Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus )

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CRYOPRESERVATION OF SPERM FROM THE ENDANGERED FORMOSAN LANDLOCKED SALMON (Oncorhynchus masou formosanus)

J.-C. Gwo, l H. Ohta, 2 K. Okuzawa 2 and H.-C. Wu, 3 'Department of Aquaculture, Taiwan National Ocean University, Keelung, Taiwan 2National Research Institute of Aquaculture, Nansei,/Vile, Japan 3Shei-Pa National Park, Taichung, Taiwan Received for publication: 20 April 1998 Accepted: 20 August 1998 ABSTRACT The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyi sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucoseDMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value &using sperm bank to safeguard this endangered species is discussed. © 1999 by ElsevierScienceInc.

Key words: salmon, semen, androgenesis, DMA, methanol, hybrid INTRODUCTION The Oncorhynchus masou species is comprised of 4 subspecies: Masu salmon ~ masou masou), Amago salmon (_Q,masou ishikawae), Biwa salmon ~ masou rhodurus), and Formosan landlocked salmon (~. masou formosanus; 31). The Formosan landlocked salmon are the southernmost distribution of the Pacific salmon and are found only in the upper streams of the Tachia River in Taiwan. The population is at a high risk &extinction due to a combination of low effective population size, overfishing and altered native habitats (26). The existing population is Acknowledgments We thank Mrs. G.-Y. Lin (EM center, Taiwan National University) for her technical advice; and the staff members &the Wu-Lin Station, Shei-Pa National Park, for providing assistance. This work was funded by the National Science Council, Taiwan (Project number NSC 810409-B-019-17). Theriogenology 51:569-582, 1999 © 1999 by Elsevier Science Inc.

0093-691X/99/$-see front matter PII S0093-691X(99)00011-4

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estimated at less than 1,000 fish. Sustained maintenance of this population in the near future may depend entirely on stocked fish originating from aquaculture systems. The use of frozen-thawed spermatozoa provides a practical means of increasing the genetically effective population size, especially when new broodstock is established for cultivation. This method can also help maintain their genetic diversity and integrity. Androgenesis (where the embryonic chromosomes come only from the father) involves inactivating the maternal genetic material by using ionizing radiation, fertilizing the treated eggs with spermatozoa, and restoring diploidy by suppressing the first cleavage (35,44). The combination of sperm cryopreservation with androgenesis offers a way of regenerating a stock using only spermatozoa as a source of nuclear genomic material. Sperm cryopreservation in salmonid fish has been intensively investigated. Empirically derived techniques have been successfully used for semen cryopreservation of many salmonid fishes (1,4,15,17,18,27,39,40,42). Various extenders ranging from mineral concentrations that mimic seminal plasma to a very unconventional extender developed by Mounlb (29) to distilled water (16,43) have been used; however, simple glucose-based extenders have also been shown to be effective (43). An extender consisting of 300 mM glucose with 20% glycerol has been successfully used for cryopreserving milt from whitefish (Coregonus muksun) and Arctic chart (Salvelinus alpinus)(36,37,38). Ohta et a1.(32,33) recently summarized their work on cryopreservation of semen from Masu and Amago salmon and recommended a simple extender consisting of 300 mM glucose supplemented with 10% dimethyl sulfoxide (DMSO). This glucoseDMSO extender also gave successful results in rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), sea trout (Salmo trutta), and landlocked salmon (Salmo salar m. sebago Girard;38). At present, dimethyl sulfoxide (DMSO) is the most widely used cryoprotectant to protect salmonid spermatozoa during the freeze-thaw process (42). With some species of salmonids, glycerol is the preferred cryoprotectant (38). Dimethyl-acetamide (DMA) has been used successfully to cryopreserve rainbow trout (28), grayling (Th~nnallus th~pnallus) and Danube salmon (I-Iucho hucho) spermatozoa (22). Methanol was the most effective cryoprotectant for grayling and Danube salmon spermatozoa (22). A DMSO/glycerol mixture was found to be an effective cryoprotectant for rainbow trout sspermatozoa (23). Minimal sperm-to-egg ratios for successful fertilization are about 3 to 6 x 10:1 for fresh salmonid semen (6,21,42). However, increased sperm-to-egg ratios (usually about 5- to 15-fold) are required for cryopreserved spermatozoa to achieve a given fertilization rate (6,42). The objectives of this research were to examine the effect of DMSO, DMA and methanol as cryoprotectants in combination with a simple extender (300 mM glucose) on the cryopreservation of Formosan landlocked salmon spermatozoa and to test if intersubspecies hybridization between Amago and Formosan landlocked salmon would be possible. Morphological changes of the spermatozoa of the Formosan landlocked salmon immediately after dilution in the 300 mM glucose-DMSO extender and after freeze-thawing were also evaluated for future development of a more optimal cryopreservation protocol for Formosan landlocked salmon spermatozoa. MATERIALS AND METHODS Mature wild populations of Formosan landlocked salmon (_Q. masou formosanus) and cultivated stocks of Amago salmon ~ masou ishikawae) were obtained from the Chichawan

