate that the energy dose of 6.12 J/cm2 significantly improved post-thaw semen quality giving rise to increases in sperm mass motility, progressive motility, and viability (P < 0.05). Contrarily, semen samples irradiated at fluences of 3.96 and 9 J/cm2 returned similar values of all the variables examined to control samples, with the exception of a lower DNA integrity recorded in the semen samples irradiated at 9 J/cm2 (P < 0.05). 3.2. Effect of He-Ne laser irradiation on sperm ATP contents The following ATP contents in nmol minute1 109 cells were recorded in the four treatment groups: 35.4 1.84 (control), 42.0 5.28 (irradiation at 3.96 J/cm2), 52.0 3.3 (irradiation at 6.12 J/cm2), and 41 2.9 (irradiation at 9 J/cm2). Thus, the sperm samples irradiated at 6.12 J/cm2 showed ATP contents that were around 125% of control values (P < 0.05) (Fig. 1).
2.6. Cytochrome c oxidase activity assay 3.3. Effect of He-Ne laser irradiation on sperm COX activity Cytochrome c oxidase reaction rates in lysated sperm were determined photometrically as described in [20,22] by monitoring the oxidation of externally added reduced cytochrome c as a decrease in absorbance at 550 nm (absorbance peak of the reduced form of cytochrome c) minus 540 nm (isosbestic point) by means of a JASCO V-560 and V-520 (Tokyo, Japan) dual-beam dualwavelength system (ε550540 ¼ 8 mM1 cm1). Briefly, sperm (about 250 106 cells) were completely lysated by the detergent Triton X-100 (0.1%) and 0.5 mg of lysate protein added to 1 mL of reaction medium consisting of Tris-HCl 50 mM, pH 7.2. The reaction was started by the addition of reduced cytochrome c. The COX reaction rate was obtained as the tangent at the initial part of experimental curve and expressed as nmol minute1 mg1 protein. Experimental data were plotted using the software package Grafit (Erithacus Software, East Grinstead UK).
Figure 2A shows a typical experiment in which the COX reaction was monitored by measuring the oxidation rate of externally added cytochrome c to lysates obtained from both control and irradiated sperm samples. As shown, rates of cytochrome c oxidation (measured as an absorbance decrease) were higher in the irradiated samples (t1 ¼ 3.96 J/cm2; t2 ¼ 6.12 J/cm2; and t3 ¼ 9 J/cm2). The addition of cyanide, which completely blocked the absorbance decrease, confirms that the oxidation of cytochrome c observed was entirely dependent on COX. The effect of He-Ne laser irradiation on COX was explored in more detail by checking the dependence of the COX reaction rate on increasing concentrations of externally added cytochrome c (Fig. 2B). In all the treatment groups, hyperbolic kinetics were found. Interestingly, laser irradiation led to a general decrease in Km values (Michaelis–Menten constant, measured as the
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Table 1 Sperm qualitative parameters (%) recorded in control (non irradiated) and He-Ne laser irradiated (3.96, 6.12 and 9 J/cm2) thawed ram sperm samples (n ¼ 7). Sperm treatment
Sperm parameter (%)
Control 3.96 J/cm2 6.12 J/cm2 9.00 J/cm2
48.78 49.07 57.28 51.60
Mass motility
1.30b 2.17b 1.76a 1.02b
Progressive motility 40.71 40.28 48.71 42.78
1.15b 1.62b 1.55a 1.16b
Viability 47.77 47.36 52.65 45.58
Osmotic resistance 1.63b 0.94b 1.59a 0.45b
39.56 40.02 43.62 38.53
1.57ab 1.76ab 1.79a 0.69b
Acrosome integrity 42.44 41.67 45.20 40.05
1.99a 2.09a 1.67a 0.63a
DNA integrity 98.53 98.08 98.58 97.82
0.15a 0.17ab 0.10a 0.27b
Sperm motility parameters were assessed in five micrographs using the methods: SYBR-14 and propidium iodide staining for sperm viability; hypo-osmotic water test (HOST) for osmotic resistance; FITC-PSA for acrosome integrity; and acridine orange test for DNA integrity (for further details see Methods Section 2.4). All qualitative parameters were determined in duplicate for each sample. a,b Different superscript letters within the same column indicate a significant difference (P < 0.05).
