The pollution by metals and metalloids represents a signi cant environmental threat, due to the a... more The pollution by metals and metalloids represents a signi cant environmental threat, due to the adverse repercussions they can have on aquatic organisms and on the health of human populations. Aquatic reptiles can be used as bioindicators of the presence of contaminants in watercourses. In the present study, water, carapace and blood samples from Trachemys dorbigni tortoises were collected in rural and urbanized areas, in order to identify the presence and consequences of exposure to metal(loid)s. The elements Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn were measured in water, blood and carapace, and erythrocitary changes were also analyzed by ow cytometry and micronucleus evaluation. In the assessment of water in the urban area, the metals evaluated exceeded all established national parameters, while in the rural area, only Cd, Hg and Ni were elevated. In the carapace evaluation, the samples collected in the urban area showed higher levels of all measured metals, when compared to animals from the rural area. In blood samples only Al, Cr, Ni and Zn showed higher values in this area. Animals from the urban area also showed a higher incidence of necrotic cells, a greater number of micronucleated cells and a high rate of DNA fragmentation, resulting in cytotoxic, genotoxic and mutagenic damage. The results highlight the importance of using bioindicators to measure metallic contaminants in urban watercourses, thus estimating their impacts on the health of animals and humans, in the short and long term.
Resumo: O presente trabalho tem como objetivo analisar o uso de lipossomas e lecitina de soja no ... more Resumo: O presente trabalho tem como objetivo analisar o uso de lipossomas e lecitina de soja no processo de criopreservação de sêmen, com foco na espécie suína. Pois a utilização do sêmen criopreservado na suinocultura representa 1% apenas, por causa dos danos que ocorrem durante o processo de criopreservação, no qual passa por três etapas antes de ocorrer o congelamento, com a diminuição da temperatura. Dessa forma, ocorrem alterações físicas e químicas dos espermatozoides, deixando-os suscetíveis a sérios danos causados pelo frio. Além disso, a redução da temperatura, leva a mudança de fase fluida para a fase gel, ocorrendo uma maior desidratação celular, decorrente da remoção de moléculas polares dos fosfolipídios, acarretando em alterações de funcionalidade. Diante disso, busca-se novos crioprotetores, antioxidantes para reduzir/ reparar os danos aos espermatozoides de suínos desde do processo de refrigeração até o processo de descongelamento do sêmen. Destacase os lipossomas, que são vesículas coloidais compostas de lipídeos anfipáticos que em excesso de água se agregam formando bicamadas lipídicas esféricas com compartimento interno aquoso, podendo carregar antioxidantes, ATP. No entanto, eles podem ser fabricados de diferentes fosfolipídios, desde da gema de ovo até lecitina de soja, e até mesmo o processo de fabricação podem ser diferentes. Nota-se que a utilização de lipossomas no processo de criopreservação de sêmen, é uma alternativa para evitar/reparar os danos que ocorrem aos espermatozoides durante esse processo.
Freezing of Equine Semen is Influenced by Exposure Time and Concentration of the Cryoprotectant Glycerol
Cryoletters
BACKGROUND: Glycerol is a cryoprotectant widely used in the freezing of mammalian species ' s... more BACKGROUND: Glycerol is a cryoprotectant widely used in the freezing of mammalian species ' semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. OBJECTIVE: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. MATERIALS AND METHODS: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Analysis (CASA) system and cell feature parameters were assessed by flow cytometry. RESULTS: Considering the total and progressive motility of the spermatozoa, we concluded that protocols using 5% glycerol for 15 and 30 min exposure, 4% glycerol for 45 min exposure and 3% glycerol for 90 min exposure generated the best results. CONCLUSION: We suggest the use ...
