CRYODILUENTS AND MORPHOLOGICAL CHANGES IN THE SPERMATOZOA
OF FISH UZA PARStA (HAMILTON-BUCHANAN)
DISSERTATIO N SUBM ITTED
IN P A R T IA L FULFILM EN T OF THE R EQ UIREM ENTS FOR T H E DEGREE O F
MASTER O F FISHERIES SCIENCE (MARICULTURE)
O F THE
C E N T R A L INSTITUTE O F FISHERIES EDUCATION (D E EM ED UNIVERSITY)
M U M B A I-400 061
by
Sandhya Sukumaran (Mori. 51)
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CENTRAL MARINE F IS H E R IE S RESEARCH INSTITUTE
INDIAN COUNCIL O F AGRICULTURAL RESEARCH C O C H IN -6 8 2 014
INDIA.
JUNE 2001
DEDICATED TO
MY PARENTS
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CENTRAL MARINE FISHERIES RESEARCH INSTITUTE
P O S T B O X No. 1603, E R N A K U L A M , COCHIN- 6 82 014 (IndiuiCouncil of Agricultursl Research)
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Dated: 30 June 2001
CERTIFICATE
C ertified that the thesis entltfed "CRYODILUENTS AND MORPHOLOGICAL CHANGES IN THE SPERMATOZOA O F FISH
LIZA P A R S /A (HAMILTON-BUCHANAN)” is a record of independentbonafide research work carried out by M s. Sandhya Sukumaran during the period of study fro m September 1999 to August 2001 under our supervision and guidance fo r the degree o f Master o f Fisheries Science (Mariculture) and th a t the thesis has not previously form ed the basis for the award of any degree, diploma, associateship, fellowship or any other sim ila r title.
M ajor Advisor/Chaftman
(D. Noble) Senior Scientist
PNP Division CIVtFRI
A dvisory committee
(A Gopalakrishnan) Scientist (Senior Scale) NBFGR, C ochin unit
(N. K. Sanil) Scientist PNP Division CMFRI
DECLARATION
1 hereby declare that the thesis entitled
“ CRYODILUENTS A N D MORPHOLOGICAL CHANGES IN TH E SPERMATOZOA OF FISH
LIZA PARSIA(H A M ILTO N -B U C H AN A N )” is an authentic record of the work done by me and no part thereof has been presented for the award o f any degree, diploma, associateship, fellowship, or any other similar title.
30 Jun e 2001
( S a n d h ^ S u k u m a ra n )
M. F. Sc. Student
Central Marine Fisheries
Research Institute
ACKNOWLEDGEMENTS
I sincerely acknowledge my extrem e sense of gratitude to my g u id e Dr. D. Noble, Senior S cientist for his inspiring guidance, tim e ly help and advise, to successfully carry out th is work.
My sincere thanks go to m y co-guide Dr. A. Gopalakrishnan, Scientist (Senior S cale) for the ideas, generous suggestions and helps given by him which w ere valuable for the successful com pletion of this work. I am extrem e ly indebted to Shri. N. K. Sanil, Scientist, who is also my co
guide fo r all his wholehearted efforts fo r getting the electron m icrographs needed for this thesis, at the right tim e .
I am also grateful to Dr. M ohan Joseph Modayil, Director, C M FRI for providing me with the facilities to carry o u t the work in th e institute. I express my sincere thanks to Dr. R. Paul Raj, O.I.C, Post graduate Program m e in M ariculture for his helps and encouragem ents throughout the M. F. Sc. course.
My deep-felt gratitude goes to Mr. M. Ayyappan Pillai, Technical Officer, CMFRI, w ho was instrumental in do in g the electron m ocroscopic w orks which were of immense help to me when I w a s really running sh o rt of time.
The timely supply of Liquid N itrogen by Kerala Livestock Developm ent Board Ltd. is also duly acknowledged.
I am also indebted to Dr. Reeta Jayasankar and Dr. L. Krtshnan for all the hatchery facilities made available during the course of this work. Words can not express my deep-felt gratitude to Dr. K. S. S obhana who was a constant sou rce of encouragem ent and help to me.
My sincere thanks go to Mr.Prashanth, Mr. P.M. M uneer, S.R.F and Mr. K. Musammilu, S. R. F for all the helps e xte nde d by them.
1 am also indebted to Ms.Nisha, S. R. P., Mr.
Unnikrishnan, S.R.F., and Mr. Anikkuttan (Ph.D , Scholar), Mr.
Ram alinga (Ph.D S cho lar),and Ms. Juliet for all the helps.
My deep fe lt gratitude also goes to Mr. Rajanna (M .F.Sc.Second semester) w ho readily helped me w h e n e ve r I needed.
I express m y thanks to all m y classmates,especially KIran, Subodh, Chandrasekhara Rao, Laxm lkant and Uya fo r all the helps and moral support to carry out this work. I also exp ress my thanks to m y room-mate, Mini Thomas who encouraged me a lot.
I acknowledge m y sincere thanks to I.C .A .R ., New Delhi, fo r awarding me w ith the Junior Research Fellowship throughout the tenure of m y M. F. Sc. course.
Last but not th e least I express my extreme gratitude to m y parents to whom this work is dedicated.
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ABSTRACT
An attempt ha s been m ade to get an in sig h t into cryolnjuries th a t occur during the process o f cryopreservation and to identify th e best cryodiluent fo r the milt o f Liza parsia, o n e of the euryhaline fish e s of im m ense culture potential. The m orphological changes o r damages caused by the cryodiluent at various stages of sperm preservation in liquid nitrogen at -196°C were studied at ultrastructural level by Transm ission Electron M icroscopy (TEM).
V 2 E + D M S 0 diluent accorded maximum protection to th e sperm atozoa and in flicte d least dam ages, retaining m axim um post-thaw motility.
Hence, V 2 E + D M S 0 appear to be the best cryodiluent for th e milt of Liza p a rsia. The present findings need further confirm ation with actual fe rtility trials.
