Asynchrony between in vivo and in vitro rabbit embryos

Background: Comparative features of embryos developed under in vitro and in vivo conditions are particularly important in designing embryo transfer procedures that fulfil embryo-recipient synchronization requirements. Objective: To determine the degree of asynchrony in rabbit embryo development between cultured and in vivo embryos. Methods: A total of 55 non- lactating multiparous female rabbits were used. Embryos were classified as 16-cells or early morulae at 48 hours post-coitum (hpc). Embryos were cultured during 30 or 32 h and embryo development was compared with in vivo embryos of 72 hpc. In vitro and in vivo embryos at 72 hpc were classified as early or compacted morulae. Bayesian statistics was used. Difference between in vivo and in vitro embryos and the actual probability of the difference between the in vivo and in vitro embryo higher than zero (P) was estimated. Results: The percentage of compacted morulae was higher in in vivo embryos than in in vitro embryos with +6 h of asynchrony (73.5 and 32.8%, P=1.00). But the percentage of compacted morulae was similar with +8 h asynchrony. Conclusions: In vitro embryos delay their development by + 8 hours compared to in vivo embryos.

Antecedentes: El desarrollo comparativo de embriones producidos in vitro e in vivo es particularmente importante para el diseño de procedimientos de transferencia de embriones cuando se requiere sincronización entre el embrión y la hembra receptora. Objetivo: Determinar el grado de asincronía en el desarrollo embrionario entre embriones in vivo y cultivados. Métodos: Un total de 55 conejas multiparas no lactantes fueron utilizadas. Los embriones se clasificaron en 16 células o mórulas tempranas a las 48 horas después del coito (hpc). Los embriones se cultivaron durante 30 ó 32 horas y el desarrollo embrionario se comparó con embriones de 72 hpc obtenidos in vivo. Los embriones in vitro e in vivo a 72 hpc se clasificaron como mórulas tempranas o compactas. Se utilizó estadística bayesiana. Se estimó la diferencia entre embriones in vivo e in vitro y la probabilidad de que la diferencia sea superior a cero (P). Resultados: El porcentaje de mórulas compactas fue mayor en embriones in vivo que en embriones in vitro con +6 horas de asincronía (73,5 y 32,8%, P=1,00), pero el porcentaje de mórulas compactas fue similar con asincronía de +8 horas. Conclusión: Los embriones cultivados retrasan +8 horas su desarrollo en comparación con los embriones in vivo.

Antecedentes: A aquisição do desenvolvimento de embriões produzidos in vitro e in vivo é particularmente importante na concepção de procedimentos de transferência de embriões em que a sincronização entre o embrião e a fêmea receptora é necessária. Objetivo: Determinar o grau de assincronia no desenvolvimento embrionário entre embriões cultivados e in vivo. Métodos: Um total de 55 coelhos multíparos não lactantes foram usados. Os embriões foram classificados em 16 células ou mórulas iniciais 48 horas de gestação (hpc). Os embriões foram cultivados por 30 ou 32 horas e o desenvolvimento embrionário foi comparado com embriões de 72 hpc obtidos in vivo. Embriões in vitro e in vivo a 72 hpc foram classificados como mórulas precoces ou compactadas. Estatísticas bayesianas foram usadas. A diferença entre embriões in vivo e in vitro e a probabilidade de que a diferença seja maior que zero (P) foi estimada. Resultados: A porcentagem de mórulas compactadas foi maior em embriões in vivo do que em embriões in vitro com +6 horas de assincronia (73,5 e 32,8%, P=1,00). Mas a porcentagem de mórulas compactadas foi semelhante com assincronia de +8 horas. Conclusão: Embriões cultivados atrasam seu desenvolvimento em +8 horas em comparação com embriões in vivo.