Exogenous ATP in the Cryopreservation of Brycon orbignyanus Spermatozoa
Cryo letters, 2020
BACKGROUND ATP exogenous (ATPe) has been used successfully in improving motility and fertility fo... more BACKGROUND ATP exogenous (ATPe) has been used successfully in improving motility and fertility for many animal species. However this has not yet been tested on Brycon orbignyamus. OBJECTIVE The objective of this study was to evaluate the use of ATPe for the cryopreservation of sperm from B. orbignyamus. MATERIALS AND METHODS The ATPe concentrations tested were 1.0 µM, 5.0 µM and 10 µM combined with Beltsville Thawing SolutionTM extender and dimethylformamide at 7.5%. The sperm were frozen in a nitrogen vapour vessel and stored in liquid nitrogen at -196 ºC. The parameters of viability post-thawing were evaluated using CASA, and flow cytometer. RESULTS The ATPe did not promote improvements in spermatic kinetics, and in the higher concentrations caused a worsening in these parameters. Also there was loss of mitochondrial functionality and greater cellular disruption with the concentration of 10 μM. CONCLUSION We do not recommend the addition of ATP for cryopreserving B. orbignyamus.
Aromatase cytochrome P450 is an enzyme that is responsible for a key step in the biosynthesis of ... more Aromatase cytochrome P450 is an enzyme that is responsible for a key step in the biosynthesis of estrogens, which is linked to reproduction. This enzyme is a product of the gene Cyp19 and catalyzes the formation of estrogen from androgen. Estrogen is essential for the development of the gonads and various other physiological processes, ranging from normal growth to reproductive behavior. This study aimed at validation of primers for microsatellite locus in the regulatory region of gene Cyp19b in Oreochromis niloticus. Thirty samples of three strains were analyzed: Chitralada and Supreme from larviculture Aquabel, and GIFT from the State University of Maringa (Parana, Brazil). After extraction of DNA, the samples were subjected to PCR using primers designed to flank a microsatellite region. After confirmation of amplification, the samples were subjected to electrophoretic separation in the apparatus Origins (Elchrom Scientific, USA) to identify the alleles. A polymorphism in the r...
Effect of Amide on Semen Cryopreservation of Curimba (Prochilodus lineatus)
Cryo letters, 2020
BACKGROUND The low molecular weight and high cellular permeability of amides make them suitable f... more BACKGROUND The low molecular weight and high cellular permeability of amides make them suitable for use as penetrative cryoprotectants for sperm cells. OBJECTIVE This study aims to evaluate the effect of dimethylformamide (DMF) and dimethylacetamide (DMA) on sperm cryopreservation of Curimba (Prochilodus lineatus). MATERIALS AND METHODS Semen samples were diluted in media containing cryoprotectants [DMF, DMA and dimethyl sulfoxide (DMSO)]. Parameters of motility, membrane integrity, DNA integrity, mitochondrial functionality, viability and fertility were assessed upon thawing. RESULTS As compared to the 10% DMSO, DMA at 5% and DMF at 2% obtained the best results for the integrity of membrane, DNA and mitochondria; the motility parameters were best in the 2% and 5% DMF treatments. The best fertilization rates were demonstrated in 2%, 5%, and 8% DMF treatment groups. CONCLUSION DMF at 2%, 5%, and 8% provided the best results for both in vitro and in vivo assessments, and can efficient...
Liquid Storage of Geophagus brasiliensis semen in the Presence of Different Extenders
BACKGROUND In order to preserve the genetic diversity of cichlid fish in gene banks, it is necess... more BACKGROUND In order to preserve the genetic diversity of cichlid fish in gene banks, it is necessary to use certain extenders to maintain the integrity of spermatozoa cells during cooling. OBJECTIVE To evaluate the effects of different extenders on the quality parameters of cooled semen of Geophagus brasiliensis. MATERIALS AND METHODS Semen samples were collected from seven adult fish and diluted with five extenders: Beltsville Thawing Solution (BTS™), Hanks' Balanced Salt Solution (HBSS), Tris-glucose, Ginsburg's Fish Ringers, and Phosphate buffered Saline. All parameters were evaluated in fresh semen samples and after cooling at 4°C at 0, 24, 48, and 96 h to evaluate cell viability (membrane integrity, DNA, and mitochondrial functionality) and motility rate and weather motility. RESULTS The BTS and Tris-glucose resulted in the best outcomes (P<0.05) in terms of membrane integrity assessments (35.1% and 30.9%, respectively), DNA integrity (71.6%; 75.7%), mitochondrial fu...
