A. Najjar, B. Benaoun, M. Ezzaouia, A. Ben Maâtoug, M. Magistrini and M. Ben Mrad

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1 Americn-Eursin J. Agric. & Environ. Sci., 8 (2): , 2010 ISSN IDOSI Pulictions, 2010 Determintion of Semen nd Sexul Behvior Prmeters of Arin Stllions to Be Selected for n Artificil Insemintion Progrm under Tunisin Conditions A. Njjr, B. Benoun, M. Ezzoui, A. Ben Mâtoug, M. Mgistrini nd M. Ben Mrd 1 Institut Ntionl Agronomique de Tunisie, 43, venue Chrle Nicolle, Cité Mhrgène, 1082, Tunis, Tunisie 2 Hrs Ntionl de Sidi Thet, Fondtion Ntionle de l Améliortion de l Rce Chevline, Tunisie 3 Lortoire BESTMOD, Institut Supérieur de Gestion de Tunis, 41, rue de l lierté, Cité Bouchouch 2000 Le Brdo, Tunis, Tunisie 4 INRA UMR 85 Physiologie de l Reproduction et des Comportements, Nouzilly, Frnce Astrct: The present study imed to evlute the sexul ehvior nd semen prmeters qulity of Tunisin Arin stllions leding in nturl reed nd sheltered under Tunisin conditions, to select them for n rtificil insemintion (AI) progrm. Semen ws collected nd nlyzed from 9 stllions ged etween 7 to 23 yers during the reeding seson (young stllions: ge <15 yers, n = 5, numer of ejcultes = 25; old stllions: ge = 15 yers, n = 4, numer of ejcultes = 18). Sexul ehvior, seminl qulittive nd quntittive prmeters were investigted immeditely fter collection. The percentge of motile sperm ws studied in the semen stored t 4 C in the semi skimmed milk extender nd ntiiotics t 0 hour nd 24, 48 nd 72 hours fter collection. ANOVA ws crried to compre vriles etween the two ge clsses, using softwre RATS (version.10). We found tht the semen qulity of young Arin stllions ws cceptle to e used in n (AI) progrm with fresh, refrigerted nd frozen semen. However, the semen of old stllions hd low qulity nd consequently they were llowed only to e used in AI progrm with fresh semen. Our study provided us the knowledge of sexul ehvior nd seminl prmeters of our Arin stllions nd the distinction etween young stllions hving high qulity of semen nd old stllions hving low qulity of semen. Key words: AI Sexul ehvior Semen qulity Tunisin Arin stllions INTRODUCTION sufertility of the stllion. This recommendtion is importnt for good mngement of stllions used in AI During the reeding seson, stllions in nturl progrm [1]. service mte gret numer of mres. This crried the The ims of the present study were to evlute the trnsmission of sexul diseses nd decrese fertility. sexul ehvior prmeters during collection nd the Therefore, to understnd the cuses for decresed semen qulity collected for the first time from Tunisin fertility in stllions, n evlution of fresh semen qulity Arin stllions known for their potentil performnces collected y n rtificil vgin cn e men to revel nd prcticed exclusively nturl service, in order to select these cuses nd to predict fertility of semen stllion them for n AI progrm. [1, 2]. It could lso suspect low fertility nd select nimls for rtificil insemintion (AI) progrm [3]. Besides, MATERIALS AND METHODS semen evlution could e utilized to determine if the semen of the stllion cn tolerte hndling procedures Generl: The study took plce in the stud frm of Sidi such s cooling or freezing [4]. Semen exmintion is Thet, situted in the north of Tunisi t recommended prior to reed mres t the eginning of N, E. Semen ws collected from 9 Arin the reeding seson or when we suspect infertility or stllions (young stllions: ge <15 yers, n = 5, numer of Corresponding Author: A. Njjr, Institut Ntionl Agronomique de Tunisie, 43, venue Chrle Nicolle, Cité Mhrgène, 1082, Tunis, Tunisie 173

