In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production

A mouse model for improving cell survival of bisected cattle embryos

Morula and blastocyst stage embryos recovered from B6D2F_1 mice were bisected with a metal microblade in M2 medium with or without sucrose and/or cytochalasin B supplementation. Cell lysis was determined by staining the embryos with Hoechst 33258 and propidium iodide. Lysed cells take up both stains but non-lysed cells only the Hoechst 33258 stain, resulting in pink fluorescence for lysed cells and blue fluorescence for non-lysed cells under UV excitation. During bisection of morulae, the presence of cytochalasin B decreased the proportion of lysed cells in both the absence (P=0.0001) and presence of sucrose (P=0.001). During bisection of blastocysts the average proportion of lysed cells was slightly lower in the presence of cytochalasin than that in the control medium, but the effect was not statistically significant (P=0.34). No effect of sucrose was observed in either demi-morulae or demi-blastocysts. These results are essentially similar to those obtained in simultaneous experiments with cattle embryos, suggesting that the simpler mouse model might be useful for developing less traumatic bisection protocols for cattle embryos

A simple culture system for time-lapse video recording of bovine embryos

Continuous observation of embryonic growth can improve understanding of the early developmental events and allow us to use parametric statistical analyses with time as a parameter. A cinematographic study such as that reported here utilizes time-lapse video recording. Previously published methods for time-lapse video recording have involved building an incubator around a microscope, a process that is both expensive and laborious. Here we present a simplified method for time-lapse video recording of early bovine embryo development. The embryos were cultured during a 24-hour period in a standard pregassed tissue culture bottle, which was darkened and placed on the heating stage of an inverted microscope for recording through a red filter. The control embryos were cultured in a conventional CO2 incubator. After 10 replicates we could not find a statistically significant difference between the cell numbers of these two treatments (P=0.95), suggesting that the culture setup is appropriate for continuous observation of early cleavage of the cattle embryo

Sex diagnosis of ovine and bovine embryos by enzymatic amplification and digestion of DNA from the ZFY/ZFX locus

A PCR-based sex determination assay for sheep and cattle embryos was developed using mouse embryos for optimizing the protocol. Samples were lysed either enzymatically or by alkaline treatment followed by enzymatic amplification of DNA from the ZFY/ZFX locus. Sex diagnosis could be done after the digestion of the amplified product by restriction endonucleases. Ovine and bovine embryos could be sexed from biopsies as small as 1-4 cells. Some embryos were split into 2-4 sections, which were amplified separately. Blind trials with such samples demonstrated that the method was highly accurate, even when embryo biopsy was done under farm conditions. The protocol involves an in-built control. This eliminates the need for autosomal control primers, which often inhibit the amplification of the Y-chromosome-specific DNA, especially when a small amount of template is used

Glucose controls sex-related growth rate differences of bovine embryos produced in vitro

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In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

Selostus: Insuliinin vaikutus naudan blastokystien tuottamiseen in vitro kemiallisesti tunnetussa liuoksess

Effect of polyvinylpyrrolidone, serum albumin and fetal calf serum on early cleavage of bovine embryos

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Insuliinin vaikutus naudan blastokystien tuottamiseen in vitro kemiallisesti tunnetussa liuoksessa

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.Naudan alkion varhaiskehitystä tutkittiin viljelemällä alkioita kemiallisesti tunnetussa viljelyliuoksessa. Kokeessa käytetty liuos CRI-PVP on muunnos CRlaa-liuoksesta, jonka proteiinilähde, naudan seerumin albumiini, korvattiin 4mg/ml polyvinyylipyrrolidonilla. Kokeessa käytetyt kolme insuliinipitoisuutta olivat 0, 2 ja 200 nM. In vitro-tuotetut alkiot jaettiin hedelmöityksen jälkeen kolmeen viljelyryhmään: CRI-PVP, CRI- PVP + insuliini (2 nM) ja CRI-PVP + insuliini (200 nM). Alkioita viljeltiin seitsemän päivää, ja neljäntenä päivänä lisättiin tuoretta viljelyliuosta ja glukoosia (lopullinen pitoisuus 5,56 mM). Viljelyn päättyessä alkiot arvioitiin kehitysvaiheen mukaan ja alkioiden solut laskettiin tumavärjäyksen jälkeen. Proteiinittomassa viljelyliuoksessa saatiin blastokysteiksi kehittymään 8,7 %. Insuliinin lisäyksellä saatiin enemmän blastokystejä (12,8 %), mutta ei tilastollisesti merkitsevästi. Jakautumisprosentti, blastokystien osuus jakautuneista alkioista sekä morula/ blastokystivaiheen alkioiden tumalukumäärä (P=0,10) olivat korkeimmat ryhmässä, jossa insuliinipitoisuus oli 200 nM

Sex-related cleavage rate difference in bovine embryos produced in vitro is controlled by glucose

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