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1 Theriogenology 79 (2013) Contents lists ville t SciVerse ScienceDirect Theriogenology journl homepge: The effect of freezing rte on the qulity of striped ss sperm T.E. Frnkel, D.D. Theisen, H.D. Guthrie, G.R. Welch, L.C. Woods III, * Deprtment of Animl nd Avin Sciences, University of Mrylnd, College Prk, Mrylnd, USA Animl Biosciences nd Biotechnology Lortory, Agriculturl Reserch Service, US Deprtment of Agriculture, Beltsville, Mrylnd, USA rticle info strct Article history: Received 29 August 2012 Received in revised form 15 Jnury 2013 Accepted 16 Jnury 2013 Keywords: Sperm cryopreservtion Freezing rte Striped ss CASA Severl studies hve een conducted in n ttempt to determine the optiml freezing rte for cryopreservtion of striped ss (Morone sxtilis) sperm. In this study, the effects of freezing rte ( 10 C, 15 C, 20 C, nd 40 C/min) on gmete qulity ws exmined, using Syr- 14 nd propidium iodide to determine viility (sperm cell memrne integrity), ATP concentrtion using luciferin-luciferse ioluminescence ssy, nd CEROS computerssisted sperm nlysis system to chrcterize striped ss sperm motion. Adult mle striped ss (N ¼ 12) were smpled once week for 5 weeks. Collected smples were extended, cryoprotected using 7.5% (vol/vol) dimethyl sulfoxide finl concentrtion solution, nd frozen using Plner Kryosve controlled-rte freezer. Smples were stored in liquid nitrogen for 49 dys, nd sperm qulity ws re-evluted fter thw (sme methods). Sperm cryopreserved t 40 C/min resulted in mens for totl motility (10.06%), progressive motility (7.14%), ATP concentrtion (0.86 pmol/10 6 cells), nd sperm viility (56.5%) tht were greter (P < 0.05) thn those for slower cooling rtes. Therefore, 40 C/min ws the optiml freezing rte (mong those tested) for cryopreservtion of striped ss sperm. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction * Corresponding uthor. Tel.: þ E-mil ddress: curry@umd.edu (L.C. Woods). Hyrid crosses with memers of the teleost fmily Moronide re widely used in fishery mngement nd quculture. The most common cross used for commercil purposes involves in vitro fertiliztion of eggs otined from white ss (Morone chrysops) femles with semen collected from striped ss (Morone sxtilis) mles. Hyrid progeny hve severl highly desirle trits (from n industry perspective), including fster growth, hrdiness, nd dptility to environmentl conditions nd stressors [1]. Although there is lck of fundmentl knowledge regrding the moleculr sis of hyrid vigor, there hve een promising results with recurrent selection of Morone roodstock, sed on performnce of their progeny [2]. To crete this hyridiztion, semen is collected from spermiting striped ss during the spring spwning seson, which cn often e prolonged y giving gondotropinrelesing hormone gonist to induce spermition nd increse milt production [3], or with the humn chorionic gondotropin product Chorulon, which ws recently pproved y the US Food nd Drug Administrtion s spwning id for ll rood fish [4]. More thn 220 species of finfish nd shellfish re now frmed, with production rtes exceeding metric tons [5]. As prt of the totl production rte, the striped ss quculture industry hs een estimted to e s high s fifth in volume nd fourth in vlue of ll food fish grown in the United Sttes. [6]. To increse production rtes, one of the mjor needs cited is efficient production nd survivl of lrvl fish to ensure tht dequte numers of fingerlings re ville on yerround sis [6,7]. The striped ss industry is very dependent on wild individuls for gmetes, creting chllenge for development of domesticted striped ss nd white ss rood stock [7], coupled with genetic improvement progrm [4]. Cryopreservtion is commonly used to preserve semen smples for long-term storge nd for use with synchronous nd geogrphiclly isolted spwning popultions, such s the white ss nd striped ss [1]. To X/$ see front mtter Ó 2013 Elsevier Inc. All rights reserved.