2 Am-Eurs. J. Agric. & Environ. Sci., 8 (2): , 2010 ejcultes = 25; old stllions: ge = 15 yers, n = 4, numer of ejcultes = 18). The collection undertook during the officilly Tunisin reeding seson, strting from 15 Ferury to 15 June Semen collection: Ejcultes were collected once every dy for six consecutive dys from ech stllion using Missouri rtificil vgin (IMV, Frnce). The rtificil vgin ws lurificted immeditely with non spermicidl luricnt (NETTEX, U.K) prior to semen collection. An estrus mre ws used for semen collection. Sexul ehvior such s preprtion durtion, numer of mounting in the rtificil vgin nd collection durtion were mesured during ech collect s descried y Clément et l. [5] nd Hrs Ntionux []. Semen evlution: The collected fresh semen ws evluted for color nd volume. When the gel frction ws present, it ws removed y suitle filter s guze with hydrophilic stitch to estimte the volume of sperm (ml) s descried y Hrs Ntionux []. Sperm concentrtion (10 spermtozo / ml) ws determined y the use of Thom cell chmer fter 1:40 dilution of rw semen using formldehyde (1.85%) s reported y Hrs Ntionux []. Moreover, sperm morphology fetures were studied from unstined smple semen fixed previously y the sme rte of dilution formldehyde solution nd oserved y phse-contrst microscope [1]. Totl sperm ws lso determined in the ejculte 9 (10 spermtozo) y multiplying sperm concentrtion y semen volume []. A suject pprisl of sperm motility ws chieved y optic microscopy s the sme used y Rousset et l. [3] nd Hrs Ntionux [] to estimte the percentge of motile spermtozo in the rw semen smples immeditely fter filtrtion. Then, the semen ws extended with semi skimmed milk nd ntiiotics Gentmicin nd Penicillin G. The percentge of ded spermtozo ws estimted y the use of n eosin-nigrosin stin t.9 ph nd counting smple of 150 spermtozo over the slide [3, ]. The percentge of motile spermtozo ws evluted gin in the semen extended under the optic microscope t 0 hour nd fter 24, 48 nd 72 hours of cool storge (4 C) fter semen collection []. Sttisticl nlysis: The sttisticl nlysis ws developed on Pnel dt [7]. The sttisticl softwre RATS (version.10) ws used for descriptive sttistics. ANOVA ws crried to compre vriles etween the two ge clsses. The Fisher s test ws used to compre the individul nd temporl prmeters of the semen. Student s t-test ws used to compre the percentge of motile spermtozo in rw nd in diluted semen. The threshold of significnce ws 5%. RESULTS AND DISCUSSION The overll mens for sexul ehvior re presented in Tle 1. The numer of mounting in the rtificil vgin ws higher in the young stllions thn in the old ones (p < 0.05). We noted tht times of collection stllions hve een less thn 200 seconds in the two ge clsses of stllions which ws in greement with Noue et l. [9]. But this prmeter ws higher in the young stllions thn the old ones (p < 0.05). A significnt individul effect found for these sexul ehvior prmeters (p < 0.01). We lso found tht the numer of mounts nd the collection time ffected the fertility of fresh semen such s the numer of ded spermtozo nd the percentge of motile spermtozo in the rw semen (p < 0.05). This result ws not in greement with those reported y Clément et l. [5] who showed tht there wsn t ny determined reltion etween fertility nd sexul ehvior. However, Sieme et l. [8] found tht semen volume, sperm concentrtion nd longevity of spermtozo were influenced y the numer of mounts. Our results showed n individul effect on seminl prmeters such s percentge of motile spermtozo in rw semen, norml sperm, totl sperm, volume of gel frction nd semen volume (p < 0.01). The individul vriility etween stllions for seminl prmeters found is in greement with the previous reports [10, 11]. This lrge difference in semen prmeters mong individuls showed tht rndom fluctutions over time re involved [12]. We lso noted tht 1% of totl collected ejculte represented volume of gel frction which vried from 0.4±0.2 ml for young stllions to 0.7±0. ml for old stllions (p < 0.01) (Tle 2). Tle 1: Sexul ehvior of Tunisin Arin stllions (mens±sem) Preprtion time Collection time Stllions Numer of mounts (seconds) (second) Age < 15 yers (n = 5) 1.± ±1 13±1 Age = 15 yers (n = 4) 1.2±0.1 89±14 117±14, : p <

