In vitro development of early porcine embryos under different culture conditions was evaluated and compared to in vivo development. First, one- and two-cell embryos were collected and cultured individually in 20- microl drops under 5% CO2 in air for 4 days. Embryos from one oviduct were cultured in NCSU-23, and those from the contralateral oviduct were cultured in KSOM/AA. The embryos developed in NCSU-23 had a higher mean number of inner cell mass (ICM) nuclei compared to those developed in KSOM/AA (p = 0.025). They also had higher trophectoderm (TE) and total nuclear number (p = 0.001), while there was no difference in the average ratio of ICM to TE nuclei (p = 0.731). When the effect of different gas atmospheres was tested, the numbers of TE and total nuclei were higher (p < 0.01 and p < 0.025, respectively) in embryos cultured in an atmosphere with 5% CO2 in air than in those developed under 5% CO2:5% O2:90% N2. Next the development of embryos cultured in NCSU-23 was compared to that of embryos incubated in vivo. By the end of the 4-day incubation, the cultured embryos had higher nuclear numbers and a higher ratio of ICM to TE nuclei than those developed in vivo (p < 0.001). Finally, the embryos that developed in NCSU-23 or in vivo were transferred into recipients. By Day 40 of pregnancy, 37.1 +/- 15.3% of the in vitro- and 53.8 +/- 15.3% of the in vivo-incubated embryos formed conceptuses. These results indicate that despite the lower nuclear numbers caused by in vitro conditions, the cultured embryos were developmentally competent.

The production of genetically engineered pigs as xenotransplant donors aims to solve the severe shortage of organs for transplantation in humans. The first barrier to successful xenotransplantation is hyperacute rejection (HAR). HAR is a rapid and massive humoral immune response directed against the pig carbohydrate Galalpha 1,3-Gal epitope, which is synthesized by alpha 1,3-galactosyltransferase (alpha1,3-GT). The Galalpha 1,3-Gal antigen also contributes to subsequent acute vascular rejection events. Genetic modifications of donor pigs transgenic for human complement regulatory proteins or different glycosyltransferases to downregulate Galalpha 1,3-Gal expression have been shown to significantly delay xenograft rejection. However, the complete removal of the Galalpha 1,3-Gal antigen is the most attractive option. In this study, the 5' end of the alpha 1,3-GT gene was efficiently targeted with a nonisogenic DNA construct containing predominantly intron sequences and a Kozak translation initiation site to initiate translation of the neomycin resistance reporter gene. We developed two novel polymerase chain reaction screening methods to detect and confirm the targeted G418-resistant clones. This is the first study to use Southern blot analysis to demonstrate the disruption of the alpha 1,3-GT gene in somatic HT-transgenic pig cells before they were used for nuclear transfer. Transgenic male pigs were produced that possess an alpha 1,3-GT knockout allele and express a randomly inserted human alpha 1,2-fucosylosyltransferase (HT) transgene. The generation of homozygous alpha 1,3-GT knockout pigs with the HT-transgenic background is underway and will be unique. This approach intends to combine the alpha 1,3-GT knockout genotype with a ubiquitously expressed fucosyltransferase transgene producing the universally tolerated H antigen. This approach may prove to be more effective than the null phenotype alone in overcoming HAR and delayed xenograft rejection.

Thimerosal (200 microM) triggered Ca2+ oscillations in 56 of 56 mature porcine oocytes. The Ca2+ oscillations were blocked by the sulfhydryl-reducing agent dithiothreitol (DTT), thus supporting the hypothesis that thimerosal acts by oxidizing critical sulfhydryl groups on intracellular Ca2+-release proteins. Thimerosal treatment alone arrested the oocytes in metaphase, probably by oxidizing tubulin sulfhydryl groups and thus destroying the spindle. However, a 10-min exposure to 200 microM thimerosal followed by a 30-min incubation in 8 mM DTT induced complete activation, as 73.8% of the oocytes formed pronuclei. The second polar body was visible in 73.3% (55 of 75) of the activated oocytes. Combined thimerosal/DTT treatment of the oocytes also induced cortical granule exocytosis, as revealed by confocal microscopy, and the subsequent hardening of the zona pellucida. After activation, some oocytes were incubated in vitro, or in vivo in a ligated porcine oviduct, for 6 days. When cultured in vitro, 42.0% (37 of 88) of the oocytes developed to the compact morula or blastocyst stage; the average number of inner cell mass (ICM) and trophectoderm (TE) nuclei in the blastocysts was 8.6 +/- 0.7 and 20.1 +/- 1.3, respectively. Culture in a ligated oviduct resulted in 42.9% development to the compact morula or blastocyst stage, with the blastocysts having a mean number of 12.5 +/- 1.0 ICM and 63.6 +/- 9.2 TE nuclei.