2 T.E. Frnkel et l. / Theriogenology 79 (2013) dte, successful fertiliztion of Morone eggs hs een chieved in severl studies using cryopreserved striped ss sperm [8 10]. Multiple experiments hve een conducted in n ttempt to determine the optiml freezing rte for cryopreservtion of striped ss semen [10 12], exmining rnge of freezing rtes from 10 Cto 40 C/min. The present study ws designed to confirm the optimum freezing rte for striped ss sperm nd to quntify dditionl informtion on the effects of cryopreservtion on cell viility, motion, nd energetics (ATP content). 2. Mterils nd methods 2.1. Collection of striped ss semen Eight-yer-old mle striped ss (Morone sxtilis) (N¼ 12) were selected from popultion mintined under computerized photo-therml control (Lutron Version 2.72 Grfix Eye GRX-PRG, Coopersurg, PA, USA) t the University of Mrylnd s Crne Aquculture Fcility. For ech of the 5 weeks, four different individuls were smpled ech dy, 3 dys week, to ensure tht ll 12 individuls were smpled once weekly. Fish were nesthetized in 100 mg/l MS-222 (Finquel, Argent Chemicl Lortories, Redmond, WA, USA) th, uffered to ph 8.0 with sodium icronte. To prevent contmintion, slight pressure ws pplied round the urogenitl vent to expel urine nd then dried with Kimwipe sorent towels. Ech semen smple (pproximtely 5 ml) ws then plced into sterile, 50-mL conicl tues nd immeditely plced on ice. An liquot (1 ml) of semen ws then removed from ech smple nd used to determine seline sperm concentrtion (totl numer of sperm per ml), viility, ATP, nd motion chrcteristics efore freezing Extender, cryoprotectnt, nd cryopreservtion protocol Freshly collected smples were extended 1:3 (v:v) using our l s striped ss extender (NCl 1400 mg, KCl 40 mg, NHCO mg, glucose 100 mg, glycine 75 mm, nd 100 ml deionized ultr-filtered wter) [13] modified to n osmollity of 550 mmol/l using NCl. Hyperosmotic extenders improved postthw qulity of cryopreserved striped ss sperm smples when quickly frozen fter dilution [14,15]. Dimethyl sulfoxide ws then dded s the cryoprotectnt to otin finl concentrtion of 7.5% (vol/ vol), which hs een reported to help preserve sperm plsm memrnes nd protect mitochondril function during cryopreservtion [16]. After 10-minute equilirtion period, six 250-mL volume liquots from single individul were pipetted into 500-mL Cssou strws for ech freezing rte nd immeditely het-seled. Ech set of six strws ws then plced into progrmmle freezer (Plner Kryosve-Model KS30, Sunury-on-Thmes, Middlesex, UK) nd frozen t 10 C, 15 C, 20 C, or 40 C/min. Once the strw s core temperture of 120 C ws reched (determined y thermistor plced into n extr strw contining the tretment mixture), the strws were removed from the freezer nd immeditely plced into dewrs contining liquid nitrogen for storge. The initil freezing rte used ws rotted for ech dy of ech smpling week (to ccount for potentil differences in the time smples were frozen reltive to when they were collected). In ll cses, semen smples were frozen no longer thn 10 minutes fter eing exposed to extender nd cryoprotectnt (equivlent to the time required to lod nd sel the strws fter extension). Ech smple ws removed from storge 49 dys fter it ws frozen, thwed for 6 seconds in 40 C wter th, nd re-evluted for postthw sperm viility, ATP, nd motion chrcteristics (using the sme procedures s descried erlier in the text) Concentrtion, viility, nd motion nlysis Concentrtion To determine initil sperm concentrtions of freshly collected semen, 10 ml of net semen ws dded to 3990 ml of deionized ultr-filtered wter (DIUF) in 5-mL conicl tue to ctivte the sperm (1:400 [v:v] dilution). To otin ccurte redings, sperm were held in DIUF for 1 minute until ll movement hd cesed. After gently homogenizing the smple, 15 ml of the diluted smple ws plced on Mkler counting chmer (Sefi Medicl Instruments, Hif, Isrel) nd nlyzed utilizing CEROS (Version 