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Animal Reproduction Science 120 (2010) 31–38

Contents lists available at ScienceDirect

Animal Reproduction Science

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

ffects of extender and equilibration time on post-thaw motility andembrane integrity of cryopreserved Gyr bull semen evaluated by

ASA and flow cytometry

iciano Guimarães Leitea, Vicente Ribeiro do Vale Filhoa, Rubens Paes de Arrudab, Andréurugen Cesar de Andradeb, Lucas Luz Emericka, Fabiane Gilli Zaffalonb, Jorge Andréatias Martinsa, Venício José de Andradec,∗

Department of Clinics and Surgery, School of Veterinary Medicine, Federal University of Minas Gerais (UFMG), BrazilLaboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, Veterinary Medicine and Animal Science School, University of Sãoaulo (USP), BrazilDepartment of Animal Science, School of Veterinary Medicine, Federal University of Minas Gerais (UFMG), Brazil

r t i c l e i n f o

rticle history:eceived 30 October 2009eceived in revised form 23 March 2010ccepted 6 April 2010vailable online 14 April 2010

eywords:attlepermemen cryopreservationquilibration timeASA

a b s t r a c t

The objectives of the present study were to investigate the effects of three equilibrationtimes (0, 2, and 4 h) and two extenders (TRIS or Bioxcell®) for cryopreservation of bullsemen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine.There were significant interactions between equilibration times and extenders for spermmotility and membrane integrity. The control treatment (0 h equilibration) had the low-est values (P < 0.05) for total (MOT) and progressive motilities (PROG), and percentage ofsperm with intact plasma and acrosomal membranes (IPIA), with no significant differencesbetween extenders. Extender TRIS had greater cryoprotective action than Bioxcell®, withgreater MOT, PROG, IPIA at 2 and 4 h, as well as the lowest proportion of damaged plasmamembrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4 h yielded the most desir-able (P < 0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6%

low cytometry for 0, 2, and 4 h, respectively). Overall, the combination of TRIS and 4 h of equilibrationwas the most desirable semen cryopreservation method, with greatest MOT, PROG, andIPIA (TRIS-T4 = 26.8%; BIO-T4 = 18.3%) and the least DPM. In conclusion, based on objectiveanalyses, equilibration during cryopreservation was essential for maintaining motility andintegrity of sperm membranes; equilibration for 4 h yielded the greatest sperm survival,

exten

independent of the

. Introduction

Protocols for freezing bull semen usually include slowooling to 4–5 ◦C, followed by a variable interval of equi-ibration (from 30 min to 24 h) at this low temperature

∗ Corresponding author at: Av. Antônio Carlos, 6627, Post-box 567,ampus da UFMG, Zip code 30123-970, Belo Horizonte, MG, Brazil.el.: +55 31 3409 2203/2168; fax: +55 31 3409 2168.

E-mail address: vejoan@gmail.com (V.J.d. Andrade).

378-4320/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.anireprosci.2010.04.005

der used.© 2010 Elsevier B.V. All rights reserved.

before freezing. Typically, glycerol is added during thisinterval, especially when egg yolk-citrate extender is used.Therefore, this period is sometimes erroneously referred toas “the glycerol equilibration time”. Although this intervalwas once considered important for glycerol equilibration,glycerol penetrates bull sperm quite rapidly and can be

added at any time during the cooling period (Berndtson andFoote, 1969; Coulter, 1992; Muino et al., 2007; Vishwanathand Shannon, 2000).

There is disagreement regarding the necessity and dura-tion of equilibration on semen cryopreservation and its

roductio

32 T.G. Leite et al. / Animal Rep

effect on sperm viability (Gao et al., 1997). In addition,there is a desire to shorten or eliminate this step, has-tening cryopreservation without compromising post-thawsperm quality (Dhami et al., 1992; Dhami and Sahni, 1993).In most studies that address this issue, post-thaw spermmotility (evaluated subjectively) was the only assessmentof sperm survival (Berndtson and Foote, 1969; Pickett andBerndtson, 1978). However, visual analysis by microscopyis not precise, cannot accurately predict fertility, andyields only superficial measures of sperm morphologyand motility, whereas fertilization involves several compli-cated biochemical and morphological events (Arruda et al.,2007; Graham et al., 1990; Jeyendran and Zaneveld, 1993;Verstegen et al., 2002).

The results of several fertility trials designed to deter-mine the optimal equilibration period for bull semenestablished the beneficial effect of a period of several hours(4–18) at 5 ◦C before freezing to obtain maximal fertility(Muino et al., 2007; Pickett and Berndtson, 1978). Despitethose findings, there are still doubts regarding the effectof equilibration time on sperm motility, integrity of spermmembranes, and mitochondrial function. Several studies(Berndtson and Foote, 1969, 1972a,b; Dhami et al., 1992;Dhami and Sahni, 1993; Foote and Kaproth, 2002; Gao et al.,1997; Muino et al., 2007) still question the necessity of per-forming the equilibration time and its real benefit to spermviability. In addition, the minimum duration of equilibriumneeded to have satisfactory results in semen cryopreserva-tion remains controversial (Dhami et al., 1992; Dhami andSahni, 1993).

