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Author: Ucar, O?mer Title: Acrosome reaction and cryopreservation of dog spermatozoa.
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ACROSOME REACTION AND CRYOPRESERVATION OF DOG SPERMATOZOA
by
Ömer Ucar
A thesis submitted in partial fulfilment of the requirements of the University of Bristol for degree of Doctor of Philosophy
May 2000
ABSTRACT The useof AI in dogs hasbeenlimited by the lack of effective and reliable meansof cryopreservationof semenand by the poor correlationbetweentraditional methodsof post-thawassessment of semenquality and fertility. In order to addresstheseproblems, the presentstudy focusesupon methodsof cold storageand cryopreservationof dog spermatozoaby undertaking comparativeevaluationsof post-thaw motility and in vitro induction of acrosome reaction. Split-ejaculate protocols were used to compare the effect of storage at +4°C and cryopreservation upon (i) the maintenance of spermatozoa! motility and (ii) spontaneous or A23187-induced acrosome reactions during incubation at 39°C (in 5% CO2 in the humidified air) for 60 or 120 min. The assessmentsof samples were made by BrightField, Phase Contrast (PC), Differential Interference Contrast (DIC), Scanning (SEM) and Transmission (TEM) Electron Microscopy.
The interactionbetweenthe processof glycerolisationand the presenceof seminalplasmais one of the key limitors for successin cryopreservationof dog spermatozoa.Interactionsbetweenthe effectsof removal of seminalplasma (by centrifugation), dilution rate, the temperatureat which glycerolisation took place and the concentrationof Spermatozoa in experiments. +4°C of split-ejaculate were studied a series of spermatozoa at glycerol upon survival were suspendedin Tris-fructose-citricacid extendercontaining20% (v/v) egg yolk and 8% (v/v) glycerol at +4°C for 48 h. Survival was assessedas the percentageof spermatozoadisplaying progressivemotility. Survival of spermatozoawas higher (P
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3.4.3- Results 3.4.3.1- Ejaculates The characteristicsof ejaculatesusedfor the experimentare given in Table 3.12:
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3.4.3.2- Results of Experiment 5: Analysis of variance Results from analysis of variance demonstratedthat there were significant effects of individual dogs (P
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232
GENERAL DISCUSSION Background Research on semen preservation has a long history over two centuries, but most of the work has been carried out in the last four decades (Watson, 1990), especially since the discovery of the use of glycerol in 1949 by Polge and his co-workers (Polge et al., 1949). Although a great deal of success has been achieved in both cryopreservation and Al with chilled/frozen semen, there is still a problem that even with the best preservation techniques to date, the post-thaw survival is restricted to about 50% of spermatozoal population (Watson, 1995; Rodriguez-Martinez et al., 1997). Moreover, most surviving from different have their unfrozen counterparts. Indeed, it is characteristics spermatozoa generally accepted that cryopreserved mammalian spermatozoa are in a state resembling partial capacitation (Watson, 1995; Rota, 1998), which accounts for their relatively reduced survival (especially in dogs: Concannon and Battista, 1989; Pena et al., 1999), and readiness to undergo acrosome reaction or egg penetration without incubation (Watson, 1995). Consequently, AI with frozen semen usually results in poorer fertility than those of fresh semen in most species (Watson, 1990; Linde-Forsberg et al., 1999), which can partially be overcome by inseminating, often on more than one occasion, greater numbers of live spermatozoa (up to a threshold) and/or precise timing of insemination. Objectives Therefore, the objectives of this study were to establish for the dog; (i) improved methods of pre-freeze handling of semen, (ii) an in vitro acrosome reaction assay using fresh and freezing data for (iii) to these of optimisation regimens on the basis chilled semen, utilise of post-thaw motility of spermatozoa, and (iv) a method of differentiation of the damage to the acrosome due to a) the acrosome reaction, b) chilling and c) freeze-thawing by means of various assessment techniques (including Bright-Field, Phase Contrast, Differential Interference Contrast, Scanning and Transmission Electron Microscopy).
