Determination of adenosine triphosphate in human semen to estimate the fertilizing potential and to quantify sperm antibodies

The measurement of the ATP content of fresh semen is as accurate as the estimation of sperm motility by conventional methods in discriminating between semen of fertile versus subfertile men. The ATP content of frozen thawed donor semen is correlated with the probability of conception per cycle of insemination. Exact quantification of cytotoxic sperm antibodies in serum is possible with the adenosine-triphosphate-release-cytotoxicity test, since measurement is free of the bias of microscopic examination. The procedure has been simplified by testing only one serum dilution and calculating the ‘sperm toxicity index’.     

Extracellular calcium is involved in egg yolk-induced head-to-head agglutination of bull sperm

Head-to-head agglutination of bull sperm occurs when semen is highly diluted in an egg yolk-citrate diluent without streptomycin. The objectives were to investigate causes of sperm agglutination and the underlying mechanism. Aliquots of bull semen were diluted in a base diluent (BD) supplemented with various test components and the percentage of agglutinated sperm (% AggSp) was quantified at 1, 5, 24, 48, and 72 h of incubation. When sperm were incubated at 22 °C, no agglutination was observed in BD for up to 72 h, whereas the % AggSp was 5.0, 41.7, 72.2, 91.1, and 92.8% in BD + 5% egg yolk (BD + EY) at 1, 5, 24, 48 and 72 h, respectively. However, no sperm agglutination was observed in BD + EY if incubation temperature was 37 °C. Addition of 5 or 10 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid to BD + EY reduced the % AggSp from 95% to <5% at 72 h (P < 0.001), but addition of 5 mM CaCl₂ to BD failed to induce sperm agglutination in the absence of egg yolk, implicating calcium and other factors in egg yolk. Addition of the citrate-soluble fraction (CSF) of egg yolk to BD induced sperm agglutination similar to whole egg yolk, whereas water- and saline-soluble fractions of egg yolk were ineffective. The sperm-agglutinating efficacy of CSF (the % AggSp = 95% at 72 h) was reduced by dialysis (20%; P < 0.05), partially restored by addition of 5 mM CaCl₂ (70%; P < 0.05), but the calcium effect was neutralized by addition of 5 mM ethylenebis (oxyethyleneni-trilo) tetra-acetic acid (1.7%; P < 0.05), again implicating calcium. Addition of 30 μM of a protein kinase A inhibitor (H-89) to an agglutinating diluent failed to inhibit sperm agglutination, whereas addition of 2 mM of a cAMP analogue, dbcAMP, to a nonagglutinating diluent failed to induce sperm agglutination. Agglutination status had no effect on sperm plasma membrane/acrosome status and mitochondrial membrane potential. In conclusion, calcium and other component(s) in the CSF of egg yolk induced head-to-head agglutination of bull sperm in a time- and temperature-dependent manner. Although the mechanism of agglutination was not determined, the cAMP- protein kinase A signaling pathway was not involved.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

Anticorps anti-spermatozoïdes: techniques de dépistage et interprétation des résultats

Since the first publication on the detection of sperm-agglutinating antibodies in infertile men, multiple assays have been described. The most useful tests are able to detect antibodies bound to the sperm membrane of motile spermatozoa. The immunobeads test (IBT) is considered to be the most advantageous in terms of its sensitivity, the low incidence of false-positive results, and its ability to localize antibodies of different immunoglobulin classes on the sperm surface. The IBT assay can be used in parallel with the mixed antiglobulin reaction (MAR), to detect sperm-associated antibodies in the ejaculates of infertile men, the most rational way to test for antisperm antibodies (ASA) in males. In view of the high level of agreement between the two assays, MAR, the easier of the two, may be used as a first step in the detection of these antibodies. A positive MAR must be confirmed by IBT, as this assay is more specific for the detection of IgA antibodies. The clinical significance of sperm-associated antibodies is usually established according to the proportion of motile spermatozoa coated with immunobeads, its class and its localization on the sperm surface. However, binding of immunobeads does not provide any information about the antigens against which the antibodies are directed. As the functional effects of sperm-associated antibodies may vary as a function of their antigenic specificities, other assays, using purified fertilization related antigens, are necessary to establish, for each individual, the specific impact of the antibodies on the fertilization process. The indirect IBT assay has recently become the most widely used test to detect the various classes of ASA in serum and cervical mucus of infertile women and in the serum and seminal fluid of infertile men, in combination with the direct assay described above. However, in most laboratories, it is performed with only one dilution of the biological fluid tested, usually a low dilution, so that antibody levels of no significance for fertility could be detected. This may explain a recente debate (Human Reprod, 1999) on the significance of ASA as a cause of infertility. At present, and in the absence of standardized assays able to identify the antigens involved in each individual immune reaction, antibody assays, as detected by the IBT assay, in the serum and/or genital secretions of infertile subjects might provide useful clinical guidance.     