3 Am-Eurs. J. Agric. & Environ. Sci., 8 (2): , 2010 Tle 2: Semen chrcteristics of Tunisin Arin stllions (mens±sem) Filtered volume Volume of gel Sperm concentrtion Totl sperm Percentge of ded Percentge of motile Stllions (ml) frction (ml) (millions spermtozo / ml) (illions spermtozo) spermtozo (%) spermtozo in rw semen Age < 15 yers (n = 5) 38±3 0.4±0.2 c 259±2 e 9±0.8 e 29±2 9±3 Age = 15 yers (n = 4) 28±11 0.7±0. d 247±41 f 7±1.1 f 41±4 53±5, : p < 0.01; c, d: p < 0.1; e, f: p < 0.05 Tle 3: Anorml sperm of Tunisin Arin stllion (mens±sem) Anorml Anorml Anorml Anorml principl Cytoplsm Anorml Stllions hed (%) flgell (%) mid-piece (%) piece (%) droplets (%) sperm (%) Age < 15 yers (n=5) 3.4± ± ±1.8 2± ± ±2.2 Age = 15 yers (n=4) 7.7± ± ±1.5 1± ± ±2.4, : p < 0.05 The sperm volume filtered (Tle 2) ws spermtozo / ml nd reported tht infertile nd norml higher in young stllions thn in the old stllions mles hd low concentrtions (93± spermtozo) (38±3 ml vs 28±11 ml, p < 0.01). The volume of old nd low percentge of motile spermtozo in diluted stllions hs een inferior to the minimum vlue indicted semen. y Tiry et l. [13] which ws 30±7.1 ml for Arin Seeing the lowest vlues of sperm concentrtion, stllions. Nevertheless young stllions pssed eyond totl sperm nd percentge of motile spermtozo in rw this vlue. In his severl studies on seminl nd the highest vlues of the percentge of ded chrcteristics of stllions, Jsko [1] found tht semen spermtozo otined in old stllions, we considered tht volume vried from 45±30 ml to 58±24 ml. In this cse, the these stllions hd low fertility in their fresh semen semen volumes tht our Arin stllions got were compred to the young stllions. inferior to those reported y Jsko [1]. This indicted In the two ge clsses of stllions, we found tht the vriility of nimls sheltered under different climtic numer of ded spermtozo ws incresed y the conditions. numer of mounting, the presence of gel frction nd the The sperm concentrtion ws inclined to go up time of collection (p < 0.01). The numer of mounting nd in the young stllions compred to the old stllions the presence of gel frction ffected the percentge of (259±29 vs 247±41 millions of spermtozo / ml, p < 0.1). motile spermtozo in the rw semen (p < 0.01). We found lso tht the totl sperm ws higher in young The percentge of norml sperm ws less in stllions thn the old stllions (9±0.8 vs 7±1.1 illions the young stllions thn in the old stllions spermtozo; p <0.05) (Tle 2). However, the percentge (25.7±2.2% vs 31.4±2.4%, p < 0.05) (Tle 3). The of ded spermtozo nd the percentge of motile percentge of norml hed, mid-piece nd cytoplsm spermtozo in rw semen were higher in the old droplets were higher in the old stllions thn in the young stllions thn the young ones (41±4 vs 29±2%, p < 0.05; stllions (7.7±1.1, 17.7±1.5 nd 3.5±0.5% vs 3.4±0.5, 9±3 vs 53±5%, p < 0.01) (Tle 2). We lso oserved 15.8±1.8 nd 2.7±0.5%). However, percentge of norml tht there ws light increse in motility when the semen principl piece ws higher in the young stllion thn the ws extended in the two ge clsses, ut our sttisticl old ones (2±0.4% vs 1±0.3). Clément et l. [15] nd Brito nlysis showed no difference etween motility in rw [1] showed when there ws high percentge of nd in extended semen. norml sperm, this led to suspect the cse of Tiry et l. [13] found tht the Arin stllions cn sufertility. Posor [17] reported lso tht some stllions hve in their semen concentrtion from 149.9±100 to hving high percentge of norml sperm could 28.8±8 10 spermtozo / ml nd Jsko [1] found tht it present normlities in the reproductive trct specilly ws vried from 173±118 to 335± spermtozo / ml. vricocele. Moreover, Jsko et l. [18] reported tht there However, Chevlier-Clément et l. [14] in her study on ws negtive correltion etween sperm hed defect nd infertile stllions defined stllion hving norml fertility the per cycle pregnncy rtes. Love et l [19] found with men sperm concentrtion of 194±10 10 tht 1% in the percentge of hed defect led reduction 175