Criteria for assessment of spermatozoa In order to obtain dataon the effectsof treatmentupon the spermatozoa,the changesin the acrosomalintegrity (for acrosomereaction only) and the motility were assessed.Wide in changes osmolality of treatment solutions are neededbefore there is damageto the
233
plasma membraneoverlying the acrosome(Gao et al., 1995; Rota, 1998). However, sperm motility will be affected by even small variations in osmolality (Gao et al., 1995). This is why sperm motility has usually been used to assessfreezing regimens (Rota, 1998). It is known that acrosomal changes are characteristic in dogs (Oettle and Soley, 1988). While substantial acrosomal damage can occur during dilution, cooling and equilibration, the major deterioration of the organelle occurs after freeze-thawing (Oettle, 1986b; Hay et al., 1997a). However, even in spermatozoa which have no apparent acrosomal damage nor reduction in motility, subtle changes may have occurred. This was concluded by Hay et al. (1997b) who found a decline in the number of spermatozoa binding to oocytes after cooling or freeze-thawing in the dog.
The effectsof seminalplasma, dilution rate and glvicerolisationregimen upon the survival of spermatozoaduring cold-storage(at +4°C) In order to freezesemenandthusprolong the fertile life of spermatozoa,semenneedsto be diluted and/orcooledat +4°C for a period of time. The choiceof Tris (Rota et al., 1995)as the extender in the presentstudy was basedon the fact that it is most widely used in practiceand that fertility resultsafter AI arewell known (Linde-Forsberg,1995). Previous studies have shown that either the removal of the first and the third fractions (England and Allen, 1992a)or the removal of the entire seminal plasma (Martin, 1963a) was beneficial for survival and morphologicalcharacteristicsof dog spermatozoa,with no adverseeffect on fertility (Platz and Seager,1977). Controversialreportsexist of the effect of dilution rates (Foote, 1964c;England, 1992),the concentration(Province et al., 1984; England, 1992; Günzel-Apel et al., 1993) and addition temperature (Martin, 1964a; Fontbonne and Badinand, 1993b) of glycerol upon semen quality. The relationship betweenthe presenceof seminalplasma,dilution rate and glycerolisationregimen is also unknown. Since pre-freeze handling steps will affect the successof freeze-thawing (Oettl6,1986b; Hay et al., 1997a),it was desirableto investigatethe effect of each step upon the survival of spermatozoa. The resultsof the presentstudy agreewell with the previous findings. Tris-fructose-citric acid extendercontaining20% (v/v) egg yolk extendedsurvival of spermatozoaat +4°C for up to 48 h dependingon dilution rates. Centrifugation was beneficial to spermatozoa!
234
motility. However, there were marked interactionswith dilution rate and glycerolisation temperature. Furthermore, spermatozoal survival was markedly higher after glycerolisation at +4°C than at room temperature. Although the beneficial effect of centrifugationupon maintenanceof spermatozoalmotility was apparentat dilution ratesup to 1:8, possible ultrastructural changesupon the plasma membrane and the acrosome should alsobe consideredbefore final recommendationscan be made. However,sincedog semen is generally diluted at 1:1 to 1:5 (or up to 1:8) in an extender depending on spermatozoalconcentration(Andersen, 1980; Christiansen,1984), centrifugation appears to be applicable. Concentrationsof glycerol that were >2% (v/v, final) resulted in markedly lower survival than at lower concentrations,although up to 14% glycerol (at a dilution rate of 1:20) hasbeenreportedin the literature (Foote, 1964a). Therefore,on the basis of spermatozoalmotility, tolerance of glycerol up to 2% in Tris-fructose based extender suggeststhat glycerol can still be used until a less toxic, thus more effective cryoprotectantis to be found. With Tris extender,the additives such as Orvus EM Paste(Nizanski et A, 1997),Equex STM Paste(Rota, 1998),sodiumdedocylsulphate(Penaet A, 1998c)and Proline (Penaet al., 1998a) have been shown to be beneficial for post-thaw motility, viability and acrosomalintegrity. However, therewas no significant increasein in vivo fertility after Al with frozen semencontainingEquex STM Paste(Rota, 1998). Nevertheless,incorporation of one of theseagentsand one antioxidant (presumablybutylated hydroxytoluene(BHT); see Watson, 1995 for detail) in the extenders may be recommended for a better maintenanceof membraneintegrity of spermatozoaespeciallyin centrifuged samples. In this