Oocyte pre-IVM with caffeine improves bovine embryo survival after vitrification

Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance

Panorâmica da produção de embriões bovinos in vitro

In vitro embryo production (IEP) is a reproducti- ve biotechnology that impacts livestock systems favoring breeding and which have also been uti- lized as research models for embryo development in other species. For the development of this method, oocytes in vivo are collected through laparoscopy and ultrasound guided follicular aspiration, or through ovaries collected from slaughterhouses. The oocytes are subjected to maturation processes in vitro using various cul- ture media, addition of gonadotropins and growth factors. Spermatozoids undergo in vitro sperm ca- pacitacion process, where fertilization capacity is adquired and live spermatozoids are separa- ted from the seminal components and Cryopro- tectants. The oocyte–sperm co-culture for the in vitro fertilization is carried out in a media that promotes maximum sperm activity and zona pe- llucida penetration. For in vitro fertilization is ca- rried out in a medium favorable to sperm activity and pellucid zone penetration. Subsequently, the collected embryos are subjected to culture media that contain energy sources, amino acids and growth factors in order to maintain their sur- vival and development. Currently, IEP in cattle has become large-scale and is working on stan- dardization process for other species. Through PIV, biotechnologies such as cloning and trans- genesis have been developed. The aim of this review is to describe the PIV phases and their relation with the physiological events of fertiliza- tion and early embryo development.A produção in vitro de embriões (PIV) é uma bio- tecnologia reprodutiva com efeitos nos sistemas de criação de gado favorecendo o melhoramento gené- tico e também tem sido usada como um modelo de pesquisa do desenvolvimento embrionário em outras espécies. Para o desenvolvimento desta técnica, são coletados ovócitos in vivo via laparoscopica: Para o desenvolvimento desta técnica,são coletados ovóci- tos in vivo por laparoscopiaou aspiração folicular por ultrassonografia ou atravésde ovários colhidos em abatedouro. Os ovócitos são submetidos a proces- sos de maturação in vitro, utilizando-se vários meios, adição de gonadotrofinas e fatores de crescimento. Os espermatozóides são submetidos a tratamento na capacitação espermática in vitro, onde adquirem a capacidade de fertilização e são separados os es- permatozoides vivos dos componentes seminais e crioprotetores. Para a fertilização in vitro é utilizada uma co-cultura de espermatozoides e ovócitos num meio que promove a atividade espermática e a pe- netração na zona pelúcida. Subsequentemente, os embriões são submetidos à cultura em meios con- tendo fontes de energia, aminoácidos e fatores de crescimento, a fim de manter a sua sobrevivência e crescimento. Na atualidade a técnica PIV em bovi- nos é massificada e se trabalha em processos de padronização em outras espécies. A partir do PIV foi dado o desenvolvimento de novas biotecnologias, como a clonagem e transgênese. O objetivo desta revisão é descrever as fases da PIV e sua relação com os eventos fisiológicos da fecundação e desen- volvimento embrionário inicial.La producción de embriones in vitro (PIV) es una biotecnología reproductiva que impacta los siste- mas ganaderos favoreciendo el mejoramiento ge- nético y que también se ha utilizado como modelo de investigación del desarrollo embrionario en otras especies. Para el desarrollo de ésta técnica, se co- lectan oocitos in vivo por medio de laparoscopia y aspiración folicular guiada por ultrasonografía ó a través de ovarios recolectados en plantas de sacri- ficio. Los oocitos son sometidos a procesos de ma- duración in vitro, utilizando diversos medios, adición de gonadotropinas y factores de crecimiento. Los espermatozoides son sometidos a tratamientos de capacitación espermática in vitro, donde adquieren capacidad fertilizante y se separan los espermato- zoides vivos de los componentes seminales y crio-protectores. Para la fertilización in vitro se realiza un cocultivo de espermatozoides y oocitos en un medio que favorece la actividad espermática y penetración de la zona pelúcida. Posteriormente, los embriones obtenidos son sometidos a cultivo en medios que contengan fuentes de energía, aminoácidos y fac- tores de crecimiento, con el fin de mantener su so- brevivencia y desarrollo. En la actualidad la técnica de PIV en bovinos se ha masificado y se trabaja en los procesos de estandarización en otras especies. A partir de la PIV se dio el desarrollo de nuevas bio- tecnologías como la clonación y transgénesis. El objetivo de esta revisión es describir las fases de la PIV y su relación con los eventos fisiológicos de la fertilización y desarrollo embrionario temprano.Incluye referencias bibliográfica

