Selection and implementation of single nucleotide polymorphism markers for parentage analysis in crossbred cattle population.

Crossbreeding is an essential way of improving herd performance. However, frequent parentage record errors appear, which results in the lower accuracy of genetic parameter estimation and genetic evaluation. This study aims to build a single nucleotide polymorphism (SNP) panel with sufficient power for parentage testing in the crossbred population of Simmental and Holstein cattle. The direct sequencing technique in PCR products of pooling DNA along with matrix-assisted laser desorption/ionization time-of-flight MS method for genotyping the individuals was applied. A panel comprising 50 highly informative SNPs for parentage analysis was developed in the crossbred population. The average minor allele frequency for SNPs was 0.43, and the cumulative probability of exclusion for single-parent and both-parent inference met 0.99797 and 0.999999, respectively. The maker-set for parentage verification was then used in a group of 81 trios with aid of the likelihood-based parentage-assignment program of Cervus software. Reconfirmation with on-farm records showed that this 50-SNP system could provide sufficient and reliable information for parentage testing with the parental errors for mother-offspring and sire-offspring being 8.6 and 18.5%, respectively. In conclusion, a set of low-cost and efficient SNPs for the paternity testing in the Simmental and Holstein crossbred population are provided.

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin.

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.