Animal Reproduction Science 159 (2015) 155162

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Animal Reproduction Science

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Liquid storage of equine semen: Assessing thd-penicillamine on longevity of ejaculated anstallion

P.T. Brogana,, M. Beitsmaa, H. Henninga, B.M. Gadellab,c, T.A.E. Stouta,b,d

a Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 114, 3584 CM Utrecht, The Netherlandsb Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CM Utrecht, The Netherlandsc Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht,The Netherlandd Section of Re

a r t i c l

Article history:Received 26 MReceived in reAccepted 13 JuAvailable onlin

Keywords:ROSAntioxidantChromatinSCSAChilled semen

1. Introdu

Articiacommon pr

CorresponE-mail add

(M. Beitsma),(B.M. Gadella)

http://dx.doi.o0378-4320/ sproduction, Faculty of Veterinary Science, University of Pretoria, Private Bag XO4, Onderstepoort 0110, South Africa

e i n f o

arch 2015vised form 12 June 2015ne 2015e 19 June 2015

a b s t r a c t

Short-term storage of equine sperm at 5 C in an extender containing milk and/or eggyolk components is common practice in the equine breeding industry. Sperm motility,viability, DNA integrity and, consequently, fertilizing ability decline over time, partlydue to reactive oxygen species (ROS) generation. We investigated whether adding theanti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decreasein semen quality. Semen was recovered on four consecutive days from eight 3-year oldWarmblood stallions. On day 5, seven of the stallions were castrated and sperm recoveredfrom the caudae epididymides. Ejaculated samples were split, and one portion wascentrifuged and re-suspended to reduce seminal plasma content. All samples were dilutedto 50 million sperm/ml and divided into two portions, one of which was supplementedwith 0.5 mM d-penicillamine. After 48 h, 96 h, 144 h and 192 h storage, sperm motility wasassessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining,and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillaminehad no effect on motility of ejaculated sperm (P > 0.05) but reduced total and progressivemotility of epididymal sperm. Sperm chromatin integrity was not inuenced by storagetime, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercialsemen extender was neither benecial nor detrimental to the maintenance of quality inejaculated semen stored at 5 C. The negative effect on motility of epididymal sperm mayreect differences in (membrane) physiology of spermatozoa that have not been exposedto seminal plasma.

2015 Published by Elsevier B.V.

ction

l insemination of liquid stored stallion semen isactice in horse breeding. While storage at 5 C

ding author. Tel.: +31 30 253 1350; fax: +31 30 253 7970.resses: P.T.Brogan@uu.nl (P.T. Brogan), M.M.Beitsma@uu.nl

H.H.W.Henning@uu.nl (H. Henning), B.M.Gadella@uu.nl, T.A.E.Stout@uu.nl (T.A.E. Stout).

for up to 72 h in an extender containing milk and/or eggyolk components generally yields acceptable pregnancyresults, sperm motility, viability and fertilizing ability haveall been shown to decline with storage time (Jasko et al.,1992; Aitken et al., 2012a; Pagl et al., 2006). The reductionin semen quality during liquid storage is partly attributableto the production of reactive oxygen species (ROS), as aresult of oxidative phosphorylation by sperm mitochon-dria (Aitken et al., 2012b; Baumber et al., 2002; Ball,

rg/10.1016/j.anireprosci.2015.06.0112015 Published by Elsevier B.V. sperme effect ofd epididymal

156 P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162

2008). Mammalian sperm contain an abundance of polyun-saturated fatty acids in their plasma membrane (Parksand Lynch, 1992) which, combined with their limitedamount of cytoplasm to carry antioxidant enzymes, ren-ders them vulnerable to oxidative damage (Aitken andClarkson, 1987; Jones et al., 1979) of various cellular com-ponents inc2015; Baum2004). ThisROS in mamoxidative stDNA integret al., 1989detrimentalead to thetions are cacopper, kno(Aitken et a2006; Bilodplasma alsooxidants (CBall et al., 2not entirelypromotes ospermatozo

Recentlylar weight t(Aitken et LWMTs areas free radPerret, 197is a low mamino acidmetal chel(Perret, 19ing the storbody tempmaintain hzoa and to d2012a). Simbeen reporaim of the penicillamiof both ejaduring storhypothesis of decline induration.