Generalidades de la producción de embriones bovinos in vitro

La producción de embriones in vitro (PIV) es una biotecnología reproductiva que impacta los sistemas ganaderos favoreciendo el mejoramiento genético y que también se ha utilizado como modelo de investigación del desarrollo embrionario en otras especies. Para el desarrollo de ésta técnica, se colectan oocitos in vivo por medio de laparoscopia y aspiración folicular guiada por ultrasonografía ó a través de ovarios recolectados en plantas de sacrificio. Los oocitos son sometidos a procesos de maduración in vitro, utilizando diversos medios, adición de gonadotropinas y factores de crecimiento. Los espermatozoides son sometidos a tratamientos de capacitación espermática in vitro, donde adquieren capacidad fertilizante y se separan los espermatozoides vivos de los componentes seminales y crio-protectores. Para la fertilización in vitro se realiza un cocultivo de espermatozoides y oocitos en un medio que favorece la actividad espermática y penetración de la zona pelúcida. Posteriormente, los embriones obtenidos son sometidos a cultivo en medios que contengan fuentes de energía, aminoácidos y factores de crecimiento, con el fin de mantener su sobrevivencia y desarrollo. En la actualidad la técnica de PIV en bovinos se ha masificado y se trabaja en los procesos de estandarización en otras especies. A partir de la PIV se dio el desarrollo de nuevas biotecnologías como la clonación y transgénesis. El objetivo de esta revisión es describir las fases de la PIV y su relación con los eventos fisiológicos de la fertilización y desarrollo embrionario temprano

Generalidades de la producción de embriones bovinos in vitro

La producción de embriones in vitro (PIV) es una biotecnología reproductiva que impacta los sistemas ganaderos favoreciendo el mejoramiento genético y que también se ha utilizado como modelo de investigación del desarrollo embrionario en otras especies. Para el desarrollo de ésta técnica, se colectan oocitos in vivo por medio de laparoscopia y aspiración folicular guiada por ultrasonografía ó a través de ovarios recolectados en plantas de sacrificio. Los oocitos son sometidos a procesos de maduración in vitro, utilizando diversos medios, adición de gonadotropinas y factores de crecimiento. Los espermatozoides son sometidos a tratamientos de capacitación espermática in vitro, donde adquieren capacidad fertilizante y se separan los espermatozoides vivos de los componentes seminales y crio-protectores. Para la fertilización in vitro se realiza un cocultivo de espermatozoides y oocitos en un medio que favorece la actividad espermática y penetración de la zona pelúcida. Posteriormente, los embriones obtenidos son sometidos a cultivo en medios que contengan fuentes de energía, aminoácidos y factores de crecimiento, con el fin de mantener su sobrevivencia y desarrollo. En la actualidad la técnica de PIV en bovinos se ha masificado y se trabaja en los procesos de estandarización en otras especies. A partir de la PIV se dio el desarrollo de nuevas biotecnologías como la clonación y transgénesis. El objetivo de esta revisión es describir las fases de la PIV y su relación con los eventos fisiológicos de la fertilización y desarrollo embrionario temprano

Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality

Cyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns

<i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.

<p><i>In vitro</i> and <i>in vivo</i> developmental rates of prepubertal and adult oocytes cultured pre- and during IVM with or without cAMP modulators.</p

Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.

<p>Total number of OPU sessions, total number of follicles, total number of oocytes per donor and suitable for IVM obtained via ovum pick up in adult and prepubertal donors.</p

Gene expression profiles in expanded blastocysts produced from adult and prepubertal oocytes treated with or without cAMP modulators prior to and during IVM.

<p>Data are shown as the mean ± SEM (n = 12). Data were analyzed using two-way ANOVA followed by a Tukey's range test. The asterisk represents statistical significance among treatments for the same transcript; <i>P</i> < 0.05. <i>In vivo</i> produced expanded blastocysts were used for comparison. The mRNA relative abundance of the EGR1 gene was lower in all <i>in vitro</i> derived blastocysts compared to <i>in vivo</i> produced counterparts. No differences among treatments were found for <i>DNMT3b</i>, <i>BCL2L1</i>, <i>PRDX1</i>, <i>SLC2A8</i>.</p