The very short duration of vigorous movement (1½ to 7 min) in fresh water and physiological solutions make trout spermatozoa difficult subjects for cryopreservation studies. Solutions consisting of 250 to 280 mmol sucrose and 5 to 12% dimethyl sulphoxide (DMSO) (4 parts) did not activate trout spermatozoa (1 part), but after dilution with fresh water vigorous motility could be fully restored. These sucrose·DMSO solutions were employed in cryopreservation studies. Using straws and a fast freezing - fast thawing procedure, post· thaw dilution with fresh water resulted in 25% - 60% of spero matozoa becoming motile, all with vigorous forward progression. Some existing methods for the cryopreservation of other freshwater fish spermatozoa were repeated on trout without success

Dott, H.M.

Foster, G.C.

van der Horst, G.

English

AJOL - African Journals Online

.. Studies on the motility and cryopreservation of rainbow trout (Salmo gairdnerl) spermatozoa G. van der Horst, H.M. Dott and G.C. Foster ARC, Institute of Animal Physiology, Animal Research Station, Cambridge, United Kingdom The very short duration of vigorous movement (1'12 to 7 min) in fresh water and physiological solutions make trout spermatozoa difficult subjects for cryopreservation studies. Solutions consisting of 250 to 280 mmol sucrose and 5 to 12% dimethyl sulphoxide (DMSO) (4 parts) did not activate trout spermatozoa (1 part), but after dilution with fresh water vigorous motility could be fully restored. These sucrose·DMSO solutions were employed in cryopreservation studies. Using straws and a fast freezing - fast thawing procedure, post· thaw dilution with fresh water resulted in 25% - 60% of spero matozoa becoming motite, all with vigorous forward progreso sion. Some existing methods for the cryopreservation of other freshwater fish spermatozoa were repeated on trout without success. S. Afr. J. Zool. 1980,15: 275-279 Die baie kort tydsduur van aktiewe beweging (1 V2 tot 7 min) in vars water en fisiologiese media, bemoeitik die bevriesing van forelspermatosoe. Oplossings bestaande uit 250 tot 280 mmol sukrose en 5 tot 12% dimetielsulfoksied (4 dele) aktiveer nie forelspermatosoe (1 deel) nie, maar na verdunning met vars water herstel aktiewe beweging van spermatosoe heeltemal. Hierdie sukrose·DMSO oplossings is gebruik vir die bevriesingstudies. Wanneer strooitjies en 'n vinnige vries -vinnige ontdooiing prosedure gebruik is, het verdunning met varswater na ontdooiing veroorsaak dat 25% - 60% van die spermatosoe beweeglik geraak het en almal het kragtige voor· waartse beweging getoon. Sommige van die bestaande metodes vir die diepvriesbewaring van ander varswatervis· spermatosoe is sonder sukses herhaal. S.·Afr. Tydskr. Dierk. 1980, 15: 275 - 279 G. van der Horst· Present address: Department of Zoology, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, Republic of South Africa H.M. Dott and G.C. Foster ARC, Institute of Animal Physiology, Animal Research Station, 307 Huntingdon Road, Cambridge CB3 OJQ, U.K. ·To whom all correspondence should be addressed Submitted 29 November 1979; accepted 18 April 1980 Although several attempts have been made during the past decade to cryopreserve trout spermatozoa, fertiliza-tion rates were either low or variable. Graybill and Hor-ton (1969) obtained a fertilization rate of 18070 using a saline-fructose-Iecithin based extender, Ott and Horton (1971) were more successful with a bicarbonate-mannitol-lecithin extender (59%), Biiyiikhatipoglu and Holtz (1978) could not repeat this latter work but used yet another extender (based upon trout seminal plasma) and obtained 2% - 80% fertilization. All of these extenders included 12% dimethyl sulphoxide (DMSO). When freshwater fish spermatozoa are collected without exposing them to water (milt) they are inactive and they can be kept in that state at 4°C for up to 24 h. During this time the spermatozoa will become motile if the milt