2. Materia

2.1. Chemic

All chemerwise statAldrich, Zw(PI) and SLIVE/DEADThe Nether

2.2. Animals and study design

Eight 3 year old Warmblood stallions from a singlestud-farm were presented for routine castration. Semencollection procedures were performed with informedowner con

al exped at Uates tater

fastedas pr

collectble dality waholog

(Van Datozo

microere cl

stallioal speded inal pur

out o

Semen

men wa. Eace to re

in a iquot erm coologieatio ofder c

d G418ethermstertranspbient

divide g forellet

106 spwas dily 50

[centtwo sastock n of 0tainedC.men qafter 4. At eaatozo

n in liqrity.luding DNA, proteins and lipids (Conrad et al.,ber et al., 2003; Ball, 2008; Baker and Aitken,

imbalance in production and degradation ofmalian sperm has been reported to result in

ress, which manifests as a loss of motility andity and a decline in fertilizing capacity (Aitken, 2012c; Baumber et al., 2003). ROS exert theirl effects via lipo-peroxidative reactions which

formation of cytotoxic aldehydes. These reac-talyzed by transition metals, such as iron andwn to be present in equine seminal plasmal., 1989; Bertelsmann et al., 2008; Pesch et al.,eau et al., 2002). On the other hand, seminal

contains ROS scavenging enzymes and anti-onrad et al., 2015; Alvarez and Storey, 1983;000; Baumber and Ball, 2005), such that it is

clear whether seminal plasma predominantlyr prevents ROS-induced damage in ejaculateda., attention has been directed to low molecu-hiols [LMWTs] for their anti-oxidant propertiesal., 2012a; Wroblewski et al., 2003). These

strong chelators of transition metals and actical scavengers (Wadhwa and Mumper, 2013;7). d-Penicillamine (3,3-dimethyl-d-cysteine)olecular weight nucleophilic thiol and an -

metabolite of penicillin with anti-oxidant andating properties but no antibiotic properties77; Shuxi Qiao et al., 2012). Moreover, dur-age of washed semen at both ambient and aterature, d-penicillamine has been reported toigher percentages of motile stallion spermato-elay the decline in sperm velocity (Aitken et al.,ilar pro-survival effects of d-penicillamine haveted for bull spermatozoa (Pavlok, 2000). Thecurrent study was to evaluate the effects of d-ne on the motility, viability and DNA integrityculated and epididymal stallion spermatozoaage at 5 C in a commercial extender. Thewas that d-penicillamine would slow the rate

semen quality and thereby extends its storage

ls and methods

als

icals were of analytical grade and, unless oth-ed, purchased from SigmaAldrich (Sigmaijndrecht, The Netherlands). Propidium iodideYBR14 were purchased as part of the

Sperm Viability Kit (Molecular Probes, Leiden,lands).

animhouscentrand wwerecise wwas a stamotimorpstainspermlight ties wOnlynorminclumentSevenday.

2.3.

SevagingauzsuredAn alof sptechnat a rextenG anThe Ning, Awas at amwas 800 the p50 part imatewaysinto (1 M tratiomainat 5

Seand tivelyspermfrozeintegsent, following consultation with the localerimentation committee. The stallions weretrecht University in individual stalls, fed con-

wice daily and had ad libitum access to hayuntil 24 h prior to castration, from when they

until full recovery from anaesthesia. Daily exer-ovided in an automated horse walker. Semened every second day for two weeks to attainily sperm output (DSO). At this point, sperms assessed and smears for evaluating sperm

y were prepared using an eosin-aniline blueer Schaaf, 1952). A minimum of two hundreda were evaluated under oil immersion with ascope at 1000 magnication, and abnormali-assied as described by Brettschneider (1948).ns with a minimum of 60% morphologicallyrmatozoa and 60% motile sperm at DSO were

the study. Thereafter, ejaculates for experi-poses were collected on four consecutive days.f the eight stallions were castrated on the fth

collection and initial processing

as collected using a Hannover model articialh ejaculate was immediately ltered throughmove debris and gel, and the volume was mea-graduated, pre-warmed (38 C) glass cylinder.was taken for spectrophotometric assessmentncentration (Accucell Equine Photometer, IMVs, LAigle, France). The semen was then diluted

