Ti

NMa

b

a

ARRAA

KRSLMS

1

iodcrl

0d

Animal Reproduction Science 119 (2010) 123–129

Contents lists available at ScienceDirect

Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

he irradiation of rabbit sperm cells with He–Ne laser prevents theirn vitro liquid storage dependent damage

icolaia Iaffaldanoa,∗, Maria Pina Rosatoa, Gianluca Paventib, Roberto Pizzutob,ario Gambacortaa, Angelo Manchisi a, Salvatore Passarellab

Department of Animal, Vegetable and Environmental Sciences, University of Molise, via De Sanctis snc, 86100 Campobasso, ItalyDepartment of Health Sciences, University of Molise, via De Sanctis, 86100 Campobasso, Italy

r t i c l e i n f o

rticle history:eceived 29 July 2009eceived in revised form 1 October 2009ccepted 6 October 2009vailable online 24 October 2009

eywords:abbitpermatozoaaser helium–neonitochondrial function

emen storage

a b s t r a c t

The aim of the study was to investigate the effects of different energy doses of helium–neon(He–Ne) laser irradiation on both mitochondrial bioenergetics functions and functionalquality of rabbit spermatozoa during 48 h of in vitro liquid storage at 15 ◦C.

11 rabbit semen pools were each divided into four aliquots: three of them were irradiatedwith He–Ne laser with different energy doses (3.96, 6.12 and 9.00 J/cm2) being the lastcontrol kept under the same experimental conditions without irradiation. Sperm motility,viability and acrosome integrity were monitored together with cytochrome c oxidase (COX)activity and the cell energy charge (EC) at 0, 24 and 48 h of storage.

Irradiated samples stored for 24 and 48 h better maintained motility (P < 0.01), acro-some integrity (P < 0.01) and viability (P < 0.05) with respect to the control, particularlywith the energy dose of 6.12 J/cm2 that showed the most intense biostimulative effect.COX activity and EC were immediately increased by irradiation particularly in the treat-ments 6.12 and 9.00 J/cm2 (P < 0.05), that maintained their levels higher with respect tothe control after 48 h of storage (P < 0.01). COX activity of rabbit sperm cells was pos-

itively correlated with EC (P < 0.05), viability (P < 0.01) and acrosome integrity (P < 0.05)parameters.

These results indicate that the effects of He–Ne laser irradiation on sperm cells are medi-ated through the stimulation of the sperm mitochondrial respiratory chain and that thiseffect plays a significant role in the augmentation of the rabbit sperm cells’ capability to

id stor

survive during liqu

. Introduction

Rabbit breeding for commercial rabbit meat productions one of the farming systems almost exclusively dependentn Artificial Insemination (AI) programs, but this proce-

ure is limited by the short length of time that rabbit sperman be stored. Up to now, almost all inseminations are car-ied out with freshly ejaculated spermatozoa, because bothong-term liquid storage and freeze-thawing techniques

∗ Corresponding author. Tel.: +39 0874 404697; fax: +39 0874 404855.E-mail address: nicolaia@unimol.it (N. Iaffaldano).

378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2009.10.005

age conditions.© 2009 Elsevier B.V. All rights reserved.

generally shorten the lifespan of sperm cells (Roca et al.,2000; Mocé and Vicente, 2002). Thus development of newprocedure aimed at improving the quality of stored sper-matozoa is a suitable goal to be pursued. In this regard,spermatozoa irradiation with helium–neon (He–Ne) lasermerits some considerations. Indeed the photostimulativeeffect of laser irradiation in various biological systemshas already been shown (Lubart and Breitbart, 2000). For

instance in somatic cells irradiation stimulates the releaseof cultured fibroblasts’ growth factor (Yu et al., 1994) andaccelerates fibroblasts mitosis (Lubart et al., 1992a), skele-tal muscle regeneration and bone repair (Bibikova et al.,1994).

roductio

124 N. Iaffaldano et al. / Animal Rep

The He–Ne laser irradiation can also improve sperm fer-tilizing potential (Ocana-Quero et al., 1997; Cohen et al.,1998).

This could occur due to a variety of factors including theacceleration in Ca2+ transport through sperm mitochondriaand plasma membrane of the irradiated sperm cells (Lubartet al., 1992b, 1997; Breitbart et al., 1996; Cohen et al., 1998)and ROS formation (Zan-Bar et al., 2005).

In addition, since He–Ne laser irradiation induced anelevation of electrochemical potential and ATP extra syn-thesis in isolated mitochondria (Passarella et al., 1984) andsince mitochondria seems to play a key role in the energyproduction and sperm motility maintenance during stor-age (Ruiz-Pesini et al., 2000; Corral-Baqués et al., 2005,2009), stimulation could be dependent on the increase ofthe energy availability. In this regard, we have shown thatHe–Ne laser irradiation can improve the quality of storedturkey semen (Iaffaldano et al., 2005).

