Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer

The aim of this study was to evaluate the efficacy of swim-up, PureSperm gradient centrifugation and glass-wool filtration methods for semen preparation and to assess the possible enhancement of the quality of the subpopulation of spermatozoa in terms of sperm concentration, morphology and chromatin condensation. Moreover, to determine the effect of this semen processing technique on the clinical outcome after in vitro fertilization embryo transfer (IVF-ET). A total of 180 semen samples of patients' husbands who were undergoing IVF therapy were prepared by swim-up (G1, n = 60), PureSperm gradient centrifugation (G2, n=60) or glass-wool (G3, n=60) methods. Chromatin condensation was assessed by Chromomycin (CMA3), whereas sperm morphology was evaluated according to strict criteria. In all three semen processing methods, the percentage of chromatin condensed and morphologically normal spermatozoa was higher after semen processing in comparison with native semen samples. The proportion of normal chromatin condensed spermatozoa prepared in glass-wool filtration was significantly higher than that in swim-up (G.I, p=0.02) or PureSperm (G.II, p=0.001). In addition semen processing with PureSperm yields significantly a higher percentage of morphologically normal spermatozoa than swim-up (p < 0.001) or glass-wool method (p < 0.002). However, the fertilization, implantation and pregnancy rates, in turn were similar in all semen preparation methods. In conclusion, PureSperm gradient centrifugation yields a higher percentage of morphologically normal spermatozoa than shown in traditional swim-up or glass-wool filtration. However, the percentage of chromatin condensed spermatozoa was significantly higher after semen processing via glass-wool in comparison with the other two methods. Nevertheless, there were no significant difference in the fertilization, implantation and pregnancy rates of sperm prepared by means of swim-up, PureSperm or glass-wool filtration. Therefore, glass-wool filtration should be recommended as the first choice for semen preparation for Intracytoplasmic sperm injection (ICSI) technique as the natural selection is bypassed. Whereas, swim-up and PureSperm should be used for semen processing in IVF programme.     

辅助生殖技术中的精液优选处理

精液处理是辅助生殖技术的重要环节。选择精液处理方法时,不仅要考虑到精液的质量问题,还要考虑处理过程可能对精液产生的影响。本文介绍了上游法、密度梯度离心法、泳动沉淀法及玻璃纤维过滤法等几种常用的精子优选方法,并分析它们对精液的处理效果,同时讨论某些特殊精液的处理方法。     

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.     

Changes in human sperm chromatin stability during preparation for in-vitro fertilization

This study was designed to define the effects of sperm preparation on sperm chromatin stability in relation to in-vitro fertilization (IVF). Semen samples used for IVF-embryo transfer (ET) in the treatment of infertility due to tubal factors were studied. Cases with semen variables below reference limits in previous samples were excluded. Sperm were prepared by a swim-up technique employing either of two different tissue culture media, Ham's F-10 or Earle's balanced salt solution. Sperm chromatin stability was tested by exposure both to sodium dodecyl sulphate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS-EDTA). Sperm head swell scores were defined under different experimental conditions and the relationship to sperm motility, morphology, fertilization rate and pregnancy occurrence was tested. No differences were seen between the chromatin stability of sperm from the original sample and that after swim-up preparation, neither immediately after completion of the swim-up procedure, nor at the time of insemination of ova. With time, the chromatin became more stable, which occurred to a similar extent both in the original sample and in swim-up preparations using Ham's F-10. Otherwise, sperm chromatin stability was unaffected by either of the two media used for swim-up. At higher incubation temperatures, decondensation in SDS was enhanced. Altogether, no correlation was found between sperm chromatin stability or enhancement of decondensation by temperature and the success of IVF treatment expressed in fertilization rates or pregnancies. The results are reassuring in that only small changes in sperm chromatin stability occurred during the preparation for IVF. As long as semen of presumably good quality is used, these changes in chromatin stability do not seem to be of clinical importance.     

Prognostic value of an automated sperm analysis in IVF or insemination therapy

Summary. During the course of sterility treatment semenograms of 271 IVF and 316 insemination patients were carried out by two automated semen analysers. The parameters of these analyses were correlated to pregnancies resulting from the treatment. Semen samples were analysed in the ejaculate and after swim-up preparation. In addition, the swim-up suspension of IVF patients was measured again 18 h after sperm preparation. Patients were divided into three groups: (1) couples who achieved a pregnancy, (2) couples who did not achieve a pregnancy, and (3) IVF patients with no fertilization of the oocytes. Because of large standard deviations in the quality of ejaculates, the results in the IVF group show no significant differences in the semen parameters of husbands of pregnant and non-pregnant women. In contrast husbands of women with no fertilization have a significantly lower sperm motility. After swim-up preparation these differences no longer occur. A further measurement, taken 18 h later, reveals that there are no differences in the sperm parameters between the pregnant and non-pregnant group. However, the semen quality in the group with no fertilization is significantly reduced. The results of the insemination patients are similar to those of the IVF group. Thus, the results from automated sperm analysers cannot replace either the microscopic or biochemical analysis of an ejaculate and, moreover, cannot be used as prognosis for the fertilization capacity of sperms or a following pregnancy.     

