Trichinocidal activity of a novel formulation of curcumin-olive oil nanocomposite in vitro.

Curcumin-olive oil nanocomposite (CO-NC), a novel formulation of nano-curcumin, was produced and characterized. By evaluating the death rate and DNA damage inflicted on adult Trichinella spiralis (T. spiralis) worms using the comet test and Scanning electron microscopy (SEM) analysis, the effectiveness of the substance against these worms was assessed in vitro. The mortality effects of CO-NC on the parasite adult worms were increased with the upgrading in the concentration and exposure time from 1 to 24 h using concentrations from 10 to 100 ppm. LC50 was determined to be 10.0 ppm/18 h, 20.0 ppm/9 h, 40.0 ppm/6 h, 80.0 ppm/2 h, and 100.0 ppm/1 h, while LC100 was 40.0 ppm/24 h, 80.0 ppm/12 h, and 100.0 ppm/6 h. The comet assay was utilized to examine DNA damage in control and dead worms exposed to varying doses. A direct correlation (P ≤ 0.05) was found between the increase in CO-NC dose and the degree of DNA damage as indicated by alterations in DNA % in the tail segment, tail length (µm), tail moment (µm), and olive tail moment with the control samples. The sub-epidermal layer was detached, the cuticle was partially sloughed off, and the usual creases, ridges, and annulations were altered in the T. spiralis exposed worms. As a result, the tested new trichinocidal drug formulation of nano-curcumin on an oil base was confirmed to be an efficient, secure, and environmentally friendly alternative. The medication has the potential to severely and irreversibly harm the DNA and ultrastructural morphology of adult worms.

Amelioration potential of Moringa oleifera extracts against sodium arsenate induced embryotoxicity and genotoxicity in mouse (Mus musculus) / Potencial de melhoria de extratos de Moringa oleifera contra embriotoxicidade e genotoxicidade induzida por arseniato de sódio em camundongos (Mus musculus)

Previous studies have suggested that arsenic crosses the placenta and affects the fetus development. The study under consideration aims to show comparative ameliorative effect of Moringa oleifera leaf and flower extracts against sodium arsenate induced fetus toxicity of mice. Pregnant mice (N=44) were kept in lab and divided into eleven group from (A to K) and were orally administered the doses 6 mg/kg, 12 mg/kg for sodium arsenate, 150 mg/kg and 300 mg/kg for Moringa oleifera leaf extracts (MOLE) and 150 mg/kg and 300 mg/kg for Moringa oleifera flower extracts (MOFE) comparing with control. The investigation revealed evident reduction in the fetuses weight, hind limb, fore limb, tail and snout length, crown rump and head circumferences well as malformations in tail, feet, arms, legs, skin and eyes in the negative control group (only administered with sodium arsenate). Co-administration of sodium arsenate with MOLE and MOFE ameliorate the reversed effect of sodium arsenate on the shape, length, body weight and DNA damage of fetus significantly at 95% confidence interval. However, Moringa oleifera leaf extract showed more significant results in comparison to Moringa oleifera flower extract. Hence concluded that Moringa oleifera leaf extract ameliorated the embryo toxic effects of sodium arsenate and can be used against environmental teratogens.

