Seasonal changes in reproductive activity, sperm variables and sperm freezability in Blanca Andaluza bucks

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1 Spnish Journl of Agriculturl Reserch 13(4), e43, 1 pges (215) eissn: Instituto Ncionl de Investigción y Tecnologí Agrri y Alimentri (INIA) RESEARCH ARTICLE OPEN ACCESS Sesonl chnges in reproductive ctivity, sperm vriles nd sperm freezility in Blnc Andluz ucks Lourdes Gllego-Clvo 1, M. Crolin Gtic 2, Julin Sntigo-Moreno 3, José L. Guzmán 1 nd Luis A. Zrzg 1 1 Universidd de Huelv, Cmpus de Excelenci Interncionl Agrolimentrio, ceia3, Deprtmento de Ciencis Agroforestles. 2 Ctr. Huelv-Plos de l Fronter s/n, Plos de l Fronter (Huelv), Spin. Universidd Arturo Prt, Fcultd de Recursos 3 Nturles Renovles. Avd. Arturo Prt, 212 Iquique, Chile. SGIT-INIA, Deprtmento de Reproducción Animl. Ctr. Coruñ km 7.5, 284 Mdrid, Spin Astrct Interest in the preservtion of endngered reeds such s the Blnc Andluz got, hs incresed nd some steps should e therefore tken to ensure it. The study ws designed to determine the sesonl reproductive pttern of Blnc Andluz ucks, nd whether this ffects the qulity of their semen nd its freezility over the yer. Seven ucks were used nd their ody weight, testiculr weight, plsm testosterone concentrtion nd fresh sperm qulity determined every week. The collected sperm ws cryopreserved nd stored; it ws then thwed nd the sme sperm qulity vriles mesured every fortnight. High plsm testosterone concentrtions were recorded during the summer nd utumn, nd low concentrtions were recorded during winter nd spring (p<.1). No differences were seen etween sesons in terms of the percentge of ucks ejculting, the percentge of ctive ucks, or ejculte volume. However, the sperm concentrtion, the totl numer of sperm per ejculte, nd the vlues for most fresh sperm vriles were lower during the winter period (t lest p<.5). After freezing-thwing, the qulity of winter-collected sperm ws etter, in some respects, thn tht of summer-collected sperm (t lest p<.5). These results revel tht Blnc Andluz ucks show sesonl reproductive ctivity in terms of their plsm testosterone concentrtion, ut no cler chnge in their sexul ehviour etween sesons ws oserved. The vlues of fresh sperm vriles lso vry over the yer, reching their lowest during winter. However, fter freezing-thwing, winter-collected sperm is of overll etter qulity thn sperm collected during the summer. Additionl key words: endngered got reed; testosterone; sesonlity; fresh semen; cooled semen; semen cryopreservtion. Arevitions used: BW (ody weight); LH (luteinizing hormone); LIN (linerity coefficient); STR (strightness coefficient); TW (testiculr weight); VAP (verge velocity); VCL (curviliner velocity); VSL (stright-line velocity); WOB (Wole coefficient); WWS (week within the seson). Cittion: Gllego-Clvo, L.; Gtic, M. C.; Sntigo-Moreno, J.; Guzmán, J. L.; Zrzg, L. A. (215). Sesonl chnges in reproductive ctivity, sperm vriles nd sperm freezility in Blnc Andluz ucks. Spnish Journl of Agriculturl Reserch, Volume 13, Issue 4, e43, 1 pges. Received: 18 Jun 215. Accepted: 29 Oct 215. Copyright 215 INIA. This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution-Non Commercil (y-nc) Spin 3. Licence, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Funding: INIA-CICYT (Spin), Grnt RZ Competing interests: All uthors declre tht there is no conflict of interest tht could e perceived s prejudicing the imprtility of the reserch reported. Correspondence should e ddressed to L.A. Zrzg: zrzg@uhu.es Introduction The Blnc Andluz reed of got, which is ntive to Spin nd dpted to Mediterrnen environmentl conditions, is endngered ccording to the Officil Ctlogue of Spnish Livestock Breeds (RD 2129/28; BOE, 29). Steps should therefore e tken to ensure its preservtion. In ddition to its use in the genetic improvement of livestock nimls, semen cryopreservtion is essentil in the preservtion of endngered genetic resources. It is not sure whether mle Blnc Andluz gots show reproductive sesonlity. Understnding how this might ffect semen qulity nd freezility over the yer could throw light on how to improve the qulity of cryopreserved sperm. The photoperiod hs een suggested the min fctor influencing sesonlity in uck reproductive ctivity (Delgdillo et l., 1993). Short dys nd decresing dylength stimulte the secretion of luteinizing hormone (LH), which in turn, induces testiculr growth nd the relese of testosterone, resulting in quntittive nd qulittive improvements in semen production plus incresed sexul ehviour. In contrst, long dys nd in-

