Swim-up Procedure Research Articles

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats.

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.

Separation of motile spermatozoa from frozen-thawed buffalo semen: Swim-up vs filtration procedures

Three experiments were conducted to maximize the recovery rate of motile spermatozoa from frozen-thawed buffalo semen. In Experiment 1, the swim-up of motile spermatozoa was performed in the presence or absence of HEPES in TALP medium and CO 2 in the environment. The recovery rate of motile spermatozoa in TALP medium (control), TALP+HEPES+CO 2, TALP+HEPES and TALP+CO 2 was 15, 18, 12 and 10%, respectively (P>0.05), with sperm motility at 87, 89, 90 and 90%, respectively (P>0.05). In Experiment 2, the pH of TALP medium was adjusted to 7.0, 7.5, 8.0, 8.5 and 9.0, and swim-up procedure was performed in the presence of HEPES and CO 2. The recovery rate of motile spermatozoa at different pH was 14, 20, 24, 27 and 16%, respectively (P<0.05). Motility of separated spermatozoa was 88, 91, 90, 89 and 90%, respectively (P>0.05). In Experiment 3, the efficiency of ion-exchange filtration and Swim-up procedure in separating motile spermatozoa from frozen-thawed buffalo semen was compared. The recoveryrate of motile spermatozoa was 95% in filtration procedure and 33% in swim-up procedure (P<0.005). In all experiments, normal acrosomes did not vary due to treatments (P>0.05). In conclusion, HEPES and C02 had no significant effect on swim-up of buffalo spermatozoa. The pH 8.5 of TALP improved the recovery rate of motile spermatozoa in swim-up procedure. The ion-exchange filtration was found superior to swim-up procedure in harvesting maximum number of motile spermatozoa from frozen-thawed buffalo semen (95 vs 33%; P<0.001).

Sperm characteristics and zona pellucida binding in relation to field fertility of frozen–thawed semen from dairy AI bulls

The present study examined the relationship between bull sperm characteristics immediately post-thaw and some characteristics registered after swim-up, including the ability of spermatozoa to bind to homologous zona pellucidae (ZP) in vitro, and fertility after artificial insemination (AI) of 9426 females. Frozen-thawed semen from 22 AI bulls of the Swedish Red and White Breed, represented by 43 different frozen batches (1-4 batches/bull, 2 consecutive ejaculates/batch), was examined with the aim of determining concentration, motility patterns, morphology and membrane integrity. In addition, the frozen-thawed spermatozoa were subjected to a swim-up procedure and those separated in this way were tested with two assays of sperm-binding to the ZP of homologous oocytes in vitro (ZBA), using either a relative ZBA index against a control bull of proven high fertility or absolute binding (Absolute ZBA). The correlations of the various sperm traits and 56-day non-return rates (NRR) after field AI were retrospectively examined as single traits and as combinations of traits (combined measures), including regression analysis of significant traits. Among the sperm characteristics, positively significant (p < 0.01) correlations with NRR were found for linear motility post-thawing (r = 0.45-0.59) and the concentration of motile spermatozoa after swim-up (r = 0.43-0.63). Results obtained with the absolute ZBA approach were significantly (p < 0.05) correlated with NRR (r = 0.50), whereas the correlation between NRR and the ZBA index was not significant. The use of combined measures of sperm traits, including the ability to bind to ZP, showed a stronger predictive correlation with NRR (r = 0.68-0.75), compared with single traits. The results suggest that the combined analysis of sperm linear-motility patterns, swim-up separated sperm motility and absolute ZBA can provide a valuable assessment of the fertilizing capacity of AI bull semen.

Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida.

The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The number of spermatozoa bound to a hemizona in the presence of 15 and 150 ng/mL progesterone was significantly higher (p < 0.05) than the number of spermatozoa bound in the absence of progesterone. A higher incidence of the AR was found in sperm-hemizona complexes incubated in the presence of progesterone (55.6 +/- 3.4% vs. 27.1 +/- 4.3%, in the presence and absence of progesterone, respectively) (n = 15, p < 0.05). It is concluded that mare FF can induce the AR in stallion spermatozoa. Progesterone is the physiological component responsible for this AR-inducing capacity. Progesterone enhances sperm-zona binding activity and exerts an additive effect on the zona-induced AR.

