Evaluation of novel SexedULTRA-4M technology for in vitro bovine embryo production

SexedULTRA-4M™ is made using an improved method of sex-sorting sperm in a less damaging environment for better retaining sperm integrity throughout the sorting process. The objective of this research was to compare conventional (CONV) and SexedULTRA-4M™ (ULTRA-4M) semen for bovine IVP using four Angus bulls. Matured slaughterhouse oocytes (n = 4000) were divided into the CONV group and the ULTRA-4M group (2000 COCs for each semen type). The IVF process was implemented with CONV and ULTRA-4M semen from the same bull. The cleavage rates, eight cell embryos and blastocysts on day 7 of culture were evaluated for each semen type and each bull. The statistical analysis was carried out with the ANOVA procedure SAS software. The results were 54.45% ± 1.03 and 58.10% ± 1.07; 35% ± 1.57 and 39.15% ± 1.62; 22.8% ± 1.09 and 27.15% ± 1.12 for CONV and ULTRA-4M, respectively, for cleavage rate, eight cell embryos and blastocysts on day 7 for the average of all bulls, comparing only the semen type. Concerning only the semen type, ULTRA-4M was significantly superior to CONV for cleavage rates (P = 0.01) and blastocysts on day 7 (P = 0.009). There were no significant differences between the CONV and ULTRA-4M groups (P>0.05) for all variables analyzed for Bull 1 and Bull 4, however, for Bull 2 ULTRA-4M was significantly superior to CONV for cleavage rates and blastocysts on day 7 (P< 0.05). In Bull 3, ULTRA-4M was significantly higher (P< 0.05) for blastocysts on day 7 compared to CONV. In conclusion, under the conditions of this research the ULTRA-4M and CONV semen produced similar bovine IVP results overall.(AU)

Desafios para a reprodução assistida em animais de vida livre / Challenges for assisted reproduction in free-living animals

A reprodução assistida se faz necessária em programas de conservação de espécies ameaçadas de extinção, sendo um facilitador de transporte e troca de material genético. Neste contexto, o acesso ao material de animais de vida livre é essencial para incrementar o banco genético da espécie em questão, no entanto adaptar os métodos possíveis à realidade do campo torna essa área de pesquisa desafiadora. Ainda hoje os espermatozoides são os gametas mais acessados em animais de vida livre, porém com pouco uso efetivo para criopreservação e produção de filhotes. É pungente a necessidade de mais pesquisas nesta área, uma vez que há centenas de espécies brasileiras ameaçadas, com especificidades fisiológicas e que habitam habitats variados, o que demanda adaptações espécie-específicas e hábitat específicas.

Assisted reproduction is necessary for conservation programs for endangered species, facilitating transport and exchange of genetic material. In this context, access to material from free-living animals is essential to increase the genetic bank of the species in question. However, adapting the possible methods to the reality of the fieldwork makes this area of research a challenge. Even today, sperm are the most accessed gametes in free-living animals, but with little effective use for cryopreservation and production of offspring. The need for more research in this area is acute, as there are hundreds of Brazilian species under threat, with physiological specificities, and that inhabit varied habitats, which demand species-specific adaptations and specific habitats.

Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines / Efeito de rolipram na maturação in vitro, expressão gênica e desenvolvimento embrionário em bovinos

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.(AU)