way, partial capacitationof spermatozoaleading to prematureacrosomereaction may be minimised. This may be particularly critical sincethe acrosomalchangesmust result in precisely `timed' acrosomereactionto fulfil fertilisation task (Tesarik, 1989). The effects of calcium ionophore (A23187) upon acrosomereaction of spermatozoaas assessedby Bright-Field andphasecontrastmicroscopy In the literature, different types of chemical (e.g. calcium ionophore, A23187) and/or biological (e.g. progesteroneand ZP) components have been used to stimulate the acrosomereactionin dog spermatozoa.It is known that extracellularcalcium is essential for motility and acrosomereaction of dog spermatozoa,which can be induced in the
235
absenceof eggs or cumulus cells (Mahi and Yanagimachi, 1978), as with many other species (Yanagimachi, 1994). Recently, Brewis et al. (1999) found that both heat solubilised bitch ZP and progesteronecan causea marked influx of calcium ions, thus, leading to acrosome reaction. In the present study, using modified TALP medium (containing 3 mM/1 Ca2'), optimum concentrationof A23187 was found to be 1 pM/l. However, the concentration was higher (i.e. 2-10 µM/1 A23187) in previous studies (Geussoväet al., 1997; Szäsz et al., 1997; Hewitt and England, 1998), using lower concentrations(51.71 mM/1) of calcium. Thesestudiesindicate that there is a relationship between constituents(especially calcium ion) of the medium and concentrationof the stimulant to induce the acrosomereactionin vitro. SinceA23187 was a suitableagentfor induction of the acrosomereactionin dogs, as with other species(e.g. bull: Christensenet it is for 1998), future use. It might be human: Baker, Liu 1994; recommended and al., possiblethat A23187-inducedacrosomereactioncould be usedto predict in vivo fertility in the dog, as is the casein the bull (Whitfield and Parkinson,1995). However, the effects of biological (i.e. ZP, hemi-zonabinding and progesterone)and non-biological (i.e. A23187) stimulantsto inducethe acrosomereactionshouldbe comparedso that a biological basisof be for dog induced can established spermatozoa. reaction chemically acrosome Although numerousassessment techniques(with or without staining) have been used for monitoring the acrosomereactionof dog spermatozoa,a novel stain, i.e. naphthol yellow S1aniline blue was found to be a useful tool in the presentstudy. Compatibility of the stain with fresh (or even diluted) samplesand distinguishablecharacteristicsof stained spermatozoamay provide further meansfor monitoring the acrosomalchangesin the dog. However, poorerstainingquality of frozen-thawedspermatozoa(in dogs:Oettl6,1986b; in fresh in 1995) Watson, 1975; boars: Paulenz to compared semenmight limit rams: et al., the distinction of the degreeof cryoinjury, as with other stains such as Spermac(Oettle, 1986b;Paulenzet al., 1995)and Giemsa(Watson,1975). The efj`ectsof glycerol upon the acrosome reaction of chilled spermatozoa as assessedby
Differential InterferenceContrastmicroscopy The presentresults demonstratedthat the acrosomalintegrity is impaired during chilling (cooling to/at +4°C). Almost half of the cells had already undergone spontaneous acrosomereaction in the incubation medium (modified TALP). This indicates marked
236
acrosomaldamageduring pre-freezehandling steps,confirming the previousfindings with light microscopy (Oettle, 1986b). Indeed, Rota (1998) showed two-fold shortening of capacitationtime in chilled-rewarmedspermatozoacomparedto fresh dog semen. This indicates that pre-freeze cooling is critical for functional integrity of spermatozoa. Therefore, a suitable diluent (e.g. Tris-based;Rota, 1998), incorporation of membrane stabilisers(i.e. detergents:Nizanski et al., 1997;Rota, 1998)and proper cooling (England, 1992; Hay et al., 1997b) may be practised for a superior maintenanceof spermatozoal integrity following chilling in future studies. By doing so, the proportion of functionally intact spermatozoaentering to harmful freeze-thawing processesmight be increased, leading to a superiorsuccessin cryopreservation. In the literature, glycerol concentrationsvaried widely (from 0.5 to 14%, v/v; England, 1992and Foote, 1964a,respectively)and inseminations,resulting in high pregnancyrates, in 2-8%. The in that the presenceof the present results showed of range used glycerol in 2% (v/v) final the extender, there was no significant of concentration glycerol at a changein the percentageof acrosomereactedspermatozoain chilled samples. Sinceusing in decrease in 2% to a non-significant