Acrosome reaction and concentration of prostaglandin E2 in semen of rams treated with flunixin meglumine (Banamine)

The objectives of this study were to determine 1) the effect of Banamine on the seminal concentration of PGE2 and 2) the ability of sperm cells from treated rams to undergo acrosome reaction in vitro as an indirect measure of their fertilizing capacity. Seven rams, approximately 55 kg bodyweight and 2 to 5 yr of age, were divided into two groups: Group 1, treated (n = 4) and Group 2, controls (n = 3). Treatment consisted of administration of 75 mg i.m. Banamine twice daily, 6 to 8 h apart, for 45 d. On Day 0 (first day of treatment) and on Days 2, 9, 11, 16, 23, 25, 28, 30, 36, 39, 43 and 46 semen samples were collected from both groups using an electroejaculator. Blood samples were obtained for determination of serum levels of Banamine using high-performance liquid chromatography. Semen samples were examined for motility and morphology. Highly motile (>/=85%), normal-appearing semen samples were pooled on each day of collection and 25 ul of the pooled sample (1x10(6)/ml) of each group were induced to undergo acrosome reaction in vitro using ionophore A23187. Acrosome reaction was demonstrated using a staining technique designed for demonstrating the process in bull sperm cells. The percentage of acrosome-reacted and non-acrosome-reacted sperm was determined by random microscopic examination of 100 sperm cells using a double-blind approach. The supernatants of the remainder of the semen samples were assayed for levels of PGE2 using RIA. Values for acrosome-reacted sperm cells and PGE2 levels on the first day of treatment from both groups were compared with corresponding values from each day of sampling using Wilcoxon Rank Sums test (P<0.05). In Group 1, the mean serum level of Banamine was 3.02+/-0.58ug/ml. There was a significant increase in the ability of sperm cells from rams in Group 1 to undergo acrosome reaction as treatment progressed compared with the sperm cells from rams in Group 2. However, there was a significant decrease in concentration of PGE2 in semen from rams in Group 1 compared with those from Group 2. The results of this study suggest an inverse relationship between the capacity of sperm cells to undergo acrosome reaction and concentration of PGE2 in semen of rams treated with a nonsteroidal anti-inflammatory drug.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

Comparison of the BullMate sperm quality analyzer with conventional means of assessing the semen quality and breeding soundness of beef bulls

An instrument called the Optibreed, BullMate sperm quality analyzer (SQA) contains a densitometer for determining sperm cell concentration and an optical sensor to evaluate light deflections caused by sperm movement. Analysis of light deflections enables the generation of a value called the sperm quality index (SQI). The SQI represents the quality of a semen sample defined by sperm motility, concentration, viability and morphology. The SQA was compared to conventional, microscopic techniques for determining percent motile sperm and sperm concentration in bull semen samples and evaluated for its ability to classify bulls as satisfactory or unsatisfactory potential breeders. Semen samples were collected from 105 mature beef bulls by electroejaculation (day 1) and from 51 of the same bulls by internal artificial vagina (IAV) on day 2. SQI values were arranged into 20 categories in increments of 50units from 0 to 1000units. Percent motile sperm and sperm concentration values from both methods were significantly positively correlated (P<0.000) with respective r values of 0.82 and 0.80. A calculation of kappa to evaluate the differences in percent motile sperm generated by each system yielded a value of 0.20 and 0.54 for unweighted and weighted determinations, respectively. The intraclass correlation coefficient used to evaluate the reliability of sperm cell concentration determinations was 0.62 (P<0.05). SQI values generated on days 1 and 2 ranged from 0-994 to 0-906, respectively. Bulls were categorized as satisfactory or unsatisfactory potential breeders in all categories. The most appropriate SQI for determining whether a bull was a satisfactory or unsatisfactory potential breeder was 500 with respective sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of 81, 65, 90.79 and 44.83%. In this experiment the BullMate SQA was not a reliable substitute for conventional semen analysis and was not useful for determining bull breeding soundness.     

A short sperm-oocyte incubation time ZBA in the dog

The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Str?m Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.     