1:1 (v:v) with a commercial milk-based semenontaining egg yolk and antibiotics (penicillin-sulphate) (Spervital EVD; Spervital, Toldijk,

lands) in 50 ml Falcon centrifuge tubes (Corn-dam, The Netherlands). The extended semenorted to the laboratory in a styrofoam box

temperature. In the laboratory, the ejaculated into two parts. One part was centrifuged at

20 min. The supernatant was discarded anddiluted to a concentration of approximatelyermatozoa/ml with Spervital EVD. The otherrectly extended to a concentration of approx-

106 spermatozoa/ml. Semen diluted in bothrifuged/non centrifuged] was further dividedmples each, to one of which d-penicillaminesolution in PBS) was added to a nal concen-.5 mM. The different semen preparations were

in 2 ml capped Eppendorf tubes in a refrigerator

uality was assessed before any treatment (0 h)8 h, 96 h, 144 h and 192 h of storage, respec-ch time point, percentages of motile and viablea were recorded, and 30 l aliquots were snap-uid nitrogen for later assessment of chromatin

P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162 157

2.4. Epididymal sperm collection and processing

The stallions were castrated under general anaes-thesia with a half-closed technique, as described bySchumachein the form(10 g/kg Dand butorpMilton Keintravenoustrafarm Seketamine (2France) andwith isouBerkshire, midine wa(10 g/kg/hdine (38 unixin memal Healthanalgesia.

The testi30 min at rther procesdeferens wof the caudof semen ature. The hemocytom50 106 spsured direcfrozen for aaddition ofdiluted samwhich wasSamples wway and at tsperm.

2.5. Compu

MotilityAnalysis (Cbach, Germpre-warmeNieuw Venaliquot of spermatozocamera adaand a cameTM-6760CLperformed Minitb, Gsperm per ssive elds iwere designparametersmotility), asperm andsperm, theof straight

path velocity (VAP), linearity (LIN = VSL/VCL), straightness(STR = VSL/VAP), wobble (WOB = VAP/VCL), amplitude oflateral head-displacement (ALH), and beat cross frequency(BCF). A spermatozoon was considered to be motile when it

ne of ttation25 Hz,/s and. A met on, (2)

, (3) VS m, (5dius greater

ssessm

erm vrm sand 3

After n a lig00 mcells w

Olymo, Japa/emis

5/O59 signa

cells t memstains iting

intacuorescged.

ssessm

e pro in apointdenatu

(SCSthe m

on a , the N

(15 m nm bm lon0 specteris

FCS engelesentat

of eveles duntageation ).r (2012). Pre-surgical sedation was provided of intravenous detomidine hydrochlorideomosedan: Orion Pharma, Espoo, Finland)

hanol (20 g/kg Dolorex: MSD Animal Health,ynes, UK). Anaesthesia was induced with

diazepam (0.06 mg/kg diazepam CF; Cen-rvices, Etten Leur, The Netherlands) and

mg/kg Narketan 10: Vetoquinol, Lure Cedex,, following orotracheal intubation, maintainedrane (isoFlo 100% w/w: Abbot Laboratories,UK) in a 1:1 oxygen:air mixture. Deto-s supplemented by constant rate infusion). All horses received post-operative detomi-g/kg i.v.) to ensure smooth recovery, andglumine (1 mg/kg iv, Cronyxin: Eurovet Ani-, Bladel, The Netherlands) for post-operative

cles and epididymides were transported withinoom temperature to the laboratory for fur-sing. A minimum of 5 cm of the distal vasas preserved to facilitate retrograde ushinga epididymides (Bruemmer, 2006) with 20 mlextender (Spervital EVD) at room temper-sperm concentration was assessed using aeter chamber and adjusted to approximately

ermatozoa/ml. Motility and viability were mea-tly (0 h) and a sample was taken and snapssessment of chromatin integrity, before the

d-penicillamine and further incubation. Theple was then divided into two parts, one of

supplemented with 0.5 mM d-penicillamine.ere stored at 5 C and examined in the samehe same time points as described for ejaculated

ter-assisted semen analysis

was assessed using a Computer Assisted SpermASA) system (Sperm Vision: Minitb, Tiefen-any) with a heated automated stage and

d (37 C) 20 m deep four-chamber slides (Leja,nep, The Netherlands). For analyses, a 2.6 lsample was used to ll the chamber, anda were examined at 200 magnication via aptor (U-PMTVC, Olympus, Hamburg, Germany)ra with a resolution of 648 484 pixels (Pulnix, JAI A/S, Glostrup, Denmark). The analysis wasusing SpermVision software (Version 3.5.6,ermany) which analyzed a minimum of 400ample at a rate of 60 Hz per eld on 12 succes-n the central part of the chamber. Sperm headsated as having a size range of 1485 m2. The