12; Hmilton-Thorne, Beverly, MA, USA) computer-ssisted sperm nlysis (CASA) system Viility Striped ss sperm were exmined for viility using LIVE/DEAD sperm viility kit (Life Technologies Invitrogen-L-7011, Crlsd, CA, USA) efore nd fter freezing. The sme procedure ws used for fresh nd postthw smples. For postthw smples, Cssou strws were plced in 40 C wter th for 6 seconds; therefter, the smple ws expelled into test tue nd immeditely diluted with our l s striped ss extender (osmollity, 350 mmol/kg) to crete sperm concentrtion of cells per ml. To ech strw s diluted semen, 9.6 mm of propidium iodide nd 0.14mM Syr-14 moleculr stin were dded. The mixture ws incuted for 5 minutes on ice, nd then run on BD-FACSVerse cytometer for nlysis vi BD- FACSuite softwre (Version 1.01; BD Biosciences, Sn Jose, CA, USA). For the purposes of this study, cells tht fluoresced oth green nd red were considered moriund ut counted s ded, ecuse red fluorescence signl cn only e emitted y cell whose memrnes hve een compromised ATP ATP concentrtion for ech smple ws determined with firefly-luciferse ioluminescence procedure, using n ATP ioluminescent ssy mix (FLAAM, Sigm-Aldrich, St. Louis, MO, USA). The sme procedure ws used for fresh nd postthw smples. For the postthw smples, Cssou strws were plced in 40 C wter th for 6 seconds, fter which the smple ws expelled into test tue in preprtion for nlysis ATP stndrd preprtion. To ech well of whiteottomed, 96-well microtiter plte, 50 ml of DIUF ws first dded. Nine ATP stndrds (0.625, 1.25, 2.5, 5, 10, 20,

3 942 T.E. Frnkel et l. / Theriogenology 79 (2013) , 80, nd 160 pmol/ml) were creted from n ATP disodium slt hydrte stock solution (FLAAS, Sigm-Aldrich) nd 25 ml of ech stndrd ws dded to wells 1 to 27 in triplicte Smple preprtion. A smple liquot equivlent to sperm per ml ws first creted using n ATP ssy mix dilution uffer (FLAAB, Sigm-Aldrich) s diluent. Immeditely fter diluting, 10 ml of Phosphtse Inhiitor Cocktil (P5726, Sigm-Aldrich) consisting of sodium vndte, sodium molydte, sodium trtrte, nd imidzole, ws dded. Addition of n inhiitor cocktil hs een shown to help preserve ATP normlly dephosphorylized y cid, lkline, nd tyrosine phosphtses [17]. The inhiited smples were mintined t room temperture for 30 minutes nd susequently plced into 80 C freezer until ll smples were collected nd prepred for the 5-week durtion. Therefter, frozen smples were plced in oiling wter th for 10 minutes. After oiling, the smples were centrifuged for 5 minutes t 23,000 g, nd the resulting superntnts removed into seprte 1.5-mL snp-cp vils. An liquot (25 ml) of ech ws then dded in triplicte to the remining empty wells. To ech well contining stndrd or smple, 100 ml of working ssy mixture (consisting of ATP ssy mix nd ATP ssy mix dilution uffer) ws dded to initite the luciferin-luciferse rection. The entire plte ws then nlyzed using SpectrFlour ioluminescence plte reder (Tecn, Morrisville, NC, USA). Smples were nlyzed within 5 minutes fter the working ssy mixture ws dded to ech well. The stndrd curve ws clculted from the fluorescence redings nd the men luminescence for ech smple ws converted to log 10 for determintion of ATP concentrtion in pmol/10 6 cells [18] Motion nlysis For motion nlysis, 15 ml of DIUF ws first dded to Mkler counting chmer (Sefi Medicl Instruments, Hif, Isrel) nd inserted onto Bionomic (Hmilton- Thorne) temperture-controlled stge mounted onto negtive phse contrst microscope (CX41, Olympus, Tokyo, Jpn). The DIUF ws llowed to chill to 4 2 C. The ddition of sperm to DIUF results in simultneous ctivtion nd using the CEROS (Hmilton-Thorne) CASA, llowed for rpid trcking of sperm motion. After the Mkler reched temperture, n undiluted fresh or postthw semen smple (contining sperm) ws dded to the Mkler chmer nd the contents quickly mixed. Sperm ctivity ws then immeditely recorded nd nlyzed using the CEROS CASA. For ech smple, single 