Use of commercial extenders and alternatives to prevent sperm agglutination for cryopreservation of brown bear semen

The objective of this study was to evaluate different bovine and canine commercial semen extenders for cryopreservation of brown bear ejaculates and the effect of semen collection directly into extender on sperm agglutination. Semen samples were obtained by electroejaculation from 13 adult males. In experiment 1, eleven ejaculates from eight bears were used to evaluate Bioxcell and Andromed as extenders, whereas in experiment 2, nine ejaculates from six bears were used to evaluate Triladyl canine, CaniPro, and Extender 2 as extenders. An extender specifically developed for brown bears (Test-Tris-fructose-egg yolk-glycerol, TTF-ULE/bear) served as a control extender in both experiments. After thawing, total and progressive sperm motility and sperm viability were greater (P < 0.05) for TTF-ULE/bear and Andromed extenders than for Bioxcell in experiment 1 and greater (P < 0.05) for TTF-ULE/bear extender than for Triladyl Canine, CaniPro, and Extender 2 in experiment 2. In experiment 3, addition of handling extender (TTF-H) to the semen collection tube for eight ejaculates from seven bears resulted in less (P < 0.05) sperm agglutination in fresh samples (score 0.5 ± 0.2 vs. 1.8 ± 0.4 in diluted and control samples, respectively) with no effect on pre-freeze and post-thawing semen quality. In conclusion, TTF-ULE/bear is the most suitable extender for brown bear semen cryopreservation, but comparable results can be obtained with the commercial extender Andromed. In addition, collection of ejaculates directly in TTF-H extender decreases sperm agglutination in fresh samples.     

The effect of nocturnal sampling on semen quality and the efficiency of collection in bovine species

This study evaluated night and day semen collection regimes in Holstein and Brahman bulls (four bulls of each breed) that were collected weekly, each during a morning and a night collection. Ejaculates (n=64) were obtained via artificial vagina over 4 weeks. The first collection of each week alternated between night and day. Two collection teams were employed. Bull behavior parameters included reaction time to first mount, time to ejaculation, a refractory period test, and a thrust intensity test. The numbers of interruptions were counted as a managerial parameter. Pre-freeze semen parameters included total volume, initial motility and concentration. Post-freeze semen parameters measured were: 0- and 3-h post-thaw motility; percent intact acrosomes; and percent sperm abnormalities. Data were analyzed by least squares methods. The bull within breed effect differed (P<0.05) for behavior parameters. The bull within breed effect for total motile sperm harvested was not significant. The bull within breed response was mixed for post-freeze semen viability parameters. Bull within breed was not significant for sperm abnormalities. The night versus day treatment was significant for the managerial parameter (P=0.002). Although a different collection schedule for Bos indicus cattle was not warranted, the efficiency of the collection process was affected by extraneous environmental conditions.     

Effects of semen plasma from different fractions of individual ejaculates on IVF in pigs

We applied IVM/IVF techniques to investigate effects of preincubation of sperm with different fractions of semen plasma harvested from fresh ejaculates on in vitro penetration and fertilization of in vitro matured oocytes. Three fractions of semen plasma were separated from the complete ejaculate of three Landrace boars and used to coincubate sperm obtained from the first sperm-rich fraction of the same ejaculates. After 14 to 16 h coincubation at room temperature, sperm were preincubated in capacitation medium and then inseminated into fertilization medium containing porcine oocytes matured in vitro. The semen plasma used for coincubation affected penetration rate (P < 0.001); Sperm coincubated with Fraction 1 semen plasma had a higher penetration rate compared with sperm coincubated with Fraction 2 (P < 0.05), but not with Fraction 3. Boar affected male pronucleus formation rates after insemination (P < 0.05), but no difference among boars was found in monospermy rate, average number of sperm penetrating into each fertilized oocyte, or the average number of sperm attached. No boar by fraction interaction was found for any parameters studied.     

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

Human sperm-cervical mucus interaction and the ability of spermatozoa to fuse with zona-free hamster oocytes

Samples of semen and cervical mucus were provided by 18 couples. Cervical mucus was obtained for each day possible and stored at 4 degrees C until all the samples were collected. Flat capillary tubes were loaded with the mucous samples and spermatozoa from the husband's semen sample were allowed to migrate through the cervical mucus (3 cm column) into culture medium. The spermatozoa recovered after migration through cervical mucus were assayed in vitro with zona-free hamster oocytes. Control experiments were carried out using spermatozoa from the same semen sample but prepared by the swimming-up technique. Altogether, 557 eggs in the control group and 1236 eggs in the experimental group were analysed, and the results demonstrated that the % of sperm penetration, the mean number of sperm decondensations per penetrated egg and the mean number of spermatozoa adhering per egg all had higher values (P less than 0.05) for the control samples than for the experimental samples. We suggest that cervical mucus modifies human spermatozoa, as measured by their interaction with zona-free hamster oocytes.     