Since turkey and rabbit semen are different in morphol-ogy and metabolic metabolism and the knowledge of theirradiation’s effect on rabbit semen preservation is lack-ing, a specific investigation of the effect of He–Ne laserirradiation on rabbit sperm cells is interesting.

Therefore, our goal was to investigate the effects ofdifferent energy doses of laser irradiation on qualitativecharacteristics and energetic profiles of rabbit spermatozoaafter 48 h of in vitro liquid storage at 15 ◦C.

2. Materials and methods

2.1. Chemicals

The LIVE/DEAD Sperm Viability Kit was purchased fromMolecular Probes Inc. (Eugene, OR, USA). The Accudenz®

was purchased from Accurate Chemical & Scientific Cor-poration (Westbury, NY 11590, USA). The other chemicalsused in this study were purchased from Sigma ChemicalCo. (St. Louis, MO).

2.2. Animals and semen treatment

40 sexual mature hybrid bucks housed in a privatebreeding at standard breeding conditions were used.During 11 weeks, semen from bucks was randomly col-lected via artificial vagina and pooled to avoid individualdifferences (7–8 ejaculates/pool; 1 pool/week). Semenwas prediluted 1:1 with Tris–citric acid–glucose (TCG)extender (Tris: 250 mmol L−1; citric acid: 88 mmol L−1;glucose: 47 mmol L−1). Sperm concentration of each poolwas measured by a Thoma–Zeiss counting cell chamber.Each pool was divided into four aliquots that were eachput in a 4 mL cuvette before being exposed to the redlaser beam. One sample was not irradiated (control), theother ones were irradiated with a single mode contin-uous He–Ne laser wave (wavelength 632.8 nm; 6 mW)and exposed to the following energy doses: 3.96, 6.12

and 9.00 J/cm2 respectively. Samples were then diluted to30×106 spermatozoa/mL with TCG and stored at 15 ◦C for48 h.

For the evaluation of sperm functional quality on bothcontrol and irradiated samples, sperm motility, viability

n Science 119 (2010) 123–129

and acrosomal integrity were measured at 0 (short timeafter irradiation), 24 and 48 h of storage.

At the same time of storage, cytochrome c oxidaseactivity (COX) and energetic charge (EC) were assessed oncontrol and irradiated samples to evaluate the energeticfunctions of spermatic cells.

2.3. Sperm motility

The motility of spermatozoa was assessed with theSperm Motility Test firstly utilized for avian semen(Froman and McLean, 1996) and then adapted to mam-malian spermatozoa (Vizcarra and Ford, 2006). The testis based on the ability of the spermatozoa with a for-ward progressive motility to penetrate a 6% Accudenz®

layer. Semen was centrifuged at 300 rpm for 5 min andresuspended to 300×106 sperm/mL in 111 mM NaClbuffered with 50 mM N-tris-[hydroxymethyl]methyl-2-amino-ethanesulfonic acid (TES), pH 7.4, containing 25 mMglucose and 4 mM CaCl2. A 60 �L sample of each sperm sus-pension was overlaid onto 600 �L of pre-warmed 6% (w/v)Accudenz® solution in a semi-micro polystyrene dispos-able cuvette according to Kennedy et al. (2003). Cuvetteswere incubated for 5 min in a 38 ◦C water bath, placedin a spectrophotometer and absorbance was measured at550 nm after 60 s.

2.4. Sperm viability

Vital staining of the spermatozoa allows quantificationof the fraction of living cells independently of their motility(Campana et al., 1995).

The sperm viability was measured using the LIVE/DEADSperm Viability Kit (Molecular Probes Inc., Eugene, OR,USA) containing the fluorescent stains SYBR-14 and pro-pidium iodide (PI), as described by Blanco et al. (2000) withminor adaptations. Seminal aliquots (5 �L) were added to50 �L solutions containing 1 �L SYBR-14 (diluted 1:100 inDMSO), incubated for 10 min at 38 ◦C, then added with5 �L PI (diluted 1:100 in the TCG diluent) and further incu-bated for 5 min at 38 ◦C. Assessment of 100 spermatozoawas made in duplicate aliquots for every sample and eval-uated microscopically at 40× by using a Leitz Aristoplan(Leica) microscope and fluorescein isothiocyanate (FITC)filter fluorescence. SYBR-14, a membrane-permeant DNAstain, stained only living spermatozoa, producing brightgreen fluorescence of the nuclei when excited at 488 nm. PIstained the nuclei of membrane-damaged cells red, so sper-matozoa that exhibit green fluorescence is considered liveand those that exhibit red fluorescence is considered dead.The percentage of membrane-intact spermatozoa was cal-culated as a fraction of the total.

