Determination of Plasma Membrane Characteristics: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Animals

Three mature Yorkshire boars (18 mo to 3 yr old, weighing 204-273 kg) were used in this study. The animals were fed, in an amount based on body weight, once a day with a mixed corn-soybean ration and were allowed free access to water. The boars could move at will inside a barn with straw on the floor or outside on a concrete slab.

Media

The media used were isosmotic Tyrode’s lactate Hepes (TL Hepes)-buffered solution (285 ± 5 mOsmol/kg) , and isosmotic and anisosmotic PBS solutions. Hyposmotic PBS solutions (< 290 mOsmol/kg) were prepared by diluting isosmotic PBS with reagent water, and hyperosmotic PBS solutions (> 290 mOsmol/kg) were prepared by diluting 10-strength PBS with reagent water. Osmolality was determined using a freezing-point depression osmometer (Model 3D2; Advanced Instruments, Needham Heights, MA). The cryoprotectant treatment solutions were prepared by mixing glycerol, DMSO, or EG to yield final CPA concentrations of 1 M (glycerol and DMSO) and 2 M (EG). All media used in the experiments were obtained from Sigma Chemical Company (St. Louis, MO). Modena extender was used in experiments 1 and 3. The extender was prepared as described by Weitze using the components shown in Table 1. buy ortho tri-cyclen

Samples

Semen samples were collected once a week by manual manipulation using a dummy and gloved hand. Filtered semen from the sperm-rich portion was diluted 1:3 in TL Hepes media in 15-ml conical tubes (Sarstedt, Newton, NC) and transported in a 37°C water bath during the 1-h transport time. Five microliters of the sample was analyzed using computer-assisted semen analysis (CASA; Cell Soft, Version 3.2/C; CRYOResources Ltd., Montgomery, NY) to determine percentage of motility and concentration. Motility was analyzed using this approach throughout experiments 1 and 3.

This entry was posted in Spermatozoa and tagged Boar, Cryopreservation, Plasma Membrane, Spermatozoa.