Seminal plasma alone provides only limited protectionfor sperms against changes in temperature. For stor-age at reduced temperature, it is necessary to extendsemen in appropriate diluents (Salamon and Maxwell,1995a). Glycerol and egg yolk are the most commonlyused cryoprotectants, but in recent years there has beena trend against the use of egg yolk in cryoprotectivemedia, due to sanitary risks. Therefore, a well-definedand pathogen-free, non-animal origin substitute for eggyolk would be preferable (Aires et al., 2003; Thun etal., 2002). Consequently, soybean lecithin-based extenderswere developed. However, previous studies that comparedboth kinds of extenders reported conflicting results (Aireset al., 2003; Gil et al., 2000; Thun et al., 2002). Furthermore,little is known regarding their interactions with equilibra-tion time (Dhami and Sahni, 1993; Muino et al., 2007).

The objectives of the present research were to investi-gate further the effects of equilibration times, extenders,and their interactions, on the cryopreservation of bullsemen, based on motility, integrity of plasma and acro-somal membranes, and mitochondrial function, usingobjective and precise methods, i.e. Computer-AssistedSemen Analysis (CASA) and flow cytometry.

2. Materials and methods

2.1. Semen collection and pre-cryopreservation analysis

Twelve andrologically normal dairy Gyr bulls, 2–4-y-old, were selected from a group of 25 bulls (belonging toGetúlio Vargas experimental farm—a property of EPAMIG;

n Science 120 (2010) 31–38

Agricultural Research Company of Minas Gerais) fortheir semen characteristics: sperm motility (total motil-ity ≥ 65%; Vigor ≥ 4) and morphology (major defects ≤ 15%;total defects ≤ 30%). There had been no previous attemptsto cryopreserve semen from these bulls. Semen wascollected by electroejaculation, and evaluated for the fol-lowing variables: volume, concentration, sperm motility,and sperm morphology. Volume was determined directlyfrom a 15-mL graduated collection tube. To determinesperm concentration, an aliquot of semen was diluted1:100 in formaldehyde-phosphate buffered saline (PBS),and cell counts were performed in a Neubauer chamberunder bright-field microscopy (magnification, ×400).

2.1.1. Assessment of sperm motility and morphologySperm motility and vigor were evaluated visually (sub-

jectively) under a phase contrast microscope equippedwith a warm stage (37◦ C) at a magnification of ×100.To assess sperm morphology, a fresh semen aliquot wasdiluted and fixed in pre-warmed (37◦ C) formaldehyde-PBS. Sperms (n = 200) were assessed under phase contrastmicroscopy (Nikon Model 50i; Nikon, Tokyo, Japan) at amagnification of ×1000, in accordance with CBRA recom-mendations (CBRA, 1998).

2.2. Semen cryopreservation

One ejaculate from each of the 12 bulls with at least65% motile sperm was used. Each ejaculate was divided intwo aliquots and diluted with extender Bioxcell® (soybeanlecithin-based extender, IMV, L′Aigle, France) or Tris-eggyolk-glycerol (TRIS) at 34 ◦C, added in single step (completeglycerol-containing extender), to a final concentration of50 × 106 sperm/mL. The semen extension was performedimmediately after sperm motility and concentration eval-uations (5–10 min after collection). The TRIS extender wasprepared as described (Gonzalez, 2004) (composition: Tris2.42 g, citric acid 1.36 g, fructose 1.0 g, egg yolk 20 mL,glycerol 7 mL, penicillin G 0.028 g, dd.H2O - q.s.p 100 mL;osmolarity = 1432 mOs/Kg H2O). After dilution, semen wasmaintained in a water bath for 10 min at 34 ◦C for sta-bilization; thereafter, it was cooled from 34 to 25 ◦C inapproximately 20 min at room temperature (22–25 ◦C), andthen packaged in 0.5 mL straws (IMV). Straws designatedfor the same duration of equilibration time were trans-ferred to the same freezing machine. Three automatedfreezing machines were used (TK-3000®, TK Congelacão,Uberaba, MG, Brazil), with the same cooling (−0.25◦ C/min;until reaching 5 ◦C; duration: 80 min) and freezing rates(−20◦ C/min; from 5 to −120 ◦C; duration: 6 min and 15 s),varying only for the equilibration time at 5 ◦C: 0 h (T0),2 h (T2), and 4 h (T4), for a total of six treatments (lengthof total cryopreservation process from 25 ◦C to −120 ◦C:T0 = 86 min and 15 s; T2 = 206 min and 15 s; T4 = 326 minand 15 s). Thereafter, the straws were transferred to liquidnitrogen (−196 ◦C) for storage.

