Influence of glucose and fructose in the extender during long-term storage of chilled canine semen

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1 Theriogenology 62 (2004) Influence of glucose nd fructose in the extender during long-term storge of chilled cnine semen Suppwiwt Ponglowhpn, Birgitt Essén-Gustvsson, Cthrin Linde Forserg,* Deprtment of Ostetrics nd Gynecology, Fculty of Veterinry Medicine, Swedish University of Agriculturl Sciences (SLU), P.O. Box 7039, SE Uppsl, Sweden Unit for Comprtive Physiology nd Medicine, Deprtment of Lrge Animl Clinicl Sciences, Swedish University of Agriculturl Sciences, P.O. Box 7018, Uppsl, Sweden Received 27 June 2003; received in revised form 11 Decemer 2003; ccepted 22 Ferury 2004 Astrct The use of chilled, extended semen in dog reeding is ecoming incresingly populr s preprtion nd trnsporttion is less expensive nd regultions re often less complicted thn for frozen semen. Sugr is one of the min constituents in semen extenders, nd glucose nd fructose re metolized in seprte pthwys y freshly ejculted dog sperm. In this study, glucose, fructose or n equl mixture of oth were used in n egg-yolk tris (EYT) extender t two different concentrtions (10 nd 70 mm). EYT extender without sugr supplementtion, providing only the glucose (3 4 mm) originting from the egg-yolk, served s control. The longevity of the chilled semen t 5 8C ws 23 dys: the qulity of physicl nd functionl chrcteristics decresing with time. Glucose nd fructose hd strong influence on motility nd movement ptterns of chilled cnine semen. The eneficil effect of 70 mm sugr concentrtions compred to 10 mm nd the control ws pronounced, nd mintined sperm motility 70% for 8 dys of storge, compred to for 4 dys in the control extender. Fructose mintined higher sperm motility thn did glucose nd the mixture. VAP vlues were higher in sugr-supplemented extenders (P < 0:05). Neither type nor concentrtion of the two sugrs influenced sperm plsm memrne, crosome integrity or the crosome rection following ionophore chllenge (ARIC). Sugr consumption y dog sperm vried etween the different periods of storge nd with sugr concentrtions provided in the extenders. Glucose consumption y dog sperm ws greter thn fructose consumption when oth sugrs were present in equl mounts, indicting tht dog sperm used glucose in preference to fructose. In conclusion, the mjor influence of the two sugrs on chilled semen ws to support motility. EYT extender supplemented with fructose t * Corresponding uthor. Tel.: þ ; fx: þ E-mil ddress: cthrin.linde-forserg@og.slu.se (C. Linde Forserg) X/$ see front mtter # 2004 Elsevier Inc. All rights reserved. doi: /j.theriogenology

2 S. Ponglowhpn et l. / Theriogenology 62 (2004) concentrtion of 70 mm ws found to e the est of the tested extenders for long-term preservtion of chilled cnine semen. # 2004 Elsevier Inc. All rights reserved. Keywords: Glucose; Fructose; Sugr concentrtions; Chilled semen; Dog; CASA 1. Introduction Frozen cnine semen cn e stored prcticlly indefinitely, nd the techniques nd extenders for dog sperm cryopreservtion developed in recent yers hve resulted in postthw motility of up to 70% [1,2] nd whelping rtes of up to 85% [3 5]. However, pregnncy rtes nd whelping rtes re generlly lower with frozen semen thn with chilled, extended semen, when the sme optiml methods re used for AI [6,7]. It is techniclly esier to chill thn to freeze semen, nd the shipment of chilled semen is less expensive nd regultions for import nd export re often less complicted thn for frozen semen. Consequently, the use nd interntionl shipment of chilled semen in dog reeding is ecoming incresingly more common. One disdvntge with chilled semen is the reltively short survivl time, which is thought to necessitte use within few dys fter collection [8 11]. Therefore, the trnsporttion of chilled semen to its destintion requires creful plnning so tht it oth remins fertile nd is ville on the most suitle insemintion dy for the itch. To preserve viility nd fertilizing cpcity of sperm cells, semen to e stored or shipped needs to e extended nd chilled. Mny extenders for chilled dog semen hve een evluted, nd egg-yolk tris (YET) extenders pper superior to the other extenders tested in vitro nd in vivo [11 19]. Mmmlin spermtozo require exogenous sustrtes for vriety of functions, e.g., to preserve intrcellulr energy reserves, cell components nd most notly to support motility [20]. They cn otin energy through mitochondril oxidtive phosphoryltion nd glycolysis y the consumption of glycolysle sugrs, such s glucose, fructose, mnnose, nd mltose [21]. Fructose is thought to e mjor energy source for ejculted spermtozo [22], nd together with glucose is found in seminl plsm in mny mmmlin species, ut not in dogs; nevertheless, dog sperm cn utilize these two sugrs. In mny species, glucose nd fructose hve een investigted for the different effects on gmetes in terms of metolizle energy nd fertility potentil, nd the eneficil effects vry etween species [23]. The effects of these two sugrs on the metolism of freshly ejculted spermtozo hs een studied in dogs, nd there is evidence tht dog sperm metolize glucose nd fructose using seprte pthwys, resulting in seprte systems of energy mngement s indicted y their different roles in glycogen metolism [24], motility ptterns [25], hexose metolism [26], nd glycogen deposition [27]. Glucose nd fructose re two of the most commonly used sugrs for cnine semen extenders; however, the concentrtion of these sugrs in the extenders for chilled cnine semen vries mrkedly from 5 mm to out 120 mm [8,13,14,28]. In order to chieve the most efficcious use of chilled semen, it is importnt to study the influence of different sugrs nd their concentrtions on chilled dog sperm. In the present study, the ims were: firstly, to determine which of the two sugrs, or their comintion, nd which sugr concentrtion (control: 10 or 70 mm) ws most eneficil

