Sperm Dimensions Research Articles

Postcopulatory sexual selection results in spermatozoa with more uniform head and flagellum sizes in rodents.

Interspecific comparative studies have shown that, in most taxa, postcopulatory sexual selection (PCSS) in the form of sperm competition drives the evolution of longer and faster swimming sperm. Work on passserine birds has revealed that PCSS also reduces variation in sperm size between males at the intraspecific level. However, the influence of PCSS upon intra-male sperm size diversity is poorly understood, since the few studies carried out to date in birds have yielded contradictory results. In mammals, PCSS increases sperm size but there is little information on the effects of this selective force on variations in sperm size and shape. Here, we test whether sperm competition associates with a reduction in the degree of variation of sperm dimensions in rodents. We found that as sperm competition levels increase males produce sperm that are more similar in both the size of the head and the size of the flagellum. On the other hand, whereas with increasing levels of sperm competition there is less variation in head length in relation to head width (ratio CV head length/CV head width), there is no relation between variation in head and flagellum sizes (ratio CV head length/CV flagellum length). Thus, it appears that, in addition to a selection for longer sperm, sperm competition may select more uniform sperm heads and flagella, which together may enhance swimming velocity. Overall, sperm competition seems to drive sperm components towards an optimum design that may affect sperm performance which, in turn, will be crucial for successful fertilization.

Comparison of different chromatin staining techniques for bull sperm

Abstract. Morphological analysis of semen is a very important step in fertility assessment, but many semen defects are not detectable at the morphological level. These include pathological changes in sperm chromatin structure. During mammalian spermiogenesis, histone proteins associated with DNA structure are replaced by specific protamines, with which chromatin does not form nucleosomal complexes. In the fully developed, mature sperm, the histones are replaced with protamines. Disruptions of nucleoprotein structure can be treated as possible indicators of the biological value of spermatozoa. The experimental material consisted of sperm from one-and-a-half-year-old bulls, isolated post mortem from the tail of the epididymis. The smears were stained with silver nitrate (AgNO3), acridine orange (AO), aniline blue (AB) and chromomycin A3 (CMA3). Sperm dimensions largely depend on individual variability among the bulls. In most cases, differences in sperm dimensions were identified between individuals, which was confirmed in the statistics. Sperm with elevated, abnormal histone levels were proportionally quite scarce (1.4 %). Studies of nuclear proteins in the context of infertility demonstrate the important influence of normal chromatin structure on sperm functions.

Do pre- and post-copulatory sexually selected traits covary in large herbivores?

BackgroundIn most species, males compete to gain both matings (via pre-copulatory competition) and fertilizations (via post-copulatory competition) to maximize their reproductive success. However, the quantity of resources devoted to sexual traits is finite, and so males are predicted to balance their investment between pre- and post-copulatory expenditure depending on the expected pay-offs that should vary according to mating tactics. In Artiodactyla species, males can invest in weapons such as horns or antlers to increase their mating gains or in testes mass/sperm dimensions to increase their fertilization efficiency. Moreover, it has been suggested that in these species, males with territory defence mating tactic might preferentially increase their investment in post-copulatory traits to increase their fertilization efficiency whereas males with female defence mating tactic might increase their investment in pre-copulatory sexually selected traits to prevent other males from copulating with females. In this study, we thus test the prediction that male’s weapon length (pre-copulatory trait) covaries negatively with relative testes size and/or sperm dimensions (post-copulatory traits) across Artiodactyla using a phylogenetically controlled framework.ResultsSurprisingly no association between weapon length and testes mass is found but a negative association between weapon length and sperm length is evidenced. In addition, neither pre- nor post-copulatory traits were found to be affected by male mating tactics.ConclusionsWe propose several hypotheses that could explain why male ungulates may not balance their reproductive investment between pre- and post-copulatory traits.