Effect of culture time of frozen embryo after thawing on clinical pregnancy rate / 第二军医大学学报

Objective To explore the effects of in vitro culture time of frozen cleavage embryo after thawing on clinical pregnancy rate during frozen-thawed embryo transfer (FET) cycles. Methods The clinical data of 692 patients were analyzed; they received FET in the Department of Reproductive Medicine Center, Changhai Hospital, Navy Medical University (Second Military Medical University) from January to December in 2016. According to the days of in vitro culture between thawing and transferring, the patients were divided into three groups: no in vitro culture group, 1-day in vitro culture group and 2-day in vitro culture group. The pregnancy outcomes were compared between the three groups. Results There were no significant differences in age, body mass index, infertile duration, basic follicle-stimulating hormone (FSH) level or endometrial preparation protocol between the three groups (all P0.05). The embryo loss rates of the no in vitro culture group, the 1-day in vitro culture group and the 2-day in vitro culture group were 0.0% (0/706), 13.3% (64/481) and 43.0% (114/265), respectively, and the difference was statistically significant (P0.01). There were no significant differences in number of transfered embryo or endometrial thickness on transplantation day between the three groups (both P0.05). The good-quality embryo transfer rates in the 2-day in vitro culture group and 1-day in vitro culture group were significantly lower than that in the no in vitro culture group (both P0.01), and the clinical pregnancy rate in the 2-day in vitro culture group was significantly higher than that in the no in vitro culture group (P0.01). Conclusion In vitro culture for 1-2 days after thawing frozen cleavage embryos may increase the embryo loss rate, but it can improve the clinical pregnancy rate by screening the embryos with developmental potential.

Pregnancy rate and live birth rate from in vitro fertilization treatment / Монголын Анагаах Ухаан

BackgroundConducting IVF treatment in laboratory environment by using ARTechnologies in idiopathic infertility,which cannot be treated by both drug treatment and surgical treatment in Mongolia, and preparinglegal environment are the crucial issues in treating infertility. The study is theoretically and practicallysignificant to properly determine pregnancy rate and live birth rate after embryo transfer treatmentin Mongolia.GoalAim of the study is to determine pregnancy rate and live birth rate after IVF treatment in coupleshaving infertility problem.Objectives1. To determine pregnancy rate in conjunction with age factor after conducting In vitro fertilizationin laboratory environment in couples with infertility problem.2. To determine live birth rate after pregnancy in conjunction with age factor.Materials and MethodsThe information of 180 couples aged 22-46 y.o., who received IVF treatment in “Unimed International”hospital’s IVF Center between March 2014 and June 2015, has been used in this study. Pregnancyrate as well as live birth rate after in vitro fertilization treatment has been determined. Statistic datawere calculated by SPSS-20 and Microsoft office excel 2013.ResultsThe average age of 180 women treated by In vitro fertilization treatment was 35.21±5.1 y.o. Sortedby age group: aged ≤30 y.o. was 34 (18.8%), aged between 31-34 y.o. was 41 (22.7%), agedbetween 35-39 y.o. was 80 (44.4%) and aged ≥40 y.o. was 25 (13.8%). 71 (39.44%) of womentreated by In vitro fertilization became pregnant. Sorted by their age: aged ≤30 y.o. was 18 (52.9%),aged between 31-34 y.o. was 20 (48.7%), aged between 35-39 y.o. was 28 (35.0%) and aged ≥40y.o. was 5 (20.0%). Resulted live birth from pregnancy is 56 (78.8%). Sorted by their age: aged≤30 y.o. was 16 (88.8%), aged between 31-34 y.o. was 18 (90.0 %), aged between 35-39 y.o. was22 (78.5%) and aged ≥40 y.o. was 2 (40.0%). Multiple births from total live birth is 16 (28.57%),amongst them 14 (25.0%) were twin births and 2 (3.51%) of them had triplet birth.Conclusions:1. IVF treatment in laboratory environment by using ARTechnologies in idiopathic infertility, whichcannot be treated by both drug treatment and surgical treatment, have good results (averagepregnancy rate was 39.44%).2. Live birth rate from total pregnancy was 56 (78.8%).3. Multiple birth from total live birth is 16 (28.57%). 14 (25.0%) were twins and 2 (3.51%) weretriplets.4. The IVF success rate depends on variable factors such as maternal age, cause of infertility,embryo status, reproductive history and lifestyle factors.

