Effect of select lipids and vitamin E on motility and viability of liquid and cryopreserved boar spermatozoa

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1 Effect of select lipids nd vitmin E on motility nd viility of liquid nd cryopreserved or spermtozo K. Merkies, L. D. Ben, K. Boehnke, nd M. M. Buhr 1 Deprtment of Animl nd Poultry Science, University of Guelph, Guelph, Ontrio, Cnd N1G 2W1. Received 17 July 2, ccepted 13 Novemer 2. Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. Merkies, K., Ben, L. D., Boehnke, K. nd Buhr, M. M. 3. Effect of select lipids nd vitmin E on motility nd viility of liquid nd cryopreserved or spermtozo. Cn. J. Anim. Sci. 83: The ojective of this work ws to test the ility of memrne-incorported lipid nd ntioxidnt constituents to improve or spermtozo function, prticulrly fter cryopreservtion. In exp. 1, fresh or spermtozo (one ejculte from ech of five ors) ws incuted t 37 C in Beltsville Thwing Solution (BTS) + select lipids with (SL), 1 or 5% vitmin E (SL + E1% or E5%). The fluorescent indictor octdecyl rhodmine (R-18) ws included to monitor fusion y flow cytometry. Spermtozo fused more redily to SL or SL + E1% thn SL + E5% (86% vs. 89% vs. 77%, respectively;, differ, P <.5). In exp. 2, fresh or spermtozo (one ejculte from ech of four ors) ws incuted with BTS uffer lone (control), or BTS with SL, SL + E1% or SL + E5%, omitting the R-18 dye, nd held t 18 C for 7 d. Overll, viility (mesured with SYBR-14/PI) ws higher in SL + E (59% vs. 51% vs. 69% vs. 67% for control, SL, SL + E1%, nd SL + E5%, respectively;, differ, P <.35). Both SL nd SL ± E1% incresed the percentge of motile spermtozo (totl nd progressive) fter 3 h of storge (P <.5); ll tretments hd similr motility y 24 h, nd % motility y 96 h. In exp. 3, or spermtozo (three ejcultes from ech of three ors) ws cryopreserved in BTS (control), SL or SL + E1% using BF5 extender. Initilly t 37 C, SL + E1% incresed motility, while viility ws similr for ll tretments. Spermtozo fused with SL + E1% hd the most motile spermtozo when cooled to 5 C during processing nd fter freezing nd thwing (P <.5); the percentge of vile spermtozo ws similr mong tretments. In conclusion, incorportion of SL + E1% into spermtozo memrnes improves motility nd/or viility of preserved or spermtozo. Key words: Freezing, α tocopherol, swine, memrnes Merkies, K., Ben, L. D., Boehnke, K. et Buhr, M. M. 3. Incidence des lipides et de l vitmine E sur l motilité et l viilité des spermtozoïdes de verrt conservés sous forme liquide ou congelés. Cn. J. Anim. Sci. 83: L recherche devit étlir si l intégrtion de lipides à l memrne et les nti-oxydnts méliorent le fonctionnement des spermtozoïdes de verrt, surtout près cryoconservtion du sperme. Dns le cdre d une première expérience, des spermtozoïdes fris (un éjcult de chcun de cinq verrts) ont été incués à 37 C dns du dilueur de Beltsville (DB) uquel on vit jouté des lipides et, 1 ou 5 % de vitmine E (L, L + E1% ou L + E5%). De l octdécyle de rhodmine (R-18), un indicteur fluorescent, devit déterminer si les lipides s étient ien fusionnés à l memrne, pr cytométrie de flux. Les spermtozoïdes fusionnent plus fcilement ux L ou ux L + E1% qu ux L + E5% (86 % c. 89 % c. 77 %, respectivement; et diffèrent; P <,5). Dns une deuxième expérience, les chercheurs ont incué des spermtozoïdes fris (un éjcult de chcun de qutre verrts) uniquement vec du DB comme tmpon (témoin) ou du DB et des L, L + E1% ou L + E5%, sns le colornt R-18. Le mélnge été conservé pendnt 7 jours à 18 C. En générl, les spermtozoïdes sont plus viles (viilité mesurée pr SYBR-14/PI) dns l solution L + E (59 % c. 51 % c. 69 % c. 67 % pour l solution témoin, L, L + E1% et L + E5%, respectivement; et diffèrent; P <,35). Les solutions L et L + E1% ugmentent l proportion de spermtozoïdes motiles (totle et grduelle) près un stockge de trois heures (P <,5). Tous les tritements donnent lieu à une motilité similire u terme de 24 heures, sns plus ucune motilité près 96 heures. Dns l troisième expérience, les spermtozoïdes (trois éjcults de chcun de trois verrts) ont été cryoconservés dns du DB (témoin), des L ou des L + E1% vec du BF5 comme gent de dilution. Au déut, à 37 C, le mélnge L + E1% ccroît l motilité, les trois tritements se ressemlnt u niveu de l viilité. Les spermtozoïdes qui fusionnent vec les L + E1% s vèrent les plus motiles près refroidissement à 5 C, lors du conditionnement, insi qu près congéltion et décongéltion (P <,5). Le pourcentge de spermtozoïdes viles est semlle pour les différents tritements. En conclusion, l incorportion de L + E1% à l memrne méliore l motilité ou l viilité des spermtozoïdes de verrt conservés, ou les deux. Mots clés: Congéltion, α tocophérol, porcs, memrne Mmmlin spermtozo re sujected to numerous environmentl chnges during their trnsit from the mle reproductive trct to the site of fertiliztion in the femle oviduct. The plsm memrne of the spermtozo t lest prtilly regultes the cell s response to these vrious environments. The exct composition of the memrne s phospholipid 1 To whom correspondence should e ddressed (e-mil: muhr@uoguelph.c). 