Development of mouse testis and epididymis following intrauterine exposure to a static magnetic field.

In order to test if the in utero exposure to static magnetic fields affects testis and epididymis development in mice, females were exposed to 0.5-0.7 T, generated by a permanent magnet, from day 7 of gestation to the day of birth. No significant differences were found between exposed and sham-exposed animals with respect to body weight gain of dam during the gestational period, litter size, body weight of male pups at the day of birth, and body or testis-epididymis weight gain of pups from birth to day 35. Histopathologic evaluation of testis and epididymis of pups of 1, 5, 15, and 35 days of age showed no detectable alterations due to in utero exposure to static magnetic fields.

Morphological characterization of ejaculated cynomolgus monkey (Macaca fascicularis) sperm.

The aim of this study was to give reference values for the frequency of morphological sperm abnormalities present in the semen from non-experimental cynomolgus monkeys as well as for the dimensions of sperm heads. Spermatozoa from the liquid portion of electroejaculates from 14 cynomolgus monkeys were air-dried as smears, fixed, and stained with Harris's Haematoxylin and subjected to visual analysis of morphology and computer-aided analysis of ten morphometric variables. The majority (83%) of sperm were morphologically normal. Tail defects were the most common (11%), and showed the highest variation between individuals, the values ranging between 4 and 23%. Head abnormalities consisted of large, tapering, and amorphous forms but were not frequent (0.4%), the values ranging between 0 and 1.3%. Midpiece imperfections were found in all the individuals; the mean percentage was 5%, and the range varied between 3 and 9%. Tail plus midpiece was the only multiple abnormality observed, with a mean value of 1.5% and a range between 0 and 8%. The majority of these double defects consisted of a coiled tail together with a coiled midpiece. Mean values for the morphometric parameters characterizing sperm heads were as follows: area 17.2 microm2, perimeter 15.2 microm, length 5.8 microm, width 4.0 microm, L/W ratio 1.5, gray-level 98, ellipticity 0.2, first shape factor 0.9, second shape factor 1.4, and third shape factor 1.1. Overall coefficients of variation for the majority of parameters were below 7%, showing the great homogeneity in the dimensions of cynomolgus sperm heads. Most useful parameters for sperm characterization, according to their low variability, were perimeter, length, width, L/W ratio, and shape factors. Differences in these parameters were, however, observed between monkeys.

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit.

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity.

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm.

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure.

Standardization of sampling and staining methods for the morphometric evaluation of sperm heads in the Cynomolgus monkey (Macaca fascicularis) using computer-assisted image analysis.

Automated sperm morphology analysis (ASMA) technology has improved the assessment of sperm morphology, but the results depend on the use of adequate and standardized procedures. In this study the Sperm-Class Analyzer (SCA) ASMA system was used to assess sperm head morphometry in the Cynomolgus monkey and to evaluate the influence of sample size, intraslide variation, and the use of three staining techniques on the accuracy of image processing and sperm head morphometry. Haematoxylin is the staining technique of choice for Cynomolgus spermatozoa, as optimum contrast of sperm heads with the surrounding background allows efficient segmentation, i.e. sperm head boundary detection, making the image analysis process more accurate. The analysis of 100 spermatozoa is recommended since a larger sample size did not result in more accurate sperm head morphometry. There were no differences in either the percentage of correctly binarized sperm heads or sperm head dimensions among samples obtained from different zones of the slides, although differences in stain intensity (grey level) were detected. The measurements made on Haematoxylin, Diff-Quik and Hemacolor-stained slides yielded different values for all of the sperm head parameters under consideration. This result demonstrates that the procedures of fixation and staining significantly affect the dimensions of sperm heads.

Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques.

The recent development of automated systems for morphometric sperm head analysis has provided a series of objective parameters which have facilitated the standardization of morphological semen evaluation. This current work attempts to establish the optimum fixing conditions for the morphometric characterization of ram spermatozoa. Ejaculates were obtained from 5 Merino rams used for periodic collection of semen and were diluted at 1:50 with TEST medium. Air-dried smears were fixed either in ethanol-ether (1:1), 50% methanol, 2% glutaraldehyde or SUZA fixative, in which case the smear was pretreated with chloramine. The samples were then stained with commercial kit Hemacolor. Once the preparations had been mounted, they were analyzed with the Sperm Class Analyzer automatic sperm morphometry analysis system (ASMA). The minimum number of sperm cells analyzed per sample was 100. The parameters evaluated were the area, perimeter, length, width, shape factor and mass. The results showed significant differences in sperm head dimensions between the 4 fixation techniques, with the lowest values for all parameters corresponding to the SUZA fixative, followed by glutaraldehyde, methanol, and finally ethanol-ether. In addition, there were significant variations between animals. It can, therefore, be concluded that the working protocol must be defined when performing morphometric analysis of ram semen and that the results obtained under different conditions of fixation cannot be entirely extrapolated. Equally, the high variability among individuals suggests that, in a species like the ram with a low index of teratozoospermia, there is a need for a revision of the classic definition of normality, which should include morphometric data.