Morphometry and Mortality of Nile Tilapia fingerlings in two different treatments with temperature
The objective of this work is to present the mortality rates of Nile Tilapia ( Oreochromis niloti... more The objective of this work is to present the mortality rates of Nile Tilapia ( Oreochromis niloticus ) subjected to two different temperature treatments, the morphometric measurements and body mass in two different phases in terms of age for each temperature and, morphologically identify differences that may be attributed to sexual dimorphism and that will determine the best age to collect animals for gonads analysis. Two treatments with water temperature were performed: 35° C and 25° C. Animals were killed in two steps: 35° C (71 and 92 days after hatching) and 25° C (112 and 131 days after hatching). The mortality record was performed by direct visualization, morphometry and weighing. The sexing was performed by gonadal histopathology. The treatment at 35° C showed lower mortality indicating that the fish would be more suited to higher temperatures. In contrast, the treatment at 25° C showed significant difference between variables and males with when the animals were 131 days aft...
High‐speed centrifugation of extender of freeze‐thaw boar semen
Reproduction in Domestic Animals, 2021
In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Muc... more In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and / or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg-yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison to the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.
Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F010... more Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRN...
Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, tran... more Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.
The effects of the cryoprotectants dimethyl sulfoxide, glycerol and methyl glycol at different co... more The effects of the cryoprotectants dimethyl sulfoxide, glycerol and methyl glycol at different concentrations on Austrolebias minuano sperm quality parameters were evaluated in this study. The cellular kinetic parameters, determined using flow cytometry, indicated the best results with the samples cryopreserved with 7.5% methyl glycol. Dimethyl sulfoxide concentrations of 7.5% and glycerol concentrations of 10%, 12.5% and 15% demonstrated the least benefit across all evaluated sperm kinetic parameters. When assessed using flow cytometry, a concentration of 7.5% methyl glycol similarly showed better sperm kinetic parameters than 12.5% dimethyl sulfoxide. We conclude that cryoprotectants, especially methyl glycol, are effective for the preservation of sperm quality in A. minuano. However, high sensitivity of spermatozoa against glycerol was observed in these studies; thus, it is not recommended for cryopreservation purposes.
h i g h l i g h t s Lifetime exposure to Cu reduced all the analyzed parameters of sperm quality ... more h i g h l i g h t s Lifetime exposure to Cu reduced all the analyzed parameters of sperm quality in the killifish Poecilia vivipara. We suggest that augmented oxidative stress, endocrine disruption and energetic disturbance are involved in these negative outcomes. Lifetime exposure to Cu may disrupt fish reproduction and negatively affect the maintenance of its populations.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2018
The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved ... more The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved weeds. The present study evaluated sperm quality parameters in zebrafish Danio rerio after 11-day exposure to nominal ATZ concentrations of 2, 10, and 100 μg L −1. All ATZ concentrations caused a decrease in motility, mitochondrial functionality, and membrane integrity, as measured using conventional microscopy or fluorescence microscopy with specific probes. The DNA integrity of sperm was not affected. The levels of expression of genes related to spermatogenesis, antioxidant defenses, and DNA repair were also investigated using RT-qPCR. The ATZ caused transcriptional repression of the spermatogenesis-related genes SRD5A2 and CFTR, the antioxidant defense genes SOD2 and GPX4B, and the DNA repair gene XPC. This is the first study to show that environmentally relevant concentrations of ATZ significantly affect the sperm quality in fish, possibly resulting in reduced fertility rates. In addition, we showed that the repression of genes related to spermatogenesis and cellular defense could be part of the mechanisms involved in the ATZ toxicity in the testes of male fish.