0.5-second cquisition ws mde within 5 seconds fter ctivtion. Prmeters mesured included: totl motility; progressive motility (defined s cells exhiiting pth velocity >80 mm/s nd strightness >80); nd curviliner velocity (mesured in mm/s). Imge cpture settings were djusted to frmes/s rte of 60 Hz nd the numer of frmes djusted to 30. Becuse striped ss sperm re smller thn those of mmmlin species, the minimum cell size ws djusted to two pixels with the minimum CEROS contrst djusted to setting of Sttisticl nlyses Dt nlysis ws performed using SAS 9.2 (SAS Institute, Cry, NC, USA). A repeted mesures, mixed model nlysis of vrince ws used, with P < 0.05 considered significnt [19]. Homogeneity of tretment vrince nd norml distriution of residul errors were exmined efore selection of the covrince structure. An LSMEANS sttement using the DIFF option ws used for comprison of lest squres mens. For the sttisticl model, freezing rte ws considered fixed effect nd week of semen smpling for ech fish s repeted mesure. All vriles were tested for rte y week interctions. For motility, progressive motility, nd viility, fresh nd thwed semen were nlyzed seprtely, ecuse of mrked differences in vrinces etween fresh nd thwed sperm. 3. Results 3.1. Viility The men percentges of vile striped ss sperm over the course of the 5-week spwning period were significntly higher in tretments using fster freezing rtes (Fig. 1). Over the sme intervl, there ws % (men SEM) vile fresh sperm (not shown). The 40 C/min freezing rte yielded the gretest men percentge of vile sperm ( %) fter thwing (significntly different from ll other freezing rtes). In contrst, the 10 C/min freezing rte hd the lowest men percentge of sperm with intct cell memrnes ( %). Men viility vlues otined from the intermedite rtes ( 15 C nd 20 C/ min) were etter thn the slower rte, ut not sttisticlly different from ech other. There were no significnt rte y week interctions for viility, or for ny other of the vriles evluted. Vile sperm (%) c Freezing rte (- o C/min) Fig. 1. Men (SEM) percentges of vile postthw mle striped ss (N ¼ 12) sperm exhiiting green fluorescence (mximum sorption t 488 nm nd emission t 518 nm) when cryopreserved t vrious freezing rtes over the 5-week spwning period. Mens without common letter differed (P < 0.05).

4 T.E. Frnkel et l. / Theriogenology 79 (2013) ATP ATP concentrtion in striped ss sperm ws determined for freshly collected nd postthw smples over the finl 3 weeks of the 5-week spwning period (Fig. 2) when semen smples were t or pproching pek qulity nd concentrtion for the entire popultion. Fresh smples hd n verge ATP concentrtion of pmol/10 6 sperm (not shown). Sperm cryopreserved using the fstest freezing rte ( 40 C/min) hd significntly higher concentrtions of ATP ( pmol/10 6 cells) thn ll other freezing rtes (which were not significntly different from ech other). Motile sperm (%) Totl motility Progressive motility Freezing rte (- o C/min) 3.3. Motion nlysis For ech freezing rte, totl motility for every postthw sperm smple ws greter thn the corresponding smple s estimte of progressive motility (Fig. 3). Overll, totl motility ws % nd progressive motility ws % for fresh smples (not shown). For postthw sperm tht were frozen t the 40 C/min rte, totl nd progressive motility ( % nd %, respectively), were significntly etter thn sperm frozen t ny of the three slower rtes (no significnt differences mong the three slower rtes for either motility end point; Fig. 3). 