Light microscopic features and morphometry of sperm in the emu (Dromaius novaehollandiae)

A comprehensive morphologic description of emu sperm at the light microscopy level, an essential prerequisite for the routine evaluation of semen quality in this species, is not currently available. In this study, sperm morphology and morphometry were evaluated using conventionally prepared Romanowsky-stained semen smears of samples collected from the distal ductus deferens from 15 adult birds and fixed in 2.5% glutaraldehyde. Examination of the smears using phase contrast under 100× magnification readily resolved the various components of the cell, namely, the acrosome, nucleus, midpiece, principal piece, and endpiece. This technique was simple to use and produced consistently reproducible results. Normal emu sperm were typically filiform in appearance and closely resembled sperm of the ostrich and other non-passerine species, particularly poultry. A previously undescribed cytoplasmic appendage, associated with the base of the head, was a novel morphologic feature. The acrosome was short (1.84 ± 0.31 μm; mean ± standard deviation), whereas the nucleus measured 11.77 ± 0.93 μm in length. The length of the segments of the flagellum were 2.91 ± 0.4 μm for the midpiece, 47.45 ± 2.8 μm for the principal piece, and 3.69 ± 0.82 μm for the endpiece. The total sperm length was 67.64 ± 3.13 μm (range, 60.14–79.49) and the head:tail ratio was 1:4. Sperm dimensions in the emu were similar to those of other ratites.

POSTCOPULATORY SEXUAL SELECTION INCREASES ATP CONTENT IN RODENT SPERMATOZOA

Sperm competition often leads to increase in sperm numbers and sperm quality, and its effects on sperm function are now beginning to emerge. Rapid swimming speeds are crucial for mammalian spermatozoa, because they need to overcome physical barriers in the female tract, reach the ovum, and generate force to penetrate its vestments. Faster velocities associate with high sperm competition levels in many taxa and may be due to increases in sperm dimensions, but they may also relate to higher adenosine triphosphate (ATP) content. We examined if variation in sperm ATP levels relates to both sperm competition and sperm swimming speed in rodents. We found that sperm competition associates with variations in sperm ATP content and sperm-size adjusted ATP concentrations, which suggests proportionally higher ATP content in response to sperm competition. Moreover, both measures were associated with sperm swimming velocities. Our findings thus support the idea that sperm competition may select for higher ATP content leading to faster sperm swimming velocity.

Nanoscale Differences in the Shape and Size of X and Y Chromosome-Bearing Bovine Sperm Heads Assessed by Atomic Force Microscopy

Sperm dimensions and the question of whether X and Y chromosome-bearing sperm differ in size or shape has been of great interest, especially for the development of alternative methods to sort or classify sperm cells. The aim of the present study was to evaluate possible differences in the shape and size of the sperm head between X and Y chromosome-bearing sperm by atomic force microscopy (AFM). One ejaculate per bull (n = 4) was used. Each ejaculate was separated into four fractions: non-sexed (NS), sexed for X-sperm (SX), sexed for Y-sperm (SY) and a pooling of SX and SY samples (SXY). Using AFM, 400 sperm heads per group were measured. Twenty three structural features were assessed including one-, two- and three-dimensional parameters and shape descriptors. These measurements determine the micro- to nanoscale features of X- and Y-bearing chromosomes in sperm cells. No differences were observed for any individual variables between SX and SY groups. Next, a simultaneous evaluation of all features using statistical discriminant analysis was performed to determine if it was possible to distinguish to which group belong each individual cells. This analysis clearly showed, a distinct separation of NS, SXY, SX and SY groups. The recognition of this structural possibility to distinguish between X and Y sperm cell might improve the understanding of sperm cells biology. These results indicated that the associations of several structural measurements of the sperm cell head are promising candidates for development of a new method of sperm sexing.

Elimination of Y chromosome-bearing spermatids during spermiogenesis in an autosomal <i>sex-ratio</i> mutant of <i>Drosophila simulans</i>

Sex ratio distortion, which is commonly abbreviated as sex-ratio, has been studied in many Drosophila species, but the mechanism remains largely unknown. Here, we report on the sex-ratio mutant of D. simulans named excess of females (exf). The third chromosomal recessive mutation results in a sex ratio of approximately 0.2 or less (males/total). Cytological observation demonstrated that meiosis appeared to be completed normally, but that most Y chromosome-bearing nuclei failed to elongate during spermiogenesis, as revealed by fluorescence in situ hybridization using sex chromosome-specific probes. These aberrant nuclei contained membranous inclusions as revealed by electron microscopic analysis. Most of the aberrant exf spermatids failed to individualize and mature, suggesting that a later stage of spermiogenesis is involved in prevention of production of sperm with abnormal morphology. On the one hand, in exf seminal vesicles, sperm nuclei with a length of 5-8.5 μm were occasionally observed, in addition to those with wild-type sperm dimensions, that is, a length of approximately 10 μm. Thus, spermatids with less severe nuclear defects can escape elimination and be released into the seminal vesicles as mature sperm. Furthermore, we constructed His2AvD-GFP and ProtamineB-eGFP transgenic lines in D. simulans, and examined the processes involved in replacement of chromatin proteins over a time course, according to nuclear morphology. We found that both normal and abnormal sperm heads demonstrated equal chromatin replacement during late spermiogenesis. Our results suggest that exf belongs to a unique class of meiotic drive systems in that (1) intranuclear membranous inclusions cause failure of nuclear shaping of Y-bearing spermatids without affecting the histone-protamine transition, and (2) a portion of the aberrant spermatids differentiate into mature sperm; these are transferred to and stored by females.