cytochrome c concentration giving half the maximum rate): 14.3 2.0 (control), 6.5 0.7 (irradiation at 3.96 J/cm2, t1), 9.4 0.9 (irradiation at 6.12 J/cm2, t2), and 10.8 1.7 mM (irradiation at 9 J/cm2, t3), indicating increased affinity between COX and its substrate. Irradiation also gave rise to increased Vmax values expressed as nmol minute1 mg1 protein of: 60.9 5.2 (control), 63.5 1.9 (irradiation at 3.96 J/cm2), 71.8 1.9 (irradiation at 6.12 J/cm2), and 66.3 3.2 (irradiation at 9 J/cm2). Thus, with respect to controls, significant (P < 0.05) increases in COX activity were recorded for semen samples irradiated at fluences of 6.12 J/cm2 (118 4.4%) and 9 J/cm2 (112 4.0%; Fig. 2C). To check whether this mitochondrial “stimulation” was somehow related to the improved quality of the cryopreserved sperm, we examined correlations between sperm bioenergetic and quality parameters for the samples in the 6.12 J/cm2 irradiation group. The results of this analysis revealed significant positive correlation between COX activity and ATP contents (P < 0.05), and more importantly, positive correlation was also detected between these variables and the sperm motility variables examined. Thus, ATP contents showed correlation with progressive motility (P < 0.05), whereas COX activity was correlated with both sperm mass and progressive motility (P < 0.01; Table 2).
Fig. 1. ATP contents recorded in control and Helium-neon laser-irradiated thawed ram sperm. ATP contents, expressed in nmol/109 sperm, are reported as the mean values standard error of the mean (7 experiments) recorded in control or irradiated sperm samples. a,bDifferent letters indicate significant differences (P < 0.05).
4. Discussion In this article, we describe how the post-thaw quality of cryopreserved ram sperm can be improved by He-Ne laser irradiation. According to our data, sperm photobiostimulation by He-Ne laser irradiation (at a fluence of 6.12 J/cm2) can improve the motility and viability of cryopreserved sperm in the sheep, an animal species that is highly susceptible to freezing and/or thawing damage [2,3,5,6]. Improved sperm quality in response to photobiomodulation has already been shown in cow [21], goat [33], dog [17], tilapia [16], turkey [19], rabbit [20], and buffalo [34] sperm. The main contribution of the present study is the finding that laser irradiation improves the quality of frozen-thawed ram sperm. In this regard, an increase in both sperm motility and acrosome integrity has been reported in cattle as the result of low-level laser irradiation before semen cryopreservation [35]. It has been well-established that in sperm cells, ATP generated by the mitochondria is mainly required for motility [17] and that a major effect of He-Ne laser irradiation is to increase ATP synthesis in both cells and isolated mitochondria (for a review see [23]). Hence, we hypothesize that the increased ATP production reported here (Fig. 1) could play a key role in the sperm quality improvement observed. Also, given that most cellular ATP is synthesized via oxidative phosphorylation, we investigated in some detail a key component of the respiratory chain, the COX [36], to determine whether and how sperm photobiostimulation could be related to mitochondrial activity. Helium-neon laser irradiation gave rise to reduced Km values, indicating increased substrateenzyme affinity and confirming the findings of a study in which a decrease in Km values for purified COX was observed in response to He-Ne laser treatment [37]. In the present study, COX activity was also enhanced as a result of He-Ne laser irradiation. Increased COX activity promotes COX proton pumping, which generates the electrochemical proton gradient that drives ATP synthesis. This notion is substantiated by the positive correlation detected here between COX activity and ATP content. Given that positive correlation was also observed between COX activity or ATP content and motility parameters, it would appear that the sperm motility increase observed here was the outcome of mitochondrial stimulation due to laser treatment.