is diluted with water or a physiological salt solu-tion. Once activated, trout spermatozoa remain vigorous-ly motile for 29 - 60 s (Scheuring 1928, Schlenk & Kahman 1937, Medem, Rotheli & Rath 1949). Either the vigorous motile life span of the sperm may be extended significantly in order to allow time for cryopreservation procedures or an extender that will not activate the sper-matozoa can be used during the freezing procedure and motility initiated by further dilution after thawing. Ginzburg (1972) indicated that a positive correlation exists between fertilization rates and good forward pro-gressive motion in trout spermatozoa. We have therefore used sperm motility as a parameter to test the effect of various extenders and procedures for cryopreservation before testing for fertilizing ability. The latter parameter would in any case also be influenced by the condition of the eggs . Materials and Methods Milt was collected from ripe, two to three year old rain-bow trout either maintained in tanks at 4°C or kept at a trout hatchery. Before stripping milt the area around the urogenital papilla was dried and pressure was exerted on the bladder thereby forcing urine out in order to avoid possible contamination with the milt. Milt was collected in precooled (4°C) test tubes and afterwards maintained at 4°C in a cold room. Of these only spermatozoan samples exhibiting Grade 3 motility (see later) and more than 90% motile spermatozoa on dilution with fresh water were used. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010). 276 Assessment of motility None of the methods used for quantifying motility of mammalian spermatozoa were suitable for trout sper-matozoa because of the long (120 s) equilibration time re-quired after the spermatozoa sample had been put on the slide (Dott & Foster 1979), therefore motility was graded using phase contrast microscopy at a magnification of 200x and 400x. To exclude bias as far as possible, the observer grading the motility was kept unaware of the solutions being tested. There were four grades of motility; grade 3 - virtually all spermatozoa move vigorously and most spermatozoa swim progressively forwards; grade 2 - although most spermatozoa move vigorously only approximately 50070 swim progressively forward; grade 1 - a few to many motile spermatozoa, although virtually no spermatozoa swim progressively forward; grade 0 - all spermatozoa immotile. The approximate percentage of motile sper-matozoa was also assessed. Experiments on duration of spermatozoan motility A stop watch was started as the milt was thoroughly mix-ed with each solution to give a final dilution of five times. The initial motility grade and the duration of each motili-ty grade was recorded. This was relatively easy because the entire population of motile spermatozoa appeared to change from one type of motility to another almost simultaneously. However, attention was primarily devoted to the duration of vigorous spermatozoan motili-ty (Grades 3 and 2). Solutions used for motility studies Table 1 outlines the concentration and composition of the solutions used for the motility studies on rainbow trout spermatozoa in this investigation. Analytical grade chemicals were used. Cryopreservation (Extenders and procedure) Milt was mixed with solutions containing 250 mmol sucrose or 280 mmol sucrose and 5,8, 10, 12, 14 and 160J0 DMSO respectively at 4°C. One to five hours was allow-ed for equilibration. Milt was also mixed 1:5 with solu-tions containing 250 mmol sucrose and 3,5 and 100J0 glycerol respectively at 4°C. The methods of Ott and Horton (1971) for the cryo-preservation of trout spermatozoa and that of Mounib (1978) for Atlantic salmon spermatozoa (freshwater spawner) were also repeated for rainbow trout sper-matozoa in this investigation. In preliminary trials with pellet freezing we obtained poor post-thaw motility, probably because the drops were too large to allow sufficiently rapid freezing and/or thawing. After preliminary trials with straw freezing, ministraws (0,25 ml) containing diluted semen were frozen in liquid N2 vapour for 7 - 9 min and then plunged into liquid N2. Fast thawing was achieved by putting the frozen straws into a water bath at 27 - 35°C for 4 - 5 s. This provided the fast thaw necessary before dilution with fresh water at 4°C to reactivate the spermatozoa. S.