2.5. Acrosome integrity

Duplicate smears with a drop from each sample were

made and air dried. After fixation in methanol for 15 min,slides were washed with water and air dried. After incu-bation with fluorescein isothiocyanate–Pisum sativumagglutinin (FITC–PSA) for 30 min, slides were mountedwith 50% glycerol and cover slipped.

roductio

ewlaiaa

2

ueAE1fcn

2

scVoaopztpu1cawmei

FD

N. Iaffaldano et al. / Animal Rep

The analyses were performed on 200 sperm fromach sample with 100× objective under oil immersionith epifluorescence illumination. The strongly applegreen

abelled acrosomal region revealed the presence of thecrosome, whereas the absence of acrosomal staining orts confinement to the equatorial segment were signs ofcrosome intact loss for spontaneous acrosome reaction orcrosome damage (Mendoza et al., 1992).

.6. Energetic charge

The EC was measured with high performance liq-id chromatography (HPLC) according to Giannattasiot al. (2003) and was defined as the sum of the ATP,DP and AMP fractions, using the following equation:C = [ATP] + 0.5[ADP]/[ATP] + [ADP] + [AMP] (Atkinson,968). Adenylates were extracted by rapid centrifugationor 1 min at 13,000 rpm in a refrigerated Eppendorf micro-entrifuge. Perchloric acid extracts were obtained andeutralized (de Bari et al., 2007).

.7. COX activity

COX activity was determined photometrically in lysatedperm cells, essentially as in Miró et al. (1998), followingytochrome c oxidation. Briefly, by using a JASCO V-560 and-520 dual-beam dual-wavelength system, the oxidationf externally added reduced cytochrome c was measureds an absorbance decrease at 550 nm (absorbance peakf cytochrome c reduced form) minus 540 nm (isosbesticoint). In order to measure total COX activity, spermato-oa (1 mg protein) were completely lysated before additiono reaction medium consisting of 1 mL of Tris–HCl 50 mMH 7.2. The reaction was started by the addition of sat-rating concentration of reduced cytochrome c (usually00 �M) and for each sample we checked that cyanideompletely inhibited the reaction in order to rule out

ny other mechanism for COX oxidation. The COX activityas obtained as tangent at the initial part of experi-ental curve. COX activities of irradiated samples are

xpressed as percentage of control sample COX activ-ty.

ig. 1. Motility (mean percentages± SEM; n = 11) of rabbit semen exposed to different letters on error bars indicate significant differences. Capital letters diffe

n Science 119 (2010) 123–129 125

2.8. Statistical analysis

Data were analyzed using a General Linear Model,followed by the Duncan for means comparison test. Pear-son correlation coefficients between qualitative seminalparameters, the COX activity and EC values were alsoinvestigated. All statistics were done with SPSS (SPSS14.0 for Windows, 2005; SPSS, Chicago, IL). The resultsare presented as non-transformed means± SEM; statisticalsignificance was set at P < 0.05 and P < 0.01.

3. Results

In this paper comparison was made in rabbit spermsamples non-irradiated (control) and the same sperm sam-ples irradiated with He–Ne laser with different energydoses with respect to semen quality as function of storagetime. As shown in Fig. 1, a slight increase in sperm motil-ity values was observed in irradiated samples with respectto the control immediately after irradiation (time 0); how-ever differences between treatments were not significant.On 24 and 48 h of storage time, irradiated samples motilitywas higher than that in the control (P < 0.01), particularlywith energy dose of 6.12 J/cm2.

When sperm viability was investigated (Fig. 2) no effectof irradiation was found at time 0 in rabbit spermatozoashowing viability higher than 80%. On 24 h of storage, thepercentage of viable spermatozoa was significantly higher(P < 0.05) in semen irradiated with 6.12 and 9.00 J/cm2 thanin control. No significant difference was found betweensamples irradiated at the energy dose of 3.96 J/cm2 com-pared to the other treatments.

On 48 h of storage samples irradiated at 6.12 J/cm2 stillretained higher viability percentages (P < 0.05) comparedto both the non-irradiated spermatozoa and the 3.96 and9.00 J/cm2 irradiation treatments.

The percentages of acrosome intact spermatozoa arereported in Fig. 3. There were no difference between control

and irradiated samples at 0 h of storage.

At 24 h of chilled storage unirradiated spermatozoashowed significant lower acrosome intact percentagescompared to those irradiated with 3.96 and 9.00 J/cm2

(P < 0.05) and 6.12 (P < 0.01) doses (Fig. 3). At 48 h of storage,

ifferent doses of He–Ne laser irradiation and stored up to 48 h at 15 ◦C.r for P < 0.01; lower case letters differ for P < 0.05.