O objetivo deste trabalho foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos, expressão gênica e embriões produzidos in vitro. Os ovários bovinos foram coletados no matadouro. Os COCs foram selecionados e divididos em cinco grupos: controle 0 tempo; controle: MIV por 24 horas; tratamentos rolipram com bloqueio MIV por 24 horas em meio de maturação contendo 100, 150 e 200µM. Após 24 horas, todos os grupos foram recolocados em MIV por mais 24 horas. Subsequentemente COCs foram submetidos ao mesmo sistema MIV e fertilizados, sendo avaliada a taxa de clivagem e de blastocisto, além da expressão dos seguintes genes: Mater, BMP15 e Bax. Nenhuma diferença foi observada na expressão gênica. Dos oócitos avaliados logo após a aspiração folicular, 79,0% estavam em GV, GVBD, MI, enquanto 13,40% estavam em MII, e 7,60% em D/NI. A diferença significativa foi observada em diferentes concentrações (T100, T200 e T150µM) em oócitos que atingiram a fase MII em comparação aos tratamentos de controle (P=0,3). Diferenças foram observadas nas taxas de clivagem (P<0,5) entre T150 e T200 quando comparadas com as taxas do grupo C/24. Uma grande diferença foi observada na taxa de blastocisto (P<0,1) entre os tratamentos em relação ao grupo controle.(AU)

Effect of Rolipram on in vitro maturation, gene expression and embryonic development in bovines / Efeito de rolipram na maturação in vitro, expressão gênica e desenvolvimento embrionário em bovinos

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.(AU)

O objetivo deste trabalho foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos, expressão gênica e embriões produzidos in vitro. Os ovários bovinos foram coletados no matadouro. Os COCs foram selecionados e divididos em cinco grupos: controle 0 tempo; controle: MIV por 24 horas; tratamentos rolipram com bloqueio MIV por 24 horas em meio de maturação contendo 100, 150 e 200µM. Após 24 horas, todos os grupos foram recolocados em MIV por mais 24 horas. Subsequentemente COCs foram submetidos ao mesmo sistema MIV e fertilizados, sendo avaliada a taxa de clivagem e de blastocisto, além da expressão dos seguintes genes: Mater, BMP15 e Bax. Nenhuma diferença foi observada na expressão gênica. Dos oócitos avaliados logo após a aspiração folicular, 79,0% estavam em GV, GVBD, MI, enquanto 13,40% estavam em MII, e 7,60% em D/NI. A diferença significativa foi observada em diferentes concentrações (T100, T200 e T150µM) em oócitos que atingiram a fase MII em comparação aos tratamentos de controle (P=0,3). Diferenças foram observadas nas taxas de clivagem (P<0,5) entre T150 e T200 quando comparadas com as taxas do grupo C/24. Uma grande diferença foi observada na taxa de blastocisto (P<0,1) entre os tratamentos em relação ao grupo controle.(AU)

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).(AU)

Intensive use of IVF by large-scale dairy programs

The number of embryos produced by in vitro fertilization (IVF) has grown exponentially in recent years. Recently, for the first time, the number of embryos produced and transferred in vitro was significantly higher than the number developed in vivo worldwide. In this context, a particular boost occurred with ovum pick-up (OPU) and in vitro embryos produced in North America, and this technology is becoming more prominent for commercial dairy farms. However, despite many advances in recent decades, laboratories and companies are looking for methods and alternatives that can be used in collaboration with the existing process to improve it. Among the strategies used to improve the dairy industry are the use of genomic analysis for the selection of animals with desired traits or as an evaluation tool of oocyte and embryo quality, the optimization of the collection and use of gametes from prepubertal females and males, the effective use of sexed semen, and improvements in culture media and methods of embryo cryopreservation. Thus, this review aims to discuss the highlights of the commercial use of IVF and some strategies to increase the application of this technique in large-scale dairy programs.

Intensive use of IVF by large-scale dairy programs

The number of embryos produced by in vitro fertilization (IVF) has grown exponentially in recent years. Recently, for the first time, the number of embryos produced and transferred in vitro was significantly higher than the number developed in vivo worldwide. In this context, a particular boost occurred with ovum pick-up (OPU) and in vitro embryos produced in North America, and this technology is becoming more prominent for commercial dairy farms. However, despite many advances in recent decades, laboratories and companies are looking for methods and alternatives that can be used in collaboration with the existing process to improve it. Among the strategies used to improve the dairy industry are the use of genomic analysis for the selection of animals with desired traits or as an evaluation tool of oocyte and embryo quality, the optimization of the collection and use of gametes from prepubertal females and males, the effective use of sexed semen, and improvements in culture media and methods of embryo cryopreservation. Thus, this review aims to discuss the highlights of the commercial use of IVF and some strategies to increase the application of this technique in large-scale dairy programs.(AU)

