G. van der Horst

Morphometric dimensions of the human sperm head depend on the staining method used

BACKGROUND Assessment of sperm morphology (including morphometry) is extensively used to determine one of the qualities of a semen sample and depends on the differential staining of spermatozoa. A staining technique should cause as little change to sperm dimensions and form as possible in order to reliably evaluate the morphometric features of the sperm. Various staining techniques have been employed, but only a few have been recommended by the World Health Organization and are amenable to automated sperm morphometry analysis. Our study was aimed at comparing the effect of three staining techniques [Papanicolaou (PAP), Rapidiff (RD) and SpermBlue (SB)] on human sperm head dimensions and to compare these with the head dimensions in fresh semen. METHODS Smears made from human semen samples (n = 24) were stained according to the three staining techniques and sperm head morphometry was assessed with the Sperm Class Analyzer. Head dimensions of fresh spermatozoa were measured with a digital calliper on a computer screen. The minimum number of spermatozoa to be analyzed to represent the sperm population and the degree of inter-laboratory variation were determined. Electron micrographs from the same semen samples were used to determine the actual acrosome coverage of the spermatozoa in the semen (n = 7) in order to verify the results of the automatic analyses. RESULTS The osmolality of human semen differs from that of the RD and PAP fixatives and stains, but is more similar to the SB fixative and stain. At least 100 spermatozoa should be analyzed to include a representative sample of the sperm population. RD caused sperm heads to swell, PAP caused them to shrink and SB had no significant effect on sperm head dimensions when compared with spermatozoa in fresh semen. Very little inter-laboratory variations were found. The percentage acrosome coverage was significantly different between the three staining techniques, as well as between the RD and PAP stains and the manual measurements obtained using the electron micrographs. CONCLUSIONS Different staining techniques change the morphometric dimensions of the human sperm head, probably due to the fact that either the fixatives or stains are not iso-osmotic in relation to human semen. Since these changes in sperm head dimensions are not uniform, care should be taken when selecting a staining technique. Ideally, stained spermatozoa should have dimensions as close to spermatozoa in fresh semen as possible, as was found with the SB staining method, resulting in accurate evaluations of sperm head morphometry.

I. The effect of p-nonylphenol, an environmental toxicant with oestrogenic properties, on fertility potential in adult male rats

Summary. Infertility is a sad reality and it is now evident that several aspects of male reproductive health have changed for the worse over the past 30–50 years. para‐nonylphenol (p‐NP), an environmental toxicant with oestrogenic properties, was tested for its effect on male fertility potential. When adult male rats were exposed to 100 mg kg−1 p‐NP the histological parameters of the seminiferous tubules were adversely affected. Although spermatogenesis was already established in these males at the time exposure commenced, p‐NP still had an effect on the histology of the seminiferous tubules. Increasing the level to 250 mg kg−1 additionally resulted in a smaller weight gain and signs of epididymal toxicity, while 400 mg kg−1 also impaired testicular mass and sperm count. In the last two groups spermatogenesis was also affected in some animals. Because p‐NP had an effect on established spermatogenesis in the rat, one could speculate that the same effects might also occur in humans. It would appear that p‐NP had toxic effects on both the testis and epididymis and both structures might be important in impairing male fertility. Bio‐accumulation may enhance the negative effects at even lower p‐NP concentrations over longer exposure periods than reported here.

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

Abstract Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 × or 1000 × magnification for most of the species studied.

The effect of cryopreservation on the survivability, viability and motility of epididymal African buffalo (Syncerus caffer) spermatozoa.

The effect of cryopreservation on the viability and motility of epididymal African buffalo spermatozoa was studied in samples obtained from 17 and 13 animals in 1995 and 1996, respectively. Cryopreservation significantly reduced the viability and motility of the epididymal spermatozoa. The average percentage of live (+/- SE) spermatozoa declined significantly from 90.4 +/- 2.0% (1995) and 84.4 +/- 1.1% (1996) in fresh epididymal samples, to 57.0 +/- 2.0% and 56.3 +/- 1.1%, respectively, in frozen-thawed samples. The acrosomal integrity (+/- SE) of spermatozoa declined from 89.3 +/- 2.3% (1995) and 93.3 +/- 2.2% (1996) to 50.2 +/- 2.3% and 37.5 +/- 2.2%, respectively. In 1995, this effect was largely associated with the thermal equilibration prior to cryopreservation.

