EFFECT OF DIFFERENT THAWING METHODS ON BULL S SEMEN CHARACTERISTICS

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1 ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS Volume Numer 3, EFFECT OF DIFFERENT THAWING METHODS ON BULL S SEMEN CHARACTERISTICS Mrtin Doležlová 1, Mrtin Ptáček 1, Luděk Stádník 1, Jromír Ducháček 1 1 Deprtment of Animl Husndry, Fculty of Agroiology, Food nd Nturl Resources, Czech University of Life Sciences Prgue, Kmýcká 957, Prgue 6 Suchdol, Czech Repulic Astrct DOLEŽALOVÁ MARTINA, PTÁČEK MARTIN, STÁDNÍK LUDĚK, DUCHÁČEK JAROMÍR Effect of Different Thwing Methods on Bull s Semen Chrcteristics. Act Universittis Agriculture et Silviculture Mendeline Brunensis, 65(3): The im of the study ws to evlute the influence of different thwing methods on ull s semen chrcteristics. The semen ws collected nd processed from 8 ulls (Holstein, n = 4; Czech Fleckvieh, n = 4) kept in privte Sire insemintion Centre. Four different thwing methods were tested: control thwing methods (temperture = 38.5 C, time = 30 seconds), slow thwing method (temperture = 30 C, time = 50 seconds), moderte thwing method (temperture = 50 C, time = 15 seconds), rpid thwing rte (temperture = 70 C, time = 3 seconds). The percentge rte of totl nd progressive motile spermtozo ove hed s well s movement chrcteristics of stright line velocity (VSL, µm/s) nd linerity (LIN, %) were recorded using CASA system immeditely fter thwing nd fter 2 hrs. of het incution (±38 C). Susequently the differences etween semen chrcteristics mesured immeditely fter thwing nd fter 2 hours of incution were clculted. The dt were nlyzed with SAS softwre. All the evluted semen trits reched significntly lowest vlues in totl motility ( 8 % to 10.5 %, P < 0.05), progressive motility ( 5.6 % to 10 %, P < 0.05) VSL ( 3.9 µm/s to 9 µm/s, P < 0.05) nd LIN ( 1.5 % to 4.8 %, P < 0.05) in control thwing method immeditely fter the thwing compred to others. However, these differences were negligile fter 2 hrs. of incution. The highest vlues of progressive motility nd movement chrcteristics fter thwing nd fter 2 hours incution were detected using slow thwing method. Moreover, the rpid method of thwing showed the significntly the lowest results in progressive motility ( 3.6 % to 6.6 %), VSL ( 3.2 to 5.6 µm/s) nd LIN ( 3 % to 4.6 %) chrcteristics ssessed fter 2 hrs. of incution compred the others (P < 0.05). Control method of thwing ws most stle during the incution nd showed the significntly lowest decrese of totl motility ( 7.6 % to 11.8 %, P < 0.05), progressive spermtozo motility ove hed ( 7.5 % to 9.8 %, P < 0.05) nd VSL chrcteristics ( 2.4 to 9.7 µm/s, P < 0.05) during incution. Therefore, this thwing protocol cn e recommended t this study sed on the lowest spermtozo chrcteristics decline during incution for using rtificil insemintion. Keywords: CASA, progressive motility, totl motility, VSL, LIN INTRODUCTION Artificil insemintion (AI) in diry cttle is provided with frozen thwed semen (Andri, 2007; Doležlová et l., 2015). Therefore, the qulity of produced insemintion doses (ID) is directly relted to fertility (Pugliesi et l., 2014). The spermtozo survivility fter cryopreservtion is influenced y mny fctors such s type of semen extender (Moore et l., 2006), pcking method, the length of equilirtion, freezing rte (Rodriguez et l., 1975; Roins et l., 1976; Doležlová et l., 2016) nd finlly thwing rte nd hndling of IDs just efore using (Moore et l., 2006). Spermtozo hving reched elevted thwed tempertures my e dmged y cold shock in cold or overheting in hot mient climtic conditions (Kproth et l., 2005). Also the thwing rte is importnt due the possiility of spermtozo intrcellulr ice recrystlliztion nd its negtive influence to spermtozo motility nd crosoml 815