In vitro development of murine embryos in presence of campylobacter fetus / Desarrollo de embriones murinos in vitro en presencia de campylobacter fetus

Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CFF (cell-free filtrate) and Brucella broth (BB). Blastocysts were cultured in MEM (CG); challenge group were constituted by the addition of Cfv 27. Differentiation, hatching, hatched, adhesion and expansion were evaluated. Results were analyzed by Chi2 test. In two-cell embryo, the differentiation rate was not modified when the study strains were added (p > 0.05). The differentiation rate at 24 h for embryos at the morula stage was lower for BB, Cfv, and CFF, compared with CG (p < 0.05). After 48 h culture, no differences were observed in blastocyst formation for Cfv and BB, compared to CG (p > 0.05). However, the differentiation rate for the CFF group was lower than for CG (p < 0.05). At 48 and 72 h, the hatching rate was higher in CFF and Cfv groups than in CG (p < 0.05). Differences were not detected in blastocyst cultures. In conclusion, under these experimental conditions, Cf was not detrimental to the development of murine embryos. Efforts will be intensified to establish in vitro infection models that reproduce their pathogenicity.

La campilobacteriosis bovina caudada por Campylobacter fetus produce pérdidas reproductivas existiendo poca información de los mecanismos de patogenicidad de dicha bacteria, por lo cual se propone un modelo utilizando ratones BALC/c. Embriones de dos células fueron cultivados en Ham-F10: grupo control (GC), los grupos experimentales fueron adicionados con las cepas Cfv 1, Cfv 3, Cff 2 y Cff 5. Mórulas fueron cultivadas en Ham-F10 (GC); los grupos tratados recibieron Cfv27, CFF (filtrado libre de células) y caldo Brucella (BB). Blastocistos fueron cultivados en MEM (GC) y MEM más Cfv 27 (grupo desafiado). Se evaluó: diferenciación, "hatching", "hatched", adhesión y expansión. Los resultados fueron analizados por Chi2. En embriones de dos células, la diferenciación no fue modificada por acción de las cepas evaluadas (p > 0,05). Para embriones en estadío de mórula, la diferenciación a las 24 h de cultivo fue menor para BB, Cfv, y CFF, comparado con el GC (p < 0,05). Luego de 48 h de cultivo, no hubo diferencias entre Cfv, BB, y CG (p > 0,05), no obstante para el grupo CFF la diferenciación fue menor al CG (p < 0,05). El porcentaje de "hatching" (48 y 72 h de cultivo), fue mayor en los grupos CFF y Cfv comparado con el GC (p < 0,05). La adición de Cfv 27 no modificó el desarrollo de blastocistos. En el modelo propuesto, Cf no afectó negativamente el desarrollo embrionario. Futuros trabajos serán necesarios para establecer un modelo de infección in vitro en pos de reproducir su patogenicidad.

Ehrlichia canis morulae and DNA detection in whole blood and spleen aspiration samples / Detecção de mórulas e DNA de Ehrlichia canis em sangue e em aspirado de baço em cães naturalmente infectados

The aim of this study was to compare the detection of Ehrlichia canis morulae and DNA by nPCR in whole blood and spleen aspiration. The sample included 40 dogs showing thrombocytopenia associated to clinical signs suggestive of canine ehrlichiosis. Morulae detection showed that in 35 of the dogs studied, 17 had morulae in spleen tissue, and two in buffy coat smears. E. canis DNA was detected in 29/40 blood samples. We verified that morulae detection is more efficient in cytological preparations from spleen aspiration. On the other hand, nPCR on spleen and blood samples were equally efficient for disease diagnosis.

O objetivo desse estudo foi comparar a pesquisa de mórulas de Ehrlichia canis e a nPCR em sangue total e em aspirado de baço. Selecionaram-se 40 cães apresentando trombocitopenia associada a sinais e sintomas sugestivos de erliquiose canina. A pesquisa de mórula mostrou que dentre 35 amostras, 17 apresentaram mórulas nas preparações do baço, e duas nos esfregaços feitos a partir da papa leucocitária. O DNA de Ehrlichia canis foi detectado em 29 de 40 amostras de baço e em 30 de 40 no sangue. No presente estudo observou-se que a pesquisa de mórula é mais eficiente nas preparações citológicas obtidas da punção aspirativa do baço e que tanto a nPCR de baço quanto a de sangue foram eficientes no diagnóstico da doença.