Estudos anteriores sugeriram que o arsênio atravessa a placenta e afeta o desenvolvimento do feto. O estudo em consideração visa mostrar o efeito melhorador comparativo de extratos de folhas e flores de Moringa oleifera contra a toxicidade fetal induzida por arseniato de sódio em camundongos. Camundongos grávidas (N = 44) foram mantidos em laboratório e divididos em 11 grupos (de A a K) e foram administrados por via oral nas doses de 6 mg/kg, 12 mg/kg para arseniato de sódio, 150 mg/kg e 300 mg/kg para extratos de folhas de Moringa oleifera (MOLE) e 150 mg/kg e 300 mg/kg para extratos de flores de Moringa oleifera (MOFE) em comparação com o controle. A investigação revelou redução evidente no peso do feto, membro posterior, membro anterior, comprimento da cauda e focinho, coroa, nádega e circunferência da cabeça, bem como malformações na cauda, pés, braços, pernas, pele e olhos no grupo de controle negativo (apenas administrado com arseniato de sódio). A coadministração de arseniato de sódio com MOLE e MOFE melhora significativamente o efeito reverso do arseniato de sódio na forma, comprimento, peso corporal e dano ao DNA do feto, com intervalo de confiança de 95%. No entanto, o extrato da folha da Moringa oleifera apresentou resultados mais significativos em comparação ao extrato da flor da Moringa oleifera. Portanto, concluiu que o extrato da folha de Moringa oleifera melhorou os efeitos tóxicos do arseniato de sódio para o embrião e pode ser usado contra teratógenos ambientais.

In vitro evaluation of zearalenone toxicity by comet assay.

The aim of this study was to reveal the potentially genotoxic effect of zearalenone on bovine lymphocytes by comet assay in vitro. The bovine lymphocytes were exposed to various zearalenone concetrations (50; 10; 2; 0.4 and 0.08 ppm). The viability and DNA damage of lymphocytes was monitored after 2 h, 24 h, 48 h and 72 h. After 2 hours of zearalenone exposure, statistically significant DNA damage occurred at all tested concentrations of 0.08 ppm (12.2±1.25; p⟨0.05), 0.4 ppm (12.7±0.88; p⟨0.01), 2 ppm (12.0±0.51; p⟨0.01), 10 ppm (11.2±0.47; p⟨0.01) and at 50 ppm (14.2±0 61; p⟨0.001). Significantly greater DNA damage was also found after 24 h, 48 h and 72 h. The obtained results showed that zearalenone may induce DNA damage of the bovine lymphocytes.

Determination of cytogenetic markers for biological monitoring in coypu (Myocastor coypu).

Cytogenetic tests are used to assess the influence of physical and chemical factors with potential mutagenic and genotoxic properties on the animal organism. The test results make it possible to eliminate mutagens, as well as helping predict possible genetic consequences in animal cells and assess animal resistance. The aim of this study was to examine, using cytogenetic tests, the spontaneous chromosome and DNA damage in coypu lymphocytes. Four tests: fragile site (FS), bleomycin (BLM), micronucleus, (MN) and comet were used for the first time in coypu cells. The averages with standard deviations obtained in the research were as follows: 3.30 ± 0.80 fragile sites/cell; 0.63 ± 0.80 BLM damage/cell; 6.10 ± 0.53% binucleated cells with MN; and 3.24 ± 0.63% DNA in tail. The present analysis showed high interindividual variation in spontaneous chromosomal and DNA damage levels. In the case of micronucleus, fragile sites, and comet assays, the differences between animals were statistically significant. The data suggest that these assays are sensitive enough to detect some effects on an individual animal and can be proposed as tools for coypu biomonitoring.

DNA Integrity Estimated via the Comet Assay Reflects Oxidative Stress and Competitive Disadvantage in Developing Birds.

AbstractIncreases in DNA degradation have been detected in numerous situations in which organisms are exposed to pollutants. However, outside of the ecotoxicological literature, few studies have investigated whether there exists important variation in DNA integrity in free-living, healthy animals. Using the alkaline version of the comet assay to estimate DNA integrity in blood samples, we aimed to evaluate whether DNA integrity during early life is associated with nestlings' age, body mass, within-brood status, and oxidative stress using nestlings from a wild population of spotless starlings (Sturnus unicolor) as a model. We found important levels of variation in DNA integrity, suggesting the possibility that DNA integrity may have implications for offspring fitness. DNA integrity was dependent on the developmental stage, being lower at hatching than at the end of the nestling period. DNA integrity was also negatively related to the levels of oxidative damage at hatching and positively associated with wing length at fledging. In addition, position within the size hierarchy of the brood at fledging explained differences in DNA integrity, with higher levels in core than in marginal nestlings. Finally, despite extensive within-individual variation along nestling's age, we found DNA integrity during early life to be moderately repeatable within broods. Hence, DNA integrity in early life appears to be mainly affected by environmental factors, such as natural stressors. Our results suggest that measuring the variation in DNA integrity may be a fruitful approach for the assessment of individual fitness in natural populations and can be applied to studies in developmental biology and ecology.

