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Theriogenology 76 (2011) 342–350

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Effects of antioxidants and duration of pre-freezing equilibrationon frozen-thawed ram semen

D.R. Câmaraa,b, S.V. Silvab, F.C. Almeidab, J.F. Nunesc, M.M.P. Guerrab,*a Department of Veterinary Medicine, Federal University of Alagoas; Fazenda São Luiz, s/n. Zona Rural de Viçosa. Viçosa-AL. Brazil. Post-

Graduate Student, Northeast Biotechnology Networkb Laboratory of Andrology, Department of Veterinary Medicine, Federal Rural University of Pernambuco; R. Dom Manoel de Medeiros, s/n,

Dois Irmãos, Recife-PE, Brazil CEP 52171-900c Laboratory of Sheep and Goat Semen Technology, Ceará State University; Av. Paranjana, 1700 - Campus do Itaperi, Fortaleza-CE, Brazil

Received 25 August 2010; received in revised form 5 January 2011; accepted 6 January 2011

Abstract

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservationof ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control),or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), orcatalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semenamples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA),crosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, andost-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were noignificant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardlessf antioxidant addition. Sperm plasma membrane integrity was worse (P � 0.05) with cryopreservation immediately afterquilibration (average 20.1 � 8.3; mean � SD) than after 12 h of equilibration (average 42.5 � 10.9); however, the addition ofOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Totalntioxidant activity was not different (P � 0.05) among treatments after sperm addition or throughout the refrigeration andost-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhancehe quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reducedembrane damage of frozen-thawed sperm.ublished by Elsevier Inc.

Keywords: Antioxidant; Equilibration time; Semen cryopreservation; Viability

www.theriojournal.com

1. IntroductionIn 1970, Lightfoot and Salamon [1] demonstrated

that the number of sperm recovered from the uterus ofan artificially inseminated sheep was lower after the useof frozen than fresh semen, regardless of sperm con-

* Corresponding author. Tel.: �055 81 3320 6412; fax: � 055 813320 6057.

E-mail address: mmpguerra@pq.cnpq.br (M.M.P. Guerra).

093-691X/$ – see front matter Published by Elsevier Inc.oi:10.1016/j.theriogenology.2011.02.013

centration. Cryopreservation causes extensive chemicaland physical damages to sperm membranes, which areattributed to alterations in the transition from the lipidphase, increases in lipid peroxidation of the membraneinduced by reactive oxygen species (ROS), and me-chanical stress on cell membranes due to osmotic stressand temperature changes [2,3].

It is well documented that ROS, formed by the

univalent reduction of oxygen [4,5], are responsible for

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343D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

maintenance of physiological processes in sperm, e.g.,capacitation and the acrosome reaction [6,7]. However,large quantities of ROS decreased the viability of cryo-preserved sperm, giving rise to lipid peroxidation dueto the large amount of polyunsaturated fatty acids pres-ent in the sperm plasma membrane [8]. Approaches toincrease the percentage of viable post-thaw cells haveincluded various extenders [9], refrigeration times[10,11], dilution rates [12], glycerol concentrations[13], and equilibration times [14–16].

Equilibration time is the interval between addition ofglycerol and freezing sperm; it is expected to be ben-eficial, as it allows a better osmotic balance after sperminteract with a cryoprotectant. Concurrently, it also hasrelevance for semen collection and processing, due totime needed prior to cryopreservation [17]. Studiescarried out since the late 1960s [18] and more recently[19] reported the beneficial effects of equilibration be-fore cryopreserving bovine sperm. Although equilibra-tion of ram semen for 24 h did not improve fertility[15], the value of an intermediate equilibration interval(12 h) for cryopreserving ram sperm has apparently notbeen reported. The objective of the present study was toassess the effects of various antioxidant substances andpre-freezing equilibration intervals on the quality ofram sperm during cryopreservation.

2. Materials and methods

2.1. Chemicals

The antioxidants (GSH, SOD and CAT) and otherchemicals used in this study were obtained from SigmaAldrich Chemicals (St. Louis, MO, USA).