assessed were the percentages of motile (totalnd progressively motile (progressive motility), within the group of progressively motile

average straight line velocity (VSL), distanceline (DSL), curved line velocity (VCL), average

met oorienthan 8 m15 mif it m15 m5 mto 20(6) raALH g

2.6. A

Spa spe(PI) adark.dry iat 10200 RFCATokytationBP54(red)of allintacand exhibbranered dama

2.7. A

Thmatintime acid Assaywith lyzedBredalasera 530670 n10,00charausingLos Afragmend samppercement2002he following three denitions; (1) average head change (AOC) higher than 7 and BCF greater

(2) DSL greater than 3.5 m, VSL greater than DSL greater than 15 m, (3) VAP greater thanotile cell was considered as progressively motilee of the following criteria; (1) DSL greater thanBCF greater than 40 Hz and radius greater thanL greater than 20 m/s, (4) DCL greater or equal) STR greater than 0.3 and LIN greater than 0.2,reater than 10 m, (7) BCF greater than 30 and

than 0.85.

ent of sperm viability

iability was evaluated by incubating 100 l ofmple with 1 l of 2.4 mM propidium iodidel of 200 M SYBR14 for 5 min at 37 C in theincubation, smears were prepared, allowed toht protected chamber and examined directlyagnication (oil immersion). A minimum ofas evaluated for each sample using a BH2-pus epi-uorescence microscope (Olympus,n) equipped with a Mercury lamp and exci-sion lters (UG-1/L420, BP490/AFC + O515 and0) to visualize the SYBR14 (green) and PIls. SYBR14 penetrates and stains the DNAand accumulates preferentially in those withbranes, whereas propidium iodide only entersthe DNA of membrane damaged cells. Cellsgreen uorescence were considered mem-t, while those that demonstrated orange toence were considered non-viable, membrane

ent of sperm chromatin structure stability

portion of spermatozoa with unstable chro-liquots snap frozen at each semen storage

was examined by testing for resistance toration using the Sperm Chromatin Structure

A) (Evenson and Jost, 2000). Samples stainedetachromatic dye, acridine orange, were ana-FACS-Calibur ow cytometer (BD Biosciences,etherlands) equipped with a 488 nm argon ion

W). Fluorescence signals were collected usingand pass lter for green uorescence and ag pass lter for red uorescence. A total ofrmatozoa, based on forward and side scattertics, were recorded per sample and analyzedxpress software (version 3: De Novo Software,, CA, USA). A semen sample with a known DNAion index (DFI) was used at the beginning andry ow cytometry session, and every twentyring measurements, as a quality control. The

of cells with a moderate or high DNA frag-index (% DFI) was determined (Larson et al.,

158 P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162

Table 1Effect of centrifugation and d-penicillamine supplementation on motility of ejaculated stallion sperm during storage at 5 C (n = 8 stallion; 4 ejaculateseach). Different superscript letters indicate signicant differences between time points within a given treatment (P < 0.05).

Penicillamine,mM

Storageduration, h

Totalmotility, %

Progressivemotility, %

VCL, m/s

Centrifuged 24.5a,b

58.2a

64.9b,c

45.9c

54.0 62.4 64.2 52.3

Notcentrifuged

64.5a 44.5 23.7a 0.32 0.11a 2.98 1.21a,b 28.7 11.6a

52.1a,b 42.2 23.0a 0.33 0.12a 3.22 1.02a 26.8 9.7a 49.6a,b 30.9 18.3b 0.23 0.10a,b* 2.44 1.18a,b 18.3 7.8b 23.4b 27.1 13.3b 0.19 0.08b 2.23 0.57b 17.2 5.2b 77.1a 44.5 32.3a 0.33 0.21 2.96 1.86 29.4 16.1a 43.4a 38.4 14.2a 0.28 0.04 2.86 0.66 21.3 5.5b 60.3a,b 36.8 21.9b 0.28 0.15* 3.02 1.09 22.9 11.4a,b 10.5b 28.7 5.0b 0.22 0.04 2.37 0.30 17.4 3.2b

VCL, curviline aight line velocity; BCF, beat cross frequency; LIN, linearity.* A signica meters, independent of treatment (P < 0.05).

