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Cryopreservation of rainbow trout (Oncorhynchus mykiss) spermatozoa using programmable freezing
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Aquaculture 143 (19%) 319-329
Cryopreservation of rainbow trout ( Oncorhynchus mykiss) spermatozoa using programmable freezing Paulette Conget *, Mireya Ferrhdez, Guillermo Herrera, JosC J. Minguell Unidad de Biologia Celular. INTA, Uniuersidad de Chile, Casilla 138, Santiago II. Chile
Accepted 6 February 1996
Abstract In an attempt to establish a protocol for the cryopreservation of the spermatozoa of the rainbow trout (Oncorhynchus mykiss), we studied the effect of various cryoprotective agents (CA) in spermatozoa motility and viability, before, during, and after freezing. Freezing was performed by using a controlled rate freezing system, which allows the accurate setting of different cooling rates, as well as a proper recording of intra-sample temperatures throughout the procedure. Results obtained indicate that before the initiation of freezing, spermatozoa motility is affected more by the length of time of exposure to CA than by the chemical nature of the agents. Exposure periods longer than 10 min affected motility irreversibly, which seems to be related to the high osmolarity of the extender solutions. To study the changes in spermatozoa motility and viability during and after cryopreservation, cells in the cryoprotective solution (glycerol, DMSO or DMSO-sucrose) were processed in a programmable biological freezer at slow (1°C and 10°C min-‘) or rapid (30°C min- ‘) cooling rates. Results obtained indicated that both during ( - 60°C) and after completion of the freezing program ( - 80°C. followed by storage under liquid nitrogen), motility and viability was well preserved only in the cells in DMSO-sucrose when subjected to a rapid cooling rate. Under these conditions, approximately 63% of spermatozoa were alive and showed progressive motility. Together, the average fertilization potential of cryopreserved semen was 58% (47% to 85%) compared with that of fresh semen. The procedure described here provides consistency and precision, and permits processing, freezing and storage of trout spermatozoa in less than 15 min. Keywords:
l
Cryopreservation; Spermatozoa; Oncorhynchus mykiss; Motility
Corresponding author. Tel: 562-678-1428; fax: 562-221-4030.
0044-8486/%/$15.00
Copyright 0 1996 Elsevier Science B.V. All rights reserved
PII SOO44-8486(96)01275-6
320
P. Conget et aL/Aquacubure
143 (1996) 319-329
1. Introduction Development of spermatozoa cryopreservation techniques has been reported for several fish species (Scott and Baynes, 1980; Stoss, 1983; Cognie et al., 1989). Cryopreserved fish sperm may enable the introduction of genes from wild or native populations into hatchery stocks when spawning times do not overlap, as well as the availability of semen for selective breeding programs, especially during seasonal shortages of males and/or females. Furthermore, cryopreserved semen can be exchanged between countries located in both hemispheres, instead of exchanging fish, a particular advantage when importation of live animals is prohibited (McAndrew et al., 1993). The increasing development of the aquaculture industry in Chile indicates a need for developing methods for the long-term storage of semen of salmonid species. For these reasons, spermatozoa preservation of trout (rainbow) and salmon (coho and sakura) is gaining importance in Chile. Several methods have been described for the short-term storage and cryopreservation of rainbow trout semen @toss and Holtz, 1981; Wheeler and Thorgaard, 1991; Holtz, 1993; Piironen, 1993). Since different authors have noticed that these methods suffered from a lack of consistency, it is important to develop alternative procedures for the establishment of a reliable method for cryopreservation of rainbow trout spermatozoa. The complexity of cryopreservation of biological material (Friedler et al., 1988) requires well-controlled freezing procedures; thus the use of programmable freezing equipment with adjustable rates of cooling and warming (Gorin, 1992) offers several advantages over manual freezing procedures. The studies reported here were done in an effort to establish, in Chile, a cryopreservation protocol that ensures the preservation of salmonid spermatozoa with high reproductive potential. In this report we have examined various factors that affected the motility, viability and the fertilization potential of rainbow trout spermatozoa, before, during, and after cryopreservation.
2. Methods 2.1. Semen collection Rainbow trout (Oncorhynchus carefully dried before extruding the separately into petri-dishes in a 2-4 stored on ice (1 to 3 h). Ejaculates were discarded. 2.2. Estimation of spermatozoa
mykiss) males, 2-3 years old, were rinsed and semen. Ejaculates of individual males were placed mm thick layer to allow proper gas exchange, and contaminated with fecal material, urine, or blood
density
Spermatozoa concentration was estimated by a spectrophotometric method (Ciereszko and Darbrowski, 1993). Briefly, semen was diluted l/600 and l/1200 with 0.7% NaCl and the optical density of each dilution was measured at a wavelength of 505 nm. The average value was used to calculate spermatozoa density by interpolating into a standard
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curve which correlates spermatozoa concentration (determined using a Neubauer counting chamber) with optical density. 2.3. Cryoprotective
agents
Fresh semen was mixed at a ratio 1:4 with the following cryoprotectants: 1.5 M propylene glycol, 1.5 M glycerol, 1.5 M dimethyl sulfoxide (DMSO), 1.5 M DMSO + 0.6 M sucrose or 1.5 M sucrose. Cells were exposed to each agent for different periods of time and then spermatozoa motility, viability and fertilization potential was evaluated. 2.4. Cryopreservation
procedures
To study the effect of cryoprotective agents on spermatozoa motility before cryopreservation, fresh semen was directly mixed with the cryoprotectant extenders (see above) and kept on ice for time periods up to 30 min, and then spermatozoa motility was evaluated. To study the effect of cryoprotectants on spermatozoa motility during and after freezing, fresh semen mixed with the different cryoprotectants, was drawn without delay into flat clear plastic straws (2.5 ml total volume) and placed in the freezing chamber (2”C), of a liquid-nitrogen programmable freezing system (CryoMed 1010, Forma Scientific, Inc.). The freezing programs used were empirically determined to give inn-a-straw freezing rates of 3°C min-’ to - 5°C and then l”C, lO”C, or 30°C min- ’ to - 80°C. During ( - 60°C) or after freezing ( - 80°C followed by a short-term storage of 15 to 30 days in liquid nitrogen), samples were thawed at 37°C and immediately used to evaluate spermatozoa motility, viability or fertilization potential. 2.5. Estimation of spermatozoa