Techniques for selection of normal-chromatin sperm preparations.

Various sperm preparation techniques, swim-up and Percoll gradient, and the newly developed Wang's tube system, were evaluated for their ability to recover normal-chromatin sperm. Twenty human semen samples, collected by masturbation, were studied simultaneously with the three methods. Analysis by Acridine orange fluorescence test was performed on all samples. Pretreated semen contains 58 +/- 22% green sperm (fertile/normal). Treatment with Wang's tube system resulted in 99 +/- 1.0% green sperm; Percoll gradient, 78 +/- 11%; and swim-up technique, 72 +/- 15%. It would appear that Wang's tube system yields a high-quality sperm preparation with enough concentration, very active forward progression, and greatly improved sperm morphology, while containing normal-chromatin, double-stranded DNA.     

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

AIM: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. METHODS: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. CONCLUSION: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.     

An evaluation of the hypo-osmotic sperm swelling test as a predictor of fertilizing capacity in vitro

The ability of sperm to swell in hypo-osmotic conditions was examined in 211 semen samples from the partners of patients about to undergo oocyte retrieval for in-vitro fertilization (IVF). The test was performed using aliquots of semen, the remainder of which was then prepared for IVF. No significant difference was found, in either the percentage of swollen sperm or the type of swelling response, between samples that achieved fertilization in vitro and those that did not, or between any of the diagnostic categories of infertility (tubal damage, unexplained infertility, oligospermia). In samples which achieved fertilization in vitro there were correlations between sperm swelling and sperm motility (r = -0.51) and abnormal morphology (r = 0.33), but no such correlations were demonstrated in samples that failed to achieve fertilization. Moreover, there was no significant difference between the percentage of swollen sperm in semen (mean motility 64%), in samples immediately after preparation for IVF (mean motility 96%) or in capacitated sperm 24 h after preparation (mean motility 91%). These results demonstrate that the hypo-osmotic sperm swelling test does not assist in the prediction of the fertilizing capacity of human sperm in vitro.     

Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology

Because morphology is regularly established in semen smears, but not in swim-up spermatozoa, we were interested in comparing some morphological parameters of semen and swim-up spermatozoa to establish if the cells selected by the swim-up method were morphologically similar to those considered normal in semen. Normal human semen samples were divided into two aliquots. One of these aliquots was washed by centrifugation with B₂ medium and sperm smears were prepared with the resulting pellet as a control. The other aliquot was used to perform swim-up separation and the spermatozoa from the supernatant were used as experimental smears. Both groups were stained according to the triple stain technique and spontaneous acrosome reaction and viability were determined. Video microscopy and computer-assisted image processing of live and non-reacted sperm cells were used to establish morphometrical parameters of the sperm head in both populations. The following set of morphometrical parameters were considered: width, length, width/length ratio, acrosome area, head area, and acrosome area/head area ratio. An increase in head width, a decrease in head length and a subsequent increase of width/length ratio were found in swim-up cells compared with the control group. A slight increase in acrosome area/head area ratio was also observed in swim-up spermatozoa. Through the swim-up methodology we were able to select a subpopulation of oval shaped heads with spermatozoa having a bigger acrosome area in comparison to semen.     

Influence of semen processing technique on human sperm DNA integrity

Objectives. To compare the effects of density-gradient centrifugation and swim-up technique on sperm DNA integrity.Methods. Semen samples (n = 22) were obtained from consecutive nonazoospermic men presenting for infertility evaluation. Individual samples were divided into three aliquots (whole semen, density-gradient centrifugation, and swim-up) for subsequent analysis of sperm motility and DNA integrity. Sperm DNA integrity was evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA.Results. Mean sperm motility (±SEM) improved significantly after processing with two-layer density-gradient and swim-up compared with whole semen (65.6% ± 4.0% and 73.0% ± 3.0% versus 52.0% ± 3.6%, respectively, P <0.005), with no significant difference in motility between Percoll-treated and swim-up-treated spermatozoa. In contrast, the percentage of spermatozoa with denatured DNA was reduced significantly in swim-up-treated but not in Percoll-treated spermatozoa compared with whole semen (4.8% ± 1.2% and 13.6% ± 3.6% versus 10.1% ± 2.3%, respectively, P <0.0001).Conclusions. Although density-gradient centrifugation is comparable to swim-up technique in recovering spermatozoa with enhanced motility, spermatozoa recovered after swim-up possess higher DNA integrity. These data urge us to reexamine our current sperm processing techniques in order to minimize sperm DNA damage.     