2 816 Mrtin Doležlová, Mrtin Ptáček, Luděk Stádník, Jromír Ducháček integrity (Roins et l., 1976; Mzur, 1984; Dhmi et l., 1992; Nur et l., 2003; Seki, Mzur, 2009 ). Tody, the most of commercil AI centers recommend wrm wter thw methods for ovine semen frozen in strws processed t their centers. The thwing procedure fetures strw eing removed from liquid nitrogen nd plced immeditely in C wter for s efore prepring the rtificil insemintion gun (DeJrnette et l., 2000; Kproth et l., 2005; DeJrnette, Mrshll, 2005). Although thwing ws initilly performed t 37 C (Slmon, Visser, 1973), higher thwing tempertures of 50 C (Pelez et l., 2006) nd 70 C (Hernández et l., 2007) were chosen to minimize the exposure time of sperm cells to potentil ice crystl nd osmotic dmge. As result, thwing durtion differed with lower volume strws ( ml) which were thwed t 10 to 20 seconds t 50 C (Amdl, Andersen, 1968) or 5 to 10 seconds t 70 C (Hernández et l., 2007). Also other studies hve proven tht thwing tempertures s high s C could further improve post thw motility (Rodriguez et l., 1975; Senger, 1980; Dhmi et l., 1992; Nur et l., 2003). The qulity of insemintion doses fter thwing used to e crried out sujectively, minly ccording to estimtion of motile spermtozo rtion (Pugliesi et l., 2014). Recently, there hs een growing interest in spermtozo motility ssessment y computer ssisted sperm nlysis (CASA) to determine spermtozo movement chrcteristics more ccurtely nd ojectively thn y sujective evlution (Šimoník et l., 2015). The spermtozo movement chrcteristics were in positively correltion with in vitro fertiliztion rte (Kumr et l., 2003). The im of presented study ws to evlute the influence of different thwing methods on spermtozo motility nd movement chrcteristics evluted y CASA system. MATERIALS AND METHODS Bulls nd semen processing The semen ws collected from the group of selected reeding ulls (Holstein, n = 4; Czech Fleckvieh, n = 4) t 3 5 yers of ge. All the ulls were commercilly used for ID production nd their semen ws collected twice week during the whole yer. All the nimls were red under the sme mngement system of privte Sire insemintion Centre (Centrl Bohemin Region, 285 m ove se level, verge nnul rinfll per yer = 650 mm, verge nnul temperture = 9 C). The semen ws collected during August 2013 using n rtificil vgin nd immeditely evluted in lortory of Sire insemintion sttion ccording to methodology pplied y trined stff. Volume of fresh semen, density of spermtozo, nd percentge rte of progressive motile spermtozo ove hed were evluted. Semen pssing the initil conditions for ordinrily commercil purpose (miniml criteri: mm 3 of semen density nd 70 % of progressive motility) ws further processed. Semen ws immeditely ordinrily diluted with phospholipid diluent AndroMed (Minitüe GmH, Tiefench, Germny) to finl spermtozo concentrtion (10 million motile spermtozo per dose). Diluted semen ws mixed up for t lest 5 minutes t room temperture (25 C). Then ws utomticlly filled in the French strws (0.25 ml, IMV Technologies, L Aigle, Frnce), spred on the rmps nd inserted into the cooling ox, cooled t n verge speed of 0.2 C per min to 4 5 C, nd equilirted for 120 minutes. After equilirtion, the strws were frozen using the controlled freezing methodology Direct Freezing in freezer ox