motility
Motility was evaluated as the percentage of spermatozoa actively swimming with progressive movement (forward movement). For this, at least two aliquots of semen samples from each male in the respective cryoprotective agent were diluted (l/320) with medium M-532 (0.55% NaCl, 0.38% Glycine, 0.24% Tris-HCl, pH 8.8) on a cold glass slide placed onto an optic microscope (Billard, 1992). For each sample, at least five microscopic fields (magnification X 400) were observed and the average of these counts was used to calculate the percentage of motile spermatozoa. 2.6. Estimation of spermatozoa
viability
Viability (VB) was estimated by the propidium iodide (PI) exclusion test (Ormerod et al., 1993). Semen samples were diluted l/1200 with 0.7% NaCl, stained with PI (1 p,g ml- ’ 1, mounted on slides and observed by epifluorescence microscopy ( X 400). While dead spermatozoa were brightly labelled with PI, viable spermatozoa were visible only by light phase. At least 200 spermatozoa were scored each time. VB was calculated as: Total number spermatozoa - (PI - stained spermatozoa) Total number spermatozoa
x 100
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2.7. Estimation
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of spermatozoa
fertilization
143 (1996) 319-329
potential
Fresh (control, diluted in the cryoprotectant mixture) or cryopreserved semen was poured over freshly collected eggs (400 per assay) pooled from four females and suspended in medium M-532 at a ratio of 3 X lo6 spermatozoa per egg. After gently stirring, spermatozoa and eggs were co-incubated for 3 min at room temperature, followed by the addition of dechlorinated tap water to water-harden the eggs. Five min later, the eggs were rinsed and finally incubated in a vertical incubator with oxygenated water at 11°C (Billard, 1992). Dead eggs were removed at regular intervals. For both fresh and cryopreserved spermatozoa, the fertilization rate (FR) was evaluated 21 days after insemination by counting the number of eyed eggs by naked eye examination, and expressed as the percentage of eyed eggs over the total number of inseminated eggs. 2.8. Electron
microscopy
studies
Samples of fresh or cryopreserved semen were fixed in 3% pamformaldehyde, 0.5% glutaraldehyde, 0.1 M cacodylate buffer and 0.3 M sucrose for 1 h, post-fixed in 1% osmium tetroxide and embedded in Epon 812 resin (Gwo and Arnold, 1992). Sections were obtained in a Sorval MT2-B microtome, stained with uranyl acetate and lead citrate and examined under a Phillips EM-300 transmission electron microscope. At least 50 spermatozoa were examined, with particular attention given to the structure of the plasma membrane, nuclear material and flagella.
3. Results 3.1. Effect of cryoprotective
agents on spermatozoa
motility before freezing
To evaluate the effect that cryoprotective agents (CA) produce in motility of rainbow trout spermatozoa, fresh semen samples were exposed to differents agents commonly employed in cryopreservation procedures (Friedler et al., 1988). As seen in Fig. 1A, when semen samples were exposed to the various CA for time periods of 10 min, all CA used except sucrose, preserved spermatozoa motility. However, when the exposure time was extended to 30 min, spermatozoa motility was extensively decreased in all cases, except with propyleneglycol (Fig. 1B). For exposure periods between 10 and 30 min, motility decreased in a time-dependent fashion (data not shown). Control semen samples in PBS exhibited a high motility (close to 100%) during the assay periods. These results indicated that to preserve motility better before freezing, spermatozoa should be exposed to CA (with the exception of the non-permeating agent, sucrose) for time periods not longer than 10 min. The above inference was supported by the observation that after exposure to DMSO-sucrose (15 min), spermatozoa motility which was low (close to 30%) fully recovered after transferring cells to PBS or seminal plasma. However, if cells were exposed to the same solution for 30 min and then transferred to PBS or seminal plasma, motility was not recovered (data not shown). The final concentration of CA used in the above experiments was 1.1 M (with the
P. Conget et al./Aquaculture
PBS
loo -
Propylene Gtycerot
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wso
DM.so+suc sucros8
DMBO
wso+auc
-
T 60-
g E = 0 I
60-
40-
20 -
01
PBS
I
sucrose
Fig. 1. Effect of cryoprotective agents on spermatozoa motility before freezing. Fresh semen was mixed (1:4) with the following extenders: phosphate buffered saline (PBS, control); 1.5 M propylene glycol (propylene); 1.5 M glycerol; 1.5 M dimethyl sulfoxide (DMSO); 1.5 M DMSO+0.6 M sucrose (DMSO+ SUC) or 1.5 M sucrose. Cells were exposed to the cryoprotective extenders for 10 min (A) or 30 mm (B) and then activated to evaluate spermatozoa motility, as indicated under Methods. Each bar represents the mean+ SD of the percentage of motile spermatozoa in semen samples obtained from two males, with each sample tested twice.
exception of DMSO-sucrose), however the contribution of each chemical to the osmolarity of the medium was different. Therefore, the dramatic decrease in spermatozoa motility observed after the 30 min exposure-period (Fig. 1B) could be the consequence of the inability of spermatozoa to adapt to the hyperosmotic medium created by the addition of the cryoprotectants. This possibility was explored by measuring motility of fresh semen exposed to solutions of cryoprotective agents with increasing osmolarities. The results in Fig. 2 show that exposure to PBS or seminal plasma (300 mOsm) resulted in no changes in spermatozoa motility, compared with fresh semen. However, as the
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Ftash Seminal plasma
PBS Gly03rOl Glycarol+sucroaa suuoae DMsG+suuoaa Glycerol+sucroaa DMSO+auaoae DMso+aucroaa 0
Fig. 2. Effect of osmolarity of cryoprotectant medium on spermatozoa motility. Fresh semen (in seminal plasma) was exposed to media prepared to the following fmal osmolarities (shown on the right of each bar): 300 mOsm (seminal plasma or PBS); 800 mOsm (1.1 M glycerol); 1000 mOsm (0.55 M glycerol + 0.1 M sucrose); 1200 mOsm ( 1.1 M sucrose or 0.55 M DMSO + 0.1 M sucrose); 1800mOsm (1.1 M glycerol + 0.1 M sucrose or 1.1 M DMSO+ 0.1 M sucrose); 2300 mOsm (1.1 M DMSO + 0.45 M sucrose). After 20 min exposure at 4°C. spermatozoa motility was evaluated. Each bar represents the mean f SD of the percentage of motile spermatozoa in semen samples obtained from two males, with each sample tested twice.