Aspectos epidemiológicos, clínicos e hematológicos de 251 cães portadores de mórula de Ehrlichia spp. naturalmente infectados / Epidemiological, clinical, and hematological aspects of 251 dogs naturally infected with Ehrlichia spp. morulae

Realizou-se um estudo retrospectivo dos aspectos epidemiológicos, sinais clínicos, dados de exame físico e alterações hematológicas da erliquiose em 251 cães naturalmente infectados por Ehrlichia spp. Dos 4407 casos atendidos em hospital veterinário no período de janeiro de 2002 a dezembro de 2003, verificou-se que 251 cães eram portadores de mórula de Ehrlichia spp. em leucócitos de sangue periférico. Destes, 48 foram eliminados das avaliações por apresentarem patologias concomitantes. Nos 203 cães restantes, verificou-se que houve maior ocorrência em fêmeas (61,1 por cento) e que a doença manteve-se constante durante todo o período avaliado. Observou-se que 38 por cento encontravam-se na faixa etária entre um e 23 meses e 58,6 por cento eram de raça definida. As principais alterações clínicas observadas foram apatia, anorexia/hiporexia, vômito, secreção oculonasal e esplenomegalia. Cento e cinco cães apresentaram temperatura retal entre 38 e 39,5ºC. As alterações observadas com maior frequência no hemograma foram anemia, predominando o tipo normocítica normocrômica (58,2 por cento); desvio nuclear de neutrófilos para a esquerda (67 por cento) e eosinopenia (58,1 por cento).

A study of epidemiological and clinical aspects, alterations of physical exams, and hematological changes of canine ehrlichiosis was performed. A retrospective study was performed in 4,407 dogs referred to a Veterinary Hospital from January 2002 to December 2003. Of all cases, 251 dogs showed Ehrlichia spp. morulae. Among these, 48 were excluded from the study due to other co-infection by other pathologies. In the other 203 evaluated dogs, females (61.1 percent) were more infected than males. The dogs aged from one to 23 months (68.6 percent) and 58.6 percent were definite breed. Emesis, apathy, anorexia/hypoxeria, spleenomegaly, and nasal discharge were the most common signs presented. Rectal temperature was 38 - 39.5ÚC in 105 dogs. The most usual changes seen during the hematological tests were normochromic and normocitic anemia (58.2 percent), a left shift of the neuthrophils (67 percent), and eosinopenia (58.1 percent).

Microhabitat use by two rocky shore gastropods in an intertidal sandy substrate with rocky fragments

Sandy beaches in some areas of the São Sebastião Channel in southeastern Brazil have unremittingly undergone a variety of impacts, including the deposition of rock fragments in the intertidal region. Consequently, these environments support a rich fauna comprising both sandy beach and rocky shore organisms. Two rocky shore gastropods, Tegula viridula and Morula nodulosa, are particularly abundant in such environments. An evaluation of the use of microhabitats by these two species revealed that they occupy the available microhabitats in different proportions and the presence of one species is associated with the absence of the other. Morula nodulosa is randomly dispersed, occupying mostly areas with rock fragments covered with sediment and branching brown algae. Tegula viridula shows a clumped dispersion associated with the patchiness of the microhabitats used: the presence of encrusting green algae and absence of sediment and branching brown algae covering the rocks. These findings suggest T. viridula has a lower tolerance than M. nodulosa to sand inundation of the rocky fragments, a stochastic event common to the environment in question.