2.3. Post-cryopreservation semen evaluation

Semen was stored frozen for a minimum of 60 d beforeassessment. Sperm motility, plasma and acrosomal mem-

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rane integrity, and mitochondrial function were evaluatedn a two-straw pool of semen, from the same bull and treat-

ent, thawed in a water bath (37 ◦C for 30 s).

.3.1. Computer-assisted assessment of sperm motilitySperm motility was evaluated using a CASA system

HTM-IVOS-Ultimate; Hamilton Thorne Biosciences, Bev-rly, MA, USA). Aliquots of 5 �L of thawed semen werelaced in a 20 �m standard count analysis chamberSC20.01.FA; Leja®, Nieuw-Vennep, The Netherlands). Tenelds were selected for analysis of total (MOT, %) and pro-ressive (PROG, %) motilities; average path (VAP, �m/s),traight-line (VSL, �m/s), and curvilinear (VCL, �m/s)elocities; amplitude of lateral head displacement (ALH,m); beat/cross frequency (BCF, Hz); straightness (STR, %);nd linearity (LIN, %), as described (Celeghini et al., 2008).

.3.2. Flow cytometry analysesThese analyses were carried out using the FACSaria

Becton-Dickinson®, Sunnyvale, CA, USA) flow cytome-er equipped with a 488-nm excitation laser (Modelapphire 488-20, Coherent®) and filters C (Long Pass95 nm/Band Pass 610/20 nm), D (LP 556 nm/BP 575/26 nm)nd E (LP 502 nm/BP 530/30 nm). The flow cytometer wasalibrated, using two aliquots of a single fresh semenample (motility ≥ 80%), with one submitted to flash-reezing in liquid nitrogen (to damage sperm membranes,esulting in a great proportion of damaged sperms) asescribed (Celeghini et al., 2007), and the other used ascontrol (great proportion of membrane-intact sperms).

ach aliquot was subdivided and stained with: (1) pro-idium iodide (PI 0.5 mg/mL; 28,707-5; Sigma–Aldrich,t Louis, MO, USA); (2) fluorescein isothiocyanate-onjugated Pisum sativum agglutinin (FITC-PSA 100 �g/mL,-0770 Sigma–Aldrich); (3) PI + FITC-PSA association; or4) 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylarbocyanine iodide (JC-1) alone (153 �M, T-3168, Molec-lar Probes, Eugene, OR, USA). Sperm suspensions stainedith PI were run through the flow cytometer and a gate was

stablished, based on forward-scatter and side-scatter lightroperties, and compensation of each probe was deter-ined. Nonsperm events were gated out of analyses, based

n scatter properties.

.3.2.1. Evaluation of plasma and acrosomal membranes anditochondrial function. For flow cytometry assessment of

he integrity of plasma and acrosomal membranes, anditochondrial function, a 200 �L frozen-thawed semen

ample was diluted in a 37 ◦C pre-warmed TALP spermedium (Bavister et al., 1983) at a concentration of

5 × 106 sperm/mL. Thereafter, two aliquots (150 �L) wereransferred to microcentrifuge tubes. One of these tubesas supplemented with 3 �L of PI (0.5 mg/mL in Dulbecco’s

BS) and 80 �L of FITC-PSA (100 �g/mL in Dulbecco’s PBS)o evaluate the integrity of plasma and acrosomal mem-

ranes, whereas the other received 6 �L of JC-1 (153 �M inimethyl sulfoxide) to evaluate mitochondrial membraneotential. Samples were incubated in the dark for 8 min at7 ◦C, and then 600 �L of TALP was added to achieve anppropriate sperm concentration.

n Science 120 (2010) 31–38 33

Two-dimensional dot-plots of FITC-PSA vs. PI fluores-cence from a total of 10,000 events were generated. Eachquadrant represented one of the following sperm sub-populations: (1) sperm with intact plasma and acrosomalmembranes (PI (−), FITC-PSA (−), unstained cells); (2)sperm with an intact plasma membrane and a damagedacrosomal membrane (PI (−), FITC-PSA (+)); (3) spermwith a damaged plasma membrane and an intact acroso-mal membrane (PI (+), FITC-PSA (−); and 4) sperm withdamaged plasma and acrosomal membranes (PI (+) andFITC-PSA (+)).

For assessment of mitochondrial function, the probeJC-1 was used alone. Filters used for analysis of PI andFITC-PSA fluorescence were the same for high (590 nm) andlow (510–520 nm) mitochondrial potential (JC-1), respec-tively. Because emission wavelengths overlapped, it wasnot possible to use these three probes concurrently forflow cytometry. This analysis generated contour-densityplots, identifying three subpopulations: sperms with low,medium, and high mitochondrial membrane potential.These protocols were adapted from previous reports(Celeghini et al., 2007; Raphael, 2007).

