Location: Reproduction Research
Title: In Vitro Elongation of Porcine Embryos Using Alginate Hydrogels as a Three-Dimensional Extracellular Matrix Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 14, 2010
Publication Date: N/A
Technical Abstract: In the pig, the pre-implantation period of pregnancy is highly influential on sow productivity and therefore the profitability of swine production. Between Day 11 and 12 of gestation, the embryo undergoes a significant morphological change, during which it transforms from an ovoid structure of about 1 cm in length to a long, thin filament that can grow to greater than 10 cm in length. This transformation process is known as elongation and is critical for maternal recognition of pregnancy. It has been found that about 20% of embryonic loss is associated with the elongation process that occurs before implantation. An effective in vitro culture system could help us develop a clear understanding of the pre-implantation period of porcine embryos, in particular elongation, which in turn can allow us to identify physiological components that could be manipulated to improve pregnancy outcomes. So far, attempts to elongate porcine embryos in vitro have been unsuccessful. We hypothesize that this failure to elongate is, at least in part, caused by an inadequate culture system which lacks three dimensional structure. Therefore, our objective is to use tissue engineering principles (i.e. scaffolds) to provide a 3D matrix in which to culture the embryos, in an attempt to establish an effective culture system that can support pig embryo elongation in vitro. In particular, we have been using alginate hydrogels as a three-dimensional scaffold. Alginate, a polysaccharide derived from brown algae that gels in the presence of a divalent cation, is an inert material that allows for gentle encapsulation of cells and tissue without any specific interaction between the cells/tissue and the surrounding hydrogel. We have investigated several different hydrogel formation and encapsulation techniques with live porcine embryos ranging from 100 µm to 1 cm in diameter. These methods have included a bead method using alginate and calcium chloride solution, a slurry method using alginate and calcium sulfate solution, and a method of boring into pre-made alginate hydrogels. For these studies, normally cycling White crossbred gilts (greater than 200 days of age) were checked for estrus daily. Following first detection of estrus (designated as Day 0), gilts were artificially inseminated with White crossbred boar semen throughout the estrus period in 24 h intervals. Gilts were then slaughtered at Days 9-10 of gestation. Following slaughter, reproductive tracts were removed immediately and each uterine horn was flushed with warmed RPMI-1640 containing antibiotics. Embryos were recovered and classified according to size and morphology and washed twice in RPMI-1640 containing antibiotics and 10% fetal bovine serum. Embryos were randomly assigned to an alginate gel of varying mechanical properties, or used as a control (i.e. not encapsulated in any gel). After encapsulation of the porcine embryos in the alginate hydrogels, using all three different hydrogel formation techniques, morphological analysis were performed daily throughout the culture period (96 h). Using live/dead staining at termination of culture, we have been able to show that embryos remain viable while encapsulated in the hydrogel throughout the experiment and a proportion of embryos that remain encapsulated within the gel demonstrate evidence of elongation, with most efficient encapsulation achieved using a bead technique with 0.7% alginate gels. Future experiments will be designed to increase encapsulation efficiency, as well as explore the addition of bioactive factors. The establishment of this culture system will allow for future studies on the specific mechanisms that regulate embryo elongation and implantation in vitro, and in the future help to determine mechanisms to improve embryo elongation and implantation, thereby increasing sow productivity and profitability of swine production.