spermmotility at +4°C (for also resulted glycerol up 48 h), the findings suggestthat glycerol can be usedas cryoprotectantof choice for further handling (freeze-thawing)until it may be substitutedby a less- or non-toxic agent (e.g. in few freezing diluents In were used previous glycerol-free species, proteins). other studies(Gibson and Graham,1969;Abdelhakeamet al., 1991a,b). Using unfrozen turkey semen, Donoghue and Walker-Simmons (1999) demonstrated that heat soluble, dehydration-inducedproteins (isolated from dry wheat seed embryos) are capable of fertility hatchability increasing during in and of eggs. protectingspermatozoa vitro storage, Thesemight alsopotentially improve long term storageof spermatozoafrom other species. The presentresultsimply that acrosomereactionassaymay be a helpful tool especiallyfor investigationof the effects of pre-freezehandling stepssuchaschilling and glycerolisation to semenquality. However, future studiesof the effect of glycerol upon the ultrastructure of dog spermatozoaor in vivo fertility arerequired. The effects of freezing regimens upon the post-thaw motility of spermatozoa
The effects of freezing rate upon the spermatozoaof virtually all species(including dog) have not yet beeninvestigatedsystematicallywith the exceptionof bovine (Parkinsonand
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Whitfield, 1987,using a semi-programmablefreezer)eventhoughmodem cryobiology has startedhalf a century ago, through the introduction of glycerol to semenbefore freezing (Polge et al., 1949). It is clear that there is no control over the freezing rate above solid carbondioxide (for pelletedsemen)andthat althoughthe adventof small-volumestrawsin 1964(Cassou,1964)allowed adaptationof more sophisticatedfreezingin vaporisedliquid nitrogen, freezingrate acrossthe straw may not be uniform (especiallyin vertical position) freezer is (McLaughlin used programmable a controlled-rate, et al., 1990). In dogs, unless the freezingmethodologieswere mainly adaptedfrom bovine (Foote, 1964a;Seager,1969; Andersen, 1975) becauseof high food/economicaldemand of this species. Therefore, there have beenvery limited systematicstudiesinvestigatingthe actual effect of freezing ratesupon dog spermatozoa(England,1993). In the presentstudy, it was found that; (i) three-stepfreezing is clearly appropriate,(ii) the optimum freezing regimen (from +4 to -120°C) is -0.5°C/min from +4 to -9°C, -40°C/min to -20°C, -100°C/min to -120°C, followed by direct immersion of the straws in liquid freezing be for (iii) the curve can of used estimationof post-thaw nitrogen, and parameters motility of spermatozoa.Theseresultsconfirm that the optimisationof freezingconditions of dog spermatozoais possible. As with the bovine (Parkinson and Whitfield, 1987), minimising the latent heatplateauphase(throughits quicker releaseby fastercooling) was beneficial for survival of spermatozoa,althoughthe initial cooling rate was also one of the main determinantsof post-thawmotility, due either to centrifugation,using smallestsize of French straws (0.25 ml French type, with high surface/volumeratio) or high dilution rate used in the presentstudies. Although England (1992) demonstratedthat the avoidanceof plateau phase during freezing was beneficial, Rota (1998) comparing fast and slow freezing (yielding different magnitudesof plateauphases)found no clear advantageof one freezingrate over the other. This was attributedto freezingratesusedthat were both in the range between -10 to -80°C/min, consideredto be suitable for freezing of spermatozoa (Watson, 1990). Therefore, it would be interesting to comparedifferent magnitudesof plateauphaseagainstits earlier induction by seeding(Hay et al., 1997b)or its avoidance by pellet freezing (England, 1992). Additionally, Olar (1984), comparing different freezing rates, demonstratedthat lectin-induced agglutination of dog spermatozoawas markedly altered after freezing. Rota (1998) found a higher incidence of head-to-head agglutination,that tendedto be higher when fast freezing rate was used. These freezing-
238
induced changes might indicate alteration in the acrosome and/or plasma membrane leading to partial capacitation(Watson, 1995, Rota, 1998). Hence, the effect of sperm agglutination during cryopreservationshould also be investigatedin detail for a better understandingof cryoinjury. It is known that glycerol decreases salt concentration (through its water binding increases fraction the thus unfrozen of water during freezing (Watson, and properties), 1995).
