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This study aimed to demonstrate the effect of conventional freezing versus cryoprotectant-free vitrification on the recovery of testicular sperm motility. Testicular samples were obtained from 50 patients with azoospermia for testicular biopsy ± potential sperm storage. We retrieved 100 spermatozoa from each patient divided equally into two straws. They were frozen using conventional freezing as a control group and cryoprotectant-free vitrification in micro-capillary system using open-pulled straws. Seven days later, cryopreserved straws were thawed and assessed in duplicate. The mean sperm motility between the original spermatozoa sample and the post warming sample was reduced after conventional freezing compared to cryoprotectant-free vitrification (4.48 ± 2.09% versus 3.25 ± 1.92%, p < 0.001; 4.48 ± 2.09% vs 3.68 ± 1.93%, p < 0.001, respectively). There was a significant difference between the two methods regarding the mean sperm motility after warming (3.38 ± 1.86% versus 3.76 ± 1.88%, p = 0.015). The mean recovery percent of testicular sperm motility from the original sperm sample was lower (p = 0.02) after conventional freezing compared to cryoprotectant-free vitrification (78.4 ± 28.17% versus 85.37 ± 23.63%). Overall, the rate of post-thaw recovery of human testicular sperm motility improved using cryoprotectant-free vitrification compared to conventional freezing.

This study was designed to confirm the previously observed relationship between response to the short hypoosmotic swelling test (sHOST) and acrosome resistance in boar spermatozoa. Ejaculates from 22 boars were incubated in a water bath at 37°C for 2 h. During the incubation period, samples were taken at 5, 20, 40, 60, 90 and 120 min and subjected to the sHOST. sHOST responses (positive HP-negative HN) and acrosomal status (normal or intact NA-damaged DA) were evaluated in 100 spermatozoa corresponding to each ejaculate and incubation time, and the results used to establish four subpopulations HPNA, HPDA, HNNA and HNDA. Over the entire incubation period, the sHOST positive subpopulation with damaged acrosomes, HPDA, was significantly smaller than the sHOST negative, damaged acrosome subpopulation, HNDA (P < 0.001). Further, proportions of HPDA spermatozoa remained stable throughout this period while the HNDA subpopulation showed a significant increase (P < 0.001) from the start to the end of incubation. These results confirm the high resistance of the plasma membrane of HP spermatozoa allowing the persistence of a higher number of intact acrosomes over time, compared to HN spermatozoa. Characterising this HPNA subpopulation may help the evaluation of ejaculate quality. © 2003 Elsevier Science Inc. All rights reserved.

The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks ( approximately 100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.

There are several reasons for a need to cryostore a smallnumber of spermatozoa. Men who require testicular biopsybenefit tremendously if the presence of spermatozoa isknown before the onset of follicular recruitment in thepartner. This can usually be achieved by cryopreserving thebiopsy material or suspension of cells. But post-thawsuccess depends on the presence of relatively largenumbers of spermatozoa, since the addition of cryo-protectant requires removal by centrifugation whichdestroys many viable cells. The cut-off level forconventional freezing in mini-straws is 1200 spermatozoa,but many men have even less cells. Freezing of multiplesamples of very few spermatozoa would neverthelessguarantee several attempts at intracytoplasmic sperminjection (ICSI) and avoid repeated testicular aspirations incase further treatments are required.The recent of use of testicular spermatozoa has greatlyemphasized the need for the storage of aspirated testiculartissue or of spermatozoa that are recovered from suchtissue (Salzbrun et al ., 1996). Other methods of cryostoringsmall numbers of spermatozoa retrieved from the testisinclude the use of small droplets of medium which areplaced on a dry ice surface before freezing in liquidnitrogen, to give pregnancy rates similar to those obtainedwith fresh testicular spermatozoa (Gil-Salom et al ., 1996).Though both techniques are elegant and extremely useful,application falters when levels drop below one thousand.Special methods are necessary to store spermatozoa incases of extreme oligozoospermia. This circumstancearises constantly during ICSI when epididymal ortesticular spermatozoa are very rare. The most extremecases involve finding

A quantitative method is described for distinguishing two types of mouse sperm motility using a new parameter, the Linear Index. One hundred spermatozoa from C57BL/6-+/+ and 100 spermatozoa from C57BL/6-tw32/ + mice were used to evaluate this parameter. Spermatozoa were tracked by videomicrography, and the following calculated: net displacement velocity, Vn; curvilinear velocity of the sperm head, Vc; the "average" velocity, Va (a 5-point moving average of the track); the progressiveness ratio, (Vn/Vc); the curvilinear progressiveness ratio, (Va/Vc); and a linear index, (Vn/Va). Frequency histograms of linear index values were bimodal for both groups and each had a clear antimode at 0.5, separating two discrete subpopulations of spermatozoa. Within each group, "linear" (greater than 0.5) and "nonlinear" (less than 0.5) tracks differed in all characteristics; between groups, linear spermatozoa were similar and nonlinear spermatozoa differed only in the curvilinear velocity of the sperm head.

An approach was made to replace the subjective evaluation of human sperm morphology by an objective method applicable in routine laboratories. 20 patients out of infertile marriages (G2 and G3) were compared with 10 normal, fertile men (G1). Two different methods were used. Highly developed instruments and techniques were used in method 1, and cheap and simple ones, designed for routine laboratories, in method 2. Head area, circumference, length, and maximal midpiece width of 100 spermatozoa/subject were determined by a semi-automatic image analysis system. Head variation and average midpiece width were clearly bigger in G2 and G3 than in G1. As a result, formulae were developed, determining the pathological alteration of sperm head and midpiece for the individual semen. In addition, an objective classification into different sperm types was elaborated. Using the developed formulae, results of methods 1 and 2 are well in agreement. Thus, method 2 is recommended for routine laboratories, allowing an objective and reproducible morphology determination within 30 min.

Two groups of men were studied to determine how much an individual's spermatozoal morphology varies and which part of the variability is due to biological and to measurement variation. A first group of ten subjects each provided five ejaculates; the other group of four subjects each provided one ejaculate, from each of which five smears were prepared, and evaluated twice. The slides were examined in random order and blindly by the same experienced technician. The total within-subject variability of the percentage of abnormal forms was large (SD = 8.8%), as was the component of this variability due to biologic fluctuation (SD = 5.8%). The distribution of the various abnormalities also varied greatly from one ejaculate to another. The results indicate that more work is needed to determine why biological variation exists, and that several ejaculates (three if possible, two at minimum) are needed for an accurate evaluation of spermatozoal morphology, whereas the examination of greater than 100 spermatozoa/ejaculate is not advantageous.