Head Morphometry Research Articles

Letter on ‘The effect of pulsed 900‐MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa’ by Falzone et al. (Int J Androl 34: 20–26, 2011)

Letter on ‘The effect of pulsed 900‐MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa’ by Falzone <i>et al.</i> (<i>Int J Androl</i> 34: 20–26, 2011)

Avaliação computacional da compactação da cromatina e de características morfométricas da cabeça de espermatozoides de coelho (Oryctolagus cuniculus)

Investigou-se a correlação entre a morfometria da cabeça e a intensidade da condensação e heterogeneidade da cromatina em espermatozoides de coelho (Oryctolagus cuniculus). Para tal, utilizaram-se 35 esfregaços de sêmen de coelhos da raça Nova Zelândia, corados com azul de toluidina e avaliados por análise de imagem computacional. As imagens foram obtidas digitalmente em tons de cinza e avaliadas por algoritmos desenvolvidos em ambiente de programação Scilab. As mensurações obtidas da cabeça dos espermatozoides foram área, perímetro, comprimento, largura, relação comprimento largura, elipsidade, fator de forma, descritores Fourier e simetria lateral e anteroposterior. Também foram avaliadas a intensidade da compactação e a heterogeneidade da cromatina espermática. Os espermatozoides de coelho apresentaram compactação e heterogeneidade cromatínica mais intensas do que os de touro e observou-se correlação significativa entre características morfométricas da cabeça e compactação e heterogeneidade cromatínica. Conclui-se que a cromatina é importante para a constituição morfológica da cabeça de espermatozoides de coelho e que a cromatina espermática de coelho é naturalmente mais heterogênea e menos compactada que a de touro

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 μm² vs. 18.8 ± 1.4 μm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds

Studies in many species indicate that variation of spermatozoan head morphology is a sensitive biomarker for abnormal chromatin structure and resultant clinical fertility. This preliminary study evaluated spermatozoan head morphometry in different dog breeds and assessed whether morphometric parameters could reflect spermatozoan DNA fragmentation in dogs. Spermatozoan morphometry and DNA quality (measured by TUNEL flow cytometry) were assessed in semen from 11 dogs of three Italian breeds (Cirneco dell'Etna, Piccolo Levriero Italiano and Segugio Maremmano). Morphometric data showed that Segugio dogs had significantly larger (33·67%) spermatozoa and that Piccolo Levrieros had a higher incidence of long (46·75%) and elliptical spermatozoan heads (11·5%) when compared with the samples from other breeds. Moreover, the predominance of elliptical spermatozoa in one dog (23%) was significantly related to the percentage of spermatozoa with fragmented DNA (12·6%), whereas in another dog, where no more than 1% of spermatozoa was elliptical, only 0·36% of spermatozoa had damaged DNA. It is noteworthy that the breeding record of the former dog in the previous 12 months showed poor fertility and fecundity. These data suggest that spermatozoan head morphometry could be breed related and that there is a significant correlation between DNA fragmentation and elliptical spermatozoa in individual animals. This finding, albeit limited in our study to a single case, is possibly related to clinical infertility.

Phenotypic Variability and Population Structure of Peridomestic Triatoma infestans in Rural Areas of the Arid Chaco (Western Argentina): Spatial Influence of Macro- and Microhabitats

Triatoma infestans, one of the main vectors of Trypanosoma cruzi, is strongly associated with rural human ecotopes. Infested peridomestic places could act as a source of house infestation as a result of the movement of vectors among habitats. The quantitative study of phenotypic traits and nutritional variables could be informative about the population structure and the relative mobility of vectors. This is the first approach toward analyzing the phenetic characteristics of T. infestans and the relationship with their potential role to invade and colonize other habitats in the arid Chaco region. This article compares the phenotypic structure of T. infestans populations living in chicken coops and goat corrals, analyzing the geometric morphometry of wings and heads, antennal phenotypes, and the nutritional status of 257 adults and nymphs. The study area remained untreated with insecticides during the 15 years before the present study. The insects collected in peridomestic structures located in two zones 80 km apart exhibited significant differences in their morphotypes, suggesting an environmental effect and/or the absence of individual exchange between zones. The population structure was clear in T. infestans from goat corrals and chicken coops in one zone and less pronounced in the other. These results suggest the existence of a different rate of individual exchange among ecotopes within each zone. Morphometric variables of heads and wings were significantly correlated in insects collected in goat corrals but not in chicken coops, suggesting a habitat effect and supporting the hypothesis of different canalization forces that affect the two organs. The nutritional status of adults indicated a low dispersal probability with zonal differences. This article provides the first combined morphometric analysis of the head of adults and fifth instar nymphs in triatomines and the first combined analysis of head and wing in T. infestans.