·Afr. Tydskr. Dierk. 1980, 15(4) Table 1 Concentration and composition of solu-tions used for trout sperm motility studies Solution Sodium chloride Sucrose Dimethyl sulphoxide (DMSO) Glycerol Egg yolk Mounib 1978 Reduced glutathione Potassium bicarbonate Ott and Horton 1971 Glucose Mannitol Sodium bicarbonate Potassium chloride Deionized water Trou t hatchery water Sucrose NaCI Composition sucrose reduced glutathione KHC03 DMSO Total concentration NaHC03 lecithin mannitol DMSO Total concentration Total concentration Concentration mmol mOsm/ 070 kg 1,2 30 53 I DO 80 150 108 2DO 118 220 150 276 162 3DO 90 50 125 lDO 150 150 180 2DO 195 220 250 273 280 3DO 70 113 140 169 197 225 41 68 136 125 6,5 lDO 5 (v/v) 8 (v/v) 10 (v/v) 12 (v/v) 14 (v/v) 16 (v/v) 3 (v/v) 5 (v/v) 10 (v/v) 3 (v/v) 5 (v/v) 169 12 (v/v) 283 6,5 7 lDO 152 8,6 0,72 (w/v) 1,5 0,75 (w/v) 1,4 0,25 (w/v) 169 12 (v/v) 187 10 9 lDO lDO lDO 125 lDO 10 230 Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010). / .. S. Afr. J. Zoo!. 1980, 15(4) Results Activation studies All concentrations of NaCI tested activated spermatozoa, as did concentrations of sucrose below 195 mmol (Table 2). If the milt was diluted at 1: 10 instead of 1 :5, sucrose above 195 mmol also activated. Distilled water activates trout spermatozoa, so solutions which activate' may be thought of as permissive while those which do not can be considered as inhibitory. If a solution did not inhibit activation, its value was assessed by comparing the durations of the various motility grades in the solution with their durations in water (see Table 3). Vigorous sperm motility lasted longer in some of the solutions than in water but none of them extended the duration of vigorous motility enough to allow pre-freeze manipulation of the diluted semen. Table 2 Activation of trout spermatozoa by dilu-tion at 4°C. (For each solution n = 5) (Motility Grade, (MG) 3 = + + +, MG 2 = + +, MG 1 = +, MG 0 = -) Osmotic concentration (mOsm/kg) NaCI (1,2 to 30 to 300 162 mmol) Sucrose (90 to 90 to 200 180 mmol) Sucrose (195 220 to 300 to 280 mmol) Sucrose (195 220 to 300 to 280 mmo\) DMSO (5-12070) DMSO (14-16%) Glycerol (3-5%) Glycerol 10% Egg yolk 5-10% Mounib 1978 283 reduced glutathione 7 6,5mmol KHC03 152 (100 mmol) (Ott and 187 Horton 1971) glucose 9 (10 mmol) mannitol (100 mmol) lecithin (1,5 mmol) NaHC03 (100 mmol) KCL (100 mmol) Sperma-tozoan dilution 5 5 5 10 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Activation after Activation dilution with and motility fresh water and grade motility grade +++ +++ +++ +++ +++ ++ +++ + + + + +++ +++ +++ +++ 277 On the other hand some solutions which inhibited ac-tivation later permitted it when further diluted with fresh water. Sucrose at 250 mmol did not activate when used as an extender at a dilution rate of 1:5 but the spermatozoa became vigorously motile when diluted further with fresh water (Table 2). The extender of Ott and Horton (1971) activated trout spermatozoa immediately whereas that of Mounib (1978) did not, even when further diluted with fresh water. Cryopreservation studies The results of the cryopreservation studies are summariz-ed in Table 4. Sucrose (250 mmol) was used as the ex-tender for most of these experiments with various con-centrations of DMSO or glycerol as cryoprotectants. Post-thaw recovery was appreciable using DMSO. All levels of DMSO resulted in about 3511/0 of spermatozoa becoming motile after the thawed extended semen was diluted with fresh water, and these spermatozoa exhibited