2.2. Rams and semen collection

Four mixed-breed Santa Inês rams, 1 to 2 y old andraised in a confinement system with natural light(8.0314 S, 34.5252 W), were used. They had ad libitumaccess to hay and good quality water, and were supple-mented with 500 g/head/d of concentrate. The ramswere previously deemed breeding sound and were inroutine semen collection (artificial vagina and a teaserfemale) thrice weekly. Four semen samples were col-lected per ram (total of 16 samples); samples from allfour rams were pooled to eliminate individual differ-ences [20]. Minimum requirements set to freeze ejac-ulates pooled were volume �3.0 mL, motility �80%,and a minimum of 3 � 109 sperm/mL. Motility wasssessed subjectively (�400) with phase-contrast mi-

roscopy (Olympus, Tokyo, Japan). Semen was diluted t

1:200) in formol citrate solution, and sperm concen-ration was calculated using a Neubauer hemocytome-er chamber [11], with bright-field microscopy (�400).

.3. Extenders and semen processing

Tris-egg yolk extender was used as the base ex-ender medium, with 6% glycerol and 10% egg yolk.ach pool was split into 10 equal aliquots and dilutedith base extender containing reduced glutathione

GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutaseSOD: 5, 10, and 20 U/mL) and catalase (CAT: 5, 10nd 20 U/mL), and no antioxidants (control), with anal concentration of 100 � 106 sperm/mL. Extendedemen samples were then manually packed into 0.25L mini straws (IMV

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Technologies, L’Aigle, Cedex,rance) at room temperature, and were immediatelyooled (average of � 0.2 °C/min) to a thermal equilib-ium of 5 °C, which occurred in approximately 90 min.ne-third of the doses of each treatment was placed inprogrammable freezer (TK-3000, TK Tecnologia emonservação Ltda., Uberaba, MG, Brazil), previously

tabilized at 5 °C. Semen was cooled from 5 to �120C at a rate of 12.5 °C/min. Once straws reached �120C, they were plunged directly into liquid nitrogen andtored until subsequent evaluation. The procedure wasepeated with remaining samples after 12 and 24 h ofquilibration. After a minimum of 15 d of frozen stor-ge, straws were thawed at 37 °C for 30 s in a waterath and evaluated.

.4. Subjective assessment of motility of fresh/dilutednd refrigerated sperm

The percentage of motile cells was subjectively as-essed by a visual estimation using a phase contrasticroscope (�400; Olympus, Tokyo, Japan) immedi-

tely after dilution of the semen in the Tris-egg yolkontrol and Tris-egg yolk, supplemented with variousoncentrations of antioxidants (fresh/diluted). Motilityas assessed when the samples had reached thermal

quilibrium at 5 °C (0 h), and after 12 and 24 h ofquilibration.

.5. Assessment of sperm viability using fluorescentrobes

Sperm viability analyses were performed in the pre-reezing periods (fresh/diluted, 0, 12, and 24 h) andfter thawing in the Tris-egg yolk control and Tris-eggolk, supplemented with various concentrations of an-

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344 D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

2.5.1. Membrane integrity

The integrity of the sperm membrane was deter-mined using a combination of propidium iodide (PI)and carboxyfluorescein diacetate (CFDA), as describedby Harrison and Vickers [21] and modified by Coleto etal [22]. Aliquots (50 �L) of each sample were dilutedn 150 �L of Tris containing 20 �L of PI (0.5 mg/mLn PBS) and 5 �L of CFDA (0.46 mg/mL in DMSO).

Using DBP 485/20 nm excitation and DBP 580-630 nmemission filters, 200 cells from each sample were ex-amined under an epifluorescence microscope (�400;Carl Zeiss, Göttingen, Germany). Green fluorescencewas interpreted as an intact membrane, whereas redindicated a damaged membrane.

2.5.2. Acrosomal integrity

For detection of acrosomal integrity, sperm werestained with fluorescein isothiocyanate conjugated topeanut agglutinin (FITC-PNA), as described [23]. Ali-quots (5 �L) of semen from each treatment were placedon microscope slides and air dried. The slides had 20�L of FITC-PNA working solution (100 �g/mL)spread over them, and were incubated at 4 °C in amoisture chamber for 15 to 20 min (in the dark). Theslides were then immersed in PBS at 4 °C twice anddried naturally in the absence of light. At the time ofevaluation, 5 �L of solution containing 4.5 mL ofglycerol, 0.5 mL of PBS and 5.0 mg of phenylenedi-amine was placed on the slide. Then the slide wascovered with a slip cover and subjected to epifluores-cence analysis (�1000; Carl Zeiss, Göttingen, Ger-many) using BP 450–490 nm excitation and LP 515nm emission filters. Among the 200 cells examined,sperm were classified as having an intact acrosome(iAC) when the acrosome region was stained fluores-cent green, and as having a reacted acrosome when thefluorescent green was absent from the head region, orwhen it was present in the equatorial region of thesperm head.