Developmental competence of immature and in-vitro matured bovine oocytes after exposure to various osmolalities

A swim-up procedure has been developed where spermatozoa from frozen-thawed bull semen were allowed to swim through a medium which contained hyaluronic acid (HA; 1 mg ml−1) to a modified Tyrode's lactate solution (Fert-TALP) commonly used for in vitro fertilization in the bovine. A control medium was prepared with the same constituents, with the exception of HA. Heparin (5 μg ml−1) was added to the Fert-TALP to induce capacitation of the spermatozoa. The separated spermatozoa from two bulls were either studied for concentration, motility and membrane integrity or, without subjecting them to further washing, used to inseminate in vitro-matured oocytes. After 20–22 h of sperm-oocyte co-incubation, the oocytes were denuded by repeated pipetting and then either morphologically examined (fixed in acetic acid-methanol (1:3) and stained with 1% orcein) for the evidence of fertilization or co-cultured with bovine oviduct epithelial cells for an additional 6 days to follow the development into morulae and blastocysts. The HA-selected spermatozoa showed better motility (P < 0.003) than the control medium-selected spermatozoa. Scanning electron microscopy showed that the surfaces of the HA-selected spermatozoa were cleaner than those of control medium-selected and pre-selected spermatozoa. The proportions of spermatozoa with intact plasma membrane and acrosome increased (P < 0.05) after swim-up through the HA-containing medium. All the 200 swim-up-separated spermatozoa scanned individually showed intact acrosome whether HA was added to the medium or not, which indicates that the acrosome-damaged spermatozoa were either moving slowly or were incapable of traversing the swim-up medium. The percentages of oocytes which were fertilized and cleaved to at least two-cell stage after insemination with HA-selected spermatozoa were, depending on the bull, 60–75% and 67–80%, respectively, whereas those of fertilized and cleaved oocytes after insemination with control medium-selected spermatozoa were 52–68% and 58–66%, respectively. The proportion of cleaved oocytes after fertilization with HA-selected spermatozoa was higher, significant in one of the two bulls (P = 0.03), than that found after fertilization with control medium-selected spermatozoa. The percentages of the morulae and blastocysts found after insemination with HA-selected and the control medium-selected spermatozoa were 22–24% and 17–31%, respectively, depending on the bull. The results indicate that the non-traumatic sperm selection following self-migration from frozen-thawed semen through a hyaluronic acid-containing medium to the fertilization medium increase the proportion of normal spermatozoa and thereby improves the percentage of in vitro fertilized oocytes.

Deleterious actions of gossypol on bovine spermatozoa, oocytes, and embryos.

Gossypol (50 and 100 micrograms/ml) decreased the percentage of sperm that completed the swim-up procedure. This effect was not blocked by glutathione monoethyl ester. Cleavage rates were not different between oocytes inseminated with gossypol-treated spermatozoa (10 or 50 micrograms/ml) and oocytes inseminated with control spermatozoa. Development to the blastocyst stage at Day 7 after insemination was reduced when spermatozoa treated with 50 micrograms/ml gossypol were used for fertilization. Gossypol toxicity was evident in cows fed cottonseed meal because erythrocyte fragility was greater than for control cows. However, there were no differences between cottonseed meal and control groups in number of oocytes collected per cow, cleavage rate after in vitro maturation and fertilization, or the proportion of oocytes or embryos that developed to blastocysts. Similarly, exposure of oocytes to 2.5-10 micrograms/ml gossypol during in vitro maturation did not affect cleavage rates or subsequent development. In contrast, addition of 10 micrograms/ml gossypol to embryos reduced cleavage rate. Moreover, development of cleaved embryos was reduced by culture with 5 or 10 micrograms/ml gossypol and tended to be reduced by 2.5 micrograms/ml gossypol. In conclusion, bovine gametes are resistant to gossypol at concentrations similar to those in blood of cows fed cottonseed meal. In contrast, the developing embryo is sensitive to gossypol.