Sperm morphology and preparation method affect bovine embryonic development

This study was conducted to evaluate the effect of sperm separation methods of semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF) and to determine whether IVF results would be affected by various heparin concentrations. Morphologically abnormal semen samples were obtained and cryopreserved from Holstein bulls following scrotal insulation for 48 hours. Standard protocols using the Percoll gradient (90%/45%) method and the swim-up method were used to separate spermatozoa fractions in experiment I. The pellet (A(p)) and the 45% layer (B(p)) were isolated from the Percoll separation, while for the swim-up separation, the supernatant (A(s)) and the interphase (B(s)) were isolated. The overall blastocyst rate for our laboratory control semen was 23.1 +/- 2.1% for Percoll separations (A(p) and B(p)) and 18.2 +/- 2.0% for swim-up (A(s) and B(s)) separations. This rate was higher (P <.01) than the rate observed for the semen from the bull that had the greatest response to scrotal insult 5 days prior to the insult, when it was 9.2 +/- 2.1% for the Percoll separation and 20.7 +/- 2.3% for the swim-up separation, while semen from 27 days after scrotal insulation (D +27) resulted in no blastocyst formation for the Percoll separation and a 4.2 +/- 2.1% rate for the swim-up separation. In experiment II, semen was sampled from the bulls that responded in the greatest and least degrees to scrotal insult 5 days before scrotal insulation (D -5) and on days 23 (D +23) and 34 (D +34) after scrotal insulation. These samples were exposed to IVF mediums with 3 different heparin concentrations (0.1, 1.0, and 10 microg/mL). There was a significant difference (P <.05) in developmental scores between the D -5 (1.08 +/- 0.08), D +23 (0.9 +/- 0.08), and D +34 (0.8 +/- 0.08) samples, but no differences were observed in blastocyst formation based on the number of cleaved embryos. Increasing the heparin concentration resulted in higher (P <.01) embryonic developmental scores. In conclusion, when semen samples with high percentages of abnormal spermatozoa are used for IVF, semen separation preparation methods affect results. Our results show that the separation methods used under these conditions were inadequate in their ability to provide potentially competent sperm for IVF. However, selecting appropriate sperm separation procedures could improve in the IVF embryonic development of semen from bulls used in artificial insemination. Also, an increase in the heparin concentration was able to partially overcome deficiencies, which suggests that morphologically abnormal spermatozoa undergo capacitation despite possible structural changes to the plasma membrane.     

精子冻存技术及上游法精子处理技术对精子DNA完整性的影响

目的通过对冷冻前后及精子处理前后精子DNA完整性的比较,探讨冷冻技术、冷冻时间及上游法精子处理技术对精子DNA完整性的影响。方法（1）精液常规检测正常的患者30例,手淫法取精,精液液化混匀后分4份,分别用于精子染色质扩散（SCD）实验检测精子DNA完整性、上游法处理精液、以及两组冻存实验（不加保护剂直接冻存的为冻存1组;添加蛋黄葡萄糖保护剂冻存的为冻存2组）;（2）上游法处理后的精液一部分用于检测精子动力和形态,一部分用于精子DNA完整性检测;（3）冻存1组分别于冻存第7天和第90天解冻,SCD实验检测精子DNA完整性;（4）冻存2组分别于第7天和第90天解冻,0．1ml用于精子DNA完整性检测,剩余精液应用上游法处理,检测上游处理前后精子DNA的完整性。结果（1）冻存1组中,冻存90d后解冻的精子DNA损伤率[（25．6土7．3）％]显著高于冻存7d者[（22．4±7．4）％]（P〈0．05）,且均显著高于新鲜精液的精子DNA损伤率[（20．6±7．3）％]（P〈O．05）;冻存2组中,冻存90d后精子DNA损伤率显著高于冻存7d者[（25．9±7．2）％VS．（23．6土7．8）％]（P〈O．05）,且均显著高于新鲜精液（P〈0．05）;而冻存7d和90d后,两个冻存组间比较,精子DNA损伤率均无显著差异（P〉0．05）。（2）新鲜精液经上游法处理后,精子DNA损伤率由处理前的（20．6±7．3）％降为（6．4±2．5）％（P〈O．05）;冻存2组中精液冻存7d和90d后复苏上游法处理后,精子DNA损伤率较未经上游法处理者均显著降低[分别为（9．38±2．8）％VS．（23．6±7．8）％和（9．7±2．6）％VS．（25．9±7．2）％]（P％0．05）。结论冻存对精子DNA有损伤,冻存时间对于精子DNA完整性有影响。不添加保护剂直接冻存和添加保护剂对精子DNA完整性的影响无显著差异。不论是新鲜精液还是冻存复苏精液,上游法处理并不会增加精子DNA的损伤,且有利于筛选出具有更好DNA完整性的精子。     