Relationship between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen

Assays based on sperm-zona pellucida binding have been developed as diagnostic tests to predict the fertilizing potential of mammalian spermatozoa. Recently, we reported on the development of a sperm-zona pellucida binding assay (SZBA) for bull spermatozoa. The aim of the present study was to develop a hemi-zona assay (HZA) for bull spermatozoa and to investigate the relationship between SZBA and HZA outcomes and in vivo fertility. Frozenthawed semen samples from 8 fertile Swedish Red and White bulls (one ejaculate per bull) designated as the test semen samples and a single ejaculate from a fertile Holstein-Friesian bull designated as the control semen sample were used in this study. In the SZBA, 2 groups of 20 oocytes per semen test sample and in the HZA a minimum of 6 matching pairs of hemizonae were used for comparison of sperm binding with control semen. Sperm binding to matching hemi-zonae of individual semen samples was equal, and clearly demonstrated the feasibility of the HZA for cattle. A significant correlation was found between the SZBA and the HZA indices obtained from the different semen test samples (r = 0.42, P < 0.001; n = 67). There was no significant relation between the SZBA indices and the 56-d nonreturn rate of the test samples. However, the HZA indices of the semen test samples and the 56-d nonreturn rate were significantly correlated (r = 0.46, P < 0.0001; n = 67). It is concluded that HZA can be regarded as a potential assay for predicting the fertilizing ability of bovine semen samples. However, further studies using more semen samples are necessary to confirm this view.     

Induction of autoantibodies against spermatozoa by injection of allogeneic sperm in the Nile Tilapia,Oreochromis niloticus

• 1.1. The sperm-agglutinating factor (SAF) could be induced in the serum of male Nile tilapias, Oreochromis niloticus, by injection of allogeneic sperm.
• 2.2. Only one class of molecules was demonstrated to be SAF in the serum.
• 3.3. Analysis on purified SAF revealed it to be a tetrameric molecule of IgM with a mol. wt of 760kD.
• 4.4. Cross reaction of the IgM with sperm of other teleost species suggests that sperm-specific surface antigens may be in evolution.

     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

Antigenic characteristics of various strains of Campylobacter jejuni. Preliminary note: technic of extraction of soluble thermostable antigens

Immune serum from rabbit has been obtained against Campylobacter jejuni ATCC no 29428 from the same strain an antigen of the outer Envelope was prepared by EDTA extraction. The specificity of antibodies against the outer antigen has been observed by direct agglutination, C.F., passive haemoagglutination and houcherlony test in agar. In eight other strains of Campylobacter isolated from patients, cross reactions with the ATCC no 29428 strain have been stored, by direct agglutination: only one strain (H) cross-reacted with the immune serum in use.     

Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1∶1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.     

Observations on the motility of rabbit spermatozoa in dilute suspension

1. Rabbit spermatozoa suspended in Baker's solution rapidly lose motility at relatively high dilutions. At a concentration of 0.4 million per ml., the spermatozoa from most ejaculates are completely immotile within 2 or 3 hours. The same phenomenon occurs with chloride-free diluents and is therefore not due to the toxic action of chlorides. 2. This rapid immobilisation may be prevented by suspension in cell-free supernatants from other more concentrated suspensions of rabbit semen and by the accessory secretions from a vasectomised buck. The most effective supernatants are those prepared from suspensions of spermatozoa which have been left overnight before centrifuging. Immobilisation may also be prevented by many other agents, the most effective of which are gum arabic, starch, glycogen, and serum proteins (carbohydrate-containing or carbohydrate-free). Yet, in no case is the action of these substances as effective as more concentrated suspension in Baker's solution. 3. The immobilisation is not prevented by catalase, gelatin, agar, or sodium silicate. It is almost certainly not due to the toxic action of traces of heavy metals and is not affected by the use of water doubly distilled over glass in the preparation of the diluent, or by treating the diluent with activated charcoal. 4. Washing with Baker's solution does not cause immobilisation of spermatozoa suspended at 20 million per ml. at a stage at which the concentration of seminal plasma has been reduced to that equivalent to dilution to 0.4 million per ml. Further washing (six repeated centrifugings of 0.2 ml. semen in 4 ml. of Baker's solution) immobilises them. This confirms the opinion that loss of intracellular, or perhaps rather paracellular, material is a responsible agent in the dilution phenomenon.