81 ilyer, with its constituent peripherl, prtilly emedded or integrl proteins, is species-specific (Mnn nd Lutwk- Mnn 1981). Cryopreservtion of spermtozo (which s used here includes the processes of dilution, cooling, freezing, nd thwing) nd long-term storge of liquid semen enhnce the Arevitions: BTS, Beltsville Thwing Solution; PI, propidium iodide; SL, select lipids

2 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. 82 CANADIAN JOURNAL OF ANIMAL SCIENCE enefits of rtificil insemintion. However, cryopreservtion compromises or kills mny spermtozo nd reduces the fertilizing ility of surviving spermtozo, lthough the percentge surviving cryopreservtion vries mong species. Cryopreservtion plces considerle stress on the plsm memrne of the spermtozo, with the ftty cids ttched to memrne phospholipids hving long een suggested s possile source of the differing success in cryopreservtion mong species (Pursel nd Grhm 1967; Poulos et l. 1973; Chow et l. 1986). The rtio of sturted to unsturted ftty cids my e ssocited with susceptiility of spermtozo to lipid peroxidtion nd cold shock (White 1993), which implies tht spermtozo with high unsturted:sturted rtio, such s found in the or nd rm, re more susceptile to cold shock thn those with low rtio like the ull nd humn (Prks nd Lynch 1992). These unsturted ftty cids in or spermtozo re prticulrly susceptile to removl during cryopreservtion (Buhr et l. 1994), nd the ctivity of the ntioxidnt superoxide dismutse ws enhnced in poor-qulity frozenthwed or semen, suggesting need to overcome peroxidtive dmge (Cerolini et l. 1). Deleterious effects of lipid peroxidtion include loss of motility nd fertility. The ddition of the ntioxidnt vitmin E to the extender improved motility nd decresed peroxidtion of ufflo spermtozo (Singh et l. 1989) nd or spermtozo (Cerolini et l. ), nd incresed the motility nd viility of oth fresh nd frozen-thwed rm spermtozo (Ollero et l. 1998). Vitmin E lso decresed the percentge of cpcitted ull spermtozo post-thw, suggesting protective effect on the plsm memrne (O Flherty et l. 1997). Success in semen cryopreservtion with species such s the pig hs een moderte to dte (Johnson et l. ). Recent reserch hs focussed on the ddition of vrious constituents during the cooling nd freezing processes to improve post-thw spermtozo function nd viility (Armstrong et l. 1998; Ollero et l. 1998; Pettitt nd Buhr 1998; Eriksson et l. 1). We hve recently shown tht crefully chosen heterogeneous mixture of lipids cn rpidly fuse with or spermtozo nd incorporte into the memrnes when dded in the form of liposomes to fresh semen (He et l. 1). These selected lipids significntly improved post-thw survivl, ut even this improved percentge of functionl spermtozo remined lower thn is commercilly desirle. Since the high content of unsturted ftty cids in or spermtozo mkes them susceptile to oxidtive dmge, ntioxidnts hve een dded to semen diluents to improve viility of stored spermtozo (Cerolini et l. ). Mny studies hve incorported vitmin E s dietry supplement to improve semen qulity (Brezezinsk- Sleodzinsk et l. 1995; Wemheuer et l. 1996; Mrin- Guzmn et l. 1997; Velsquez-Pereir et l. 1998). The current study tested our hypothesis tht incorportion of select lipids with or without vitmin E directly into spermtozo memrnes would improve the qulity of stored or semen. The first experiment estlished the concentrtion of vitmin E, which, when incorported into liposomes, would mximize liposome fusion to spermtozo. Susequent experiments ssessed the motility nd viility of semen stored in the presence of select lipids nd vitmin E either in liquid extender over 7 d, or fter cryopreservtion. MATERIALS AND METHODS Experiment 1: Fusion Efficiency Select lipids (SL) were prepred t 25 C s previously descried (He et l. 1). A mixture of 15 lipids (Sigm, Okville, ON; Avnti, Alster, AL, USA; Mtrey, Plesnt Gp, PA, USA) ws comined in chloroform with nd without vitmin E (α-tocopherol; Sigm) t, 1, nd 5% (vol:vol; finl concentrtion,.8 or 4 µg ml 1, respectively). The lipid mixture ws composed of phosphtidylcholine, phosphtidylethnolmine, phosphtidylserine, phosphtidylinositol, nd sphingomyelin in the rtio of 21:26:5:5:42, respectively. The fluorescent dye octdecyl rhodmine B-chloride (R18) ws included t 2% (vol:vol; finl concentrtion.28 µmol ml 1 ; Moleculr Proes, Eugene, OR). The mixture ws then dried under nitrogen gs nd desiccted for 1 h efore eing rehydrted in Beltsville Thwing Solution (BTS; mm dextrose, mm N citrte, 15 mm N icronte, 3.3 mm EDTA, 1 mm potssium chloride, 1.3 mm dihydrostreptomycin; ll chemicls from Sigm, ph 7.3; Johnson et l., ) t 6 C to finl concentrtion of.693 µmol lipids ml 1. This solution ws sonicted for 1 h to crete smll unilmellr vesicles consisting of single lipid ilyer, seled in cryo vils under nitrogen gs nd stored t 7 C until use, t which time liposomes (1 ml) were thwed to 37 C, resonicted for 1 h nd the ph djusted to 7.2. The sperm-rich frction ws collected from five mture, helthy Yorkshire ors of proven fertility using the gloved hnd technique. Bors were housed, hndled nd mintined ccording to the Guidelines of the Cndin Council on Animl Cre. Semen ws held t 37 C nd filtered through doule lyer of Mircloth guze (Cliochem, L Joll, CA) immeditely fter collection. Motility ws visully ssessed nd concentrtion determined y clirted spectrophotometer. Semen ws diluted to sperm ml 1 in BTS. Fusion of liposomes to spermtozo ws evluted s efore (He et l. 1), using Coulter Epics ESP flow cytometer (Coulter, MI), nd confirmed y microscopic monitoring of the spermtozo cells. To determine fusion efficiency (n = 8 ejcultes), excittion nd emission wvelengths were set t 488 nd 57 to 58 nm, respectively. Spermtozo nd liposomes were ech evluted individully prior to fusion to estlish the size nd fluorescence distriution of ech. Voltge of fluorescent intensity ws djusted to 6 to otin the mximum seprtion of spermtozo nd R18-lelled liposomes. The fusion rection ws strted y mixing spermtozo (1 µl), SL (9 µl), nd BTS (1 ml) t 37 C nd reding fluorescent emissions of prticles t ech of 1, 1, 3, nd 6 min. The resulting histogrms (Fig. 1) were used to determine the percentge of spermtozo fused to liposomes (% fused sperm), nd the percentge of ville liposomes tht were fused to spermtozo (% fused liposomes) y the following equtions:

3 i) ii) MERKIES VITAMIN E TO PRESERVE BOAR SPERM 83 C C: liposomes A A Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. iii) C B : Sperm A: fusion B B Fig. 1. Fusion histogrms generted y flow cytometry of or spermtozo incuted for 1 min with liposomes ± vitmin E using R18 s mrker fluorescent dye. (i) Spermtozo only. The spermtozo popultion ws scttered y size informtion (FS). (ii) Liposomes only. The liposome popultion ws scttered y the fluorescent intensity of the R18 dye (PMT3). (iii) Fusion product. In ll histogrms, Region A designtes the fusion product of spermtozo + liposomes, region B is the popultion of spermtozo only, nd region C is the popultion of liposomes only. % fused spermtozo = [prticles in re fused/prticles in (re fused + re of unlelled sperm)] 1 OR = (prticles in re A/prticles in res A + B); A, B re res in the histogrm (Fig 1) % fused liposomes = [prticles in re fused/prticles in (re fused + re of liposomes)] 1 OR = (prticles in re A/prticles in res A + C); A, C re res in the histogrm (Fig 1)

4 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. 84 CANADIAN JOURNAL OF ANIMAL SCIENCE Experiment 2: Function of Extended Fresh Spermtozo over 7 d A single ejculte ws collected from ech of four ors s ove nd diluted to sperm ml 1 in 37 C BTS contining 5 IU penicillin L 1. Select lipids were prepred s ove, omitting the R18. Aliquots of spermtozo (15 µl) were comined with 135 µl of either BTS or SL contining, 1 or 5% vitmin E in BTS. Ech of these four tretments ws cpped under 5% CO 2 /95% ir nd cooled in progrmmle cooler (Biocool II, FTS Systems Inc., NY) to 18 C over 3 h. Smples were held t 18 C over 7 d, turning nd exposing ech smple to fresh 5% CO 2 /95% ir once every 24 h. Viility ws determined t 3, 24, 96, nd 168 h using the LiveDed Kit (Moleculr Proes), which uses the fluorescent dye SYBR-14 to stin live spermtozo green, nd propidium iodide (PI) to stin memrne-disrupted spermtozo red. Viility ws determined using