The current study assessed a semen cryopreservation protocol in the Amazonian catfish Leiarius ma... more The current study assessed a semen cryopreservation protocol in the Amazonian catfish Leiarius marmoratus, a freshwater fish, of rheophilic behavior, and of great importance for Brazilian fish farming. Eight males (n=8) were stripped and the semen was cryopreserved if total motility in fresh semen was higher than 80%. The external cryoprotectant Trehalose was then diluted in Beltsvile Thawing Solution (BTS) extender in the following concentrations: 50, 100, 150, and 200 mM. Semen samples were diluted in the media (1:9 v/v) being tested, then frozen in a container with nitrogen
Cadmium is a natural element found in the earth's crust; it is usually associated with other meta... more Cadmium is a natural element found in the earth's crust; it is usually associated with other metals, but due to the impacts caused by human activity, its concentration has increased in the aquatic environment. This metal may damage aquatic animal reproduction, decreasing the rate of fertilization of organisms such as fish. Thus, this study aimed to evaluate the in vitro toxicity of different concentrations of cadmium (0 (control), 0.5, 5, and 10 g/L) using sperm cells of model organism zebrafish, Danio rerio. Structural parameters, including integrity and fluidity of the plasma membrane, concentration of oxygen species, mitochondrial function and DNA fragmentation were measured by flow cytometry. The following sperm movement parameters were also measured using the computer assisted sperm analysis (CASA) system: motility, time of motility, curvilinear velocity, average path velocity and straight line velocity in m/s. Significant effects were observed on path speed, straight speed, curvilinear velocity, motility time, progressive and total motility, and plasma and DNA integrity. The results showed that cadmium can negatively affect some reproductive parameters in D. rerio, which may reduce the fertility rate of these animals.
The pollution by metals and metalloids represents a signi cant environmental threat, due to the a... more The pollution by metals and metalloids represents a signi cant environmental threat, due to the adverse repercussions they can have on aquatic organisms and on the health of human populations. Aquatic reptiles can be used as bioindicators of the presence of contaminants in watercourses. In the present study, water, carapace and blood samples from Trachemys dorbigni tortoises were collected in rural and urbanized areas, in order to identify the presence and consequences of exposure to metal(loid)s. The elements Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn were measured in water, blood and carapace, and erythrocitary changes were also analyzed by ow cytometry and micronucleus evaluation. In the assessment of water in the urban area, the metals evaluated exceeded all established national parameters, while in the rural area, only Cd, Hg and Ni were elevated. In the carapace evaluation, the samples collected in the urban area showed higher levels of all measured metals, when compared to animals from the rural area. In blood samples only Al, Cr, Ni and Zn showed higher values in this area. Animals from the urban area also showed a higher incidence of necrotic cells, a greater number of micronucleated cells and a high rate of DNA fragmentation, resulting in cytotoxic, genotoxic and mutagenic damage. The results highlight the importance of using bioindicators to measure metallic contaminants in urban watercourses, thus estimating their impacts on the health of animals and humans, in the short and long term.
Resumo: O presente trabalho tem como objetivo analisar o uso de lipossomas e lecitina de soja no ... more Resumo: O presente trabalho tem como objetivo analisar o uso de lipossomas e lecitina de soja no processo de criopreservação de sêmen, com foco na espécie suína. Pois a utilização do sêmen criopreservado na suinocultura representa 1% apenas, por causa dos danos que ocorrem durante o processo de criopreservação, no qual passa por três etapas antes de ocorrer o congelamento, com a diminuição da temperatura. Dessa forma, ocorrem alterações físicas e químicas dos espermatozoides, deixando-os suscetíveis a sérios danos causados pelo frio. Além disso, a redução da temperatura, leva a mudança de fase fluida para a fase gel, ocorrendo uma maior desidratação celular, decorrente da remoção de moléculas polares dos fosfolipídios, acarretando em alterações de funcionalidade. Diante disso, busca-se novos crioprotetores, antioxidantes para reduzir/ reparar os danos aos espermatozoides de suínos desde do processo de refrigeração até o processo de descongelamento do sêmen. Destacase os lipossomas, que são vesículas coloidais compostas de lipídeos anfipáticos que em excesso de água se agregam formando bicamadas lipídicas esféricas com compartimento interno aquoso, podendo carregar antioxidantes, ATP. No entanto, eles podem ser fabricados de diferentes fosfolipídios, desde da gema de ovo até lecitina de soja, e até mesmo o processo de fabricação podem ser diferentes. Nota-se que a utilização de lipossomas no processo de criopreservação de sêmen, é uma alternativa para evitar/reparar os danos que ocorrem aos espermatozoides durante esse processo.