Protein profile study in European eel (Anguilla anguilla) seminal plasma and its correlation with sperm quality

Summary Along with sperm quality parameters, the protein profile of European eel seminal plasma was analyzed during induced spermiation (n = 56 samples). Motility, Percentage of live cells, spermatozoa head morphometry and concentration showed low values during the initial weeks of spermiation and maintained high levels throughout the rest of the experiment. The protein profile gradient by SDS-PAGE (4–15%) registered four important electrophoretic bands around 80, 40, 26 and 12 KDa. Three of them showed significant differences in concentration during treatment (80, 40 and 12 KDa), and all of them showed the highest value on the 8th week. Both 80 and 12 KDa bands increased until the 8th week, followed by a progressive decline. One possible explanation for these profiles is that, in the first weeks of treatment, proteins originated from blood plasma are accumulated in the seminal plasma, and from the 8th week some of these proteins are incorporated into the spermatic membranes. The 40 KDa protein band also increased during the first 8 weeks, but maintained high concentrations in the seminal plasma for the rest of the experiment. One result confirms the theory that the presence of proteins in the seminal plasma having a molecular weight lower than 50 KDa increased spermatozoa motility, since the 40 KDa band displayed significantly higher values coinciding with the high percentages of spermatozoa motility. Seminal plasma proteins seem to have an important role in spermatogenesis and spermatozoa movement, but further studies are necessary to discover the identity of these proteins and their precise functions.

Prenatal development of Crocodylus niloticus niloticus Laurenti, 1768

Prenatal development in crocodilians represents a very interesting model for comparative studies. As the speed of prenatal development of crocodilians varies depending on incubation conditions, the staging of embryos and fetuses is a very important prerequisite for data correlation. To establish a background for future developmental studies on Crocodylus niloticus, we characterized its prenatal development in a collection comprising 169 animals during embryonic/incubation days 9-70. The characteristics included external morphology, head morphometry, and wet body weight determined before fixation. We documented the external morphology of prenatal Nile crocodiles in a large collection of photographs and described landmarks during the morphogenesis of the head, face and limbs. In the development of the facial processes (medial nasal, lateral nasal, maxillary), three phases could be distinguished: union, separation, reunion. At the free jaw margin, a regular series of prominences was present. The outer aspect of a prominence gave rise to a labial scale, the inner aspect to a tooth. In contrast to mammals (humans and mice), the hindlimbs of C. niloticus developed faster than the forelimbs. We also determined changes in basic measures of the head and of the wet body weight. Both morphological and morphometric characteristics showed an apparent inter-individual variability among animals of the same age. This variability decreased among animals of a similar body weight (irrespective of their age). Body weight can be considered as the most representative and complex parameter for crocodile staging reflecting the overall growth of a whole embryo/fetus.

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.

Characterization of ram ( Ovis aries) sperm head morphometry using the Sperm-Class Analyzer

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams ( Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter 2/[4 × π × area]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.

Poor Prediction Value of Sperm Head Morphometry for Fertility and Litter Size in Rabbit

This study was conducted to investigate the predictive capacity of fertility and litter size of sperm head morphometric measurements when the ejaculates fulfilled the minimum requirements commonly used in artificial insemination (AI). Semen samples from 11 rabbits (77 ejaculates) were evaluated for sperm motility, abnormal spermatozoa and sperm head morphometry using computer automated sperm analysis system. Morphometric dimensions for length, width, area and perimeter were analysed. Only ejaculates with more than 70% of motility rate and <15% of abnormal sperm were used for AI. A total of 1031 individual AI were performed in commercial rabbitries. Our results showed significant differences among animals for all sperm head measurements. The mean values for fertility and litter size obtained were 68.4 ± 0.01% and 9.3 ± 0.1% respectively. To assess the predictive value of morphometric dimensions in fertility, a logistic regression analysis was applied. Moreover, multiple linear regression analyses were used to examine the relationship between litter size and sperm head morphometric parameters. Logistic regression analysis rendered a significant model between fertility and area and perimeter, explaining the 0.65% variation. Multiple linear regression analysis rendered a significant model between litter size and width, area and perimeter that explained the 1.3% variation. By conclusion, the sperm head morphometric parameters assay showed low potential to predict fertility and litter size when the ejaculates fulfilled the minimum requirements commonly used in AI (motility and abnormal spermatozoa) in rabbit.