CONTENTS
1. INTRODUCTION 1
2. REVIEW O F LITERATURE 4
2.1 .Sperm S tructure and Milt Composition 4
2.1.1.Nucleus 5
2.1.2. M itochondria 5
2.1.3. C entrioles 5
2.1.4. A xonem e 5
2.2. Cryopreservation of Sperm atozoa 5
2.3. M orpliological Changes O f Fish Sperm 9 2.4. Post- th a w Motility and Fertility 12
3. MATERIALS AND METHODS 15
3.1. Coffection o f Milt 15
3.2. Prelim inary Motility Studies 15
3.3. Preparation of Extenders 17
3.4. Procedure 19
3.5. Treatm ents 19
3.6. Processing o f Sperms F o r Electron M icroscopy Studies 20 3.7. Counting o f Post- thaw In ta ct Spermatozoa 21
4. RESULTS 22
4.1. Ultrastructure of Untreated Spermatozoa 22 4.2. The ultrastructural C hanges o f L/za pars/a 22
sperm a fte r Osmotic S hock
4.3. The Ultrastructural Changes after Treatm ents 23
4.3.1. Chao’s Extender and D M S O 23
4.3.2. Mixture C and DMSO 23
4.3.3. Rana and Me Andrew Extender and DMSO 24
4.3.4. V2E Extender and D M SO 25
5. DISCUSSION 3 8
SUMMARY 44
REFERENCES 45
Table 1. Chem ical composition of d iffe re n t extenders 18
T a b le 2. Percentage o f intact sperm atozoa of Liza pa rsia and their m otility
score after each step in various extenders + DtvlSO (10% ) 26
Figure 1. D iagram m atic representation of sperm atozoa of L/za species 14 Figure 2. Percentage of intact sperm atozoa of Liza p a rsia after each 37 step in various extenders +DM SO
Plate 1. E lectron micrograph of Untreated spermatozoa(20000x) 27 Plate 2. E lectron micrograph of untreated spermatozoa(40000x) 27 Plate 3. E lectron micrograph of spermatozoa a fte r osmotic shock 28 Plate 4. E lectron micrograph of spermatozoa a fte r osmotic shock 28 Plate 5. E lectron micrograph o f spermatozoa treated with Chao’s
extender + DMSO, after step 1 29
Plate 6. E lectron micrograph o f spermatozoa treated with Chao’s
extender + DMSO, after step 2 29
Plate 7. E lectron micrograph of spermatozoa treated with Chao’s
extender + DMSO, after step 3 30
Plate 8. E lectron micrograph of spermatozoa tre ate d with Chao’s
extender + DMSO, after step 4 30
Plate 9. E lectron micrograph of spermatozoa treated with Mixture C
+ DMSO, a fte r step 1 31
Plate 10. E lectron micrograph of spermatozoa treated with M ixture 0
+ DMSO, a fte r step 2 31
Plate 11. E lectron micrograph of spermatozoa treated with M ixture 0
+ DMSO. a fte r step 3 32
Plate 12. E lectron micrograph of spermatozoa treated with M ixture C
+ DMSO, a fte r step 4 32
Plate 13. Electron micrograph o f spermatozoa treated with Rana
and Me A ndrew + DMSO, a fte r s te p i 33
Plate 14. E lectron micrograph o f spermatozoa treated with Rana
and Me A n d re w + DMSO, after step2 33
Plate 15. E lectron micrograph of spermatozoa treated with Rana
and Me A n d re w + DMSO, a fte r step3 34
Plate 16. E lectron micrograph o f spermatozoa treated with Rana
and Me A n d re w + DMSO. a fte r step4 34
Plate 17. E lectron micrograph o f spermatozoa treated with V2E
+ DMSO, a fte r step 1 35
Plate 18. E lectron micrograph o f spermatozoa treated with V2E
+ DMSO, a fte r step 2 35
Plate 19. E lectron micrograph o f spermatozoa treated with V2E
+ DMSO. a fte r step 3 36
Plate 20. E lectron micrograph o f spermatozoa treated with V2E
+ DMSO, a fte r step 4 36
INTRODUCTION
1. INTRODUCTION
F o r more than a century, scientists have investigated m ethods to preserve ga m ete s o f fish, The ea rly studies were directed at prolonging th e life of gametes in non-frozen state. W ith th e advances in th e field of Cryobiology, the focus has sh ifte d to develop cryoprotective techniques fo r preservation o f viable gametes in fro z e n condition at - 1 96°C in Liquid Nitrogen. Most of th e work in literature relate to fishes of S alm on id group or fre shw ate r species. N o t much work has been reported on m arine cultivable fishes o f commercial importance
like seabass, m ullets, seabreams etc.
Developments in th e cryopreservation o f fish sperms depend upon the identification and testing of su ita b le cryodiluents. Undiluted sperm s cannot withstand the rigours of freezing and thawing. T h e y must be dilute d with a suitable exte n d e r which is a solution of balanced sa lts and sometimes organic compounds. Although there have been many extender media reported in literature, it ha s been claimed th a t there is little difference in post-thaw fertility between them , w hile some w orkers claim superiority o f certain extenders.
W h e n biological cells are cooled in an aqueous solution, both cells and solution su p e r cool to som e extent. Then heterogeneous nucleation takes place usually in the extracellular solution. As water is frozen out the extracellular solution be co m e s progressively m o re concentrated. If cooling rate is low there is sufficient tim e fo r the cells to lose enough water to rem ain in osmotic equilibrium with the concentrating extracellular solution. This causes dehydration. If cooling rate is high, th e re Is Insufficient tim e fo r water to d iffu s e ou t of the ceIJs to the ice crystal. The c e lls will equilibrate by intracellular freezing initiated e ith e r by hom ogenous o r heterogeneous nucleation. Intracellular freezing is also fatal.