Therefore, it minimises cryoinjury (mainly during slow cooling) and as its
increased freezing be (Farrant, 1980). This can is decreased rate must concentration normally be achieved in pellet freezing or by using programmable freezers (McLaughlin et incorporation Although Rota, 1998). 1993; the of glycerol in freezing extender could al., have been avoided completely (Gibson and Graham, 1969; Abdelhakeam et al., 1991a,b), the variation in post-thaw motility of spermatozoa between individual sires then became substantially greater (Gibson and Graham, 1969). However, since dog spermatozoa may in % 21 freezing (i. the absence of glycerol or any to post-thaw motility) up e. survive cryoprotectant other than egg yolk (Olar et al., 1989), the optimisation of freezing development The in might also yield promising extender results. regimens a glycerol-free freezing (providing fast (with coupled glycerol) with pellet cooling no of a newer extender be 1978) Graham needed, as with ram spermatozoa (Abdelhakeam et might rates; et al., lambing 1991a, b; >50% >60% and post-thaw motility rate after cervical who achieved al., insemination in natural oestrus). If the results of Abdelhakeam et al. (1991a,b) are confirmed with further studies, it is possible that glycerol might not be essential (Watson, 1995). Therefore, the methodology of England (1992), using a combination of alcohol bath and LN2 coupled with straw packaging (0.25 ml, French type), may be considered for future studies of cryopreservation of semen in the dog.
In cryobiology, prior attentionhasusually beengiven to the effectsof freezing,but the role been ignored. from Apart has thawing, to also which contributes of generally cryoinjury the cryoinjury during freezing, substantial damage to the cells occurs during or after thawing (Woolley and Richardson, 1978). The effects of warming rate dependon prior cooling rate and cell type (Watson et al., 1992a). However, Rota (1998) found a highly between interaction glycerol concentrationand thawing rate but not between significant freezing rate and thawing rate for dog spermatozoa.For cooling rate, the major concernis
239
whether it was high enough to induce intracellular freezing (requiring fast thawing) or low enough to produce cell dehydration (requiring slow thawing) in cell survival (Mazur, 1988).
Woolley
and Richardson (1978), using human spermatozoa, found that
ultrastructural changes of the plasma membrane, acrosome and mitochondria occur either during or after thawing (to be discussed later). Osmotic studies upon the ram and human damaged by ingress (during thawing) rather that the are water cells spermatozoa showed than by its egress (during freezing) (Gao et al., 1992; Curry et al., 1994; Holt and North, 1994).
