Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Corpus luteum diameter and embryo developmental stage are associated with pregnancy rate: data analysis from 17, 521 embryo transfer s from a commercial in vitro bovine embryo production program

The objective of th is study was to assess the association between CL diameter , embryo developmental status and recipient pregnancy rate after in vitro - produced embryo transfer. Data from a total of 17 , 521 embryo transfer s from a commercial program were analyzed. The information was organized in pivot tables and the frequency of variables was determined with contingency tables. Additionally, a logistic regression analysis was performed to determine the o dds ratio and the degree of association between the variables. Higher pregnancy rates were achieved when the CL was larger than 24 mm (39.7%) and 22 mm (33.7 %) in diameter , whereas lower pregnancy rates were associated with a CL of 14 mm ( 24.2 %). Moreover, pregnancy rate was 30.5% when the recipients had a CL greater than 20 mm in diameter , whereas pregnancy rate was 32.0% with a CL smaller than 20 mm (P ˂ 0.01). Furthermore, an association between high conception rate s and CL diameter was found. With regard to embryo developmental status , higher pregnancy rates were associated with expanded blastocyst (37.0 %) and blastocyst (28.8%) transfer. Low pregnancy rates were achieved when morula e were transferred (15.4%). The present results suggest that variables such as CL diameter and embryo developmental status are significantly important and must be considered for the success of an in vitro - produced bovine embryo transfer program.

Corpus luteum diameter and embryo developmental stage are associated with pregnancy rate: data analysis from 17, 521 embryo transfer s from a commercial in vitro bovine embryo production program

The objective of th is study was to assess the association between CL diameter , embryo developmental status and recipient pregnancy rate after in vitro - produced embryo transfer. Data from a total of 17 , 521 embryo transfer s from a commercial program were analyzed. The information was organized in pivot tables and the frequency of variables was determined with contingency tables. Additionally, a logistic regression analysis was performed to determine the o dds ratio and the degree of association between the variables. Higher pregnancy rates were achieved when the CL was larger than 24 mm (39.7%) and 22 mm (33.7 %) in diameter , whereas lower pregnancy rates were associated with a CL of 14 mm ( 24.2 %). Moreover, pregnancy rate was 30.5% when the recipients had a CL greater than 20 mm in diameter , whereas pregnancy rate was 32.0% with a CL smaller than 20 mm (P ˂ 0.01). Furthermore, an association between high conception rate s and CL diameter was found. With regard to embryo developmental status , higher pregnancy rates were associated with expanded blastocyst (37.0 %) and blastocyst (28.8%) transfer. Low pregnancy rates were achieved when morula e were transferred (15.4%). The present results suggest that variables such as CL diameter and embryo developmental status are significantly important and must be considered for the success of an in vitro - produced bovine embryo transfer program.(AU)

High throughput non-invasive oocyte quality assessment: the search continues

Non-invasive oocyte quality assessment remains a major challenge of routine bovine in vitro embryo production (IVP). There is a major need for techniques allowing early selection of developmentally competent oocytes on the basis of a simple, quick, economic and feasible protocol. The availability of such a technique would clearly in crease IVP efficiency since only competent oocytes would be used, maximising blastocyst yield by ameliorating culture conditions. This mini-review summarizes briefly the currently available techniques that allow high throughput non-invasive oocyte quality assessment and indicates their possibilities and limitations.

High throughput non-invasive oocyte quality assessment: the search continues

Non-invasive oocyte quality assessment remains a major challenge of routine bovine in vitro embryo production (IVP). There is a major need for techniques allowing early selection of developmentally competent oocytes on the basis of a simple, quick, economic and feasible protocol. The availability of such a technique would clearly in crease IVP efficiency since only competent oocytes would be used, maximising blastocyst yield by ameliorating culture conditions. This mini-review summarizes briefly the currently available techniques that allow high throughput non-invasive oocyte quality assessment and indicates their possibilities and limitations.(AU)

Desenvolvimento de embriões de camundongas após manutenção em diferentes soluções de manipulação / Development of mice embryos after maintenance in different manipulation solutions