A balanced situation m ay exist which allows sun/ival w hen the cooling rate is high enough to m in im ise the tim e o f exposure to concentrated
solutions and y e t is low enough to minimise the intracellular ice below a damaging level. Certain chem icals can increase th e dimensions of this balance between the e ffe c ts of intracellular ice and concentrated solution thus improving survival. T h ese chemicals are term ed cryoprotectants. There are perm eating and non-perm eating cryoprotectants. D im ethyl sulphoxide( DMSO) is a perm eating cryoprotectant w hile glycerol is a non permeating one. Cryoprotectants suppress most of the cryoinjuries and the effectiveness of cryoprotectants in suppressing cryoinjuries v a rie s at different concentrations and along with different extenders.
The prevention o f gamete activation is most critical in both short and long-term storage as fe rtility declines soon a fte r sperm activation. Reduction o f fertility because of lo ss of motility by exhaustion has be en postulated. DM SO at concentrations o f 5-20% has be en used successfully fo r cryopreservation of fish sperm atozoa and appears to be better cryoprotectant than glycerol or methanol. Ten percent DMSO a p p e a rs to be the optim al level.
T h e ultimate success o f cryopresen/atlon depends on the fertilising ability of cryopreserved sperms. A p a rt from the extender and cryoprotectant, standardisation o f equilibration tim e is also found to be an important fa c to r to avoid the e ffe ct o f temperature sho ck on sperm viability. Although cooling rate is the most critical factor in cryopreservation, it is the least standardised varia ble in fish sperm cryopreservation studies and no result yet on the theoretical optim um - freezing rate. H ence, sperm injuries are bound to occur.
D espite advances to da te, at best, only 30-50% of the sperm atozoa retain their po st-th aw motility and have little insight into when th e damage occurs, (t is im perative to study th e structural changes o f the sperm atozoa in order to a rrive at the right extender, cryoprotectant, equilibration tim e and freezing rate to retain post-thaw spe rm fitness and fertility. Hence th e present work is taken u p to study the m orphological changes In the sperm atozoa o f the brackishwater fish Liza parsia (Ham ilton-Buchanan, 1822) using Transm ission Electron M icroscopy (TEM) in different diluents w ith 10% DM SO as a cryoprotectant during various stages o f cryopreservation protocol.
Liza parsia is a sm a lle r variety of m ullet w ith restricted distribution commonly fo u n d in south west co a s t o f Indian peninsula south of Bom bay, in Kerala they a re found mainly in Vembanad and Kayamkulam lakes. The maximum size attained by the sp e cie s is 330 mm. T h e size at first m aturity is found to be 120 mm in males and 129 mm in females. Females are the dominant sex.
L iza parsia is having Immense culture potential and a preferred table fish com m anding prem ium price in the m arket. Identification of a cryodiluent th a t accord maximum protection to the spermatozoa of L/za parsia will greatly e n h a n ce the farming potential of this spe cie s by ensuring constant supply of ‘s e e d ’ fo r culture. H ence th e present work ha s been taken up.
REVIEW OF
LITERATURE
2. REVIEW OF LITERATURE
The history o f cryogenic preservation of gam etes is a long one. The application o f the sam e fo r fish sperm s is of recent origin. Studies on th e cryopresen/ation o f milt of m arine and brackish w a te r fishes are limited (Cnao e ra /., 1975). Since the first work o f Blaxter in 1953, fish sperm cryopresen/ation ha s been attempted on about 30 m a rin e species (Jam ieson, 1991). Compared with fre s h w a te r species, a high percentage o f spermatozoa survives cryopresen/ation In m arine fishes (Suquet e t a!., 2000).
Dilution of m ilt w ith cryodiluents, freezing and subsequent thawing re s u lt in structural changes such a s deformed n u cle us and acrosome and rupture o f membranes in the sperm atozoa of various te le o sts including rainbowtrout, A tlantic croaker and grayling (Billard, 1983). Although ce llu la r damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this dam age and th e fertility rates o f eggs with post th a w e d milt (C abrita eia/.,1998).
T h e ultra-structural images of fish sperm are useful in (1) prelim inary screening and selection of suitable cryodiluents for freezing. (2) determining th e quality and freezing ability o f milt (3) predicting post - th a w sperm fitness and fertilizing ability (Gopalakrishnan et al., 2000).
2.1 SPERM STRUCTURE A N D MILT COMPOSITION
Mullet sperm atozoan is com posed of a h e a d part measuring 2 .Smicron X I .4 m icro n and a ta il part four to fiv e times as long as head. There w e re about .5.3’ ° sperms in each m l of semen and the pH value o f mullet sem en is 7.4 on th e average (Chao e f a/.,1975 ) . The biochemical constituents o f mullet
semen are glucose 31.66 %, protein 33.8%, lipid 38.83%, sod iu m 124.3 + /' 5.4 and potassium 164 .4+/- 7,6 m Eq / 1 in fresh milt (Diwan and Nandakum ar, 1992).
The sperm o f mugiloids (Liza and Gaieoids) studied are similar. (F ig .l), described by Jam ieson (1991).
2.1.1. Nucleus: The nucleus, bilobed or kidney shaped in longitudinal section is tilted relative to th e axoneme. The chromatin is ve ry coarsely granular. In L/za dumerilii a dense body has been observed lying in contact w ith the nucleus in som e sperm; the nuclear and overlying plasma m em brane covering th e ventral and dorsal tip of th e nucleus forms b u lb o u s evaginations containing chrom atin.
2.1.2. Mitochondria: Four sub spherical m itochondria are arranged in a ring but eccentrically around th e base of the axonem e from which th e y are seperated by a long cytoplasm ic canal.
2.1.3. Centrioles: Two fu lly developed centrioles are present. Despite the ecce ntric emergence o f th e flagellum, the proximal cen trio le and
in one plan e the distal cen trio le lies in the nu cle ar fossa.
2.1.4. Axonem e: Two classes o f flagella were observed. Eighty percent varied betw een 30 and 43 m icro m ete rs. In length, while the remainder, considered to indicate abnorm al sperm, were much shorter a n d varied in length (21-30 micrometers).