Sperm count and motility influence the results of human fertilization in vitro

In order to select sperm characteristics that can predict the outcome of in-vitro fertilization-embryo transfer (IVF-ET), semen samples delivered in conjunction with this treatment were studied carefully. We have analysed these data retrospectively in relation to the outcome of treatment. Ninety-one couples were treated for tubal infertility by IVF-ET. Fifteen women became pregnant. Sperm were isolated from semen using a swim-up technique and in most cases 40-80 x 10(3) (range 20-120 x 10(3)) motile sperm per ovum were used for insemination. The couples were divided into three groups: group A who achieved pregnancies, group B who achieved cleaved ova but no pregnancies, and group C who achieved no ova that were cleaved 48 h after oocyte recovery. Comparisons between these groups showed that some characteristics of the native semen samples and the swim-up preparations were significantly different: the sperm concentration (P = 0.001) and total sperm count (P = 0.003) in the native sample, the number of sperm recovered during 30 min of swim-up (P = 0.001), and the specific progressive motility of sperm in the swim-up preparation, both at the time of insemination and on each day, up to 5 days thereafter (P = 0.002-0.028). No pregnancy was achieved with a sperm concentration below 26 x 10(6) ml-1 in the native sample. Some of the sperm characteristics studied in this paper may be of value in the pretreatment evaluation for IVF treatment.     

两种精子处理方法的卵细胞胞质内单精子显微注射结局的比较

目的:评价密度梯度离心和改良上游法两种处理活动精子的分离方法在卵细胞胞质内单精子显微注射(ICSI)中的效果,从而指导临床应用。方法:选取2004年10月~2005年4月在本中心完成的42例患者42个周期为研究对象,前瞻性比较了两种精子分离方法的受精率、卵裂率、优质胚胎率、临床妊娠率、精子畸形率、精子回吸收率等。结果:两种方法分离精子所获得的ICSI胚胎移植(ICSI-ET)中,受精率、卵裂率、优胚率、临床妊娠率均无明显差异,但是改良上游法所获得的精子畸形率明显高于密度梯度离心法(P<0.01);重度少精子症密度梯度离心法回吸收精子优于改良上游法(P<0.01)。结论:在辅助生育技术ICSI-ET中,密度梯度离心法分离活动精子临床妊娠结局与改良上游法无明显差异;除对重度少精子症者外,均可以采用改良上游法。     

Less NO production and better motion parameter in human sperm by swim-up processing

Fifteen semen specimens were obtained from men for semen analysis; each was divided into two aliquots for prepararation. The motile sperm recovery rate, percentage motility, and motion parameters were measured for each semen specimen (n = 15) before and after preparation with the use of the two methods, and cultured with different time courses (1 hr, 3 hr, and 6 hr). Nitric oxide (NO) was measured using the chemiluminscence method after centrifugation. Recovery rate of motile cell was significantly higher in direct swim-up method (69.5 +/- 42.4% versus 49.3 +/- 29.3%, p < 0.05). In motility, direct swim-up method in the different time courses was significantly better than IxaPrep method. (1 hr: 91.1 +/- 5.2% vs 65.6 +/- 16.4%, 3 hr: 87.2 +/- 7.9% vs 65.2 +/- 16.5%, 6 hr: 86.1 +/- 7.5% vs 60.8 +/- 17.6% and prewash: 61.6 +/- 16.2%, p < 0.05). In VAP and VSL, the sperm prepared by the above two methods all improved compared to pre-wash sperm (p < 0.05), but there was no statistical significance between the two methods. NO production in the direct swim-up group was significantly lower than IxaPrep group in the first hour of culture (0.09 +/- 0.09 uM vs 0.15 +/- 0.09 uM, p < 0.05). NO production increased as the culture time increased in swim-up group, but conversed in IxaPrep group. The lower level of NO produced in the swim-up group may suggest that better sperm quality achieved is due to the decreased NO production.     