4. Discussion Motility, viility, nd ATP quntifiction re some of the most widely used stndrds to determine sperm qulity nd hve een correlted with fertiliztion success (see lter in text). For the purpose of our study, n optiml freezing rte ws defined s the rte tht produced the highest percentges of totl nd progressive motility, percentge viility, nd ATP concentrtion in postthw striped ss sperm. Unfortuntely, we were unle to exmine fster freezing rtes, ecuse our progrmmle freezer ws only le to crete consistent, repetle freezing rtes up to ATP (pmol/million cells) Freezing rte (- o C/min) Fig. 2. Men (SEM) ATP concentrtion of postthw mle striped ss (N ¼ 12) sperm when cryopreserved t vrious freezing rtes over 3-week intervl. Mens without common letter differed (P < 0.05). Fig. 3. Men (SEM) percentge of totl nd progressive motility estimtes for postthw mle striped ss (N ¼ 12) sperm when cryopreserved t vrious freezing rtes over 5-week spwning period. Progressive motility ws defined s cells with pth velocity >80 mm/s nd strightness >80%. Mens without common letter differed (P < 0.05). 40 C/min. Bsed on the present results, using the welldocumented, preferred cryoprotectnt DMSO long with pproprite isosmotic extenders for striped ss semen, freezing rte of 40 C/min ws optiml for cryopreservtion of striped ss sperm. For viility, there ws n incresing percentge of striped ss sperm with intct cell memrnes or vile sperm s the freezing rte incresed, with the highest memrne integrity otined from the 40 C/min freezing rte. Becuse the freezing process hs een previously shown to cuse vrying degrees of dmge to sperm cell memrnes [20], xonemes [21], nd mitochondri [22] ecuse of osmotic chnges nd ice crystl formtion, it is vitl for the optiml freezing rte to demonstrte the cpcity to produce sperm with intct memrnes. Postthw sperm ATP concentrtions in species such s the se ss Dicentrrchus lrx [23] nd crp [24] were positively correlted with sperm cell motility nd fertiliztion cpcity. In the present study, sperm from mle striped ss cryopreserved using the 40 C/min rte hd significntly greter concentrtions of ATP fter thwing. Preservtion of these limited ATP resources is prmount, ecuse of severl limiting fctors unique to striped ss sperm. Compred with mmmlin nd other teleost species, striped ss sperm contin reltively smll mount of ATP, commensurte with the presence of only two mitochondri [10]. In ddition, striped ss sperm ppered unle to replenish endogenous energy nd to use exogenous energy sources for motion, s demonstrted y previous study in which dememrnted striped ss sperm incuted with excess exogenous ATP were unle to mintin norml durtion of motility [15]. This ws in contrst to study performed using dememrnted rinow trout sperm cells, in which durtion of motility ws incresed (from 30 seconds to pproximtely 20 minutes) with the ddition of ATP to the extender solution [25]. These differences highlighted not only the inility for striped ss sperm to crete new ATP vi mitochondril function nd oxidtive phosphoryltion [26] when ctivted, ut to dte, hve lso not een shown to e le to ctively trnsport or use the energy sustrtes in ny

5 944 T.E. Frnkel et l. / Theriogenology 79 (2013) mesurle wy, when they hve een provided to the cells (efore or fter freezing) vi extenders or cryomedi. This experiment ws pprently the first to use CEROS CASA system, not only to quntify striped ss sperm motion chrcteristics fter thwing in controlled nd repetle mnner, ut to use the dt in comprtive method to evlute the est freezing rtes for cryopreservtion of striped ss sperm. Totl nd progressive motilities were significntly higher in smples frozen using the 40 C/min rte thn ll other rtes. Striped ss sperm frozen t 40 C/min hd the highest ATP concentrtions nd the highest percentges of cell memrne integrity ws consistent with this freezing rte yielding the highest percentges of motile sperm. Although there ws significnt difference etween postthw motility from the 40 C/min rte nd other freezing rtes, ll cryopreserved smples were very low (<10%) when compred with the corresponding fresh smples (see section 3.3). An unusully high degree of strightness (%) ws oserved from fresh nd postthw striped ss sperm smples, surpssing mny species, e.g., horse [27], turkey [28], nd even in other teleost species such s the