Similarly, Holt et al. (1992), studying the membrane permeability of ram
injury, freeze-thawing, found the that as a result of was observed membrane spermatozoa, only during warming in the range of 2-30°C (well after dissolution of ice and re-dilution of the solute that was concentrated during freezing). Hence, they considered that membrane lipid changes might be involved (as also suggestedby Quinn, 1985), but concluded that the freezing injury is different from cold shock injury (see Watson, 1981 for detail). However, due to the limited number of ultrastructural observations (e.g. Woolley and Richardson, 1978; Courtens and Paquignon, 1985) on fresh, frozen and frozen-thawed spermatozoa, freezing injury has not yet been distinguished clearly from those that occur during and/or dog for Therefore, thawing. post-thaw survival of spermatozoa, the actual a greater after isotonic investigated dilution have be to to thawing an solution effect of or post-thaw for 'fixed' (i. 37°C 1 thawing than rate at min) single, e. using simply systematically rather for for 37°C 1 70 (e. +4°C minutes, several min, and at g. rates only or comparing several below). 1990 Watson, (also for 6,8 75°C 12 and see or either or sec) The effects of freezing regimens upon the acrosome reaction ospermatozoa as assessed by Differential Interference Contrast microscopy
The results of the present study demonstratedthat the acrosomalintegrity deteriorates during chilling (cooling to/at +4°C) and especiallyafter freeze-thawing.This confirms the previous findings with light microscopy (Oettl6,1986b, Rota, 1998). Considering the between it differences the various that there changes, was acrosomal noted were no freezingregimensusedin the presentstudy,while the motility differed betweenthem. This indicatesthat spermatozoalmotility is more sensitiveto osmotic gradient,as generatedby (Gao rates cooling et al., 1995; Liu and Foote, 1998a,b), than the plasma various membrane(with underlying acrosome)of the head(Rota, 1998). This provesthe accuracy but useful, not a single criterion of cryoinjury during optimisation of as a of motility
240
freezing conditions. Lack of correlation between the acrosomal(and overlying plasma membrane)damageand post-thawmotility (dog: Oettle, 1986b;Rota, 1998;boar: Purselet al., 1972;human:McLaughlin et al., 1993;Gao et al., 1995)requiresfurther consideration of both attributesof spermatozoafor a superiorcryosurvival. The previous studies using light microscopy techniques demonstratedthat acrosomal changes(i.e. 77 to >80%) are common following freeze-thawingby various regimens (Oettl6,1986b; Hay et al., 1997b). Regardlessof freezing regimen applied in the present study, the incidence of acrosomaldeteriorationwas too high (>90%), possibly reducing potentially viable spermatozoaavailable for acrosome reaction. Considering other difficulties in performing the inseminationitself and precisetiming of insemination (see Linde-Forsberg,1995; England, 1998 for detail), future studiesare neededto reduce the acrosomaldamageto increasethe viable populationof spermatozoacapableof undergoing 'true' (physiological) acrosomereaction. However, it is possible that thawing itself, as it can exert effects comparableto those of cooling, or post-thaw handling processes(e.g. further centrifugation, re-suspension in an isosmotic medium, TALP containing extracellularcalcium, etc.) contributedto substantialchangeson the acrosome. The effects of chilling and freeze-thawing upon the acrosome reaction of spermatozoa as Electron Microscopy by Transmission Scanning and assessed SEM
SEM provided spectacularimagesof the entire integrity and surfacecharacteristicsof dog spermatozoa,although visualisation of the sub-compartments(e.g. the outer and inner acrosomalmembranesand the mitochondria)was usually impossible. Therefore,in future studies it may not be recommendedfor monitoring the sequenceof acrosomereaction especiallyfor the intermediatestages(i.e. the early stagesof partial reaction). However, it may be used for determiningthe presenceof the acrosomebefore or after freeze-thawing aswell as assessment of grossmorphologicalabnormalitiesof spermatozoa. TEM Although it was laborious and time demanding,virtually every single compartment of spermatozoacould be visualised by TEM, which confirms its value for assessmentof dog spermatozoaas a standardtest (Oettle and Soley, 1988). Using this of ultrastructure technique, the initial stages of acrosome reaction could be illustrated rather easily.
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Therefore, for future studies upon cryopreservationof dog spermatozoa,as with other species(Yanagimachi, 1994), priority should be given to TEM to illustrate the actual ultrastructural changes on the acrosome due either to chilling, freeze-thawing or acrosome reaction.