2.8. Statisti

Data ansis System NC, USA). Pusing the Sessary, dattransformeence of censemen quaanalysis of vGLM). Pair-dents t-tesmeans stato be signi

3. Results

3.1. Effect oejaculated a

Reducinsemen by ceffect on alexcept BCF ative effectsperm (P < ameliorate.

On the odetrimentasive motilitime 0, all ephologicallsperm popsive motilitwith regarddroplets wpared to ej

0

10

20

30

40

50

60

70

1921449648

% M

ole

Storage Time (hrs)

Control

Penicillamin e

40

50

60

70

80

90

100

% L

ive

Cells

Control

*P=0.10

*

* 0 48 56.5 16.8a 32.6 14.4a 168.6 96 47.0 26.0b 24.3 15.1b 168.2

144 33.8 15.0c 15.1 7.7c 161.0 192 22.5 25.1d 9.6 14.8d 162.1

0.5 48 55.8 27.9a 33.6 22.3a 165.9 96 46.6 26.6b 24.2 20.9b 166.5

144 33.7 23.0c 16.2 15.7c 167.8 192 23.0 24.4d 10.5 14.7d 155.9

0 48 22.9 25.1a* 9.4 15.2a 119.1

96 14.2 30.2b 5.3 21.3,b 116.4 144 9.1 21.2c 3.0 12.3b 92.1 192 6.9 20.6d 2.2 12.1c 80.6

0.5 48 21.4 29.6a* 8.6 24.1a 115.9 96 16.0 20.0a 6.1 11.2a 104.8

144 9.7 26.3b 3.4 16.7b 105.8 192 6.7 19.1c 2.1 11.4c 84.0

ar velocity; ALH, average amplitude of lateral head displacement; VSL, strnt effect of 0.5 mM. Centrifugation had a signicant inuence on all para

cal analysis

alysis was performed using Statistical Analy-software (SAS Version 9.4: SAS Inst., Cary,arameters were tested for normal distributionhapiro Wilk test (PROC UNIVARIATE). If nec-a were square root or logarithmically (log10)d to achieve equivalence of variances. The inu-trifugation, penicillamine and storage time onlity parameters was tested by multifactorialariance (ANOVA) for repeated measures (PROCwise comparisons were performed using Stu-t (PROC UNIVARIATE). All data are presented asndard deviations. Differences were consideredcant when P < 0.05.

f d-penicillamine on motility and viability of

A

Bnd epididymal sperm

g the amount of seminal plasma in ejaculatedentrifugation and re-suspension had a positivel motility parameters (P < 0.05; Tables 1 and 3)(P = 0.056) and viability. Storage time had a neg-

on total and progressive motility of ejaculated0.05), which d-penicillamine was not able to

ther hand, d-penicillamine had an unexpectedl effect on total motility (Fig. 1A) and progres-ty (Table 2) of stored epididymal sperm. Atpididymal samples had greater than 60% mor-y normal sperm and were characterized by aulation exhibiting 47% total and 25% progres-y. Viability was high at approximately 91% and,

to morphology, high numbers of cytoplasmicere seen in epididymal sperm (33.4%) com-aculated sperm (10.6%). After 48 h of storage,

0

10

20

30

Fig. 1. Motilitdata of epididysignicant diff

penicillamilower total ples (39.9 was appareeffect of pesperm kine96 h of storeffect on thVSL, m/s LIN VSL/VCL ALH, m BCF, Hz

68.6 11.7a 0.40 0.04a* 4.03 0.43 34.9 3.8a62.9 20.4b 0.37 0.12b 4.18 1.14 32.3 13.0b56.3 18.9c 0.34 0.10c 4.05 1.42 30.1 9.7c55.9 23.4c 0.34 0.14c 4.03 1.10 30.1 10.5c70.2 21.3a 0.42 0.09a* 3.96 1.14a 35.9 10.9a64.8 30.0a 0.39 0.20b 4.17 1.53b 33.1 14.3b60.3 22.9b 0.36 0.11c 4.15 1.28a,b 31.8 13.0b,c54.5 20.8b 0.32 0.10c 3.80 1.10a,b 29.2 10.6c1921449648Storage Time (hrs)