fluorescence microscopy, chrcterizing minimum of spermtozo per tretment s hving green (live), red (ded) or dul (moriund) fluorescence. To test the conclusions of the first tril, replicte experiment treted liquots from single ejculte from ech of three ors with SL, SL plus 1% vitmin E, or BTS lone (control). Viility nd motility were determined t 3, 24, 96, nd 168 h. Viility ws ssessed s previously descried. For oth experiments, motility ws ssessed in duplicte y light microscopy, chrcterizing minimum of sperm per tretment s progressively motile, nonprogressively motile or non-motile. Progressive nd nonprogressive counts were comined to give reding of totl motility. Experiment 3: Motility nd Viility of Cryopreserved Spermtozo A minimum of three ejcultes ws otined from ech of three ors, Lincospectin ws dded (Upjohn, Orngeville, ON;.6 ml ml 1 semen) nd motility nd concentrtion were ssessed s ove. Semen with > 7% motile spermtozo ws diluted to sperm ml 1 in BTS. Semen ws then further diluted to sperm ml 1 (totl volume 4 ml) in one of three tretment groups: either (1) Control (2) SL, or (3) SL + E1%. Immeditely fter dding lipids, viility nd motility redings were tken in duplicte s ove. Spermtozo were then cryopreserved s efore (Pettitt nd Buhr 1998). Briefly, semen ws llowed to cool from 37 to 25 C in progrmmle cooler t rte of.1 C min 1. At this point, semen ws centrifuged (1 min, 8 g, 25 C) nd the superntnt ws removed. The spermtozo pellet ws gently resuspended in frction A of the Beltsville F5 (BF5) extender to concentrtion of sperm ml 1 (totl volume 1.5 ml). Smples were returned to the progrmmle cooler nd llowed to cool to 5 C t the rte of.1 C min 1. Once the smples hd reched this temperture, the glycerolted frction B of the BF5 extender ws dded (1:1, vol:vol; finl glycerol concentrtion 3%) to give finl concentrtion of sperm ml 1. Motility nd viility were gin smpled in duplicte. To cryopreserve the semen,.5 ml strws were filled nd seled t 5 C. Strws were frozen in progrmmle freezer from 5 C to 7 C t rte of 3 C min 1 efore eing plunged into liquid nitrogen. For post-thw nlysis, strws were thwed for 5 s in 6 C wter th. Strws were emptied into tues held t 37 C, nd diluted with 1 mm cffeine (Sigm) in BTS. Motility nd viility redings were determined s ove. Sttisticl Anlysis Percentge of fused sperm, percentge of fused liposomes, nd percentge of live nd ded spermtozo were treted to rcsine trnsformtion nd nlysed using Generl Liner Models (SAS Institute, Inc. 1987) with or, dte of collection (s mesure of within-or vriility), tretment, time, nd ll interctions in the model. Differences mong tretments were determined y protected Lest Significnt Differences (LSD) test, nd P vlue of <.5 ws considered significnt. Percent dt of progressively motile, totl motile, green- (live), nd red- (ded) stined spermtozo were treted to rcsine trnsformtion nd nlysed using Generl Liner Models (SAS Institute, Inc. 1987) with or, dte of collection, tretment, nd ll interctions in the model. Differences mong tretments were determined y the Bonferroni t-test, nd P vlue of <.5 ws considered significnt. RESULTS Experiment 1: Fusion Efficiency Histogrms generted y flow cytometry hd regions corresponding to spermtozo lone (Fig. 1 i, Region B), liposomes lone (Fig. 1 ii, Region C), nd the fusion product of spermtozo plus liposomes (Fig. 1 iii, Region A). The prticle count in the re of the region depicting the fusion product (Region A) ws djusted y sutrcting ny prticles found in Region A when spermtozo or liposomes lone were present (Fig. 1 i nd ii). Liposome composition significntly ffected the percentge of spermtozo tht fused to lipids (P <.5), with more spermtozo fusing to SL nd SL + E1% liposomes thn to SL + E5% liposomes (Fig. 2A). There ws no significnt difference mong time periods s to the verge percentge of spermtozo fused to liposomes (P >.58), indicting tht fusion ws mximl nd stle immeditely. The percentge of ville liposomes (ll tretments) fusing to spermtozo decresed over time nd ecme stle t % usge fter 3 min (Fig. 2B; P <.1). Experiment 2: Function of Extended Fresh Spermtozo Over 7 d Viility decresed slightly ut not significntly in ll tretment groups over 7 d (73 vs. 63% live; dy to dy 7, P >.58), nd vlues over time were therefore pooled to ssess tretment effects. Liposomes contining vitmin E enhnced viility compred to the controls or SL lone (Fig. 3A; P <.35). In the replicte experiment, viility gin did not chnge significntly over time, with vlues rnging from 64 to 58%