3D morphometry of the human optic nerve head

Human optic nerve head (ONH) anatomy is of interest in glaucoma. Our goal was to carry out a morphometric study of the human ONH based on 3D reconstructions from histologic sections. A set of 10 human ONHs (from four pairs of eyes plus two singles) were reconstructed in an iterative procedure that required the resulting geometries to satisfy a set of quality control criteria. Five models corresponded to eyes fixed at 5 mmHg and the other five models to eyes fixed at 50 mmHg. Several aspects of ONH morphology were measured based on surface and point landmarks: the thicknesses of the lamina cribrosa (LC), the peripapillary sclera and the pre-laminar neural tissue (peripapillary and within the cup); the minimum distance between the anterior surface of the LC and the subarachnoid space; the surface area of the anterior and posterior surfaces of the LC; and the diameter of the scleral canal opening. Our results showed that about one third of the anterior LC surface was obscured from view from the front by the sclera. In all eyes the LC inserted into the pia mater, and not only into the sclera. The variations in ONH morphology between eyes of a pair exceeded, or were of the same order as, changes in morphology due to acute changes in IOP. The reconstruction and morphometry techniques introduced are suitable for application to the ONH. Comparison of measurements in eyes fixed at different pressures suggested small effects on geometry of the increase in IOP. A large variability in ONH morphology, even between contralateral eyes of different IOP, was observed. We conclude that reconstruction of human ONH anatomy from 3D histology is possible, but that large inter-individual anatomic variations make morphometric analysis of the ONH very difficult in the absence of large sample numbers. The insertion of the pia mater into the LC may have biomechanical implications and should be further investigated. Emerging clinical imaging techniques such as deep-scanning OCT will be limited to investigation of the central and mid-peripheral regions of the LC due to optical “occluding” by the peripapillary sclera.

Head morphometry and isoenzymatic profile of two Triatoma infestans Klůg, 1834 (Hemiptera, Reduviidae) populations

The triatomine bug Triatoma infestans was probably originated in Bolivia and dispersed passively over large areas of South America, where it is the major vector of Trypanosoma cruzi. In its probable origin area this species shows two different patterns of behaviour, being found both in sylvatic and human related habitats. Such behaviour is not observed in other areas of its distribution, where it is exclusive to human related habitats. The main objective of the present study was to evaluate head morphometry characters and isoenzymes patterns of two T. infestans populations, one collected in Minas Gerais (Brazil) and other in the Cochabamba Valley (Bolivia), in order to elucidate the factors that could have an implication with the different colonization behaviour. The two populations presented differences in both morphometric (head size) and isoenzymes (specially PGM enzyme profile). The insects from the colonies reared for several years in laboratory conditions, showed reduction in head size starting from the third generation, maintaining this same size in all generations after, until 10 years after generation. The laboratory rearing conditions could be similar to human associated habitats, inducing certain level of genetic homogeneity. Our results showed increase of genetic homogeneity in Brazilian and laboratory-reared colonies from Bolivia, that may be due to a selection process due to passive dispersion, followed by several founder effects episodes and genetic drift.

Compactação da cromatina e morfometria da cabeça de espermatozoides na produção de embriões &lt;em&gt;in vitro&lt;/em&gt; utilizando touros zebuínos

The objective of this work was to evaluate the relationship between sperm head morphometry and chromatin packaging with the cleavage ratio and blastocyst formation during in vitro fertilization programs (IVF). Data of 38 IVFs using semen of zebu bulls with normal spermogram, carried out at two laboratories in Uberaba, Minas Gerais, Brazil, were used in this study. Sperm smears stained with toluidine blue and analyzed by computer image analysis were used to evaluate the morphometry and chromatin packaging. The packaging intensity and homogeneity of sperm chromatin were evaluated. The results made it possible to conclude that light variations in the head morphometry and chromatin packaging had little influence on the cleavage and number of morphologically normal blastocysts during IVF.

THE EFFECT OF DIFFERENT FREEZING PROCEDURES ON SPERM HEAD MORPHOMETRY IN STALLIONS

The evaluation of sperm quality is useful in predicting the fertility of sperm and is of great importance in maximizing reproductive efficency. This work used semen samples collected from 3 stallions and assessed for sperm morphometry (length, width, area, perimeter and ellipticity) before and after cryopreservation with two freezing extenders and procedures and then compared. Sperm freezability was judged according to the sperm quality parameters assessed. Cryopreservation succes was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreservated samples that in the fresh ones. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury during cryopreservation.