Freezing of Equine Semen is Influenced by Exposure Time and Concentration of the Cryoprotectant Glycerol
Cryoletters
BACKGROUND: Glycerol is a cryoprotectant widely used in the freezing of mammalian species ' s... more BACKGROUND: Glycerol is a cryoprotectant widely used in the freezing of mammalian species ' semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. OBJECTIVE: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. MATERIALS AND METHODS: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Analysis (CASA) system and cell feature parameters were assessed by flow cytometry. RESULTS: Considering the total and progressive motility of the spermatozoa, we concluded that protocols using 5% glycerol for 15 and 30 min exposure, 4% glycerol for 45 min exposure and 3% glycerol for 90 min exposure generated the best results. CONCLUSION: We suggest the use ...
Exogenous ATP in the Cryopreservation of Brycon orbignyanus Spermatozoa
Cryo letters, 2020
BACKGROUND ATP exogenous (ATPe) has been used successfully in improving motility and fertility fo... more BACKGROUND ATP exogenous (ATPe) has been used successfully in improving motility and fertility for many animal species. However this has not yet been tested on Brycon orbignyamus. OBJECTIVE The objective of this study was to evaluate the use of ATPe for the cryopreservation of sperm from B. orbignyamus. MATERIALS AND METHODS The ATPe concentrations tested were 1.0 µM, 5.0 µM and 10 µM combined with Beltsville Thawing SolutionTM extender and dimethylformamide at 7.5%. The sperm were frozen in a nitrogen vapour vessel and stored in liquid nitrogen at -196 ºC. The parameters of viability post-thawing were evaluated using CASA, and flow cytometer. RESULTS The ATPe did not promote improvements in spermatic kinetics, and in the higher concentrations caused a worsening in these parameters. Also there was loss of mitochondrial functionality and greater cellular disruption with the concentration of 10 μM. CONCLUSION We do not recommend the addition of ATP for cryopreserving B. orbignyamus.
Aromatase cytochrome P450 is an enzyme that is responsible for a key step in the biosynthesis of ... more Aromatase cytochrome P450 is an enzyme that is responsible for a key step in the biosynthesis of estrogens, which is linked to reproduction. This enzyme is a product of the gene Cyp19 and catalyzes the formation of estrogen from androgen. Estrogen is essential for the development of the gonads and various other physiological processes, ranging from normal growth to reproductive behavior. This study aimed at validation of primers for microsatellite locus in the regulatory region of gene Cyp19b in Oreochromis niloticus. Thirty samples of three strains were analyzed: Chitralada and Supreme from larviculture Aquabel, and GIFT from the State University of Maringa (Parana, Brazil). After extraction of DNA, the samples were subjected to PCR using primers designed to flank a microsatellite region. After confirmation of amplification, the samples were subjected to electrophoretic separation in the apparatus Origins (Elchrom Scientific, USA) to identify the alleles. A polymorphism in the r...
Effect of Amide on Semen Cryopreservation of Curimba (Prochilodus lineatus)
Cryo letters, 2020
BACKGROUND The low molecular weight and high cellular permeability of amides make them suitable f... more BACKGROUND The low molecular weight and high cellular permeability of amides make them suitable for use as penetrative cryoprotectants for sperm cells. OBJECTIVE This study aims to evaluate the effect of dimethylformamide (DMF) and dimethylacetamide (DMA) on sperm cryopreservation of Curimba (Prochilodus lineatus). MATERIALS AND METHODS Semen samples were diluted in media containing cryoprotectants [DMF, DMA and dimethyl sulfoxide (DMSO)]. Parameters of motility, membrane integrity, DNA integrity, mitochondrial functionality, viability and fertility were assessed upon thawing. RESULTS As compared to the 10% DMSO, DMA at 5% and DMF at 2% obtained the best results for the integrity of membrane, DNA and mitochondria; the motility parameters were best in the 2% and 5% DMF treatments. The best fertilization rates were demonstrated in 2%, 5%, and 8% DMF treatment groups. CONCLUSION DMF at 2%, 5%, and 8% provided the best results for both in vitro and in vivo assessments, and can efficient...