2.5.3. Mitochondrial membrane potential

Aliquots (50 �L) of semen from each sample werediluted in 150 �L of Tris containing 5 �L of lipophilicationic JC-1 (0.15 mM in DMSO), incubated for 10in, fixed with glutaraldehyde, and subjected to epi-uorescence microscopy (�400; Carl Zeiss, Göttingen,ermany) using BP 450–490 nm excitation and LP15 nm emission filters. From each sample, 200 cellsere examined and classified as having high mitochon-

rial membrane potential (H�m) when emitting or-

nge fluorescence in the region of the midpiece, and asaving low mitochondrial membrane potentialL�m) when emitting green fluorescence.

.6. Computer Assisted Sperm Analysis (CASA)

The CASA system consisted of an optical phase-ontrast microscopy system (Nikon™ H5505, Eclipse0i, Japan) with stroboscopic illumination, a warmingtage (37 °C), video camera (Basler Vision Tecnolo-ie™ A312FC, Ahrensburg, Germany), and a PC withperm Class Analyzer (SCA™) software (Microptics,.L., Version 3.2.0, Barcelona, Spain). Motility endoints were assessed from two thawed straws/treat-ent/equilibration time, previously pooled and homog-

nized in a 1.0 mL microcentrifuge tube, diluted inodium citrate solution at 2.94% (v/v), and incubated inwater bath at 37 °C for 5 min. A pre-warmed Maklerhamber® (Sefi Medical Instrument, Haifa, Israel) was

oaded with 5 �L of diluted sample; at least four non-consecutive, randomly selected microscopic fields persample were scanned, recording at least 400 motilesperm. Events not related to sperm were removed, andimage sequences were saved and analyzed later. Thefollowing end points were analyzed: total motility(TM), progressive motility (PM), path velocity (VAP),progressive velocity (VSL), curvilinear velocity(VCL), amplitude of lateral head displacement (ALH),beat frequency of the tail (BCF), straightness (STR),and linearity (LIN). Motility end points were measuredwith the following settings: temperature 37 °C; framesacquired, 25; frame rate, 25 s; minimal contrast, 75;frame number, 25 per field; sperm velocity that can beanalyzed, 0 to 180 �m/s; and threshold STR, 75%.

2.7. Total antioxidant capacity

Total antioxidant capacity (TAC) was determinedthrough spectrophotometry of extenders in natura(without the addition of semen), as well as fresh/dilutedsemen samples, after 0, 12, and 24 h of equilibration at5 °C, and after thawing. The content of two straws/treatment/equilibration time in refrigerated and thawedsamples was pooled and well mixed in a 1.0 mL mi-crocentrifuge tube before analysis. For this, the sampleswere subjected to centrifugation at 10,000 � g for 15min. Then 5 �L of the supernatant of each semenample was placed in each well of the reading plate,long with 10 �L of myoglobin working solution and5 �L of ABTS working solution, and were incubatedt room temperature for 5 min. Then 50 �L of stop

solution was added. All readings were performed

within 30 min after finalizing the preparation.

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345D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

Prior to analysis, a calibration curve was determinedusing Trolox (6-hydrohy-2, 5, 7, 8-tetramethylchro-mane-2-carboxylic acid) as the standard antioxidantfollowing the kit (Antioxidant Assay Kit, code CS0790,Sigma Aldrich Co.) and manufacturer’s recommenda-tions. The concentration of antioxidants of each samplewas calculated based on the equation obtained from thelinear regression of the standard curve, using the equa-tion X (mM) [Y(A405) � 0.549/-1.2361] � 10, inwhich X (mM) was the concentration of the antioxidantrelative to the standard Trolox concentration andY(A405) was the mean absorbance of each sample at405 nm.