3 1500 S. Ponglowhpn et l. / Theriogenology 62 (2004) for long-term preservtion of chilled cnine semen. Secondly, to investigte the effects of n EYT extender contining glucose nd/or fructose on cnine spermtozo s evluted y motility, plsm memrne nd crosome integrity, crosome rection following ionophore chllenge, ph chnge nd sugr consumption during long-term storge t 5 8C. 2. Mterils nd methods 2.1. Animls Five privtely owned, cliniclly helthy stud dogs etween 2.5 nd 8 yers old were used in this study: one Appenzell Mountin Dog, one Bvrin Schweisshund, one Brird, nd two Golden Retrievers. For the dogs to e included in the study, the percentge of motile nd morphologiclly norml spermtozo of the fresh semen hd to e 70% Extenders The extenders were used to dilute the smples of semen to e chilled. Two different concentrtions (10 nd 70 mm) of sugrs in the extenders were tested. Seven EYT extenders were prepred in single tch nd stored frozen to void vrition mong tches (Tle 1). An extender contining no sugr supplementtion served s control. Ten egg-yolks from hen s eggs were initilly smpled nd nlyzed for differences in glucose nd fructose content. Prior to sugr mesurement, ech egg-yolk ws mixed in distilled wter to the sme finl concentrtion s in the extenders, i.e., 20% (v:v). To mke the extenders, fresh egg-yolks were pooled nd mixed to reduce the iologicl vrition found in the eggs. Prticles of size similr to tht of sperm heds re confounding fctor with Computer Assisted Sperm Anlysis (CASA) nd results in over-estimted dt of immotile sperm, therefore, ll extenders were clrified from prticles y centrifugtion t 3,310 g for 20 min t room-temperture. The osmolrity nd ph of the extenders were determined (Tle 1). A smple from ech extender ws lso sumitted for glucose nd fructose nlysis prior to use. Tle 1 Compositions of chilled semen extenders used in the experiments Ingredients I II III IV V VI VII Tris (g) Citric cid (g) N-enzylpenicillin (g) Streptomycin sulfte (g) Egg-yolk (ml) D-Glucose (g) D-Fructose (g) Distilled wter (ml) to 100 to 100 to 100 to 100 to 100 to 100 to 100 Osmolrity (mosm) ph

4 2.3. Experimentl design Three different series of experiments with the seven different extenders (Tle 1) were performed to evlute the most eneficil sugr type nd concentrtion for chilled cnine semen stored t 5 8C. Experiment Series A included extenders I III. Experiment Series B included extenders I, IV, nd V. Experiment Series C included extenders I, VI, nd VII. All series of experiments (A C) were repeted in triplicte. The semen smples were collected nd pooled from three of the dogs on ech of the nine occsions Semen collection nd processing The sperm-rich frction of the ejcultes ws collected y digitl mnipultion into pre-wrmed clirted polypropylene vil [16]. The semen smples were pooled to llow for sufficient numer of sperm nd to reduce n individul vrition mong the evluted smples [1,3,29]. The sperm concentrtion ws mesured with clirted photocolorimeter (SpermCue, Minitü, Tiefench, Germny). The totl numer of spermtozo in the pooled semen rnged etween nd 2: cells. The percentge of motility in the pooled semen, ssessed y Computer Assisted Sperm Anlysis (Strömerg-Mik Cell Motion Anlyzer SM-CMA, MTM Medicl Technologies, Montreux, Switzerlnd), ws 88:9 4:2 (men S:D: rnge: ). The pooled semen ws divided into three equl liquots nd plced in three screw cp closed sterile plstic tues. The three liquots were then centrifuged t 700 g for 8 min nd the superntnts were removed, collected, frozen in Liquid N 2 nd stored t 80 8C for glucose nd fructose nlysis. The resultnt sperm pellets were re-suspended in the different extenders (Tle 1) to give finl sperm concentrtion of cells/ml. To void cooling too rpidly nd to reduce the effects of cold shock to the spermtozo, the extended semen smples were plced in roomtemperture cooler, which reched temperture of 5 8C within 45 min. The cooling rte in the cooler ws 0.5 8C/min. Susequently, ll smples were stored t 5 8C in refrigertor throughout the whole period of study, i.e., until sperm motility ws down to 0%. Chilled semen smples were evluted dily during the first 4 dys nd every second dy from Dy 6 to Dy 14, nd then on Dy 17, Dy 20, nd Dy Semen evlution S. Ponglowhpn et l. / Theriogenology 62 (2004) A 300-ml liquot of ech chilled semen smple ws tken on ech experimentl dy nd ws re-wrmed to room-temperture for 15 min efore evlution Assessment of sperm motility nd movement ptterns Spermtozol motility ws evluted using CASA nd t 37 8C. For ech smple, t lest 200 sperm cells from four rndomly selected fields were evluted. The following prmeters were mesured: the percentge of totl motility (TM%); the percentge of liner motile spermtozo (progressive motility; PM%); verge pth velocity (VAP, mm/s); stright line velocity (VSL, mm/s); nd curviliner velocity (VCL, mm/s). PM% ws clculted from mong the motile spermtozo nd the velocities were clculted from

