2006 merck/merial - School of Veterinary Medicine - Louisiana State ...

the gravid and non-gravid females to determine whether reproductive success is affected by these factors. Continuousvariables (complete blood counts, plasma biochemistries, corticosterone) will be assessed for normality using the Shapiro-Wilk test statistic. Non-normally distributed data will be log transformed for analysis. Comparisons between gravid and nongravidfemales for continuous data will be made using a one-way analysis of variance. Categorical data (herpes virus status,Mycoplasma status, and parasite status) will be made using chi-square tests or Fisher exact tests, depending on cell size. Ifneeded, stratified analysis will be done to assess for interaction or effect modification. This field research project is ongoing.Evaluation of the insulin gene and DLA alleles as genetic markers for diabetes mellitus in Samoyed andAustralian Terrier dogsRachel Toaff-Rosenstein*, Paula S. Henthorn, Rebecka HessDepartment of Clinical Studies-Philadelphia, University of Pennsylvania School of Veterinary MedicineCanine diabetes mellitus (DM) is a common disorder for which the etiology is unknown. Samoyeds are about 12times more likely and Australian Terriers are about 32 times more likely to develop DM compared to dogs of mixedbreeding. The goal of this summer project is to analyze allelic variations in and flanking the insulin gene region and in exon2 of the Dog Leukocyte Antigen class II genes to determine if there is any association between allelic frequencies in diabeticcompared to non-diabetic dogs in each breed. Dogs are uniformly considered to have type I DM, which is characterized bydestruction of pancreatic beta-cells and hypoinsulinemia. Genetic susceptibility and immune mediated destruction ofpancreatic beta-cells are among the important possible causes for canine type I DM. In humans, the two most importantgenetic determinants of type I DM are the major histocompatibility complex class II (MHC-II) genes and the insulin generegion. MHC-II genes coding for HLA class II proteins are estimated to determine about 40% of type I DM heritability inhumans, and the insulin gene region contributes about 10% to this heritability. Analysis of the insulin and DLA class IIgenes in Samoyed and Australian Terrier dogs will enable testing of the hypothesis that allelic variations in the insulin geneand DLA class II gene are associated with increased incidence of DM in these breeds. To do so, DNA has been purified fromdiabetic and control dogs of each breed. The insulin gene and promoter will be sequenced, in addition to the sequencing ofthe DLA class II genes. The results of this study will contribute to the identification of genetic factors contributing to theonset of DM in Samoyeds and Australian Terriers, and may make it possible to develop DNA-based tests useful for theidentification of dogs predisposed to developing diabetes or producing diabetic offspring.Zona Pellucida Penetration Assay to evaluate sperm cells integrity and fertilization ability in the dog.Georgina Ushi*, Tameka Phillips, Karine Onclin, John VerstegenUniversity of Florida College of Veterinary Medicine, Large Animal Clinical Sciences, P.O. Box 100136Gainesville, FloridaWhen a spermatozoa penetrates an oocyte, it must go through an essential step for fertilization and oocyte survivalof zona pellucida (ZP) penetration. The binding rate of spermatozoa to the ZP represents a critical event in gameteinteraction and is predictive of sperm fertilizing potential (Ivanova et al., 1998). For this reason, assays (zona pellucidabinding, zona penetration and in vitro fertilization assays) have been developed to determine binding capacity of spermatozoaand to predict fertility in several species. Zona pellucida binding assays have also been tested in the dog as a tool to correlatespermatozoas’ capacity to bind to the ZP in vitro and to determine fertility (Fazeli et al., 1993). In the present study, wedetermined whether the Zona Pellucida Penetration Assay can be used to assess semen fertility in the dog. Oocytes havebeen collected from canine ovaries and purified. The ovaries were obtained from the spay clinic of the Veterinary TeachingHospital of the University of Florida from dogs aged 3 months to 2 years (n= 5). Some oocytes have been frozen before use(n=87) while some have been used fresh immediately after collection (n=30). After capacitation (Mahi et al., 1978), spermcells obtained from 4 fertile male dogs were incubated in vitro at 37ºC and 5% CO2 in air for 90 minutes with the fresh orthawed oocytes and their ability to penetrate the zona pellucida were evaluated. The effects of semen preservation were alsoevaluated by using either preserved (5 days chilled or after freezing/thawing) or fresh sperm cells (100,000 sperm cells/5oocytes). The experimentations were repeated 3 times. Our purposes were to correlate penetration ability with the quality ofthe oocytes and their technique of preservation (fresh versus frozen) and the preservation of semen. The objective is topropose this technique as a tool in the evaluation of the semen integrity in presence or absence of preservation procedures orin pathological processes.90