osmolarity of the medium was increased, spermatozoa motility was concomitantly decreased. Thus, at osmolarities of 800 to 1000 mOsm, motility ranged between 80% and 50%, respectively. Exposure to solutions of higher osmolarity, resulted in semen with low spermatozoa motility (less than 30%). Thus, the exposure of semen to non-isosmotic conditions for time periods longer than 10 min, proved to be critical for the preservation of spermatozoa motility. The exposure of fresh semen to solutions with the same osmolarity, but containing different CA, did not affect motility (see mixtures at 1200 or 1800 mOsm in Fig. 2), thus excluding a cytotoxic effect, per se of cryoprotectants on spermatozoa motility. 3.2. EfSect of cryoprotective
agents on spermatozoa
motility during and after freezing
Programmable freezing equipment was used to establish proper experimental conditions for rainbow trout spermatozoa cryopreservation. The use of such methodology allows the evaluation of spermatozoa motility at different rates of cooling during freezing. As seen in Table 1, at freezing rates of 1°C or 10°C min- ’ (slow freezing) there was a complete loss of motility, which was independent of the CA used. When cells were processed at 30°C min-’ (rapid freezing), semen diluted with DMSO-sucrose demonstrated a 63% motility, both during freezing (- 60°C) and after completion of freezing (- 80°C) followed by storage under liquid nitrogen. Spermatozoa motility for samples
P. Conget et aI./Aquaculture Table 1 Effect of cooling rates on spermatozoa
A.F.
325
motility during and after freezing Motility (o/o)
Cooling rate
D.F.
143 (1996) 319-329
l°C min-’ 10°C mm’ 30°C min30°C mm-’
’
Glycerol
DMSO
0 15* 12 15*10 lo*11
0 0
DMSO + sucrose n.d. n.d 63kl9 63fl2
5flO IX*11
Fresh semen was mixed ( 1:4) with 1.5 M glycerol, 1.5 M DMSO or 1.5 M DMSO + 0.6 M sucrose. Samples in the freezing chamber of a programmable freezing equipment were subjected to cooling rates of l”C, 10°C or 30°C min-‘. Spermatozoa motility was evaluated during the freezing procedure (DF, at -60°C) and after completion of the freezing program (AF, at -80°C followed by storage under liquid nitrogen).Data correspond to the mean motility (o/o) f SD determined in semen samples obtained from five males, being each sample tested twice.
cryopreserved in glycerol or DMSO alone was negligible. Propyleneglycol, which proved to be an adequate agent to preserve spermatozoa motility under pre-freezing conditions (Fig. l), failed to preserve motility during freezing, both at slow and rapid freezing conditions (not shown). 3.3. Fertilization potential of cryopreserved
spermatozoa
To evaluate the fertilization capacity of spermatozoa cryopreserved under the conditions described above, aliquots of cryopreserved semen were used to inseminate rainbow trout eggs. Fertilization potential was evaluated 21 days after insemination, by counting eyed eggs. As seen in Fig. 3, cryopreserved semen samples (average motility of 64 f 15% after thawing) exhibited a high fertilization potential, which ranged from 47% to 85% (average fertilization rate 58 4 15%). On the other hand, semen samples cryopreserved under the same conditions, except that glycerol or DMSO was used instead of DMSO-sucrose, exhibited spermatozoa with low motility and a negligible fertilization potential (data not shown). 3.4. Ultrastructural
changes produced
in spermatozoa
by cryopreservation
The damage produced on rainbow trout spermatozoa by cryopreservation was observed at the structural level by TEM analysis. For these studies, samples of fresh and cryopreserved spermatozoa were used. Fresh spermatozoa exhibited a well defined and continuous cell membrane, in close contact with the cell nucleus. Chromatin was homogenous without evidence of high electron density zones (Fig. 4a). Non-damaged cryopreserved spermatozoa presented the same characteristics of fresh spermatozoa, except for chromatin which was extensively clumped (Fig. 4b). Spermatozoa that were damaged by cryopreservation exhibited a swelled and non-continuous membrane with a clumpered chromatin, showing high electron density zones (Fig. 4c). The flagella of fresh spermatozoa show a continuous and well organized membrane which surrounds the typical 9 + 2 structure of the axonema (Fig. 4a and d). While the same flagella
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80
-
';; b 9) 60
5 .z
-
-
40
g .r I!! 20
-/
I Il-i -
H
i
-
0
1
2
3
4
5
6
7
a
i 10
Male number
Fig. 3. Fertilization potential of cryopreserved spermatozoa. Semen samples from ten fish were rapidly (10 min or less) diluted (1:4) in a mixture of DMSO (1.5 M) and sucrose (0.6 M) and cryopreserved at a cooling rate of 30°C mm-‘. After storage in liquid nitrogen, semen from each male was thawed and used for motility and fertilization assays. The day of the fertilization assay, cryopreserved spermatozoa as well as a selected sample (motility and viability = 100%) of freshly collected semen were co-incubated with eggs (3X lo6 sperm per egg) and fertilization evaluated. as indicated under Methods. For the studied population, the average post-freezing motility was 64 f 15% and the average post-freezing fertilization rate was 58 f 15%. The results are expressed as percentages of the control (90 f 10% eyed). Note that cryopteserved samples from each male were tested once.
structure was observed in non-damaged cryopreserved spermatozoa (not shown), in spermatozoa damaged by cryopreservation, a break and a displacement of the axonema structure, along with changes in the flagella membrane were observed (Fig. 4e). Note that the shape of mitochondria was not altered by cryopreservation. The notion that the damage caused by cryopreservation modifies motility and viability (Moccia and Munkittrick, 1987), prompted us to analyze the relationship between motility and viability in samples subjected to conditions that will not affect (control, fresh samples at 4°C) or will affect (exposure to cryoprotective agents, followed or not by cryopreservation) spermatozoa structure, as suggested by the results in Fig. 4. As seen in Fig. 5, the linear relationship ( rz = 0.86) obtained for both spermatozoa survival parameters strengthens the concept that as a consequence of the disruption of the structure of the flagella and/or cell membrane, spermatozoa motility and viability are affected.
