RESUMO
This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).
Assuntos
Ácido Tióctico , Técnicas de Cultura de Tecidos , Animais , Feminino , Ácido Tióctico/farmacologia , Ovinos , Técnicas de Cultura de Tecidos/veterinária , Ovário/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Antioxidantes/farmacologia , Vitrificação , Criopreservação/veterináriaRESUMO
Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.
Assuntos
Antioxidantes , Técnicas de Maturação in Vitro de Oócitos , Bovinos , Feminino , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oogênese , Oócitos , Fertilização in vitro/veterinária , BlastocistoRESUMO
This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.
Assuntos
Eugenol , Técnicas de Maturação in Vitro de Oócitos , Animais , Antioxidantes/farmacologia , Blastocisto , Calreticulina , Bovinos , Contagem de Células/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.
Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas.
Assuntos
Animais , Bovinos , Blastocisto/fisiologia , Desenvolvimento Embrionário , Embrião de Mamíferos/ultraestrutura , Clonagem de Organismos/veterinária , Embrião de Mamíferos/anatomia & histologia , Partenogênese , Técnicas In Vitro/veterináriaRESUMO
In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles(AU)
Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas(AU)
Assuntos
Animais , Bovinos , Desenvolvimento Embrionário , Blastocisto/fisiologia , Embrião de Mamíferos/ultraestrutura , Técnicas In Vitro/veterinária , Embrião de Mamíferos/anatomia & histologia , Clonagem de Organismos/veterinária , PartenogêneseRESUMO
This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk).
Assuntos
Animais , Bovinos , Criação de Animais Domésticos , Genômica/classificação , Melhoramento Genético , Transferência Embrionária/veterináriaRESUMO
This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk). (AU)
Assuntos
Animais , Bovinos , Melhoramento Genético , Criação de Animais Domésticos , Genômica/classificação , Transferência Embrionária/veterináriaRESUMO
The aim of the present study was to evaluate the effect of bovine somatotropin (bST; 500mg) administration on lactating buffalo donors submitted to two different ovum pick-up (OPU) and in vitro embryo production schemes with a 7 or 14d intersession OPU interval. A total of 16 lactating buffalo cows were randomly assigned into one of four experimental groups according to the bST treatment (bST or No-bST) and the OPU intersession interval (7 or 14d) in a 2×2 factorial design (16 weeks of OPU sessions). The females submitted to OPU every 14d had a larger (P<0.001) number of ovarian follicles suitable for puncture (15.6±0.7 vs. 12.8±0.4) and an increased (P=0.004) number of cumulus-oocyte complexes (COCs) recovered (10.0±0.5 vs. 8.5±0.3) compared to the 7d interval group. However, a 7 or 14d interval between OPU sessions had no effect (P=0.34) on the number of blastocysts produced per OPU (1.0±0.1 vs. 1.3±0.2, respectively). In addition, bST treatment increased (P<0.001) the number of ovarian follicles suitable for puncture (15.3±0.5 vs. 12.1±0.4) but reduced the percentage (18.9% vs. 10.9%; P=0.009) and the number (1.4±0.2 vs. 0.8±0.1; P=0.003) of blastocysts produced per OPU session compared with the non-bST-treated buffaloes. In conclusion, the 14d interval between OPU sessions and bST treatment efficiently increased the number of ovarian follicles suitable for puncture. However, the OPU session interval had no effect on embryo production, and bST treatment reduced the in vitro blastocyst outcomes in lactating buffalo donors.
Assuntos
Búfalos/fisiologia , Fertilização in vitro/veterinária , Hormônio do Crescimento/farmacologia , Recuperação de Oócitos/veterinária , Folículo Ovariano/fisiologia , Óvulo/fisiologia , Animais , Bovinos , Células do Cúmulo , Técnicas de Cultura Embrionária/veterinária , Feminino , Lactação , Recuperação de Oócitos/métodos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacosRESUMO
The number of follicles recruited in each estrous cycle has gained practical importance in artificial reproductive technology, as it determines the oocyte yield from ultrasound-guided ovum pickup for in vitro embryo production. We aimed to identify single nucleotide polymorphisms (SNPs) in bovine genes related to reproductive physiology and evaluate the association between the candidate SNPs and the number of oocytes collected from ultrasound-guided ovum pickup. We sequenced genomic segments of GDF9, FGF8, FGF10 and BMPR2 and identified seventeen SNPs in the Bos taurus and Bos indicus breeds. Two SNPs cause amino acid changes in the proteins GDF9 and FGF8. Three SNPs in GDF9, FGF8 and BMPR2 were genotyped in 217 Nelore cows (B. indicus), while two previously identified mutations in LHCGR and mitochondrial DNA (mtDNA) were genotyped in the same group. The polymorphisms in GDF9, FGF8, BMRP2 and LHCGR were significantly associated (P<0.01) with the number of oocytes collected by ovum pickup, whereas the SNP in the mtDNA was not. In addition, we estimated an allelic substitution effect of 1.13±0.01 (P<0.01) oocytes for the SNP in the FGF8 gene. The results we report herein provide further evidence to support the hypothesis that genetic variability is an important component of the number of antral follicles in the bovine ovary.