Comparing the chilled and the frozen-thawed cells, there were obvious differences betweenthe integrity of the entire head and mid-piece. On the head,both the equatorial membraneand plasmamembranewere generally intact in chilled cells, however various degrees of acrosomal changes usually proceededtowards these regions after freezethawing. This acceleratesthe acrosomereactionin frozen thawedcells, eventuallyleading to cell death, which may therefore impair the ability of spermatozoato fuse with the plasmamembraneof the ovum at the end of the acrosomereaction (Bedford et al., 1979). In the mid-piece, the plasma membraneand mitochondrial structureswere affected by freeze-thawing,although they were well preservedin chilled spermatozoa. Despite the fact that the cooling regimen (given above)was optimal for post-thawmotility, this might not be the case for the plasma membrane or the acrosomal integrity of the head of spermatozoa,as previously shown (Oettle, 1986b;Rota, 1998). Therefore,during freezethawing, it is relevantto maintain the entire integrity of spermatozoonto achievesuperior post-thawsurvival. An optimal cooling regimen should be followed by an optimal warming regimen as the latter is as important as the former in contributing to the eventual cryoinjury (Mazur, 1988). Thawing at around body temperature,i. e. 35-37°C (as widely used in many species;Watson, 1990),might not simply 'fit' for every single cooling regimen,even it is an `optimal' one. Therefore,systematiccomparisons,or using at leastthree different rates of thawing (i.e. slow, intermediateand fast rates,as given above)are required. In this way, it is possible to overcome excessivecryoinjury to spermatozoaduring or after thawing (Woolley and Richardson, 1978; Courtens and Paquignon, 1985). It is known that the processof dehydrationof cells accompanyingslow freezing (which might be an 'optimal' one) is potentially associatedwith cell survival, whereasat more rapid rates cell death is more likely due to likelihood of intracellular ice, which is usually lethal (Watson, 1995). Unless thawing is `optimised', swelling of the entire head of spermatozoacould become inevitable even it was combinedwith an `optimum' freezingregimen. Indeed,this was the
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case in the presenttrials and many other studiesin mammalsduring thawing (involving rehydration)of spermatozoa(Watson, 1995). Therefore,using a small amountof glycerol (such as 2%, v/v, final concentration)in the extender(Tris) may becomerelevant,so that the cell volume excursionscould be minimised during or after cryopreservation. Obviously, during post-thaw handling processes(i.e. either re-dilution of semeninto an 'isosmotic' medium, acrosomereaction in vitro or direct introduction of semeninto the genital tract of femaleduring Al) care shouldbe practised. Post thaw re-dilution of semen in homologousprostatic fluid, that may result in maximum pregnancyrate (100%) after intravaginal insemination (Nöthling and Volkmann, 1993) should also be considered. However, the actual effect of prostatic fluid upon the frozen-thawedcells is not known with certainty (Nöthling and Volkmann, 1993). Nevertheless,the addition of prostatic fluid doesincreasethe volume of the inseminate.This may overcomethe difficulties in Al allowing a substantiallylarger proportion of spermatozoato enter into the uterus through the rather tortuouscervix of the bitch. Using this technique,when coupledwith a properly timed insemination schedules (that includes monitoring the plasma LH and/or inseminations be decreased the to only one during oestrus of might number progesterone), (Nöthling et al., 1997). Finally, therefore,appropriateprecautionsareneededduring both pre-freezeand post-thaw handling (e.g. centrifugation,dilution into an extender,cooling and glycerolisationat +4°C, freezing and thawing regimen, re-dilution) and inseminationprocedures(e.g. timing and the number of inseminations, deposition site of the inseminate) when the preserved (especiallyfrozen-thawed)semenis to be used. In this way, the thermoresistanceof dog spermatozoaat body temperaturemight be improved substantiallywhich allows using a more flexible inseminationschedule,yet resultsin high in vivo fertility.
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FINAL CONCLUSIONS For the in vitro induction of the acrosome reaction, cold storage and freeze-thawing of dog spermatozoa, the present study indicated that:
1. The removal of seminal plasma was beneficial for the survival of spermatozoain Tris-fructosecitric acid extendercontaining 20% egg yolk and 8% glycerol (v/v, final concentration)at dilution higher dilutions At (>1: 8) (semen: 8 the presence of seminal plasma was