2 2 Lourdes Gllego-Clvo, M. Crolin Gtic, Julin Sntigo-Moreno, José L. Guzmán nd Luis A. Zrzg cresing dylength reduce LH secretion nd testiculr growth, leding to fll in the plsm testosterone concentrtion, reduced sperm qulity, nd diminished sexul ehviour (Rouger, 1974; Muduuli et l., 1979; Corteel, 1981; Thimonier et l., 1986; Pelletier et l., 1988; Delgdillo & Chemineu, 1992; Zrzg et l., 29). The informtion in the literture regrding the freezility nd fertilizing cpcity of uck sperm collected during the reeding nd non-reeding seson is contrdictory. Some uthors report sperm freezility, frozenthwed sperm vriles (Nunez et l., 1982; Boue & Corteel, 1992; Muhuyi et l., 1992; Pintdo et l., 1992; Tuli & Holtz, 1995) nd the fertilizing cpcity of frozen-thwed sperm (Corteel et l., 1978) to e etter in sperm collected during the reeding seson. However, other uthors (Peskovtskov et l., 1974; mtter & Flukiger, 1982) report no sesonl differences. The ims of the present work were to: 1) determine whether Blnc Andluz ucks show sesonl pttern of reproductive ctivity; nd 2) exmine the qulity of frozen-thwed sperm collected t different times of the yer. The results otined could e of use in progrmmes designed to preserve this reed of got. Mteril nd methods Generl All procedures were performed y trined personnel in strict ccordnce with Spnish guidelines for the protection of experimentl nimls (RD 53/213; BOE, 213), nd in greement with Europen Union Directive 86/69. The study ws conducted t the University of Huelv experimentl frm (37º 2ºN, 6º 54 W), which meets the requirements of the Europen Community Commission for Scientific Procedure Estlishments (21/63; OJEU, 21). The nimls exmined were seven Blnc Andluz ucks (8 months old t the strt of the experiment), previously trined to mount teser doe nd to ejculte into n rtificil vgin. These nimls were fed dily with rley strw (d liitum), lucerne hy nd commercil concentrte, ccording to their ody weight nd in greement with INRA stndrds (Mornd-Fehr & Suvnt, 1988). All nimls hd free ccess to wter nd minerl locks contining trce elements nd vitmins. Experimentl design Dt collection egn on 14 th Novemer 212 nd ended on 9 th July 214. However, since the ucks were young t the strt of the experiment, only dt from the lst yer (July 213 to July 214) were used in nlyses. The experiment ws designed to determine the effect of seson on reproductive sttus nd sperm vriles., utumn, winter nd spring were defined s the periods etween June 21 st nd Septemer 22 nd, Septemer 23 rd nd Decemer 2 th, Decemer 21 st nd Mrch 2 th, Mrch 21 st nd June 2 th, respectively. Body weight, testiculr weight, nd plsm testosterone concentrtions Body weight (BW), testiculr weight (TW) nd plsm testosterone concentrtions were recorded weekly throughout the study. Testiculr weight ws ssessed y comprtive plption using n orchidometer; the sme opertor lwys performed this tsk (Oldhm et l., 1978). Blood for determining the plsm testosterone concentrtion ws otined y jugulr venipuncture, employing vcuum tues contining heprin. This ws performed once per week t 9: h over the entire experimentl period. Plsm ws otined y centrifuging the collected lood t 3 g for 3 min. It ws then stored t -2ºC until nlysis for testosterone using commercil enzymelinked immunossy (ELISA) kit (Demeditec Dignostics, Kiel-Wellsee, Germny). All plsm smples were nlysed t the sme time t the end of the experiment. The intr-ssy coefficient of vrition, estimted from plsm stndrds, ws 8.9% for smples contining.5 ng/ml testosterone, 5.1% for smples with 2 ng/ml, nd 7.3% for smples contining 16 ng/ml. Semen collection nd sexul ehviour Semen ws collected weekly. On ech occsion, the sexul ehviour of ech uck ws ssessed y presenting it with n intct oestrus-induced doe, llowing 5 min for the mle to ejculte. Oestrus ws induced in teser does vi sucutneous injection of 2 mg of oestrdiol cypionte (Sigm-Aldrich Químic, S.A., Spin) (Delgdillo et l., 1999; Zrzg et l., 29). The ejcultion ltency, the percentge of ucks tht ejculted, nd the percentge of ctive mles (ucks tht ttempted to ejculte t lest twice, ut did not chieve ejcultion within 5 min), were recorded. Animls were lwys tested in the sme order nd y the sme hndlers. Sperm evlution A totl of 324 ejcultes were evluted. The volume of ejculted semen ws recorded immeditely fter Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

