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AIM: To study whether additional measurements of motility characteristics of spermatozoa by computer assisted semen analysis (CASA) were more sensitive indicators of reduced semen quality than estimates of percentages of motile, rapid or progressive cells. METHODS: Intermittent scrotal insulation was applied to 6 rams for 16 h per day for 21 days or to 2 of these for 12 h per day for 28 days in the following year. Semen was collected and evaluated by CASA immediately and either frozen or stored at 30 degrees Celsius or 5 degrees Celsius before re-evaluation. RESULTS: Intermittent scrotal insulation caused falls in the percentage of motile, progressive and rapid sperm, as did freezing-thawing and storage at 30 degrees Celsius or 5 degrees Celsius. Motility characteristics (amplitude of lateral head displacement, mean path velocity, mean progressive velocity and curvilinear velocity), as determined by CASA fell only when the percentage of motile sperm was already reduced. Freezing and thawing or liquid storage of the semen from insulated rams caused a greater fall in the percentage of motile and rapid sperm than control semen, but only affected the motility characteristics when the percentage of motile sperm was already reduced. CONCLUSION: Intermittent scrotal insulation affected not only the motility of the freshly collected sperm, but also their ability to withstand the additional stress of storage. The additional data on motility characteristics obtained by CASA appeared to be no more a sensitive indicator than the percentage of motile cells of reductions in semen quality.

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Many of the proteins and their encoding genes involved in spermatogenesis are unknown, making the specific diagnosis and treatment of infertility in males difficult and highlighting the importance of identifying new genes that are involved in spermatogenesis. Through genome-wide chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and a three-generation breeding scheme to isolate recessive mutations, we have identified mouse lines with a range of abnormalities relevant to human male fertility. Abnormal phenotypes included hypospermatogenesis, Sertoli cell-only (SCO) seminiferous tubules, germ-cell arrest and abnormal spermiogenesis and were accompanied, in some, with abnormal serum levels of reproductive hormones. In total, from 65 mouse lines, 14 showed a reproductive phenotype consistent with a recessive mutation. This study shows that it is feasible to use ENU mutagenesis as an effective and rapid means of generating mouse models relevant to furthering our understanding of human male infertility. Spermatozoa and genomic DNA from all mouse lines, including those with abnormal reproductive tract parameters, have been cryopreserved for the regeneration of lines as required. This repository will form a valuable resource for the identification and analysis of key regulators of multiple aspects of male fertility.

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The fertility of ewes after artificial insemination and the relationship between fertility and motility characteristics assessed by a computerized motility analysis system were examined with ram semen frozen in diluents reported to improve postthaw motility. The percentages of motile and progressive spermatozoa were better when frozen in proline- or glycine betaine-containing or HEPES-based, rather than Tris-based, diluents (P < 0.01). The fertility of spermatozoa frozen in diluents containing proline or glycine betaine was slightly reduced, whereas when both compatible solutes were present, the reduction was more pronounced, in comparison with semen frozen in Tris- or HEPES-based diluents (9.5 versus 71.1 and 66.6%; P < 0.01). Fertility of frozen-thawed spermatozoa was higher after laparoscopic insemination than after cervical or transcervical insemination (P < 0.01). Similarly, higher fertility was obtained after cervical insemination with fresh than with frozen-thawed semen (32.4 versus 11.3%; P < 0.01). Furthermore, loss of embryos was lower after laparoscopic insemination of ewes with semen frozen in a Tris diluent than with semen frozen in proline diluents, in glycine betaine diluents, or in proline-plus-glycine betaine diluents (0.0 versus 26.0, 38.5, and 60.0%; P < 0.001). A wide variation in the postthaw percentage of motile (31.6-59.7%) and progressive (22.6-43.1%) spermatozoa and in the fertility of spermatozoa from individual rams was also observed after laparoscopic (29.2-59.7%) or cervical insemination (8.7-30.5%). Postthaw motility results from immediately after thawing and fertility results from experiments where intrauterine insemination was performed with semen frozen in proline- or glycine betaine-containing or HEPES- or Tris-based diluents were pooled and subjected to a pairwise correlation procedure. The correlation analysis showed relationships between some of the motility characteristics (P < 0.01), but there were no relationships between the motility characteristics and fertility.

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The effect of the compatible solutes proline, glycine betaine and trehalose in Tris-based diluents at varying pH, concentrations of egg yolk or glycerol on the post-thaw motility characteristics and fertility of ram sperm was examined. In addition, the amino acid glycine was compared with proline, glycine betaine and a standard Tris-based diluent. Post-thaw motility was assessed using a Hamilton-Thorn motility analyser. In the presence of glycerol and egg yolk, proline and glycine betaine improved the post-thaw motility characteristics of ram sperm. Regardless of the pH of the diluent at which semen was frozen, the percentage of motile sperm was higher when frozen in the presence of proline or glycine betaine than in their absence, whereas proline and glycine betaine only improved the progressive and rapid percentages of sperm for semen frozen in diluents at pH lower than 7.0. When semen was frozen in the absence of egg yolk or glycerol all the motility characteristics were reduced. Increasing the concentration of egg yolk in the diluent from 5% to 10, 15 or 20% had no effect on the post-thaw motility of sperm. The addition of 27 mM of proline or glycine betaine to the diluent also improved post-thaw motility. However, at a concentration of 81 mM, proline and glycine betaine had a detrimental effect on the percentage of motile sperm. Trehalose had no effect on the motility of sperm frozen in glycerol-containing diluents, but motility was lower after cryopreservation in glycine than in Tris-, proline- or glycine betaine-based diluents. There were no differences in the fertility of sperm frozen in Tris-, proline or glycine betaine diluents after cervical or laparoscopic insemination of ewes.

