An auto-controlled study in in-vitro fertilization reveals the benefit of Percoll centrifugation to swim-up in the preparation of poor-quality semen

The efficiency of spermatozoa prepared by swim-up or by Percoll centrifugation was assessed in an in-vitro fertilization programme on 71 semen samples of a well-defined quality [total number of type A (WHO criteria) motile spermatozoa]: category I (n = 21) with > 100 x 10(6), II (n = 31) with 15-100 x 10(6), III (n = 11) with 5-15 x 10(6) and IV (n = 8) with < 5 x 10(6) type A motile spermatozoa. Oocytes were inseminated 4 h after oocyte retrieval, alternately with spermatozoa derived from swim-up and Percoll preparation. Both selection procedures resulted in a significantly higher (P < 0.001) percentage motility as compared to fresh semen. For low-quality samples (III and IV), however, swim-up was more effective in selecting highly motile (P = 0.004) and morphologically normal spermatozoa (P < 0.05). For high-quality samples, this difference might have been masked by introducing a swim-up step to remove Percoll particles. Regardless of the initial sperm quality, the mean fertilization rate was significantly higher (P = 0.003) when Percoll-treated spermatozoa were used for insemination (51.3 versus 37.8%). For semen of groups I and II, no difference in fertilization capacity was observed according to the sperm preparation method. Despite the lower percentage motility and normal morphology for the Percoll compared to the swim-up treatment in groups III and IV, fertilizing capacity was significantly (P < 0.001) in favour of this selection method (65.3 versus 26.5% in group III, 47.6 versus 11.6% in group IV).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rotational digital subtraction carotid angiography: technique and comparison with static digital subtraction angiography

To compare the efficiency of sperm preparation between the two-layer Percoll gradient and mini-Percoll methods, 50 normal and 33 abnormal semen samples from male partners of infertile couples were studied. The number of recovered spermatozoa, percentage of motility, percentage of normal morphology, and their survival at 24 and 48 hours were assessed. Both Percoll gradient techniques resulted in a significantly higher percentage of motility and percentage of normal morphology compared with the original semen samples (p < 0.0001). The two-layer Percoll gradient showed a higher sperm recovery than the mini-Percoll method (p < 0.001), but the latter resulted in a higher percentage of motility (p > 0.001) and a higher sperm survival rate at 24 hours (p < 0.05) than the former, regarding normal semen samples. These differences did not appear with abnormal semen samples when analyzed as a group. Considering each of the abnormal parameters separately, sperm recovery was significantly higher after the two-layer Percoll gradient in the case of astheno- and teratozoospermia (p < 0.05), but sperm survival at 48 hours was higher after the mini-Percoll gradient in the case of teratozoospermia (p < 0.05). It is concluded that both the two-layer Percoll gradient and mini-Percoll method can be used effectively for sperm preparation. The former yields a higher sperm recovery, but the latter should be considered regarding teratozoospermic samples and semen samples of very low volume.     

Comparison of single- and two-layer Percoll separation for selection of motile spermatozoa

OBJECTIVE: Sperm recovery using a single-layer Percoll procedure is significantly better than using the swim-up technique for infertile men and patients with normal sperm characteristics; however, in normal men results have been contradictory. Some studies have shown further improvement in semen quality with multiple layers. Therefore, this study compared the effect of single-layer and two-layer Percoll procedures on sperm characteristics of normozoospermic men. METHODS: Semen specimens from 10 normal donors were processed by layering 1 mL of the liquefied ejaculate on a single layer of 80% Percoll or on a two-layer (47% and 90%) Percoll gradient. Computer-assisted semen analysis was done to examine total motile sperm, percentage of recovery of motile cells, percent motility, curvilinear velocity, linearity, and amplitude of lateral head displacement. Each specimen was evaluated by the hypo-osmotic swelling (HOS) test, bovine cervical mucus penetration test, viability (eosin-nigrosin stain), and sperm morphology (World Health Organization and Kruger's strict criteria). RESULTS: Specimens processed with the two-layer Percoll procedure had significantly better recovery of spermatozoa, and significantly better percentage motility, linearity, amplitude of lateral head displacement, percentage tail swelling, and percentage viability than those separated on single-layer Percoll. Results for sperm morphology using WHO and Kruger's criteria were similar between the two methods (P = 0.92 for both sets of criteria). CONCLUSIONS: In normozoospermic men, the two-layer Percoll separation procedure significantly improves semen characteristics compared with separation on a single layer.     