3 Sesonlity of Blnc Andluz ucks 3 collection in grduted collection vil. Overll motility ws immeditely ssessed y trnsferring drop of undiluted semen to wrm slide (35ºC), plcing cover slip on it, nd oserving it under microscope t 4. Results were recorded on n ritrry scle of to 5 ( = no motility, 5 = 1% motility) (Bril et l., 1993). Sperm concentrtion nd kinemtic motility vriles were mesured using CASA system (ISAS, Proiser SL, Vlenci, Spin). Sperm concentrtion ws mesured employing Bürker chmer fter diluting n liquot of semen with.5% formldehyde sline solution (1:4) nd oserving t 4 mgnifiction. The totl numer of spermtozo per ejculte ws clculted from the ejculte volume nd sperm concentrtion. The kinemtic motility vriles recorded y the CASA system were: percentge of sttic, motile nd progressive motile spermtozo, curviliner velocity (VCL, µm/s, i.e., the velocity of the ctul trjectory of the sperm), stright-line velocity (VSL, µm/s, i.e., the velocity clculted using the stright-line distnce etween the eginning nd end of the sperm trck), verge velocity (VAP, µm/s, i.e., the velocity over the smooth clculted pth), nd the linerity coefficient (LIN), strightness coefficient (STR) nd wole coefficient (WOB). Sperm were clssified s eing of medium velocity when their VCL ws etween 45 nd 75 µm/s nd rpid when their VCL ws >75 µm/s. Sperm were deemed progressively motile when their trjectory ws stright for t lest 8% of the pth tken, s suggested y the mnufcturer of the CASA system. Sperm processing, chilling nd sperm freezing Selected semen smples, i.e., those with sperm concentrtion of > spermtozo/ml, nd n overll motility of 4 ccording to the ove criteri, were frozen every fortnight. These criteri determined tht totl of 128 ejcultes were processed. After checking tht these criteri were met, the chosen smples were diluted in wshing solution t 1:1 (v:v) (25 mm Tris, 28 mm glucose, 14 mm citric cid,.5% streptomycin, 5 UI penicillin/ml) t 37ºC, nd then centrifuged once (7 g for 15 min) to eliminte the seminl plsm. The superntnt ws then removed (now t room temperture [2ºC]) nd the pellet diluted in Tris-yolk extender (25 mm Tris, 28 mm glucose, 14 mm citric cid, 12% egg yolk,.5% streptomycin, 5 UI penicillin/ml nd distilled wter to 1 ml). After 5 min, similr volume of second extender (25 mm Tris, 28 mm glucose, 14 mm citric cid, 12% egg yolk, 8% glycerol,.5% streptomycin, 5 UI penicillin/ml, nd distilled wter to 1 ml) ws dded t room temperture (2ºC), resulting in finl sperm concentrtion of spermtozo/ml, 6% egg yolk nd 4% glycerol. Lecithin in the egg yolk ws inctivted y sujecting the ltter to 56ºC for 3 min efore its use in the extenders. Both extenders were prepred in the lortory using regent-grde chemicls purchsed from Pnrec Químic S.A. (Brcelon, Spin) nd the Sigm Chemicl Co. (St. Louis, MO, USA). The sperm ws then chilled from room temperture to 5ºC in cooler over period of lest 3 h, efore eing pcked in.25 or.5 ml plstic strws. Finlly, the strws were plced horizontlly on rck situted 4 cm ove the surfce of liquid nitrogen th for 15 min efore eing plunged into the sme th. The frozen strws were then stored in liquid nitrogen. Post-chilling nd post-thwing ssessment of sperm vriles Sperm qulity ws evluted fter the 3 h chilling period nd gin fter freezing-thwing; thwing ws performed no lter thn one dy fter freezing. The frozen strws were thwed in wter th t 37ºC for 3 s. Both the chilled nd frozen-thwed sperm ws trnsferred to wrm slide nd the kinemtic motility vriles mesured s descried ove. Sttisticl nlyses The