For sperm membrane integrity, only percentages of cellswith intact plasma and acrosomal membranes (IPIA), dam-aged plasma membrane (DPM), and damage acrosomalmembrane (DA) were used. For mitochondrial function,only sperms with high mitochondrial membrane potential(HMP, %) were used for the data analysis.

2.4. Statistical analysis

A completely randomized block design in a 2 × 3 fac-torial arrangement (2 extenders × 3 equilibration times),with 12 replications per experimental unit was used. Datawere analyzed using SAEG 9.1 software (Statistical AnalysisSystem, UFV, Vicosa, MG, Brazil) (SAEG 9.1, 2001). Normal-ity of data was tested by Lilliefors test and homogeneityof variances by Cochran and Bartlett tests. Dependent vari-ables that did not meet the assumptions of equal varianceand normal distribution were subjected to a logarithmictransformation [Log (X + 1)]. Effects of extender, equilibra-tion time and block (bull) were evaluated by ANOVA, withmeans compared by Duncan’s test at a 5% level. A non-parametric test was used to analyze STR, as it did not meetassumptions even after transformations. For that, effects oftreatments were tested with a Friedman’s test, consideringbull as block, and treatments compared by Wilcoxon’s testat a 5% level, using Stat-View 5.0 program (Stat-View, 1998,SAS Institute Inc., Cary, NC, USA) (Stat-View, 1998).

3. Results

3.1. Post-thaw motility evaluated by CASA

There were significant interactions between extenderand equilibration time for total (MOT, %) and progres-

sive (PROG, %) motilities. When analyzed within extender,treatments with no equilibration time (T0) had lowervalues (P < 0.05) for MOT and PROG and differed fromtreatments T2 and T4; however, there was no difference(P > 0.05) between T2 and T4 for both extenders. Within

34 T.G. Leite et al. / Animal Reproduction Science 120 (2010) 31–38

Table 1Mean ± SD total and progressive motilities (evaluated by CASA) of bull semen cryopreserved using two extenders and three equilibration times (0, 2, and4 h).

Variable Extender Equilibration time

T0 T2 T4

Total motility (%) TRIS 14.5 ± 12.7bA 36.7 ± 13.9aA 41.0 ± 11.8aA

BIO 16.8 ± 15.5bA 24.2 ± 15.4aB 28.6 ± 16.0aB

Progressive motility (%) TRIS 10.8 ± 9.3bA 26.6 ± 11.1aA 30.8 ± 10.4aA

BIO 13.8 ± 11.9bA 19.8 ± 12.6aB 22.9 ± 12.2aB

a,bWithin a row, means without a common superscript differed (P < 0.05). A,BWithin a column and end point, means without a common superscript differed(P < 0.05).

Table 2Mean ± SD computer-assisted semen analysis of sperm motility parameters of bull semen cryopreserved using two extenders and three equilibration times(0, 2, and 4 h).

Variable Extender Equilibration time Average by extender

T0 T2 T4

VSL (�m/s)* TRIS 56.9 ± 4.4 55.2 ± 6.4 55.8 ± 6.1 56.0 ± 5.6B

BIO 69.1 ± 11.7 63.0 ± 6.3 62.1 ± 7.0 64.7 ± 9.0A

ALH (�m) TRIS 6.2 ± 1.2 6.2 ± 1.0 6.0 ± 0.9 6.1 ± 1.0A

BIO 5.8 ± 1.3 5.6 ± 0.8 5.4 ± 0.9 5.6 ± 1.0B

BCF (Hz) TRIS 22.3 ± 5.2 22.5 ± 4.7 22.0 ± 4.9 22.3 ± 4.8B

BIO 29.4 ± 5.9 30.4 ± 4.3 29.2 ± 5.0 29.7 ± 5.0A

LIN (%) TRIS 50.3 ± 3.3 47.3 ± 5.0 48.6 ± 4.8 48.7 ± 4.5B

BIO 53.3 ± 6.3 53.4 ± 4.3 54.4 ± 4.6 53.7 ± 5.0A

STR (%) TRIS 81.6 ± 4.8aB 78.3 ± 6.3bB 79.6 ± 5.7abB 79.8 ± 5.7aA 86.1 ± aA aA

A,BWithi

nt; BCF

BIO 85.4 ± 4.6

a,bWithin a row, means without a common superscript differed (P < 0.05).(P < 0.05).

* VSL–straight-line velocity; ALH–amplitude of lateral head displaceme

equilibration time, TRIS had greater values (P < 0.05) forMOT and PROG than Bioxcell® at T2 and T4, but with nodifference (P > 0.05) between extenders at T0 (Table 1).

There were no interactions (P > 0.05) for the otherparameters evaluated by CASA (VAP, VSL, VCL, ALH, BCF,

STR, LIN). For sperm velocities, only VSL was affected byextender (P < 0.05; Bioxcell® had higher values), whereasVAP and VCL were not affected (P > 0.05) by extender orequilibration time. Amplitude of lateral head displace-ment (ALH), beat cross frequency (BCF), and linearity

Table 3Mean ± SD effects of extender and equilibration time (0, 2, and 4 h) on integrity of pby flow cytometry.