Failure of multitube sperm swim-up for sex preselection

Objective: To use double-label fluorescence in situ hybridization to evaluate a modified swimup procedure that is purported to be effective for preconceptual sex selection. Design: Controlled, blinded study. Setting: University hospital laboratories. Patient(s): Donor males reporting for routine semen analysis. Main Outcome Measure(s): Percentages of X- and Y-bearing spermatozoa in neat semen and in two swim-up fractions, determined using double-label fluorescence in situ hybridization. Result(s): No clinically significant change from a 1:1 ratio was found in the distribution of X- or Y-bearing spermatozoa after double-label fluorescence in situ hybridization following a modified swim-up procedure and irrespective of the time (15, 30, 45, and 60 minutes) allowed for swim-up. Conclusion(s): Using fluorescence in situ hybridization, a modified swim-up procedure was evaluated for its purported ability to skew the relative percentages of X- and Y-bearing spermatozoa. No clinically significant change in the ratio of X- to Y-bearing spermatozoa was detected independent of time. Therefore, clinical application of this procedure should be strongly discouraged.

Monoclonal antibodies as direct probes for human sperm acrosome reaction.

To develop simple and rapid assay procedures for determining human sperm acrosome reaction under various experimental conditions. Specific monoclonal antibodies against human sperm acrosome were generated and utilized as probes for acrosome reaction assays by direct labelling of antibodies with fluorescein-isothiocyanate (FITC). Among the generated monoclonal antibodies, HS-63 was shown to react with antigens in the acrosome content of permeabilized acrosome-intact human sperm, but not with those of the live ones. HSA-10 was found to recognize antigens on the surface of sperm inner acrosome and to react with acrosome-reacted human sperm in suspension. Following a swim-up procedure, highly motile sperm were recovered and incubated in BWW medium at 37 degrees C for 18 h. The percentages of acrosome-reacted sperm were determined at various incubation times by using FITC-labeled HS-63 or HSA-10 as the indicators in 10 min direct immunofluorescent assay. With the FITC-labeled HS-63 probe, the percentage of positively stained human sperm fixed in methanol decreased significantly after 18 h of incubation from > 90 to 70-80%. In contrast, positively stained live human sperm by HSA-10 increased significantly from < 1% to 15-30%. A high correlation was obtained between the use of monoclonal antibodies and PSA (Pissum sativum agglutinin) for direct assessment of human sperm acrosome reaction. In view of the simplicity in assay procedures and evaluations, these FITC-labeled monoclonal antibodies are reliable probes for direct assessment of acrosomal status of human sperm.

Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes

Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment, either compact or expanded, were randomly assigned to IVF or ICSI trials and separately cultured for IVM. Frozen-thawed stallion spermatozoa were prepared for IVF with a swim-up procedure conducted in Talp-Hepes with heparin or for ICSI in Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA). Oocytes for IVF were partially decumulated by pipetting, whereas those for ICSI were totally denuded with 80 UI/ml hyaluronidase. Oocytes were fixed, stained and examined for signs of fertilization the day after IVF or ICSI. The percentage of normally fertilized oocytes showing 2 pronuclei or cleavage was significantly higher with ICSI than IVF (29.8%, 17 57 vs 8.7%, 9 103 ; P < 0.01). Significantly higher fertilization rates were observed in oocytes retrieved with an expanded cumulus when submitted to ICSI procedure as compared with IVF (52.2%, 12 23 vs 17.1%, 6 35 ; P < 0.01), whereas in oocytes recovered with a compact cumulus, fertilization rates were low (14.7%, 5 34 with ICSI and 4.4%, 3 68 with IVF; NS). Embryonal development did not occur after culture following IVF, as indicated by absence of cleavage in any of the 93 inseminated oocytes. Following ICSI, 7 of 55 injected oocytes cleaved, 5 of which had shown expanded cumuli; of the 5, 2 were at the 16-cell stage and one each at the 8-, 3- and 2-cell stage, respectively. The other 2 fertilized oocytes, originating from compact cumuli, reached 4- and 8- cell stages, respectively. These results indicate that ICSI can be applied successfully to in-vitro matured equine oocytes to increase the fertilization rates. In addition, it seems that in vitro cytoplasmic maturation of oocytes issuing from a compact cumulus may not be complete enough to lead to a successful fertilization and that ICSI may be a tool to evaluate ooplasmic maturation.