2.8. Statistical analysis

Four replicates were performed. The experimentalunit was each of the four pools formed by the ejaculateof four rams. The variables used for comparison pur-poses were the various media (control; GSH 0.5, 1.0, or2.0 mM; SOD 5, 10, or 20 U/mL; and CAT 5, 10, or 20U/mL) and pre-freezing equilibration times (fresh/di-luted, 0, 12, and 24 h). Differences between treatmentsor equilibration times were assessed using one-wayANOVA, followed by Tukey’s test, with SPSS Version11.0 for Windows (SPSS Inc., Chicago, IL, USA). Forall analyses, P � 0.05 was considered significant. Thepercentage values of motile cells during the refrigera-tion period, plasma membrane and acrosome integrity,and mitochondrial membrane potential were arcsinetransformed prior to analysis, although all data were

Fig. 1. Mean � SD percentages of total motility of ram sperm dilutedand subjected to incubation at 5 °C for 24 h (four replicates, with foa–cWithin an end point (fresh/diluted, 0 h, 12 h, 24 h), means witho

GSH, reduced glutathione; SOD, superoxide dismutase; CAT, catala

expressed as non-transformed means � SD. (

3. Results

The effects of refrigeration on subjective sperm mo-tility are shown (Fig. 1). There were no differences(P � 0.05) between treatments within each incubationtime. However, in all treatments, there was a reductionin motility throughout 24 h of refrigeration (P � 0.05).

In all treatments, there were no differences (P �0.05) in the percentages of sperm with an intact plasmamembrane following dilution in Tris-egg yolk aloneand supplemented with antioxidants throughout refrig-eration at 5 °C, relative to fresh/diluted samples (Table1). Likewise, there were no differences (P � 0.05)between treatments considering the same equilibrationtime and type of sample (refrigerated or frozen-thawed). However, thawed sperm had a lesser propor-tion (P � 0.01) of intact plasma membranes in com-parison to the fresh and refrigerated semen samples,regardless of treatment or duration of equilibration (Ta-ble 1). Moreover, there was a greater percentage ofsperm with an intact plasma membrane (P � 0.05) insamples frozen after 12 h of equilibration time (5 °C)than in the absence of equilibration time (0 h), with theexception of treatments SOD 10 and 20 U/mL and CAT10 and 20 U/mL (Table 1). There was no significantdifference in the percentage of plasma membrane in-tegrity between sperm frozen after 12 and 24 h ofequilibration, whereas with SOD 20 U/mL medium, the24-h equilibration time treatment resulted in betterpreservation (P � 0.05) of the sperm plasma membraneompared to the sample cryopreserved without any

s-egg yolk supplemented with various concentrations of antioxidantss per replicate).

mon superscript differed (P � 0.05).

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346 D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

For percentages of sperm with iAC and H�m,there were no differences (P � 0.05) among fresh/diluted, refrigerated, and frozen-thawed semen sam-ples, regardless of treatment. Combined for all treat-ments, iAC and H�m were 68.82 � 8.26 and63.74 � 6.38%, respectively for fresh/diluted semen,and were 63.79 � 4.57 and 50.74 � 8.14% for frozen-thawed samples.

There were no differences (P � 0.05) among treat-ments and duration of pre-freezing equilibration forPM, VAP, VSL, and BCF in thawed semen samples.Combined for all treatments and equilibration times,overall averages were 10.13 � 3.85% (PM), 45.66 �7.58 �m/s (VAP), 28.26 � 5.48 �m/s (VSL), and.95 � 0.84 Hz (BCF). There were no differences (P �.05) among treatments with regards to TM, VCL,LH, STR, and LIN within the same pre-freezing

quilibration time. However, the absence of equilibra-ion time (0 h) reduced (P � 0.05) TM in the GSH 1.0M and SOD 20 U/mL; the VCL in the GSH 1.0 mM

nd SOD 20 U/mL; and the ALH in the GSH 1.0 and.0 mM, SOD 5 and 20 U/mL; in comparison to 12 h ofquilibration. In contrast, the absence of equilibration0 h) increased (P � 0.05) STR and LIN in mostreatments (control; GSH 1.0 and 2.0 mM; SOD 5 and0 U/mL; CAT 20 U/mL) when compared to 12 hquilibration time at 5 °C prior to cryopreservationTable 2). The post-thaw motility parameters of spermamples frozen after 24 h of equilibration time werenaffected (P � 0.05) by the addition of antioxidantsnd were similar (P � 0.05) to those with 12 h equil-

Table 1Mean � SD percentages of ram sperm with intact plasma membranwith various concentrations of antioxidants during refrigeration at 5various equilibration times at 5 °C (0 or 12 h) prior to freezing (fou