Thirty years of assisted reproductive technology in the domestic cat: A selected summary / Trinta anos de tecnologia de reprodução assistida no gato doméstico: Um resumo selecionado

The first kittens to be born after embryo transfer (ET) in the cat (Schriver and Kraemer, 1978) were reported four decades ago. The births of kittens after in vitro fertilization (IVF)/ET (Goodrowe et al., 1988) and after embryo cryopreservation/ET (Dresser et al., 1988) were described 10 years later. In an early report it was established that embryo donors had the ability ot exhibit repeated ovarian stimulatory response to multiple gonadotropin treatments (porcine FSH; Dresser et al., 1987). After studies on gonadotropin-induced hyperstimulation of follicular development for recovery and ET of fresh and cryopreserved in vivo derived uterine stage embryos from mated donors (Dresser et al., 1988; Pope et al., 1989), we transitioned into developing methods for in vitro production of domestic cat embryos for the purpose of applying the technology to support conservation of threatened and endangered felid species (Pope et al., 1993). Since then, techniques for the in vitro production of cat embryos have been developed sufficiently to allow births of kittens after transfer of embryos derived by an assortment of in vitro techniques, including cryopreservation (Pope et al., 1994; Gómez et al., 2003a; Pope et al., 2012a, b; Galiguis et al., 2014), intracytoplasmic sperm injection (ICSI; Pope et al., 1998; Gómez et al., 2000; Pope et al., 2012a), gender selection (Pope et al., 2009) and somatic cell nuclear transfer (SCNT) (Gómez et al., 2004; 2008; 2009).

Estratégias para aumentar a produção de embriões em bovinos / Strategies to increase embryo production in cattle

As biotecnologias para produção de embriões in vivo e in vitro são de grande importância para acelerar o ganho genético dos rebanhos de corte e de leite. No entanto, o sucesso desses processos depende diretamente da quantidade e da qualidade dos oócitos da doadora de embrião. Diversos fatores influenciam a produção de embriões como a população folicular, a idade da doadora, o estresse térmico, o status metabólico e o status reprodutivo. Na atualidade, vários estudos têm sido desenvolvidos para elaborar estratégias que contornem estes desafios e aumentem a eficiência das técnicas de produção de embriões. Dentre os tratamentos estabelecidos para melhorar a produção de embriões das doadoras destacam-se a utilização de FSH, rBST, propilenoglicol e células tronco mesenquimais.(AU)

Biotechnologies for producing in vivo and in vitro embryos are very important to accelerate the genetic gain of beef and dairy herds. However, the success of these processes depend directly on the quantity and quality of the donors oocytes. Many factors influence the embryo production, such as follicle population, age, heat stress, metabolic status and reproductive status. Recently, many studies have been conducted to elaborate strategies that can overcome these issues and increase the efficiency of the embryo production techniques. Among the treatments developed to improve the embryo production, the use of FSH, rBST, propylene glycol and mesenchymal stem cells stand out.(AU)

Estratégias para aumentar a produção de embriões em bovinos / Strategies to increase embryo production in cattle

As biotecnologias para produção de embriões in vivo e in vitro são de grande importância para acelerar o ganho genético dos rebanhos de corte e de leite. No entanto, o sucesso desses processos depende diretamente da quantidade e da qualidade dos oócitos da doadora de embrião. Diversos fatores influenciam a produção de embriões como a população folicular, a idade da doadora, o estresse térmico, o status metabólico e o status reprodutivo. Na atualidade, vários estudos têm sido desenvolvidos para elaborar estratégias que contornem estes desafios e aumentem a eficiência das técnicas de produção de embriões. Dentre os tratamentos estabelecidos para melhorar a produção de embriões das doadoras destacam-se a utilização de FSH, rBST, propilenoglicol e células tronco mesenquimais.