5 1502 S. Ponglowhpn et l. / Theriogenology 62 (2004) spermtozo showing liner motility. The settings for the motion nlyzer were s follows: frmes per second, 32; minimum numer of frmes, 16; cquisition time, 20 ms (50 Hz); minimum nd mximum re of ojects, 35 nd 350 pixels; velocity clss width, 5 mm/s; velocity limit for immotile ojects, 10 mm/s; velocity limit for locl motile ojects, 25 mm/s; mximum rdius for circles, 30 mm; minimum nd mximum re for the immotile ojects, 20 nd 100 pixels; nd depth of mesuring chmer, 10 mm Assessment of plsm memrne integrity Sperm plsm memrne ws ssessed with dul fluorescent stining technique descried y Grner et l. [30] nd Hrrison nd Vickers [31], nd modified y Rot et l. [17], using 6-croxyfluorescein dicette (C-FDA; Cliochem, LKemi, Stockholm, Sweden) nd propidium iodide (PI; Sigm Chemicl Compny Ltd, St. Louis, MO, USA). A stining medium for the fluorescent proes ws prepred within 1 h efore use. For ech smple stined with C-FDA/PI, totl of 200 spermtozo were evluted nd clssified into two ctegories (intct or dmged plsm memrne) [17]. In the results, dt on dmged plsm memrne were presented s percentge Assessment of crosoml integrity After dul fluorescent stining with fluorescein isothiocynte conjugted penut gglutinin (FITC-PNA, Sigm) nd propidium iodide, crosoml integrity ws chrcterized with protocol modified y Axnér et l. [32] from the procedure for FITC-PNA stining of ct spermtozo [33], nd the procedure descried y Cheng et l. [34], for FITC-PNA stining of stllion spermtozo. Two hundred spermtozo were ssessed in ech smer nd ctegorized ccording to their FITC-PNA ptterns [34]. Spermtozo with rected crosome or crosome loss were presented s percentge Induction of the crosome rection y clcium ionophore chllenge Clcium ionophore-induced crosome rection ws oserved s descried y Hewitt nd Englnd [35] nd Brewis et l. [36], slightly modified. The stock solution of clcium ionophore A23187 (C-7522, Sigm) ws prepred in dimethyl sulfoxide (DMSO) (Sigm) t 5 mm concentrtion. This ws divided into 10 ml liquots nd plced into 0.5 ml microcentrifuge tues nd further diluted in 90 ml phosphte-uffered sline (PBS) nd stored frozen prior to use. A control solution ws prepred with 10 ml of DMSO diluted with 90 ml of PBS, plced in 0.5-mL microcentrifuge tue nd stored frozen. The chilled semen smple ws djusted to finl sperm concentrtion of cells/ml into cnine cpcittion medium (CCM) [37] nd incuted for 2 h t 37 8C. Following incution n liquot (10 ml) of the smple ws oserved under light microscope to confirm tht the spermtozo displyed hyperctivted motility nd therefore, were cpcitted. A 20-mL liquot of cpcitted spermtozo ws further incuted with 20-mL ionophore chllenge solution (10 mm A23187) or 20-mL control solution for 1 h t 37 8C. After incution, the cultures were evluted for the presence of crosome-rected spermtozo nd stined with FITC-PNA/PI s descried ove. The percentge of spontneous

6 rection rtes (control) nd of A23187 induced rection rtes were then determined s descried y Cummins et l. [38]. The difference etween the two vlues ws considered the percentge of sperm in the popultion tht ws cple of responding to ionophore chllenge, which ws termed the crosome rection following ionophore chllenge (ARIC). In cses with low response, the oserved incidence in ionophore-stimulted suspensions ws less thn oserved in the control. In these cses, ARIC ws negtive nd, ws therefore, considered essentilly zero ph mesurement The ph vlues of the extenders efore use (Tle 1) nd of the chilled semen smples on Dy 23 were mesured y ph meter (PerpHect 1 METER, model 330, Orion Reserch, Inc., MA, USA) Glucose nd fructose mesurement A preliminry study indicted tht the concentrtion of spermtozo in the incution medi ffected the mount of sugr utiliztion, therefore, stndrdized sperm concentrtion of cells/ml ws used in this study. On semen ssessment, the chnges in glucose nd fructose concentrtions in the extenders during cool storge were mesured fluorometriclly (Fluorometer FL 600 1, Bio-Tek Instruments, Inc., VT, USA), with modified method y Lowry nd Pssonneu [39]. The sme method ws used in the preliminry studies on sugr content in egg-yolk. All enzyme components used in this study (glucose-6-phosphte dehydrogense, hexokinse, nd phosphoglucose-isomerse) were purchsed from Roche Dignostics Corportion (IN, USA). The sugr concentrtions were expressed s mm. Both sugrs were mesured y smpling 50 ml of the superntnt from ech smple, plcing it into 1 ml microcentrifuge tues tht were lowered into liquid N 2 for 30 s nd stored t 80 8C prior to sugr mesurements. Sugr consumption y spermtozo over time ws clculted nd compred Sttisticl nlyses S. Ponglowhpn et l. / Theriogenology 62 (2004) Dt were sttisticlly nlyzed using the SAS (Ver. 8e, SAS Institute Inc., Cry, NC, USA). Anlysis of vrince (PROC MIXED) ws pplied to the dt. Except for ph, the sttisticl model for ll prmeters included: the fixed effects of sugr types (glucose, fructose, nd mixture of oth); sugr concentrtions (control: 10 nd 70 mm); nd time (Period I: Dys 1 þ 2 þ 3; Period II: Dys 4 þ 6 þ 8; Period III: Dys 10 þ 12 þ 14; nd Period IV: Dys 17 þ 20 þ 23). Dys were nested within periods nd the interction etween sugr types, sugr concentrtions nd periods. The rndom effect of replictions, nested within sugrs, ws lso included in the model. For the nlysis of ph vlues (on Dy 23), the sttisticl model included the fixed effects of sugrs, dded concentrtions nd their interction, s well s the rndom effect of replictions, nested within sugrs. In the results, men stndrd devitions were presented, however, pirwise t-tests were performed to compre lest-squres mens. Chnges in consecutive periods were compred to Period I. A vlue of P < 0:05 ws considered sttisticlly significnt.