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Modified comet assay with Giemsa staining: a suitable method for assessing genotoxicity in rats / Ensaio cometa modificado com coloração de Giemsa: um método adequado para avaliar a genotoxicidade em ratos

The present study tested a comet assay that was modified for compatibility with Giemsa staining to assess the drug genotoxicity in the peripheral blood of rats. We analysed the peripheral blood of 16 female Wistar rats (N=8 rats/group) from a control group and from a group that was treated with an intraperitoneal injection of 50mg cyclophosphamide/kg. The comet assay was carried out with modifications of the blood volume and immersion time in the lysing solution and different combinations of electrophoresis conditions (running time, voltage and current), to Giemsa staining. The lysing time and electrophoresis conditions allowed for the expression of all classes of DNA damage during the electrophoresis run, and the comets were efficiently stained with Giemsa. The technique showed high reproducibility for the DNA classes. The results demonstrate that the modified comet assay with Giemsa staining can be standardized for routine laboratory procedures using a 20µL blood sample, 3h and 30min immersions in the lysing solution and electrophoresis runs with 23 to 25 V and 310 and 360mA of electrical current. The modified comet assay with Giemsa staining that was described in the present study was standardized to be applied in the laboratory routine.(AU)

O presente estudo testou um ensaio cometa modificado para a coloração de Giemsa para avaliar a genotoxicidade de fármacos no sangue periférico de ratos. Analisou-se o sangue periférico de 16 ratas Wistar (n=8 ratas/grupo) de um grupo controle e de um grupo que foi tratado com uma injeção intraperitoneal de 50mg/kg pv. de ciclofosfamida. O ensaio cometa foi realizado com modificações do volume sanguíneo e do tempo de imersão na solução de lise, bem como com diferentes combinações de condições de eletroforese (tempo de corrida, tensão e corrente), para coloração de Giemsa. O tempo de lise e as condições de eletroforese permitiram a expressão de todas as classes de danos no DNA durante a corrida de eletroforese, e os cometas foram eficientemente corados com Giemsa. A técnica mostrou alta reprodutibilidade para as classes de DNA. Os resultados demonstram que o ensaio cometa modificado com coloração de Giemsa foi padronizado para procedimentos laboratoriais de rotina usando-se uma amostra de sangue de 20µL, 3h30min de imersão na solução de lise e eletroforese com 23 a 25 V e 310 e 360mA. O ensaio cometa modificado com coloração de Giemsa descrito foi padronizado para ser aplicado na rotina laboratorial.(AU)

Seminal plasma fractions can protect common carp (Cyprinus carpio) sperm during cryopreservation.

This study aimed to investigate the effect of fractionated seminal plasma on characteristics of common carp Cyprinus carpio cryopreserved sperm. Nanosep® centrifugal devices yielded four seminal plasma fractions with different total protein content ranging in molecular weight from less than 17 to almost 74 kDa. Each protein fraction was added to semen extender medium prior to freezing. Spermatozoon motility characteristics and DNA integrity were analyzed in supplemented and non-supplemented cryopreserved samples. The cryopreservation process strongly affected the swim-up sperm quality. Treatment with fractions 1, 2, 3, and 4 was associated with significantly higher spermatozoon motility rate and curvilinear velocity than seen in extender only, with highest values obtained with fraction 4 (78.21 ± 2.41% and 168.05 ± 4.46 µm/s, respectively). Significantly less DNA damage, expressed as percent tail DNA (12.23 ± 1.27) and olive tail moment (0.68 ± 0.12), was recorded in fraction 4. The findings indicated that addition of fractionated seminal plasma to cryopreservation medium can preserve the quality of common carp sperm. The protective effect of each fraction varied, suggesting the presence of distinct components exerting different effects on cryopreserved sperm function.