5 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. A % fused sperm B % ville liposomes fused to sperm SL SL + E1% SL + E5% Intervl (min) Fig. 2. Fusion efficiency of liposomes with or sperm. Spermtozo (n = 8 ejcultes) were incuted over 6 min with liposomes (SL liposomes with, 1, or 5% vitmin E) lelled with the fluorescent proe R18. Fusion ws determined y flow cytometer t 1, 1, 3, nd 6 min y prticle count of liposomesized prticles nd spermtozo-sized fusion products. A. Percentge of or spermtozo (± SEM) fused to liposomes with or without vitmin E t 1 or 5%. The percentge of spermtozo tht fused to liposomes ws similr t ll times (1, 1, 3, 6 min; P >.); thus, vlues over time for ech tretment were verged (, differ, P <.5). B. Percentge of ville liposomes fused to or spermtozo (± SEM). There ws no difference due to liposome composition (P >.); thus, tretments were pooled t ech time point (,, c differ; P <.1). (P >.5). Percent of vile spermtozo ws verged over time s efore. More spermtozo remined vile when exposed to SL with or without vitmin E thn in the control (54% vs. 64% vs. 68% for control, SL, nd SL + E1%, respectively; P.6; Fig. 3B). Both progressive nd totl motility declined over 7 d incution t 18 C, nd were virtully zero y 96 h in ll tretments. At 3 h, oth progressive nd totl motility were higher in tretments with SL thn in the controls (Fig. 4, A nd B; P <.5). By 24 h, there ws no difference in either progressive or totl motility in ny tretment (P >.51). c c MERKIES VITAMIN E TO PRESERVE BOAR SPERM 85 Experiment 3: Motility nd Viility of Cryopreserved Spermtozo Prior to cryopreservtion, there ws lwys significnt difference in spermtozo motility mong ors (P.3). Post-thw, however, this difference tended to e less significnt in progressive motility (P =.57), nd ws not significnt t ll in totl motility (P.21). To ccount for this individulity, we evluted the percent chnge in motility nd viility for ech or from 37 C to post-thw. This nlysis produced the sme conclusions regrding tretment effects s did the simple post-thw % motile nd % vile, which re therefore presented here. At 37 C, totl motility ws similr for spermtozo fused to SL nd SL + E1%, nd ws higher thn in the control (P <.5). The SL + E1% ws significntly etter thn either the control or SL tretments in progressive motility t 37 C, nd in totl nd progressive motility t 5 C nd postthw (Fig. 5 A nd B; P <.5). Progressive motility ws higher t 5 C thn t wrmer tempertures, proly due to the ddition of Frction B of the BF5 extender contining egg yolk nd glycerol. Viility mong tretments did not differ initilly t 37 C (Fig. 5; P <.5). The controls nd SL + E1% provided the est viility t 5 C nd post-thw, equlling or exceeding viility in SL. DISCUSSION Select lipids contining 1% vitmin E significntly improved the motility nd viility of cooled (stored over 7 d) or cryopreserved or spermtozo. Lipid dynmics within the plsm memrne of the spermtozo influence not only functionl chnges such s mturtion nd cpcittion (Berer nd Friend 199), ut lso memrne integrity during cooling nd cryopreservtion (Buhr et l. 1994). The current results support the hypothesis tht simultneously compensting for lipids known to e lost in cryopreservtion (Buhr et l. 1994; Kelso et l. 1997), nd interfering with peroxidtion should improve spermtozo qulity. Vitmin E redily incorportes into memrnes contining lrge mounts of phospholipids (Ptel et l. 1991) such s found in our pure phospholipid liposomes nd, indeed, in or spermtozo. Adding vitmin E directly to BTS incresed its concentrtion in extrcts of pelleted whole spermtozo (Cerolini et l. ), ut the nture of its ssocition with spermtozo ws unknown. Inclusion of R18 mrker dye in the lipid mixture llowed, s efore (He et l. 1), visul confirmtion with fluorescence microscope (dt not shown) tht lipids did indeed fuse to spermtozo, nd monomeric lipid trnsport ws not involved (Gdell et l. 1999). Flow cytometery quntified the fusion dynmics, quntifying more thn 85% of the spermtozo popultion incorporting liposomes. The percentge of spermtozo fused to liposomes remined constnt over time, nd significntly more spermtozo fused to liposomes contining 1% vitmin E thn 5% vitmin E (Fig. 2A). The higher concentrtion of the vitmin might force it to disperse more evenly through the memrne, which would counter its nturl preference for non-uniform distriution (Ptel et l. 1991), nd could reduce its fusogenicity.