grade 3 motility. On the other hand, glycerol proved a much less effective cryoprotectant; only about 311/0 of the spermatozoa became motile and these only showed grade I motility. When 280 mmol sucrose was compared to 250 mmol sucrose, in combination with DMSO, the higher concentration did not improve post-thaw recovery. No recovery at all was obtained when the DMSO containing extenders of Ott and Horton (1971), or Mounib (1978) were used. Discussion Trout spermatozoa in milt remain inactive at 4°C for at least 24 h (unpublished observations). The spermatozoa become vigorously motile when fresh water at 4°C is add-ed at any time during this period, but the vigorous motili-ty persists for less than I min. We have observed that if dilution of the milt is 1:5 the presence of NaCl, NaHC03, mannitol, lecithin, or up to 1211/0 DMSO, does not pre-vent activation; on the other hand, KCI, KHC03, reduced glutathione, 14 -1611/0 DMSO, or glucose inhibit activa-tion, and only feeble motility is obtained in the presence of glycerol or egg yolk (Table 2). At a dilution rate of 1:5, up to 190 mmol sucrose per-mitted activation, whereas concentrations greater than 195 mmol inhibited it. On the other hand, if the milt was diluted I: 10, the higher concentrations of sucrose were no longer inhibitory. In view of this it was not surprising to find that the inhibition of motility by high concentrations of sucrose could be overcome by further dilution with fresh water. Inhibition by glycerol was also reversed by further dilution with fresh water, whereas spermatozoa in KCI, KHC03 or glucose remained inactive after further dilution. One explanation of these results is that there are 2 types of inhibition, an 'active' inhibition exhibited by K+ or glucose, and a 'passive' inhibition by sucrose; the latter might result from an endogenous inhibitor in or on the trout spermatozoon which is soluble in water but only slightly soluble in a sucrose solution. Vigorous motility following activation of trout sper-matozoa is short-lived yet it is apparently a pre-requisite for good fertilizing ability Ginzburg (1972). It seems logical therefore to carry out cryopreservation either in an extender that prolongs vigorous motility or in an ex-tender that inhibits activation in a reversible fashion. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010). 278 So-Afro Tydskro Dierko 1980,15(4) Table 3 The duration of trout spermatozoon motility in various solu-tions (spermatozoa diluted x 5) at 4°C Duration of vigorous motility (s) Grade 3 Grade 2 Osmotic Range Average Range Total dura-Sperm concentration Average tion of diluent mOsm/kg n = 2 n = 2 motility (s) Deionized 90 82-98 24 20-28 457 water Fresh water 10 86 81-90 27 21-33 428 Sucrose 50 III 107-115 42 35-43 309 100 109 108-110 26 20-32 340 150 110 105-115 37 29-45 280 200 123 120-126 17 10-24 245 220 0 0 250 0 0 Sodium chloride 30 75 74-78 35 30-40 157 100 84 80-88 117 105-130 350 150 104 91-118 91 60-121 370 200 143 121-165 104 74-134 >600 220 144 133-155 118 69-167 >600 276 165 159-172 209 202-216 >600 300 157 137-178 117 74-160 >600 Sucrose: 230 122 118-126 116 105-127 >600 NaCI(I:1) Huxley (1930) found that the motile life of trout sper-matozoa could be prolonged in saline solutions. Our results confirmed this (Table 3, final column) but the duration of vigorous motility could be increased only to approximately 6 min, not long enough for freezing to be attempted. Sucrose at concentrations greater than 195 mmol inhibited activation reversibly and in combination with DMSO enabled trout spermatozoa to be frozen and thawed with a recovery of motility sufficiently high (25 - 60070) to suggest a high fertilization rate. Similar recovery rates were reported by Mounib (1978) with frozen Atlantic salmon spermatozoa, when an 85 % fer-tilization rate was obtained. The cryopreservation procedure reported by BOyukhatipoglu and Holtz (1978) for trout spermatozoa which