Spatial and temporal variation of three biomarkers in Mytilus edulis.

Environmental conditions and xenobiotic exposure can be sources of stress to living organisms. Biological markers are measurable indicator of changes which may happen at any biological level and which can be considered an early warning signal of some biological or environmental state or condition. A structured field study was undertaken to investigate the relationship between three biomarker assays and the spatial and temporal variation of each biomarker in samples of Mytilus edulis. The three biomarkers were the neutral red retention assay, micronucleus assay and comet assay, which indicate damage at different cellular/molecular levels. Three sites in Poole Harbour, an area on the South coast of the UK were sampled on six separate occasions at least three weeks apart. The results for the comet assay showed a significant difference between sites and between sampling dates whereas the results for the other two assays did not show a significant difference for either factor. There was no significant correlation between the results of any pair of the three biomarkers. The results of the micronucleus assay showed a significant correlation with water temperature. This temperature effect, as well as induced repair, may contribute to explain the lack of a strict correspondence between pollution gradients and biomarkers responses.

The Comet assay for detection of DNA damage in canine sperm.

Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p < .001). There was a negative correlation between progressive motility and oxidative damage (r = -.792; p = .001). Basal and oxidative DNA damage of spermatozoa are increased in dogs with non-normospermic semen. In conclusion, and considering the elevated correlation with classical tests of sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs.

Cytogenetic tests for animal production: state of the art and perspectives.

Cytogenetic tests are effective tools for monitoring the health status of livestock and improving their genetic value. Cytogenetic screening allows for the detection of animals carrying chromosomal aberrations and to avoid using them as breeders. Progress in karyotype monitoring, with new molecular probes and automation, has greatly increased the productivity of this procedure. Several genotoxicity tests are available to detect the possible presence and effects of pollutants or drugs. Among these, the micronucleus test and the Comet assay are the most convenient in terms of costs and benefits. Finally, analysis of telomeres, the end of chromosomes and markers of genomic instability, may be developed into a new marker of stress and genetic value.

The effect of flash-freezing temperature on stallion sperm DNA structure.

The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-αt. The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-αt and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting.

Genotoxicity assessment of fipronil (frontline plus®) in Canis familiaris / Avaliação de genotoxicidade do fipronil (frontline plus®) em cães

Fipronil is a pesticide widely used for controlling fleas and ticks in domestic animals, and its short-term exposure can lead to serious effects on animals. However, the possible genotoxic effect of this compound has not been investigated in target animals. Based on the hypothesis that fipronil can induce genotoxicity, this study evaluated the effect of fipronil on DNA damage in peripheral blood cells. For that purpose, ten dogs of both sexes were used in the study. The product (6.7mg/kg) was applied on the dorsal neck region of each animal. Peripheral blood samples were collected immediately prior to application of the product, and at 3, 8 and 24 hours after the application. Samples were processed for comet assay. No statistically significant differences were found among the four time points. The current study suggests for the first time that a single exposure to this pesticide does not induce systemic genotoxic effect in dogs.(AU)

O fipronil é um inseticida/herbicida amplamente utilizado para controle de pulgas e carrapatos em animais domésticos. Sua exposição a curto prazo tem acarretado efeitos deletérios em animais. Entretanto, o possível efeito genotóxico deste composto ainda não foi investigado em animais alvo. Baseando-se na hipótese de que o fipronil pode induzir genotoxicidade, o presente estudo avaliou o efeito deletério do fipronil no material genético de células de sangue periférico. Para isso, dez cães sadios, de ambos os sexos, foram utilizados neste estudo. O produto (6,7mg/kg) foi aplicado na região dorsal do pescoço de cada animal. As amostras de sangue foram coletadas imediatamente antes da aplicação do produto (controle) e após três, oito e 24 horas da aplicação. As amostras foram imediatamente processadas para condução do teste do cometa, a fim de se avaliar os danos basais no DNA. Não houve diferença significativa entre os quatro momentos de coleta em relação aos danos no material genético. O estudo sugere, pela primeira vez, que uma exposição única a este pesticida não induz efeito genotóxico sistêmico em cães.(AU)

Evaluation of Stage-Dependent Genotoxic Effect of Roundup® (Glyphosate) on Caiman latirostris Embryos.