Variable Extender Equilibration time

T0 T2

IPIA1 (%) TRIS 8.8 ± 3.8cA 20.9BIO 6.9 ± 4.6bA 15.2

DA2 (%) TRIS 65.4 ± 14.8bA 44.7BIO 60.9 ± 24.7aA 65.0

DPM3 (%) TRIS 80.7 ± 11.6 71.8BIO 92.3 ± 4.7 84.3

Average by time 86.5 ± 10.5c 78.0

a,bWithin a row, means without a common superscript differed (P < 0.05). A,BWithi(P < 0.05).

1 IPIA—intact plasma membrane and intact acrosome.2 DA—damaged acrosome.3 DPM—damaged plasma membrane.

3.5 86.6 ± 3.0 86.0 ± 3.7

n a column and end point, means without a common superscript differed

–beat/cross frequency; LIN–linearity; STR–Straightness.

(LIN) were affected (P < 0.05) only by extender, withTRIS having higher values for ALH and Bioxcell® for BCFand LIN. Straightness of sperm movement (STR) wasaffected (P < 0.05) by extender and equilibration time,with Bioxcell® values greater than TRIS at all equilibra-

tion times. However, STR values did not differ amongequilibration times, within Bioxcell® (P > 0.05). Conversely,within TRIS, T4 did not differ from the other times(P > 0.05), but T0 had greater values than T2 (P < 0.05;Table 2).

lasma and acrosomal membranes in cryopreserved bull semen, evaluated

Average by extender

T4

± 6.1bA 26.8 ± 7.6aA 18.8 ± 9.6± 8.2aB 18.3 ± 5.9aB 13.5 ± 7.9

± 13.0aB 44.4 ± 12.5aA 51.5 ± 16.4± 20.7aA 55.8 ± 17.5aA 60.6 ± 20.9

± 10.7 64.2 ± 12.7 72.2 ± 13.3A

± 8.2 81.0 ± 6.0 85.8 ± 7.9B

± 11.3b 72.6 ± 13.0a

n a column and end point, means without a common superscript differed

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.2. Integrity of plasma and acrosomal membranes anditochondrial function

There were interactions between equilibration time andxtender (P < 0.05) for IPIA and DA. The lowest values of IPIAere for T0, for both extenders (P < 0.05). However, withinioxcell®, there was no difference (P > 0.05) between T2nd T4. Within TRIS, there were differences among all equi-ibration times (P < 0.05), with T4 having the greatest IPIAalues (i.e. best preservation of sperm membranes). Withinquilibration times T2 and T4, TRIS was more efficient, withreater values of IPIA (P < 0.05) than Bioxcell®; however,here was no difference (P > 0.05) between extenders wheno equilibration time (T0) was used.

For DA, within each equilibration time, differencesetween extenders (P < 0.05) were detected only for T2,ith Bioxcell® having a greater percentage. When using

RIS, T0 had the greatest DA value (P < 0.05); however, thereas no difference (P > 0.05) between T2 and T4. Conversely,ithin Bioxcell®, there was no difference (P > 0.05) among

ll three equilibration times (Table 3).For DPM, there were no interactions between equi-

ibration time and extender (P > 0.05), despite isolatedffects. Semen frozen with Bioxcell® had the high-st percentages of sperms with DPM. In relation tohe equilibration time, there were differences (P < 0.05)mong all treatments, with T4 having the least percent-ges of DPM (Table 3). Regarding mitocondrial function,here were no effects (P > 0.05) of extender or equilibra-ion time for HMP (%; TRIS = 20.9 ± 9.7; BIO = 17.7 ± 14.1;0 = 20.3 ± 15.9; T2 = 20.0 ± 9.9; T4 = 17.7 ± 10.0).

. Discussion

.1. Post-thaw motility evaluated by CASA

The total and progressive motilities were lower at TOhan T2 and T4 confirmed the need for equilibration toreserve sperm motility, consistent with several othereports (Berndtson and Foote, 1969, 1972a,b; Dhami etl., 1992; Dhami and Sahni, 1993; Foote and Kaproth,002; Graham et al., 1957; Pickett and Berndtson, 1978).tilizing egg yolk-Tris and whole milk-egg yolk-glycerolxtenders, Dhami et al. (1992), and Dhami and Sahni (1993)eported that slow cooling and at least 2 h of equilibra-ion at 5 ◦C significantly improved post-thaw motility (44.0ompared to 35.5%) and fertility (57.9 compared to 47.2%,0-d non-return rates) in comparison with no equilibra-ion. Furthermore, there was speculation that results couldave been improved if longer equilibration times weresed. These previous studies differed from the presentnly in methods used, i.e. empiric cooling and freezingates, manual cryopreservation and subjective assessmentf motility, compared to an automated freezing machinend post-thaw motility evaluated by CASA. Therefore, theurrent study provided additional support for the previous

onclusions.

When bull semen freezing technology was being devel-ped, many aspects of the cryopreservation protocol,ncluding glycerol concentration, rate and temperature oflycerol addition, and duration of equilibration were care-

n Science 120 (2010) 31–38 35

fully evaluated. In an extensive review of the literature,Pickett and Berndtson (1978) concluded, based on resultsof several fertility trials, that slow cooling and equilibrationat 5 ◦C were necessary to obtain maximal fertility. Based onthese results, an equilibration lasting 4–18 h at 5 ◦C beforefreezing was recommended. Experiments were conductedwith various equilibration times at 5 ◦C, in the presence orabsence of glycerol, to determine optimal interval neededfor glycerol to equilibrate on both sides of the spermplasma membrane. It was noteworthy that several experi-ments yielded evidence that glycerol penetrated bull spermrapidly (2–5 min) (Berndtson and Foote, 1969, 1972a,b),suggesting that equilibration time was more important forsperm membranes to adapt to low temperatures, ratherthan for glycerol penetration and equilibration.

Several investigators used subjective assessment ofsemen in an attempt to establish the optimal durationof equilibration. However, there were conflicting reportsregarding survival and fertility of frozen-thawed bullsemen (see Foote and Kaproth, 2002), particularly whenworking with various extenders, semen packaging, andrates of cooling and freezing (see Graham et al., 1957). Forexample, Miller and Vandemark (1954) compared 2, 6, and18 h using egg yolk-citrate (EYC) and found that 6 h wassuitable for acceptable sperm survival. Conversely, O’Delland Hurst (1955), using EYC and skimmed milk, reporteda greater recovery rate for 0 h than for 18 h, in contrastwith the present study. Ennen et al. (1976), working withstraws, did not find significant differences for motility afterpost-thaw incubation (38 ◦C/2 h) among 1, 2 and 4 h ofequilibration time, but these times were superior to 16 h.The authors observed that the optimum equilibration timefor semen storage in straws ranged from 2 to 10 h and wasdependent on cooling time, and that slowly cooled semenseemed to require less equilibration for optimal motility,similar to the present study.

Sperm motility is one of the most important featuresassociated with semen fertilizing capacity, and for manyyears has been recognized as essential for sperm trans-port and fertilization in the female reproductive tract(Januskauskas et al., 1999; Verstegen et al., 2002). How-ever, visual assessment of post-thaw motility, the mostlywidely used single parameter to determine bovine semenquality, has been reported to be strongly or poorly corre-lated with fertility (see Tartaglione and Ritta, 2004). Thismethod is inexpensive and rapid, but is also subjectiveand inaccurate. A more objective and precise assessmentof sperm motility can be achieved utilizing a computer-assisted semen analyzer (CASA) (Christensen et al., 2005);it provides more precise calculations of velocity and pathof each sperm cell. It is noteworthy that some CASA vari-ables have been correlated with fertility (MOT, r2 = 0.34)and combinations of several CASA variables resulted ingreater r2 values (0.68–0.98) (Farrell et al., 1998).

The recommendations of using equilibration time werebased on fertility trials. However, fertility data presented

in most publications have severe limitations when used tovalidate a diagnostic assay. The ideal fertility trial should beperformed ‘on the edge of the dose–response curve’, as theeffect of a compensable sperm trait can be masked by usinga large number of sperm per insemination dose (Amann

roductio

36 T.G. Leite et al. / Animal Rep

and Hammerstedt, 2002). A large-scale, low-dose insemi-nation trial is a laborious and very expensive way to testnew extenders. A simpler approach would be to use flowcytometry to screen extenders for post-thaw sperm viabil-ity and longevity of a large number of bulls and ejaculates;if initial results were encouraging, an insemination trialcould be subsequently conducted. Flow cytometry enablesanalysis of thousands of sperms per sample, with muchgreater precision than is possible with subjective micro-scopic assessment or even CASA systems (Christensen etal., 2005; Muino et al., 2007).

An important aspect of the present study was the useof a totally automated freezing machine, which providedconsistent cooling and freezing rates. In addition, the use ofCASA and flow cytometry to evaluate motility and integrityof sperm membranes, respectively, facilitated comparisonof the shortest equilibration times possible with higherprecision than previous studies. These methods were pur-posefully chosen to minimize or eliminate the subjectivityand confounding factors that presumably have contributedto the uncertainty regarding the effects of equilibrationtime on survival and fertility of cryopreserved bull semen.We are unaware of any study that used this methodology(CASA and flow cytometry) to assess equilibration times.