6 86 CANADIAN JOURNAL OF ANIMAL SCIENCE % vile sperm A) B) control SL SL + E1% SL + E5% control SL SL + 1%E Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. Fig. 3. Percentge of vile or spermtozo (± SEM) in replicte experiments (A nd B) determined y fluorescence microscopy fter stining with SYBR 14 nd PI. Spermtozo were exposed nd incuted t 18 C in BTS contining no liposomes (control), or liposomes mde of select lipids contining vtmin E t (SL), 1% (SL + E1%), or 5% (SL + E5%). There ws no difference in viility over 7 d (P >.58), thus vlues over time were pooled for ech tretment (, differ; P <.35). Totl motility (%) A: Totl Motility B: Progressive Motility hours The percentge of the liposomes tht incorported into the spermtozo ws similr for ll three types of liposomes, even though fewer spermtozo took up the SL + E5% liposomes. Therefore, it seems likely tht there ws supopultion of the spermtozo tking up reltively greter mount of the SL + E5% liposomes. Bor semen is known to hve supopultions of spermtozo with different motility chrcteristics (Aigr et l. 1999), different shpe of the hed (Thurston et l. 1), nd most pertinently, differing responses of memrne lipids to temperture chllenge (Cerolini et l. 1). Spermtozo supopultions tht cn incorporte different mount of lipids from the environment (current results), nd regulte memrne lipid metolism differently during cryopreservtion (Cerolini et l. 1), my explin why some spermtozo survive cryopreservtion nd some do not, nd why individul ors produce spermtozo of differing cryosensitivity. We lso noticed n pprent decrese over time in the % of the ville liposomes tht hd fused to spermtozo (Fig. 2B). Progressive motility (%) 8 6 control SL SL + E1% 3 24 hours Fig. 4. Percentges ± SEM of totl (A) nd progressive (B) motility of or spermtozo (n = 3) incuted t 18 C in BTS contining no liposomes (control), or liposomes mde of select lipids contining vitmin E t (SL) or 1% (SL + E1%) over 7 d t 18 C. Motility ws higher in SL thn in controls t 3 h (, differ within time period; P <.5). Motility ws virtully zero y 96 h. This my suggest tht, initilly, high numers of liposomes dhere to sperm, ut only portion of those remin nd ctully fuse with the plsm memrne while the unfused liposomes slough off. In exp. 2, or spermtozo retined their viility over 7 d t 18 C, nd the ddition of SL plus vitmin E overll improved viility. The SL lone provided protection in one experiment ut not the other, while SL contining vitmin E consistently preserved viility (Fig. 3). Therefore, the protective effect of liposomes my e due to the SL, the vitmin E, or n interction of these components. We previously (He et l. 1) showed positive effect of SL lone on viility of or sperm, leding to the conclusion tht vitmin E stilizes or enhnces the protective effect of the SL lipids on the viility of fresh or spermtozo in storge. This concurs with the findings of Cerolini et l. () tht direct inclusion of vitmin E in BTS improved the viility of stored or sperm. The SL with or without vitmin E lso riefly preserved progressive nd totl motility, which is

7 A: Totl Motility B: Progressive Motility MERKIES VITAMIN E TO PRESERVE BOAR SPERM 87 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. Totl motility (%) C: Viility Vile sperm (%) control SL SL + E1% similr to previous results otined with SL lone t 24 C (He et l. 1). However, this eneficil effect quickly diminished, nd there ws no difference mong tretments nd control fter 24 h, likely due to the decrese in spermtozo metolism induced y the reduction of energy sustrtes ville in the BTS medium. When cryopreserving spermtozo (exp. 3), SL + E1% prevented the decrese of totl nd progressive motility upon cooling to 5 C, thus vitmin E is effective ginst oth short-time- nd cold-temperture-induced injury. Progressive motility in ll