2.2 CRYOPRESERVATION O F SPERMATOZOA
Sperm motility was preserved fo r up to 2 3 days in the case o f raw milt at 5°C (Chao e ta /., 1975). Undiluted m ilt with or without a cryoprotectant ad ded directly to it is not s u ita b le for
freezing. M ilt must be diluted with a suitable extender {Scott &
Baynes, 1980; Gopalakrishnan e t al., 1999), M otility was u se d to evaluate th e quality of raw milt and to verify the e ffe c t of extender - cryoprotectant com bination on pre-freezing and frozen thawed sam ples (Chao eta l., 1986) .The intensity of motility decreased when th e osmotic pressure of diluent w as reduced, and was zero o r significantfy inhibited when osm otic pressure o f d ilu e n t was close to th a t of fish se m in al plasma (Chamberyon & 2 o h a r,1 9 9 0 ).
A diluent in which the sperm rem ains Immotile w ould always be preferable for cryopreservation (Jamieson, 1991; G opalakrishnan et al.. 2000).
Ability of the d ilu e n t used fo r cryopreservation to maintain the sperm atozoa in quiscent state is a critical requirem ent as activation prior to cryofreezing can result in loss of c a p a c ity to fertilize (Jam eson, 1991). S perm cell density, m otility and v ia b ility are the potential predictors related to fertility of sperm after cryopreservation (Paul W heeler and Thorgaard, 1991). Milt of European catfish stripped into an immobilizing solution was successfully cryopreserved after a stepw ise freezing and fast thawing procedure (Linhart e?a/., 1993). Sperm m otility is a routine indicator of th e fertilization ability of fresh sem en (Babiak e f a/., 1994). T h e motility of frozen thaw ed semen is a reliable param eter fo r quality determ ination since there is a statistically greater correlation with po st- thaw fertilization rate (Lahnsteiner ef a/., 1995). Sperm suspended in extender solution retained motility significantly lo n g e r than did sperm in in ta c t testis (Michael christensen & T e rre n ce Tiersch, 1996). In case o f Liza parsia spe rm motility co u ld be preserved successfully fo r more than 240 days using a su ita b le cryoprotectant (Dlwan & Nandakum ar, 1998). An increase in viable m o tile sperm atozoa to egg w o u ld result in better fertilization and hatching of paddle fish (George Brown
and Steven Mims, 1999). T h e extenders used fo r cryopreservation of spermatozoa of various cyprinid and salm onid fishes were found to be unsuitable for Tenualosa ilisha as they activated the sperm on dilution (Kuldeep Lai ef a/., 1999). Osmolality o f seminal p lasm a and concentrations of sodium, potassium and magnesium ions had low variability, w hich suggests th a t they are im portant for creating a stable environm ent for sperm storage in sperm duct (Glogowski et a i, 1999).
Cryoprotective agents are needed at the ultra low tem perature o f preservation in liquid nitrogen (Chao e fa /., 1975).
DMSO repeatedly provides the best protection of salmonid sperm atozoa from dam age by freezing (Horton and O tt, 1976;
G opalakrishnan e f a/.. 1999). G lycerol and DM SO bind electrolytes thereby preventing these substances fro m concentrating in the residual unfrozen solution in and around th e cell during freezing (Scott and Baynes, 1980). No fertilization w as obtained when methanol w as the cryoprotectant (Gwo e fa /., 1991). The mechanism of cryoprotection of fish sperm by DMSO lies in its fast penetration into the sperm and increase of osmotic pressure of the spe rm in Atlantic croaker, Micropogonias undu/afus(2hang et a/., 1994). Methanol and DM SO have proven m ost satisfactory as cryoprotective agents, often yielding post - th a w m otility value of greater than 40% in m any species (Terrence Tiersch, 1995), Studies in turbot sperm atozoa showed that a high rate o f success obtained in a sucrose solution with 10% DMSO and 10% egg yolk (Olvidio ChereguinI e fa /., 1997). In tilapia sperm atozoa the optimum concentrations of cryoprotectant glycerol was found to be between 10 to 20 %, w h ile maximum post-thaw m o tility of sperm atozoa w as observed in 20% concentration (Rexaline Sumathi and Sukum aran, 1998). Sperm atozoa frozen with a com bination of an intracellular cryoprotectant (methanol) and an extracellular
cryoprotectant (skim milk) produced no fertilization in blue catfish (Bart e f a/., 1998). Fertilization and larval hatch rates w ere higher for milt frozen with a diluent containing DMSO than containing glycerol In striped trum peter, Latris fineata(R\tar et a!., 1999), Forty percent po st- thaw motility w a s achieved using a cryodiluent of 5%
glucose as an extender and 5% glycerol as a cryoprotectant in African ca tfish sperm cryopreservation (Urbanyi e ta /.,1999). Presence of DMSO in milt extender was essential for protecting the sperm fro m dying during freeze and th a w and 20% o f DMSO yielded th e highest post - thawed sperm moti(ity of 20-25 % o f the total cells (Y ao etal., 2000).
M ost workers use a dilution ratio o f one part sem en to 3-4 parts exte nde r (Scott & Baynes. 1980) .The post-thaw fertilisation rates decrease a t dilution ratios o f less than 3 fold in Onchorhynchus mykiss and of less than five to seven fold in Salm o trutta f.lacustris and Salmo trutta f.fario which demonstrates th a t the optimal dilution ratio m u st not exceed a critical value estim ated to 2.0 to 2.5x10®
sperm atozoa /m l diluent. H igher cell concentrations in the extender significantly decrease th e post-thaw fe rtility of milt, lower sperm concentrations do not affect its post-thaw fertility (W eism ann et a i, 1995).
Equilibration tim e should be kept to a minimum (minutes to less tha n one hour) to avoid exhaustion (Billard, 1978; C h a o e t a!., 1975). The ra te of cooling is a critical variable during cryopresen/ation (Scott & Baynes, 1980). O ptim al cooling rate is 30°C per minute (Harvey, 1982). French straw method w a s designed to speed up the collection, freezing a n d distribution of sperm (Scott and Baynes, 1980). Fresh semen diluted with cryoprotective a g ents was dispensed into 0.5 m l straws, which w ere th e n sealed, T h e s e pre treatm ents prior to cryopreservation had to be done within th e correct equilibration tim e o f one
hour o r )ess. The optim um effect of oryoprotectlve agents was found w ith 5-10 % glycerine /DMSO at 1:1, 1:5, 1:10 dilution. In this condition both good motility and fertility before freezing and cryoprotection were obtained (Chao a/., 197S).