7 1504 S. Ponglowhpn et l. / Theriogenology 62 (2004) Tle 2 Glucose nd fructose concentrtions (mm) in the semen extenders Sugr concentrtions Semen extenders I II III IV V VI VII Glucose Prepred concentrtion Mesured concentrtion Fructose Prepred concentrtion Mesured concentrtion Extender I contining no dditionl sugrs served s control. Extenders II nd III contined 10 nd 70 mm glucose, respectively. Extenders IV nd V contined 10 nd 70 mm fructose, respectively. Extenders VI nd VII contined equl comintions of glucose nd fructose t finl concentrtions of 10 nd 70 mm, respectively. 3. Results 3.1. Glucose nd fructose contents in cnine seminl plsm nd in hen s egg-yolk Glucose nd fructose concentrtions in pooled seminl plsm could not e detected. The men glucose concentrtion (mm) in the 20% egg-yolk/distilled wter mixture ws 3:5 0:5 (rnge: ) nd for fructose 0:4 0:2 (rnge: 0 0.8) Glucose nd fructose contents in chilled semen extenders Prior to use, ll lortory-prepred semen extenders were sujected to glucose nd fructose nlyses (Tle 2) Sperm motility nd movement ptterns The men TM% in freshly pooled semen ws 88:9 4:2 nd PM% ws 69:6 12:6 (n ¼ 9). TM% decresed with storge time in ll tretments (Tle 3, Fig. 1). There were no differences in TM% etween sugrs t the sme concentrtion nd in the sme period. When pooled cross time, however, type nd concentrtion of sugr influenced TM% giving 55:9 28:7 with fructose, 53:8 28:9 with the mixture nd 53:7 29:1 with glucose. The ddition of fructose to EYT extenders provided significntly etter TM% thn the other two extenders (P < 0:05). The overll percentges of TM were 58:9 29:0 with 70 mm, 54:9 28:9 with 10 mm nd 49:1 28:1 for the control (P < 0:001). Higher mounts of sugr showed tendency of etter TM% nd this ws more pronounced when the comprisons of TM% etween the control nd 70 mm sugrs were mde (Tle 3). Storge time hd detrimentl effect lso on PM%. The decrese in PM% ws found from Period II onwrds (P < 0:05) (Tle 4). The eneficil effect of higher sugr concentrtions in mintining higher PM% ws consistent, nd significnt differences were found etween 70 mm sugrs nd the control in Period I. The type of sugr tested hd no effect on PM%.

8 Tle 3 Men percentges (S.D.) over time of totl motile spermtozo in chilled cnine semen in extenders contining glucose, fructose or mixture of oth t vrious concentrtions Periods Sugr types nd concentrtions (mm) Glucose Fructose The mixture Control Control Control I c II c III c c IV Period I included Dys 1, 2, nd 3. Period II included Dys 4, 6, nd 8. Period III included Dys 10, 12, nd 14. Period IV included Dys 17, 20, nd 23. Significnt differences were found etween oservtion periods in ll tretments (P < 0:05). Different superscripts ( d) in the sme row indicte differences etween sugr concentrtions of ech sugr (P < 0:05). S. Ponglowhpn et l. / Theriogenology 62 (2004)

9 1506 S. Ponglowhpn et l. / Theriogenology 62 (2004) control 10G 70G Totl motility (%) (A) Dys Totl Motility (%) (B) control 10F 70F Dys control 10M 70M Totl motility (%) (C) Dys Totl motility (%) (D) G 70F 70M Dys Fig. 1. Men (S.D.) vlues of totl motility y oservtion dy of chilled cnine semen in extenders contining vrious concentrtions (control: 10 nd 70 mm) of glucose (A), fructose (B), nd the mixture (C). Results of the est motility rte in ech sugr were lso compred (D).