A dextran swim-up procedure for separation of highly motile and viable ram spermatozoa from seminal plasma

Although washing of sperm cells by centrifugation is a procedure in widespread use, there have been indications that centrifugation may be harmful to the cells. The objective of this study was to develop a modified swim-up technique, without centrifugation, to get a selection of highly motile and viable ram spermatozoa free of semen plasma. Semen collected from 3 rams over a period of a year was pooled into a low, medium and high motility group, and aliquots from each pool were placed beneath a dextran solution and overlaid with medium. The top layer of the medium was collected (and replenished) 4 times at 15-min intervals. Evaluated were the pre- and post-swim-up progressive individual motility, membrane integrity and resistance to a hypoosmotic swelling test (HOS). Semen samples with initial motility < 60% showed the highest relative improvement in all 3 parameters; samples with 65 to 70 and > 70% initial motility improved less but showed final absolute values similar to those in the low motility group and to each other. The first swim-up layer had the highest contamination with semen plasma (17% beta-N-acetylglucosaminidase (NAG), 13% citric acid content) and the lowest motility score. The second, third and fourth fractions were pooled and showed low plasma contamination (2% NAG and 5% citrate), 80% motility, 70% HOS, and 72% viability, up from the pre-swim-up values of 68, 66 and 59%, respectively. Our data suggest that the dextran swim-up procedure is suitable for evaluating ram spermatozoa for in vitro and in vivo procedures in assisted reproduction.

The combination of two semen preparation techniques (glass wool filtration and swim-up) and their effect on the morphology of recovered spermatozoa and outcome of IVF-ET.

The aim of this prospective study was to relate sperm quality, especially sperm morphology, to the outcome of IVF results, when glass wool filtration (GWF) and swim-up were used in combination for the preparation of spermatozoa. A total of 60 ejaculates were analysed. GWF was compared with glass wool filtration/swim-up (GWF-SU) by using aliquots from the same semen samples to increase the precision of the comparison and to establish the cumulative effect of these two semen preparation methods on the morphology of the recovered spermatozoa. Sperm parameters were examined in native semen, in semen preparation samples after GWF and GWF-SU. The mean percentages for motility, morphology and velocity were improved significantly over those in fresh semen only when GWF-SU was used. GWF alone resulted only in a significantly higher recovery of motile spermatozoa. In semen preparation, 10% of spermatozoa with normal morphology appeared to be the cut-off point as there was a significantly higher recovery in fresh semen samples containing > 10% morphologically normal spermatozoa after GWF-SU. Outcome of IVF-ET following preparation with GWF-SU showed better results in comparison to the swim-up procedure alone, though this difference was not statistically significant. The cut-off point was also > 10% morphologically normal spermatozoa. Improvement in all aspects of IVF-ET occurred when native semen contained > 10% morphologically normal spermatozoa. It is concluded that GWF alone did not produce significantly better results but, in combination with swim-up, it resulted in significantly better sperm morphology and in an improved outcome of IVF-ET for fresh semen samples with > 10% morphologically normal spermatozoa.