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The epididymal compounds taurine, hypotaurine and inositol, and the antioxidants carnosine and ascorbic acid, were added to Tris-based diluents containing varying concentrations of glycerol, and their effect on the post-thaw motility characteristics and fertility of ram spermatozoa was examined. Overall, the post-thaw motility characteristics of spermatozoa were better when semen was frozen in the presence rather than in the absence of glycerol. Only taurine protected spermatozoa during cryopreservation; the presence of 25 mM or 50 mM taurine significantly improved the post-thaw percentage of motile spermatozoa but this had no effect on fertility after cervical or laparoscopic insemination of ewes. Increasing the concentration of taurine to more than 100 mM significantly reduced the percentage of motile spermatozoa, compared with the lower concentrations of the amino acid. The presence of more than 50 mM carnosine or ascorbic acid significantly reduced all motility characteristics compared with the control diluent. Given that hypotaurine, carnosine, or ascorbic acid did not improve post-thaw motility, the cryoprotective effect of taurine may be attributable to its osmoregulation rather than to its antioxidant properties.

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Post-thaw characteristics of ram semen frozen as pellets were assessed using biochemically (amidase activity) or motility-based (Hamilton Thorn Motility Analyzer) techniques. The total variation associated with each semen characteristic measured was partitioned between rams (5), ejaculates within rams (5), pellets within ejaculates (5) and within pellets (2). A variety of variance distributions were observed for the characteristics measured. Of the 18 post-thaw characteristics examined, 10 had > 50% of variance distributed between within-ejaculate components. This has important implications for the way in which such measurements may be used in post-thaw semen analysis.

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The motility, acrosome integrity and fertility of ram spermatozoa were examined after treatment with five compounds (caffeine, pentoxifylline, cAMP, 2-deoxyadenosine and kallikrein). Semen was extended with a Tris-based medium and frozen in pellet form. The compounds were added at various concentrations to the semen before freezing or after thawing. Motility and acrosome characteristics were assessed over a 6-h post-thaw period of incubation at 37 degrees C. Of the five compounds examined, only pentoxifylline, when added after thawing, stimulated motility but had not effect on the acrosome integrity of spermatozoa. Pregnancy rates for ewes inseminated in the uterus with semen treated after thawing with Dulbecco's phosphate-buffered saline (PBS, control) or PBS containing pentoxifylline (15 mM in the thawed semen), were 16/39 (41%) and 22/42 (52%) respectively. Motility characteristics of spermatozoa assessed by image analysis (Hamilton-Thorne analyser) were initially better after treatment of thawed semen with pentoxifylline than with PBS ("percent motility', "percent progressive motility', "percent rapid', "percent moderate' and "percent static', P < 0.01 or P < 0.001), but there was a greater deterioration in these characteristics during post-thaw incubation in semen treated with pentoxifylline than in the controls. The deterioration in motility characteristics occurred within 4 h of thawing and this may have been responsible for the modest improvement in fertility of spermatozoa treated with pentoxifylline.

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In vertebrate animals, the duration of storage of viable spermatozoa in the epididymis varies from a few hours in birds to many months in reptiles and bats. The available information on the unusual composition of the epididymal luminal fluid is summarized, and the effect of the various constituents on sperm motility is described. Spermatozoa would probably be best stored in an immotile state and some constituents of epididymal luminal fluid may be able to inhibit the motility of mammalian spermatozoa during storage in vitro. If this effect can then be removed at the time of insemination, by changing the spermatozoa to a different medium, such a procedure may allow storage of spermatozoa at room or even body temperature for extended periods.

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Fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 x 10(6) frozen spermatozoa from four control rams and from four rams submitted to a moderate (1.4-2.2 degrees C), but repeated, intermittent (16 h/day for 21 consecutive days) increase in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe from concentration of progesterone in blood plasma at 17 days and by ultrasound at 65 days after insemination. No differences were observed in the pregnancy rate at 17 days between ewes inseminated with semen collected from control rams (56.0, 65.2, 66.7 and 60.3%) and from heated rams (60.6, 71.8, 63.6 and 48.2%) before or after 4, 15 and 21 days of heating, respectively. In contrast, the rate of embryonic mortality between 17 and 65 days after insemination was significantly higher at days 4, 15 and 21 in the heated rams (78.7, 78.6 and 93%) than in the control rams (55, 59 and 65.7%). These results indicate that an intermittent slight, but repeated, increase in the subcutaneous scrotal temperature could induce a significant increase in the embryonic mortality rate. As these changes were apparent on day 4 of heating, an effect must have occurred on sperm stored in the epididymis.

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