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Fig. 2. Cytochrome c oxidase (COX) activity assay in control and Helium-neon (He-Ne) laser–irradiated thawed ram sperm. (A) Lysated sperm (0.5 mg protein) from samples not irradiated (ctrl B) or irradiated with He-Ne laser for different time periods with resultant energy doses of 3.96 (t1 C), 6.12 (t2 :), and 9 (t3 -) J/cm2 were incubated at 25 C in 1 mL of standard medium consisting of Tris-HCl 50 mM, pH 7.2. Absorbance (l ¼ 550–540 nm) was continuously monitored (for details see Methods Section). Arrows indicate the following additions: reduced cytochrome c (cyt c, 0.05 mM), potassium cyanide (KCN, 1 mM). (B) Dependence of the cyt c oxidation rate on increasing concentration of externally added cyt c. The reaction rate was measured as tangent to the initial part of the progress curve obtained as in (A), and expressed as nmol cyt c oxidized minute1 mg1 protein. (C) COX activity values (mean standard error of the mean; seven experiments), expressed as percentages of control values (nonirradiated sperm) recorded in thawed sperm samples subjected to three different energy doses of He-Ne laser irradiation. a,bDifferent letters indicate significant differences (P < 0.05).
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Table 2 Pearson correlation coefficients recorded for sperm qualitative versus bioenergetic parameters in thawed ram sperm subjected to Helium-neon laser irradiation at a fluence of 6.12 J/cm2. Parameter
Mass motility
Progressive motility
Viability
Osmotic resistance
Acrosome integrity
DNA integrity
ATP
ATP COX
0.488 0.689**
0.549* 0.736**
0.307 ns 0.352 ns
0.155 ns 0.375 ns
0.096 ns 0.338 ns
0.002 ns 0.242 ns
1 0.545*
Pearson correlation coefficients were calculated for the sperm qualitative parameters (Table 1) versus ATP contents (Fig. 1) or COX activity values (Fig. 2C) provided for sperm samples in the 6.12 J/cm2 irradiation group in the indicated table and figures (for further details see Methods Section 2.6). Values in bold represent significant correlation detected: *at the 0.05 level; **at the 0.01 level. Abbreviations: COX, Cytochrome c oxidase; ns, not significant.
Despite these observations, further aspects related to laser irradiation warrant consideration: (1) a cellular stress response [38] induced by laser irradiation; (2) Ca2þ transport across both the mitochondrial and plasma membrane, which plays a major role in controlling sperm motility and proved to be stimulated in irradiated sperm [14]; and (3) the formation of reactive oxygen species [16], known to mediate the effects of light [23]. Thus, besides ATP synthesis, COX could play also a role in ATP-independent Ca2þ transport, as reported in ram sperm [39] and could also act as a potent antioxidant-scavenging reactive oxygen species in the mitochondria, as suggested in [20]. The positive effects of He-Ne laser irradiation observed were dependent on the energy dose used. Effectively, both the lower (3.96 J/cm2) and higher (9 J/cm2) doses used in this study were ineffective at improving post-thaw sperm quality. Moreover, DNA integrity was impaired by the 9 J/cm2 treatment. We propose that this higher energy dose adds to the DNA damage incurred by the ram sperm during cryopreservation [2]. In contrast, other authors have noted a progressive decline in the mortality of bull sperm subjected to increasing doses of He-Ne laser irradiation (fluence 2–16 J/cm2) although sperm motility was not investigated [21]. In stored turkey semen, as in the present study, we observed that while lower energy doses were ineffective, higher doses of He-Ne laser irradiation could even be damaging [19]. Whether sperm photobiostimulation via laser light is a new method to be used in artificial insemination remains a matter of debate. Further work is needed to confirm in vivo that sperm quality is effectively improved by laser irradiation. Moreover, because laser irradiation could affect numerous biomolecules thus modifying a variety of biochemical processes (for references see [23]), we need to take a cautious approach to avoid, e.g., possible modification of the mammalian genome. Our findings nevertheless indicate that He-Ne laser irradiation is an innovative tool to be considered in future investigations aimed at improving the quality of cryopreserved mammalian sperm. Acknowledgments The work was supported by the Marie Curie Actions for International Research Staff Exchange (FP7-PEOPLE-2011IRSES, project no. 295137). The authors thank APA, Potenza for kindly providing the cryopreserved ram semen, the PhD student Martina Rocco for help with the laboratory analyses, and Ana Burton for editorial assistance. The authors N.I., G.P., designed the experiments reported in Tables 1 and 2 and Figs. 1 and 2, respectively; M.D.I., R.P., and G.P.
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