Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue

Development and optimization of a fluorescent differential display PCR system for studying bovine embryo development in vitro

Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.