A comparative study of the sperm nuclear morphometry in cattle, goat, sheep, and pigs using a new computer-assisted method (CASMA-F)

This study was designed to compare the sperm nuclear morphometry of four species of domestic artiodactyls (cattle, sheep, goats, and pigs), using the newly developed automatic computer-assisted sperm morphometry analysis-F. The study was divided into two experiments. In the first experiment, samples from 20 males from each species were collected, diluted, and divided into four sample aliquots. The first was labeled directly with Hoechst 33342, and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying, and the other was fixed either with glutaraldehyde (GLUT), or with methanol, and afterward labeled with Hoechst. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and at least 200 sperm cells per sample were processed using the Image J analysis open software. Air-drying significantly reduced nuclear sperm dimensions in ruminant species, whereas no effect was observed in pigs. For most of the primary morphometric parameters, the relationship between the four species for the sperm nuclear dimensions can be described as follows: bull > ram ≥ boar > goat. However, ram sperm nuclei had greater width than those of the other species studied. For the secondary morphometric parameters, ram sperm nuclei were clearly less elliptical and elongated and showed greater regularity than in the other studied species. In the second experiment, ejaculates from 10 males per species were used to compare the sperm head morphometric results obtained with the computer-assisted sperm morphometry analysis-F system (using the GLUT treatment as reference) to a more conventional CASMA method (semen smears stained with Harris's hematoxylin and processed with the Integrated Sperm Analysis System [ISAS] commercial software [Proiser R&D SL, Buñol, Spain]). Spermatozoa displayed a bigger size when processed with Harris's hematoxylin than with the GLUT method in all primary sperm head morphometric parameters for the four species studied. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters in the four species studied. It was concluded that drying and fixation has little effect on sperm nuclear morphometry, with differences between species, and that there are significant variations in size of the sperm nucleus and in the hydrodynamic properties between the four species studied.

Artificially selected human sperm morphology after swim-up processing

The swim-up technique is a clinical practice used to select highly motile sperm cells from patient ejaculates to use in assisted fertilization. The aim of this study was to investigate whether the length of different sperm-cell components is related to gamete function. Thus, we explored whether swim-up technique selects for longer sperm cells than mean sperm cells from unprocessed ejaculates. Sperm midpiece, tail endpiece, and total length were measured before and after the swim-up selection by means of contrast-phase and electron microscopy. Correlations between sperm dimensions, sperm motility, and sperm concentration were also investigated. Swim-up selected cells with longer midpiece compared with the unprocessed fractions (5.8 μm (CI 5.52–6.16 μm) vs. 5.3 μm (CI 4.97–5.61 μm), p < 0.05) and shorter tail endpiece (7.8 μm (CI 7.11–8.44 μm) vs. 8.5 μm (CI 7.81–9.14 μm), p < 0.05 after meta-analysis), whereas no effect of swim-up selection was detected on the total sperm cell length. Individuals producing high sperm concentrations had longer sperm midpiece than had men producing lower sperm concentrations. It is concluded that short sperm flagellar tips with long midpieces may be used as biomarkers in infertility therapy.

Baboon spermatology: basic assessment and reproducibility in olive baboons (Papio anubis)

Development of a reproducible baboon in vitro fertilization (IVF) system require optimized and reproducible sperm parameters. The objective of this study was to document basic spermatology values and investigate the reproducibility of these variables in the same baboons 1 or 3months later in a larger number of baboons. In this prospective study, sperm quality (semen volume, pH, concentration, motility, morphology and size) was evaluated in 27 sperm samples obtained from 9 baboons electroejaculated three times with a time interval of 1month (between first and second sample collection) and 3months (between second and third round sample collection). Baseline sperm values for semen volume (0.5±0.3ml), pH (7.5±0.3), concentration (54.2±19.3million/ml), motility (67.3±18.5%) and morphology (89.2±4.8%) were similar to sperm samples obtained after 1 or 3months (P>0.05). Head, midpiece and tail abnormalities were rarely observed (0-9%). Sperm dimensions were characterized by a tail length of 69.6±13.9μm, a head width of 2.41±0.43μm and a head length of 3.49±0.6μm. Sperm quality was not affected by repeated electroejaculation with time intervals of 1 or 3months, suggesting that the same baboon can participate multiple times in reproductive research.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): A model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.