Effect of Cumulus Cells on In Vitro Fertilization of Bovine Oocytes

Bovine follicular oocytes (n=265) collected from abattoir ovaries were matured for 24 hours in modified Menezo's medium (MMM) supplemented with Na-pyruvat, glucose, FSH and oestrus cow serum (ECS). After maturation, oocytes were randomly divided into three groups. One group of oocytes (Group 1) was denuded from cumulus cells prior to fertilization and another group of oocytes (Group 2) was denuded 20 hours after fertilization. In the third group, oocytes were remained with cumulus cells intact (Control group). All of the oocytes were fertilized with frozen-thawed bull spermatozoa treated with swim-up procedure in a modified MenezoÕs medium supplemented with Na-pyruvat, HEPES, BSA, essential and non-essential amino acid solutions and vitamin solutions. Modified Tyrode-lactate medium supplemented with heparin, Na-pyruvat, BSA and PHE was used for fertilization medium. At the end of the maturation and fertilization in vitro, presumptive zygotes were transferred to the culture medium (modified MenezoÕs medium) and cultured for 7 days. After culture all of the embryos were fixed with 3:1 ethanol: acetic acid for 24 hours and stained with aceto-orcein for microscopic evaluation. Of the 80 oocytes in Group 1, 45 were fertilized (56.25 %). Embryos reached to morula and blastocyst stages were 8 (17.78 %) and 3 (6.67 %) respectively. In Group 2, of the 89 oocytes, 55 were fertilized (61.80 %). Embryos reached to morula and blastocyst stages were 8 (14.55 %) and 3 (5.45 %) respectively. In control group (n=96), 49 oocytes were fertilized (51.04 %). Embryos reached to morula and blastocyst stages were 12 (24.49 %) and 9 (18.37 %) respectively.

Detection of feline immunodeficiency virus in semen from seropositive domestic cats (Felis catus).

Electroejaculates from experimentally infected domestic cats were evaluated for the presence of feline immunodeficiency virus (FIV). Virus was isolated from cell-free seminal plasma and seminal cells by cocultivation with a feline interleukin-2-dependent CD4+ T-cell line, in which productive infection was demonstrated by syncytium formation and FIV gag p26 antigen secretion. In addition, an 868-bp segment of the FIV gag provirus gene was identified in cocultured cells by PCR and Southern analysis. A 582-bp fragment of the FIV gag provirus genome was detected by nested PCR and Southern analysis in nonfractionated seminal cells and in sperm purified by a swim-up procedure. This is the first report describing the detection of replication-competent FIV in cell-free and cell-associated forms in domestic cat semen.

Fertilization and early embryology: Intracytoplasmic sperm injection of mouse oocytes with 5 mM Ca2+ at different intervals

The objective of this investigation was to determine whether intracytoplasmic sperm injection (ICSI) can be performed in the mouse. Metaphase II oocytes were obtained from F1 hybrid mice (C57BL x CBA) by i.p. injections of 10 IU pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) administered 48 h apart. Oocytes with cumulus oophorus were retrieved 13-14 h post HCG. Cumulus was dispersed with 0.1% hyaluronidase. Mouse spermatozoa were obtained from the cauda epididymides of males of the same strain. The spermatozoa were processed by the standard swim-up procedure. The harvested spermatozoa were then incubated for 1.5 h to allow capacitation. Healthy oocytes were injected with 3-4 pl 5 mM Ca2+, followed by one live morphologically normal spermatozoon into the cytoplasm at intervals of 0, 0.5, 1, 2 and 3 h. The proportion of 2-cell embryos that developed from oocytes injected with Ca2+ and spermatozoa ranged between 29.5 and 36.5% in all groups, with no statistical difference between treatments. Chromosomal analysis showed that two-thirds of the ICSI-derived 2-cell embryos were diploid. The proportion of parthenogenetically activated embryos in the ICSI groups was similar to that in the control group (8-10%) which was injected with Ca2+ and polyvinyl pyrrolidone only. The proportion of blastocysts that developed in culture from the ICSI-derived 2-cell embryos was of the order of 36-42%. Some blastocysts were used for cell number counts. There was a significant increase in total and inner cell mass counts of blastocysts in which the spermatozoon was injected at 2 and 3 h following Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Sperm recovery and survival: two tests that predict in vitro fertilization outcome