Penicillamine

y (A) and viability (B, % live cells stained PIve/SYBR14+ve)mal spermatozoa held at 5 C (n = 7). An asterisk indicates aerence between treatments at a given time point (P < 0.05).

ne-treated samples showed approximately 10%motility values (29.5 14.5%) than control sam-

19.2%) (Table 2). A similar depressive effectnt at all subsequent time points. An adversenicillamine was also evident on the epididymalmatic parameters, VCL and ALH, after 48 h andage (Table 2). However, penicillamine had noe percentage of membrane intact epididymal

P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162 159

Table 2Effect of d-penicillamine supplementation on stallion epididymal sperm motility during storage at 5 C (n = 7). Different superscript letters indicatesignicant differences between time points within a treatment (P < 0.05).

Penicillamine,mM

Storageduration, h

Totalmotility, %

Progressivemotility, %

VCL, m/s VSL, m/s LIN VSL/VCL ALH, m BCF, Hz

0 60.7 58.1 48.2 37.3

0.5 54.6 50.1 56.0 31.6

VCL, curvilinea aight lin* A signica

spermatozoover time in

3.2. Effect oindex of ejac

Contrarythe presenpenicillamimeasured btion in % Dpenicillamifact of sper

This is bahave a highagglutinatevidual lesswill be biasbetter chro

4. Discussi

In this tive effect and viabilitpreservatioprior to storthe motilityis in agreemAlghamdi epositive effuid stored swas not repthe conditioet al. (2012aBiggers, W1 mg/ml poof penicillastudy was a practical extender cponents. Thyolk proteinsufcient Rneed for fu

; Samades arvitin isture (in theition mming aarawesecona) stu, whee that ice). C

lipid enzymree pea lowred (Hed chcial e.ti-oxi

ts in sg ROSidase)tion oVitamBall, 2; Ball eives, ehia etlk baseoveme48 39.9 19.2a* 21.3 12.3a* 166.2 33.7a*96 31.3 16.5a* 15.8 12.5a* 170.4 16.1a*

144 14.4 11.5b 6.4 6.4b 146.4 65.3a.b192 12.3 11.0b* 4.0 4.6b 109.8 76.4b

48 29.5 14.6a* 15.9 8.6a* 146.0 66.9*96 14.4 7.4b* 6.7 3.9b* 140.3 62.6*

144 5.6 5.9c 2.0 2.3c 152.8 32.1 192 4.0 5.4c* 1.3 2.5c 83.6 79.5

r velocity; ALH, average amplitude of lateral head displacement; VSL, strnt effect of supplementation with 0.5 mM penicillamine.

a (Fig. 1B), with viability decreasing similarly its presence or absence (Fig. 1B).

f d-penicillamine on DNA fragmentationulated and epididymal sperm

to expectations, none of time in storage,ce of seminal plasma, or the addition of d-ne inuenced sperm chromatin integrity asy SCSA (Table 3). A slight, but signicant reduc-FI for non-centrifuged semen without addedne after 144 h and 192 h was most likely an arte-m agglutination.sed on the assumption that agglutinated spermer incidence or likelihood of damage; since thed cells are not gated in the same region as indi-

damaged cells, the associated SCSA analysised towards individual cells assumed to have

matin integrity and stability.

on

study, we failed to demonstrate a protec-of d-penicillamine on the decline in motilityy of ejaculated stallion sperm during liquid-n at 5 C. A reduction in seminal plasma contentage, i.e. by centrifugation, did however improve

and viability of ejaculated spermatozoa, whichent with previous reports (Jasko et al., 1991;

t al., 2002; Picket et al., 1975). However, theect of d-penicillamine on the longevity of liq-tallion sperm reported by Aitken et al. (2012a)licated. This difference in result may relate to

2002peptiphosin nayolk transwhel(Sam

A (2012ature(sincpractbraneMostto thfore, occurinducbeneused

AneffecenginperoxoxidaROS [als] (2006addit(Coccto miimprns under which sperm were preserved. Aitken) used chemically dened conditions (modiedhitten and Whittingham medium containinglyvinyl alcohol: BWW) to study the direct effectmine and other thiols on spermatozoa. Ourdesigned to translate the earlier ndings intosetting by incorporating a commercial semenontaining skimmed milk and egg yolk com-e addition of casein and other milk and eggs to the extender may however have provided