126 N. Iaffaldano et al. / Animal Reproduction Science 119 (2010) 123–129

Fig. 2. Viability (mean percentages± SEM; n = 11) of rabbit semen exposed to different doses of He–Ne laser irradiation and stored up to 48 h at 15 ◦C.Different letters on error bars among the same time of storage indicate significant differences (P < 0.05).

f rabbittime of

Fig. 3. Acrosome intact spermatozoa (mean percentages± SEM; n = 11) ostored up to 48 h at 15 ◦C. Different letters on error bars among the samelower case letters differ for P < 0.05.

in spite of better acrosome integrity values in irradiatedsamples, only those exposed to the dose of 6.12 J/cm2 hadsignificantly higher percentages (P < 0.01) of intact acro-somes compared to the control.

Having established that rabbit semen irradiationresulted in a preservation of the time dependent decreasein feature markers of semen quality, we investigatedwhether this is accompanied with a prevention of dam-age in the energy producing pathways. This was made

Fig. 4. Energetic charge (mean± SEM; n = 11) of rabbit spermatozoa exposed toDifferent letters on error bars among the same time of storage indicate significanP < 0.05.

spermatozoa exposed to different doses of He–Ne laser irradiation andstorage indicate significant differences. Capital letters differ for P < 0.01;

by calculating the semen energy charge and COX activ-ity. The results of the effect of laser He–Ne on energeticstatus of rabbit semen are shown in Figs. 4 and 5. Immedi-ately after irradiation, samples exposed at energy doses of

6.12 J/cm2 of He–Ne laser showed a significantly increasedEC charge (P < 0.05; Fig. 4) with respect to control. At 24 hof storage no significant differences existed between treat-ments. After 48 h of chilled storage, the EC in the groupsirradiated at 6.12 and 9.00 J/cm2 was found to be signif-

different doses of He–Ne laser irradiation and stored up to 48 h at 15 ◦C.t differences. Capital letters differ for P < 0.01; lower case letters differ for

N. Iaffaldano et al. / Animal Reproduction Science 119 (2010) 123–129 127

F differel differen

it

(ldwascu9t

tvbCbNg

4

d6aul

TPpH

Ed

ig. 5. COX activity (mean± SEM; n = 11) of rabbit spermatozoa exposed toetters on error bars among the same time of storage indicate significant

cantly higher (P < 0.01) than the control and 3.96 J/cm2

reatment.Since COX proved to be a specific target of He–Ne laser

Pastore et al., 1994, 2000) COX activity was measured inysated spermatozoa. Unlike the control and the group irra-iated with 3.96 J/cm2, COX activity in the groups irradiatedith 6.12 and 9.00 J/cm2 increased significantly immedi-

tely after irradiation (P < 0.01; Fig. 5). All the irradiatedamples maintained higher values after 24 h of storageompared to the control (P < 0.01) and after 48 h of liq-id chilled storage the groups irradiated with 6.12 and.00 J/cm2 registered again higher COX activity comparedo the remaining groups (P < 0.05).

Positive correlations (Table 1) were found between allhe sperm functional qualitative characteristics as motility,iability and acrosomal integrity (P < 0.01) and between theioenergetics parameters of COX activity and EC (P < 0.05).OX activity was also positively correlated with sperm via-ility (P < 0.01) and sperm acrosome intact (P < 0.05) values.o significant correlations were found between bioener-etics and motility parameters.

. Discussion

Here we show that as a result of rabbit sperm cells irra-iation with He–Ne laser (in particular with energy dose of

.12 J/cm2), their motility, viability and acrosome integrityre mostly preserved in time. Such a conclusion is notnique: in our previous work we have already shown that

aser irradiation (fluences ranging from 3.24 to 5.40 J/cm2)

able 1earson correlation coefficients between sperm qualitative and energeticarameters of liquid-stored rabbit sperm cells exposed to low doses ofe–Ne laser irradiation.

Motility Viability AI COX activity

EC 0.032 ns 0.042 ns −0.100 ns 0.181*

Motility 0.632** 0.662** 0.086 nsViability 0.692** 0.245**

AI 0.181*

C: energetic charge; AI: acrosome integrity; n = 132 (degrees of free-om = n−2); ns: no significant.