To determine if human sperm recovery during swim-up and sperm survival after 24 hours, as obtained from a screening semen specimen, are predictive of subsequent IVF and clinical pregnancy rates (PRs) and to determine if these techniques can identify men with normal semen analysis parameters and poor IVF success. Historical prospective study. All semen evaluations and IVF cycles were performed at the University of Pittsburgh, Magee-Womens Hospital, Pittsburgh, Pennsylvania. Couples undergoing IVF at Magee-Womens Hospital from August 1988 through June 1993. A screening semen analysis and swim-up procedure were performed on all couples undergoing IVF. The number of spermatozoa recovered after swim-up and the percentage of motile spermatozoa present after a 24-hour incubation were recorded. Fertilization and PRs were compared according to the parameters obtained from routine semen analysis, the number of spermatozoa obtained with swim-up, and the percentage of motile spermatozoa at 24 hours. Using chi2 or Fisher's exact test, fertilization rates were significantly different according to the number of spermatozoa recovered after swim-up (< or = 2.0 and > 2.0 x 10(6) spermatozoa recovered, 48.3% versus 71.4%) as were PRs (16.9% versus 29.8%). Similarly, the percentage of motile spermatozoa present at 24 hours (< or = 20% and > 20%) discriminated between fertilization rates (45.9% versus 65.8%) and PRs (16.4% versus 36.5%). Among a subset of men with normal semen analyses and total motile sperm counts > or = 40 x 10(6), the results from swim-up and survival discriminated between men with high and low fertilization and PRs. Receiver operating characteristic analysis revealed that swim-up results better discriminated between pregnant and nonpregnant IVF patients than sperm motility, but that the percentage of motile spermatozoa present at 24 hours was no better in this regard than sperm motility. The number of spermatozoa recovered after swim-up and the percentage of spermatozoa that maintain their motility after 24 hours were both helpful in assessing IVF and PRs and may be helpful in altering physicians to a subset of men having normal semen analysis parameters yet poor IVF success.

Effects of antioxidants on human sperm preparationtechniques

The effect of two different sperm preparation techniques, Percoll gradient centrifugation and swim-up from a washed pellet were tested on the functional competence of the selected spermatozoa. Percoll gradient centrifugation brought about an improvement in sperm motility parameters such as curvilinear velocity and straight-line velocity, an increase in the rates of hyperactivation and the acrosome reaction and an increase in the percentage of motile spermatozoa after 24 h of incubation compared to the centrifugation, swim-up procedure. The effects of antioxidants such as dithiothreitol (DTT) or reduced glutathione (GSH), and reactive oxygen species-scavenging enzymes such as catalase or superoxide dismutase (SOD) during the stage of centrifugation before the swim-up procedure were also studied. Though all of these agents prevented the fall in sperm motility after 24 h incubation, only DTT and SOD improved the rates of both hyperactivation and the acrosome reaction. GSH also improved the acrosome reaction, whereas catalase was without significant effect on the rates of hyperactivation or the acrosome reaction. These results indicate that Percoll gradient centrifugation selects spermatozoa with better functional competence than does centrifugation swim-up. The damage caused by the centrifugation can be prevented by the addition of antioxidants, suggesting that the differences noted with the Percoll gradient method was due to better protection against peroxidative damage due to the centrifugation of unselected spermatozoa. However, the use of DTT is limited by virtue of the fact that this sulphydryl reducing agent leads to destabilization of the sperm chromatin. In contrast, GSH and SOD could have therapeutic potential.

A simple, non-traumatic swim-up method for the selection of spermatozoa for in vitro fertilization in the bovine