OS scavenging phosphopeptides to obviate therther addition of anti-oxidants (Foote et al.,

no positiveet al., 2001;et al., 2005;

Unexpecpenicillamiepididymald-Penicillamcan readilycellularly, itin thioredNADPH oxiperoxidase 14.1a 0.36 0.05 3.93 0.75* 33.7 5.0 11.6a 0.33 0.05 4.07 0.38* 33.0 4.8 22.0a.b 0.28 0.12 3.54 1.58 26.9 12.0 26.2b.c 0.24 0.16 2.85 1.98 22.1 15.2 24.5 0.32 0.15a,b 3.38 1.58* 31.0 14.1 22.8 0.30 0.14a 3.38 1.55* 28.4 13.1 6.9 0.38 0.13c 4.20 0.86 31.1 4.7 29.6 0.22 0.20a.b 2.21 2.10 19.3 18.3e velocity; BCF, beat cross frequency; LIN, linearity.

raweera et al., 2011). In this respect, phospho-e also effective metal chelators, and egg yolk

one of the most phosphorylated proteins foundSamaraweera et al., 2011). Therefore, the egg

semen extender is likely to have bound anyetals present preventing oxidation, and over-ny potential benets of added d-penicillamineera et al., 2011; Lu and Baker, 1986)d major difference was that, in Aitken et al.sdy, the semen was stored at room temper-reas in our study semen was stored at 5 Cis the most common storage temperature inhilling spermatozoa results in lowered mem-uidity and associated membrane alterations.atic processes are slowed by a factor of two

r 10 C reduction in temperature and, there-ered rate of ROS propagation would haveammerstedt et al., 1990). Therefore, chilling-anges may have masked or prevented anyffects of d-penicillamine at the concentration

dants that have been reported to have benecialemen extenders act either by directly scav-

(catalase, superoxide dismutase, glutathione or by preventing the propagation of lipid per-n the cell membrane by blocking the effect ofin E, C, B complexes, polyphenols, trace miner-008; Fernndez-Santos et al., 2009; Pagl et al.,t al., 2001; Agarwal et al., 2014). Although some.g. superoxide dismutase, seem to be benecial

al., 2011), the majority of anti-oxidants addedd semen extenders have yielded, at best, minornts in motility and membrane integrity and had impact on preservation of DNA integrity (Ball Aurich et al., 1997; Pagl et al., 2006; Kankofer

Baumber et al., 2005).tedly, in our study the presence of d-ne had a negative effect on the motility of

sperm at all time-points during storage at 5 C.ine is a small-molecular weight thiol that

pass through the plasma membrane. Intra-s actions are complex and may involve changesoxin or glutathione availability, increaseddase activity, reduced NO availability, impaired

activity, increased mitochondrial electron

160 P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162

Table 3Effect of d-penicillamine supplementation or seminal plasma removal on the percentages of ejaculated (n = 8 stallions; 4 ejaculates each) and epididymal(n = 7 stallions) sperm recorded as viable PIve/SYBR14+ve) or as having damaged DNA (DNA fragmentation index: DFI) during storage at 5 C.

Penicillamine, mM Storage duration, h % Live cells % DFI

ed

Centrifuged ab

c* .4da

b

c* .3d

Not centrifu .8a

.3b

.7c*

.3d

.2a

.7b

.6c*

.7d

n/a, not applic a.

leakage, meelectrophiledifferent tohigher numthe mid anentering tha nal conenabled bybridges (Soenging procell permeametals) maprocess antion of momajor modexplain whplasma (i.e.d-penicillamgone the nhave also bethe accesso(Sostaric etcomponentof the sperdered themd-penicillampenicillamitilization mmitochondrmotility of washing sthave remoseminal plastate susceOn the othaffect viabialthough vaprolonged samples.

e alsoooled sturatiod of 8C was nicillame perc

methles froh the normae curreles wen. On

chroted toged eqeyers

nclusEjaculat

0 48 75 3.896 70 5.3

144 62 8.1192 53 11

0.5 48 76 6.996 71 5.8

144 66 9.0192 56 13

ged 0 48 60 1496 48 17

144 38 16192 26 18

0.5 48 61 1596 51 14

144 40 16192 26 14

able as epididymal sperm was not centrifuged as it has no seminal plasm

tal chelation, and the elimination of cytotoxics (Aitken et al., 2012a). Epididymal sperm are

ejaculated sperm, having reduced motility andbers of cytoplasmic droplets associated withd principle piece of the spermatozoa. Sperme caput epididymis are immotile and requiredensation maturational step, which is in part

oxidation of free thiol groups into disuldestaric et al., 2008). Hypothetically the scav-perties of d-penicillamine (either by offeringble free thiol groups or by chelating transfer

y inhibit this intrinsic disulde bridge formationd consequently prevent or delay the acquisi-tility (Wroblewski et al., 2003). If this is thee of action of d-penicillamine, it would largelyy spermatozoa previously exposed to seminal

ejaculated) were not affected by the addition ofine. In addition, ejaculated sperm have under-al stages of maturation before ejaculation anden exposed to various components secreted byry sex glands, which bind to the sperm surface

Wing cdenaperioat 5

d-pein thSCSAsampwhicof abin thsampsemespermrepordamaand M

5. Co al., 2008). The presence of bound cell surfaces is known to affect reactivity and interactionm with other compounds, and may have ren-

less susceptible to any detrimental effects ofine. If so, it may further explain why adding

ne, hypotaurine and epinephrine to in vitro fer-edium was previously reported to reduce spermial membrane potential and compromise thebull spermatozoa (Gonc alves et al., 2014); theeps used to prepare spermatozoa for IVF mayved the protective components derived fromsma, thereby returning the spermatozoa to aptible to the negative effects of penicillamine.er hand, d-penicillamine did not signicantlylity in either epididymal or ejaculated sperm,lues for d-penicillamine-treated samples afterstorage were somewhat lower than untreated

The addiin a milk/etively nor nviability anwere storenegatively presumablyerties of spplasma.

Conict of

None.

Acknowled

The autassistance wEpididymal Ejaculated Epididymal

n/a 9.71 2.95* n/an/a 9.24 2.48 n/an/a 10.33 3.62 n/an/a 8.72 2.23 n/an/a 10.81 3.53* n/an/a 9.94 3.08 n/an/a 9.65 2.87 n/an/a 10.09 2.98 n/a90 5.4a 11.56 3.42a 5.8 2.883 5.7b 10.13 3.27a,b 6.1 1.769 12.3c 9.49 2.83b 6.7 3.057 14.9d 9.78 3.45b 5.5 1.690 4.9a 10.87 3.68 6.3 2.081 2.4b 9.45 2.94 6.1 1.157 16.4c 9.13 2.87 6.1 1.943 11.8d 9.06 2.65 5.7 3.0

found no increase in chromatin fragility dur-torage. The susceptibility of sperm DNA to acidn (assessed with the SCSA procedure) over a

days in either ejaculated or epididymal spermno different in either the presence or absence of

ine. Signicant storage-time related increasesentage of sperm with damaged DNA using theod were previously reported when applied tom stallions known to be sub-fertile, and frominitial ejaculates contained a high proportionl spermatozoa (Love et al., 2002). By contrast,nt study the ejaculated and epididymal spermre obtained from stallions with good quality

the other hand, another technique for revealingmatin aberrations, the COMET assay, has been

detect signicant increases in numbers of DNAuine sperm after 48 h of chilled storage (Linfor, 2002).

iontion of d-penicillamine to equine semen dilutedgg yolk based semen extender neither posi-egatively affected the maintenance of motility,d DNA integrity when ejaculated spermatozoad at 5 C. On the other hand, d-penicillamineinuenced the motility of epididymal sperm,

reecting differences in cell (membrane) prop-erm that have not been exposed to seminal

interest

gements

hors would like to thank Arend Rijneveld forith semen assessment.

P.T. Brogan et al. / Animal Reproduction Science 159 (2015) 155162 161

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Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion...1 Introduction2 Materials and methods2.1 Chemicals2.2 Animals and study design2.3 Semen collection and initial processing2.4 Epididymal sperm collection and processing2.5 Computer-assisted semen analysis2.6 Assessment of sperm viability2.7 Assessment of sperm chromatin structure stability2.8 Statistical analysis

3 Results3.1 Effect of d-penicillamine on motility and viability of ejaculated and epididymal sperm3.2 Effect of d-penicillamine on DNA fragmentation index of ejaculated and epididymal sperm

4 Discussion5 ConclusionConflict of interestAcknowledgementsReferences