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Stream in the Shei-Pa National Park, Taiwan, and from captive brood fish at the Inland Station of the National Research Institute of Aquaculture in Japan. The semen of both subspecies was stripped from 8 anesthetized (MS-222) males and pooled in equal volume during the breeding season (late October). Special care was taken to avoid contamination of semen with urea, feces, blood or water. The semen was stored on crushed ice, and sperm motility and concentration were determined within 3 to 4 h of sampling. After 100-fold dilution (v/v) with 0.7% NaCI, sperm concentration was determined under a light microscope using a Thoma hemocytometer. Cryopreservation of Sperm Fresh semen showing more than 90% progressively motile spermatozoa and vigorous forward motility when diluted with ISER (Isotonic Solution for Egg Rinsing; NaCI 154.7 raM, KCI 3.2 raM, CaCI2 2.3 raM;34) was used in this study. Three cryoprotectants at 10% concentration in 300 mM glucose solution were compared: DMSO (dimethyl sulfoxide), DMA (dimethyl-acetamide), and methanol. All chemicals were reagent grade (Sigma, St. Louis, MO, USA), and water was deionized and then glass-distilled. Semen dilution ratio was 5"1 for all cryoprotectants. All semen samples were processed within 10 rain of sampling. The general freezing protocol followed the methods ofOhta et a1.(32). Briefly, 1 part semen was diluted with 5 parts extender. Extended semen was immediately pelleted (0.1 mL) on dry ice. The elapsed time from dilution of milt to dropping on dry ice was around 30 sec. After being frozen for about 5 rain the samples were immersed in liquid nitrogen. Evaluation of Sperm Motility One pellet (0.1 mL) was rapidly thawed with 5 mL of thawing solution (120 ram NaHCO3) at room temperature (25°C). Thawing of the pellet was accelerated by mixing the thawing pellets with a vortex mixer. A drop of sperm suspension was put on a glass slide placed on the stage of a microscope at a magnification of x 200. The movement of spermatozoa in the thawing solution was recorded by videomicroscopy for 5 to 10 sec immediately after thawing the pellet, and the percentage of motility was measured by tracing the location of the heads of over 50 randomly chosen sperm cells on a TV monitor (33). Other aliquots of frozen Formosan landlocked salmon semen pellets were saved for 4 d in liquid nitrogen and shipped to Japan where only fresh Amago salmon ova are available. Using eggs from a different subspecies (Amago salmon) of the same species (~. masou), androgenesis may recover rare genotypes from cryopreserved sperm cells (Formosan landlocked salmon). Evaluation of Sperm Fertility Formosan landlocked salmon. Since the most effective cryoprotectant DMSO at 10% concentration in 300 mM glucose was determined in our preliminary experiments, this 300 mM glucose-10%DMSO extender was chosen to be used as the sole extender in sperm fertility trials with Formosan landlocked salmon eggs. The eggs stripped from 4 mature Formosan landlocked salmon females were pooled and rinsed with ISER. Two pellets of frozen Formosan landlocked salmon semen were thawed and immediately mixed with 5 g of eggs (about 50 eggs) by gentle stirring. The fertilization capacities of the fresh and cryopreserved semen were determined by