4. Discussion Effective storage of gametes is essential whenever selective breeding is carried out and is also a prerequisite for the establishment of gene banks for endangered wild stocks. Salmonids, like the rainbow trout and the coho salmon are commercially
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327
Fig. 4. Transmission electron microscopy of cross-sections of fresh and cryopreserved rainbow trout spermatozoa. Heads from fresh spermatozoa (a) and from non-damaged (b) and damaged (c) cryopresetved spermatozoa; flagella from fresh spermatozoa (d) and from damaged cryopreserved spermatozoa (e). Final magnification for heads is X 2OooO and for flagella X 42000.
important species in Chile. Since at present no routine long-term storage techniques for fish semen are available in Chile, we attempted to develop a protocol for the cryopreservation of rainbow trout spermatozoa by using programmable instead of manual freezing. Programmable freezing allows the pre-setting of different freezing programs, the monitoring of precise temperature during the cooling sections, and the continuous biological examination of cells, during the freezing stages (Gorin, 1992). By using programmable freezing equipment and after evaluating spermatozoa motil-
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60
0
20
40
60
80
100
Viability (%) Fig. 5. Relationship between motility and viability for rainbow trout spermatozoa. Linear regression analysis of percentage of motility and viability of fresh (Cl), fresh exposed to cryoprotective agents (0) and cryopreserved(+)spennatozoa(n=84). y=-6.76+1.15x; R*=0.86; P
ity. viability and fertilization potential, we have been able to define the following conditions for the successful cryopreservation of rainbow trout spermatozoa: 1. The use of a mixture of permeating (DMSO) and non-permeating (sucrose) cryoprotectant agents; 2. A short-term exposure of fresh semen (less than 10 min at 4°C) to the cryoprotective mixture, and finally; 3. Freezing should be performed at a cooling rate of 30°C min-‘, once the phase change has been attained. In this respect, it is important to emphasize that the most critical point for rainbow trout spermatozoa freezing is the rate selected for solid phase cooling (from - 5°C to - 80°C) and not the rate at which the liquid sample is cooled (from 4°C to - 5°C). This practical approach is supported by the theoretical concept that ice nucleation within cells occurs at - 40°C (Friedler et al., 1988). As deduced from our TEM studies, as well as those performed in the Atlantic croaker spermatozoa (Gwo and Arnold, 1992), the major damage produced by cryopreservation occurred in the spermatozoa cell membrane and flagellum. These observations, together with the correlation between spermatozoa viability (as indicated by the PI staining method) and motility, and between motility and fertilization potential (Moccia and Mtmkittrick, 1987) suggest that spermatozoa motility might be used as an initial predictive index to evaluate the efficiency of a cryopreservation protocol. A full
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correlation between the viability assessed by the PI exclusion method and fertilization success is required in future work. The freezing conditions described here, offer consistency, precision, and accuracy. Furthermore, the whole procedure is rapid, since after obtaining the ejaculates, cells are processed, frozen and ready to be stored under liquid nitrogen in less than 15 min. Although too sophisticated to be employed at the hatchery site, this method of cryopreservation should be also useful for the establishment of spermatozoa banks to handle material that has been obtained after selective breeding programs or genetic manipulation. Acknowledgements
This work was supported by Grant FONDEF (PI-lo), Chile. PC is the recipient of an A. Stekel Scholarship, INTA, University of Chile. References Billard, R., 1992. Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture, 100: 263-298. Ciercszko, A. and Darbrowski, K., 1993. Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using a spectrophotometric technique. Aquaculture, 109: 367-373. Cognie, F., Billard, R. and Chao, N.H., 1989. La Cryoconservation de la laitance de la carpe Cyprinus curpio . J. Appl. Ichthyol., 5: 165-176. Friedler, S., Giudice, L.C. and Lamb, E.J., 1988. Cryoprcscrvation of embryos and ova. Fertil. Steril., 49: 743-764. Gorin, N.C., 1992. Cryopreservation and storage of stem cells. In: E.M. Amman, H.J. Deeg and R.A. Sacher (Editors), Bone Marrow and Stem Cell Processing: A Manual of Current Techniques. F.A. Davis, Philadelphia, PA, pp. 293-362. Gwo, J.C. and Arnold, C.R., 1992. Cryopreservation of Atlantic croaker spermatozoa: evaluation of morphological changes. J. Exp. Zool., 264: 444-453. Holtz, W., 1993. Cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: practical mcommendations. Aquaculture, 110: 97-100. McAndrew, B.J., Rana, KJ. and Penman, T.J., 1993. Conservation and preservation of genetic variation in aquatic organisms. In: J.F. Muir and R.J. Roberts (Editors), Recent Advances in Aquaculture, Vol 4. Blackwell Science, Oxford, pp. 295-336. Moccia, R.D. and Munkittrick, K.R., 1987. Relationship between the fertilization of rainbow trout (Sulmo gairdneri) eggs and the motility of spermatozoa. Theriogenology, 27: 678-688. Ormerod, M.G., Sun, X.M., Snowden, RT, Davies, R., Fearnhead, H., and Cohen, G.M., 1993. Increased membrane permeability of apoptotic thymocytes: A flow cytometric study. Cytometry, 14: 595-602. Piironen, J., 1993. Cryopresevation of sperm from brown trout (Salmo rrurra m. lacusrris L.) and Attic chart (Salvelinus alpinus 15.1. Aquaculture, 116: 275-285. Scott, A.P. and Baynes, S.M., 1980. A review of the biology, handling and storage of salmonid spermatozoa, J. Fish Biol., 17: 707-739. Stoss, J. and Holtz, W., 1981. Cryopreservation of rainbow trout (Salmo gairdneri) sperm. I. Effect of thawing solution, sperm density and interval between thawing and insemination. Aquaculture, 22: 97-104. Stoss, J., 1983. Fish gamete preservation and spermatozoan physiology. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IX, Reproduction. Part B, Behaviour and Fertility Control. Academic Press, London, pp. 305-330. Wheeler, P.A. and Thorgaard, G.H., 1991. Cryopteservation of rainbow trout semen in large straws. Aquaculture, 93: 95- 100.