Effect of age on the dimensions and shape of spermatozoa of Large White Polish boars

Abstract. Studies were conducted on 190 ejaculates collected from 16 Large White Polish boars, 7-8 months old. In order to examine spermatozoa morphology, an ejaculate sample was taken from boar monthly microscopic slides were prepared. In each preparation morphometric measurements were taken of fifteen randomly selected spermatozoa, using a computer image analysis software (Screen Measurement v. 4.1), to determine basic sperm dimensions and morphology parameters. Moreover, evaluation of frequency of spermatozoa morphological abnormalities was made. It was found that boar age was associated with sperm dimensions and shape. As boars grew older, sperm length, width and head area increased. However, young boars, under 11 months old, had slightly longer spermatozoa than older boars, in terms of both the length of the whole spermatozoon and the length of the flagellum. The spermatozoa’s morphological build indicators show that boars aged 16 months approx possess semen that is characterised by an elongated head. This assertion is based on the proportions between the head’s width and its length. This proportion proved to be smaller when compared to that present in the sperm of boars over the age of 17 months (P≤0.01).

Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates

It is widely accepted that sperm morphology is a good indicator of fertility and it has been proposed that sperm quality may be related to subtle changes in sperm head morphology. However, a precise estimation of the morphology of ram sperm would be very useful to improve reproductive success in ovine. Computer-assisted morphometric analysis and clustering analysis have been important tools to study sperm subpopulations in domestic animals. However, to the best of our knowledge, no data exist studing morphometric differences regarding to sperm subpopulations within the ovine ejaculate. The aim of this study was to test the presence and distribution of sperm morphometric subpopulations in cryopreserved ejaculates from yearling and mature rams using an objective method by computer analysis system and to establish the relationship between the distribution of the subpopulations found and sperm quality in each individual ram. Principal component analysis revealed that three principal components for yearlings and four components for mature rams that represented more than 84% of the cumulative variance in both cases. After cluster analysis, three sperm morphometric subpopulations for yearlings (CLY) and four for mature (CLM) rams were identified with defined sperm dimensions and shapes. CLY1 included big, round and short sperm (37%), CLY2 included average size and slightly elliptical and elongated sperm (48%), CLY3 included small, long, elliptical and elongated sperm cells (15%). CLM1 consisted of average size and moderate elliptical and elongated (26%), CLM2 consisted of small, long, elliptical and elongated (31%), CLM3 consisted of small and round (32%) and CLM4 included big, short and round (8%) spermatozoa respectively. There were significant differences in the distribution of the three subpopulations (P < 0.001) as well as in the sperm concentration, total motility (%), sperm viability (%) and the overall (P < 0.05) in the ejaculates among the four yearling rams tested. Same results were found for the four subpopulations and the different sperm quality parameters in the ejaculates among the four mature rams tested. In conclusion, cryopreserved ram semen showed a specific structure with regard to sperm morphometric subpopulations. In addition, the distribution of these subpopulations seems to be related to stud maturity age and the ejaculate quality which would be a very important indicator of sperm function. Thus, analysis of sperm morphometric subpopulation structure together with functional tests could provide valuable information to assess the cryoresistence of ram spermatozoa.

Sperm dimensions differ between two Coal Tit Periparus ater populations

The evolutionary forces that shape and maintain variation in sperm traits across species in birds have recently attracted increased attention. Much less is known about the patterns, the causes and the consequences of variation in sperm traits within and, in particular, between populations of the same species. We here analyse intraspecific variation in sperm dimensions within and between two populations of the Coal Tit Periparus ater, a socially monogamous passerine with a high frequency of extra-pair paternity. Spermatozoa from a Norwegian population had a greater mean total length compared to those from a German population, mainly as a result of longer sperm heads. Sperm head length in the Norwegian population also accounted for a larger percentage of sperm total length suggesting differences in sperm proportions between populations. Furthermore, spermatozoa from the Norwegian population showed lower mean within-male variation in sperm length and there was significant between-male variation in sperm total length within each of the two populations. We discuss these results in the light of recent comparative evidence suggesting relationships between sperm length, and variation in sperm length, and the frequency of extra-pair paternity across passerine birds.