tretments incresed upon the ddition of the B frction of the BF5 extender t 5 C. This my e due to the ddition of more egg yolk, which improves spermtozo function nd cts s n extrcellulr lipid source providing significnt protective effect on the spermtozo during cooling nd freezing (Wtson nd Mrtin 1975; Pettitt nd Buhr 1998). Lipid protection ginst low-temperture injury ws evident even in the fce of the mjor chllenge of cryopreservtion in liquid nitrogen. The presence of select lipids plus vitmin E in the initil semen diluent incresed the percentge of motile nd vile or spermtozo during cryopreservtion nd post-thw. The SL contining vitmin E 37 C 5 C posthw Progressive motility (%) 37 C 5 C posthw C 5 C posthw Fig. 5. Percentges ± SEM of totl (A) nd progressively (B) motile sperm, nd vile (C) or spermtozo (n = 3) ejcultes for ech of three ors t 37 C, 5 C, or post-thw, incuted with SL + or 1% vitmin E (SL, SL + E1%) or in extender lone (control). Progressive motility ws higher in SL + E1% t ll times. Totl motility ws higher in SL ± vitmin E t 37 C nd higher in SL + E1% t 5 C nd postthw. Viility ws similr mong ll tretments t 37 C, nd somewht lower in SL t 5 C nd post-thw (, differ within temperture period; P <.5). consistently nd significntly incresed the percent of totl nd progressively motile spermtozo during cooling, nd fter freezing nd thwing, without ffecting viility s mesured y memrne integrity. Therefore, incorportion of low mount of vitmin E into memrnes ppers to protect or spermtozo ginst some of the dmges cused y cryopreservtion. In conclusion, select lipids nd vitmin E preserve the motility nd viility of stored or semen, prticulrly improving the function of cryopreserved sperm. These lipid constituents my prove to e importnt fctors in successful cryopreservtion of or semen. ACKNOWLEDGEMENTS The uthors wish to thnk the Nturl Sciences nd Engineering Reserch Council of Cnd, Ontrio Pork nd the Ontrio Ministry of Agriculture, Food nd Rurl Affirs for their finncil support. Aigr, T, Holt W. V., Hrrison R. A. nd del Brrio, G Spermtozo supopultions in or (Sus scrof) nd gzelle (Gzell dm mhorr) semen s reveled y pttern nlysis of computer-ssisted motility ssessments. Biol. Reprod. 6:

8 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. 88 CANADIAN JOURNAL OF ANIMAL SCIENCE Armstrong, J. S., Rjsekrn, M., Hellstrom, W. J. nd Sikk, S. C Antioxidnt potentil of humn serum lumin: role in the recovery of high qulity humn spermtozo for ssisted reproductive technology. J. Androl. 19: Berer, E. L. nd Friend, D. S Morphology of mmmlin spermtozo memrnes during differentition, mturtion, nd cpcittion. J. Electron Microsc. Tech. 16: Brezezinsk-Sleodzinsk, E., Sleodzinski, A. B., Pietrs, B. nd Wieczorek, G Antioxidnt effect of Vitmin E nd glutthione on lipid peroxidtion in or semen plsm. Biol. Trce Elem. Res. 47: Buhr, M. M., Curtis, E. F. nd Kkud, N. S Composition nd ehvior of hed memrne lipids of fresh nd cryopreserved or sperm. Cryoiology 31: Cerolini, S., Mldjin, A., Pizzi, F. nd Gliozzi, T. M. 1. Chnges in spermtozo qulity nd lipid composition during cryopreservtion of or semen. Reproduction 121: Cerolini, S., Mldjin, A., Suri, P. nd Nole, R.. Viility, susceptiility to peroxidtion nd ftty cid composition of or semen during liquid storge. Anim. Reprod. Sci. 58: Chow, P. Y. W., White, I. G. nd Pickett, B. W Stllion spermtozo seminl plsm phospholipids nd glycerylphosphorylcholine. Anim. Reprod. Sci. 11: Eriksson, B. M., Vzquez, J. M., Mrtinez, E. A., Roc, J., Lucs, X. nd Rodriguez-Mrtinez, H. 1. Effects of holding time during cooling nd of type of pckge on plsm memrne integrity, motility nd in vitro oocyte penetrtion ility of frozenthwed or spermtozo. Theriogenology 55: Gdell, B. M., Miller, N. G. A., Colenrnder, B., Vn Golde, L. M. G. nd Hrrison, R. A. P Flow cytometric detection of trnsilyer movement of fluoresecent phospholipid nlogues cross the or spermtozo plsm memrne: elimintion of lelling rtifcts. Mol. Reprod. Dev. 53: He, L., Biley, J. L. nd Buhr, M. M. 1. Incorporting lipids into or spermtozo decreses chilling sensitivity ut not cpcittion potentil. Biol. Reprod. 