10 S. Ponglowhpn et l. / Theriogenology 62 (2004) Tle 4 Percentge chnge of progressive motility nd movement ptterns (VAP, VSL, nd VCL) of chilled cnine semen compred with the initil vlues of freshly pooled semen Sugrs (mm) Periods PM% VAP (mm/s) VSL (mm/s) VCL (mm/s) Glucose Control I II c III 23.7 c 75.9 c 70.4 ce IV 17.2 c 61.3 d 55.6 de I II c c III 20.1 c ce c IV 13.5 c 65.9 c 58.1 de I II III 23.4 c 92.4 c IV 13.9 c 69.6 d 62.9 c Fructose Control I II c III 33.9 c 77.6 c 72.8 c IV 16.8 d 54.0 d 44.4 d I II c c III 36.7 c 86.4 c 81.4 c IV 16.5 d 56.4 d 47.9 d I II III 34.3 c 86.3 c IV 26.1 c 60.0 d 51.8 c 93.9 c The mixture Control I II III d IV c 54.7 cd I II c III 20.2 c 86.6 c IV 13.8 c 54.9 d 47.8 c I II III 21.9 c IV 13.2 c 61.3 c 54.3 c Period I included Dys 1, 2, nd 3. Period II included Dys 4, 6, nd 8. Period III included Dys 10, 12, nd 14. Period IV included Dys 17, 20, nd 23. Dt tht do not hve common superscript in the sme sugr nd concentrtion indicte differences etween oservtion periods (P < 0:05).

11 1508 S. Ponglowhpn et l. / Theriogenology 62 (2004) In the freshly pooled semen, the movement chrcteristics of VAP, VSL, nd VCL were 107:3 15:0 mm/s, 96:0 14:6 mm/s, nd 139:5 16:7 mm/s, respectively. After extension nd chilling, these vlues significntly incresed in Period I, compred to initil vlues. Therefter, in ll tretments the vlues of VAP nd VSL significntly decresed with storge time (Tle 4). Supplementtion with sugrs (10 or 70 mm) produced etter VAP results (P < 0:05), the iggest difference eing seen etween the control nd 70 mm concentrtions. Sugr concentrtions hd no effects on VSL nd VCL vlues (P > 0:05), however, it ws oserved tht incresing sugr concentrtions induced higher VSL over time. The men vlues of VCL incresed in Period II (Tle 4). At concentrtions of 10 nd 70 mm, the presence of glucose provided higher vlues of VCL thn fructose in Period III nd Period IV (P < 0:05) did Plsm memrne integrity Extension, chilling nd storge over time induced reduction in plsm memrne integrity (P < 0:05). The men percentge of spermtozo with dmged plsm memrne in the freshly pooled semen ws 19:2 3:0, nd fter chilling the percentges incresed over the periods (P < 0:05). When dt ws pooled cross sugr nd concentrtion, the respective men percentges of spermtozo with dmged plsm memrne were: 32:6 5:7 (Period I), 40:74:1 (Period II), 54:2 6:7 (Period III), nd 76:1 5:7 (Period IV). Neither type nor concentrtion of sugr hd protective effect on plsm memrne integrity Acrosoml integrity Men percentge of crosoml loss in pooled ejcultes ws 4:2 1:0. When dt ws pooled cross sugr nd concentrtion, the respective men percentges of crosoml loss were 5:8 2:6 (Period I), 9:6 4:1 (Period II), 14:0 5:5 (Period III), nd 34:7 15:0 (Period IV). There ws no significnt increse in percentges of crosoml loss from tht in freshly pooled semen compred to Period I. In most extenders, n increse in percentges of spermtozo with crosoml loss ws found in Period III (P < 0:05). No significnt differences in the percentges of crosoml loss were found etween sugr types or concentrtions throughout the study Induction of crosoml rection y clcium ionophore chllenge The crosome rection following ionophore chllenge (ARIC) in fresh pooled semen ws 27:8 15:0. After extension nd chilling, there ws significnt decrese in men ARIC vlues in Period I. Neither type nor concentrtion of sugrs significntly ffected the ARIC vlue (Fig. 2). However, the eneficil effect of glucose on this vlue ws most pronounced t 70 mm sugr concentrtion Chnge in ph Pooled seminl plsm hd men ph vlue of 6:4 0:2 (n ¼ 9): the ph vlues of the seven different extenders re shown in Tle 1. Men ph vlues recorded on Dy 23 were

12 S. Ponglowhpn et l. / Theriogenology 62 (2004) ARIC vlue (%) I II III IV 0 control 10G 70G control 10F 70F control 10M 70M Glucose Fructose The mixture Fig. 2. The effect over time on the men percentges (S.D.) of the crosome rection following ionophore chllenge (ARIC) of glucose (G), fructose (F), or the mixture (M) extenders t concentrtions of 10 nd 70 mm, compred to control extenders; (I IV) represent the periods. Period I included Dys 1, 2, nd 3. Period II included Dys 4, 6, nd 8. Period III included Dys 10, 12, nd 14. Period IV included Dys 17, 20, nd 23. Different letters (, ) indicte significnt differences compred to Period I (P < 0:05). ltered from the men ph vlue in the extenders prior to use (Fig. 3). The men ph in 70 mm sugr extenders ws significntly lower thn t 10 mm sugr. The men ph vlue ws lower in the presence of glucose thn in the presence of fructose (P < 0:05) Consumption of glucose nd fructose y cnine spermtozo During the 23 dys, men sugr consumption (mm) in glucose contining extenders ws 1:5 2:0 nd in fructose contining extenders 1:3 2:9 (P > 0:05) (Fig. 4). In the extenders contining equl mounts of oth sugrs, men consumption of glucose ws 1:0 1:3 nd of fructose 0:4 0:7(P ¼ 0:005) (Fig. 5). Sugr consumption ws greter in Period I thn in the following periods. Glucose nd fructose consumption y chilled dog sperm incresed with the incresing sugr concentrtions provided in the extenders, ph vlue * * * * * * * control 10G 70G control 10F 70F control 10M 70M Glucose Fructose The mixture D0 D23 Fig. 3. Chnges in ph vlue (men S:D:) of extenders contining three different sugrs nd two different concentrtions (10 nd 70 mm) efore use (D0) nd of chilled cnine semen stored t 5 8C in corresponding extenders on Dy 23 (D23). The sterisks indicte significnt differences (P < 0:05).