Treatments Fresh/diluted Refrigerate

0 h

Control 80.9 � 5.3A 71.2 � 4.9A 71.GSH 0.5 mM 77.9 � 7.1A 71.0 � 11.1A 73.GSH 1.0 mM 77.6 � 5.6A 69.6 � 9.5A 71.GSH 2.0 mM 76.6 � 8.6A 73.4 � 6.7A 73.SOD 5 U/mL 82.6 � 4.8A 73.6 � 8.2A 64.SOD 10 U/mL 79.5 � 4.5A 65.5 � 5.9A 70.SOD 20 U/mL 79.2 � 3.9A 65.9 � 13.9A 65.CAT 5 U/mL 77.7 � 6.8A 72.7 � 8.2A 64.CAT 10 U/mL 76.8 � 2.9A 73.6 � 4.7A 68.CAT 20 U/mL 78.6 � 6.8A 71.2 � 5.9A 64.

a,b Within a line, means without a common superscript differed (PA,B Within a line, means without a common superscript differed (P

GSH, reduced glutathione; SOD, superoxide dismutase; CAT, c

bration. Conversely, 24 h of pre-freezing equilibration r

esulted in greater percentages (P � 0.05) of TM (GSH.0 mM and SOD 20 U/mL) and VCL (SOD 20 U/mL),nd lesser (P � 0.05) percentages of STR (Control,OD 5 and 10 U/mL) and LIN (control, GSH 1.0 and.0 mM, SOD 5 and 10 U/mL) compared to 0 h equil-bration (data not shown).

Total antioxidant capacity in the in natura Tris-eggolk was numerically proportional to the concentrationf the supplemented antioxidant in the medium, with aifference (P � 0.05) between the in natura Tris-eggolk treatment (control) and the Tris-egg yolk supple-ented with GSH 2.0 mM. However, there were no

ifferences (P � 0.05) in total antioxidant capacityetween the media supplemented with antioxidants fol-owing the addition of the semen (fresh/diluted) or evenhroughout refrigeration and freezing (Table 3).

. Discussion

The reduction in the percentage of motile spermuring refrigeration in the present study was similar torevious reports [10,12,24]. This phenomenon has beenttributed to changes in the kinetic properties of en-ymes induced by the reduction in temperature [25].upplementation with antioxidants (SOD, CAT, orSH) did not prevent this effect (at the concentrations

ested). Similarly, no improvement in sperm motility inhe presence of GSH (5 or 10 mM) was reported during0 h of refrigeration, when compared to control (nodditives) [24]. Furthermore, in that study, there was a

wing dilution in Tris-egg yolk extender (control), supplemented12, or 24 h) and in frozen-thawed semen samples subjected toates, four rams per replicate).

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347D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

increased. Although those authors used a greater con-centration of GSH than in the present study, the dilutionrate of semen described by these authors was four timeslower than that in our work, resulting in a similarsperm/GSH rate in both experiments. In contrast, theaddition of SOD and CAT to ram semen in a Tris-eggyolk base extender enhanced sperm motility duringrefrigeration [26]. However, the concentrations of an-tioxidants used (100, 200, 400, and 800 U/mL) weregreater than in the present study. Unfortunately, finalsperm concentration in that study was not reported.

A stable percentage of cells with an intact plasmamembrane during the equilibration period (5 °C) priorto freezing, regardless of the treatment, contrasted withprevious findings [25]. In that study, it was stated thatexposure of sperm to subphysiological temperaturesprior to freezing can induce alterations in the organi-zation of the lipid bilayer of the membrane, particularlyin species with high concentrations of polyunsaturatedfatty acids in the membrane [25], including ram sperm.However, current results were similar to those obtainedby O=Hara et al [9]. Using a combination of SYBR andPI, they demonstrated that the integrity of the spermmembrane remained relatively stable for up to 72 hduring storage at 5 °C. There was a significant reduc-tion in the percentage of sperm with intact plasmamembrane after thawing, when compared to fresh/di-luted and refrigerated sperm, which corroborated a pre-vious report [27]. Perhaps phospholipids of the spermmembrane exhibited different phase transition temper-atures, inducing the transition to the gel phase in othermolecules, which in turn influenced its diffusion coef-ficient [28] and fusion capacity of the membrane [29].

he 12 h pre-freezing equilibration (at 5 °C) signifi-antly enhanced preservation of plasma membrane in-egrity relative to no equilibration (0 h). Nonetheless,4 h equilibration was better than no equilibration (0 h),ut this difference did not achieve statistical signifi-ance, similar to findings described by Purdy [14].

