Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture.
Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day=D0), zygotes were cultured on granulosa cells+M199+10% serum+100microM GSH supplemented with 100microM of t10, c12 CLA (CLA group, n=1394) or without supplementation (control group, n=1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n=24; group B-CLA, n=23). Non-biopsied embryos were also frozen (group NB-Control, n=49; group NB-CLA, n=45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P<or=0.003) and a smaller (P<0.001) fat embryo index being leaner (P=0.008) than control embryos. Embryo postwarming survival was higher in B-CLA than in B-control group (95.0+/-7.0% versus 62.5+/-7.9%; P<0.001). After 24h of culture, the viability (expansion rate) of biopsied embryos and nonbiopsied embryos, cultured with t10, c12 CLA was higher than control embryos (B-CLA=64.6+/-4.4% and B-control=27.5+/-2.5%, P=0.01; NB-CLA=86.0+/-3.5% and NB-Control=68.6+/-7.0%, P=0.05). Results showed that supplying t10, c12 CLA to serum-containing media decreases embryo cytoplasmic lipid deposition during in vitro culture and significantly improves resistance of IVP embryos to micromanipulation and cryopreservation.
Pereira
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RM
Estação Zootécnica Nacional - INIAP, 2005-048 Vale de Santarém, Portugal. rosalnp@gmail.com
Carvalhais
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Pimenta
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Baptista
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MC
Vasques
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Horta
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Santos
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Marques
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MR
Reis
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Pereira
M Silva
MS
Marques
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D000818:Animals; D001706:Biopsy; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media / Q000494:pharmacology; D046149:Embryo Culture Techniques; D004624:Embryo Transfer / Q000379:methods; D004622:Embryo, Mammalian / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*; D005260:Female; D044243:Linoleic Acids, Conjugated / Q000494:pharmacology*; D008055:Lipids / Q000032:analysis; D008297:Male; D019849:Sex Determination Processes
D003470:Culture Media; D044243:Linoleic Acids, Conjugated; D008055:Lipids; C496197:trans-10,cis-12-conjugated linoleic acid