* Correlation is significant at the 0.05 level.** Correlation is significant at the 0.01 level.

nt doses of He–Ne laser irradiation and stored up to 48 h at 15 ◦C. Differentces. Capital letters differ for P < 0.01; lower case letters differ for P < 0.05.

increased the quality of stored turkey semen (Iaffaldano etal., 2005). Moreover a positive effect of low dose of He–Nelaser irradiation on fertilizing potential of spermatic cellshave also been reported by other authors, with differentsensitivity to He–Ne laser exhibited by the investigatedspecies. Ocana-Quero et al. (1997) showed an increase inthe acrosome reaction of bull sperm and a decrease ofthe spermatozoa mortality at fluences ranging from 2 to16 J/cm2. Wenbin et al. (1996) found that laser irradia-tion increased sperm fructose fermentation, respiration,the amount of 32P uptake and the Ca2+ absorption, thusincreasing the motility and survival time of buck sperma-tozoa. Corral-Baqués et al. (2005) reported that irradiationof dog sperm with continuous wave 655 nm diode laser at 4,6 and 10 J/cm2 improves the speed and linear coefficient ofsperm. Zan-Bar et al. (2005), working with ram and tilapiasperm, found in tilapia sperm higher motility and fertilityfollowed red light (660 nm) and white light (400–800 nm)irradiation, whereas in ram spermatozoa both motility andfertility were slightly increased only by red light.

Our findings have led us to sustain that low doses ofHe–Ne laser irradiation retain better the rabbit semen qual-ity during storage time.

The mechanism by which this occurs must be a matter ofspeculation. According to Zan-Bar et al. (2005) the effects oflight are mediated through reactive oxygen species (ROS).Indeed, although high ROS level can lead cells to death(by ATP depletion and lipid peroxidation), at low levelROS can play a major role in the activation of many cellprocesses. In the case of spermatozoa, ROS, including thesuperoxide anion and H2O2, and reactive nitrogen speciesas nitric oxide (NO) can cause sperm hypercapacitation andthe acrosome reaction (Aitken, 1995; Aitken et al., 1995;Martínez-Pastor et al., 2009).

On the other hand, an increase of intracellular Ca2+ lev-els and transport in the irradiated sperm cells of bull (Lubartet al., 1992b; Breitbart et al., 1996), and mouse (Cohen et al.,1998) were shown. More recently, intracellular Ca2+ move-ments were shown to control sperm motility, capacitation

and the acrosome reaction (Darszon et al., 2007).

Our study indicated that the time-related decline insemen quality during chilled storage was in fact sloweddown by laser irradiation treatment and this eventappeared to be linked to an increased COX activity and

roductio

128 N. Iaffaldano et al. / Animal Rep

a consequent EC augment in spermatic cells for energysupply. The EC augment was particularly evident in sper-matozoa irradiated with 6.12 and 9.00 J/cm2, due to anextra ATP synthesis in the samples subjected to these treat-ments.

Iaffaldano et al. (2005) also showed an increased cellenergetic charge in stored turkey semen after laser irradi-ation.

Previous studies reported that He–Ne laser irradiationon isolated mitochondria resulted in the increase of ATPlevel (Passarella et al., 1984), RNA (Greco et al., 1989) andDNA synthesis (Vacca et al., 1993), generation of new mito-chondria (Greco et al., 1991), enzyme activation (Pastore etal., 2000) and modifications in the substrate enzyme inter-action (Gagliardi et al., 1997). In sperm cells ATP, generatedin the mitochondria, is primarily required for sperm motil-ity (Minelli et al., 1999).

Thus the mitochondria can play roles other than justenergy supply which are needed to maintain the con-tractibility of the tail, e.g. the regulation of the calcium fluxand membrane potential (Eddy and O’Brien, 1994; Rossatoet al., 2001).

Also in somatic cells laser light seems to affect themitochondrial respiratory chain by changing the electricpotential of cell membranes and, consequently, their selec-tive permeability for sodium, potassium and calcium ions,or by increasing the activity of certain enzymes such as COXand ATP (Passarella et al., 1984; Karu, 1988; Friedmann etal., 1991; Tamura, 1993; Koutná et al., 2003).

He–Ne laser irradiation generated an extra COX activityas measured in rabbit spermatic lysated cells, particularlywith fluences of 6.12 and 9.00 J/cm2. Notice that an increasein cytochrome c oxidase activity as measured in intact cellmirrors a damage of both cellular and outer mitochon-drial membranes (see Schober et al., 2007). In our case theincrease of COX activity is assumed to result in increaseof the COX proton pumping which generates the elec-trochemical proton gradient driving to ATP synthesis. Infact, sperm motility largely depends on the aerobic energymetabolism of the mitochondrial compartment. In partic-ular, sperm motility is related with enzymatic activitiesof the sperm mitochondrial respiratory chain (MRC) com-plexes, in which the apparent rate-limiting step is catalyzedby COX enzyme (or complex IV). Although COX and ECwere not significantly correlated with motility parameterof stored semen, COX resulted positively correlated withviability and acrosome intact spermatozoa. On reports inhuman semen correlations between mitochondrial func-tionality and the overall seminal quality are also reported(Ruiz-Pesini et al., 2000). COX is responsible for the vastmajority of oxygen consumption in cells and essential forthe efficient generation of cellular ATP: providing an effi-cient mechanism for dioxygen reduction on one hand,COX also acts as a redox-linked proton pump, couplingthe free energy of water formation to the generation ofa trans-membrane electrochemical gradient to eventually