13 1510 S. Ponglowhpn et l. / Theriogenology 62 (2004) Sugr consumption (mm) Glucose consumption Fructose consumption control 10G 70G control 10F 70F Glucose extenders Fructose extenders I II III IV Fig. 4. Glucose or fructose consumption (men S:D:) over time in the extenders contining glucose (G) or fructose (F) t concentrtions of 10 nd 70 mm; (I IV) represent the periods. Period I included Dys 1, 2, nd 3. Period II included Dys 4, 6, nd 8. Period III included Dys 10, 12, nd 14. Period IV included Dys 17, 20, nd 23. Different letters (, ) indicte significnt difference compred to Period I (P < 0:05). Sugr consumption (mm) Glucose consumption Fructose consumption control 10M 70M control 10M 70M The mixture extenders I II III IV Fig. 5. Glucose or fructose consumption (men S:D:) over time in the mixture extenders (M) contining equl mounts of glucose nd fructose to give finl concentrtions of 10 nd 70 mm; (I IV) represent the periods. Period I included Dys 1, 2, nd 3. Period II included Dys 4, 6, nd 8. Period III included Dys 10, 12, nd 14. Period IV included Dys 17, 20, nd 23. Different letters (, ) indicte significnt difference compred to Period I (P < 0:05). lthough the difference etween the control nd the 10 mm concentrtion ws not significnt (P > 0:05). 4. Discussion Two different concentrtions (10 nd 70 mm) of sugrs were tested nd compred to the control, ecuse it hs een climed tht low concentrtions (1 10 mm) hve more intense nd stronger positive effects on motility ptterns nd hexose metolism thn higher concentrtions (50 mm) do [25,26]. The hexose metolism is intimtely correlted with intrcellulr energy reserves in freshly ejculted dog sperm. In contrst, for semen

14 S. Ponglowhpn et l. / Theriogenology 62 (2004) preservtion, the reltively high concentrtions (for exmple, 70 mm) of sugrs commonly used in EYT extenders hve given stisfctory results when evluted y sperm motility, plsm memrne nd crosome integrity, survivl time [13,17,40] nd whelping rte fter AI [16]. The results from the present study clerly demonstrted tht the mjor effect of glucose nd fructose in semen extenders on chilled cnine semen ws to support sperm motility nd movement ptterns. Motility is n importnt indictor of sugr utiliztion y spermtozo s the sugrs provide the externl energy source essentil for mintining motility. In this study, TM% (81.4%) nd PM% (58.0%) remined high during the first 3 dys nd susequently, s could e expected, decresed with storge time. The decrese in the percentge of totl motile spermtozo in Period I (81.4%) compred to in freshly pooled semen (88.9%) my e due to tht there re supopultions of dog spermtozo with different sensitivities to chnges in the environment, prticulrly reduced temperture. Decresed sperm motility fter chilling nd re-wrming hs een postulted to e due to temperture sensitivity of the ATPse-linked sodium potssium pump nd susequently lekge of ions [41]. Preservtion with incresing mounts of sugrs resulted in etter mintennce of sperm motility nd movement ptterns. The ddition of 70 mm sugrs to EYT extenders produced notle eneficil effects on chilled semen. Sperm motility, considered n importnt criterion in cnine semen qulity evlution, should exceed 70% in norml smple [10]. Overll TM% ws significntly higher when 70 mm ws dded to the extenders nd TM of >70% ws mintined until Dy 8 of storge. This is in greement with previous study [40] using similr sugr concentrtion. Although our results still showed high motility rte fter 8 dys of storge, fluctutions in continer temperture during semen trnsporttion might ffect semen qulity [42]; thus, the results my differ in vitro nd in vivo. A 70- mm extenders provided significntly higher PM% over time, nd significntly higher VAP. The men vlues of VAP, VSL, nd VCL incresed significntly in Period I fter extension nd preservtion, compred to the freshly pooled semen, thus suggesting tht sugrs in the extenders ctivte sperm velocity. Fructose ppered preferle to glucose or the mixture y inducing significntly higher percentges of TM% compred to glucose nd the mixture, nd, from Dy 1 onwrds, ddition of 10 mm fructose to the extender produced etter effect on TM% thn the ddition of 10 mm glucose. Fructose provided higher sperm motility over time compred with glucose nd the mixture t ll concentrtions. It hs een stted [25] tht neither glucose nor fructose modify dog sperm motility in rw ejcultes oserved for 60 min t 37 8C, nd tht the most positive effect is oserved t the reltively low concentrtion of 10 mm. The difference in results compred to our study could e due to differences in oservtion time (60 min versus 23 dys) nd in sugr metolism of fresh spermtozo compred to spermtozo preserved t 5 8C. Lowering of the temperture is widely ccepted mens of slowing down oth cell metolism nd chemicl rections, thus prolonging cell life spn [21]. As motility is n indictor of the viility of spermtozo, the results in this study demonstrted tht dog sperm stored in suitle semen extender, nd in cool conditions, cn survive for pproximtely 3 weeks. Dog sperm preserved in utologuous seminl plsm t 4 8C survive for only 2 dys [17]. In most studies on chilled cnine semen

