Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated boar spermatozoa incubated in a protein-free chemically defined medium

Abstract

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen-thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO3 l-1 and 5 mmol caffeine l-1. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml-1, more penetrated oocytes were observed 14 h after insemination with 7-12 x 10(6) cells ml-1 than with 4-5 x 10(6) cells ml-1 and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO3 concentration but maintaining the sperm concentration at 8 x 10(6) cells ml-1 resulted in significantly more oocytes being penetrated in media containing 45.83-50.25 than 37.0-41.42 mmol NaHCO3 l-1; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO3 l-1, the inclusion of PVA at 0-5 mg ml-1 had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine sperm capacitation involves tyrosine phosphorylation and activation of aldose reductase

“…The sperm were incubated in Cap medium, and capacitation was induced, as assessed by the CTCfluorescence staining pattern of the sperm head (Wang et al 1995, Tardif et al 1999. Under the conditions of the present experiment, over 70% of the sperm represented pattern B fluorescence (fluorescence-free band in the postacrosomal region), thus proving them to be capacitated sperm (Fig.…”

“…The washed sperm were incubated either in 15 ml of Non-cap medium or in Cap medium (5!10 6 cells/ml) at 37 8C for 3 h in a 5% CO 2 atmosphere. Capacitated sperm were assessed on the basis of the changes in the pattern of the CTC fluorescence staining, according to the method of Wang et al (1995).…”

Insulin affects sperm capacity in pig through nitric oxide

“…The chlortetracycline (CTC) staining assay was performed according to Wang et al 8 Briefly, 45 ml of the sperm suspension (5310 6 ml 21 ) was treated with an equal volume of a CTC solution for 30 s, followed by the addition of 8 ml of 12.5 paraformaldehyde in 0.5 mol l 21 TrisHCl (pH 7.4). Ten microlitres of the fixed sperm suspension were placed on a microscope slide and examined with an epifluorescence microscope (Olimpus BX41) with multiple fluorescent filters.…”

Histochemical Demonstration of Lipids in Cultured Porcine Oocytes and Preimplantation Embryos

“…Ejaculated boar semen was treated by the method of Wang et al [11], in order to induce capacitation of spermatozoa. The semen was washed three times in Brackett and Oliphant (BO) [12] medium containing 5 mM caffeine (Sigma-Aldrich) and 0.3% BSA (SigmaAldrich).…”