Praias arenosas em algumas partes do Canal São Sebastião, região sudeste do Brasil, têm sido constantemente submetidas a diferentes tipos de impacto como deposição de fragmentos rochosos na região entremarés. Como conseqüência, estes ambientes abrigam uma rica fauna com organismos tanto de costões rochosos quanto de praias arenosas. Em especial, duas espécies de gastrópode típicas de costões rochosos, Tegula viridula e Morula nodulosa, são muito abundantes nestes ambientes. Uma avaliação do uso de microhabitats por estas duas espécies revelou que elas ocupam os microhabitats disponíveis em diferentes proporções e que a presença de uma espécie esteve associada à ausência da outra. Morula nodulosa apresentou uma dispersão ao acaso ocupando áreas com sedimento e algas marrons ramificadas recobrindo os fragmentos de rocha. Tegula viridula apresentou uma dispersão agrupada associada à característica agrupada dos microambientes ocupados: presença de algas verdes incrustantes e ausência de sedimento e algas marrons ramificadas recobrindo os fragmentos de rocha. Os resultados indicam que T. viridula pode ser menos tolerante que M. nodulosa à inundação dos fragmentos rochosos por sedimento, um evento estocástico comum ao ambiente estudado.

Vitrification of Mouse Embryos in Ethylene Glycol-based Solutions / 대한불임학회지

OBJECTIVE: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. MATERIALS AND METHODS: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. RESULTS: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. CONCLUSION: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

Cryopreservation of mouse morula and blastocysts by vitrification / 中华妇产科杂志

Objective To compare the survival and developmental potential of mouse morula, early blastocysts and blastocysts cryopreserved by vitrification. Methods One hundred and forty-two mouse morula, 135 early blastocysts and 148 blastocysts were cryopreserved by 6 mol/L ethylene glycol and 1 mol/L sucrose vitrification solutions. The survival rates and blastocysts hatching rates after thawing were observed. Results The survival rates of morula, early blastocysts and blastocysts groups were 88. 0% ,73. 3% ,and 60. 1% respectively. The blastocyst hatching rates were 73. 9% , 61. 5% ,and 49. 3% respectively. Both the survival rates and blastocyst hatching rates in morula group were higher than those in early blastocysts group (P

Timed Analysis of Mouse 2 - Cell Embryo Development in Dulbecco's Modified Eagle Medium ( DMEM ) in The Presence of Glutamine With of Without Glucose / 대한산부인과학회잡지

OBJECTIVE: To evaluat the effects of a culture medium with glucose in the presence of glutamine on the development of mouse embryos. METHODS: Two-cell embryos recovered from ICR mice at 48 hrs after hCG injection (mated just after hCG injection) were cultured in DMEM (with 20% hFF) supplemented with or without glucose on the presence of glutamine. Embryos were cultured under three different glucose regimens: (1) 0 mM (control); (2) 0.5 mM (group I); or (3) 3.15 mM (group II), and were analyzed at 24, 48, 72 and 96 hours intervals. Chi-square test (x2-test) was used to compare values of groups. RESULTS: No differences were found in the number of embryos showing morula (control: 37.5%; group I: 51.0%; group II: 48.4%), blastocyst (control: 21.5%; group I: 33.3%; group II: 34.4%) and blastocyst and hatching or hatched blastocyst (control: 81.9%; group I: 83.3%; group II: 82.8%) between groups at 24 hrs, 48 hrs or 72 hrs respectively. However at 96 hrs, the number of hatched and attached blastocyst was significantly higher in group I (82.3%) and II (78.5%) than control (63.2%; P<0.05). CONCLUSION: The addition of glucose (0.5 mM) to the DMEM, as energy source, improved the rate of development of late stage embryos in mice.

THE STUDY OF THE OPTIMUM CONDITION OF TWO FREEZING METHODS FOR PRESERVATION OF RABBIT EMBRYOS / 解剖学报

This paper reports the effects of the temperature control freezing and the liquid nitrogen vapour freezing on the viability of rabbit morulae. After rapid thawing, 70.27% embryos were able to develope well in vitro in the temperature control freezing group and in the liquid nitrogen vapour freezing group, the developmental rate was 80.56% when the freezing medium contained higher concentration of glycerol. After synchronous embryos were transplanted, the birth rate was 13.64% and 13.33%, respectively, in both freezing groups, and 11.11% in the control group. The results indicate that both freezing methods have almost the same good results, but the method of liquid nitrogen vapour freezing is more useful than the temperature control freezing method in some aspects.