15 1512 S. Ponglowhpn et l. / Theriogenology 62 (2004) [11,14,15,17,19], motility is oserved for up to 4 dys, nd the motility rte on Dy 4 of storge vries etween 4 nd 80%. There re few oservtions over longer period. Chilled cnine semen stored in n EYT extender t 4 5 8C displyed zero motility on Dy 10 [43], Dy 17 [40] nd, in the present study, Dy 23. The vrition in motility rte nd storge life of chilled cnine semen could e due to severl fctors. These include: differences in motility in the fresh ejcultes prior to processing (rnging etween 75 nd 90% in the studies cited ove); differences in composition of semen extenders; differences in preservtion procedures, such s the cooling rte, prosttic fluid removl nd sperm concentrtion; nd differences in methods of estimting the percentge of motility (sujective or ojective). Once cpcitted, spermtozo express n ltered motility pttern clled hyperctivtion [44]. Cpcittion-like chnges re found in chilled cnine semen s determined y the chlortetrcycline ssy together with ltertions of motility chrcteristics (minly VCL nd lterl hed displcement; LHD) [28]. In dogs, VCL nd LHD reflect hyperctivted movement, usully occurring during cpcittion, nd the chilling procedure initites nd ccelertes these chnges [28]. Similrly, the VCL vlues in this study incresed with storge time from Dy 1 onwrds, compred to the initil vlue. From Dy 10 onwrd, ddition of glucose mintined higher VCL vlues thn fructose did. Although the interction mong cpcittion, storge time nd sugrs ws not evluted, reltion etween the higher men vlues of VCL, longer storge time nd the presence of glucose ws found. If these results indicte tht dog sperm undergo cpcittion when they re stored nd exposed to cool conditions, thus reducing longevity, insemintions using chilled semen should e undertken in the most fertile period of the itch, i.e., 2 5 dys fter ovultion, nd the semen should e deposited in the uterus to fcilitte sperm migrtion. This theory is supported y recent finding tht intruterine, s compred to intrvginl, insemintion with chilled cnine semen increses whelping rte nd litter size [45]. Egg-yolk hs een reported to contin some glucose [46], nd in 20% egg-yolk solution in distilled wter, the glucose concentrtion ws found to e 3 4 mm. The ddition of eggyolk to the extenders ws considered necessry, even though this incresed the glucose content, ecuse egg-yolk is n essentil ingredient in semen extenders. The mechnisms y which egg-yolk provides protective ction to spermtozo hve een extensively studied [47 51]. Interestingly, dog sperm, which were preserved in control extender providing only the glucose originting from egg-yolk, were le to mintin 70% motility during the first 4 dys of oservtion nd to survive for up to more thn 21 dys of storge, thus demonstrting tht the smll mount of glucose contined in the egg-yolk component of the EYT extender my e sufficient to preserve chilled dog sperm over considerle period of time. Supplementtion with glucose or fructose, however, resulted in etter motility rtes over time, thus, if semen is to e preserved for longer period, sugrs should e dded to the EYT extenders. Dog sperm re sensitive to osmotic stress [52], however, spermtozo cn tolerte moderte rnge of osmolrities without reduction in fertility [53]. In this study, the osmolrities of semen extenders were within the rnge of physiologicl vlues for cnine seminl plsm (315:9 21:8 mosm) [17,54] nd elow tht proven to suppress dog sperm motility [52,55]. Therefore, the difference in osmolrities mong the extenders is not expected to hve ny detrimentl effect on the chilled semen.