effect of seson (spring, summer, utumn nd winter) nd week within the seson (WWS, with 13 weeks per seson) on BW, TW nd plsm testosterone concentrtion ws nlysed using the repeted mesures option of the Generl Liner Model procedure provided y the SPSS pckge for Windows (28). In this model, the different sesons nd WWS were tken s intr-suject fctors. Significnt differences in the influence of WWS were nlysed using the Bonferroni test. ANOVA including the mle s fixed fctor ws used to exmine the effect of seson on ll the studied vriles in fresh, chilled nd frozen-thwed sperm. Vriles expressed s percentges (sperm motility, ucks tht ejculted, nd percentge of ctive mles), nd the vlues for overll motility were rcsine-trnsformed efore nlysis. When differences etween sesons were detected, they were exmined using the Tukey t-test. ANOVA ws lso used to compre the effect of seson on the motility vriles of the rpid nd medium velocity fresh, chilled nd frozen-thwed sperm. Significnce ws set t p<.5. Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

4 4 Lourdes Gllego-Clvo, M. Crolin Gtic, Julin Sntigo-Moreno, José L. Guzmán nd Luis A. Zrzg Results Body weight, testiculr weight nd plsm testosterone concentrtion Repeted mesures nlysis showed seson to hve significnt effect on BW nd plsm testosterone concentrtion (p<.1). The BW ws higher during spring thn ny other seson, nd plsm testosterone ws higher during summer nd utumn thn during spring or winter (Tle 1). Moreover, the interction Seson WWS hd significnt (p<.1) effect on plsm testosterone, with concentrtions incresing over the weeks of summer, nd decresing over the weeks of utumn. During spring nd winter, testosterone concentrtions remined t low levels, during summer testosterone concentrtions incresed, nd during utumn they decresed (Fig. 1). Sexul ehviour nd fresh sperm vriles No differences were seen (Tle 2) etween sesons in terms of the percentge of ucks tht ejculted 9 Autumn Winter 8 Body weight (kg) Autumn Winter 35 Testiculr weight (g) Autumn Winter Testosterone concentrtions (ng/ml) July Aug. Sep. Oct. Nov. Dec. Jn. Fe. Mrch April My June July Month Figure 1. Weekly mens (±SEM) over one yer for ody weight (kg, top), testiculr weight (g, middle) nd plsm testosterone concentrtion (ottom, ng/ml) for seven Blnc Andluz ucks mintined under nturl photoperiod conditions. Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

5 Sesonlity of Blnc Andluz ucks 5 Tle 1. Live weight, testiculr weight nd plsm testosterone concentrtion over the four sesons of the yer. Vlues for ech seson re mens ± SEM. Autumn Winter Live weight (kg) 67.1 ± ±.7c 71.7 ±.8d 76.8 ±.7 Testiculr weight (g) ± ± ± ± 7.7 Testosterone concentrtion (ng/ml) 1. ± ± ± ±.36 Different letters in the sme row indicte significnt differences etween groups (p<.5). Tle 2. Sexul ehviour nd vlues for fresh sperm vriles over the four sesons of the yer. Vlues for ech seson re mens ± SEM. Autumn Winter Bucks tht ejculte (%) Active mles (%) Ejcultion ltency (s) 51.8 ± ± ± ± 3.6 Ejculte volume (ml).87 ±.4.91 ±.4.93 ±.9.95 ±.5 Semen concentrtion (1 6 sperm/ml) ± ± ± 449.4c ± 448.6c Totl sperm per ejculte (1 6 sperm/ejculte) ± ± ± ± Glol motility 4.52 ± ± ± ±.24 Different letters in the sme row indicte significnt differences etween groups (p <.5). (85.6%), the percentge of ucks considered ctive (93.%), ejcultion ltency (47. ± 2.8 s), or ejculte volume (.91 ±.3 ml). However, differences were seen etween sesons in terms of sperm concentrtion nd the numer of sperm per ejculte, with vlues lower during winter nd higher in summer (t lest, p<.1; Tle 2). Finlly, overll motility vried etween sesons (p<.1; Tle 2), with the lowest vlues recorded during winter. Fresh sperm kinemtic motility vriles The VSL, LIN, STR nd WOB vlues, nd the percentges of motile, rpid nd progressive spermtozo, vried etween seson, with the lowest vlues recorded during winter (t lest p<.5; Fig. 2). For the rpid spermtozo, only the vlues of VSL nd LIN vried etween sesons (Tle 3), with the lowest vlues recorded in winter nd the highest in summer. For the medium velocity spermtozo, the vlues of ll the kinemtic motility vriles vried etween sesons, gin with the