Digitcool (IMV Cryo Bio System, L Aigle, Frnce). The 3-phse stndrd freezing curve ws used (Muino et l., 2007). The ull s strws were stored in liquid nitrogen ( 196 C) up to their thwing. Semen thwing The thwing of strws ws performed in preheted wter th. Four different thwing methods sed on vrious wter th temperture nd length of thwing were tested: Control method of thwing in the wter th heted t 38.5 C for 30 seconds Slow thwing in the wter th heted t 30 C for 50 seconds Moderte thwing in the wter th heted t 50 C for 15 seconds Rpid thwing in the wter th heted t 70 C for 3 seconds Evlution of semen chrcteristics The 2 strws per ull per thwing method were used to crete the mixed smple for the next evlution of spermtozo motility. Ech smple ws ssessed repetedly, such tht overll of 64 oservtions were monitored during the whole tril. After thwing the volume of strws were positioned into the 500 µl of physiologicl solution nd plced in the dry heter (Thermo lock Flc, Treviglio, Itly, ±38 C). The percentge rte of totl (Totl, %) nd progressive (Prog, %) motile spermtozo ove hed ws evluted nd recorded using CASA system (SCA Production v. 5.3.; MICROPTIC S.L., Brcelon, Spin) with phse contrst microscope Eclipse E200 (Nikon, Tokyo, Jpn) t mgnifiction when five fields of view per ech strw were evluted t lest (Tuncer et l., 2011). As supplementry spermtozo movement chrcteristics were selected stright line velocity (VSL, µm/s) nd linerity (LIN, %). Semen smples were evluted immeditely fter thwing (Totl0, Prog0, VSL0, LIN0) nd fter 2 hours of incution (Totl2, Prog2, VSL2, LIN2). Susequently the difference etween semen chrcteristics immeditely fter thwing (0 hrs.) nd fter 2 hours of incution (Totl0 2, Prog0 2, VSL0 2, Lin0 2) were clculted.

3 Effect of Different Thwing Methods on Bull s Semen Chrcteristics 817 I: Bsic chrcteristics of the dt structure N AM MIN MAX Sd CV Totl Prog VSL LIN Prog Totl VSL LIN Prog Totl VSL LIN N = numer of oserving; AM = rithmetic men; Sd = stndrd devition; MIN = miniml vlue; MAX = mximl vlue; CV = coefficient of vrince (%); Totl0, 2 = totl spermtozo motility t time 0, 2 hrs. (%); Prog0, 2 = progressive spermtozo motility t time 0, 2 hrs. (%); VSL0, 2 = stright line velocity t time 0, 2 hrs. (µm/s); LIN0, 2 = linerity t time 0, 2 hrs. (%);Totl2 0 = decrese of totl spermtozo motility from 0 to 2 hrs. of incution (%); Prog2 0 = decrese of progressive spermtozo motility from 0 to 2 hrs. of incution (%); VSL2 0 = decrese of stright line velocity from 0 to 2 hrs. of incution; (µm/s); LIN2 0 = decrese of linerity from 0 to 2 hrs. of incution (%) Sttisticl nlysis All sttisticl nlyses were conducted using SAS 9.3. (SAS/STAT 9.3., 2011), MEANS, CORR nd GLM procedures. Correltion nlysis mong vriles (Totl0, Prog0, VSL0, LIN0, Totl2, Prog2, VSL2, LIN2) ws performed using Person correltion coefficients. The nlysis of vrince (ANOVA) ws used to evlute influence of fixed effect of ull nd thwing method on dependent vriles (Totl0, Prog0, VSL0, LIN0, Totl2, Prog2, VSL2, LIN2, Totl0 2, Prog0 2, VSL0 2, Lin0 2). The effects of ull thwing procedure interction nd repetility of mesurement were lso tested during ongoing nlysis. However, oth these fctors were non significnt for ll the evluted trits, nd therefore excluded in the finl model. The chrcteristics of ctul spermtozo motility t time 0 nd 2 hrs. were presented in the form of tles, while chrcteristics of spermtozo persistence (Totl0 2, Prog0 2, VSL0 2, Lin0 2) were expressed s figures using of MS Excel softwre. The model eqution