Production of viable trout offspring derived from frozen whole fish

Long-term preservation of fish fertility is essential for the conservation of endangered fishes. However, cryopreservation techniques for fish oocytes and embryos have not yet been developed. In the present study, functional eggs and sperm were derived from whole rainbow trout that had been frozen in a freezer and stored without the aid of exogenous cryoprotectants. Type A spermatogonia retrieved from frozen-thawed whole trout remained viable after freezing duration up to 1,113 days. Long-term-frozen trout spermatogonia that were intraperitoneally transplanted into triploid salmon hatchlings migrated toward the recipient gonads, where they were incorporated, and proliferated rapidly. Although all triploid recipients that did not undergo transplantation were functionally sterile, 2 of 12 female recipients and 4 of 13 male recipients reached sexual maturity. Eggs and sperm obtained from the salmon recipients were capable of producing donor-derived trout offspring. This methodology is thus a convenient emergency tool for the preservation of endangered fishes.

examine the effects of frozen whole trout body weight on type A spermatogonia (ASG) survival, orange-colored pvasa-Gfp rainbow trout weighing 0.9 ± 0.1 g at 3-month-old (a), 18.8 ± 1.6 at 10-month-old (b), 101.6 ± 5.7 g at 15-month-old (c), and 203.9 ± 8.0 g at 18-month-old (d) were frozen in a 80°C standard deep freezer for 8, 372, and 735 days (n = 5). (e) No viable ASG were retrieved from whole trout weighing 0.9 g frozen in a freezer for 735 days. (f-h) Testicular cells retrieved from whole trout weighing 18.8 g (f), 101.6 g (g), and 203.9 g (h) frozen in a freezer for 735 days. (i) Viability of ASG retrieved from whole trout weighing 18.8, 101.6, and 203.9 g frozen in a freezer for 8, 372, and 735 days. There were no significant differences in ASG viability among different freezing periods. (n = 4-5). (j) GFP (+) ASG retrieved from 203.9 g frozen whole trout (arrow) were incorporated into the genital ridges of wild-type trout. (k,l) Incorporated ASG (arrows) began to proliferate within the recipient gonads (k) and the gonad of a nontransplanted control (l). (m-o) Percentage of recipients that contained donor ASG within recipient gonads (m), number of ASG incorporated into recipient gonads (n), and percentage of recipients having proliferating donor ASG (o) were not significantly different between 203.9 g frozen trout and fresh control groups (n = 26-33). Data are shown as mean ± SEM. Scale bars, 5 cm (a-d); 20 μm (e-h, j-l). All images were taken by Seungki Lee. WT trout, and WT salmon at 1 year of age. (g,h) Fertilization rates (g) and hatching rates (h) of eggs inseminated with milt obtained from fresh, F 7, F 30, F 189, F 371, F 738, and WT trout at 2 years of age. Milt obtained from trout recipients at 1 and 2 years of age were inseminated with eggs obtained from WT trout of the same ages. There were no significant differences within each developmental stage. Data are shown as mean ± SEM (n = number of mature fish within each group).        (-196) Liquid nitrogen (-196) 3.5 Incubator (10) 0 (0) f Fresh control b 15,467 ± 395 -Incubator (10) 3.5 -15,306 ± 526 (98.8 ± 1.1) g a Type A spermatogonia with 100 ml Eagle's minimum medium (EMEM) in cryotube frozen by direct plunging into liquid nitrogen followed by slow thawing.

Supplementary
b Type A spermatogonia with 100 ml EMEM in cryotube as a control of frozen group.
c Initial GFP-lebeled type A spermatogonia prepared from pvasa-Gfp rainbow trout was maintained with EMEM in a 10°C incubator. d Frozen cryotube were slowly thawed in pre-frozen Bicell slow-freezing container (Nihon Freezer) located in a 10°C incubator for 3 hours.
e Viability of GFP-labeled type A spermatogonia was analyzed by Guava PCA-96 flow cytometry (Millipore) immediately after thawing.
f,g Values in a column with different superscripts are significantly different (P < 0.05).
Data are shown as mean ± standard error of the mean values derived from three independent experiments.
Statistical significance was determined using Student's t-test for comparisons between two groups.