Liquid Storage of Geophagus brasiliensis semen in the Presence of Different Extenders
BACKGROUND In order to preserve the genetic diversity of cichlid fish in gene banks, it is necess... more BACKGROUND In order to preserve the genetic diversity of cichlid fish in gene banks, it is necessary to use certain extenders to maintain the integrity of spermatozoa cells during cooling. OBJECTIVE To evaluate the effects of different extenders on the quality parameters of cooled semen of Geophagus brasiliensis. MATERIALS AND METHODS Semen samples were collected from seven adult fish and diluted with five extenders: Beltsville Thawing Solution (BTS™), Hanks' Balanced Salt Solution (HBSS), Tris-glucose, Ginsburg's Fish Ringers, and Phosphate buffered Saline. All parameters were evaluated in fresh semen samples and after cooling at 4°C at 0, 24, 48, and 96 h to evaluate cell viability (membrane integrity, DNA, and mitochondrial functionality) and motility rate and weather motility. RESULTS The BTS and Tris-glucose resulted in the best outcomes (P<0.05) in terms of membrane integrity assessments (35.1% and 30.9%, respectively), DNA integrity (71.6%; 75.7%), mitochondrial fu...
Morphometry and Mortality of Nile Tilapia fingerlings in two different treatments with temperature
The objective of this work is to present the mortality rates of Nile Tilapia ( Oreochromis niloti... more The objective of this work is to present the mortality rates of Nile Tilapia ( Oreochromis niloticus ) subjected to two different temperature treatments, the morphometric measurements and body mass in two different phases in terms of age for each temperature and, morphologically identify differences that may be attributed to sexual dimorphism and that will determine the best age to collect animals for gonads analysis. Two treatments with water temperature were performed: 35° C and 25° C. Animals were killed in two steps: 35° C (71 and 92 days after hatching) and 25° C (112 and 131 days after hatching). The mortality record was performed by direct visualization, morphometry and weighing. The sexing was performed by gonadal histopathology. The treatment at 35° C showed lower mortality indicating that the fish would be more suited to higher temperatures. In contrast, the treatment at 25° C showed significant difference between variables and males with when the animals were 131 days aft...
High‐speed centrifugation of extender of freeze‐thaw boar semen
Reproduction in Domestic Animals, 2021
In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Muc... more In semen cryopreservation, egg yolk is still widely used as a non-penetrating cryoprotectant. Much has been developed in the search for alternatives for this biological product. This work aimed to evaluate the processed egg yolk through ultracentrifugation and / or sonication in the cryopreservation of swine semen. Twenty-seven semen doses were purchased from a commercial boar stud and processed for cryopreservation using egg-yolk lactose 11% (control) extender, processed using two different methods: high-speed centrifugation and sonication. Then, they were submitted to freeze-thawing protocol and were assessed for kinematic and cell structural parameters. Samples in which extenders underwent centrifugation had better results in velocity parameters, meanwhile those that only sonication was performed had poorest results in this parameter. The preservation of the membrane and mitochondria structure had better results when the diluent was only centrifuged in comparison to the other treatments. Therefore, centrifugation of extender containing egg yolk is important for better cryopreservation of swine semen.
Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F010... more Overexpression of growth hormone (GH) in gh-transgenic zebrafish of a highly studied lineage F0104 has earlier been reported to cause increased muscle growth. In addition to this, GH affects a broad range of cellular processes in transgenic fish, such as morphology, physiology, and behavior. Reports show changes such as decreased sperm quality and reduced reproductive performance in transgenic males. It is hypothesized that microRNAs are directly involved in the regulation of fertility potential during spermatogenesis. The primary aim of our study was to verify whether gh overexpression disturbs the sperm miRNA profile and influences the sperm quality in transgenic zebrafish. We report a significant increase in body weight of gh-transgenic males along with associated reduced sperm motility and other kinetic parameters in comparison to the non-transgenic group. MicroRNA transcriptome sequencing of gh-transgenic zebrafish sperms revealed expressions of 186 miRNAs, among which six miRN...
Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, tran... more Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.