Aquaculture 143 (19%) 319-329
Cryopreservation of rainbow trout ( Oncorhynchus mykiss) spermatozoa using programmable freezing Paulette Conget *, Mireya Ferrhdez, Guillermo Herrera, JosC J. Minguell Unidad de Biologia Celular. INTA, Uniuersidad de Chile, Casilla 138, Santiago II. Chile
Accepted 6 February 1996
Abstract In an attempt to establish a protocol for the cryopreservation of the spermatozoa of the rainbow trout (Oncorhynchus mykiss), we studied the effect of various cryoprotective agents (CA) in spermatozoa motility and viability, before, during, and after freezing. Freezing was performed by using a controlled rate freezing system, which allows the accurate setting of different cooling rates, as well as a proper recording of intra-sample temperatures throughout the procedure. Results obtained indicate that before the initiation of freezing, spermatozoa motility is affected more by the length of time of exposure to CA than by the chemical nature of the agents. Exposure periods longer than 10 min affected motility irreversibly, which seems to be related to the high osmolarity of the extender solutions. To study the changes in spermatozoa motility and viability during and after cryopreservation, cells in the cryoprotective solution (glycerol, DMSO or DMSO-sucrose) were processed in a programmable biological freezer at slow (1°C and 10°C min-‘) or rapid (30°C min- ‘) cooling rates. Results obtained indicated that both during ( - 60°C) and after completion of the freezing program ( - 80°C. followed by storage under liquid nitrogen), motility and viability was well preserved only in the cells in DMSO-sucrose when subjected to a rapid cooling rate. Under these conditions, approximately 63% of spermatozoa were alive and showed progressive motility. Together, the average fertilization potential of cryopreserved semen was 58% (47% to 85%) compared with that of fresh semen. The procedure described here provides consistency and precision, and permits processing, freezing and storage of trout spermatozoa in less than 15 min. Keywords:
l
Cryopreservation; Spermatozoa; Oncorhynchus mykiss; Motility
Corresponding author. Tel: 562-678-1428; fax: 562-221-4030.
0044-8486/%/$15.00
Copyright 0 1996 Elsevier Science B.V. All rights reserved
PII SOO44-8486(96)01275-6
320
P. Conget et aL/Aquacubure
143 (1996) 319-329
1. Introduction Development of spermatozoa cryopreservation techniques has been reported for several fish species (Scott and Baynes, 1980; Stoss, 1983; Cognie et al., 1989). Cryopreserved fish sperm may enable the introduction of genes from wild or native populations into hatchery stocks when spawning times do not overlap, as well as the availability of semen for selective breeding programs, especially during seasonal shortages of males and/or females. Furthermore, cryopreserved semen can be exchanged between countries located in both hemispheres, instead of exchanging fish, a particular advantage when importation of live animals is prohibited (McAndrew et al., 1993). The increasing development of the aquaculture industry in Chile indicates a need for developing methods for the long-term storage of semen of salmonid species. For these reasons, spermatozoa preservation of trout (rainbow) and salmon (coho and sakura) is gaining importance in Chile. Several methods have been described for the short-term storage and cryopreservation of rainbow trout semen @toss and Holtz, 1981; Wheeler and Thorgaard, 1991; Holtz, 1993; Piironen, 1993). Since different authors have noticed that these methods suffered from a lack of consistency, it is important to develop alternative procedures for the establishment of a reliable method for cryopreservation of rainbow trout spermatozoa. The complexity of cryopreservation of biological material (Friedler et al., 1988) requires well-controlled freezing procedures; thus the use of programmable freezing equipment with adjustable rates of cooling and warming (Gorin, 1992) offers several advantages over manual freezing procedures. The studies reported here were done in an effort to establish, in Chile, a cryopreservation protocol that ensures the preservation of salmonid spermatozoa with high reproductive potential. In this report we have examined various factors that affected the motility, viability and the fertilization potential of rainbow trout spermatozoa, before, during, and after cryopreservation.
2. Methods 2.1. Semen collection Rainbow trout (Oncorhynchus carefully dried before extruding the separately into petri-dishes in a 2-4 stored on ice (1 to 3 h). Ejaculates were discarded. 2.2. Estimation of spermatozoa
mykiss) males, 2-3 years old, were rinsed and semen. Ejaculates of individual males were placed mm thick layer to allow proper gas exchange, and contaminated with fecal material, urine, or blood
density
Spermatozoa concentration was estimated by a spectrophotometric method (Ciereszko and Darbrowski, 1993). Briefly, semen was diluted l/600 and l/1200 with 0.7% NaCl and the optical density of each dilution was measured at a wavelength of 505 nm. The average value was used to calculate spermatozoa density by interpolating into a standard
P. Conget et aL/Aquaculture
321
143 (1996) 319-329
curve which correlates spermatozoa concentration (determined using a Neubauer counting chamber) with optical density. 2.3. Cryoprotective
agents
Fresh semen was mixed at a ratio 1:4 with the following cryoprotectants: 1.5 M propylene glycol, 1.5 M glycerol, 1.5 M dimethyl sulfoxide (DMSO), 1.5 M DMSO + 0.6 M sucrose or 1.5 M sucrose. Cells were exposed to each agent for different periods of time and then spermatozoa motility, viability and fertilization potential was evaluated. 2.4. Cryopreservation
procedures
To study the effect of cryoprotective agents on spermatozoa motility before cryopreservation, fresh semen was directly mixed with the cryoprotectant extenders (see above) and kept on ice for time periods up to 30 min, and then spermatozoa motility was evaluated. To study the effect of cryoprotectants on spermatozoa motility during and after freezing, fresh semen mixed with the different cryoprotectants, was drawn without delay into flat clear plastic straws (2.5 ml total volume) and placed in the freezing chamber (2”C), of a liquid-nitrogen programmable freezing system (CryoMed 1010, Forma Scientific, Inc.). The freezing programs used were empirically determined to give inn-a-straw freezing rates of 3°C min-’ to - 5°C and then l”C, lO”C, or 30°C min- ’ to - 80°C. During ( - 60°C) or after freezing ( - 80°C followed by a short-term storage of 15 to 30 days in liquid nitrogen), samples were thawed at 37°C and immediately used to evaluate spermatozoa motility, viability or fertilization potential. 2.5. Estimation of spermatozoa