lowest vlues recorded in winter nd the highest in summer (Tle 4). Chilled sperm vriles Only the vlues of VCL, LIN, STR nd WOB vried etween sesons (t lest p<.5; Fig. 3), with the highest VCL nd lowest LIN, STR nd WOB vlues recorded in utumn. For the rpid spermtozo, VSL, LIN, STR nd WOB ll vried etween sesons, with the highest vlues recorded in spring (Tle 3). For the medium velocity spermtozo, the vlues of ll the kinemtic motility vriles (except for VCL) vried etween sesons, with the lowest recorded in utumn (Tle 4). Frozen-thwed sperm vriles The VCL nd VAP results differed etween sesons, with higher vlues recorded in winter thn in summer. Very lrge differences were lso oserved in terms of the percentge of motile, rpid nd progressive spermtozo, with the est results recorded in winter (Fig. 4). When exmined seprtely, neither the rpid nor medium velocity spermtozo differed etween sesons in terms of ny kinemtic motility vrile (Tles 3 nd 4). Discussion The present results show tht, when mintined under nturl photoperiod conditions, Blnc Andluz ucks show mrked sesonl vrition in their reproductive ctivity, s mesured y the plsm testosterone concentrtion nd fresh sperm qulity nd freezility. When plsm testosterone concentrtions were high the ucks showed their lowest ody weight nd vice vers. In generl the lowest vlues for fresh semen vriles were recorded in winter. However, winter-collected sperm returned etter qulity results fter freezing-thwing thn summer-collected sperm. Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

6 6 Lourdes Gllego-Clvo, M. Crolin Gtic, Julin Sntigo-Moreno, José L. Guzmán nd Luis A. Zrzg Tle 3. Vlues for kinemtic motility vriles for rpid velocity spermtozo in fresh, chilled nd freeze-thwed sperm over the four sesons of the yer. Vlues for ech group re mens ± SEM. Vrile Autumn Winter Fresh semen VCL 18.3 ± ± ± ± 6. VSL 86.7 ± ± ± ± 5. VAP 1.6 ± ± ± ± 5.7 LIN 79.9 ± ± ± ± 4.4 STR 86. ± ± ± ± 4.6 WOB 92.8 ± ± ± ± 5. Chilled semen VCL 1.7 ± ± ± ± 1.5 VSL 54.6 ± ± ± ± 3.3 VAP 77.7 ± ± ± ± 2.2 LIN 54.2 ± ± ± ± 2.9 STR 69.7 ± ± ± ± 2.1 WOB 77.1 ± ± ± ± 1.6 Frozen-thwed semen VCL 85.1 ± ± ± ± 1. VSL 56.2 ± ± ± ± 2.1 VAP 69.7 ± ± ± ± 1.3 LIN 62.7 ± ± ± ± 2.3 STR 76.4 ± ± ± ± 1.9 WOB 77.8 ± ± ± ± 1.1 VCL, curviliner velocity, µm/s; VSL, stright-line velocity, µm/s; VAP, verge pth velocity, µm/s; LIN, linerity coefficient, %; STR, strightness coefficient, %; WOB, Wole coefficient, %. Different letters in the sme row indicte significnt differences etween groups (p<.5). Tle 4. Vlues for kinemtic motility vriles for medium velocity spermtozo in fresh, chilled nd freeze-thwed sperm over the four sesons of the yer. Vlues for ech group re mens ± SEM. Vrile Autumn Winter Fresh semen VCL 63. ± ± ± ± 2.8 VSL 5.4 ± ± ± ± 2.8 VAP 57.9 ± ± ± ± 2.9 LIN 8.3 ± ± ± ± 4.1 STR 87.1 ± ± ± ± 4. WOB 92. ± ± ± ± 4. Chilled semen VCL 62.6 ± ± ± ±.8 VSL 36. ± ± ± ± 1.2 VAP 48.5 ± ± ± ± 1.1 LIN 57.4 ± ± ± ± 1.9 STR 73.6 ± ± ± ± 1.1 WOB 77.4 ± ± ± ± 1.4 Frozen-thwed semen VCL 59.6 ± ± ± ±.7 VSL 39.8 ± ± ± ±.7 VAP 49.3 ± ± ± ±.6 LIN 66.3 ± ± ± ± 1.3 STR 79.6 ± ± ± ± 1. WOB 82.5 ± ± ± ±.7 VCL: curviliner velocity, µm/s; VSL: stright-line velocity, µm/s; VAP: verge pth velocity, µm/s; LIN: linerity coefficient, %; STR: strightness coefficient, %; WOB: Wole coefficient, %. Different letters in the sme row indicte significnt differences etween groups (p<.5). The plsm testosterone concentrtions recorded were clerly ssocited with the nturl photoperiod; high testosterone concentrtions were recorded during summer nd utumn (decresing dylength), nd low concentrtions were recorded during winter nd spring (incresing dylength). These results revel the existence of well-defined reeding seson chrcterised y high testosterone production in these nimls. The sesonl chnges in testosterone secretion seen were very similr to those reported for Pyoy ucks living Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