Fertilizing ability o f previously frozen and thawed rainbow trout sperm was high when pH was 7-8 p rio r to addition o f DMSO and sem en. Fertilization rates o f eggs from individual fe m a le s with frozen thaw ed sperm were found to be significantly different ( Stoss and H oftz,198 1).
2.3 MORPHOLOGICAL CHANG ES OF FISH SPERM
Cryoinjuries occurs to biological material during cryopreservation. In trout sperm morphological changes occur w hen they are diluted in fresh water. W ithin seconds th e plasma m em brane around th e head becom es swollen and blisters form a t the insertion of flagellum and m idpiece. The flagellum axis subsequently becomes coiled up within its membrane. T h e se changes a re due to rapid uptake of w ater, as a consequence of the osmotic imbalance o f the sperm and the medium. This is evidenced by the fact th a t phy'siological salines result in less pronounced changes (Billard, 1978).
In rainbow tro u t and brown trout in thawed sperm atozoa after cryopreservation considerable ultra-structural alterations w a s observed in chromatin structure (Billard, 1983).
W ithin the spermatozoan, plasm a m em brane is probably th e site most susceptible to dam age due to w a te r flux during freezing and thawing (Baynes and S c o tt ,1987).
In rainbow tro u t Oncorhynchus mykiss, sperm atozoa went through morphological changes during storage, like w inding of
flagella, detachm ent of n u cle a r envelope and plasma m em brane from the nucleus of the sperm head. There were 1% abnormal sperm atozoa in fresh spe rm and about 15% during storage (Park and Yoon, 1992). Electron m fcroscopy indicated obvioas structural damage, especially in the trout sperm atozoa (Gwo e ta /., 1993).
In ocean pout, cryopresen/ation caused sperm ultra structural changes Including the shrinkage of cell m embranes leading to the exposure o f mitochondria and death of th e sperm (Yao e ta l., 1995}.
Osmolality is an external trig g e r for the initiation of sperm m o tility in marine teleosts. Following the activation o f the 3 m arine fish sperm in a rtificia l seawater, th e mitochondria of the sperm w e re shrunken and completely disappeared at the end of m otility duration, with no obvious cha nge in other sperm ultrastructures. On exposure to a hypotonic solution (distilled water) severe morphological distortions including swelling of the nucleus, m itochondria completely disappeared and bursting of plasma m em branes occurred. A fte r 1-minute activation in artificial seawater, th e intracristal spaces were grossly dilated, th e matrix space reduced to a m inim um , and bud like evaginations o f cristae m em branes grew into in tra cristae spaces. Marine fish sperm m otility is closely related to existence o f mitochondria in the m id-piece (Gwo, 1995).
In rainbow tro u t {Oncorhyncus mykiss ) sem en, after cryopreservation, about 10-20% of the spermatozoa had an unchanged structure th a t w a s similar to untreated sperm atozoa;
about 20-40% showed in te n sive signs of sw elling of the head and midpiece regions and o f the m itochondria. The remaining sperm atozoa were da m ag ed in som e w ay. Creatine phosphate
and a d e n o sin e tri ph osph ate levels w ere significantly low er in frozen th a w e d sem en tha n in untreated semen (Lahnsteiner e ta l.. 1996).
In rainbow tro u t non-dam aged cryopresen/ed sperm atozoa presented th e same characteristics of fresh spermatozoa, except for chrom atin , w hich was e x te n s iv e ly clumped. Spermatozoa th a t were dam aged b y cryopreservation exhibited a sw elled and non-continuous m e m b ra n e with a c lu m p e re d chromatin, showing high electron de n sity zo n e s. The fla g e lla of fresh sperm atozoa sho w e d a c o n tin u o u s and well organ ize d membrane which surroun ds the typical 9 + 2 structure o f th e axoneme, w h ile the sam e flagella s tru ctu re w a s observed in non-damaged cryopresen/ed sperm atozoa.
In s p e rm a to z o a damaged b y cryopreservation, a break and a d is p la c e m e n t of axonem e, along with changes in flagella m e m b ra n e w ere observed. T h e shape o f mitochondria w as not altered by cryopreservation (C onget etal., 1996).
A low m e m b ra n e cholesterol or phospholipid ratio w as s tro n g ly correlated w ith better fre e zin g resistance o f the c o rre s p o n d in g sperm (Labbe e t al.. 1996). M o st of the dam age occurred w ithin 10 m in u te s of a d d in g DMSO to th e sperm suspension in carps (L u b z e n s etal., 1997).
In freeze-fracture electron microscopy stud ies of ra in bow tro u t {O ncorhynchus m ykiss), it w as revealed th a t the changes w e re induced in the organization of the plasma m e m b ra n e s o f sp e rm a to zo a when th e y were cryopreserved, Electron m icrographic im a g e s of sperm atozoa that had n o t been exposed to cryopreservation showed a surface with particles h o m o g e n o u sly distributed. T h e concentration o f particles w a s low in the m e d ia n portion o f th e tail. The authors obtained longitudinal strips, c o n s is tin g of p a rtic le s along the bands. In the neck of
the spermatozoan the particfes were aggregated in chaotic manner. Electron micrographs of trout sperm atozoa that had been cryopreserved showed particles grouped in rounded clusters on the protoplasm ic surface o f both head and tail. In some spermatozoa, folding of protoplasm ic m em brane, with the particle free sites w e re found (Drokin eta l.,19 98).
Studies in European catfish sperm , the best protection was given by dimethylacetam ide (lO a n d lS % ) in sucrose solution.
Under th e se conditions, the percentage of cells with intact m em brane was high (90%) and the protection of the activity of the m itochondria was m edium (47%). It was shown that addition of dimethylacetamide increased th e ATP content o f spermatozoa (Ogier-de*baulney e t a!.,
1999).