drive ATP synthesis (Ruiz-Pesini et al., 2000; Richter andLudwig, 2003). For this reason COX acts as a potent antiox-idant that scavenges exceeding ROS in the mitochondria.This may explain the best viability and acrosome integrityobtained in irradiated stored semen of rabbit and the pos-

n Science 119 (2010) 123–129

itive correlation between the two parameters and COXactivity.

In previous studies Maxwell and Stojanov (1996)showed that COX addition to semen improved the survivalof ram spermatozoa in liquid storage and Faustini et al.(2004) showed a positive correlation between COX activ-ity and boar sperm preservation during 72 h of storage at18 ◦C.

Our findings suggest that the interaction between632.8 nm He–Ne laser irradiation and rabbit sperm func-tions is mediated through the generation of extra COXactivity and subsequent increase of EC by the spermatozoaand that this effect plays a significant role in the augmen-tation of the sperm cells’ capability to survive during liquidstorage conditions.

In conclusion, the helium–neon laser irradiationimproved rabbit sperm preservation during liquid storagemodulating sperm qualitative functions. This effect may berelated to the evidence of energetic biostimulation of rabbitspermatic cells and to an improved cytochrome c oxidaseactivity.

Acknowledgments

This work was supported by a grant from the Univer-sity of Molise (FAR 2003) and partially financed by FIRBRBNE03B8KK 003. The authors thank Innocenzo Gentile ofAzienda Cunicola Gentile (Collesannita, BN, Italy) for thetechnical assistance with semen collection.

References

Aitken, R.J., 1995. Free radicals, lipid peroxidation and sperm function.Reprod. Fertil. Dev. 7, 659–668.

Aitken, R.J., Paterson, M., Fisher, H., Buckingham, D.W., Van Duim, M., 1995.Redox regulation of tyrosine phosphorylation in human spermatozoaand its role in the control of human sperm function. J. Cell Sci. 108,2017–2025.

Atkinson, D.E., 1968. The energy charge of adenylate pool as a regula-tory parameter. Interaction with feedback modifiers. Biochemistry 7,4030–4034.

Bibikova, A., Belkin, V., Oron, U., 1994. Enhancement of angiogenesis inregenerating gastrocnemius muscle of the toad (Bufo viridis) by lowenergy laser irradiation. Anat. Embryol. 190, 597–602.

Blanco, J.M., Gee, G., Wildt, D.E., Donoghue, A.M., 2000. Species variation inosmotic, cryoprotectant, and cooling rate tolerance in poultry, eagle,and peregrine falcon spermatozoa. Biol. Reprod. 63, 1164–1171.

Breitbart, H., Levinshal, T., Cohen, N., Friedmann, H., Lubart, R., 1996.Changes in calcium transport in mammalian sperm mitochondria andplasma membrane irradiated at 633 nm (HeNe laser). J. Photochem.Photobiol. B: Biol. 34, 117–121.

Campana, A., de Agostini, A., Bischof, P., Tawfik, E., Mastrorilli, A., 1995.Evaluation of infertility. Hum. Reprod. Update 1, 586–606.

Cohen, N., Lubart, R., Rubinstein, S., Breitbart, H., 1998. Light irradiation ofmouse spermatozoa: stimulation of in vitro fertilization and calciumsignals. Photochem. Photobiol. 68, 407–413.

Corral-Baqués, M.I., Rigau, T., Rivera, M.M., Rodríguez, J.E., Rigau, J., 2005.Effect of 655 nm diode laser on dog sperm motility. Lasers Med. Sci.20, 28–34.

Corral-Baqués, M.I., Rivera, M.M., Rigau, T., Rodríguez-Gil, J.E., Rigau, J.,2009. The effect of low-level laser irradiation on dog spermatozoamotility is dependent on laser output power. Lasers Med. Sci. 24,703–713.