7 Sesonlity of Blnc Andluz ucks 7 VSL (µm/s) STR (%) WOB (%) LN (%) Motile espermtozo (%) Autumn Winter Seson Rpid espermtozo (%) Autumn Winter Seson Progressive espermtozo (%) c Autumn Seson Winter Figure 2. Men vlues (±SEM) for stright-line velocity (VSL, µm/s), linerity coefficient (LIN, %), strightness coefficient (STR, %), Wole coefficient (WOB, %), motile spermtozo (%), rpid spermtozo (%) nd progressive spermtozo (%) in fresh semen over the four sesons of the yer (: n=86; Autumn: n=8; Winter: n=76; : n=82). Different letters ove the histogrm rs, indicte significnt differences etween groups (p<.5). c VCL (µm/s) STR (%) Autumn Winter Seson Autumn Winter Seson Figure 3. Men vlues (±SEM) for curviliner velocity (VCL, µm/s), linerity coefficient (LIN, %), strightness coefficient (STR, %), Wole coefficient (WOB, %) in chilled sperm over the four sesons of the yer (: n=4; Autumn: n=4; Winter: n=24; : n=24). Different letters ove the histogrm rs, indicte significnt differences etween groups (p<.5). LIN (%) WOB (%) Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

8 8 Lourdes Gllego-Clvo, M. Crolin Gtic, Julin Sntigo-Moreno, José L. Guzmán nd Luis A. Zrzg VCL (µm/s) VAP (µm/s) Motile spermtozo (%) Autumn Winter Seson Rpid spermtozo (%) c c Autumn Winter Seson Progressive spermtozo (%) Autumn Winter Seson Figure 4. Men vlues (±SEM) for curviliner velocity (VCL, µm/s), verge pth velocity (VAP, µm/s), motile spermtozo (%), rpid spermtozo (%) nd progressive spermtozo (%) in frozen-thwed sperm over the four sesons of the yer (: n=4; Autumn: n=4; Winter: n=24; : n=24). Different letters ove the histogrm r, indicte significnt differences etween groups (p<.5). t the sme ltitude (Zrzg et l., 29), for Creole ucks in Mexico (Delgdillo et l., 1999; 24), for Blnc Andluz ucks under rtificil photoperiod (Gllego-Clvo et l., 215) nd ucks of other Mediterrnen got reeds (Todini et l., 27). However, in Alpine ucks living t 46ºN, increses in plsm testosterone re delyed until lte August Septemer (Delgdillo & Chemineu, 1992). This is proly due to longer lg time etween the perception of the photoperiodic signl nd the expression of physiologicl responses in these nimls (Delgdillo et l., 24). The chnges seen in plsm testosterone were inversely ssocited with chnges in BW. Such BW chnges hve previously een reported in oth sexes of this species (Delgdillo et l., 1991; Wlkden- Brown et l., 1994; Delgdillo et l., 1999, 24; Zrzg et l., 29). It hs een suggested tht differences in food intke might explin them (Wlkden- Brown et l., 1994; Argo et l., 1999). It my e tht, s the mles show more reeding ctivity due to their higher testosterone concentrtions (even homosexul ehviour), their rowsing time is reduced. Lower sperm concentrtions, smller totl numers of spermtozo per ejculte, nd lower overll motility were lso seen during winter when plsm testos- terone ws low. These findings re similr to those otined y Krginnidis et l. (2) who worked with Alpine, Snen nd Dmscus gots, nd tht reported y Roc et l. (1992), Pérez & Mteos (1996), Zrzg et l. (29) nd Dordo et l. (21) for other Spnish got reeds. However, the sesonl chnges in plsm testosterone were not ssocited with ny vrition in TW, the percentge of ejculting ucks or ctive ucks, ejcultion ltency, or ejculte volume. This contrsts with results otined y our group for Pyoy ucks (Zrzg et l., 29), nd suggests tht Blnc Andluz ucks re less sesonl thn other Spnish got reeds t lest in terms of the ove vriles. Recently, Gllego-Clvo et l. (214), working with femles of this reed, reported tht round 1% of does showed ovrin ctivity throughout the yer; this hs not een descried for other Spnish got reeds. For the fresh sperm, the vlues for the kinemtic motility vriles nd the percentges of motile, rpid nd progressive sperms were lower during winter compred to summer, with no differences seen etween the other sesons. This, long with the lower sperm concentrtion, suggests winter to e the worst period during which to collect Blnc Andluz semen. This Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