The effects of the cryoprotectants dimethyl sulfoxide, glycerol and methyl glycol at different co... more The effects of the cryoprotectants dimethyl sulfoxide, glycerol and methyl glycol at different concentrations on Austrolebias minuano sperm quality parameters were evaluated in this study. The cellular kinetic parameters, determined using flow cytometry, indicated the best results with the samples cryopreserved with 7.5% methyl glycol. Dimethyl sulfoxide concentrations of 7.5% and glycerol concentrations of 10%, 12.5% and 15% demonstrated the least benefit across all evaluated sperm kinetic parameters. When assessed using flow cytometry, a concentration of 7.5% methyl glycol similarly showed better sperm kinetic parameters than 12.5% dimethyl sulfoxide. We conclude that cryoprotectants, especially methyl glycol, are effective for the preservation of sperm quality in A. minuano. However, high sensitivity of spermatozoa against glycerol was observed in these studies; thus, it is not recommended for cryopreservation purposes.
h i g h l i g h t s Lifetime exposure to Cu reduced all the analyzed parameters of sperm quality ... more h i g h l i g h t s Lifetime exposure to Cu reduced all the analyzed parameters of sperm quality in the killifish Poecilia vivipara. We suggest that augmented oxidative stress, endocrine disruption and energetic disturbance are involved in these negative outcomes. Lifetime exposure to Cu may disrupt fish reproduction and negatively affect the maintenance of its populations.
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2018
The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved ... more The herbicide atrazine (ATZ) is used worldwide in the control of annual grasses and broad-leaved weeds. The present study evaluated sperm quality parameters in zebrafish Danio rerio after 11-day exposure to nominal ATZ concentrations of 2, 10, and 100 μg L −1. All ATZ concentrations caused a decrease in motility, mitochondrial functionality, and membrane integrity, as measured using conventional microscopy or fluorescence microscopy with specific probes. The DNA integrity of sperm was not affected. The levels of expression of genes related to spermatogenesis, antioxidant defenses, and DNA repair were also investigated using RT-qPCR. The ATZ caused transcriptional repression of the spermatogenesis-related genes SRD5A2 and CFTR, the antioxidant defense genes SOD2 and GPX4B, and the DNA repair gene XPC. This is the first study to show that environmentally relevant concentrations of ATZ significantly affect the sperm quality in fish, possibly resulting in reduced fertility rates. In addition, we showed that the repression of genes related to spermatogenesis and cellular defense could be part of the mechanisms involved in the ATZ toxicity in the testes of male fish.
The current study assessed a semen cryopreservation protocol in the Amazonian catfish Leiarius ma... more The current study assessed a semen cryopreservation protocol in the Amazonian catfish Leiarius marmoratus, a freshwater fish, of rheophilic behavior, and of great importance for Brazilian fish farming. Eight males (n=8) were stripped and the semen was cryopreserved if total motility in fresh semen was higher than 80%. The external cryoprotectant Trehalose was then diluted in Beltsvile Thawing Solution (BTS) extender in the following concentrations: 50, 100, 150, and 200 mM. Semen samples were diluted in the media (1:9 v/v) being tested, then frozen in a container with nitrogen
Cadmium is a natural element found in the earth's crust; it is usually associated with other meta... more Cadmium is a natural element found in the earth's crust; it is usually associated with other metals, but due to the impacts caused by human activity, its concentration has increased in the aquatic environment. This metal may damage aquatic animal reproduction, decreasing the rate of fertilization of organisms such as fish. Thus, this study aimed to evaluate the in vitro toxicity of different concentrations of cadmium (0 (control), 0.5, 5, and 10 g/L) using sperm cells of model organism zebrafish, Danio rerio. Structural parameters, including integrity and fluidity of the plasma membrane, concentration of oxygen species, mitochondrial function and DNA fragmentation were measured by flow cytometry. The following sperm movement parameters were also measured using the computer assisted sperm analysis (CASA) system: motility, time of motility, curvilinear velocity, average path velocity and straight line velocity in m/s. Significant effects were observed on path speed, straight speed, curvilinear velocity, motility time, progressive and total motility, and plasma and DNA integrity. The results showed that cadmium can negatively affect some reproductive parameters in D. rerio, which may reduce the fertility rate of these animals.
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Papers by Izani Acosta