The greater effectiveness of TRIS in preserving total andprogressive motility in T2 and T4 differed from a previousreport (Celeghini et al., 2008) of satisfactory motility withno equilibration time. These authors, working with Bostaurus, found differences between extenders with no equi-libration time. They reported that Botu-bov® (MOT = 40.1%;PROG = 20.9%) was more effective at preserving totaland progressive motility than Bioxcell® (MOT = 24.2%;PROG = 10.7%). Apparent differences between their resultsand those of the present study may have been due to differ-ences in cooling and freezing rates, and type of EY-extenderemployed, but was also likely due to differences in bullbreeds. There are breed differences in semen freezability;in particular, European breeds have superior freezabilitythan Zebu breeds (Anchieta et al., 2005), probably due to alonger selection time for reproductive traits.

Although equilibration time significantly affected totaland progressive motility in the present study, there wasno significant effect on other characteristics of spermmovement (i.e. VAP, VSL, VCL, ALH, BCF, and LIN). It wasnoteworthy that extender had a significant effect on VSL,ALH, BCF, LIN and STR; except for ALH, all these variableswere greater in semen frozen with Bioxcell®. These resultswere apparently contradictory, since indicate better spermmovement features for Bioxcell®, even thought this exten-der showed lesser MOT and PROG. However, these findingscould have been due to differences in extender density, vis-cosity or even the presence of large particles, as previouslysuggested (Celeghini et al., 2008; Mortimer, 1997).

That ALH was greater in egg yolk than in a soybeanlecithin extender was in direct contrast with a previousreport (Celeghini et al., 2008). In general, greater ALH

is not desired, because it denotes lesser sperm qualityand can interfere with cell cycle progression, as demon-strated by Arruda et al. (2003). However, the ALH value ismathematically determined, and no minimum and maxi-mum limits have been yet established regarding the extent

n Science 120 (2010) 31–38

to which it influences progressive motility or fertiliz-ing ability (Celeghini et al., 2008). Semen cryopreservedwith Bioxcell® had greater BCF values, suggesting thatthis extender was more effective at preserving flagellarstructures, or that compounds present in this extenderstimulated ATP production and consequently beat fre-quency, as previously suggested by Celeghini et al. (2008).

For LIN and STR, Bioxcell® had greater values at all times.A greater value of STR indicates that the trajectory of spermis more uniform and with low amplitude, whereas a greaterLIN indicates a lesser amplitude of curvilinear path anddirection similar to the path in a straight line. Differencebetween extenders for STR and LIN indicated that move-ment of sperms cryopreserved with TRIS was more circular,but it is uncertain if these differences could affect fertility.Januskauskas et al. (2003) found no correlation betweenLIN and fertility rate in cattle, whereas Celeghini et al.(2008) found that VAP, VSL, VCL, ALH were greater in soylecithin than in egg yolk-based extenders, but BCF, STR andLIN were greater in the latter. Clearly, further studies areindicated.

4.2. Plasma and acrosomal membranes integrity andmitochondrial function

There was a beneficial effect of equilibration time forpost-thaw integrity of these membranes, and its lackwas prejudicial, because T0 had the least percentage ofcells with both membranes intact (IPIA) and the greatestdamaged plasma membrane (DPM). Despite no differ-ences between T2 and T4 for motility (MOT and PROG),there were significant differences for plasma membraneintegrity. The least percentage of DPM cells for T4 indicatedthat a longer equilibration time was more effective at pre-serving the plasma membrane, independent of extender.

For preserving both membranes, TRIS was more effi-cient, because there was a progressive increase in spermsurvival from T0 to T4, based on percentage of IPIA cells,despite the lack of a difference between extenders at T0. Incontrast, for Bioxcell®, there were no differences betweenT2 and T4 in percentage of IPIA cells (although both werebetter than T0, with greater cryoprotection when equili-bration was used). For preservation of acrosome integrity,there was a significant difference between extenders onlyat T2 (TRIS was better), probably due to the great varia-tion in DA in semen diluted in Bioxcell®. In all treatments,the percentage of cells with plasma membranes damagedby cryopreservation was greater than that with motil-ity loss, consistent with previous reports (Celeghini et al.,2008; Salamon and Maxwell, 1995b) that frozen-thawedsperm cells with damaged membranes may remain motile,although they probably have substantially reduced fertil-izing capacity.

Previous studies comparing soybean lecithin and eggyolk-based extenders on bull and buffalo sperm cry-opreservation yielded conflicting results. Some authors

reported no differences (Aires et al., 2003; Gil et al., 2000),whereas others (Christensen et al., 2005; Herold et al.,2006; Thun et al., 2002; Van Wagtendonk-De Leew etal., 2000) found significant differences for in vitro spermquality, post-thaw sperm motility, plasma and acrossomal

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T.G. Leite et al. / Animal Repr

embrane integrity, capacitation status, or 56 d non-eturn rate (56 d NR) with semen cryopreserved withoybean lecithin or with egg yolk. Other research groupstilizing a single (18 h) (Muino et al., 2007) or more thanne equilibration time (Herold et al., 2006) and compar-ng both types of extenders registered higher post-thaw

otility and acrossomal integrity for egg yolk; however,ost-thaw quality of epididymal sperm was not affectedy varying the equilibration time (range, 2–9 h) for eitherxtender (Herold et al., 2006). Despite the apparent dis-repancies, the advantages of one type of extender relativeo another have generally been small, and may be due inart to differences in bull breeds (Muino et al., 2007).