16 S. Ponglowhpn et l. / Theriogenology 62 (2004) Dmges to the plsm memrne occurred erlier nd were more pronounced thn dmges to the crosome. This indicted tht the sperm memrne ws more sensitive to the cool storge conditions thn the crosome ws. Our findings were similr to those of previous studies [56,57] tht used scnning electron microscopy (SEM) nd trnsmission electron microscopy (TEM). Although stimultion of the AR y the ionophore A23187 is non-physiologicl process, it hs een widely used in mny species to dignose potentil fertility [33,38,58]. In dogs, this test hs een estlished s simple method of predicting cryopreservility of semen from individul donors [59]. In the present study, this vlue ppered to decrese with time. This suggested tht the ility of dog sperm to elicit AR following longer storge time ws reduced. On Dy 23 of the experiment, the men ph in the sugr-supplemented extenders hd decresed, compred to the initil vlues. The ph vlues in 70 mm extenders were lso lower thn in 10 mm extenders. This indicted tht ddition of sugrs in extenders prolonged sperm life nd metolism. Incution of freshly ejculted dog sperm t 37 8C with fructose, which is intimtely ssocited with sperm motility, induces greter mounts of CO 2, n cidic end-product, thn glucose does [26]. In the present study, sperm preservtion with glucose t 5 8C resulted in more pronounced decline of ph thn with fructose. This implied tht when the motility ws depressed y cold storge, dog sperm utilized more glucose for other cellulr ctivities thn fructose. Clerly, sugr consumption y dog sperm depended on type nd concentrtion of sugrs ville in their environment. The consumption in Period I ws greter thn tht in the following periods (P < 0:05), indicting tht the slowing down of the sperm metolic rte in the erly stge of preservtion ws not s efficient s during the lter periods, nd could lso reflect decrese in the percentge of living cells. Sugr concentrtion ffected the rte of consumption. This could depend on the sturtion kinetic of monoschrides, which is chrcteristic feture of crrier-medited trnsport system [60]. The preference of dog sperm for the consumption of glucose rther thn fructose ws demonstrted when oth sugrs were present in equl mounts nd the utiliztion of glucose ws greter thn tht of fructose. This is in greement with previous studies on other species [22,61,62 64]. The higher cpcity of the glucose trnsport system possily explins the preference for glucose utiliztion. Independent glucose nd fructose trnsport systems, nd loctions in the dog spermtozoon, hve een reported [26]. It is still uncler why extenders contining reltively lrge mounts of sugr preserved dog sperm etter. In contrst, we demonstrted tht reltively smll mounts of sugrs were utilized y dog sperm throughout the study period. The greter mounts of sugrs in extenders for chilled semen my ply prticulr nd different role in cold stored dog sperm, compred to in freshly ejculted spermtozo. The mechnisms of dog sperm for mnging intrcellulr energy when preserved in cool conditions wrrnt further investigtion. Bsed on the results in this study, it is possile to use chilled dog sperm, stored t 5 8C for t lest up to 8 dys, when spermtozo re extended in n EYT extender contining 70 mm glucose or fructose. However, further studies re needed to test the fertility of longterm chilled dog sperm in vivo. One study on chilled cnine semen, preserved in n EYT extender nd stored t 4 8C for 4 dys, demonstrted tht the men numer of spermtozo tht ound to homologous zon pellucid in vitro ws reduced from 4.8 on Dy 1 to 0.9 on

17 1514 S. Ponglowhpn et l. / Theriogenology 62 (2004) Dy 4 [19]. Although this difference ws non-significnt, the dt indictes tht with incresing storge time the inding cpcity to zon pellucid of chilled, stored dog sperm is reduced. Cliniclly, it would e useful if chilled semen could e preserved while mintining its fertilizing cpcity for longer thn few dys so tht it could e stored witing the optiml insemintion time of the itch, which is importnt considering the extended fertile period (2 4 dys) in this species, nd to llow for sufficient time for semen trnsporttion. Furthermore, repeted insemintions in dogs hve een reported to result in higher pregnncy rtes thn single insemintion [4,65,66]. Therefore, it would e of dvntge if the numer of spermtozo in chilled semen from one ejcultion could e divided into, t lest, two insemintion doses, to e kept stored t 5 8C for repeted insemintions in the sme cycle of the itch. Acknowledgements The skilled technicl support of Krin Selin-Wretling, Annik Rikerg, nd Kristin Krlström is grtefully cknowledged. The uthors thnk Nils Lundeheim nd Annop Suriysomoon for excellent sttisticl ssistnce. The Swedish Foundtion for Interntionl Coopertion in Reserch nd Higher Eduction (STINT) nd Chullongkorn University re cknowledged for providing study leve to Suppwiwt Ponglowhpn. References [1] Peñ A, Linde-Forserg C. Effects of Equex, one- or two-step dilution, nd two freezing nd thwing rtes on post-thw survivl of dog spermtozo. Theriogenology 2000;54: [2] Peñ AI, Lugilde LL, Brrio M, Herrdon PG, Quintel LA. Effects of Equex from different sources on post-thw survivl, longevity nd intrcellulr C 2þ concentrtion of dog spermtozo. Theriogenology 2003;59: [3] Rot A, Iguer-oud M, Verstegen J, Linde-Forserg C. Fertility fter vginl or uterine deposition of dog semen frozen in tris extender with or without Equex STM pste. Theriogenology 1999;51: [4] Linde-Forserg C, Ström Holst B, Govette G. Comprison of fertility dt from vginl vs intruterine insemintion of frozen-thwed dog semen: retrospective study. Theriogenology 1999;52: [5] Thomssen R, Frstd W, Krogenes A, Fougner JA, Berg KA. Artificil insemintion with frozen semen in dogs: retrospective study. J Reprod Fertil Suppl 2001;57: [6] Frstd W. Bitch fertility fter nturl mting nd fter rtificil insemintion with fresh or frozen semen. J Smll Anim Prct 1984;25: [7] Linde-Forserg C. Biology of reproduction nd modern reproductive technology. In: Ruvinsky A, Smpson J, editors. The genetics of the dog. New York: CABI Pulishing; p [8] Gill HP, Kufmn CF, Foote RH, Dirk RW. Artificil insemintion of egle itches with freshly collected, liquid-stored nd frozen-stored semen. Am J Vet Res 1970;31: [9] Morton DB, Bruce SG. Semen evlution, cryopreservtion nd fctors relevnt to the use of frozen semen in dogs. J Reprod Fertil Suppl 1989;39: [10] Linde-Forserg C. Achieving cnine pregnncy y using frozen or chilled extended semen. Vet Clin North Am: Smll Anim Prct 1991;21: [11] Kumi-Dik J, Bdtrm G. Effect of storge on sperm memrne integrity nd other functionl chrcteristics of cnine spermtozo: in vitro iossy for cnine semen. Theriogenology 1994;41: [12] Hrrop AE. Artificil insemintion in itch with preserved semen. Br Vet J 1954;110:424 5.

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