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inseminating eggs at a sperm-to-egg ratio of 4.2 x 10~:1 (calculated from the average sperm concentration of 6.4 x 109/mL for studied population of Formosan landlocked salmon). Amago salmon eggs was evaluated for the 3 cryoprotectants (DMSO, DMA, and methanol) at 10% concentration in 300 mM glucose. Amago salmon. Amago salmon semen was also frozen by the same process as described above. Twenty captive 2-yr-old Amago salmon brood fish at the Japan Inland Station of National Research Institute of Aquaculture were used as egg donors. Ovulated Amago salmon eggs were obtained from 10 mature females; they were then pooled and rinsed with ISER. The following fertilization tests were performed. Amago salmon egg x Amago salmon semen; Amago salmon egg x cryopreserved Amago salmon semen; and Amago salmon egg x cryopreserved Formosan landlocked salmon semen. Amago semen was also thawed with the same process as described above. For each extender, 2 pellets were thawed and immediately mixed with 5 g of eggs (about 50 eggs) by gentle stirring. The same sperm-to-egg ratio for both Formosan landlocked and Amago salmon was used for the fertilization trials. The fertilized eggs were rinsed in hatchery water and incubated in flow trays at 8 to 10°C. The fertilization rate was determined by the percentage of eyed embryos. Some juveniles obtained by intersubspecies hybridization (Amago salmon eggs x cryopreserved Formosan landlocked salmon semen) were reared for 4 too. Statistical Analysis Three replicates were used for each treatment in motility evaluations and fertility trials, and experiments were performed 4 times for each treatments. Each fertilization trial was done with the same batch of eggs. Percentages of fertilization rates were subjected to arcsine transformation before statistical evaluation. One-way analysis of variance was used to compare treatments, with significance at P = 0.05. Electron Microscopy Study Studies were conducted on Formosan landlocked salmon spermatozoa: when freshly collected, at 30 sec after being diluted in the 300 mM glucose-10% DMSO extender, and after freeze-thawing at the room temperature. Semen samples were fixed for 1 h in 2% glutaldehyde in phosphate buffer (0.12 M), and postfixed in 1% OsO4 in the same buffer for 1 h. The samples were dehydrated in a graded alcohol series. The dehydrated samples were either embedded in Spurr resin for transmission electron microscopy or prepared for scanning electron microscopy (11,14). About 50 spermatozoa from each sample were examined utilizing photoprints obtained with electron microscopy. Particular attention was given to damage of the plasma membrane, mitochondria, axoneme and nuclear material. RESULTS The percentage of motility of the cryopreserved spermatozoa were significantly lower than those of fresh spermatozoa. The percentages of motility of Formosan landlocked salmon were 14.5, 9.9, and 4.6% for semen frozen in cryoprotectants DMSO, DMA and methanol, respectively (Table 1). The lowest post-thaw motility rate for semen of Formosan landlocked salmon was observed with 10% methanol as the cryoprotectant (Table 1). However, semen frozen using

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methanol had a significantly higher post-thaw motility rate for Amago salmon than for Formosan landlocked salmon (Tables 1 and 2). Dimethyl-acetamide (DMA) provided better protection for Amago salmon semen than for the Formosan landlocked salmon. High percentages of fertility were obtained with fresh semen of both species. Formosan landlocked salmon semen cryopreserved in DMSO, DMA and methanol resulted in 84.2, 72.1, and 71.0% fertilities, respectively, with Amago salmon eggs (Table 1). The highest post-thaw fertilization rate for semen of Formosan landlocked salmon was obtained with 10% DMSO as the cryoprotectant; methanol and DMA were less effective cryoprotectants. The fertilization capacity of frozen-thawed Formosan landlocked salmon with Formosan landlocked salmon eggs was comparable to that of fresh Formosan landlocked salmon semen (94.5 to 96.2%). As for Amago salmon semen, all 3 cryoprotectants showed no significant differences in post-thaw fertilization rates (Table 2). The intersubspecies fertilities of the cryopreserved spermatozoa were 71 to 84% (Amago salmon eggs x cryopreserved Formosan landlocked salmon semen), which were comparable to those obtained with Amago salmon egg x cryopreserved Amago salmon semen (83 to 88%). Morphologically, Amago salmon have red spots on their body sides, whereas Formosan landlocked salmon do not. The intersubspecies hybrid, produced with frozen spermatozoa, appeared to be normal and looked superficially like the paternal Formosan landlocked salmon ( i.e, no red spots were observed on their bodies; data not shown). The sperm morphologies of Formosan landlocked salmon in the control (Figures 1A, 2AC) observed in this experiment were similar to the structures described in an earlier study (14). The morphology of spermatozoa was about the same in diluted and undiluted semen before freezing; only a slight shrinkage of the midpiece was observed when the spermatozoa were suspended in the 300 mM glucose-10% DMSO extender. Drastic morphological changes were found only in frozen-thawed spermatozoa (Figures 1B-F, 2D-K). Frozen-thawed spermatozoa dispersed evenly without the formation of clumped aggregateds. Freezing and thawing caused structural damage to all types of spermatozoal organelles examined. The damage appeared greatest to the plasma membrane and mitochondria. The plasma membrane became loose, swollen, ruptured, or vesiculated, and blisters formed around the spermatozoa, especially in the nucleus, midpiece and flagella (Figures 1B-F, 2D-K). Mitochondria were occasionally missing (Figures 1D-F, 2H) and were swollen (Figures 1C, 2J), with a corresponding decrease in density of the matrix (Figures 2J, 2K). Cristae were disrupte d, and normal profiles were only occasionally seen. Flagella were broken or become detached in some specimens (Figures 1D-F, 21). The 9+2 pattern of the axoneme was generally unchanged. Clumping of chromatin was seldom observed (Figure IE).

