We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca2+ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: "F," characteristic of uncapacitated, acrosome-intact cells; "B," characteristic of capacitated, acrosome-intact cells; "AR," characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently approximately 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.

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)

Summary. Pig oocytes matured in culture were inseminated with frozen\p=n-\thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85\p=n-\89%) and increased incidence of polyspermy were obtained at 25\p=n-\100\m=x\106 spermatozoa/ml. Wide variation in penetration rates (16\p=n-\89%) was observed in oocytes inseminated in medium containing 5mm caffeine and at 25\p=n-\50\m=x\106 spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25\p=n-\50\m=x\106 spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mm caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5\p=n-\40 \g=m\g/ml. When heparin was included in the medium with 5mm caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.

Bovine oocytes that had been matured and fertilized in vitro were cultured in a simple, chemically defined, protein-free medium (mTLP-PVA). When the medium was supplemented with 19 amino acids, development to the 8-cell (14-20% vs. 38-46%), morula (0-6% vs. 27-32%), and blastocyst (0-1% vs. 9-13%) stages 96, 144, and 192 h after insemination, respectively, was significantly greater in the absence than in the presence of glucose (5.56 mM) regardless of the presence of phosphate (1.05 mM). However, blastocyst development was difficult in medium with any combination of glucose and phosphate without amino acids. In mTLP-PVA with amino acids and different concentrations of phosphate, the highest proportions of embryos reaching the > or = 8-cell (56%), morula (44%), and blastocyst (24%) stages were obtained at a 0.35 mM concentration. When lactate and pyruvate were omitted from mTLP-PVA (mT-PVA) supplemented with amino acids and 0.35 mM phosphate, the first cleavage was completely inhibited. Although lactate or pyruvate alone could support blastocyst development to a limited extent (10-15%), a significantly higher proportion (22%) of blastocysts was obtained in medium with both lactate (10 mM) and pyruvate (0.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

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Rat one-cell embryos recovered from naturally mated females were cultured in modified hamster embryo culture medium 1 without amino acids. In