Improved semen collection method for wild felids: Urethral catheterization yields high sperm quality in African lions (Panthera leo)

For wild and domestic felids, electroejaculation (EE) is the most common semen collection method. However, the equipment is expensive, there is a risk of urine contamination and animals usually show strong muscular contraction despite general anesthesia. Accordingly, we tested the feasibility of a different approach using urethral catheterization (UC) in seven African lions, previously described for domestic cats only. After general anesthesia with the α2-agonist medetomidine (which also stimulates semen release into the urethra) and ketamine, a transrectal ultrasound was performed to locate the prostate. A commercial dog urinary catheter (2.6 or 3.3 mm in diameter) was advanced approximately 30 cm into the urethra to allow semen collection into the lumen of the catheter by capillary forces. After retraction, sperm volumes between of 422.86 ± 296.07 μl yielded motility of 88.83 ± 13.27% (mean ± SD) with a mean sperm concentration of 1.94 × 10(9)/ml. Here we describe a simple, field friendly and effective method to attain highly concentrated semen samples with excellent motility in lions and potentially other wild felid species as an alternative to electroejaculation.

The effect of oleanolic acid on sperm motion characteristics and fertility of male Wistar rats

The study was designed to examine the effect of oleanolic acid on cauda epididymal sperm motion using a computer-aided sperm analysis system and to elucidate the relationship between sperm motion and fertility, as a tool for contraceptive studies. Oleanolic acid-polyvinylpyrrollidone suspension was orally administered to adult male Wistar rats for 30 days, followed by a 14-day drug withdrawal from half of the rats in the group. Control rats received only polyvinylpyrrollidone. All males were mated with untreated females. Treated males failed to impregnate females, whereas control and oleanolic acid withdrawn males achieved 100% pregnancies. Sperm motion analysed on the Sperm Motility Quantifier (SMQ) showed significant differences in linearity (P < 0.001) and wobble (P < 0.01) between control and treated groups. However, the curvilinear velocities were not significantly different (P > 0.05) among all the groups. Sperm motility patterns verified differences among kinematic parameters.

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed

Abstract Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.

Wheat germ agglutinin receptors on human sperm membranes and sperm morphology

Summary. Sperm membrane components play an important role in the determination of sperm fertilizing ability. During spermatogenesis, epididymal transit, and capacitation, the sperm membrane undergoes various subtle changes which are important maturational events for the production of viable spermatozoa and hence partly determine fertility. Localization and intensity of wheat germ agglutinin (WGA) receptors are reported to be directly associated with male fertility. We examined the correlations and differences between the distribution patterns and intensities of WGA receptors of 75 semen samples, classified according to the strict criteria described for sperm morphology, namely P‐pattern (0–4% normal forms, n = 19), G‐pattern (5–14% normal forms, n = 41) and normal samples (>15% normal forms, n = 15). Results indicate distinct differences in WGA receptor intensity in all of the three groups as well as significantly (P < 0.05) lower per‐cent staining of the equatorial segment amongst the P‐pattern group when compared to the other two groups. It would appear that there is a close relationship between WGA receptors on human sperm membranes and sperm morphology.

Sperm Swimming Velocity as Evaluated by Frame Lapse Videography and Computer Analysis

A videographic method is described to measure the swimming velocity of fresh human, frozen-thawed bull and white mussel (Donax serra) sperm. The method can be accurately controlled. The pattern of sperm movement can be analyzed on a frame-by-frame basis or over 0.0206-sec intervals. Sperm velocities can be standardized per time unit. Sperm swimming velocity of different species ranging from very slow to very fast erratically moving sperm can be measured. Parameters such as swimming speed, progressive swimming speed, progressiveness ratio, and percentage motility can be evaluated.

The effect of the breeding season, cryopreservation and physiological extender on selected sperm and semen parameters of four ferret species: implications for captive breeding in the endangered black-footed ferret

In the present investigation, comparative baseline information on selected sperm characteristics of ejaculate spermatozoa of the domestic (Mustela putorius furo), fitch (Mustela sp.) and black-footed ferrets (Mustela nigripes) and the Siberian polecat (Mustela eversmanni) are presented. The main emphasis was to establish differences and similarities among these species in relation to semen and sperm quality during the breeding season, in cryopreservation success and in supporting sperm motility in different extenders or physiological media. The results confirm that most sperm morphology abnormalities were evident during the beginning of the breeding cycle in all four species. No significant interspecies differences were apparent in the sperm attributes examined, for all sampling months during the breeding season. Moreover, all species exhibited comparable patterns of reproductive seasonality. Cryopreservation suppressed sperm characteristics equally in all species studied. Ejaculate spermatozoa of closely related ferret species shared many similar motion characteristics using computer-aided sperm motility analysis. These results suggest that the basic sperm physiology of the ferret species under examination is very similar. Disparate to the interspecies comparisons, there were significant differences for most sperm motion parameters when spermatozoa of any of the ferrets were compared in different extenders. Assisted reproductive technologies developed for use in domestic ferret, fitch ferret or Siberian polecat may be successfully applied to captive breeding of the black-footed ferret using semen during any of the functional breeding months.