Stimulation of protein synthesis and expansion of pig blastocysts by insulin in vitro

Present evidence indicates that insulin may act as a growth factor during preimplantation development. This hypothesis has been tested on pig blastocysts by determining the effect of insulin on protein synthesis and blastocyst expansion over 24 h. Blastocysts were collected from superovulated gilts or sows on Day 5 or 6 and incubated overnight in a modified BMOC2 medium. Those that were cultured with 1. 7 nM insulin had 14% larger radii, and were 36% more active in their incorporation of [H]leucine (protein synthesis) than those that had been cultured in non-supplemented medium. There was a significant linear correlation between the rate of protein synthesis and the radius of blastocysts when all blastocysts and only those cultured with insulin were examined, but the correlation for the blastocysts in non-supplemented medium was just outside statistical significance. The regression coefficient for the insulin-treated blastocysts was 132% of that for blastocysts cultured in unsupplemented medium; this suggests that insulin increased the size of blastocysts and the rate of protein synthesis per unit size. The results indicate that pig blastocysts respond to physiological levels of insulin in similar fashion to those of mice and cattle, supporting the hypothesis that insulin may act as a general embryonic growth factor. Because of the cross reaction between the insulin receptor and the ligands, insulin and insulin­like growth factor 1 (IGF-1), the results also suggest that IGF-1, reported to be present in pig uterine fluid, could be involved in this stimulation in utero

An updated method for the jugular catheterization of grower pigs for repeated blood sampling following an oral glucose tolerance test

Jugular catheterization is a common procedure used under experimental conditions. However, there is considerable variation in the reported techniques, particularly for grower pigs (>40 kg and 10 mL) are required. This paper provides a complete methodology including the use of current equipment and anaesthetic regimen for grower pigs. This surgical jugular catheterization method was carried out in 30 large white grower pigs. Firstly, the pigs were habituated to human handling for at least two weeks prior to surgery. Animals were sedated and anesthetized. Following intubation, an incision was made in the jugular fossa, and the jugular vein was located. A catheter was then inserted and fixated. The wound was stapled and the catheter line secured to the back of the neck. The pigs recovered fully from the surgery and the catheters remained patent for the duration of the blood sampling period (min 72 h). Twenty millilitres of blood were collected every 15 min, taking approximately 2 min per pig. No haemolysis was detected in any samples. Jugular catheterization of pigs using this procedure proved successful both in terms of animal recovery and quality of samples. Catheters remained patent and pigs remained calm during sampling

Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification

The present study examined whether delipated porcine oocytes and embryos at various stages of development can be cryopreserved by conventional slow cooling or vitrification. Most (93%) of the 27 delipated morulae developed to blastocysts after freezing with 1.5 M propanediol + 0.1 M sucrose. Late morulae and early blastocysts delipated at 2-4 cell stage and cultured in vitro survived freezing either with 1.5 M glycerol + 0.25 M sucrose (10/18, 56%) or 1.8 M ethylene glycol + 0.25 M sucrose (14/19, 74%). Delipated 2-4 cell stage embryos and oocytes could be cryopreserved by vitrification with 40% ethylene glycol, 1 M sucrose and 20% fetal calf serum. Half (7/14) of the vitrified, delipated embryos developed to blastocysts after thawing. Of 48 delipated oocytes, 27 (56%) maintained an intact outline of the ooplasm after vitrification and underwent subzonal sperm injection. Fertilization was confirmed in 12 (25%) of these oocytes and 3 (6%) developed to morula stage. This study also aimed at developing a non-invasive method for cryopreserving porcine embryos after reducing their cytoplasmic lipid content without micromanipulation. Morulae and early blastocysts were centrifuged in the presence of cytochalasin B and cryoprotectants and then frozen immediately. More than half (14/24, 58%) of the centrifuged morulae developed to blastocycts after freezing with 1.5 M propanediol + 0.1 M sucrose. Greater than 70% of centrifuged early blastocysts survived freezing either with 1.5 M propanediol (30/31, 97%), 1.5 M glycerol (22/29, 76%) or 1.8 M ethylene glycol (21/29, 72%). These results demonstrated that delipation (lipid removal) from porcine oocytes and embryos at various stages enables their cryopreservation. A new insight into the development of a non-invasive method for cryopreserving porcine embryos was also provided