Physical changes in specimens of five species of Cyprinidae preserved in ethanol and frozen.

Preservation in 30% ethanol and freezing to a temperature of -20 ± 2° C is an appropriate method for measurement of fish eggs, larvae and juveniles. Egg diameter of the common carp Cyprinus carpio increased insignificantly by 1·32% after preservation compared with live size. The total length (LT ) of 1 day post-hatching (dph) larvae as well as the standard length (LS) of 16 dph larvae of C. carpio increased significantly (2·95 and 1·50%, respectively) after preservation. Egg diameter as well as the LT of 1 dph larvae of barbel Barbus barbus increased significantly after preservation, by 1·74 and 1·96%, respectively over their original size. The standard length (LS ) of 14 dph larvae of B. barbus as well as juveniles of B. barbus, crucian carp Carassius carassius, common nase Chondrostoma nasus and tench Tinca tinca decreased significantly after preservation (-0·56 to -5·54%), whereas their body mass increased significantly (11·46-18·57%). Preserved eggs of C. carpio and B. barbus were hard, round and transparent. The larvae and juveniles of examined fishes, preserved in frozen ethanol, were straight, flexible and easily measurable after 60 days. Integrity of body surface and fins, as well as preservation of colours were much better in larvae or juveniles frozen and thawed only once than in specimens frozen and thawed thrice. Cooling in 30% ethanol to a temperature of 6 ± 2° C and freezing in water to a temperature of -20 ± 2° C are not appropriate preservation methods for eggs and larvae of C. carpio (1 and 16 dph).

Effects of preactivation of ooplasts or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos.

Blastomeres from eight-cell-stage rabbit embryos have been fused with enucleated metaphase II oocytes (ooplasts) or with ooplasts that were preactivated before fusion. Preactivation of ooplasts before nuclear transfer (NT) raises the rate of preimplantation development from 15% to 56%, which remains elevated in the next series of NT (48.6% and 47.2% in the second and third rounds, respectively). Transfer of eight-cell embryos from the third round to the recipient resulted in the birth of normal young. Synchronization of blastomere nuclei in the G1 phase with nocodazole before fusion results in 42% morula/blastocyst formation. However, in the second generation of NT embryos, the yield drops to as low as 17%, indicating deleterious effects of the second nocodazole treatment on blastomeres. The calculated number of clones per one round of cloning was 4.5, 3.9, and 3.8 in subsequent series; the highest number of morulae and blastocysts that developed from individual donor embryos after three rounds were 26 and 27, respectively.

Development of isolated sheep inner cell masses/embryonic discs in vitro.

Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10-11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55-60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12-13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.