Darszon, A., Trevino, C.L., Wood, C., Galindo, B., Rodríguez-Miranda, E.,

Acevedo, J.J., Hernandez-González, E.O., Beltrán, C., Martínez-López,P., Nishigaki, T., 2007. Ion channels in sperm motility and capacitation.Soc. Reprod. Fertil. Suppl. 65, 229–244.

de Bari, L., Valenti, D., Pizzuto, R., Atlante, A., Passarella, S., 2007. Phos-phoenolpyruvate metabolism in Jerusalem artichoke mitochondria.Biochim. Biophys. Acta Bioenerg. 1767, 281–294.

roductio

E

F

F

F

G

G

G

G

I

K

K

K

L

L

L

L

M

M

M

on the secretion of TGF and PDGF from fibroblasts in vitro. Lasers Surg.

N. Iaffaldano et al. / Animal Rep

ddy, E.M., O’Brien, D.A., 1994. The spermatozoon. In: Knobil, E., Neill, J.D.(Eds.), The Physiology of Reproduction. Raven Press, New York, pp.29–77.

austini, M., Torre, M.L., Stacchezzini, S., Norberti, R., Consiglio, A.L.,Porcelli, F., Conte, U., Munari, E., Russo, V., Vigo, D., 2004. Boarspermatozoa encapsulated in barium alginate membranes: a micro-densitometric evaluation of some enzymatic activities during storageat 18 ◦C. Theriogenology 61, 173–184.

riedmann, H., Lubart, R., Laulicht, I., 1991. A possible explanation of laser-induced stimulation. J. Photochem. Photobiol. B: Biol. 11, 87–95.

roman, D.P., McLean, D.J., 1996. Objective measurement of sperm motil-ity based upon sperm penetration of Accudenz. Poult. Sci. 75, 776–784.

agliardi, S., Atlante, A., Passarella, S., 1997. A novel property of ade-nine nucleotides: sensitivity to helium–neon laser in mitochondrialreactions. Biochem. Mol. Biol. Int. 41, 449–460.

iannattasio, S., Gagliardi, S., Samaja, M., Marra, E., 2003. Simultaneousdetermination of purine nucleotides, their metabolites and beta-nicotinamide adenine dinucleotide in cerebellar granule cells byion-pair high performance liquid chromatography. Brain Res. Protoc.10, 168–174.

reco, M., Guida, G., Perlino, E., Marra, E., Quagliariello, E., 1989. Increasein RNA and protein synthesis by mitochondria irradiated withhelium–neon laser. Biochem. Biophys. Res. Commun. 163, 1428–1434.

reco, M., Perlino, E., Pastore, D., Guida, G., Marra, E., Quagliariello, E., 1991.Helium–neon laser irradiation of rat liver mitochondria gives rise toa new subpopulation of mitochondria: isolation and first biochemicalcharacterization. J. Photochem. Photobiol. B: Biol. 10, 71–78.

affaldano, N., Meluzzi, A., Manchisi, A., Passarella, S., 2005. Improvementof stored turkey semen quality as a result of He–Ne laser irradiation.Anim. Reprod. Sci. 854, 317–325.

aru, T., 1988. Molecular mechanism of the therapeutic effect of low inten-sity laser irradiation. Laser Life Sci. 2, 53–74.

ennedy, J.H., Korn, N., Thurston, R.J., 2003. Prostaglandin levels in sem-inal plasma and sperm extracts of the domestic turkey, and theeffects of cyclooxygenase inhibitors on sperm mobility. Reprod. Biol.Endocrinol. 1, 74.

outná, M., Janisch, R., Veselská, R., 2003. Effects of low-power laser irra-diation on cell proliferation. Scr. Med. 76, 163–172.

ubart, R., Breitbart, H., 2000. Biostimulative effects of low-energy lasersand their implications for medicine. Drug Dev. Res. 50, 471–475.

ubart, R., Wollman, Y., Friedmann, H., Rochkind, S., Laulicht, I., 1992a.Effects of visible and near infra-red lasers on cell cultures. J. Pho-tochem. Photobiol. B: Biol. 12, 305–310.

ubart, R., Friedmann, H., Levinshal, T., Lavie, R., Breitbart, H., 1992b. Effectof light on calcium-transport in bull sperm cells. J. Photochem. Pho-tobiol. B: Biol. 15, 337–341.

ubart, R., Friedmann, H., Sinyakov, M., Cohen, N., Breitbart, H., 1997.Changes in calcium transport in mammalian sperm mitochondria andplasma membranes caused by 780 nm irradiation. Laser Surg. Med. 21,493–499.

artínez-Pastor, F., Aisen, E., Fernández-Santos, M.R., Esteso, M.C.,Maroto-Morales, A., García-Álvarez, O., Garde, J.J., 2009. Reactiveoxygen species generators affect quality parameters and apopto-sis markers differently in red deer spermatozoa. Reproduction 137,225–235.

axwell, W.M.C., Stojanov, T., 1996. Liquid storage of ram semen in

the absence or presence of some antioxidants. Reprod. Fertil. Dev. 8,1013–1020.

endoza, C., Correras, A., Moos, J., Tesarik, J., 1992. Distinction betweentrue acrosome reaction and degenerative acrosome loss by a one-stepstaining method using Pisum sativum agglutinin. J. Reprod. Fertil. 95,755–763.

n Science 119 (2010) 123–129 129

Minelli, A., Moroni, M., Castellini, C., Lattaioli, P., Mezzasoma, I., Ronquist,G., 1999. Rabbit spermatozoa: a model system for studying ATP home-ostasis and motility. J. Androl. 20, 259–266.