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Table 1. Effects of the type of cryoprotectant on post-thaw Formosan landlocked salmon sperm motility and fertility rates Cryoprotectant Motility (%) DMSO 10% 14.5 ± 0.6 b DMAE 10% 9.9±1.0 ° Methanol 10% 4.6 ± 1.0 d Control (Fresh semen) 95.1 ± 4.6" Control (Frozen semen'S) 18.2 ± 3.7 b

Fertilization rate (%) 84.2 ± 3.4 b I, 72.1±5.7 "1" 71.1 ± 2.2 ~ I, 96.2 ± 1.4" * 94.5 ± 2.4" **

Values are means ± standard deviations, n = 4 in all experiments. Values in the same column with different superscripts differ significantly (P < 0.05). The diluent was 300 mM glucose. Weight of eggs per experiment was 5 g (about 50 eggs); sperm to egg ratio was 4.2 x 10 cells per egg. •

•

6

•~ 300 mM glucose-10% DMSO extender Amago salmon egg x cryopreserved Formosan landlocked salmon semen. • Formosan landlocked salmon egg x Formosan landlocked salmon semen. • * Formosan landlocked salmon egg x cryopreserved Formosan landlocked salmon semen.

Table 2. Effects of the type of cryoprotectant on post-thaw Amago salmon sperm motility and fertility rates Cryoprotectant Motility (%) Fertilization rate (%) DMSO 10% 17.1 ±0.7 b 88.0 ± 2.2 b+ DMAE 10% 15.4 ± 1.2 b 83.4 ± 3.8 b+ Methanol 10% 12.4 + 1.6b 88.0±5.5 ~÷ Control (Fresh semen) 95.1 + 1.1 • 97.9 ± 1.6 ' + Values are means + standard deviations, n = 4 in all experiments. Values in the same column with different superscripts differ significantly (P < 0.05). The diluent was 300 mM glucose. Weight of eggs per experiment was 5 g (about 50 eggs); sperm to egg ratio was 4.2 x 106 cells per egg. + Amago salmon egg x Amago salmon semen. + Amago salmon egg x cryopreserved Amago salmon semen.

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Figure I(A). Fresh, untreated, nondiluted, unfrozen spermatozoa of Formosan landlocked salmon (Oncorhynchus masou formosanus). Sagittal longitudinal section of a spermatozoon showing the nucleus (n) with the heterogeneously granular chromatin. The centriolar complex, has proximal (pc) and distal centrioles (de) located at a right angle to each other. A fibrous body (arrow) adheres to the 2 eentrioles laterally. The cytoplasmic canal (cc) separates the midpiece from the flagellum (f) and 1 mitochondrion (m) surrounds the cytoplasmic canal. The plasma membrane (pm) is applied tightly to the spermatozoon x 77,000. (B to F) Changes in the ultrastructure of spermatozoa diluted in 300 mM glucose-10% DMSO extender and freeze-thawed at room temperature. A considerable range in type and extent of morphological changes ranging from no apparent damage to virtual disintegration of the spermatozoa was found within the same post-thaw sample of spermatozoa. (B) Ruptured plasma membrane (pm) in midpiece region of frozen sperm, m: mitochondrion x 60,000. (C) Swelling of the mitoehondrion (m) in frozen sperm. Mitochondrion exhibits vacuolization (*) and cristae are disrupted x 37,000. (D to F) The plasma membrane (pm) appearing loose and ruptured in the midpiece region. No mitochondrion can be observed and flagella (f) are bent or broken. The centriole complex is generally unchanged x 49,000. (E) Clumping (arrow head) of chromatin was seldom observed in the nucleus (n) x 40,000. (F) The plasma membrane and nuclear envelope have become wrinkled (arrow head) in the head region x 35,000.