Biotechnologies for producing in vivo and in vitro embryos are very important to accelerate the genetic gain of beef and dairy herds. However, the success of these processes depend directly on the quantity and quality of the donors oocytes. Many factors influence the embryo production, such as follicle population, age, heat stress, metabolic status and reproductive status. Recently, many studies have been conducted to elaborate strategies that can overcome these issues and increase the efficiency of the embryo production techniques. Among the treatments developed to improve the embryo production, the use of FSH, rBST, propylene glycol and mesenchymal stem cells stand out.

Melatonin and tannic acid supplementation in vitro improve fertilization and embryonic development in pigs

The objective of this study was to determine the effects of melatonin supplementation during maturation and tannic acid supplementation during IVF on fertilization kinetic sand early embryonic development. Experiment 1 determined the optimum concentration of melatonin supplemented to the oocytes for subsequent embryonic development. Oocytes (n = 400)were supplemented at 22 h of maturation with 0, 75, 100, or 150 nm melatonin and then subjected to IVF and embryo culture. After IVF, a portion of the embryos were evaluated for penetration, polyspermy, and male pronuclear (MPN) formation rates. Embryos were evaluated 48 h after IVF for cleavage and 144 h for blastocyst formation. There were no significant differences between treatment groups with respect to penetration and polyspermy. Supplementation of 150 nm melatonin produced a significantly greater (P < 0.05)percent of embryos with MPN compared to those supplemented with 75 nm or 100 nm. Supplementation of 150 nm melatonin produced significantly less (P < 0.05)embryos cleaved by 48 h after IVF while 75 nm melatonin supplementation had a significantly higher(P < 0.05) percentage of blastocyst formation by 144 h after IVF. Based on the optimal concentration of melatonin observed in experiment 1, experiment 2 determined the effects of supplementing 75 nm melatonin to the maturation media and 5.0 μg/ml tannic acid supplementation during IVF on oxidative stress, fertilization kinetics, and embryonic development. Oocytes (n = 720) were supplemented at 22 h of maturation with or without 75 nm melatonin and then fertilized with frozen-thawed sperm supplemented with or without 5 μg/ml tannicacid.(AU)

Melatonin and tannic acid supplementation in vitro improve fertilization and embryonic development in pigs

The objective of this study was to determine the effects of melatonin supplementation during maturation and tannic acid supplementation during IVF on fertilization kinetic sand early embryonic development. Experiment 1 determined the optimum concentration of melatonin supplemented to the oocytes for subsequent embryonic development. Oocytes (n = 400)were supplemented at 22 h of maturation with 0, 75, 100, or 150 nm melatonin and then subjected to IVF and embryo culture. After IVF, a portion of the embryos were evaluated for penetration, polyspermy, and male pronuclear (MPN) formation rates. Embryos were evaluated 48 h after IVF for cleavage and 144 h for blastocyst formation. There were no significant differences between treatment groups with respect to penetration and polyspermy. Supplementation of 150 nm melatonin produced a significantly greater (P < 0.05)percent of embryos with MPN compared to those supplemented with 75 nm or 100 nm. Supplementation of 150 nm melatonin produced significantly less (P < 0.05)embryos cleaved by 48 h after IVF while 75 nm melatonin supplementation had a significantly higher(P < 0.05) percentage of blastocyst formation by 144 h after IVF. Based on the optimal concentration of melatonin observed in experiment 1, experiment 2 determined the effects of supplementing 75 nm melatonin to the maturation media and 5.0 μg/ml tannic acid supplementation during IVF on oxidative stress, fertilization kinetics, and embryonic development. Oocytes (n = 720) were supplemented at 22 h of maturation with or without 75 nm melatonin and then fertilized with frozen-thawed sperm supplemented with or without 5 μg/ml tannicacid.