The agricultural expansion over the past decades, along with the associated increase in the use of pesticides, represents a high risk for many wild species. Caiman latirostris is a South American caiman with many features that make it highly vulnerable to pesticide exposure. Considering previous finding on the genotoxicity of the glyphosate-based formulation Roundup® in this species, the aim of this study was to evaluate the possible stage-dependent effect of this compound on C. latirostris embryos through the Comet assay (CA), micronuclei (MN), and nuclear abnormalities (NA) tests. Caiman eggs were exposed to three effective concentrations of Roundup® (750, 1250, 1750 µg/egg) in three different stages of the incubation period (total duration 70 ± 3 days at 31 ± 2 °C) of approximately 23 days each. A statistically significant difference in DNA damage determined by the CA was found between groups exposed to different concentrations of RU (p < 0.05) and the negative control, but no difference was observed among the three stages of exposure within any treatment (p > 0.05). There was no differences in the MN or NA frequencies between the different groups and the negative control (p > 0.05), nor among the different stages within each treatment. The results obtained in this study indicate that RU produce DNA damage on C. latirostris embryos independently of the developmental stage where the exposure occurs, implying an important risk for the species during all its period of development, when pesticide application is at maximum rate.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis.

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd2+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd2+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

Longitudinal profile of the genotoxic potential of the River Danube on erythrocytes of wild common bleak (Alburnus alburnus) assessed using the comet and micronucleus assay.

The Joint Danube Survey 3 (JDS3; the biggest river expedition in 2013) had offered the unique opportunity for a large-scale monitoring approach for biomarker response in feral fish collected along a Danube stretch from Kehlheim (DE) to Sulina (RO). The advantage of genotoxicity as a marker for pollution exposure in fish is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was in the focus of the biomarker investigations in fish during the expedition. Blood samples of common bleak (Alburnus alburnus) for the investigation of the micronucleus frequency and comet tail intensity of fragmented DNA material in erythrocytes were collected at 18 and 12 sampling sites, respectively. For 9 sampling sites same samples were used to compare the in-situ data for the comparable genotoxic endpoint in the micronucleus (MN) and comet assay (CM). The data of both in-situ assays showed a significant correlation, indicating the strength and comparability of the data sets. Significant variation in DNA damage in fish along the longitudinal profile of the Danube was demonstrated for both assays compared to reference sites. The results suggest that DNA damage in erythrocytes of fish was mainly affected by wastewater of highly populated regions. No linkage between the results and the general health/dietary status of the fish were revealed, whereas correlation with some genotoxicity drivers in the water phase, suspended particulate matter and sediments could be demonstrated.

Assessing genotoxic effects in fish from a marine protected area influenced by former mining activities and other stressors.

The goal of the current study was to evaluate different genotoxicity tools in order to assess a marine protected area (MPA) affected by former mining activities and urban settlements. A catfish (Cathorops spixii) was analyzed for genotoxic effects at the (i) molecular and at the (ii) chromosomal levels. Through factor analysis, genotoxicity was found to be linked to levels of metals bioaccumulated and PAH metabolites in the bile. Micronucleus and nuclear alteration were less vulnerable to the effects of confounding factors in mildly contaminated areas since they were more frequently associated with bioaccumulated metals than the DNA analysis. The different genotoxicity responses allowed for the identification of sources of pollution in the MPA. This approach was important for detecting environmental risks related to genotoxic contaminants in a mildly contaminated MPA.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd²+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd²+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.

Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P<0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P<0.05). The % Ghosts and COMP-αt were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality.