Gene expression of estrogen and oxytocin receptors in the uterus of pregnant and parturient bitches

In the canine species, the precise mechanisms of pregnancy maintenance and the initiation of parturition are not completely understood. The expression of genes encoding the receptors for estrogen (ERα mRNA) and oxytocin (OTR mRNA) was studied in the endometrium and myometrium during pregnancy and parturition in dogs. Real-time PCR was performed to quantify the levels of ERα mRNA and OTR mRNA in the uterus of bitches during early (up to 20 days of gestation), mid (20 to 40 days) and late pregnancy (41 to 60 days), and parturition (first stage of labor). All tissues expressed ERα and OTR mRNA, and are thus possibly able to respond to eventual estrogen and oxytocin hormonal stimuli. No statistically significant differences in the expression of ERα mRNA were verified in the endometrium and myometrium throughout pregnancy and parturition, but expression of OTR mRNA increased at both parturition and late pregnancy. We concluded that the increase of endometrial and myometrial OTR mRNA expression in dogs is not an event dependent on estrogenic stimulation. Moreover, the contractility response of the canine uterus to oxytocin begins during pregnancy and maintains myometrial activity. The expression of OTR mRNA in canine uterine tissues varied over time, which supports an interpretation that the sensitivity and response to hormone therapy varies during the course of pregnancy and labor. Further studies are needed to elucidate the factors underlying the synthesis of uterine oxytocin receptors and the possible role of ERβ rather than ERα in the uterine tissues during pregnancy and parturition in dogs.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5 percent) were successfully fused and 24 (28.9 percent) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8 percent) of which were pregnant on day 35 and 3 (16.7 percent) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).