9 Sesonlity of Blnc Andluz ucks 9 grees with previous results otined y our group on Pyoy ucks (Zrzg et l., 29). In tht erlier experiment, the vlues for VCL, VSL nd VAP were t their lowest in Decemer. It ws surprising to see, therefore, tht winter-collected sperm showed etter post-thw qulity thn summer-collected sperm. It my e tht the lrger numer of sperms in the summer ejcultes impeded the removl of the seminl plsm y the wshing solution (the sme volume ws used in oth sesons), leding to its poorer cryopreservtion. Some enzymes produced in the ulourethrl glnd re responsile for the degrdtion of egg yolk nd skimmed milk products, producing compounds toxic to sperm (Roy; 1957; Iritni & Nishikw, 1961). The vlues for the kinemtic motility vriles nd the percentge of motile spermtozo decresed progressively from fresh to chilled to frozen thwedsperm. This might e expected since this kind of processing cn e hrmful to sperm ultrstructure, iochemistry nd function (Wtson, 2), resulting in reduced motility, memrne integrity, nd fertilizing cpcity (Purdy, 26). In conclusion, the results of this work support the ide tht Blnc Andluz ucks re sujected to mrked sesonlity in terms of their plsm testosterone concentrtion, with more intense secretion occurring during summer nd utumn (decresing dylength). However, no cler chnges in their sexul ehviour re seen over the yer. The lowest fresh semen qulity ws otined in winter, period with very low testosterone concentrtions. However, fter freezing-thwing, winter-collected sperm returned etter sperm qulity results thn summer-collected sperm. Acknowledgments The uthors thnk the Asocición Ncionl de Cridores de Gndo Cprino de Rz Blnc Andluz (ABLANSE) for supplying the nimls used. References Argo CM, Smith JS, Ky RNB, Sesonl chnges of metolism nd ppetite in Soy rms. Anim Sci 69: Bril G, Chemineu P, Cognie Y, Guerin Y, Leoeuf B, Orgeur P, Vllet JC, Mnuel de formtion pour l insémintion rtificielle chez les ovins et les cprins. FAO, Rome. 193 pp. BOE, 29. Royl decree 2129/28, of 26th Decemer, out the conservtion, improvement nd promotion of livestock reeds Ntionl Progrm. Boletín Oficil de Estdo, No. 23, 27/1/29, pp [In Spnish]. BOE, 213. Royl decree 53/213, of 1st Ferury, out the niml protection policy for nimls used in scientific experiments. Boletín Oficil del Estdo No. 34, 8/2/213, pp [In Spnish]. Boue P, Cortee1 JM, Aptitude of mle got sperm to withstnd freezing: Comined effects of seson nd time of sexul rest etween two successive collections. In: Recent dvnces in got production; Lokeshwr RR (ed.). pp: Nutn Printers, New Delhi, Indi. Corteel JM, Collection, processing nd rtificil insemintion of got semen. In: Got production; Gll C (ed). pp: Acdemic Press, London. Corteel JM, Bril G, Leoeuf B, Mrcellier N, Voies disponiles pour ugmenter l utilistion des meilleurs oucs. 4ème Journées Rech. Ovine et Cprine. INRA- ITOVIC, Pris, pp: Delgdillo JA, Chemineu P, Aolition of the sesonl relese of luteinizing hormone nd testosterone in Alpine mle gots (Cpr hircus) y short photoperiodic cycles. J Reprod Fertil 94: org/1.153/jrf Delgdillo JA, Leoeuf B, Chemineu P, Decrese in the sesonlity of sexul ehvior nd sperm production in ucks y exposure to short photoperiodic cycles. Theriogenology 36: Delgdillo JA, Leoeuf B, Chemineu P, Mintennce of sperm production in ucks during third yer of short photoperiodic cycles. Reprod Nutr Dev 33: Delgdillo JA, Cnedo GA, Chemineu P, Guillume D, Mlpux B, Evidence for n nnul reproductive rhythm independent of food vilility in mle Creole gots in sutropicl northern Mexico. Theriogenology 52: qwq12 Delgdillo JA, Cortez ME, Durte G, Chemineu P, Mlpux B, 24. Evidence tht the photoperiod controls the nnul chnges in testosterone secretion, testiculr nd ody weight in sutropicl mle gots. Reprod Nutr Dev 44: Dordo J, Muñoz-Serrno A, Hidlgo M, 21. The effect of cryopreservtion on got semen chrcteristics relted to sperm freezility. Anim Reprod Sci 121: Gllego-Clvo L, Gtic MC, Guzmán JL, Zrzg LA, 214. Role of ody condition score nd ody weight in the control of sesonl reproduction in Blnc Andluz gots. Anim Reprod Sci 151: org/1.116/j.nireprosci Gllego-Clvo L, Gtic MC, Sntigo-Moreno J, Guzmán JL, Zrzg LA, 215. Exogenous meltonin does not improve the freezility of Blnc Andluz got semen over exposure to two months of short dys. Anim Reprod Sci 156: Iritni A, Nishikw Y, Studies on the egg yolk cogulting fctors in got semen: II properties of the cogulting fctor nd influentil conditions for cogultion. Proc. Silver Juilee Lortory of Animl Husndry, Kyoto University (Jpn), pp: Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43