motility
Motility was evaluated as the percentage of spermatozoa actively swimming with progressive movement (forward movement). For this, at least two aliquots of semen samples from each male in the respective cryoprotective agent were diluted (l/320) with medium M-532 (0.55% NaCl, 0.38% Glycine, 0.24% Tris-HCl, pH 8.8) on a cold glass slide placed onto an optic microscope (Billard, 1992). For each sample, at least five microscopic fields (magnification X 400) were observed and the average of these counts was used to calculate the percentage of motile spermatozoa. 2.6. Estimation of spermatozoa
viability
Viability (VB) was estimated by the propidium iodide (PI) exclusion test (Ormerod et al., 1993). Semen samples were diluted l/1200 with 0.7% NaCl, stained with PI (1 p,g ml- ’ 1, mounted on slides and observed by epifluorescence microscopy ( X 400). While dead spermatozoa were brightly labelled with PI, viable spermatozoa were visible only by light phase. At least 200 spermatozoa were scored each time. VB was calculated as: Total number spermatozoa - (PI - stained spermatozoa) Total number spermatozoa
x 100
322
2.7. Estimation
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fertilization
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potential
Fresh (control, diluted in the cryoprotectant mixture) or cryopreserved semen was poured over freshly collected eggs (400 per assay) pooled from four females and suspended in medium M-532 at a ratio of 3 X lo6 spermatozoa per egg. After gently stirring, spermatozoa and eggs were co-incubated for 3 min at room temperature, followed by the addition of dechlorinated tap water to water-harden the eggs. Five min later, the eggs were rinsed and finally incubated in a vertical incubator with oxygenated water at 11°C (Billard, 1992). Dead eggs were removed at regular intervals. For both fresh and cryopreserved spermatozoa, the fertilization rate (FR) was evaluated 21 days after insemination by counting the number of eyed eggs by naked eye examination, and expressed as the percentage of eyed eggs over the total number of inseminated eggs. 2.8. Electron
microscopy
studies
Samples of fresh or cryopreserved semen were fixed in 3% pamformaldehyde, 0.5% glutaraldehyde, 0.1 M cacodylate buffer and 0.3 M sucrose for 1 h, post-fixed in 1% osmium tetroxide and embedded in Epon 812 resin (Gwo and Arnold, 1992). Sections were obtained in a Sorval MT2-B microtome, stained with uranyl acetate and lead citrate and examined under a Phillips EM-300 transmission electron microscope. At least 50 spermatozoa were examined, with particular attention given to the structure of the plasma membrane, nuclear material and flagella.
3. Results 3.1. Effect of cryoprotective
agents on spermatozoa
motility before freezing
To evaluate the effect that cryoprotective agents (CA) produce in motility of rainbow trout spermatozoa, fresh semen samples were exposed to differents agents commonly employed in cryopreservation procedures (Friedler et al., 1988). As seen in Fig. 1A, when semen samples were exposed to the various CA for time periods of 10 min, all CA used except sucrose, preserved spermatozoa motility. However, when the exposure time was extended to 30 min, spermatozoa motility was extensively decreased in all cases, except with propyleneglycol (Fig. 1B). For exposure periods between 10 and 30 min, motility decreased in a time-dependent fashion (data not shown). Control semen samples in PBS exhibited a high motility (close to 100%) during the assay periods. These results indicated that to preserve motility better before freezing, spermatozoa should be exposed to CA (with the exception of the non-permeating agent, sucrose) for time periods not longer than 10 min. The above inference was supported by the observation that after exposure to DMSO-sucrose (15 min), spermatozoa motility which was low (close to 30%) fully recovered after transferring cells to PBS or seminal plasma. However, if cells were exposed to the same solution for 30 min and then transferred to PBS or seminal plasma, motility was not recovered (data not shown). The final concentration of CA used in the above experiments was 1.1 M (with the
P. Conget et al./Aquaculture
PBS
loo -
Propylene Gtycerot
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wso
DM.so+suc sucros8
DMBO
wso+auc
-
T 60-
g E = 0 I
60-
40-
20 -
01
PBS
I
sucrose
Fig. 1. Effect of cryoprotective agents on spermatozoa motility before freezing. Fresh semen was mixed (1:4) with the following extenders: phosphate buffered saline (PBS, control); 1.5 M propylene glycol (propylene); 1.5 M glycerol; 1.5 M dimethyl sulfoxide (DMSO); 1.5 M DMSO+0.6 M sucrose (DMSO+ SUC) or 1.5 M sucrose. Cells were exposed to the cryoprotective extenders for 10 min (A) or 30 mm (B) and then activated to evaluate spermatozoa motility, as indicated under Methods. Each bar represents the mean+ SD of the percentage of motile spermatozoa in semen samples obtained from two males, with each sample tested twice.
exception of DMSO-sucrose), however the contribution of each chemical to the osmolarity of the medium was different. Therefore, the dramatic decrease in spermatozoa motility observed after the 30 min exposure-period (Fig. 1B) could be the consequence of the inability of spermatozoa to adapt to the hyperosmotic medium created by the addition of the cryoprotectants. This possibility was explored by measuring motility of fresh semen exposed to solutions of cryoprotective agents with increasing osmolarities. The results in Fig. 2 show that exposure to PBS or seminal plasma (300 mOsm) resulted in no changes in spermatozoa motility, compared with fresh semen. However, as the
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Ftash Seminal plasma
PBS Gly03rOl Glycarol+sucroaa suuoae DMsG+suuoaa Glycerol+sucroaa DMSO+auaoae DMso+aucroaa 0
Fig. 2. Effect of osmolarity of cryoprotectant medium on spermatozoa motility. Fresh semen (in seminal plasma) was exposed to media prepared to the following fmal osmolarities (shown on the right of each bar): 300 mOsm (seminal plasma or PBS); 800 mOsm (1.1 M glycerol); 1000 mOsm (0.55 M glycerol + 0.1 M sucrose); 1200 mOsm ( 1.1 M sucrose or 0.55 M DMSO + 0.1 M sucrose); 1800mOsm (1.1 M glycerol + 0.1 M sucrose or 1.1 M DMSO+ 0.1 M sucrose); 2300 mOsm (1.1 M DMSO + 0.45 M sucrose). After 20 min exposure at 4°C. spermatozoa motility was evaluated. Each bar represents the mean f SD of the percentage of motile spermatozoa in semen samples obtained from two males, with each sample tested twice.