In the present study, TRIS was superior in preserv-ng the motility and integrity of sperm membranes. Wenferred that this beneficial effect of TRIS was probablyue to a better cryoprotective action of egg yolk lipopro-eins than soybean lecithin during equilibration, althoughhe influence of differences between extenders in glyc-rol concentrations (TRIS 7%; Bioxcell® 3.19%, v/v) shouldot be ignored. Notwithstanding, the optimum concentra-ion of glycerol for freezing bull semen is also influencedy other extender components (Vishwanath and Shannon,000). A new mechanism for sperm protection by low-ensity lipoproteins (LDL) of egg yolk has been proposedBergeron et al., 2004; Manjunath and Thérien, 2002),nvolving the binding of LDL to bovine seminal plasmaroteins (BSP) and to the sperm plasma membrane, withgain of cholesterol and choline phospholipids by the

perm membrane. These mechanisms of action of LDLre time- and concentration-dependent, and furthermorehey are influenced by temperature. This might be onef the mechanisms involved in the enhanced protectionf egg yolk relative to soybean lecithin-based extenders,s observed in this study. Because this mechanism isime- and temperature-dependent, this could account forRIS requiring an adequate equilibration interval to man-fest a greater cryoprotective action. We inferred that thenteraction between equilibration time and extender wasecessary to preserve sperm motility and integrity of spermembranes during cryopreservation.Mitochondrial function did not follow the same pattern

bserved for the other variables, because it was affectedeither by equilibration time nor by extender, suggest-

ng that equilibration time did not have a direct effect.owever, further studies are necessary to confirm these

esults, because there was a difference between extenders,nd positive correlations between mitocondrial functionnd total and progressive motilities in a previous studyCeleghini et al., 2008). However, the importance of mito-hondria for sperm motility has been recently reconsideredTurner, 2006). Some studies suggested that the sperm pro-uces most of the ATP necessary for flagellar movement bylycolysis (anaerobic ATP production) (see Turner, 2006),ut another study provided some evidence to the contraryFord, 2006). Nevertheless, mitochondria provide energy

or the midpiece, and presumably the sperm head (for

aintenance of important membrane processes). There-ore, the functional integrity of the mitochondria may bemportant for the sperm survival in the female genital tractSilva and Gadella, 2006). A more complete evaluation of

n Science 120 (2010) 31–38 37

mitochondrial function before and after cryopreservationmay provide information to clarify the results observed.

The 4 h equilibration time was chosen, since it is a con-venient interval to have large volumes extended semenprepared for freezing. However, in some studies, holdingsemen overnight increased fertility (see Foote and Kaproth,2002; Pickett and Berndtson, 1978). A study reported(Graham et al., 1957) 75 d NR of 63.4, 65.2, and 67.8%,respectively, for 4, 8, and 12 h of equilibration, but the onlysignificant difference was between 4 and 12 h, suggestingthat more than 4 h was beneficial. Further studies are nec-essary to clarify whether semen quality is improved withequilibration times exceeding 4 h.

In the current study, equilibration was beneficial forsperm survival during cryopreservation, since it increasedsperm motility and integrity of membranes, favored byegg yolk-based extenders (in agreement with most semencryopreservation protocols). We inferred that equilibra-tion time facilitated adaptation of sperm membranes andmetabolism to cold rather than to glycerol, allowing spermto become less susceptible to cryopreservation damage, aspreviously proposed (Berndtson and Foote, 1969, 1972a,b).

5. Conclusion

Equilibration time is necessary for preservation ofmotility and integrity of sperm membranes. Equilibrationfor 4 h resulted in the greatest preservation of total andprogressive motility, as well as the integrity of plasma andacrosomal membranes during cryopreservation. Further-more, there were some significant interactions betweenequilibration time and extender which lead to greatersperm survival. Overall, TRIS provided enhanced cryopro-tection than Bioxcell®, resulting in greater motility andintegrity of sperm membranes, and when combined with4 h equilibration (TRIS-T4), resulted in the greatest qualityfrozen-thawed semen.

Acknowledgements

Thanks to CNPq for the scholarship grants, to FAPEMIGfor financial support, to IMV (IVP Brazil), TK Congelacão(Mr. Carlos C. Motta) and EPAMIG for their support. Thanksto Dr. Marcos Brandão D. Ferreira (EPAMIG) for his help inthe experiment and to Dr. Andréia K. R. Santos and Prof.John Kastelic for their friendship and suggestions for themanuscript.

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