dpted to explin the vriility in spermtozo motility nd movement chrcteristics ws s followed: y ijk = µ + BULL i + THW j + e ijk y ijk = dependent vrile (Totl0, Prog0, VSL0, LIN0, Totl2, Prog2, VSL2, LIN2, Totl0 2, Prog0 2, VSL0 2, Lin0 2) µ = overll men vlue BULL = fixed effect of i th ull (i = 8 clsses, n = 8 oservtions in ech clss) THW = fixed effect of j th thwing method (j = 4 clsses, n = 16 oservtions in ech clss) e ijk = residul error The differences etween the vriles estimted were tested y the Tukey Krmer method t the level of significnce P < RESULTS Bsic sttistics nd model description Short overview of sic chrcteristics of the dtset structure is presented in T. I. Model used to explin the vrition in ull s semen chrcteristics ws significnt for ll the evluted trits. Also fctors in the model were significnt in the mjority, except of effect of thwing method on Totl2, Prog2, VSL2 nd LIN2 chrcteristics s documented in T. II. Correltion nlysis Results of correltion nlysis performed mong dependent vriles re presented in T. III. Totl0 nd Prog0 trits were significntly correlted mutully (r = 0.93; P < 0.001). These oth chrcteristics (Prog0, Totl0) were lso significntly correlted with Prog2 nd Totl2 trits (r = ; P < 0.001), indicting positive reltions mong sic spermtozo motility tht persisted from time 0 up to 2 hrs. fter incution. This thesis ws demonstrted lso y positive correltion (r = 0.92; P < 0.001) etween Totl2 nd Prog2 trits. No or negtive reltion ws oserved mong spermtozo movement chrcteristics t time 0 (VSL0, LN0) nd Totl0, Prog0, Totl2 or Prog2 trits. As interesting VSL2 ws significntly correlted with spermtozo motility t time 0 nd fter 2 hrs. of incution, while non significnt correltion ws detected in reltion with VSL0 or LIN0. Contrry, solutely opposite reltions to VSL2 chrcteristic were detected in LIN2 prmeter.

4 818 Mrtin Doležlová, Mrtin Ptáček, Luděk Stádník, Jromír Ducháček Influence of thwing procedure on the ull s semen chrcteristics The influence of different thwing method on the ull s spermtozo motility nd selected movement chrcteristics is presented in T. IV. Significntly lowest results of Totl0 ( 8.0 to 10.5 %) nd Prog0 ( 5.6 to 10.0 %) were detected in control thwing in comprison with ll the others used procedures. However, no significnt differences were ovious mong slow, moderte nd rpid thwing methods in these trits (Totl0, Prog0). Significntly highest VSL0 nd LIN0 were oserved in slow thwing, which differed significntly with control thwing (+9.4 µm/s in VSL0, +4.8 % in LIN0) nd moderte thwing (+5.5 % µm/s VSL0, +3.3 % in LIN0) methods. No significnt differences were oserved in Totl2 prmeter in reltion to different methods of thwing; however, numericlly highest vlues (+1.8 to +3.9 %) were detected in slow method. Others semen chrcteristics evluted fter 2 hrs. (Prog2, VSL2 nd LIN2) were lso highest in slow method of thwing with significnt differences to rpid thwing. More exctly expressed these differences were 6.6 % in Prog2, 5.6 µm/s in VSL2 nd 4.6 % in LIN2. The other im of the study ws to evlute decrese of spermtozo motility nd movement chrcteristics from time 0 to 2 hrs. fter thwing in reltion to prticulr vritions of thwing method. The results re presented in Figures 1 to 4. The significntly lowest decrese of totl nd progressive motility from time 0 to 2 hrs. of incution ws oserved in control thwing (Totl0 2 = 7.5 %; Prog0 2 = 9.9 %) in comprison with slow (Totl0 2 = 15.1 %; Prog0 2 = 17.4 %), moderte (Totl0 2 = 19.3 %; Prog0 2 = 19.0 %) nd rpid