From reproductive technologies to genome editing in small ruminants: an embryo’s journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.

From reproductive technologies to genome editing in small ruminants: an embryos journey

The beginning of this century has witnessed great advances in the understanding of ovarian physiology and embryo development, in the improvement of assisted reproductive technologies (ARTs), and in the arrival of the revolutionary genome editing technology through zygote manipulation. Particularly in sheep and goats, the current knowledge on follicular dynamics enables the design of novel strategies for ovarian control, enhancing artificial insemination and embryo production programs applied to genetic improvement. In vitro embryo production (IVEP) has evolved due to a better understanding of the processes that occur during oocyte maturation, fertilization and early embryo development. Moreover, interesting advances have been achieved in embryo and oocyte cryopreservation, thereby reducing the gap between the bench and on-farm application of IVEP technology. Nevertheless, the major breakthrough of this century has been the arrival of the CRISPR/Cas system for genome editing. By joining diverse disciplines such as molecular biology, genetic engineering and reproductive technologies, CRISPR allows the generation of knock-out and knock-in animals in a novel way never achieved before. The innumerable applications of this disruptive biotechnology are challenging the imagination of those who intend to build the animals of the future.(AU)

Estado da arte e perspectivas da produção in vitro de embriões em caprinos / State of art and prospects of in vitro embryo production in goats

A produção in vitro de embriões (PIVE) é uma biotecnologia reprodutiva com diferentes etapas:colheita e maturação in vitro (MIV) de oócitos, fecundação in vitro (FIV) ou coincubação de espermatozoidescapacitados in vitro com oócitos maturados e cultivo in vitro (CIV) de zigotos até ao estádio de blastocisto. APIVE, em conjunto com a criopreservação de embriões, pode permitir a comercialização de embriões em largaescala, o transporte de embriões livres de patógenos e transações comerciais de germoplasma mais fáceis ebaratas. No entanto, ainda são poucas as pesquisas sobre a PIVE em caprinos em comparação com outrasespécies de produção. O objetivo desta revisão é fornecer uma visão geral do estado da arte da PIVE em caprinoscom ênfase na descrição das principais metodologias atualmente utilizadas para colheita de oócitos, MIV, FIV eCIV de embriões e apresentar as perspectivas da pesquisa nesta biotécnica.(AU)

In vitro embryo production (IVEP) is a reproductive biotechnique with a multi-step methodologycomprising: in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) or co-incubation of capacitatedspermatozoa with in vitro matured oocytes and in vitro culture (IVC) of zygotes up to the blastocyst stage. TheIVEP together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-freegenetic movement and easier and cheaper germplasm commercial transactions. There is less IVEP research ingoats compared to other farm animals. The aim of this review is to provide an overview of the state of art ofIVEP in goats with an emphasis on description of the main methodologies currently used for oocyte recovery,IVM, IVF and IVC of embryos and the future research perspectives of this biotechnique.(AU)

Estado da arte e perspectivas da produção in vitro de embriões em caprinos / State of art and prospects of in vitro embryo production in goats

A produção in vitro de embriões (PIVE) é uma biotecnologia reprodutiva com diferentes etapas:colheita e maturação in vitro (MIV) de oócitos, fecundação in vitro (FIV) ou coincubação de espermatozoidescapacitados in vitro com oócitos maturados e cultivo in vitro (CIV) de zigotos até ao estádio de blastocisto. APIVE, em conjunto com a criopreservação de embriões, pode permitir a comercialização de embriões em largaescala, o transporte de embriões livres de patógenos e transações comerciais de germoplasma mais fáceis ebaratas. No entanto, ainda são poucas as pesquisas sobre a PIVE em caprinos em comparação com outrasespécies de produção. O objetivo desta revisão é fornecer uma visão geral do estado da arte da PIVE em caprinoscom ênfase na descrição das principais metodologias atualmente utilizadas para colheita de oócitos, MIV, FIV eCIV de embriões e apresentar as perspectivas da pesquisa nesta biotécnica.