64: Johnson, L. A., Weitze, K. F., Fiser, P. nd Mxwell, W. M.. Storge of or semen. Anim. Reprod. Sci. 62: Kelso, K. A., Redpth, A., Nole, R. C. nd Speke, B. K Lipid nd ntioxidnt chnges in spermtozo nd seminl plsm throughout the reproductive period of ulls. J. Reprod. Fertil. 19: 1 9. Kok, J. W. nd Hoekstr, D Fluorsecent lipid nlogues: Applictions in cell nd memrne iology. Pges in W. T. Mson, ed. Fluorescent nd luminescent proes for iologicl ctivity. Acdemic Press Inc., Hrcourt Brce & Co., Sn Diego, CA. Mnn, T. nd Lutwk-Mnn, C Mle reproductive function nd semen. Springer-Verlg, Berlin nd Heidelerg, Germny. Mrin-Guzmn, J., Mhn, D. C., Chung, Y. K., Pte, J. L. nd Pope, W. F Effects of dietry selenium nd Vitmin E on or performnce nd tissue responses, semen qulity, nd susequent fertiliztion rtes in mture gilts. J. Anim. Sci. 75: O Flherty, C., Beconi, M. nd Beorlegui, N Effect of nturl ntioxidnts, superoxide dismutse nd hydrogen peroxide on cpcittion of frozen-thwed ull spermtozo. Andrologi. 29: Ollero, M., Perez-Pe, R., Muiño-Blnco, T., nd Cerin- Perez, J. A Improvement of rm spermtozo cryopreservtion protocols ssessed y spermtozo qulity permeters nd heterogeneity nlysis. Cryoiology 37: Prks, J. E. nd Lynch, D. V Lipid composition nd thermotropic phse ehvior of or, ull, stllion, nd rooster spermtozo memrnes. Cryoiology 29: Ptel, J. M., Sekhrm, M. nd Block, E. R Vitmin E distriution nd modultion of the physicl stte nd function of pulmonry endothelil cell memrnes. Exp. Lung Res. 17: Pettitt, M. J. nd Buhr, M. M Extender components nd surfctnts ffect or spermtozo function nd memrne ehvior during cryopreservtion. J. Androl. 19: Poulos, A., Drin-Bennett, A. nd White, I. G The phospholipid-ound ftty cid nd ldehydes of mmmlin spermtozo. Comp. Biochem. Physiol. B46: Pursel, V. G. nd Grhm, E. F Phospholipids of ovine spermtoz nd seminl plsm. J. Reprod. Fertil. 14: SAS Institute, Inc SAS system for elementry sttisticl nlysis. SAS Institute, Inc., Cry, NC. Singh, P., Chnd, D. nd Georgie, G. C Effect of Vitmin E on lipid peroxidtion in ufflo Bulus ulis Indin. J. Exp. Biol. 27: Thurston, L. M., Wtson, P. F., Milehm, A. J. nd Holt, W. V. 1. Morphologiclly distinct spermtozo supopultions defined y Fourier shpe descriptors in fresh ejcultes correlte with vrition in or semen qulity following cryopreservtion. J. Androl. 22: Velsquez-Pereir, J., Chenoweth, P. J., McDowell, L. R., Risco, C. A., Stples, C. A., Prichrd, D., Mrtin, F. G., Clhoun, M. C., Willims, S. N. nd Wilkinson, N. S Reproductive effects of feeding gossypol nd Vitmin E to ulls. J. Anim. Sci. 76: Wtson, P. F. nd Mrtin, I. C Effects of egg yolk, glycerol nd the freezing rte on the viility nd crosoml structures of frozen rm spermtozo. Aust. J. Biol. Sci. 28: Wemheuer, W., Steinrink, J., Fuhrmnn, H., Schmidt, F. W. nd Sllmnnn, H. P The effect of vitmin A nd etcrotene on the Vitmin E sttus, ejcultion prmeters nd helth of or used for insemintion. Dtsch. Tierrztl. Wochenschr. 13: White, I. G Lipids nd clcium uptke of spermtozo in reltion to cold shock nd preservtion: review. Reprod. Fertil. Dev. 5:

9 Cn. J. Anim. Sci. Downloded from y on 2/18/18 For personl use only. This rticle hs een cited y: 1. L Rdomil, MJ Pettitt, KM Merkies, KD Hickey, MM Buhr. 11. Stress nd Dietry Fctors Modify Bor Sperm for Processing. Reproduction in Domestic Animls 46, [Crossref] 2. Elizeth Breininger, Norm B. Beorlegui, Cristián M. O Flherty, Mrth T. Beconi. 5. Alph-tocopherol improves iochemicl nd dynmic prmeters in cryopreserved or semen. Theriogenology 63:8, [Crossref]