thwing (Totl0 2 = 17.6 %; Prog0 2 = 19.7 %) methods. The highest decrese of VSL prmeter from 0 to 2 hrs. fter thwing ws detected in rpid thwing method (VSL0 2 = 11.8 µm/s), which significntly differed to control (+9.7 µm/s) or II: Description of the model MODEL THAWING BULL R2 Pr > F F-test P F-test P Totl P< P< P<0.001 Prog P < P < P < VSL P < P < P < LIN P < P < P < Totl P < P < P < Prog P < P < P < VSL P < P < P < LIN P < P < P < Totl P < P < P < Prog P < P < P < VSL P < P < P < LIN P < P < P < Totl 0, 2 = totl spermtozo motility t time 0, 2 hrs. (%); Prog0, 2 = progressive spermtozo motility t time 0, 2 hrs. (%); VSL0, 2 = stright line velocity t time 0, 2 hrs. (µm/s); LIN0, 2 = linerity t time 0, 2 hrs. (%); Totl2 0 = decrese of totl spermtozo motility from 0 to 2 hrs. of incution (%); Prog2 0 = decrese of progressive spermtozo motility from 0 to 2 hrs. of incution (%); VSL2 0 = decrese of stright line velocity from 0 to 2 hrs. of incution; (µm/s); LIN2 0 = decrese of linerity from 0 to 2 hrs. of incution (%) III: Correltion nlysis of ull s semen chrcteristics Totl0 Prog0 VSL0 LIN0 Totl2 Prog2 VSL2 LIN2 Totl *** 0.08 n.s ** 0.63 *** 0.61 *** 0.27 * 0.20 n.s. Prog n.s * 0.55 *** 0.58 *** 0.31 * 0.13 n.s. VSL *** 0.30 * 0.20 n.s n.s * LIN n.s n.s n.s *** Totl *** 0.37 ** 0.12 n.s. Prog *** 0.01 n.s. VSL *** LIN2 1 Totl0, 2 = totl spermtozo motility t time 0, 2 hrs. (%); Prog0, 2 = progressive spermtozo motility t time 0, 2 hrs. (%); VSL0, 2 = stright line velocity t time 0, 2 hrs. (µm/s); LIN0, 2 = linerity t time 0, 2 hrs. ( %); n.s. = non-significnt; * = significnt on P < 0.05; ** = significnt on P < 0.01; *** = significnt on P < 0.001

5 Effect of Different Thwing Methods on Bull s Semen Chrcteristics 819 IV: Influence of different thwing process on the ull s semen chrcteristics Totl0 Prog0 VSL0 LIN0 Totl2 Prog2 VSL2 LIN2 C S c 50.8 c M R c 49.5 c RMSE Totl0, 2 = totl spermtozo motility t time 0, 2 hrs. ( %); Prog0, 2 = progressive spermtozo motility t time 0, 2 hrs. (%); VSL0, 2 = stright line velocity t time 0, 2 hrs. (µm/s); LIN0, 2 = linerity t time 0, 2 hrs. (%); C = thwing in wter th 38.5 C for 30 s; S = thwing in wter th 30 C for 50 s; M = thwing in wter th 50 C for 15 s; R = thwing in wter th 70 C for 3 s; RMSE = root men squre error; Mens within columns with different letters differed significntly (,,c = P <0.05) moderte (+7.3 µm/s) thwing methods. These two vritions hd generlly the lowest decrese for the evluted period of 2 hrs. of incution. Higher results of LIN chrcteristic ws detected fter 2 hrs. of incution thn t time 0 in ll the evluted thwing methods, such tht LIN0 2 prmeter cquired negtive numers. Therefore, the results presented in Fig. 4 re expressed in solute vlues for the etter projection. Anywy, the lowest decrese of LIN0 2 during fter 2 hrs. of incution ws monitored in rpid method (0.64 %), while the opposite results ws detected in control method (7.2 %). DISCUSSION The fctors of ull s individulity or thwing method were detected s importnt in presented study s well s in mny previous (Bern et l., 2013; Pldusová et l., 2016; Doležlová et l., 2016). Both these criteri should e therefore tested nd proven in specified conditions to optimize methods of IDs processing. Positive effect on spermtozo motility ws descried y Rodriguez et l. (1975), Senger (1980), Dhmi et l. (1992) or Nur et l. (2003) while using high temperture (reching C) protocols. Dmge of the plsm memrne nd cell orgnelles occurs during slow thwing methods, due to the smll, innocuous