Sperm morphological abnormalities appearing in the male rabbit reproductive tract.

The role of the excurrent duct system in producing and/or eliminating morphologically abnormal spermatozoa may modify the semen parameters and interfere with sperm fertilizing capacity. To study this process, changes in the morphology of spermatozoa during their transit through the reproductive tract in sexually mature rabbits were investigated. The incidence of head, midpiece and tail abnormalities as well as of multiple defects in a single spermatozoon, and the position of the cytoplasmic droplet along the sperm midpiece were evaluated in samples from the testis, 6 regions of the epididymis and the vas deferens. Spermatozoa were characterized by rapid migration of the cytoplasmic droplet when passing from the proximal to the distal caput of the epididymis, and spermatozoa with no droplet predominated in the distal epididymis and vas deferens. In passing from the testis to the proximal caput of the epididymis, the incidence of spermatozoa with an abnormal midpiece and those with multiple defects decreased significantly. The proportion of spermatozoa with abnormal heads was also lower in the testis, but no statistically significant differences were found, whereas there was no change in the proportion of those with abnormal tails. These results indicate that there must be a mechanism for the disposal of defective spermatozoa. No evidence of spermiophagy by luminal macrophages was observed in the extracts, although a few spermatozoa exhibited signs of degeneration, suggesting, that although intraepithelial phagocytosis has not been clearly demonstrated in the nonexperimental rabbit, sperm cells may undergo a form of autolysis within the lumen of the duct.

Is sperm motility maturation affected by static magnetic fields?

Kinematic parameters were evaluated in mouse epididymal extracts to monitor maturation of sperm movement in animals exposed to static magnetic fields using the Sperm-Class Analyzer computerized image analysis system. For this purpose, animals were exposed to a field of 0.7 T generated by a permanent magnet over 10 or 35 days for either 1 or 24 hr/day. The values of the motion endpoints were similar in animals used as controls and in those exposed to the nonionizing radiation, whatever the period of exposure or daily dosage. Changes in motility were observed in all groups: the percentage of total motile and progressive motile spermatozoa increased during passage through the epididymis, with major changes between the caput and corpus epididymides, and the pattern of swimming changed clearly towards more rapid and straighter trajectories. The processes of initiation of sperm motility and maturation of displacement patterns were not then affected by magnetic treatment. Moreover, it appears that sperm production is unaffected because no changes were observed in testicular or epididymal weights after exposure to static magnetic fields.

Changes in the motility patterns of spermatozoa from the rabbit epididymis as assessed by computer-aided sperm motion analysis.

Sperm maturation in the epididymis includes changes in their potential for motility that enables spermatozoa to reach the egg and penetrate its investments. The motility characteristics of spermatozoa from the testis, the epididymis, and vas deferens of the rabbit were investigated by computer-assisted sperm analysis (CASA). Various forms of motility were displayed by sperm from different regions of the epididymis released into incubation medium. Testicular sperm were motile, although nonprogressive. The maximum percentage motility was expressed by sperm in the proximal cauda epididymidis, and forward progression was developed by spermatozoa from the distal caput. Once forward progression was established, the curvilinear velocity was about the same for sperm from all regions of the tract, whereas straight-line velocity increased between the mid-corpus and cauda and paralleled the decline in lateral displacement of the head. The maintenance of motility in vitro was best maintained by sperm from the distal regions of the tract although sperm from the distal caput maintained motility better than sperm from the proximal and midcorpus regions. Analysis of the motile sperm cells revealed several types of trajectories ("irregular," "small circular," "large circular and arcs," "jagged" and "straight-line") that were analyzed by discriminant analysis using the variables generated by CASA. Accuracy of classification varied from 70% to 96%, depending on the type of track. The classification function was then applied to the changes that occurred during incubation and showed that irregular trajectories gave way to small and then large circular tracks and progressive forms as sperm matured.