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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.

Sperm morphometry, in combination with other morphological traits and sperm parameters, can be useful for developing a fertility index. In present study various morphological and morphometric characters of cauda epididymal spermatozoa from 30 house rats were measured. About 100 spermatozoa of each rat with normal morphology and abnormalities like abnormal head, midpiece swelling, coiled tail, bent tail, detached heads and cytoplasmic droplets were counted in different fields and percentage of each was calculated. About 50 spermatozoa of each rat were measured for head length (HL), mid piece length (MPL), tail length (TL), head area (HA) and head perimeter (HP) using Magnus pro analytic software. In cauda epididymal spermatozoa of rat primary abnormalities included abnormal heads and mid piece swelling and secondary abnormalities included cytoplasmic droplet, coiled tails, bent tails, free heads. Variation among the sperm abnormalities was considerably high (>50 %). There was little variation in HL, MPL, TL, HA and HP of cauda epididymal spermatozoa of 30 rats (

The fertility of a rooster is not only influenced by motility, viability and DNA integrity of the spermatozoa but also by the morphological integrity of the spermatozoa. The objective of the study was to determine the morphological and morphometric description of spermatozoa in Nunukan rooster, one of Indonesian germplasm. Rooster’s semen was collected with dorso-abdominal massage method. The semen was stained with a combination of Eosin and Nigrosin prior to the observation of the morphologic and morphometric parameters. A microscope with 100 times magnification, as many as 100 spermatozoa, was used to evaluate the morphologic aspects. Hereafter, the morphometric aspects were analyzed using the Raster Image program to identify the head dimension, the head length and width, the mid-piece length, the tail length, and the ratio of the head and tail. The spermatozoa of Nunukan rooster showed the average total length of 86.724±12.496 μm. Furthermore, the head length, the mid-piece length and the tail length are 12.766±2.307 μm, 4.220±1.127 μm, and 75.738 ± 9.061 μm, respectively, while the head width is 1.391±0.244 μm. On the other hand, the ratio of head length and the tail lenght is 1: 5.9. The morphologic normal percentage is 80.50±9.40%. In conclusion, the characteristics of spermatozoa of Nunukan rooster due to the aspects of morphologic, morphometric and morphologic normal percentage are within the range of other types of rooster in many previous studies.

The reproductive tract of Chu-Lin, a giant panda that had died at Madrid Zoo, was removed. Four hours postmortem the epididymides was cut with a blade, and the spermatozoa were collected and immersed in a freezing medium. The sample was kept under refrigeration at 5°C. After 4 hr the sperm suspension was aspirated into 0.25-mL straws, which were held for 7 min in nitrogen vapors before they were plunged into liquid nitrogen. To characterize sperm population and distribution, a morphometric study of the triple-stained smears of the thawed semen sample was undertaken with the aid of computerized image analysis. Spermatozoa with intact acrosomes were selected and assessed using an immersion lens (1,000×) and bright-field illumination. The software made it possible to take six basic linear measurements of each spermatozoon: maximum head width (HW), base of the head (BH), maximum head length (HL), midpiece length (ML), midpiece width (MW), and terminal piece length (TL). The system was connected to a data base from which derived parameters could be calculated: head form factor (HL/HW), total flagellum length (TFL), total sperm length (TSL), and the proportion of each of the sections in relation to the whole. The results of the morphometric study did not demonstrate the existence of sperm subpopulations, as the measurements of the 100 spermatozoa analyzed were relatively homogeneous with regard to their distribution for each of the parameters considered. The sperm head of the giant panda displayed a relatively rounded morphology, and the HL/HW was 1.24±0.03. Mean head length was 5.05±0.08 µ, and mean head width was 4.07±0.08 µ. The mean total length of the giant panda spermatozoon was 49.26±0.67 µ. Zoo Biol 22:355–364, 2003. © 2003 Wiley-Liss, Inc.

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

Eggs and sperm of the sea urchin Paracentrotus lividus of the Mediterranean are used for an in vitro study of fertilization kinetics. The results are analyzed in terms of two models. One of these models assumes that all sperm-egg encounters lead to permanent attachment; the other (less realistically) assumes that sperm continue their random search after an unsuccessful encounter. More than 100 spermatozoa per egg are needed to achieve a fertilization ratio of more than 95%. There are two explanations for this: only 1% of the egg surface is subject to fertilization, or only 1% of spermatozoa are intrinsically able to fertilize. In the same context, chemotactic attraction and the role of the jelly are discussed. Comparison with earlier work of Rothschild and Swann and of Hultin and Hagstrom clarifies some discrepancies between and within these papers.

Spermatozoa were relatively inactive and did not enter the spermatheca of female boll weevils, Anthonomus grandis, whose spermathecal glands were removed as teneral adults. However, these females were able to lay fertile eggs for a 2 week period. When the spermathecal gland was removed from older females, spermathecal filling occurred, and although the spermatozoa retained their fertilizing capacity for extended periods, spermathecal emptying did not occur. Spermatozoa gradually lost their motility and fertilizing capacity, indicating that spermathecal secretions are effective in very small amounts. Spermatozoa were not activated by any of the materials contained in the normal male ejaculate. These materials alone did not effect spermathecal filling nor were they capable of maintaining the fertilizing capacity of the spermatozoa for very long. Sperm economy is low with less than 1 fertile egg laid per 100 spermatozoa used.

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.

The structural changes induced by X-rays in cells at different stages of spermatogenesis were analysed in salivary gland chromosomes of Drosophila melanogaster and compared with the changes induced by formaldehyde added to the food (FF) of the larvae.The different stages of spermatogenesis vary in sensitivity to X-rays when measured by the percentage of sex-linked lethals, by the percentage of spermatozoa carrying structural changes, and by the number of changes in 100 spermatozoa. The proportions of the different types of change (T, In, Rp, Df), however, are fairly similar in all stages of spermatogenesis, but entirely different from those found after FF treatment. This suggests that it is the mutagen and not the sensitive stage which is responsible for the characteristic pattern of the FF effects.The differences between the effects of X-rays and of FF are attributed to the different proportions of potential breaks induced by these two mutagens. Evidence has been presented indicating that while most of FF induced breaks are potential (about 73%), most of the X-rays induced breaks are immediate. For the dose rate used in the present experiment (below 1000 r/min.) only a small proportion (4–10%) of breaks induced by X-rays was found to be potential.

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.

Computer-assisted sperm morphometry analysis has improved the assessment of sperm morphology, but the results depend on the use of adequate evaluation and staining procedures of spermatozoa from individual species. In this study, the morphological module of the Sperm Class Analyzer ® was used for the morphometric analysis of stallion sperm heads and midpieces. Semen samples were obtained from six fertile stallions in order to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, and the effect of the sample size on sperm morphometric measurements. Harris' Haematoxylin was the staining technique of choice on the accuracy of the image process- ing with an optimum contrast of sperm cells with the surrounding background that allows an efficient bound- ary detection and segmentation which results in the highest proportion of sperm heads and midpieces assessed (80.47%). The results indicate that the staining methods affected significantly the sperm dimensions with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin respectively (Diff-Quik > Hemacolor > Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. In conclusion, to obtain objective and accurate sperm morphometric measurements by the Sperm Class Analyzer ® system in the stallion, it's recommended the analysis of 100 spermatozoa from slides which have been previously stained with Harris' Haematoxylin.