A swim-up procedure has been developed where spermatozoa from frozen-thawed bull semen were allowed to swim through a medium which contained hyaluronic acid (HA; 1 mg ml −1) to a modified Tyrode's lactate solution (Fert-TALP) commonly used for in vitro fertilization in the bovine. A control medium was prepared with the same constituents, with the exception of HA. Heparin (5 μg ml −1) was added to the Fert-TALP to induce capacitation of the spermatozoa. The separated spermatozoa from two bulls were either studied for concentration, motility and membrane integrity or, without subjecting them to further washing, used to inseminate in vitro-matured oocytes. After 20–22 h of sperm-oocyte co-incubation, the oocytes were denuded by repeated pipetting and then either morphologically examined (fixed in acetic acid-methanol (1:3) and stained with 1% orcein) for the evidence of fertilization or co-cultured with bovine oviduct epithelial cells for an additional 6 days to follow the development into morulae and blastocysts. The HA-selected spermatozoa showed better motility ( P < 0.003) than the control medium-selected spermatozoa. Scanning electron microscopy showed that the surfaces of the HA-selected spermatozoa were cleaner than those of control medium-selected and pre-selected spermatozoa. The proportions of spermatozoa with intact plasma membrane and acrosome increased ( P < 0.05) after swim-up through the HA-containing medium. All the 200 swim-up-separated spermatozoa scanned individually showed intact acrosome whether HA was added to the medium or not, which indicates that the acrosome-damaged spermatozoa were either moving slowly or were incapable of traversing the swim-up medium. The percentages of oocytes which were fertilized and cleaved to at least two-cell stage after insemination with HA-selected spermatozoa were, depending on the bull, 60–75% and 67–80%, respectively, whereas those of fertilized and cleaved oocytes after insemination with control medium-selected spermatozoa were 52–68% and 58–66%, respectively. The proportion of cleaved oocytes after fertilization with HA-selected spermatozoa was higher, significant in one of the two bulls ( P = 0.03), than that found after fertilization with control medium-selected spermatozoa. The percentages of the morulae and blastocysts found after insemination with HA-selected and the control medium-selected spermatozoa were 22–24% and 17–31%, respectively, depending on the bull. The results indicate that the non-traumatic sperm selection following self-migration from frozen-thawed semen through a hyaluronic acid-containing medium to the fertilization medium increase the proportion of normal spermatozoa and thereby improves the percentage of in vitro fertilized oocytes.

Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance.

Experiments were conducted to determine the water volume and osmotic behaviour of mouse spermatozoa using an electron paramagnetic resonance technique using the spin label tempone, and the broadening agent potassium chromium oxalate. After a swim-up procedure, an average water volume of 43.3 micron3 of individual spermatozoa was obtained at 290 mosmol. If a water compartment of 59% is assumed, the total volume of mouse spermatozoa is 73.4 micron3. A plot of the relative water volume of mouse spermatozoa versus the reciprocal of buffer osmolality (Boyle van't Hoff plot) is linear in the range 250-900 mosmol of sodium chloride solutions (r2 = -.96). The Boyle van't Hoff plot intercept indicates that 13% of the spin-label accessible isotonic water is osmotically inactive.

In vitro fertilization of follicular oocytes of domestic cat using fresh and cryopreserved epididymal spermatozoa

The in vitro fertilizing competence of fresh and frozen epididymal sperm of domestic cat was compared. The average portions of intact and motile spermatozoa amounted to 42.8% and 75%, respectively, immediately after preparation, and decreased after cryopreservation to values of 27.8% and 53%, respectively. Enrichment of vital frozen spermatozoa by a swim-up procedure provided a motility level similar to that of fresh spermatozoa (74%). The spermatozoa showed a high rate of spontaneous acrosome loss, especially after thawing. Therefore, the number of intact frozen cells with induced acrosome reaction (0.005% hyamine for 20 min at 38°C) was reduced significantly. Oocytes were obtained from antral follicles of ovariectomized cats, matured in vitro for 48 h and inseminated in vitro with fresh or frozen spermatozoa. The insemination doses contained comparable sperm concentrations and percentages of motile spermatozoa. However, the cleavage rates were significantly lower for zygotes produced by frozen spermatozoa ( 43 170 = 25.3% ) than for zygotes produced by fresh spermatozoa ( 61 150 =40.7% ). The rates of production of cat embryos using epididymal sperm might be a useful basis for application of these techniques in other feline species.

Evaluation of different sperm separation techniques for harvesting equine spermatozoa intended for in vitro culture

Summary Sperm separation techniques have been utilized for enhancing success of in vitro fertilization in humans. Similar techniques were evaluated in an attempt to isolate populations of highly motile equine spermatozoa for research investigating in vitro fertilization and spermatozoal maturation. Spermatozoa were harvested from ejaculates (n = 9) of three stallions. Ejaculates were split to compare the efficacy of Sperm Selec a (sodium hyaluronate), SpermPrep b (sephadex), Perception c (percoll) and swim-up procedures in the recovery and maintenance of a population of highly motile spermatozoa. The recovery of motile and viable spermatozoa was similar (P>.05) among Perception, SpermPrep and swim-up procedures, whereas the Sperm Select procedure yielded a lower (P