Perhaps adoption of a 12 h pre-freezing equilibrationenhanced organization of the cell membrane during thephase transition of the microdomains, thereby minimiz-ing cryoinjury during semen processing. The additionof SOD and CAT at concentrations of 10 or 20 U/mLpartially reduced the harmful effects of the absence ofpre-freezing equilibration on the sperm plasma mem-brane. In that regard, high concentrations of these an-tioxidants were capable of compensating for the effectsof dilution [4]. Furthermore, that GSH was not protec-tive may stem from its dependence on the adequate

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348 D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

tase system [30]. Moreover, GPx had limited availabil-ity in ovine semen [31] and was further limited by thedilution inherent to semen processing, which explainsthe inability of GSH to protect the sperm membrane, aspreviously reported in pigs [32].

Reports of the antioxidant activity of specific en-zymes in sperm cells and seminal plasma [20,33–35]may not adequately reflect the balance between theproduction of ROS and their detoxification in the ex-tender in which the sperm are suspended; this wasconsidered an important factor to cell survival before,during, and after freezing [4]. Thus, total antioxidantcapacity of the diluting medium was determined. Themaintenance of total antioxidant capacity during vari-ous treatments and processing times was consistentwith a previous report [36]. In spite of a reduction in thectivity of specific antioxidant substances (CAT, SODnd GPx) after the dilution and freezing of stallionemen, they detected no significant alterations in theotal antioxidant capacity of fresh/diluted, refrigeratedr frozen-thawed semen [36]. The Tris-egg yolk ex-ender (in natura) in the control sample had substantialntioxidant capacity; this was likely due to the antiox-dant properties of egg yolk proteins, which are capablef preventing the oxidation of polyunsaturated fattycids [37]. As these acids are the main target of ROS,his additional biological property of egg yolk empha-ized the difficulty of replacing it with other compo-ents in the extender for freezing ovine semen [38].imilarly, Brouwers and Gadella [39] demonstrated

Table 3Mean � SD values of total antioxidant capacity of in natura Tris-egantioxidants, in fresh/diluted and refrigerated (0, 12, or 24 h at 5 °Cequilibration times at 5 °C (0, 12, or 24 h) prior to freezing (four re

Treatment Tris-egg yolk(in natura)

Fresh/Diluted

Refrige

0 h

Control 11.1 � 0.3bcd 11.8 � 0.7 12.2 � 0.4GSH 0.5 mM 11.3 � 0.2bcd 12.3 � 0.5 12.2 � 0.8GSH 1.0 mM 11.5 � 0.2abc 12.2 � 0.5 11.7 � 0.7GSH 2.0 mM 12.3 � 0.2a 12.1 � 0.6 12.6 � 0.3SOD 5 U/mL 10.6 � 0.4d 11.8 � 1.1 12.0 � 0.3SOD 10 U/mL 10.8 � 0.1cd 11.6 � 0.7 12.2 � 0.2SOD 20 U/mL 11.5 � 0.1abc 12.1 � 0.3 11.6 � 0.7CAT 5 U/mL 10.8 � 0.1cd 11.4 � 0.3 11.9 � 0.5CAT 10 U/mL 11.3 � 0.1bd 12.3 � 0.4 12.3 � 0.1CAT 20 U/mL 11.7 � 0.1ab 12.1 � 0.6 12.4 � 0.1

a–d Within a column, means without a common superscript differedGSH: reduced glutathione, SOD: superoxide dismutase, CAT: cugation at 10,000 � g for 15 min.The content of two straws/treahomogenized in a 1.0 mL microcentrifuge tube before analysis.

that lipid aggregates in egg yolk adsorbed on to the

sperm membrane and prevented the occurrence of per-oxidative damage.

The percentage of cells with iAC and H�mthroughout refrigeration and freezing remained similarto the values found for fresh/diluted semen, whichcorroborated a previous report [38] regarding iAC inovine sperm. However, latent damage may have oc-curred; in that regard, perhaps the degree of injuryduring the freezing/thawing process is manifested dur-ing the post-thaw incubation, with a greater degree oflatent damage decreasing sperm longevity in the femalegenital tract [40].

There is a negative correlation between productionof ROS and �m [41]. The main source of ROS isoxidative phosphorylation, which occurs in the mito-chondria. Since there was no significant differenceamong treatments throughout semen processing in mi-tochondrial membrane potential and total antioxidantcapacity, we inferred the system was able to maintain abalance between generation and removal of ROS.

