Preimplantation development of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture under low oxygen atmosphere.
01 Apr 1994-Biology of Reproduction (Society for the Study of Reproduction)-Vol. 50, Iss: 4, pp 715-724
TL;DR: Two distinct culture environments are characterized, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro, and survival of the embryo under a reduced oxygen atmosphere is investigated.
Abstract: The roles of medium composition, serum source, embryo coculture, and culture under low O2 conditions on the development of in vitro-matured and in vitro-fertilized (IVMF) ovine zygotes were investigated in three separate experiments. In the first experiment, the proportion of cocultured IVMF zygotes developing to the blastocyst stage was significantly higher (38.0% vs. 3.5%; p < 0.05) than that of non-cocultured zygotes treated within three embryo culture media (TCM-199 + 10% fetal bovine serum [FBS]; bicarbonate-buffered, glucose-free synthetic oviduct fluid medium [mod-SOFM] + 10% FBS; and bicarbonate-buffered BSA-free Tyrode's salt solution [mod-TALP] + 10% FBS) under a 5% CO2 atmosphere in air. In a second experiment, a significantly higher (p < 0.05) proportion of cocultured zygotes placed in TCM-199 medium survived to the blastocyst stage (37.4% blastocysts vs. 23.4% in mod-SOFM). No significant effect of serum (FBS vs. human serum [HS]) was observed on embryonic development, but coculture was confirmed to exert a significant influence on development to the blastocyst stage. In the final experiment, survival of the embryo under a reduced oxygen (5% CO2:5% O2:90% N2) atmosphere was investigated. In contrast to results in the initial experiments, embryonic survival was significantly higher (p < 0.05) in the non-cocultured treatment groups (21.9% blastocysts vs. 0.4% for cocultured zygotes). Serum source also had a significant (p < 0.05) influence upon the development of non-cocultured zygotes: 32.3% of zygotes cultured with HS progressed to the blastocyst stage vs. 11.5% of zygotes cultured in FBS-supplemented medium. These results have characterized two distinct culture environments, each capable of supporting the development of high frequencies of unselected IVMF zygotes to the blastocyst stage in vitro.
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TL;DR: When treatment with P4 commenced only 2 d before oocyte collection, rates of IVF were reduced in both experiments, and progestin treatment protocols used in ovine IVF programs should be carefully designed to minimize adverse effects on fertilization rates.
20 citations
Cites background from "Preimplantation development of in v..."
...During seasonal anestrus, the rates of IVF (about 10–60%) are much lower than during the normal reproductive season (about 70–80%) [3,16,37,38]....
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Journal Article•
TL;DR: In-vitro development of mammalian embryos usually remains inferior to development than invivo produced embryos and it is needed to standardize the culture conditions that mimic in-vivo embryo development.
Abstract: Pre-implanted growth and development of embryo is deficient in many aspects as is evidenced by the great difficulty in growing embryos in-vitro, while maintaining viability as shown by development of early embryos in-vitro generally delayed or completely blocked at 8‐16 cell stage embryo and hardly small percentage of the in-vitro fertilized embryo develop to morula and blastocyst, which is probably due to minor physio- chemical variation and lack of certain factors in the culture system. In-vitro development of mammalian embryos usually remains inferior to development than invivo produced embryos. The mechanism regulating the in-vitro acquisition of developmental competence of oocytes is still obscure. Therefore it is needed to standardize the culture conditions that mimic in-vivo embryo development. To know the exact mechanism of developing embryos, there should be a defined system that caters the needs of developing embryos. The current review describes the criteria and factors affecting in-vitro maturation, fertilization and embryo development in goat.
19 citations
TL;DR: The results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 μm EGCG, but the effects vary with individual boars.
Abstract: Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen-thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen-thawed spermatozoa were co-incubated with in vitro-matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen-thawed spermatozoa from six boars were co-incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co-incubation with 50 μm EGCG, but the effects vary with individual boars.
19 citations
Cites background from "Preimplantation development of in v..."
...…proteins, lipids and nucleic acid components, resulting in mitochondrial alterations (Guerin et al. 2001), sperm axonemal protein phosphorylation (Aitken et al. 1993), 2-cell block of embryos (Nasr-Esfahani and Johnson 1992) and reduced embryo development (Watson et al. 1994; Blondin et al. 1997)....
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TL;DR: This study demonstrates that P4 applied on anEstradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.
Abstract: Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17β (E 2 ), progesterone (P 4 ), E 2 +P 4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P 4 alone or in combination with E 2 significantly reduced steady-state RBP mRNA levels compared to those in E 2 -treated animals. Oviductal explants from E 2 - and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P 4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E 2 and E2+P4 treatments. This study demonstrates that P 4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.
18 citations
TL;DR: The data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.
15 citations
Cites methods from "Preimplantation development of in v..."
...supplemented with 2% of oestrus ovine serum [21], 50 mg/ml streptomycin and 50 IU/ml penicillin (SOFs)....
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