Morphometric characteristics and chromatin integrity of spermatozoa in three Italian dog breeds (original) (raw)
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Theriogenology, 2006
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Animal Reproduction Science, 2017
The aim of this study was to compare sperm DNA fragmentation of frozen-thawed epididymal sperm of dogs using the SCSA (Sperm Chromatin Structure Assay) and SCDt (Sperm Chromatin Dispersion test). For this purpose, epididymis from neutered dogs were minced and incubated in a Tris-based extender. The recovered sperm were frozen in a two-step cooling protocol with Trisbased, egg yolk extender and increasing glycerol concentrations, and stored in liquid nitrogen. After thawing, each replica was incubated at 38°C for 24 h. Sperm DNA fragmentation index (sDFi) was assessed by SCSA and SCDt at 0, 3, 6 and 24 h of incubation and compared within treatments. The relationship and agreement between techniques were evaluated by Pearson's coefficient and Intraclass Correlation Coefficient (ICC). The results were expressed as mean ± standard error of the mean (SEM). Both techniques indicated there was a significant increase of DNA fragmentation after 24 h of incubation. Moderate correlation (r = 0.65; P < 0.01) but lack of agreement (ICC = 0.451; P > 0.05) was found between SCSA and SCDt. The lack of agreement indicates that SCSA and SCDt measure different aspects of DNA fragmentation. Four halo morphologies were detected after 24 h of incubation using the SCDt: un-fragmented DNA with a small halo, fragmented DNA with large halo and two new halo presentations never described before for dog sperm: receding sperm with a disappearing halo and "bald" sperm without chromatin dispersion halo around the core. Sperm without a halo of chromatin dispersion are not described by the manufacturer and are similar to un-fragmented sperm, which could lead to erroneous results when using the SCDt. Further studies with different incubation periods and including the new morphologies described in this study should be performed. In conclusion, although SCSA and SCDt can evaluate the changes in the sperm DNA fragmentation dynamics of frozen-thawed epididymal dog sperm, these provided different findings on sperm DNA fragmentation.
Animal reproduction science, 2018
When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtaine...
Dog sperm DNA: Raw semen evaluation with Toluidine blue stain
Reproduction in Domestic Animals, 2019
The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty‐one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue‐violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min)...
Reproduction in Domestic Animals, 2009
Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys.
Dog sperm head morphometry: its diversity and evolution
Asian Journal of Andrology, 2017
possible relevant differences among different brands, for example, comparing normozoospermic (implying the general concept of a dog) and teratozoospermic dogs, when differences could be explained as a consequence of samples taken from different breeds. 15 The aim of the present work was to define the morphometric relationship between sperm morphological characteristics of several well-defined dog breeds by the use of the ISAS®v1 CASA-Morph system and advanced clustering techniques. It includes the analysis of cluster distance to define a morphometric sperm phylogeny. MATERIALS AND METHODS Collection and preparation of the samples In this study, only the animals which had a clear pedigree as a true representative of their breeds were used. In total, 39 animals were included, comprising four British Bulldog, five Chihuahua, five German Shepherd, five Labrador Retriever, six Spanish Mastiff, seven Staffordshire Terrier, and seven Valencian Rat Hunting dogs. Sampling and analysis were carried out on the REPROVALCAN and Clínica Veterinaria Sangüeso veterinary clinics, located in Valencia (Spain). Following the routine semen extraction by manual masturbation after 14 days ejaculatory abstinence, one ejaculate was
Bioscience Journal
This paper proposes to classify the sperm chromatin compaction alterations in bulls, according to the affected area location and its objective is evaluating the correlation of the intensity, the heterogeneity and these kinds of chromatin decompaction with the rates of cleavage and the formation of blastocysts of in vitro production of embryos (IVPE). It was used several subfertile animals sperm samples, which were evaluated using the toluidine blue staining and computer image analysis, making possible the categorization of the chromatin decompaction according to their location. The percentages of chromatin decompaction and heterogeneity were also evaluated. IVPEs were done and the rates of cleavage and of blastocysts were correlated with the chromatin characteristics. It made possible the classification of the chromatin decompaction according to the head affected part in at least four types: base decompaction, basal half decompaction, central axis decompaction, total decompaction. B...
Canine testicular disorders and their influence on sperm morphology
The objective of this study was to evaluate the influence of testicular disease on sperm morphology. The reproductive tracts of 33 dogs were evaluated clinically and with ultrasound, followed by orchiectomy and harvesting of fluid from the vas deferens to evaluate sperm morphology. A section from each testis was used to conduct histological analyses. Histological changes were noted in 71.2% of testes (47/66). Regardless of dog age, the most frequent pathology was testicular degeneration (80.8%; 38/47), whereas testicular tumors were observed only in adult and old dogs (25.9%; 7/27). Harvesting fluid from the vas deferens for sperm morphology assessment was effective in 87.9% of cases (58/66), and severe testicular degenerative processes induced an increase (P < 0.05) in the percentage of abnormal sperm when compared with normal testes or those with moderate testicular degeneration (special attention given to detached heads). In conclusion, regardless of dog age, breed or origin, a severe testicular degeneration process led to a significant increase in detached heads. Furthermore, the collection of sperm from the vas deferens proved to be an alternative and reliable technique for future research.
BMC Genomics, 2013
Background: Infertility affects~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors.
Sperm Global DNA Methylation (SGDM) in Semen of Healthy Dogs
Veterinary Sciences
Male infertility is an emerging problem in both humans and animals, and the knowledge of its causes is the first step to identifying new diagnostic and therapeutic strategies. In humans, alteration of sperm DNA methylation have been related to poor quality semen, impaired seminal parameters, azoospermia and reduced fertility. Although semen analysis is routinely used to evaluate the male reproductive potential in the canine species, no authors have attempted to relate semen characteristics to the sperm global DNA methylation (SGDM). The aim of this study was to evaluate the SGDM level in healthy dogs and to correlate it with semen parameters that are currently used in dog semen analyses. Conventional and unconventional (sperm DNA fragmentation and SGDM) seminal parameters of thirty dogs from different breeds were evaluated. A positive correlation was found between SGDM and sperm concentration (r = 0.41; p < 0.05), and total sperm count (r = 0.61; p < 0.001); SGDM was significa...