E

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Figure 2(A). Fresh, untreated, nondiluted, unfrozen spermatozoa of Formosan landlocked salmon (Oncorhynchus masou tbrmosanus). Scanning electron micrograph of a spermatozoon showing the ovoid-shaped head (h), midpiece (mp), and flagellum (f) x 35,000. (B) A ring-shaped mitochondrion (m) surrounding the cytoplasmic canal (co) in the midpiece region, t2 flagellum. Inset: enlarged portion from 2B showing midpiece (mp) region, cc: cytoplasmic canal x 33,000. (C) Transverse section of a spermatozoon showing the mitochondrion (m) bounded by an outer and inner membrane, exhibiting transverse cristae. The flagellum has an axoneme (a) with the classical 9+2 microtubular pattern. The plasma membrane (pm) is closely apposed to the axoneme, n: nucleus x 47,000. (D to K) Changes in the ultrastructure of spermatozoa diluted in 300 mM glucose-10% DMSO extender and freeze-thawed at room temperature. Morphological changes ranged from no apparent damage to disintegration. D x 35,000; E x 37,000; F x 32,000; G x 32,000; H x 48,000. (E to H) Blisters (*) formed in the head (h), midpiece (mp), and flagellum (t). (D to F) The midpiece (mp) is greaterly reduced or (G to I) totally missing. (I) Loss of the flagellum by detachment from the sperm head was seldom observed x 36,000. (J, K) Wrinkled and non-continuous plasma membrane (pro) are observed. The 9+2 pattern of the axoneme (a) was unchanged. Mitochondrion (m) exhibits severe vacuolization (*) and cristae are disrupted J x 70,000; K x 62,000.

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DISCUS SION Fertilization success for Formosan landlocked salmon semen cryopreserved in a glucoseDMSO extender was 84.2% with Amago salmon eggs. This result confirms that of the data of Ohta et al.(32,33), who reported a 90% fertilization rate for Masu salmon semen cryopreserved in the same extender. The freezing protocol developed for Masu salmon semen (32,45) appears to be applicable to Formosan landlocked salmon. In addition, DMA and methanol were found to be less effective cryoprotectants for Formosan landlocked salmon semen, as previously reported by Baulny et al.(3) in fleeting rainbow trout semen. The use of DMA and methanol as cryoprotectants with 300 mM glucose resulted in low post-thaw sperm motility rates, but in high fertilization rates. One possible reason for the high fertilization rate with the low percentage of motility of cryopreserved spermatozoa in our present study may be that few eggs were inseminated. Fertilization capacity of cryopreserved spermatozoa is clearly lower than that of flesh spermatozoa; however, increasing the number of flozen-thawed spermatozoa appears to compensate for their decrease in fertilizing ability (10,21,22,23,24,25). Lahnsteiner et a1.(21,22,23) recommended a 3 x 106:1 sperm-to-egg ratio for various salmonid species. In the present study, we used a 4.2 x 106:1 sperm-to-egg ratio. With Formosan landlocked salmon, the lowest percentage of post-thaw motility was obtained with 10% methanol, which also gave the best postthaw fertilization rates for grayling and Danube salmon (22). Dimethyy-acetamide (DMA) provided better protection for Amago salmon semen than for the Formosan landlocked salmon in our present study. Species-specific modifications of semen cryopreservation protocols are needed for different fish species (9,12,13,21,22,36,37,38). The pellet method is most widely used in salmon hatcheries because of its favorable postthaw fertility, convenience and simplicity. Pellet size and the freezing temperature (temperature reached before immersion in liquid nitrogen) influence the post-thaw fertility in the pellet method (16,32). Ohta et a1.(32) estimated the optimum fleeting rate to be between 29.9 and 92.6°C/rain, and that reaching a temperature of-70°C before plunging into liquid nitrogen is a prerequisite for both Masu and Amago salmon spermatozoa. In general, the freezing rate of 50 uL extended semen dropped on a dry ice block (-79°C) is about 30°C/rain (8,39), which may explain the successful results achieved by the pellet technique in most salmonids. Recently the straw method also has become popular for freezing salmonid spermatozoa (21,22,23,28). The extenders developed for the pellet method may, however, need to be adjusted for the straw method. Piironen (38) compared the fertilization rate of landlocked salmon using the same glucose- and saccharose-based 10% DMSO extender but different freezing techniques (straw and pellet) with similar freezing rates. He found the post-thaw fertility of the straw method to be significantly lower than that of the pellet method. In contrast, consistently higher fertility rates were obtained using the straw method rather than the pellet (8). Conversely, Steinberg et al.(41) observed similar results with both methods. The possible cause &the discrepancies between the 2 freezing techniques (pellet and straw) requires further study. Even with the best cryopreservation techniques developed by empirical methods to date, post-thaw morphological intact spermatozoa are restricted to less than 20% (3,21,24,25). Moreover, most surviving spermatozoa have characteristics which distinguish them from spermatozoa before cryopreservation such as the mitochondrial architecture being severely altered by cryopreservation with their substance being less electron dense as in the present study and