Sierin sturgeon [29] nd Chinook slmon [30]. However, it ws not possile to determine whether freezing rte hd n effect on this prticulr motion nlysis chrcteristic in striped ss sperm. The use of CASA to determine motility might provide n explntion for the differences etween the results pulished y erlier sources [10 12], lthough direct comprison is difficult ecuse of the much smller concentrtion of cells needed for CASA nlysis compred with previous methods. In previous studies, motility ws determined sujectively, using only visul ssessment of n entire screen of sperm (in some cses, recorded nd viewed more thn once). In contrst, CASA enles collection of unised, controlled dt, nd multiple other chrcteristics (i.e., stright line velocity, curviliner velocity, strightness, pth velocity, etc.) tht cnnot e evluted sujectively. Becuse successful fertiliztion might require multiple, positive sperm chrcteristics (motility, cell memrne integrity, ATP concentrtion, etc.) in order to successfully trnsfer its genetic informtion to the egg, it is importnt tht studies exmining the optiml conditions under which sperm re cryopreserved nd stored use multiple methods of evlution. Interestingly, when striped ss sperm hve een cryopreserved in the presence of DMSO nd t freezing rtes of 10 C, 20 C, 30 C, nd 40 C/min, the fstest rte ( 40 C/min) yielded significntly higher postthw motility thn the slower rtes tested nd when used in fertiliztion trils, provided fertiliztion rte tht ws not sttisticlly different from fresh semen control smples [10] Conclusions The results of previous studies exmining the effects of freezing rtes on striped ss sperm cells were not concordnt with previous recommendtions of oth slower nd fster freezing rtes [10 12]. Wheres the present results of 40 C/min were comprle with those otined y He nd Woods [10], their study ws limited y pooled smple of three individuls tht were only smpled over the course of single week during the spwning seson. In ddition, sperm cell qulity ws sed solely on ATP concentrtion nd mesurements of motility otined without CASA. Therefore, the current study ws performed to reffirm the optiml freezing rte, using multiple ssy techniques, including CASA motion chrcteriztion, to further define nd delinete differences in sperm qulity of striped ss sperm collected from sustntilly lrger smple size (N ¼ 12) over n extended intervl (5 weeks). For future studies, it would e desirle to exmine the effect of controlled, repetle freezing rtes fster thn 40 C/min with DMSO s the cryoprotectnt, nd novel cryoprotectnts on postthw qulity of striped ss sperm. Acknowledgments This work ws supported y grnt from the Mrylnd Agriculturl Experiment Sttion (MAES) Competitive Grnt Progrm (grnt ). All niml use ws conducted under the pproved, University of Mrylnd Institutionl Animl Cre nd Use Protocol (Reserch Protocol R-10-27). The uthors thnk Smuel Adler, Christine Brdley, Christopher Slmon, nd Willim Dvid Shughnessy of our lortory for providing cre nd husndry of the dult striped ss, nd for their ssistnce with the collection, preprtion, nd nlysis of striped ss semen in this study. References [1] Hrrell RM, Kery JH, Smith TIJ, Stevens RE. Striped ss nd striped ss hyrid culture: the next twenty-five yers. In: Hrrell RM, Kery JH, Minton RV, editors. Culture nd propgtion of striped ss nd its hyrids. Bethesd, MD: Americn Fisheries Society; p [2] Grer AF, Sullivn CV. Selective reeding for the hyrid striped ss (Morone chrysops, Rfinesque M. sxtilis, Wlum) industry: sttus nd perspectives. Aquculture Res 2006;37: [3] Woods LC, Sullivn C. Reproduction of striped ss, Morone sxtilis (Wlum) roodstock: monitoring mturtion nd hormonl induction of spwning. Aquculture Res 1993;24: [4] Joling M, Peruzzi S, Woods C. The temperte sses (Fmily: Moronide). In: Le Frncois N, Joling M, Crter C, Blier P, editors. 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