Sperm morphology using strict criteria after Percoll density separation: influence on cleavage and pregnancy rates after in-vitro fertilization

The main purpose of the study was to evaluate the use of sperm morphology assessment by strict criteria on the post-Percoll separated spermatozoa used for oocyte insemination in an in-vitro fertilization programme. This study included a consecutive unselected series of 213 oocyte aspirations in 159 women. In 177 aspirations the patient had tubal infertility and in 36 unexplained infertility. Data have been analysed from 197 aspirations where the semen sample used for insemination had a normal sperm concentration (> or = 20 x 10(6)/ml). A total of 1413 oocytes were aspirated, resulting in 863 oocytes which were fertilized and cleaved (cleavage rate 61%). In all, 492 pre-embryos were transferred in 193 cycles, resulting in a pregnancy rate of 42% per transfer. Sperm morphology evaluation using strict criteria showed that Percoll separation significantly increased the percentage of sperm cells with normal morphology from 7.7 to 11.3%. Sperm morphology analysis showed that Percoll separation decreased the number of sperm samples in the 'poor prognosis pattern' group from 31 to 13% and increased the number of sperm samples classified as 'normal' from 16 to 33%. After Percoll separation the poor prognosis pattern group had a cleavage rate of 46%, which was significantly lower than in the good prognosis pattern and the normal groups. However, the poor prognosis pattern group had a significantly higher pregnancy rate than the normal group (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsatellite deletion mapping on chromosome 10q and mutation analysis of MMAC1, FAS, and MXI1 in human glioblastoma multiforme

The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore-induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore-induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.     

Enhancement of motility by treating spermatozoa with an antioxidant solution (Sperm-Fit) following ejaculation

Oxygen radical generation is known to be detrimental to sperm function, especially motility, through the lipid peroxidation of the membranes. Generation of reactive oxygen species can be induced by leukocyte contamination, sperm centrifugation and the presence of abnormal spermatozoa with excess residual cytoplasm. This study aims to evaluate the effect on sperm motility of incubation in an antioxidant-containing solution, during liquefaction and centrifugation. Thirty semen samples were each divided into two equal parts: one mixed with Tyrode's solution, the other with a salt solution containing antioxidants (Sperm-Fit; Ellios Bio-Media, Paris, France). All the procedures were identical in the two groups. The ratio of leukocytes to spermatozoa was significantly correlated with the motility after liquefaction and after a 24 h incubation in routine in-vitro fertilization (IVF) medium and with the number of motile spermatozoa recovered after Percoll preparation. Moreover, when this ratio was > or = 0.2, all motility parameters were lowered. Incubation with Sperm-Fit allowed a higher percentage of motility after Percoll preparation when the ratio was > or = 0.2 (48 +/- 5% versus 41 +/- 6% for Sperm-Fit and Tyrode's solution respectively; P < 0.05) and a greater number of motile spermatozoa recovered after Percoll preparation, whatever the ratio (3.2 +/- 1.0 x 10(6) versus 2.4 +/- 0.7 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio > or = 0.2; 18.1 +/- 3.4 x 10(6) versus 14.4 +/- 2.9 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio < 0.2; P < 0.05). These results show that incubation with antioxidants during liquefaction and centrifugation increases recovery of motile spermatozoa.     

Spermatozoa selection by the swim-up procedure and two-layer percoll gradient centrifugation