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

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. 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. 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. 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.

Size determination of Acipenser ruthenus spermatozoa in different types of electron microscopy

In this study three types of scanning electron microscopes were used for the size determination of spermatozoa of sterlet Acipenser ruthenus – high vacuum scanning electron microscope (SEM, JEOL 6300), environmental scanning electron microscope (ESEM, Quanta 200 FEG), field emission scanning electron microscope (FESEM, JEOL 7401F) with cryoattachment Alto 2500 (Gatan) and transmission electron microscope (TEM, JEOL 1010). The use of particular microscopes was tied with different specimen preparation techniques. The aim of this study was to evaluate to what degree the type of used electron microscope can influence the size of different parts of spermatozoa. For high vacuum SEM the specimen was prepared using two slightly different procedures. After chemical fixation with 2.5% glutaraldehyde in 0.1 M phosphate buffer and post-fixation by 1% osmium tetroxide, the specimen was dehydrated by acetone series and dried either by critical point method or by means of t-butylalcohol. For ESEM fresh, unfixed material was used, which was dropped on microscopic copper grids. In FESEM working in cryo-mode the specimen was observed in a frozen state. Ultrathin sections from chemically fixed and Epon embedded specimens were prepared for TEM observation. Distinct parts of sterlet spermatozoa were measured in each microscope and the data obtained was statistically processed. Results confirmed that the classical chemical procedure of specimen preparation for SEM including critical point drying method led to a significant contraction of all measured values, which could deviate up to 30% in comparison with values measured on the fresh chemically untreated specimen in ESEM. Surprisingly sperm dimensions determinated on ultrathin sections by TEM are comparable with values obtained in ESEM or FESEM.

Morphometric changes in boar spermatozoa induced by cryopreservation

Computer-assisted sperm morphometry analysis was used to determine the effects of cryopreservation on boar sperm head and midpiece morphometry. Sperm-rich fractions were collected from five mature boars. Three microscope slides were prepared from single extended sperm samples prior freezing and post-thawing. All slides were stained with Hemacolor, and 250 sperm images were obtained from each slide. The sperm head dimensions for length, width, area, perimeter and four shape factors and sperm-midpiece dimensions for area, width, angle and distance were determined in each spermatozoa. The effects of sperm freezing on sperm dimensions within and among boars were determined. A previous discriminant analysis of the results was able to correctly classify a 78.3 and 82% of fresh and frozen-thawed spermatozoa respectively. Sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for length, width, area and perimeter. Sperm midpieces were also significantly smaller in cryopreserved spermatozoa for width and area. The highest changes in morphometric dimensions after the freeze-thawing process were found in the midpiece of spermatozoa. The variability of morphometric measurements only was significantly different between fresh and thawed samples for head rugosity and midpiece area. The effects of cryopreservation on morphometric parameters were similar in the boars, which allow us to conclude that cryopreservation process does not have a different effect in each individual boar. In summary, morphometric changes associated with the cryopreservation process on boar spermatozoa do not apparently depends on an effect at individual level.

Comparative study of sperm ultrastructure of five species of teiid lizards (Teiidae, Squamata), and Cercosaura ocellata (Gymnophthalmidae, Squamata)

Sperm ultrastructure of five teiid lizards ( Callopistes flavipunctatus, Crocodilurus amazonicus, Dicrodon guttulatum, Dracaena guianensis, and Teius oculatus), and the gymnophthalmid Cercosaura ocellata is described for the first time. Comparisons of sperm ultrastructure among these species and with those of previously examined teiids and gymnophthalmids revealed that the two groups of Teiioidea (Gymnophthalmidae and Teiidae), and the two subfamilies of Teiidae (Teiinae and Tupinambinae) could be distinguished on the basis of sperm ultrastructure data. Significant differences in sperm dimensions between Cnemidophorus and Aspidoscelis support the recent splitting of these two lineages into different genera. Our results revealed high levels of inter-generic variability in sperm ultrastructure within Teiidae, which produces a data set useful in analyzing relationships between genera and families. In phylogenetic analyses, however, sampling multiple species within teiid genera is essential and recording sperm measurements may profitably complement qualitative ultrastructural characters, maximizing the information content of these structures.

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer® ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris’ Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa.Diff-Quik and Harris’ Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) 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. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts.In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer® system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.