Morphology and head morphometric characters of sperm in Thai native crossbred stallions

BackgroundOne of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions.MethodsSemen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Standard-bred; F2 was a Warm-blood; F3 and F4 were Thoroughbreds). All the animals were aged between 5 and 12 years. Sperm morphological examination was performed using formaldehyde-fixed samples under phase-contrast microscopy (1000×). Normal sperm head morphometry characteristics were measured by Computer-Assisted Semen Analysis (Hamilton Thorne, USA.) after applying the Harris' haematoxylin staining technique.ResultsThe percentages of morphologically normal and abnormal sperm varied among individual stallions in both the T and F groups. The mean percentage of morphologically normal sperm was not significantly different (P > 0.05) between T and F stallions (mean ± SE, 49.7 ± 1.3 and 48.1 ± 2.8, respectively). A comparison between the T and F sperm heads revealed that all the dimensional parameters were significantly different (P < 0.05). The coefficients of within-animal variation (CVs) ranged from 2.6 (shape factor 1) to 7.5 (elongation) and 2.9 (shape factor 1) to 8.1 (elongation) in T and F, respectively. In the case of the T group, those sperm head parameters that featured a low within-animal CV and a high between-animal CV were perimeter (2.9, 19.1), shape factor 1 (2.6, 25.8) and shape factor 3 (3.8, 32.0). In the case of the F group, only shape factor 1 (2.9, 26.1) featured such characteristics.ConclusionWe found variability in the percentage of morphologically normal and abnormal sperm, as well as in sperm head dimensions among Thai native crossbred stallions, and these results were similar to those of purebred stallions. Our findings demonstrate that the heads of the T sperm specimens were larger and rounder than that of the F sperm. Perimeter, shape factor 1 and shape factor 3 could be used as parameters for the identification of individual T stallions based on a sperm sample.

Semen characteristics and sperm morphology of serow ( Capricornis sumatraensis)

The serow ( Capricornis sumatraensis) is a critically endangered species. The objectives of this study were to evaluate ejaculate quality in captive males, and to investigate and characterize sperm morphology. Semen was collected using electroejaculation. Mean (±S.D.) seminal characteristics were: semen volume 2.3 ± 0.8 mL, pH 7.8 ± 0.4, and osmolality 329.9 ± 32.9 mOsmol/kg; sperm concentration 515.8 ± 263.1 × 10 6 cells/mL; wave motion score (1–5) 3.9 ± 0.4; motile sperm 60.5 ± 22%; viable sperm 68.3 ± 9.4%; morphologically normal sperm 70.8 ± 19.3%; and an opacity that was yellowish to milky-white. Sperm head length, width, degree of elongation, area, and perimeter were 6.0 ± 0.6 μm, 4.3 ± 0.3 μm, 71.7 ± 8.6%, 19.8 ± 2.5 μm 2, and 17.9 ± 2.1 μm. Based on these measurements, we categorized sperm head morphometry as small, medium, or large. In addition, sperm morphology was examined by light and scanning electron microscopy; overall, morphologically normal and abnormal sperm were similar to those reported for other bovidae. In summary, this study provided baseline data regarding semen characteristics of C. sumatraensis, which should be of value in the preservation of this endangered species.

Pre-freeze bull sperm head morphometry related to post-thaw fertility

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. The ability to determine post-thaw fertility from pre-freeze sperm function parameters would be of major benefit to producers. In this study computer-aided sperm head morphometry was used to determine whether there is any association between pre-freeze bull sperm head measurements and post-thaw non-return to oestrus rates. Sixteen commercial artificial insemination bulls were used for this study. Of the 16 bulls, eight had ≥69% non-return to oestrus rates while the remaining eight bulls had <69% non-return to oestrus rates, based on artificial insemination of cryopreserved semen from a minimum of 748 inseminations per bull. Microscope slides of extended or cryopreserved and thawed semen were prepared and stained by the MGZIN procedure. The morphometric dimensions for length, width, width/length, area and perimeter for a minimum of 200 sperm heads were analyzed from each slide by ASMA and the mean measurements and intra-analysis coefficients of variation (CV) recorded. The post-thaw non-return to oestrus rates for all bulls were correlated with pre-freeze measurements of width ( r = 0.53, P < 0.05), and the change in width/length after cryopreservation ( r = 0.61, P < 0.05). There were no significant differences in the mean pre-freeze or post-thaw sperm head measurements between the two groups. However, the mean post-thaw change in width/length was significantly ( P = 0.003) different between the groups. The mean post-thaw intra-analysis variability for width was significantly lower ( P = 0.03) in bulls with non-return to oestrus rates (NRR) ≥ 69%. Overall, the data suggests that pre-freeze and post-thaw bull sperm head morphometry measurements of individual bulls may have minimal predictive value of post-thaw fertility as determined by non-return to oestrus rates between groups of bulls with limited variance in non-return to oestrus rates. The clinical relationship of sperm head morphometry and fertility in bulls with a greater, clinically significant range of non-return to oestrus, remains to be studied.

Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen

Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear ( Ursus arctos) spermatozoa

The Cantabrian brown bear ( Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/ W, circularity: 4 πA/ P 2, elongation: ( L − W)/( L + W), regularity: πLW/4 A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area ( r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter ( r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer-assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time-consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm2 vs 4.50 ± 0.04 μm2 and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm2 vs 3.18 ± 0.06 μm2 and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.