intrcellulr ice crystls tht cn grow nd ecome recrystllized (Pnyorin et l., 2016). Therefore, using low thwing tempertures is usully not recommended (DeJrnette et l., 2000; DeJrnette, Mrshll, 2005). Contrry, Seidel (1986) reported tht spermtozo dmges my occur fter high thwing temperture protocols due to reduced spermtozo efflux of cryoprotective gents out of the spermtozo nd therefore suggested slow thwing protocols s more suitle. Vriety of ove mentioned uthors ws underlined y Muino et l. (2008) who found no significnt effect on spermtozo motility t time 0 in reltion of vrious thwing rtes (control 35 C for 40 seconds, moderte 50 C for 15 seconds nd rpid 70 C for 5 seconds). Our results monitored immeditely fter thwing re miguous in this connection, when oth too slow thwing method s well s rpid thwing method reched etter spermtozo motility thn control method of thwing. The individulity of ull nd lso the semen processing influenced the results of spermtozo motility fter thwing (Roins et l., 1976; Moore et l., 2006; Bern et l., 2011; Doležlová et l., 2016). Therefore, these fctors should serve s possile explntion of vrious results presented in this study with those of opposite. Nevertheless, results of presented study detected fter 2 hrs. of incution re definitely in ccordnce with Seidel (1986) when rpid thwing using high tempertures decresed spermtozo motility. The rpid method of thwing strws is not suitle for rtificil insemintion, ecuse of the lowest vlues of semen chrcteristics evluted fter 2 hrs. of incution. Similr results to ours were descried y Rstegrni et l. (2013) who noticed higher proportion of progressive spermtozo motility nd movement chrcteristics fter thwing using higher temperture thwing protocols. However, this reltive dvntge hd disppered fter 2 hrs. incution. Decrese of sperm motility during incution ws dominntly influenced y thwing method (DeJrnette, Mrshll, 2005), which is lso in greement with mny previous studies (Linford et l., 1976; Scke et l., 1980; DeJrnette et l., 2000). Just the lower decline of motility during incution is lso importnt in prticulr time periods from the viewpoint of susequent insemintion (Doležlová et l., 2015). Our results clerly indicted tht the lowest decline of motility chrcteristics ws demonstrted during control method of thwing, despite the significntly lowest vlues t time 0 nd comprle results fter 2 hrs. of incution. Therefore, this thwing protocol cn e recommended t this study for using rtificil insemintion due the lowest decline of semen chrcteristics during incution. These results lso corresponded with previously pulished studies of Nrsimh Ro et l. (1986) or Lrson Cook et l. (2003).

6 820 Mrtin Doležlová, Mrtin Ptáček, Luděk Stádník, Jromír Ducháček 1: Totl spermtozo motility decrese during incution sed on thwing method C = thwing in 38.5 C wter th for 30 s; S = thwing in 30 C wter th for 50 s; M = thwing in 50 C wter th for 15 s; R = thwing in 70 C wter th for 3 s; Different letters within columns men significnce (, = P < 0.05) 2: Progressive spermtozo motility decrese during incution sed on thwing method C = thwing in 38.5 C wter th for 30 s; S = thwing in 30 C wter th for 50 s; M = thwing in 50 C wter th for 15 s; R = thwing in 70 C wter th for 3 s; Totl0-2= decrese of totl spermtozo motility during 2 hrs. of incution; Prog0-2= decrese of progressive spermtozo motility during 2 hrs. of incution Different letters within columns men significnce (, = P < 0.05) c c 3: VSL decrese during incution sed on thwing method C = thwing in 38.5 C wter th for 30 s; S = thwing in 30 C wter th for 50 s; M = thwing in 50 C wter th for 15 s; R = thwing in 70 C wter th for 3 s; Different letters within columns men significnce (,,c = P < 0.05)