The presence of different intracellular Ca2+ release mechanisms in porcine oocytes and their involvement in mediating Ca2+ transients in different developmental stages were investigated. Metaphase II arrested oocytes showed an increase in intracellular Ca2+ concentration after injection of inositol 1,4,5-trisphosphate (InsP3), the InsP3 receptor agonist. Similar Ca2+ spikes could be detected after injection of ryanodine and cyclic ADP ribose, the ryanodine receptor agonists. The InsP3-induced Ca2+ release was inhibited by heparin, the InsP3 receptor antagonist, whereas procaine, the ryanodine receptor antagonist, blocked the Ca2+ transients generated by ryanodine and cyclic ADP ribose. In germinal vesicle-stage oocytes, intracellularly stored Ca2+ could also be mobilized by agonist treatment, though the effective concentration to generate the Ca2+ spikes was higher. After in vitro fertilization, repetitive Ca2+ transients were generated in oocytes starting 2.5-3 h after insemination. They ceased around the time of pronuclear formation when the oocytes entered first interphase. At this stage, the receptors were still capable of mediating Ca2+ release upon agonist treatment; in many cases these spikes were of longer duration, suggesting that in interphase it takes a longer time for the Ca2+ stores to resequester the mobilized Ca2+ from the cytosol. These results suggest that porcine oocytes possess both InsP3 and ryanodine Ca2+ channel receptors and that the properties of the Ca2+ release mechanisms change during oocyte development.

During the past 6 years, considerable advancement has been achieved in experimental embryology of pigs. This process was mainly generated by the rapidly increasing need for transgenic pigs for biomedical research purposes, both for future xenotransplantation to replace damaged human organs or tissues, and for creating authentic animal models for human diseases to study aetiology, pathogenesis and possible therapy. Theoretically, among various possibilities, an established somatic cell nuclear transfer system with genetically engineered donor cells seems to be an efficient and reliable approach to achieve this goal. However, as the result of unfortunate coincidence of known and unknown factors, porcine embryology had been a handicapped branch of reproductive research in domestic animals and a very intensive and focused research was required to eliminate or minimise this handicap. This review summarises recent achievements both in the background technologies (maturation, activation, embryo culture) and the actual performance of the nuclear replacement. Recent simplified methods for in vivo development after embryo transfer are also discussed. Finally, several fields of potential application for human medical purposes are discussed. The authors conclude that although in this early phase of research no direct evidence can be provided about the practical use of transgenic pigs produced by somatic cell nuclear transfer as organ donors or disease models, the future chances even in medium term are good, and at least proportional with the efforts and sums that are invested into this research area worldwide.

Calcium ionophore A23187 can parthenogenetically activate oocytes in many animals. The present study was designed to analyze functionally the mechanism of A23187 activation of pig oocytes matured in vitro. In experiment 1, effects of the concentration of A23187 on intracellular calcium transients, cortical granule (CG) exocytosis, nuclear activation, and zona reaction, which was determined by zona hardening and sperm penetrability, were examined. Cumulus-free oocytes were exposed to 0-100 microM A23187 for 5 min. It was found that the amplitude of the intracellular calcium transients, percentage of CG exocytosis, and percentage of pronuclear formation were increased in a concentration-dependent manner. The time for dissolution of zona pellucida (ZP) was increased in the oocytes treated with 25-100 microM A23187. Penetration of ZP-intact oocytes by spermatozoa was decreased and only 3-4% of oocytes were penetrated by spermatozoa after 50-100 microM A23187 treatment. In experiment 2, oocytes were treated with 100 microM A23187 for 5 min and then cultured for 10 min or 3.5 h before insemination. No difference in penetration rates was observed between the two groups of oocytes (12.0% vs. 12.2%), but the penetration rates were significantly lower than those in controls (85.2% vs. 82.4%). In experiment 3, treatment of oocytes with 100 microM A23187 for 5 min was followed by removal of the ZP from a portion of the oocytes. ZP-intact and ZP-free oocytes were then inseminated for examination of sperm penetration. One of 65 (2%) oocytes with ZP and 48 of 52 (92%) oocytes without ZP were penetrated by spermatozoa. These results indicate that activation of pig oocytes by A23187 is the result of A23187-induced intracellular calcium increase and that A23187-induced cortical reaction can prevent sperm penetration of ZP-intact oocytes, but not ZP-free oocytes.