Better sperm selection for intracytoplasmic sperm injection with the side migration technique

The side migration technique (SMT) is a recent method for preparing very poor-quality semen samples to be used in intracytoplasmic sperm injection (ICSI). In most centres, the washing swim-up and Percoll gradient columns techniques have been routinely used. The present study is aimed at comparing the quality of oligozoospermic semen samples selected after these three methods. All three methods were found to select better percentage motility, normal morphology, viability, functional integrity of plasma membrane and nuclear chromatin integrity compared with the original semen samples. Among the three methods, however, SMT yielded better sperm quality, including morphology, viability, membrane integrity and nuclear chromatin integrity. The results of this study and our experience have confirmed that SMT is an effective and physiological method to prepare sperm for ICSI.     

Glass wool filter preparation of cryopreserved spermatozoa

Summary: Oligozoospermic and asthenozoospermic semen ejaculates, as well as cryopreserved sperm samples prepared by the wash and swim-up procedure often result in unsatisfactory sperm recovery rates. In this study the glass wool filter and the wash and swim-up preparation procedures were compared on the basis of their 'effective' (number of live sperm per millilitre) recovery rates. The glass wool filter procedure consistantly produced significantly ( P = 0.0002) higher viable sperm concentrations, making it the preferred method for the preparation of cryopreserved sperm to be used in assisted reproduction techniques. The use of this preparation procedure has also been shown to have no adverse affect on the fertilizing potential of human spermatozoa in our unit.     

Comparison between a swim-up and a Percoll gradient technique for the separation of human spermatozoa

Two methods of separating human sperm were compared using twenty-two semen samples. The sperm were separated by a swim-up technique or by self-migration on a Percoll gradient followed by medium change. After separation, the sperm obtained were assessed for progressive motility, ATP content, energy charge index ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) and morphology. In general, and especially for semen samples containing less than 20 X 10(6) sperm/ml, separation by Percoll gradient selected sperm that were superior to those separated by the swim-up technique. The relatively high energy charge index (greater than 0.8) showed that the sperm tolerated the separation conditions well. It is suggested that self-migration on a Percoll gradient should prove useful for obtaining sperm of high quality.     

Temperature controlled centrifugation improves sperm retrieval

Sperm retrieval techniques form an integral part of the assisted reproductive programme. The success of sperm separation is measured by the number of motile sperm retrieved from a given semen sample. The study aimed to evaluate the effect of temperature during sperm preparation events on the number and percentage motile sperm retrieved following a double wash swim-up procedure. Thirty semen samples were obtained from 10 normozoospermic donors. After collection samples were divided into two aliquots, one aliquot was placed in an incubator at 34 °C, while the second aliquot was left at room temperature (25 °C). Sperm motility assessments were recorded with a computer assisted sperm analyser. Motile sperm fractions were retrieved from the semen samples following a double wash swim-up technique. Two tubes were prepared for each experiment. Tubes were placed in two different centrifuges: (i) SpermFuge (Shivani Industries, India) with temperature centrifuge control (34 °C) and (ii) Sigma with no temperature control facilities. Both centrifuges were set at 484 g for 5 min. Following the second wash, sperm pellets were layered with culture medium, and sperm was allowed to swim up. Supernatants were removed and analysed for sperm concentration and motility values. Percentage motile sperm was transformed to ARCSIN values and results of the two centrifugation methods at 34 °C and room temperature were compared with Mann-Whitney test for independent samples. The mean sperm concentration retrieved at 34 °C was 43.8 ± 50 (SpermFuge) and 32.7 ± 21 (Sigma) (P < 0.05), compared to retrieved concentration at room temperature namely, 30.9 ± 33 (SpermFuge) and 30.6 ± 17 (Sigma) (P ≥ 0.05). The mean percentage motile sperm at 34 °C was 64.0 ± 19 (SpermFuge) and 44.2 ± 24 (Sigma) (P = 0.02), while at room temperature the percentage motile sperm was 54.7 ± 17 (SpermFuge) compared to 46.5 ± 14 (Sigma) (P ≥ 0.05). Centrifuge temperature and incubation temperature significantly influenced the percentage retrieved motile sperm. The use of temperature-controlled sperm preparation might have clinical value for men with poor sperm motility values.     

Comparative study of four different sperm washing methods using apoptotic markers in ram spermatozoa

The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this study, we investigate the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides the evaluation of viability and the capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the phosphatidylserine translocation and caspase-3 and -7 activities. This is the first report, to our knowledge, that evidences the presence of active caspases in ram sperm. The results confirm the better ability of the dextran/swim-up procedure to select nonapoptotic spermatozoa, in addition to viable and noncapacitated sperm, compared with other sperm preparation methods. This should be considered to enhance results of artificial insemination techniques in ovine reproduction.