yielded variable results (2 - 80% fertilization) but seems to be the most successful method so far reported, appeared too late to be tested. Although our method can not yet be regarded as suc-cessful, it appears from this investigation and the literature that future studies should involve more detailed research of basic trout spermatozoon physiology. Special attention should be devoted to activating and inhibiting substances on spermatozoon motility. It was for example shown that the monosaccharides glucose and mannitol have opposite effects on trout spermatozoon motility (Table 2). The former renders spermatozoa immotile while mannitol as used in the extender of Ott and Horton (1971) can induce vigorous spermatozoon motility. The underlying principles of phenomena like these should be investigated as well as the quantification of trout sper-matozoon motility in relation to fertilization in order to have a better scientific basis and rationale for developing cryopreservatives in future studies. Table 4 Cryopreservatives used aAd spermato-zoan motility results after thawing and dilution with fresh water (n = 5 for each cryopreservative) (% v/v) Percentage motile Motility grade spermatozoa after thawing Cryopreservative and dilution Average Range 250 mmol sucrose, 5070 DMSO 3 40 30-60 , 8070 DMSO 3 35 25-55 ,10070 DMSO 3 35 30-55 ,12070 DMSO 3 35 30-50 280 mmol sucrose, 5070 DMSO 3 35 30-55 , 8070 DMSO 3 40 30-60 ,10070 DMSO 3 30 25-55 ,12070 DMSO 3 35 30-55 250 mmol sucrose, 3 to 10070 3 0-5 glycerol Mounib 1978 0 0 Ott and Horton 1971 0 0 Acknowledgements We gratefully acknowledge financial support by the Council for Scientific and Industrial Research (South Africa), the Fisheries Corporation of South Africa, The Department of Environmental Planning and Energy (South Africa) and the University of Port Elizabeth for this research project as well as the research facilities of the Animal Research Station, Cambridge, U.K.; Mr P Miles and Mr F Head for technical assistance, Mr Harden of the Trout Hatchery at Empingham and Mrs A J Gerber for typing the manuscript. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010). S. Afr. J. Zool. 1980, 15(4) References BOYOKHATIPOGLU, S. & HOLTZ, W. 1978 Preservation of trout spermatozoa in liquid or frozen state. Aquaculture 14: 49 - 56. DOTT, H.M. & FOSTER, G.C.A. 1979. The estimation of sperma-tozoa motility in semen, on a membrane slide, by measuring the area change frequency with an image analysing computer. J. Reprod. Fert. 55: 161 -166. GINZBURG, A.S. 1972. Fertilization in fishes and the problem of polyspermy, Ed. T.A. DetIaf p. 140, Israel Program for Scientific Translations, Jerusalem. GRAYBILL, J.R. & HORTON, H.F. 1969. Limited fertilization of steelhead trout eggs with cryo-preserved spermotozoa. J. Fish. Res. Bd. Can. 26: 1400-1404. 279 HUXLEY, J .S. 1930. The maladaption of trout spermatozoa to fresh water. Nature Lond.: 125(3152),494. MEDEM, F., ROTHELl, A. & ROTH, H. 1949. Biologischer Nach-weis von Befruchtungsstoffen bei Felchen und Flussforelle. Schwei<.. Z. Hydrol. II: 361 - 377. MOUNIB, M.S. 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fert. 53: 13 - 18. OTT, A.G. & HORTON, H.F. 1971. Fertilization of stee\head trout (Salmo gairdnerl) eggs with cryopreserved spermotozoa. J. Fish. Res. Bd. Can. 28: 1915-1918. SCHEURING, L. 1928. Weitere biologische und physiologische Unter-suchungen an Salmonidensperma. Zool. Jb. 45: 651 - 706. SCHLENK, W. & KAHMANN, H. 1937. Reaktionkinetische Unter-suchungen der Bewegung der Forellenspermatozoen. Z. vergl. Physiol. 24: 518 - 531. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2010). 

Studies on the motility and cryopreservation of rainbow trout (Salmo gairdneri) spermatozoa

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Studies on the motility and cryopreservation of rainbow trout (Salmo gairdneri) spermatozoa

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