osmolarity of the medium was increased, spermatozoa motility was concomitantly decreased. Thus, at osmolarities of 800 to 1000 mOsm, motility ranged between 80% and 50%, respectively. Exposure to solutions of higher osmolarity, resulted in semen with low spermatozoa motility (less than 30%). Thus, the exposure of semen to non-isosmotic conditions for time periods longer than 10 min, proved to be critical for the preservation of spermatozoa motility. The exposure of fresh semen to solutions with the same osmolarity, but containing different CA, did not affect motility (see mixtures at 1200 or 1800 mOsm in Fig. 2), thus excluding a cytotoxic effect, per se of cryoprotectants on spermatozoa motility. 3.2. EfSect of cryoprotective
agents on spermatozoa
motility during and after freezing
Programmable freezing equipment was used to establish proper experimental conditions for rainbow trout spermatozoa cryopreservation. The use of such methodology allows the evaluation of spermatozoa motility at different rates of cooling during freezing. As seen in Table 1, at freezing rates of 1°C or 10°C min- ’ (slow freezing) there was a complete loss of motility, which was independent of the CA used. When cells were processed at 30°C min-’ (rapid freezing), semen diluted with DMSO-sucrose demonstrated a 63% motility, both during freezing (- 60°C) and after completion of freezing (- 80°C) followed by storage under liquid nitrogen. Spermatozoa motility for samples
P. Conget et aI./Aquaculture Table 1 Effect of cooling rates on spermatozoa
A.F.
325
motility during and after freezing Motility (o/o)
Cooling rate
D.F.
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l°C min-’ 10°C mm’ 30°C min30°C mm-’
’
Glycerol
DMSO
0 15* 12 15*10 lo*11
0 0
DMSO + sucrose n.d. n.d 63kl9 63fl2
5flO IX*11
Fresh semen was mixed ( 1:4) with 1.5 M glycerol, 1.5 M DMSO or 1.5 M DMSO + 0.6 M sucrose. Samples in the freezing chamber of a programmable freezing equipment were subjected to cooling rates of l”C, 10°C or 30°C min-‘. Spermatozoa motility was evaluated during the freezing procedure (DF, at -60°C) and after completion of the freezing program (AF, at -80°C followed by storage under liquid nitrogen).Data correspond to the mean motility (o/o) f SD determined in semen samples obtained from five males, being each sample tested twice.
cryopreserved in glycerol or DMSO alone was negligible. Propyleneglycol, which proved to be an adequate agent to preserve spermatozoa motility under pre-freezing conditions (Fig. l), failed to preserve motility during freezing, both at slow and rapid freezing conditions (not shown). 3.3. Fertilization potential of cryopreserved
spermatozoa
To evaluate the fertilization capacity of spermatozoa cryopreserved under the conditions described above, aliquots of cryopreserved semen were used to inseminate rainbow trout eggs. Fertilization potential was evaluated 21 days after insemination, by counting eyed eggs. As seen in Fig. 3, cryopreserved semen samples (average motility of 64 f 15% after thawing) exhibited a high fertilization potential, which ranged from 47% to 85% (average fertilization rate 58 4 15%). On the other hand, semen samples cryopreserved under the same conditions, except that glycerol or DMSO was used instead of DMSO-sucrose, exhibited spermatozoa with low motility and a negligible fertilization potential (data not shown). 3.4. Ultrastructural
changes produced
in spermatozoa
by cryopreservation
The damage produced on rainbow trout spermatozoa by cryopreservation was observed at the structural level by TEM analysis. For these studies, samples of fresh and cryopreserved spermatozoa were used. Fresh spermatozoa exhibited a well defined and continuous cell membrane, in close contact with the cell nucleus. Chromatin was homogenous without evidence of high electron density zones (Fig. 4a). Non-damaged cryopreserved spermatozoa presented the same characteristics of fresh spermatozoa, except for chromatin which was extensively clumped (Fig. 4b). Spermatozoa that were damaged by cryopreservation exhibited a swelled and non-continuous membrane with a clumpered chromatin, showing high electron density zones (Fig. 4c). The flagella of fresh spermatozoa show a continuous and well organized membrane which surrounds the typical 9 + 2 structure of the axonema (Fig. 4a and d). While the same flagella
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80
-
';; b 9) 60
5 .z
-
-
40
g .r I!! 20
-/
I Il-i -
H
i
-
0
1
2
3
4
5
6
7
a
i 10
Male number
Fig. 3. Fertilization potential of cryopreserved spermatozoa. Semen samples from ten fish were rapidly (10 min or less) diluted (1:4) in a mixture of DMSO (1.5 M) and sucrose (0.6 M) and cryopreserved at a cooling rate of 30°C mm-‘. After storage in liquid nitrogen, semen from each male was thawed and used for motility and fertilization assays. The day of the fertilization assay, cryopreserved spermatozoa as well as a selected sample (motility and viability = 100%) of freshly collected semen were co-incubated with eggs (3X lo6 sperm per egg) and fertilization evaluated. as indicated under Methods. For the studied population, the average post-freezing motility was 64 f 15% and the average post-freezing fertilization rate was 58 f 15%. The results are expressed as percentages of the control (90 f 10% eyed). Note that cryopteserved samples from each male were tested once.
structure was observed in non-damaged cryopreserved spermatozoa (not shown), in spermatozoa damaged by cryopreservation, a break and a displacement of the axonema structure, along with changes in the flagella membrane were observed (Fig. 4e). Note that the shape of mitochondria was not altered by cryopreservation. The notion that the damage caused by cryopreservation modifies motility and viability (Moccia and Munkittrick, 1987), prompted us to analyze the relationship between motility and viability in samples subjected to conditions that will not affect (control, fresh samples at 4°C) or will affect (exposure to cryoprotective agents, followed or not by cryopreservation) spermatozoa structure, as suggested by the results in Fig. 4. As seen in Fig. 5, the linear relationship ( rz = 0.86) obtained for both spermatozoa survival parameters strengthens the concept that as a consequence of the disruption of the structure of the flagella and/or cell membrane, spermatozoa motility and viability are affected.
