Summary. The effects of dialysis fluids containing blood serum, serum albumin or activated charcoal on the storage of boar semen were examined. The effects of antibiotics were also tested by including them in the dialysis fluids and semen samples. Undiluted semen was stored for 7 days at 15°C by means of dialysis across a cellulose membrane. A combination of sulbenicillin and streptomycin was superior to that of penicillin and streptomycin in reducing bacteria and maintaining sperm motility and normal acrosomes. Serum albumin exerted a beneficial effect on the stored spermatozoa which may be due to its capacity to adsorb the metabolic products from bacteria and spermatozoa; it could be replaced with activated charcoal.
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K. Bamba and M. Sone
K. Bamba and D. G. Cran
Summary. Boar spermatozoa acquired resistance to cold shock immediately after exposure to 2·0mmol butylated hydroxytoluene (BHT) l−1 when Beltsville thawing solution was used as a basic diluent, as judged by motility (the proportion of motile spermatozoa) and acrosomal integrity. The concentration of BHT could be reduced to 0·2 mmol l−1 without decreasing the protective action. However, motility was altered in the presence of >0·15 mmol BHT l−1. Beltsville freezing 5 (BF5) diluent was more effective than Beltsville thawing solution in protecting spermatozoa from cold shock, but addition of BHT to BF5 diluent did not affect the motility and acrosomal morphology of spermatozoa before or after cold shock. Dilution of BHT-treated spermatozoa with BF5 diluent did not restore motility and did not afford further protection against cold shock; it was detrimental to spermatozoa treated with 2 mmol BHT l−1 for > 15 min. Egg yolk or lecithin had a detrimental effect.
When spermatozoa were treated with 0·05–0·10 mmol BHT l−1 before slow cooling to 5°C, the progressive motility and acrosomal integrity were maintained better after storage for 6 days than in untreated spermatozoa.
Keywords: spermatozoa; cold shock; boar; butylated hydroxytoluene
K. Bamba and D. G. Cran
Summary. The effect of rapid dilution (1:8 with BTS or 1:6·5 with KRP) and temperature change on sperm morphology and physiology were studied using boar spermatozoa pre-diluted in BF5 diluent. Rapid dilution of cold semen (5°C) with a warm solution (37°C) caused marked acrosomal changes which were most prominent in the anterior region. The acrosomal damage appeared to be caused mainly by rapid warming. In contrast to rapid cooling, rapid warming had little effect upon motility, glutamic-oxaloacetic transaminase release and respiration.
K. Bamba and D. G. Cran
Summary. Rapid warming of semen from 5 to 37°C caused visible damage to the acrosomes of bull and rabbit spermatozoa. The degree and type of damage varied with the species, the bull being the more resistant. While vesiculation was observed in rabbit spermatozoa, neither warm nor cold shock resulted in this defect in the bull. Warm shock of bull spermatozoa caused acrosomal knobbing in an anterior region of the head. Spermatozoa with thread and/or droplet-like structures were frequently observed in bull semen after cold shock.
Keywords: bull; rabbit; rapid warming; spermatozoa; morphology
K. Bamba and D. G. Cran
Summary. Studies have been carried out to investigate factors related to the induction of warm shock in boar spermatozoa. Rapid dilution per se caused visible damage to acrosomes when the sample contained 7·5% or more glycerol. This dilution effect was greater at lower temperatures. Acrosomal damage was greatly reduced by raising the dilution temperature from 15 to 25°C, suggesting that a change in the physico-chemical characteristics of the acrosomal membrane occurred between these temperatures. During rapid dilution with warming, the dilution rate, the magnitude of the temperature change and the terminal temperature had a significant influence on acrosomal integrity; a terminal temperature of 35°C was much more detrimental than one of 25°C. The first sign of acrosomal damage was observed 15 sec after rapid dilution + warming and the damage was nearly maximal by 60 sec. An antioxidant, butylated hydroxytoluene (BHT), was effective against both rapid cooling and warming, while glycerol, dimethylsulphoxide and propylene glycol were ineffective in preventing warm shock.
Keywords: boar spermatozoa; warming and dilution; acrosome; structure
M. Yoshida, Y. Ishizaki, H. Kawagishi, K. Bamba, and Y. Kojima
Summary. This study examines the effects of pig follicular fluid on the maturation of pig oocytes and on their subsequent fertilizing and developmental capacity in vitro. The addition of pig follicular fluid or its fractions obtained by ultrafiltration, gel filtration and ion-exchange chromatography to maturation medium significantly increased the rates of nuclear maturation, normal fertilization and normal cleavage of pig oocytes after fertilization in vitro: the rates of normal fertilization and cleavage were 2–4 times higher than those in the control medium. The efficacy of pig follicular fluid was lost after heating at 56°C for 30 min, whereas no significant decrease in activity was observed after defatting. In addition, the effective component(s) was partially purified by ultrafiltration, gel filtration and ion-exchange chromatography: the activity was observed in the fraction (UF2; M r 10 000–20 000) obtained by ultrafiltration. Activity was found in the first fraction (G1) obtained by gel filtration of UF2. Among three fractions obtained by ion-exchange chromatography of Gl, only the third fraction had the activity. The results indicate that pig follicular fluid contains an acidic substance(s) (M r 10 000–200 000) that promotes oocyte maturation.
Keywords: follicular fluid; maturation; fertilization; development; pig; oocytes
T. Kohsaka, H. Takahara, H. Sasada, T. Kawarasaki, K. Bamba, J. Masaki, and S. Tagami
Summary. Light-microscope immunocytochemistry using the peroxidase–antiperoxidase technique and a polyclonal rabbit antiserum raised against purified porcine relaxin showed that cytoplasmic immunostaining for relaxin could be visualized in the epithelial cells of the seminal vesicle. No relaxin immunoreactivity was seen in the testis, epididymis, ductus deferens, prostate or bulbo–urethral gland. A ten times higher concentration of porcine relaxin antiserum was necessary to achieve immunostaining in the seminal vesicle comparable to that in the corpora lutea of pregnant sows.
Ultrastructural examination showed that the epithelial cells of the boar seminal vesicle resembled typical protein-secreting cells with prominent rough endoplasmic reticulum and well-developed Golgi apparatus. The most striking feature of these cells was the accumulation of granules with a limiting membrane, which ranged from 200 to 600 nm in diameter and contained flocculent material of moderate electron density. Electron-microscope immunocytochemistry using the protein A-gold technique and relaxin antiserum demonstrated that the granules were the only intracellular organelles that showed immunoreactivity for relaxin.
These results indicate that a relaxin-like substance is present in boar seminal vesicles and that the subcellular site of its localization is the granules, suggesting that the seminal vesicle produces and stores a relaxin-like substance, but that it is present at much lower concentrations than in the corpora lutea of pregnant sows.
Keywords: relaxin; seminal vesicle; immunocytochemistry; boar