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many previous reports (5,7,9,20,24). Since oxidative phosphorylation and proton transport are membrane-bound in mitochondria, frozen-thawed spermatozoa usually markedly reduced motility characteristics. Motility is a simple, convenient, and important characteristic for estimating the quality of cryopreserved spermatozoa. A close relationship between the sperm velocities of frozen-thawed semen and post-thaw fertility rates has been reported (24,25). The sperm motility velocity and the percentage of motility of cryopreserved Amago salmon spermatozoa were lower than those of fresh spermatozoa (33). In contrast, Lahnsteiner et al.(24,25) found that the motility velocity of spermatozoa was not influenced by the cryopreservation process, whereas the sperm motility pattern was changed from circular to linear in post-thaw salmonid spermatozoa. Duration of sperm motility is shortened in post-thaw sperm (39,42). Lahnsteiner et aL(24,25) have also noted a close relationship between the percentage of sperm motility and the percentage (%) of structurally intact spermatozoa. They suggested that motility can only be initiated aRer thawing when spermatozoa are morphologically intact. It may be the survivors (less than 20%) that escaped major damage were able to fertilize ova in the present study and in previous experiments by other researchers. Motility rating, especially the percentage of motility, appears to be a valuable method for evaluating the efficiency of a cryopreservation protocol. The percentage of motility was a reliable predictor for the post-thaw fertilization rate in rainbow trout semen (25). However, fertility trials are necessary to present a complete assessment of viability of sperm after cryopreservation. Gwo (9) reported that as long as the nucleus of fish spermatozoa is intact, the correlation between the mean percentage of progressively motile spermatozoa and fertility was 0.739 in Atlantic croaker (Micropogonias undulatus). A full correlation between the post-thaw motility rate and the fertilization rate needs to be determined. Demographic instability, inbreeding depression, and loss of genetic diversity are significant problems facing the continued survival of small populations. Natural catastrophe, agricultural pollution, and sand-retention dams constructed along the native habitat of Formosan landlocked salmon, have severely affected Formosan landlocked salmon populations and restricted its distribution (26). A considerable genetic homogeneity in mtDNA has been confirmed in Formosan landlocked salmon (31). Although successful cryopreservation of Formosan landlocked salmon eggs and embryos is still elusive, sperm banks evidently can play a crucial role in conservation management of this endangered species. Sperm banks extend the lifetime of an individual for as long as its stored germ plasma lasts. With this technology, propagation goals could substantially lengthen generation times and retain higher levels of genetic diversity. Androgenesis is used in the production of homozygous inbred lines in various fish species (2,30,35,44). It can be induced by treating the eggs with radiation before fertilization, then fertilizing these eggs with normal spermatozoa, followed by pressure treatment to suppress the first cleavage division (2,30,35,44). Androgenesis has a major advantage in that it can be combined with sperm cryopreservation, allowing for the recovery of strains from cryopreserved spermatozoa. Onozato (35) successfully induced diploid androgenetic Masu salmon from crossing female Amago salmon with male Masu salmon. The optimal conditions for mass production of androgenetic diploid Amago salmon have been established (30). Successful fertility between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs has been demonstrated in this study, and this combination clearly offers a method for storing Formosan landlocked salmon spermatozoa for long periods of time and then regenerating a stock by using eggs from closely related subspecies such as Amago or Masu salmon as receptacles for the stored nuclear genomes.

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