4. Discussion Effective storage of gametes is essential whenever selective breeding is carried out and is also a prerequisite for the establishment of gene banks for endangered wild stocks. Salmonids, like the rainbow trout and the coho salmon are commercially
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Fig. 4. Transmission electron microscopy of cross-sections of fresh and cryopreserved rainbow trout spermatozoa. Heads from fresh spermatozoa (a) and from non-damaged (b) and damaged (c) cryopresetved spermatozoa; flagella from fresh spermatozoa (d) and from damaged cryopreserved spermatozoa (e). Final magnification for heads is X 2OooO and for flagella X 42000.
important species in Chile. Since at present no routine long-term storage techniques for fish semen are available in Chile, we attempted to develop a protocol for the cryopreservation of rainbow trout spermatozoa by using programmable instead of manual freezing. Programmable freezing allows the pre-setting of different freezing programs, the monitoring of precise temperature during the cooling sections, and the continuous biological examination of cells, during the freezing stages (Gorin, 1992). By using programmable freezing equipment and after evaluating spermatozoa motil-
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60
0
20
40
60
80
100
Viability (%) Fig. 5. Relationship between motility and viability for rainbow trout spermatozoa. Linear regression analysis of percentage of motility and viability of fresh (Cl), fresh exposed to cryoprotective agents (0) and cryopreserved(+)spennatozoa(n=84). y=-6.76+1.15x; R*=0.86; P
ity. viability and fertilization potential, we have been able to define the following conditions for the successful cryopreservation of rainbow trout spermatozoa: 1. The use of a mixture of permeating (DMSO) and non-permeating (sucrose) cryoprotectant agents; 2. A short-term exposure of fresh semen (less than 10 min at 4°C) to the cryoprotective mixture, and finally; 3. Freezing should be performed at a cooling rate of 30°C min-‘, once the phase change has been attained. In this respect, it is important to emphasize that the most critical point for rainbow trout spermatozoa freezing is the rate selected for solid phase cooling (from - 5°C to - 80°C) and not the rate at which the liquid sample is cooled (from 4°C to - 5°C). This practical approach is supported by the theoretical concept that ice nucleation within cells occurs at - 40°C (Friedler et al., 1988). As deduced from our TEM studies, as well as those performed in the Atlantic croaker spermatozoa (Gwo and Arnold, 1992), the major damage produced by cryopreservation occurred in the spermatozoa cell membrane and flagellum. These observations, together with the correlation between spermatozoa viability (as indicated by the PI staining method) and motility, and between motility and fertilization potential (Moccia and Mtmkittrick, 1987) suggest that spermatozoa motility might be used as an initial predictive index to evaluate the efficiency of a cryopreservation protocol. A full
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correlation between the viability assessed by the PI exclusion method and fertilization success is required in future work. The freezing conditions described here, offer consistency, precision, and accuracy. Furthermore, the whole procedure is rapid, since after obtaining the ejaculates, cells are processed, frozen and ready to be stored under liquid nitrogen in less than 15 min. Although too sophisticated to be employed at the hatchery site, this method of cryopreservation should be also useful for the establishment of spermatozoa banks to handle material that has been obtained after selective breeding programs or genetic manipulation. Acknowledgements
This work was supported by Grant FONDEF (PI-lo), Chile. PC is the recipient of an A. Stekel Scholarship, INTA, University of Chile. References Billard, R., 1992. Reproduction in rainbow trout: sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture, 100: 263-298. Ciercszko, A. and Darbrowski, K., 1993. Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using a spectrophotometric technique. Aquaculture, 109: 367-373. Cognie, F., Billard, R. and Chao, N.H., 1989. La Cryoconservation de la laitance de la carpe Cyprinus curpio . J. Appl. Ichthyol., 5: 165-176. Friedler, S., Giudice, L.C. and Lamb, E.J., 1988. Cryoprcscrvation of embryos and ova. Fertil. Steril., 49: 743-764. Gorin, N.C., 1992. Cryopreservation and storage of stem cells. In: E.M. Amman, H.J. Deeg and R.A. Sacher (Editors), Bone Marrow and Stem Cell Processing: A Manual of Current Techniques. F.A. Davis, Philadelphia, PA, pp. 293-362. Gwo, J.C. and Arnold, C.R., 1992. Cryopreservation of Atlantic croaker spermatozoa: evaluation of morphological changes. J. Exp. Zool., 264: 444-453. Holtz, W., 1993. Cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: practical mcommendations. Aquaculture, 110: 97-100. McAndrew, B.J., Rana, KJ. and Penman, T.J., 1993. Conservation and preservation of genetic variation in aquatic organisms. In: J.F. Muir and R.J. Roberts (Editors), Recent Advances in Aquaculture, Vol 4. Blackwell Science, Oxford, pp. 295-336. Moccia, R.D. and Munkittrick, K.R., 1987. Relationship between the fertilization of rainbow trout (Sulmo gairdneri) eggs and the motility of spermatozoa. Theriogenology, 27: 678-688. Ormerod, M.G., Sun, X.M., Snowden, RT, Davies, R., Fearnhead, H., and Cohen, G.M., 1993. Increased membrane permeability of apoptotic thymocytes: A flow cytometric study. Cytometry, 14: 595-602. Piironen, J., 1993. Cryopresevation of sperm from brown trout (Salmo rrurra m. lacusrris L.) and Attic chart (Salvelinus alpinus 15.1. Aquaculture, 116: 275-285. Scott, A.P. and Baynes, S.M., 1980. A review of the biology, handling and storage of salmonid spermatozoa, J. Fish Biol., 17: 707-739. Stoss, J. and Holtz, W., 1981. Cryopreservation of rainbow trout (Salmo gairdneri) sperm. I. Effect of thawing solution, sperm density and interval between thawing and insemination. Aquaculture, 22: 97-104. Stoss, J., 1983. Fish gamete preservation and spermatozoan physiology. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IX, Reproduction. Part B, Behaviour and Fertility Control. Academic Press, London, pp. 305-330. Wheeler, P.A. and Thorgaard, G.H., 1991. Cryopteservation of rainbow trout semen in large straws. Aquaculture, 93: 95- 100.