In this study, antioxidants did not appear to have anyspecific protective effect on TM and PM (relative to thecontrol). We inferred that there was no associationbetween lipoperoxidation of sperm cells following thefreeze-thawing process and post-thaw motility param-eters, as previously reported [42]. In the present study,the TM seemed equal to or higher than that usuallyreported for frozen ovine sperm [15,20,31,43–45]. Fur-thermore, overall average of PM (10.13 � 3.85 %),regardless of treatment and equilibration, was lower

(control) extender, supplemented with various concentrations ofemen, and in frozen-thawed semen samples subjected to various, four rams per replicate).

mples (5 °C) Thawed samples

Equilibration time 5 °C

h 24 h 0 h 12 h 24 h

0.7 11.6 � 0.6 11.7 � 1.0 12.3 � 0.5 11.8 � 1.00.3 11.1 � 1.0 11.5 � 0.1 13.1 � 0.5 12.7 � 0.50.5 12.4 � 0.2 12.5 � 0.2 12.5 � 0.3 12.6 � 0.60.8 12.6 � 0.6 13.0 � 0.5 13.6 � 0.8 14.4 � 1.10.4 12.3 � 0.3 12.4 � 1.0 12.6 � 0.3 12.6 � 0.10.8 11.8 � 0.5 12.1 � 0.6 12.0 � 0.3 12.1 � 0.70.4 12.4 � 0.4 12.0 � 0.7 12.1 � 0.7 12.1 � 0.70.4 12.1 � 0.7 12.0 � 0.4 12.4 � 0.2 12.3 � 0.60.7 12.2 � 0.7 11.7 � 0.1 12.4 � 0.5 12.2 � 0.10.5 12.4 � 0.7 12.7 � 0.1 12.4 � 0.5 11.9 � 0.3

.05).Total antioxidant capacity was assessed in supernatant after centrif-quilibration time in refrigerated and thawed samples was pooled and

g yolk) ram splicates

rated sa

12

12.1 �12.3 �11.8 �12.5 �12.3 �12.0 �11.6 �12.2 �11.9 �11.8 �

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349D.R. Câmara et al. / Theriogenology 76 (2011) 342–350

to others [13,44,46]. For unknown reasons, additionalmotility end points assessed in thawed semen samples(VAP, VSL, VCL, ALH, BCF, STR and LIN) seemedlower than those previously reported [15,28,43,44].However, despite the numerous advantages of CASA[47], comparing sperm kinematics between studies isdifficult, since CASA settings, sperm concentrationsand diluents used can affect these end points [48,49],and furthermore, CASA settings are frequently notspecified.

It was not possible to identify any factor that couldexplain the reduction in TM in GSH 1.0 mM and SOD20 U/mL; in VCL in the control, GSH 1.0 mM andSOD 20 U/mL treatments; and in ALH in the GSH 1.0and 2.0 mM and SOD 5 and 20 U/mL treatmentssubjected to 0 h of equilibration in comparison to 12 hpre-freezing equilibration. Perhaps this variation wasrelated to subpopulations of sperm with different anti-oxidant enzyme distribution patterns and the proportionof each cell subtype may be altered by the processing[50], which was reflected in the metabolic and func-tional characteristics of the sample. The greater LIN inthe sperm in the absence of equilibration relative to12 h equilibration in the control and samples supple-mented with GSH (1.0 and 2.0 mM), SOD (5 and 10U/mL) and CAT (10 U/mL) may be indicative of lowerin vivo fertility rates. Verstegen et al. [47] stated that anncrease in LIN values may be related to reduced fer-ilization capacity.

In summary, the addition of GSH (0.5, 1.0, or 2.0M), SOD (5, 10, or 20 U/mL) and CAT (5, 10, or 20/mL) did not significantly influence the total antioxi-ant capacity of the Tris-egg yolk extender throughoutefrigeration and freezing. However, maintaining ovineemen at 5 °C for 12 h prior to cryopreservation re-uced cell membrane damage. Moreover, the harmfulffects of cryopreserving semen once equilibration waseached were partially overcome by the addition ofOD (10 or 20 U/mL) and CAT (10 or 20 U/mL).

cknowledgements

The authors are grateful to the following Brazilianostering agencies: Coordenação de Aperfeiçoamentoe Pessoal de Nível Superior (CAPES) and Conselhoacional de Desenvolvimento Científico e Tecnológico

CNPq) for their financial support.

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