In Rohu sperm atozoa dam ages were observed in head, m idpiece, and tail of about 51% of spermatozoa im m ediately after dilution in one o f the cryodiluents. After 10 m inutes of equilibration period, the frequency of damaged sperm is increased to 85% (Gopalakrishnan eta!., 20 00).
2.4 PO ST-THAW MOTILITY A ND FERTILITY
In a series o f fertility tests in carps, the chemical constituency of the medium in which fertilization was attem pted influenced the fertility o f cryopreserved sperm (Kurokura e fa /., 1984).
Addition o f egg yolk to th e extenders increased the fertilization rates in brown tro u t (Piironen, 1993). Cryopreserved sperm showed motility percentages between 20 and 30% (0tem e e fa /., 1996). A strong positive correlation was fo u n d between the post-thaw m otility and fertility of frozen sperm securing high fertilization in com m on carp (Magyary e t al., 1996). Experiments on improvem ent of sperm atozoa motility and stabilization o f spermatozoa m otility
after tha w in g of frozen spermatozoa w ith Na super (+) and K super (+) ions in solutions were initiated with com m on carp, Cyprinus carpio for th e purpose of long-term preservation of sperm atozoa. The m aintenance of capacity fo r motility o f post
thawed sperm atozoa was tested with KCI and NaCI solutio ns 200 -4 0 0 mM w ith 30mM T r is - H C I buffer, pH 7. T h e 275 mM NaCI solution appeared to be the best one with 80% pos -thaw sperm atozoa motility a g a in st 23% sperm atozoa m otility o f the control. (Linhart and C osson, 1997). In Liza macrolepis sperm atozoa, which displayed a p o s t-th a w m otility of 30-40% , w ere selected fo r inseminating th e ova (Sultana e t a i , 1998).
In studies in Muskellunge spermatozoa, although sperm atozoa cryopresen/ed w ith extender containing 10% methanol had only traces of m otility o r no m otility at all after thawing, fertilization trials indicated that their fertilizing a b ility was preserved (Cierezko etal., 1999).
nucleus
rrijtochondrbn
flagellum-
proximal centrbld dismal centriole
cytoplasmic canal
Fig 1. Diagrammatfc representation o f S perm atozoa o f L iz a spe cie s (Jamieson, 1991)
MATERIALS AND
METHODS
3.MATERIALS AND METHODS
The candidate species fo r this cryopreservation experiments w as the gold spot mullet, Liza parsia. Ripe males with milt oozing on slight pressure of the abdomen were collected from Chinese dipnets operated in Vypeen region. T h e collected fishes were then transported in small bins with aeration and stocked in fibre tanks. They w ere maintained at a salinity o f 20 °/oo.
3.1 C O LL E C T IO N OF M ILT
T h e stocked Liza parsia m ales were given an intramuscular injection of Ovaprim (Syndel laboratories. Canada, marketed in India by Agrivet Farm Care Division of Glaxo) at the rate of 0.3 ml per kg body weight. The fishes after injection were given a brief disinfection treatm ent in povidone iodine solution. After 24 hours they were dry stripped manually and milt collected in vials, without contamination of blood, urine and faeces.
3.2 P R E LIM IN A R Y MOTILITY STUDIES
O ne drop of the milt collected was placed on a glass slide and its motility checked in fre sh water and seawater.
For this a compound m icroscope w a s used and motility observed in lO xlO x m agnification. The sp e rm s were im m o tile in fresh w ater. T h e sperms a fte r treatm ent in freshwater were collected and fixed in glutaraldehyde fo r studying the e ffe c t of osmotic sh o ck on the m orphology o f sperms e le ctro n microscopically.
Sperm s were a c tiv e ly motile in seawater o f salinity 35°/oo’ M otility in seawater lasted for ab o u t 6 minutes .T he milt
samples fo r further treatm ents were te ste d for their q u ality taking motility as the criteria.
A drop o f m ilt taken from a live ripe specim en of Liza p a rs ia was placed on a clean dry glass slid e with a drop o f seawater. A co ve r slip was carefully placed over it and th e material w a s immediately observed u n d e r a com pound microscope a t a m agnification of 10x1 Ox. Three main typ e s of m ovem ents could be observed.
• Rapid progressive o r shooting m ovem ent.
• S luggish o r fethargic movement.
• V ibratio n in loco.
A quick eye estimation of the approximate percentage of sperm atozoa belonging to each of the above categories was done. On the basis of this a motility score was given to each sample as per the table below. The m ethod was standardised by repeating the process w ith a number o f samples.
CRITERIA MOTILITYSCORE
90% or above of the sp e rm s 5
exhibiting rapid progressive or shooting m o v e m e n t.
75% or m ore exhibiting rapid 4
progressive m ovem ent, 10%
sluggish and the rest im m o tile .
50% exhibiting rapid progressive 3
m o ve m e n t, 25% sluggish and 10%
vibrating in loco and th e rest immotile.
25% exhibiting shooting m o v e m e n t, 2
50% m oving sluggishly, 10% vibrating in loco and the rest im m o tile ,
O ccasional sperm shooting, 10% 1
Showing sluggish m ovem ent, 50%
Vibrating in loco and th e rest immotile.
No shooting movement, occasionally sperm 0.5
Moving sluggishly. About 10% vibrating in Loco. M ajo rity immotile.
CompJetely immotile. 0
O nly samples with a motility score of 4 and above were chosen fo r treatments.
3.3 PREPARATION OF EXTENDERS
Extenders are sim ple salt solutions which kee p the sperms in viable but in inactive condition. The chem ical composition o f diiferent extenders Is presented in T a b le 1.