The efficiency of the swim-up procedure and two-layer Percoll gradient centrifugation in procession of spermatozoa was assessed in ejaculates from 47 infertile men. A significantly higher total number of spermatozoa was harvested from Percoll gradients than from the swim-up procedure, the loss rates in concentration being -13.6 +/- 6.4% and -70.8 +/- 5.8%, respectively (p < 0.0001). Recovery in per cent motility was significantly higher after the Percoll gradient than after the swim-up procedure (34.8 +/- 10.2% versus -10.4 +/- 17.2%, p < 0.05). No significant difference was noted between the mean motility grades of the final solutions obtained by the two methods (2.7 +/- 0.2 and 2.0 +/- 0.4, respectively, p > 0.05). When evaluation was conducted within three initial fresh sample concentration categories such as severe oligospermia (lower than 5 x 10(6)/ml), moderate oligospermia (5 to 10 x 10(6)/ml) and mild oligospermia (higher than 10 x 10(6)/ml), the Percoll technique recovered significantly higher number of spermatozoa than the swim-up procedure through all concentration categories (p < 0.05 for each range). Despite being statistically insignificant, Percoll gradients produced final spermatozoa pools with higher per cent motility and motility quality within all concentration ranges. The results suggest that the Percoll gradient centrifugation should be the preferred selection method regardless of the initial fresh sample concentration.     

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods

The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep II column. The Percoll density gradient method was compared with the SpermPrep II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morphometric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swimup method.     

Dynamic nuclear polarization at very low magnetic fields

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.     

Full-term delivery following intracytoplasmic sperm injection with frozen-thawed immotile testicular spermatozoa

We present one of the very few deliveries occurring following intracytoplasmic sperm injection of thawed immotile testicular spermatozoa from a testicular biopsy of a man with bilateral congenital absence of the vas deferens. A first attempt in the 33 year old woman with fresh testicular biopsy extracted spermatozoa was unsuccessful. The supernumerary spermatozoa were cryopreserved for later use. After thawing, testicular spermatozoa were immotile. From 11 intact oocytes injected with frozen-thawed immotile testicular spermatozoa, a two pronuclear fertilization rate of 27% and a cleavage rate of 100% were obtained. A total of three embryos was transferred resulting in a singleton pregnancy and the birth of a normal female baby.     

Comparison of various methods of processing human cryopreserved-thawed semen samples

We compared the efficacy of various methods of processing cryopreserved-thawed samples for the recovery of functionally adequate spermatozoa as assessed by the response to the sperm stress test (SST), an index of temperature activated sperm membrane lipid peroxidation, and immediate and delayed changes in sperm viability and motion parameters. Donor semen samples (n = 28) were cryopreserved-thawed and divided into six equal parts, one part was used as control and the remaining parts were used to compare five methods of sperm processing as follows: direct Percoll gradient processing, washing by one-step or stepwise addition of the washing medium followed by Percoll processing, and washing by one-step or stepwise addition of the washing medium. Additional samples (n = 10) were evaluated for the immediate and delayed (6 h at 37 degrees C) impact of one-step and stepwise washing (without Percoll separation). Compared with wash-only methods, samples processed using Percoll had a significantly higher SST score (P = 0.001), motility, rapid spermatozoa (>50 microm/s), curvilinear velocity and motility index (P < 0.001). Comparing various Percoll methods, direct Percoll processing resulted in the highest number of motile spermatozoa recovered (P < 0.00001) and a higher SST score based on curvilinear velocity (P = 0.001). Stepwise washing gave a significantly higher number of motile spermatozoa (P < 0.001) but with a significantly lower SST score based on the concentration of motile spermatozoa (P = 0.001), motility (P = 0.001) and motility index (P = 0.01). Sperm viability and motion parameters after 6 h of incubation showed no difference between one-step and stepwise washing. In conclusion, compared with wash-only methods, Percoll processed samples resulted in the recovery of spermatozoa with superior quality as assessed by SST and motion analysis. One-step washing of the samples gave an overall comparable recovery compared to the samples prepared stepwise. Having significantly higher SST scores, similar viability and the maintenance of motility, one-step washing may be a better method of processing thawed samples than the stepwise washing.     

Neuropilin is a semaphorin III receptor

Testicular biopsy has been widely used for the diagnosis of male infertility. Since the introduction of intracytoplasmic sperm injection (ICSI), sperm recovered from a testicular biopsy specimen can be successfully used for establishing pregnancies. Testicular spermatozoa may be recovered from testicular tissue in patients with excretory azoospermia but also in many patients with secretory azoospermia. In the latter patients spermatozoa may be recovered only after multiple excisional testicular biopsies, irrespective of follicle-stimulating hormone level, testicular size or medical history. Less invasive techniques such as percutaneous fine-needle aspiration have been introduced and may yield comparable success rates in patients with normal testicular function. The use of cryopreserved testicular spermatozoa may become an alternative to repeated surgery for obtaining testicular tissue for subsequent ICSI treatment cycles if larger series confirm the preliminary case reports. The introduction of the use of testicular spermatozoa for ICSI has raised new concerns because potentially genetically immature germ cells are being used from patients who may carry genetic defects causing their infertility problems.     

Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods

Using three-colour fluorescence in-situ hybridization, sex chromosome ratios and frequencies of diploidy and disomy for chromosomes X, Y and 18 were compared in spermatozoa of good and poor motility after separation by swim-up, glass-wool and two-layer discontinuous Percoll methods. Semen samples were collected from seven normal males aged 26-31 years. A minimum of 6000 sperm nuclei per sample were evaluated for each chromosome for a total of 308,432 sperm nuclei. Hybridization efficiency was 99.8%. A slight change in the ratio of X- to Y-bearing spermatozoa was noted after Percoll separation (from 49.3:49.5 to 50.0:48.9; P = 0.036 and P = 0.046), but not after separation by the other two methods. We did not observe significant differences in the disomy rates for sex chromosomes or chromosome 18 or in the diploidy rate between spermatozoa with good and poor motility after separation by any of the three methods. Our data indicate that separation of motile spermatozoa does not alter the ratio of X- to Y-bearing spermatozoa to a degree that represents sex chromosome selection.     

Morphology, fine structure, biochemistry, and function of the spermatic ducts in marine fish

The spermatic ducts and the testicular efferent ducts were investigated in different marine teleost fish species (Diplodus sargus, Mullus barbatus, Thalassoma pavo, Trachinus draco, Uranuscopus scaber, Sparisoma cretense, Synodon saurus). From the morphological, histological, fine structural and biochemical investigations it appeared that the testicular main ducts and spermatic ducts of the investigated marine fish have the following functions: storage of spermatozoa, monosacharide synthesis for nutrition of spermatozoa, synthesis of steroid glucuronides, synthesis of seminal plasma proteins, formation of a ionic gradient in the seminal fluid and phagocytotic activity. Species-specific differences were only found in the morphology of the gonads and in the histology of the spermatic duct epithelium.     

Testicular morphological changes in children with acute lymphoblastic leukemia following chemotherapy

Morphological changes in the testis induced by chemotherapy given according to the Tokyo Children's Cancer Study Group (TCCSG) regimens were studied in children with acute lymphoblastic leukemia (ALL). After informed consent, testicular biopsies were performed 14 times in 12 patients at the end of treatment. The testicular morphology in all cases had sustained a degree of damage. The tubular fertility index (TFI), calculated as the percentage of seminiferous tubules containing identifiable spermatogonia, was from 0 to 42.8% (mean 33.4%) below the normal value. Infiltration of leukemic cells was the most significant factor contributing to the decrease in TFI. There were no differences in the TFI among the TCCSG protocols. Formation of sperm was recognized in six cases, whose ages were 7, 8, 9, 10, 15 and 19 years. In two children, testicular biopsy was performed twice. In the second biopsy, TFI was elevated and sperm formation with the maturation of Leydig cells was observed. A number of other pathological changes were observed: modification of spermatogonia, Sertoli cells and inclusion bodies in spermatogonia, abnormal maturation of Leydig cells, evidence of interstitial fibrosis and thickening of the basement membrane. These results suggest that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue. As chemotherapy escalates, more investigation of testicular function will be necessary.     

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.     

Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia

The efficiency of testicular sperm retrieval by testicular fine needle aspiration (TEFNA) was compared with open biopsy and testicular sperm extraction (TESE), in 37 rigorously selected patients with non-obstructive azoospermia. All patients underwent TEFNA and TESE consecutively. Thus, each patient served as his own control. The case was regarded as successful if at least one testicular spermatozoon was found allowing intracytoplasmic sperm injection (ICSI) of at least one oocyte. The mean age of the male patients was 32.7 years (range 24-47). Whereas by TEFNA spermatozoa enabling performance of ICSI were found in only four patients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16 cases (43%). The negative predictive value of high serum follicle stimulating hormone (FSH) concentrations (> or =10 IU/l) (predicting failure to find spermatozoa for ICSI) was low (38.4%). The positive predictive value (predicting the chance to find spermatozoa for ICSI) of normal-sized testicle was not different from that of small-sized (<15 ml) testicle (50%). Complications included one case of testicular bleeding following fine needle aspiration, treated locally, and two cases of extratunical haematomata following TESE requiring no intervention. In patients with non-obstructive azoospermia, TEFNA has a significantly lower yield compared to TESE. Performance of ICSI with testicular sperm in these cases resulted in satisfactory fertilization and high embryo transfer rates. The implantation and pregnancy rates per embryo transfer were 13 and 29% respectively. Neither serum FSH values nor testicular size were predictive of the chances to find spermatozoa for ICSI. Some complications may occur even following TEFNA.     

Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection

The microinjection of completely immotile spermatozoa may impair the outcome of intracytoplasmic sperm injection (ICSI). Eleven couples underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa. Two-pronuclear fertilization ensued in 18 of 145 (12.4%) successfully injected oocytes. None of these cycles resulted in a pregnancy. Nine couples underwent ICSI in subsequent cycles (n = 16). Ejaculated spermatozoa were injected in 15 cycles and testicular spermatozoa in one cycle. In 10 of the 15 cycles, motile spermatozoa were available at the time of injection. Motile testicular spermatozoa could also be injected. In the subsequent cycles, 91 of 176 (51.7%) successfully injected oocytes fertilized normally and four patients became pregnant. In the subsequent cycles where again immotile spermatozoa had to be injected no pregnancies occurred. In four subsequent cycles embryo cryopreservation was carried out. After replacement of two frozen-thawed embryos one additional pregnancy was obtained. In all, five healthy infants were born. It has been ascertained that motile spermatozoa can be detected either in repeated ejaculates or after testicular biopsy. The causes of total asthenozoospermia are variable and the problem is a sporadic rather than a permanent condition.     

Discontinuous Percoll gradient preparation for donor insemination: determinants for success

The use of cryopreserved donor spermatozoa for insemination has become necessary to decrease the risk from sexually transmitted infectious diseases. Lower fecundity rates have been reported with this practice. Efforts have been applied to increase success, including identification of those sperm characteristics which correlate with increased fecundity. Data from in-vitro fertilization have revealed sperm morphology, motility and zona pellucida binding as important sperm parameters. Discontinuous Percoll gradient preparation yields a high concentration of motile spermatozoa. Using this preparation for thawed donor spermatozoa, we have identified post-preparation motility and progression as factors associated with increased fecundity. Consideration should be given to screening sperm donors with a freeze-thaw Percoll gradient preparation prior to acceptance into a donor bank.     

External ionic conditions, internal pH and motility of ram and boar spermatozoa

Internal pH and motility of testicular, epididymal and ejaculated ram and boar spermatozoa were studied as a function of external ionic composition. Internal pH was estimated by the amine distribution method and motility was characterized by percentage of cells that were motile and flagellar beat frequency. Upon dilution in media at different external pH values, internal pH of boar and ram spermatozoa changed rapidly towards the external pH. High external concentrations of Na+ or K+ had no effect on the rate of equilibration and only a slight effect on the final internal pH value, ruling out a role of Na(+)-H+ or K(+)-H+ exchange mechanisms in this process. In both species, a linear relationship was observed between internal and external pH but equilibration was incomplete suggesting that there is a complex regulatory mechanism. This result was unaffected by epididymal maturation and ejaculation. Ram and boar testicular spermatozoa showed no increase in movement after dilution, suggesting that simple changes in internal pH are not a sufficient trigger for motility. At high external pH, internal pH increased and motility of epididymal boar spermatozoa was initiated. Motility of ejaculated boar spermatozoa, and epididymal and ejaculated ram spermatozoa was less dependent upon external pH and affected only very slightly by the internal pH changes. K+ or Na+ had almost no effect on motility just after dilution. After 1 h of incubation, movement decreased. Maintenance of motility in sodium or potassium showed a sharp external pH optimum. Media without Na+ and K+ allowed a better conservation of motility at external pH > 8 for ram epididymal and ejaculated spermatozoa and at external pH > 6 for boar ejaculated spermatozoa.