Miró, O., Cardellach, F., Barrientos, A., Casademont, J., Rötig, A., Rustin, P.,1998. Cytochrome c oxidase assay in minute amounts of human skele-tal muscle using single wavelength spectrophotometers. J. Neurosci.Methods 80, 107–111.

Mocé, E., Vicente, J.S., 2002. Effect of cooling and freezing, the two firststeps of a freezing protocol, on the fertilizing ability of the rabbitsperm. Reprod. Nutr. Dev. 42, 189–196.

Ocana-Quero, J.M., Gomez-Villamandos, R., Moreno-Millan, M.,Santisteban-Valenzuela, J.M., 1997. Biological effects of helium–neon(He–Ne) laser irradiation on acrosome reaction in bull sperm cells. J.Photochem. Photobiol. B: Biol. 40, 294–298.

Passarella, S., Casamassima, E., Molinari, S., Pastore, D., Quagliariello, E.,Catalano, I.M., Cingolani, A., 1984. Increase of proton electrochemicalpotential and ATP synthesis in rat liver mitochondria irradiated invitro by helium–neon laser. FEBS Lett. 175, 95–99.

Pastore, D., Greco, M., Petragallo, V.A., Passarella, S., 1994. Increase in←H+/e− ratio of the cytochrome c oxidase reaction in mitochon-dria irradiated with helium–neon laser. Biochem. Mol. Biol. Int. 34,817–826.

Pastore, D., Greco, M., Passarella, S., 2000. Specific helium–neon laser sen-sitivity of the purified cytochrome c oxidase. Int. J. Radiat. Biol. 76,863–870.

Richter, O.M.H., Ludwig, B., 2003. Cytochrome c oxidase – structure,function, and physiology of a redox-driven molecular machine. Rev.Physiol. Biochem. Pharmacol. 147, 47–74.

Roca, J., Martínez, S., Vázquez, J.M., Lucas, X., Parrilla, I., Martínez,E.A., 2000. Viability and fertility of rabbit spermatozoa diluted inTris-buffer extenders and stored at 15 ◦C. Anim. Reprod. Sci. 64,103–112.

Rossato, M., Di Virgilio, F., Rizzuto, R., Galeazzi, G., Foresta, C., 2001. Intra-cellular calcium store depletion and acrosome reaction in humanspermatozoa: role of calcium and plasma membrane potential. Mol.Hum. Reprod. 7, 119–128.

Ruiz-Pesini, E., Lapena, A.C., Díez, C., Alvarez, E., Enríquez, J.A., López-Pérez,M.J., 2000. Seminal quality correlates with mitochondrial functional-ity. Clin. Chim. Acta 300, 97–105.

Schober, D., Aurich, C., Nohl, H., Gill, L., 2007. Influence of cryopreservationon mitochondrial functions in equine spermatozoa. Theriogenology68, 745–754.

Tamura, M., 1993. Non-invasive monitoring of the redox state ofcytochrome oxidase in living tissue using near infra-red laser lights.Jpn. Circ. J. 57, 817–824.

Vacca, R.A., Marra, E., Quagliariello, E., Greco, M., 1993. Activation ofmitochondrial DNA replication by He–Ne laser irradiation. Biochem.Biophys. Res. Commun. 195, 704–709.

Vizcarra, J.A., Ford, J.J., 2006. Validation of the sperm mobility assay inboars and stallions. Theriogenology 66, 1091–1097.

Wenbin, Y., Wenzhong, L., Mengzhao, L., Baotian, Z., Laizeng, A.I., Tongya,L., 1996. Effects of laser radiation on Saanen buck’s sperm energymetabolism. In: Proceedings of the Sixth International Conference onGoats, Beijing, China, 5–11 May.

Yu, M., Naim, J.O., Lanzafama, R.J., 1994. The effects of photo-irradiation

Med. Suppl. 6, 8.Zan-Bar, T., Bartoov, B., Segal, R., Yehuda, R., Lavi, R., Lubart, R., Avtal-

ion, R.R., 2005. Influence of visible light and ultraviolet irradiation onmotility and fertility of mammalian and fish sperm. Photomed. LaserSurg. 23, 549–555.