Table 1. Chemical composition of different extenders
Extender /
v ' Chemical composition (mg)
CC1 (Kuroku ra et al,
1984)
Rana and
Me Andrew
(1989)
Chao (19751
Marine teleost ringer solution
fAixture B rEliza-
beth, 1987)
Mixture C [Eliza
beth, 1987)
V2E (Scott &
Baynes, 1980)
NaCI 750 650 1350 1350 600 600 750
KCI 2 0 300 60 60 38 38 38
CaCl2 2 0 30 - - - 23 -
NaHCOs 2 0 20 20 20 200 100 200
N a H P O t W • • - - - 41 -
MgSO,, 7HsO - • 35 35 23 23 -
MgClz - - - - - - •
NasHPO^ - - - - 53 - -
Glucose - - 5 00 0 - - - 100
E g g yolk - - - - - - 20
Distilled
w ater/seaw ater
100 100 100 100 100 100 100
p H 7.3 7.3 7.2 6.8 7.0 7.3 7.0
A s the sperm s were m o tile in seawater only, seawater w as identified as th e spawning m edium and all these extenders (except C hao's extender and 0.9% NaCI) w e re also prepared w ith seawater as base solution.
M otility of sp e rm s in these extender solutions was observed usin g a com pound microscope, and extenders were selected ba se d on their c a p a c ity not to a ctiva te spermatozoa after mixing. T h e sperms were actively m otile in Marine Fish Ringer solution (pH 6.8), Chao’s E x te n d e r (pH 7.2), CC 1 Extender (pH 7.3), CC1 E xtender with seawater as base solution (pH 7.3), Rana and Me Andrew E xte n d e r (pH 7.2), M ixtu re B (pH 7). Mixture B w ith seaw ater as base solutio n (pH 7.2), M ixtu re C (pH 7.3) and V2E Extender (pH 7).
T h e sperms w e re feebly active in (motility score 1 and 0 .5 ) V2E w ith seawater as b a se solution, M ixture C with sea w a ter as b a se solution and Rana a n d Mo Andrew e xte n d e r with se a w a te r as b a se solution. These exte nde rs were selected for the present experim ents.
C hao's extender was also selected e ve n though sp e rm s w e re motile w hen diluted, as it is one of the com m only u s e d exte nde rs for cryopreserving sperm s of mullets. Dimethyl sulphoxide (D M SO ) at a fin a l concentration o f 10% was used as the c ry o p ro te c ta n t.
3 .4 PRO CEDURE
T h e ratio of m ilt; cryodiluent (Extender + DMSO) fo r this e xp erim e nt w as fixed as 1:3, with a final concentration o f DMSO, 10% . So fo r every 1ml of m ilt, 2.6ml of extender and 0 .4 m l of D M S O w as taken,
3 .5 TR EA TM E N TS
T h e spermatozoa were treated with cryodiiuents as fo llow s:
1) Zero seconds after m ixing with cryodiiuents.
2) After an equilibration tim e of 10 m in u te s with cryodiiuents including the time ta k e n for filling diluted milt into 0.5 m l French straws.
3 ) Exposing to liquid nitrogen vapours (-100°C) after step 2.
4 ) After vapour phase, plunging straws into liquid nitrogen and keeping overnight.
A fte r each tre a tm e n t the diluted m ilt was thawed fo r 20 seconds in a water bath m aintained a t 37°C and processed for
Transmission electron m icroscopy studies follow ing the method o f Elizabeth (1987), Diwan & Nandakumar (1998) and Gopalakrishnan etal., (2000).
3.6 PROCESSING OF SPERMS FOR ELECTBON MICROSCOPY STUDIES
The samples after all the treatm ents were primarily fixed in 2% buffered glutaraldehyde for 2 hours at 4'^C following immersion fix a tio n . Then th e supernatant w as decanted and the pellets w ere washed thrice for 15 minutes each using sodium cacodylate buffer. Then the samples were centrifuged for 10 minutes at 5,000 rpm. The pellets were post - fixed In 1 % osmium tetroxide fo r 1 hour at 4°C and centrifuged at 5000 rpm fo r 10 minutes . A fte r the fixations th e pellets were transferred to 2% agar. Then the agar blocks were trimmed to Im m ^ size. The agar embedded samples were transferred stepwise through a concentration series of acetone (30%, 50% , 70% , 90% and 100 % v A /) following standard time schedule. Following the dehydration steps, infiltration is carried out in spurr m edium with acetone in 3 steps. First in Spurr/acetone ratio 1 :3 f o r i hour, second step in1:1 and finally in 3:1 for I hour each at 4°C. Embedding was carried in Spurr’s medium in plastic mould and kept in incubator for 12 hours at 70°C , fo r polymerisation.
The polymerised blocks were cu t into ultra thin sections in the LKB ultra tome Nova .The thin sections were double stained in uranyl acetate and lead citrate for 10 minutes. The stained thin sections w ere mounted on the grid and observed In the STEM model Hitachi (H 600) electron microscope and recorded the fmage.
The ratio o f intact and damaged spermatozoa for each treatm ent was calculated by counting sperms under low magnification (4000 x) in TEM . In this process, random ly selected portions o f grids (at least Sgrids for each treatment) were brought under magnification and all together 150 spermatozoa were screened fo r each treatment. TEM images of untreated raw mitt served as control.
RESULTS
4. RESULTS
4.1 ULTRASTRUCTURE O F UNTREATED SPERMATOZOA
T h e sperm o f Liza parsia is a typical anacrosomal aquasperm. T h e nucleus is bilobed or kidney shaped in longitudinal section {Plate 1). It is also tilted relative to the axoneme. Chromatin is very coarsely g ra n u la r and not condensed. Matrix spaces are clearly visible.
A cytoplasm ic c o lla r, which exte nds around the b a se o f the flageKum, Is p resen t and it Is separated fro m the flagellum b y a periaxonemal space, the c y to p la sm ic canal (P la te 1). Small cristate mitochondria are present and th e y m ay be situ a te d In the cytoplasmic collar.
The flageilum is parallel to th e b a s e of the n u c le u s and a depression is present a t th is point, the n u c le a r fossa (Plate 1). A plasma m em brane surrounding th e whole stru c tu re is also present (Plate 2).
