Freezing of in vitro produced bovine embryos in animal protein-free medium containing vegetal peptones.
ABSTRACT Successful cryopreservation is essential for a large-scale dispersal of bovine in vitro produced (IVP) embryos that have been shown to be more sensitive to cryopreservation than their in vivo counterparts. On the other hand, the use of animal proteins in freezing media increases sanitary risks. We first replaced animal proteins, such as bovine serum albumin (BSA) in the freezing medium by plant-derived peptides (vegetal peptones). A batch of wheat peptones was selected after a preliminary experiment showing the absence of toxicity of concentrations<18 mg/mL on in vitro bovine blastocysts. Increasing concentrations of peptones were then added in the freezing medium. The surviving and hatching rates were not affected by comparison with those observed with BSA. No significant difference was observed between groups either for the total number of cells or for the ratio ICM/Total cell, nor for the rate of apoptosis in surviving embryos. When embryos were cryopreserved in 1.8 mg/mL peptone, the hatching rate and embryo quality as assessed at 48 h post-thawing were not significantly different from those of unfrozen embryos. In a second experiment two additives were added in this animal protein-free freezing medium containing 1.8 mg/mL peptones. No beneficial effect of adding 1 mg/mL sodium hyaluronate or 100 microM beta-mercaptoethanol was observed on embryo survival or quality. In conclusion, we have demonstrated that vegetal peptones can replace BSA in freezing media without affecting blastocyst survival and quality.
- SourceAvailable from: Kaladhar SVGK Dowluru[Show abstract] [Hide abstract]
ABSTRACT: Sheep play a vital role within the Indian economy and therefore the production from native breeds is comparatively low because of their poor generative potency. A great challenge of analysis is concerned to beat the constraints within the technology like costly and sophisticated nature of the technology and low success rate. Fetal bovine serum can be supplemented in IVM and IVF media to achieve better maturation and cleavage. Wheat peptone may not produce desired maturation and cleavage when compared to FBS and BSA, and may not be a useful supplement in IVM and IVF. BSA supplementation also yields better maturation and embryo development when compared to wheat peptone but would involve high cost in embryo production. Supplementation of wheat peptones in IVC of embryos yielded better embryo production suggesting that wheat peptones can be an alternative to animal protein in IVC and would help to minimize the cost in embryo production. The studies have shown that CR1aa medium with α-tocopherol at 200 μM is a better media for in vitro embryo production in sheep.02/2013;
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ABSTRACT: In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.Theriogenology 10/2013; · 1.85 Impact Factor
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ABSTRACT: In vitro embryo-production procedures developed for sheep have tremendously been improved from decades, but many factors influencing their efficiency still need to be investigated. The overall of this study showed the production of the sheep embryos till blastocyst stage from the ovaries of the slaughtered ewes for IVM, IVF of the oocytes and then IVC in the complex culture media like TCM-199, TCM-199-, TCM-199+ and CR1aa. The morula yield was significantly higher in the wheat peptone and BSA supplemented group. The nuclear maturation rates of ovine oocytes matured in FBS (69.15 ± 1.07) group was more compared to BSA (56.47 ± 0.73) and wheat peptone (35.26 ± 0.79). The nuclear maturation of oocytes was significantly higher in the FBS group when compared to BSA and wheat peptone supplemented groups. The lowest maturation rate was observed in wheat peptone supplemented group. The development rates in 2, 4, 8, 16 and morula stages of ovine embryos produced from BSA group were high (74.22 ± 2.56, 60.93 ± 2.55, 42.97 ± 2.20 and 24.21 ± 1.28) compared to FBS group (73.58 ± 2.92, 58.77 ± 2.01, 41.22 ± 1.68 and 20.17 ± 0.76) and wheat peptone (72.89 ± 1.09, 57.01 ± 0.76, 42.05 ± 0.36 and 24.29 ± 0.55). The cleavage, morula and blastocyst percent in CR1aa was found significantly high compared with TCM- 199, TCM-199- and TCM-199+. The cleavage, morula and blastocyst percent in L-Ascorbic acid at 100 μM with CR1aa is found more significant compared with other compositions used in the experimentation.tissue science and engineering. 01/2014;