10 1 Lourdes Gllego-Clvo, M. Crolin Gtic, Julin Sntigo-Moreno, José L. Guzmán nd Luis A. Zrzg Krginnidis A, Vrskeli S, Krtzs G, 2. Chrcteristics nd sesonl vritions in the semen of Alpine, Snen nd Dmscus got ucks orn nd rised in Greece. Theriogenology 53: org/1.116/s93-691x()272-7 Mornd-Fehr P, Suvnt D, Got nutrition. In: Alimenttion des ovins, ovins et cprins; Jrrige R (ed.). pp: INRA, Pris. Muduuli S, Snford LM, Plmer WM, Howlnd BE, Secretory ptterns nd circdin nd sesonl chnges in luteinizing hormone, follicle stimulting hormone, prolctin nd testosterone in the mle Pygmy got. J Anim Sci 49: Muhuyi EZ, Dronis EZ, Nelxon EA, Lin TY, Seson, reed nd ge influence on production nd freezility of diry got semen. Proc. 3rd Int Conf on Got Production nd Diseses, Tucson, AZ (USA). Jn p: 283. Nunez JF, Corteel JM, Commous Y, Bril G, Study of the involvement of seminl plsm constituents in the sesonl vritions of got spermtozo motility. Proc. 3rd Int Conf on Got Production nd Diseses, Tucson, AZ (USA). Jn p: 285. OJEU, 21. Directive 21/63/EU of the Europen Prliment nd of the Council of 22 Septemer 21 on the protection of nimls used for scientific purposes. Officil Journl of the Europen Union, No. 276: Oldhm CM, Adms NR, Gherrdi PB, Lindsy DR, McKintosh JB, The influence of level of food intke on sperm-producing cpcity of testiculr tissue in the rm. Aust J Agric Res 29: AR Pelletier J, Chemineu P, Delgdillo JA, Sesonlity of sexul ctivity nd its photoperiodic control in the dult rm nd he-got. Proc. 11th Int Congr Anim Reprod & A. I., Dulin (Irelnd). June Vol 5, pp: Pérez B, Mteos E, Effect of photoperiod on semen production nd qulity in ucks of Vert nd Mlgueñ reeds. Smll Ruminnt Res 22: org/1.116/s (96)887-5 Peskovtskov AP, Vsitjev GI, Dzumghiev M, First results of rtificil insemintion with frozen semen. Zhivotnovodstvo 8: [In Russin]. Pintdo B, Perez B, Mteos E., Effect of seson on freezility of Verto uck spermtozo. In: Recent dvnces in got production; Lokeshwr RR (ed.). pp: 142- l45. Nutn Printers, New Delhi, Indi. Purdy PH, 26. A review on got sperm cryopreservtion. Smll Ruminnt Res 63: org/1.116/j.smllrumres Roc J, Mrtínez E, Vázquez JM, Coy P, Chrcteristics nd sesonl vritions in the semen of Murcino-Grndin gots in the Mediterrnen re. Anim Reprod Sci 29: Rouger Y, Etude des interctions de l environnement et des hormones sexuelles dns l régultion du comportement sexuel des Bovide. Doctorl thesis, Université de Rennes, Frnce. Roy A, Egg yolk cogulting enzyme in the semen nd Cowper s glnd of the got. Nture 179: dx.doi.org/1.138/ SPSS, 28. SPSS sttistics se user s guide 17.. Sttisticl Pckge for the Socil Sciences, SPSS Inc., Chicgo, USA. 64 pp. mtter P, Flukiger A, Buck semen processing during off reeding seson. Proc. 3rd Int Conf on Got Production nd Diseses, Tucson, AZ (USA). Jn p:.284. Thimonier J, Terqui M, Chemineu P, Conduite de l reproduction de petits ruminnts dns les differentes prties du monde. Proc Inter Atomic Energy Agency, pp: 135-l47. Todini L, Mlftti A, Terzno GM, Borghese A, Pizzillo M, Deenedetti A, 27. Sesonlity of plsm testosterone in mles of four Mediterrnen got reeds nd in three different climtic conditions. Theriogenology 67: Tuli RK, Holtz W, Effect of seson on the freezility of Boer got semen in the northern temperte zone. Theriogenology 43: Wlkden-Brown SW, Restll BJ, Norton BW, Scrmuzzi RJ, Mrtin GB, Effect of nutrition on sesonl ptterns of LH, FSH nd testosterone concentrtion, testiculr mss, seceous glnd volume nd odour in Austrlin cshmere gots. J Reprod Fertil 12: Wlkden-Brown SW, Norton BW, Restll BJ, Sesonl vrition in voluntry feed intke in Cshmere ucks fed d liitum diets of low or high qulity. Aust J Agric Res 45: Wtson PF, 2. The cuses of reduced fertility with cryopreserved semen. Anim Reprod Sci 6-61: dx.doi.org/1.116/s ()99-3 Zrzg LA, Guzmán JL, Domínguez C, Pérez MC, Prieto R, 29. Effects of seson nd feeding level on reproductive ctivity nd semen qulity in Pyoy uck gots. Theriogenology 71: Spnish Journl of Agriculturl Reserch Decemer 215 Volume 13 Issue 4 e43