In vitro embryo production (IVEP) is a reproductive biotechnique with a multi-step methodologycomprising: in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) or co-incubation of capacitatedspermatozoa with in vitro matured oocytes and in vitro culture (IVC) of zygotes up to the blastocyst stage. TheIVEP together with embryo cryoconservation would allow large-scale embryo marketing, a pathogen-freegenetic movement and easier and cheaper germplasm commercial transactions. There is less IVEP research ingoats compared to other farm animals. The aim of this review is to provide an overview of the state of art ofIVEP in goats with an emphasis on description of the main methodologies currently used for oocyte recovery,IVM, IVF and IVC of embryos and the future research perspectives of this biotechnique.

Effects of chlorogenic acid on the maturation and fertilisation of bovine oocytes and their embryonic development with a comparative bovine granulosa cell co-culture

Chlorogenic acid (CGA) plays several biological roles, but lacks studies that demonstrate how this phenolic compound affects animal reproduction. The aim of the present study was to evaluate the effects of different CGA concentrations on bovine oocyte maturation and embryo development in vitro. This study also evaluates co-culture systems involving bovine granulosa cells (BGC) from fed with CGA containing plant, Pittosporum Undulatum. The ovaries were recovered after slaughter and the oocytes were removed, maturated, in vitro fertilized and cultured in medium containing CGA in 5 different concentrations 1.25; 2.5; 5; 10; 20 µm and a control group (0 µm) for seven days. Selected oocytes (n = 1040) were maturated in any of the 5 treatment or control groups. Significantly lower (P 0.05) were identified between control and experimental groups in relation to the progesterone production by BGC. These results suggest that CGA may affect oocyte maturation and inhibit the progression of meiosis and consequently the entire embryo development in vitro.

Intracytoplasmic sperm injection after vitrifcation of immature oocytes in follicular fluid increases bovine embryo production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes. Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitroembryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).[...]

Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set

Effects of chlorogenic acid on the maturation and fertilisation of bovine oocytes and their embryonic development with a comparative bovine granulosa cell co-culture

Chlorogenic acid (CGA) plays several biological roles, but lacks studies that demonstrate how this phenolic compound affects animal reproduction. The aim of the present study was to evaluate the effects of different CGA concentrations on bovine oocyte maturation and embryo development in vitro. This study also evaluates co-culture systems involving bovine granulosa cells (BGC) from fed with CGA containing plant, Pittosporum Undulatum. The ovaries were recovered after slaughter and the oocytes were removed, maturated, in vitro fertilized and cultured in medium containing CGA in 5 different concentrations 1.25; 2.5; 5; 10; 20 µm and a control group (0 µm) for seven days. Selected oocytes (n = 1040) were maturated in any of the 5 treatment or control groups. Significantly lower (P < 0.05) maturation rates were observed for the highest CGA concentrations 10 µm, and 20 µm, compared to the control group (Control = 93.4 ± 2.1% vs. 10 µm = 80.9 ± 2.2%; 20 µm 77.9 ± 3.3%). We observed that the higher the concentration of CGA present, the lower the rate of cleavage and development after 3 and 7 days, respectively. It was observed that the significant difference recorded in regards to embryonic development were evident between control and group (20; 51.1 ±5.6 vs. 19.4 ± 2.2%). In respects to the study involving co-culture of embryos with BGC the only difference recorded involved the block rate. No differences (P > 0.05) were identified between control and experimental groups in relation to the progesterone production by BGC. These results suggest that CGA may affect oocyte maturation and inhibit the progression of meiosis and consequently the entire embryo development in vitro.(AU)