Avaliou-se a eficácia de duas soluções de manipulação (SM) de embriões de camundongas nos estádios de blastocisto inicial (Bin), mórula compacta grau I (McI) e II (McII), distribuídos aleatoriamente em três tratamentos (T), de acordo com a solução de manutenção. No T1 usou-se PBS modificado (controle); no T2, SME e no T3, SME enriquecida. Os embriões foram mantidos durante quatro horas na solução de manutenção e posteriormente classificados quanto ao estádio de desenvolvimento e à qualidade embrionária. Logo após, foram cultivados em meio TCM 199 e classificados novamente quanto ao estádio de desenvolvimento e à qualidade embrionária. A taxa de desenvolvimento dos embriões após manutenção por quatro horas em solução de manipulação foi menor (P<0,05) nos embriões do controle, comparada à de embriões do SME e SME enriquecida, diferença esta não observada (P>0,05) após o cultivo in vitro. Os embriões McII do T3 tiveram maior desenvolvimento (P<0,05) em relação aos embriões do T1 e T2, indicando o efeito benéfico do enriquecimento da solução SME. Conclui-se que as soluções de manipulação SME e SME enriquecida influenciaram beneficamente o desenvolvimento de embriões.(AU)

The effect of embryo manipulation solution followed by in vitro culture in mice embryos was studied. The embryos at early blastocyst (Bin), and compact morula grades I (McI) and II (McII) were randomly assigned into three treatments. T1 used modified PBS (control), T2 used EMS, and T3 used EMS supplemented. In each treatment, the embryos were kept in manipulation solution for four hours. Finishing the manipulation period, the embryos were classified according the development stage and quality. Following, the embryos were cultured in TCM 199. After the culture period, the embryos were evaluated according to quality and development stage. The development rate for Bin, McI, and McII after maintenance for four hours in manipulation solution was lower for control embryo (P>0.05) as compared to EMS and EMS supplemented embryos. After in vitro culture, no differences (P>0.05) on embryo development rate among control, EMS, and EMS supplemented were observed. Moreover, McII from EMS supplemented had a higher development (P<0.05) (93 percent) as compared to control (82.5 percent) and EMS (83.9 percent), suggesting a beneficial effect of EMS supplemented. EMS and EMS supplemented embryos had a positive effect on embryo development, showing higher embryo development than those in PBS solution.(AU)

Desenvolvimento e sobrevivência de embriões no período de pré-implantação: enfoque em ruminantes / Embryo development and survival during preimplantation period: focus in ruminants

O período de pré-implantação é considerado crítico para o desenvolvimento dos embriões em ruminantes, apresentando elevados índices de perda gestacional. Nesse período, uma série de eventos coordena o crescimento e a sobrevivência dos embriões, envolvendo a atuação de citocinas, esteroides, metabólitos e fatores de crescimento, responsáveis por garantir o sucesso da gestação e o subsequente desenvolvimento fetal. Esta revisão destacou os principais mecanismos e hormônios relacionados ao crescimento embrionário, utilizando os ruminantes como foco de estudo.(AU)

Preimplantation is considered a critical period for embryo development in ruminants, characterized by high pregnancy loss rates. In this period, many events coordinate the embryo growth and survival, involving cytokines, steroids, metabolites and growth factors, required to assure the pregnancy success and fetal development. This review aimed to highlight the major mechanisms and hormones related to embryo growth, using the ruminants as study focus.(AU)

Desenvolvimento e sobrevivência de embriões no período de pré-implantação: enfoque em ruminantes / Embryo development and survival during preimplantation period: focus in ruminants

O período de pré-implantação é considerado crítico para o desenvolvimento dos embriões em ruminantes, apresentando elevados índices de perda gestacional. Nesse período, uma série de eventos coordena o crescimento e a sobrevivência dos embriões, envolvendo a atuação de citocinas, esteroides, metabólitos e fatores de crescimento, responsáveis por garantir o sucesso da gestação e o subsequente desenvolvimento fetal. Esta revisão destacou os principais mecanismos e hormônios relacionados ao crescimento embrionário, utilizando os ruminantes como foco de estudo.

Preimplantation is considered a critical period for embryo development in ruminants, characterized by high pregnancy loss rates. In this period, many events coordinate the embryo growth and survival, involving cytokines, steroids, metabolites and growth factors, required to assure the pregnancy success and fetal development. This review aimed to highlight the major mechanisms and hormones related to embryo growth, using the ruminants as study focus.