The development of sensitive measurements to analyze individual cells is of relevance to elucidate specialized roles or metabolic functions of each cell under physiological and pathological conditions. Lipids play multiple and critical roles in cellular functions and the application of analytical methods in the lipidomics area is of increasing interest. In this work, in vitro maturation of porcine oocytes was studied. Two independent sources of chemical information (represented by mass spectra in the positive and negative ion modes) from single oocytes (immature oocytes, 24-h and 44-h in vitro matured oocytes) were acquired by using desorption electrospray ionization-mass spectrometry (DESI-MS). Low and mid-level data fusion strategies are presented with the aim of better exploring the large amount of chemical information contained in the two mass spectrometric lipid profiles. Data were explored by principal component analysis (PCA) within the two multi-block approaches to include information on free fatty acids, phospholipids, cholesterol-related molecules, di- and triacylglycerols. After data fusion, clearer differences among immature and in vitro matured porcine oocytes were observed, which provide novel information regarding lipid metabolism throughout oocyte maturation. In particular, changes in TAG composition, as well as increase in fatty acid metabolism and membrane complexity were evidenced during the in vitro maturation process. This information can assist the improvement of in vitro embryo production for porcine species.

Abstract. The effects of resveratrol (a phytoalexin with a wide variety of pharmacological activities) on pig embryos produced by parthenogenesis and/or in vitro fertilization have been investigated. First, parthenogenetic embryos were generated and cultured in PZM-3 medium supplemented with various amounts of resveratrol (0, 0.05, 0.1, 0.5, 1.0 and 25 μM final concentrations). In the presence of 0.5 μM resveratrol a significantly higher percentage of parthenogenetic embryos reached the blastocyst stage by day 7 compared to non-treated control (43.5 ± 6.3% vs. 33.0 ± 5.4%; P<0.05). The total cell number of blastocysts also increased as a result of incubation with 0.5 μM resveratrol; the difference was statistically significant between treated and non-treated embryos on day 5 of culture (35.8 ± 0.9 vs. 32.1 ± 1.1; P<0.05). Resveratrol incubation affected the expression levels of apoptosis-related genes in parthenogenetic blastocysts: the level of Bax transcripts was similar but lower expression of Bcl-2 and Caspase-3 was observed in embryos treated with 0.5 μM resveratrol when compared to control blastocysts (P<0.05). The results of the TUNEL assay were similar in blastocysts developing with or without resveratrol supplementation. In addition, when embryos produced by in vitro fertilization were incubated with 0.5 μM resveratrol, the treatment led to higher frequencies of blastocyst formation (8.6% vs. 13.3%) and elevated total cell numbers (37.1 ± 2.4 vs. 43.2 ± 1.7) by the end of the 7-day culture period (P<0.05). The results indicate that 0.5 μM resveratrol during culture has a positive effect on early embryonic development of porcine embryos. Key words: Development, Embryo, Pig, Resveratrol (J. Reprod. Dev. 56: [330][331][332][333][334][335] 2010) mbryo culture is valuable in the study of preimplantation embryonic development and is indispensable for the in vitro production of transferable embryos. In spite of significant improvements in the culture conditions, the development of in vitro-produced embryos is still suboptimal [1]. Because the preimplantation embryo is extremely sensitive to environmental factors, deficiencies in culture conditions often lead to aberrant embryo development that manifest in lower frequency of blastocyst formation and lower cell numbers and can affect fetal as well as postnatal life [2]. Modifications in the culture system can potentially improve the development of the cultured embryos.Resveratrol (3,4',5-trihydroxystilbene) is a phytoalexin identified in more than 70 plant species including grapes, plums, and peanuts [3]. Its biological function is to protect the plant in case of a parasitic attack or environmental stress [4]. Originally, the search for plant-derived compounds with pharmacological properties identified resveratrol as a blocker of enzymes involved in arachidonate metabolism [5] and later, an inhibitor of partially purified kinases [6]. Based on its ability to block the cyclooxygenase COX-1 resveratrol was suggested to have anticancer properties and it was demo...