7 Effect of Different Thwing Methods on Bull s Semen Chrcteristics 821 4: LIN decrese during incution sed on thwing method C = thwing in 38.5 C wter th for 30 s; S = thwing in 30 C wter th for 50 s; M = thwing in 50 C wter th for 15 s; R = thwing in 70 C wter th for 3 s; Different letters within columns men significnce (, = P < 0.05) CONCLUSION Higher progressive motility nd spermtozo movement chrcteristics immeditely fter the thwing nd fter 2 hours of incution were detected in slow thwing method (30 C, 50 s). The lowest spermtozo motility ws recorded in rpid thwing fter 2 hrs. of incution. This method is difficult to perform in field conditions nd therefore could not e recommended for rtificil insemintion. Contrry, methods using lower thwing tempertures were more stle during the incution nd usully esier to perform in field conditions. Therefore they were ssessed s more pproprite for rtificil insemintion. However, the modified thwing methods lso could e tested for potentil ppliction for the specific purposes of in vitro mnipultion. Also evlution of semen chrcteristics during incution period longer thn 2 hours should e other im of further reserch. Acknowledgments This study ws supported y S grnt of MEYS of the Czech Repulic nd NAZV [No.QJ ]. REFERENCES AAMDAL, J. nd ANDERSEN, K Fst thwing of semen frozen in strws. Reprod. Domest. Anim., 3(1): ANDRABI, S. M. H Fundmentl principles of cryopreservtion of Bos turus nd Bos indicus ull spermtozo. Int. J. Agric. Biol., 9: BERAN, J., STÁDNÍK, L., DUCHÁČEK, J., TOUŠOVÁ, R., LOUDA, F. nd ŠTOLC, L Effect of ulls reed, ge nd ody condition score on quntittive nd qulittive trits of their semen. Act Univ. Agric. et Silvic. Mendel. Brun., 59(6): BERAN, J., ŠIMONÍK, O., STÁDNÍK, L., RAJMON, R., DUCHÁČEK, J., KREJCÁRKOVÁ, A., DOLEŽALOVÁ, M. nd ŠICHTAŘ, J Effect of ull, diluter nd LDL-cholesterol concentrtion on spermtozo resistnce ginst cold shock. Act Univ. Agric. et Silvic. Mendel. Brun., 61(6): DEJARNETTE, J. M., BARNES, D. A. nd MARSHALL, C. E Effects of pre- nd post-thw therml insults on viility chrcteristic of cryopreserved ovine semen. Theriogenology, 53(6): DEJARNETTE, J. M. nd MARSHALL, C. E Strw-thwing method intercts with sire nd extender to influence sperm motility nd conception rtes of diry cows. J. Diry Sci., 88(11): DHAMI A. J., SAHNI K. L. nd MOHAN G Effect of vrious cooling rtes (from 30 degrees to 5 degrees C) nd thwing tempertures on the deep-freezing of Bos turus nd Bos ulis semen. Theriogenology, 38(3): DOLEŽALOVÁ, M., STÁDNÍK, L., BINIOVÁ, Z., DUCHÁČEK, J. nd BERAN, J Effect of freezing curve type on ull spermtozo motility fter thwing. Act Vet. Brno, 84(4): DOLEŽALOVÁ, M., STÁDNÍK, L., BINIOVÁ, Z., DUCHÁČEK, J. nd STUPKA, R Equilirtion nd freezing interction ffecting ull sperm chrcteristics fter thwing. Czech J. Anim. Sci., 61(11): HERNÁNDEZ, M., ROCA, J., GIL, M. A., VÁZQUEZ, J. M. nd MARTÍNEZ, E. A Adjustments on the cryopreservtion conditions reduce the incidence of or ejcultes with poor sperm freezility. Theriogenology, 67(9): KAPROTH, M. T., RYCROFT, H. E., GILBERT, G. R., ABDEL-AZIM, G., PUTNAM, B. F., SCHNELL, S. A., EVERETT, R. W. nd PARKS, J. E Effect of semen thw method on conception rte in four lrge commercil diry heifer herds. Theriogenology, 63(9):

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