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Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including their morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of both in vitro and in vivo differentiation have been established (Shim H et al. 1997 Biol. Reprod. 57, 1089-1095. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide an inexhaustible source of karyoplasts in nuclear transfer (NT). This would be particularly advantageous in maintaining nuclear donor cells carrying a transgene. In addition, genome-wide demethylation of DNA occurs in pre-implantation embryos as well as PGC. Nuclear transfer embryos using EG cells rather than somatic cells may be close to embryos from normal fertilization in their DNA methylation status. If combined with NT technique, EG cells may potentially be useful for genetic manipulation in pigs. In this study the efficiencies of transgenesis and NT using porcine fetal fibroblast and EG cells were compared. Two different techniques were used to perform NT. When conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes was used, the rates of development to the blastocyst stage were 16.8% (59/351) and 14.1% (50/354) in EG and somatic cell NT, respectively. In piezo-driven micromanipulation (Honolulu method) involving direct injection of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 7.5% (12/160), respectively. Although the differences between EG and somatic cell NT were statistically insignificant, the rates of blastocyst development in EG cell NT were comparable to the somatic cell counterpart regardless of NT methods used in the present study. To investigate if EG cells can be used for transgenesis in pigs, GFP gene was introduced into porcine EG cells. Nuclear transfer embryos using transfected EG cells gave rise to blastocysts (29/137, 21.2%), and all embryos that developed to the blastocyst stage expressed GFP, based on observation under fluorescence microscope. In this study, the possibility of using EG cells as karyoplast donors in NT procedure was tested. The results suggest that EG cell NT may be used as an alternative to somatic cell NT, and transgenic pig embryos may be produced using EG cells. It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304-5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3-5 were used following culture in serum-starved medium for 5-7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264-272). NT embryos were cultured in a serum-free me...

Lypsykarjalla sperman sukupuolilajittelulla on monta etua: nuorten sonnien testityttärien tuottaminen pienemmillä siemennysmäärillä, huippusonnien X-sperman käyttö parhaille lehmille ja lihasonnien Y-sperma keskitason lehmille. Tehokkain tapa varmistaa naudan alkioiden ja niistä syntyvien vasikoiden sukupuoli on käyttää X- ja Y-fraktioihin lajiteltua spermaa hedelmöityksessä, jolloin lähes kaikki tuotetut alkiot ovat haluttua sukupuolta. Lajittelemattomalla spermalla tuotetuista alkiosta haluttua suku-puolta on teoriassa noin puolet. Yhdysvaltalaisen XY Inc.:n kehittämä ja patentoima virtaussytometriaan perustuva siittiöiden lajittelumenetelmä on tällä hetkellä maailmalla tutkituista menetelmistä ainoa, joka on kaupallistettu. Lajiteltua spermaa voidaan myös pakastaa, mikä on suuri etu sperman logistiikan kannalta. Maa- ja metsätalousministeriön ja jalostus- ja elintarviketeollisuuden rahoittamassa tutkimushankkeessa selvitettiin XY Inc.:n patentoimalla menetelmällä lajitellun sperman laatua ja lajitteluun käytettyjen sonnien välisiä eroja alkioiden laboratoriotuotannossa ja alkiohuuhtelusiemennyksissä. Kokeissa käytetty holstein-friisiläissonnien sperma ostettiin Cogent Ltd:ltä Englannista. Laboratoriossa lajitellun sperman sulatuksen jälkeinen elävyys oli silmämääräisessä tarkastelussa lajittelematonta spermaa heikompi. X-sperma penetroi munasoluja heikommin ja X-spermalla hedelmöitetyt munasolut jakautuivat lajittelemalla ja Y -spermalla hedelmöitettyjä munasoluja heikommin. Y-sperma tuotti eniten ja laadullisesti parhaita siirtokelpoisia alkioita. X- ja Y-spermalla tuote-tuista siirtokelpoisista alkioista keskimäärin 10% oli väärää sukupuolta. Hiehoilla ja lehmillä tehdyissä alkionhuuhteluissa verrattiin lajittelematonta ja X-spermaa. Vain X-spermalla siemennetyistä luovuttajista löydettiin hedelmöitymättömiä munasoluja. Siirtokelpoisten tai kehityksessä pysähtyneiden alkioiden osuuksissa ei lajittelemattoman ja X-sperman välillä ollut vastaavaa eroa.Vastaavana ajanjaksona tilatasolla yhteensä 30 karjanomistajaa käytti kolmen Englannista tuodun sonnin lajiteltua X-spermaa hiehojen ja lehmien alkionhuuhtelusiemennyksissä. Myös tässä aineistossa X-spermalla saatiin lehmillä vähemmän siirtokelpoisia alkiota ja enemmän hedelmöitymättömiä munasoluja kuin vastaavana ajanjaksona suomalaisilla holstein-friisiläissonneilla tehdyissä huuhteluissa. Alkiontuotantotuloksissa oli myös sonnien välisiä eroja. Tulokset osoittavat, että XY Inc.:n kehittämä lajittelumenetelmä on erittäin luotettava. Lehmille tarvittanee useampia siemennyksiä ja/tai isompia siemennysannoksia kuin hiehoilla. Keinosiemennyssonnien soveltuvuus lajitteluun on arvioitava yksilökohtaisesti.

Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696-1701. The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579-585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 µM BLI (B) or 6.25 µM BLI + 12.5 µM ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 µg mL −1 FSH, 50 µg mL −1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40-50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL −1 EGF). After IVM, all groups (140-150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 × 10 6 sperm cells mL −1 ) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5 • C under 5% CO 2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 ± 1%, P < 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 ± 2 and 80 ± 1%, respectively; P > 0.05) than C (100%, P < 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 ± 1-83 ± 2%, P > 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 ± 1-89 ± 1 for 21 h and 85 ± 2-96 ± 8 for 24 hP > 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 ± 3-84 ± 3%, P > 0.05) and Day 7 blastocyst rates (26 ± 4-37 ± 4%, P > 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 ± 4-40 ± 4%, P > 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 ± 4%) only when compared with C (40 ± 4%, P < 0.05). Hatching rates were similar (10 ± 3-16 ± %3, P > 0.05) as were total cell numbers (141 ± 5-170 ± 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in...

We have studied the effect of suppressed IVM on the developmental competence of bovine oocytes, aiming at elucidating the importance of cytoplasmic maturation in fertilization and embryo development. Six replicates of abattoir-derived oocytes were randomly divided into three IVM groups. Control (n = 950): TCM-199 with glutamax-I (Gibco, Grand Island, NY, USA), 0.25 mM Na-pyruvate, 100 IU mL−1 penicillin and 100 μg mL−1 streptomycin, 50 ng mL−1 FSH, and 10% fetal bovine serum (FBS) (Gibco); Serum+FSH-free (n = 944): same as control but without FSH and FBS; α-amanitin (n = 977): same as control but with 10 μg mL−1 α-amanitin. Nuclear maturation of oocytes was studied 24 h after the onset of IVM, the formation of sperm aster structure 10 hours post-insemination (hpi) and the formation of pronuclei 20 hpi. Sperm aster was visualized with β-tubulin antibody (modified from Navara et al. 1999 Dev. Biol. 162, 29–40). Presumptive zygotes were cultured until Day 7 in modified SOFaaci + 4 mg mL−1 fatty acid-free BSA in 5% O2. Cumulus cell expansion was seen only in the control group. The results of nuclear maturation, fertilization, and embryo development are summarized in Table 1. Serum and FSH deprivation did not have a statistically significant effect on the parameters studied (vs. control). α-amanitin exposure during IVM reduced nuclear maturation, fertilization, and Day 3 embryo cleavage vs. control, and resulted in total blockage of Day 7 blastocyst development. The treatment groups had significantly smaller mean diameters of male pronuclei (control: 14 ± 0.6 μ­m; serum+FSH-free: 12 ± 0.5 μ­m, P < 0.05; α-amanitin: 10 ± 0.6 μ­m, P < 0.001) and sperm asters (control: 86 ± 4 μ­m; serum+FSH-free: 82 ± 4 μ­m, P < 0.01; α-amanitin: 49 ± 7 μm, P < 0.001) (nonparametric Kruskall Wallis and Mann-Whitney U tests) vs. control group. Despite reduction in pronucleus and sperm aster diameter, serum and FSH deprivation during IVM did not affect in vitro developmental competence of bovine oocytes, suggesting a need for re-evaluation of the components of IVM. α-Amanitin exposure in IVM disturbed nuclear maturation, fertilization, and embryo development, indicating the essence of early transcription. Table 1. Average percentages ± (n) for nuclear maturation, fertilization (min two pronuclei), embryo cleavage, and blastocyst development