4 .2 THE U L T R A S T R U C T U R A L CHANGES O F L iza pars/a SPERM AFTER O S M O T IC S H O C K . (ZER O S E C O N D S AFTER DILUTION WITH F R E S H W A T E R )
T h e most c o n s p ic u o u s change a fte r the osmotic sho ck is bursting o f p la s m a m em brane a n d it's subsequent loss (Plates 3 & 4). The nucleus b e c o m e s rounded in 80% of the sp e rm s and the nuclear fossa becomes le ss conspicuous (P la te s 3 & 4). F lageilum gets separated from tl*e nucleus a n d gets coiled u p w ithin its m e m b ra n e (Plates 3 & 4).
4.3 THE ULTRASTRUCTURAL CHANGES A FTER TREATMENTS
4.3.1 C H A O ’S EXTENDER A N D DMSO
4.3.1.1 Z e ro seconds after dilution (Plate 5): Mitochondria and plasma membrane is intact in 80% o f sperms. A condensed nucleus with a clear nuclear fo s s a is present.
4.3.1.2 10 m in u tes after dilution- after an equilibration tim e (plateS) Nucleus is d ila te d and nuclear fossa becam e less visible. Plasm a membrane is present. The chrom atin material ha s becom e more condensed.
About 33% o f sperm s exhibited such abnormalities.
4.3.1.2 A fte r exposure to Liquid Nitrogen v a p o u rs (Plate?): T h e gross morphology of the sperm atozoa remained m ore or less sim ila r as compared to th e second step, but the percentage of damaged sperm atozoa increased to 42% .
4.3.1.4 After plunging into Liquid Nitrogen (PlateS): The sa m e intact nucleus is p re se n t as in step 3 ,But the plasm a membrane a p p e a re d to be ruptured. T h e percentage o f damaged sperm s Increased to 52%.
4.3.2 M IX T U R E C AND DMSO
4.3.2.1 Zero seconds after dilution (Plate 9): T h e spermatozoa appeared similar to th e ra w m ilt in 70% o f sperm s. Rest 30% of sperms exhibited slight disruption o f mid-piece.
A.3.2.2 10 m in u te s after dilution- after an equilibration time (platelO ):
After an equilibration time m itochondria are less conspicuous. T h e plasm a
membrane exhibited corrugated appearance. T h e chromatin g o t more condensed. T h e s e damages o ccu rred in almost 6 0 % o f sperms.
4.3.2.3 A fte r exposure to L iq u id Nitrogen v a p o u rs (P!ate11): Plasma membrane is to ta lly disrupted b u t the shape o f th e nucleus rem ained more or less sa m e as in the a b o v e case, The m id -p ie ce and m itochondria became a lto g e th e r ruptured.
4.3.2.4 After plunging Into Liquid Nitrogen (PIate12): A lm ost 75% of sperm atozoa becam e abnorm al a n d exhibited s tru c tu ra l changes a s in step 3. In addition th e nucleus exhibited vacuoles inside chromatin m aterial.
4.3.3 RANA A N D MC A NDREW EXTENDER A N D DMSO
4.3.3.1 Zero seconds after d ilu tio n (Plate 13): A lm o s t 70% of sperm atozoa became a b n o rm a l and exhibited following stru c tu ra l alterations. Nucleus remained in ta ct but the plasm a membrane e xh ib ite d undulations and the chromatin m a terial got condensed and vacuoles w e re present in it. T h e mid
piece and m itochondria were deform ed.
4.3.3.2 10 m in u te s after d ilu tio n - after an equilibration time (plate14):
The structure is almost sim ila r to the a b o v e treatm ent (1), bu t th e rate damaged sp e rm s increased to 85% .
4.3.3.3 A fte r exposure to Liquid N itro g en vapours(Plate15):
Mitochondria in the m idpiece are totally da m ag ed and th e plasm a membrane sh o w e d severe undulations.
4.3.3.4 After plunging into Liquid Nitrogen (Plate16): alm ost 95% of sperms exhibited complete d a m a g e s in head a n d mid-piece re gion as in previous tre a tm e n t (3).
4.3.4 V2E EXTENDER AND D M SO
4.3.4.1 Z e ro seconds after dilution (Plate17): A lm o st all the sperm atozoa appeared s im ila r to sperms in ra w milt without m u ch damages. A ppearance of flagellum , mitochondria, nucle us, nuclear fo s s a etc, are m ore o r less similar to th e ra w milt In 90% o f th e sperms.
4.5.4.2 10 m in u te s after dilution^ after an equilibration time (plate18): All the stru cture s remained m ore or less similar to the above step in 76%
sperms. R e s t o f the sperms sh o w e d disintegration o f plasma m em brane and rupture m id -p ie ce .
4.5.4.3 A fte r exposure to liquid nitrogen vapours (PlatelS): All m o s t 60%
sperms n e a r norm al structure. T h e plasma m em brane is more o r less intact and did no t e xh ib it much evaginations. Nucleus is intact. M itochondria also appeared n e a r normal,
4.3.4.4 A fte r plunging Into liq uid nitrogen (Plate 20): Structural fe a tu re of sperm atozoa did not differ fu rth e r after freezing and thawing protocol. The plasma m e m b ra n e appeared to be more disrupted compared to th e previous stage and th e mitochondria de tache d. The percentage of damaged sperms increased to 46% .
Table 2. Percentage of intact spermatozoa of liza parsia and their m otility score after each step in various extenders+DMSO (10%)
Extender
Treatments
CHAO’ S EXTENDER
+DMSO
MIXTURE C +DMSO
RANA&
MC ANDREW
+DMSO
V2 E +DMSO
Step-1 (0 seconds a fte r
dilution)
80.67±5.33 (5)
70.34±5.86 (4)
29.98±5.11 (3)
89.23±6.68 (5)
Step-2 (10 min a fte r
exposure)
66.38±5,61 (4)
40,04*3,34 (3)
15.56±1.67 (2)
76.G4±2.98 (5)
Step-3 (After va p o u r
pfiase)
58,10*3.89 (4)
30,66±6.94 (3)
10,89±5.32 (1)
59.87±4,91 (4)
Step-4 (After Liquid N2
exposure)
47.83±0.64 (3)
25,06±9.91 (2)
5.01±0.2 (5)
54.0±1.76 (4)
Figures in parenthesis indicate m otility score
