Although acceptable rates of blastocyst formation are achieved with in vitro production of bovine embryos, several problems still compromise the subsequent development of the fetus and newborn, especially in embryos originating from somatic cell nuclear transfer. Routinely, the potential development of a bovine conceptus is predicted either on blastocyst quality or on various parameters related to the embryonic-fetal development in a foster mother. These methods are either imprecise or costly, highlighting the need for more reliable and practical methods to evaluate early embryonic development and differentiation. Thus, our aim was to improve the in vitro culture of embryos post hatching and to define a stable and repeatable system to monitor the development of bovine embryos. For that, in vitro-derived embryos were cultured in agarose gel tunnels in a modified culture medium (SOFaaci within 10% fetal bovine serum and 27.7 mM glucose). Daily monitoring of embryo length revealed that 56%-67% of the embryos in culture showed rapid growth and elongated until Day 13. Electron microscopy of elongated embryos at Day 14 confirmed successful localization of differentiated cells forming the trophoblast and hypoblast, with the definition of the Rauber layer. In conclusion, a stable culture system of post hatching embryos was first defined and can be used as a model for rapid growth, elongation, and initial differentiation of bovine post hatching embryos produced entirely in vitro.

200 Reproduction, Fertility and DevelopmentDevelopmental Biology for the first experiment, loaded into straws, and assigned to one of 4 treatment groups. Half the straws from each bull were exposed to 90 MPa/30 min, 90 MPa/90 min, 30 MPa/30 min, or 30 MPa/90 min, and then cryopreserved. Controls consisted of straws that were cryopreserved without pressure treatment. Cryopreservation steps were 60 min equilibration at 5 • C, followed by 10 min at −110 • C, and then plunging into liquid nitrogen. Straws were thawed in a 35 • C water-bath for 30 s. Each treatment and control group was replicated 8 times (8 samples per bull). The average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized groups compared to the samples frozen without previous pressurization (P < 0.001) (Bull I: 2-3% without pressurization vs. 17-33% with pressurization; Bull II: 0% without pressurization vs. 21-35% with pressure pre-treatment). Among the pressure/time parameters used, 30 MPa/90 min proved significantly superior (33 and 35%; P < 0.05) for each of the bulls. Expt. 2 clearly demonstrates the beneficial effect of a previous pressure treatment on post-thaw motility of bull semen cryopreserved in our experiment. Further investigations are needed, including samples from different bulls, different freezing protocols, and the biological background of the process. Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253-260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5-to 10-µL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN 2 ) for 15 s and then plunged into LN 2 . The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN 2 -submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2...

Com bioeficiência comprovada em equinos, foi realizado no Uniceub-LABOCIEN o primeiro estudo em Rattus novergicus (linhagem Wistar) utilizando gonadotrofina (hormônio LH:FSH) e somatotrofina (hormônio do crescimento ou GH) extraídos de hipófises equinas coletadas em abatedouro.Para testar a gonadotrofina equina, 35 fêmeas pré-púberes com peso vivo médio de 54,5 g foram divididas nos tratamentos Placebo, 0,025UI, 1UI ou 10UI de gonadotrofina equina, e 10UI de eCG (Novormon®), por dose, por 3 dias consecutivos, via subcutânea. Todos os tratamentos foram associados com 4UI/dia de hCG conforme protocolo da farmacopeia internacional. Já a somatotrofina foi testada com 28 machos pré-púberes com peso vivo médio de 50,7g nos tratamentos Placebo, 0,025UI, 1UI ou 10UI de somatotrofina equina por dose via subcutânea, 3 dias/semana, durante 30 dias. Os animais foram mantidos em gaiolas individuais em estantes ventiladas, com ciclo de 12h escuro e 12h claro, água e ração ad libitum. Ambos os hormônios tiveram delineamento inteiramente casualizado com 7 repetições, sendo as médias resultantes comparadas pelo teste de Tukey (p<0,01). Ao final do experimento os animais foram eutanasiados, pesados e necropsiados. O peso médio relativo dos ovários das fêmeas Placebo (0,46g) não diferiu das doses 0,025UI (0,63g) ou 1UI (0,63g). As médias nos tratamentos 10UI de gonadotrofina equina (1,01g) e 10UI do produto comercial eCG (0,83g) foram as mais elevadas, mas não diferiram entre si. Não houve diferença entre as médias de peso corporal final (54,5g) e médias de ganho de peso relativo (29,0g). Nos machos foram pesados fígado, rins, coração e baço, bem como o aparelho reprodutor intacto (excluindo pênis) para avaliação macroscópica. Não houve diferença entre as médias de peso corporal final (50,7g), comprimento (35,8cm), ou peso médio relativo dos órgãos fundamentais, bem como seu aspecto. Já o aparelho reprodutor dos machos tratados apresentou modificação macroscópica no padrão de irrigação sanguínea, com maior ramificação e espessamento aparente nos vasos dos testículos, epidídimo, ducto deferente, vesícula seminal e plexo pampiniforme. Houve também aparente incremento de gordura justaposta aos órgãos fundamentais e plexo pampiniforme. Nenhum grupo tratado apresentou reação tecidual, alteração dos órgãos ou efeitos colaterais aparentes, indicando assim inocuidade das moléculas. Todos os órgãos avaliados em todos os tratamentos foram fixados em formaldeído 10% para análise 4 microscópica. Os resultados avaliados confirmaram a atividade das gonadotrofinas equinas, com incremento de peso ovariano em fêmeas pré-púberes. O GH equino apresentou indicadores de suas atividades angiogênica e metabólica, devendo ser estudado em tratamentos mais prolongados. Sendo o equino doador universal de hormônio proteico, incluindo para humanos, o trabalho indica a bioatividade dos hormônios hipofisários de origem equina também nos ratos, espécie heteróloga fundamental nos estudos científicos. Reforçada pelos príons, tem início a frenética era da síntes...

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Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: &gt;1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: &lt;0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P&lt;0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P&lt;0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P&lt;0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P&lt;0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.