4 % Am-Eurs. J. Agric. & Environ. Sci., 8 (2): , 2010 ge < 15 yers, : p<0.01 ge = 15 yers Yers Percentge of motile spermtozo t 0h Percentge of motile spermtozo fter 24h Percentge of motile spermtozo fter 48h Percentge of motile spermtozo fter 72h Fig. 1: Percentge of motile spermtozo during storge for 72 hours fter collection t 4 C (mens±sem) of 0.7% in the per cycle pregnncy rtes. Other uthors CONCLUSION [20, 21] reported tht the percentge of detched hed in norml ejculte should e low thn 5%. Crd [22] Our study ws tken to prove tht semen ffirmed tht if there ws high percentge of detched evlution is importnt to identify the fertile Tunisin sperm hed, it indicted norml spermtogenesis or Arin stllions nd to select them for n AI progrm. It senescence in the trct. For the percentge of cytoplsm provided us the knowledge of sexul ehvior nd droplets, it s lso correlted negtively with the per cycle seminl prmeters of our Arin stllions sheltered pregnncy rtes [23]. Jsko et l. [18] showed tht there under Tunisin conditions nd the distinction etween ws negtive correltion etween proximl cytoplsm young stllions whose semen cn e used in AI droplets nd per cycle pregnncy rtes. However, progrm with fresh, refrigerted or frozen semen nd Love et l. [19] showed tht it wsn t correltion old stllions whose semen cn e used only in AI etween proximl cytoplsm droplets nd fertility. We cn progrm with fresh semen. dd tht Colenrnder et l. [2] showed when the percentge of norml nd motile sperm ws elow 40% the ACKNOWLEDGEMENT fertility ws compromised. So, for the old stllions of our study, it ws lso The uthors thnk the personnel of the stud frm of necessry to exmine the orgns of their reproductive Sidi Thet for their help during this work. trct in order to etter understnd the origin of the lowest fertility in their fresh semen. REFERENCES The percentge of motile spermtozo extended in semi-skimmed milk with ntiiotics stored t 4 C ws 1. Jsko, D.J., Evlution of stllion semen. decresed during its conservtion for 72 hours fter Veterinry Clinics of North Americ, Equine Prctice, collection in the two ge clsses of stllions (Figure 1). 8(1): The decrese in the percentge of motile spermtozo ws 2. Colenrnder, B., B. Gdell nd T.A.E. Stout, higher in the old stllions compred to the young ones The predictive vlue of semen nlysis in the (p < 0.01). Consequently, the semen of old stllions were evlution of stllion fertility. Reprod. Dom. Anim., llowed only to e used for n AI progrm with fresh 38: semen ccording to the work of Hrs Ntionux [], 3. Rousset, H., Ph. Chnteloue, M. Mgistrini nd Clément et l. [15] nd Clément et l. [5]. For the young E. Plmer, Assessment of fertility nd semen stllions under study, the motility of their spermtozo evlutions of stllions. J. Reprod. Fert. Suppl., during 24, 48 nd 72 hours llowed them to e exploited 35: for n AI progrm with fresh, refrigerted or frozen semen 4. Mgistrini, M., Semen evlution. In: nd this ccording to Mgistrini [24], who reported tht Equine Breeding Mngement nd Artificil motility of extended semen fter 24 nd 48 hours is n Insemintion. Smper JC, editor. Americn College of importnt prmeter in order to select stllions for AI. Theriogenologists, pp:

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