Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.(AU)

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.(AU)

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Sêmen refrigerado ­ estado da arte em diferentes espécies / Chilled semen ­ state of the art in different species

Na atual conjuntura da criação artificial de bovinos e bubalinos, o material genético masculino de qualidade superior de reprodutores é explorado ao máximo possível através da inseminação artificial em tempo fixo de um grande número de fêmeas com apenas um único ejaculado. Para isso, é necessário um sêmen de boa qualidade que desempenhe um papel indispensável na melhoria das taxas de fertilidade, independente de qual tipo seja utilizado (fresco, refrigerado e congelado). Porém, o processo de congelação/descongelação causa uma série de injúrias aos espermatozoides, ocasionando resultados inferiores para percentuais de viabilidade espermática, motilidade, membrana plasmática e integridade acrossomal, potencial de membrana mitocondrial, cinemática do esperma, quando comparado ao sêmen refrigerado. Assim, o objetivo desta revisão é disseminar o conhecimento sobre o uso sêmen refrigerado na preservação de germoplasma de reprodutores bovinos e bubalinos para melhorar as taxas de concepção em propriedades. Para isso, serão abordados comentários sobre o armazenamento do sêmen refrigerado, com ênfase nas diferenças entre curvas de refrigeração, suas vantagens e desvantagens relativas para procedimentos de uso na IATF, identificando o método mais indicado por diversos autores, o estado atual da biotécnica, seus méritos e possibilidades futuras.(AU)

In the current conjuncture of the artificial creation of bovines and buffaloes, the male genetic material of superior quality of sires is exploited to the maximum possible through the fixed-time artificial insemination of a large number of females with only a single ejaculate. For this, a good quality semen is needed that plays an indispensable role in improving fertility rates, regardless of which type is used (fresh, chilled and frozen). However, the freezing/thawing process causes a series of injuries to spermatozoa, causing lower results for percentages of sperm viability, motility, plasma membrane and acrosomal integrity, mitochondrial membrane potential, sperm kinematics, when compared to refrigerated semen. Thus, the objective of this review is to disseminate knowledge about the use of chilled semen in the preservation of germplasm of bovine and buffalo breeders to improve conception rates in properties. For this, comments on the storage of refrigerated semen will be addressed, with emphasis on the differences between refrigeration curves, their relative advantages and disadvantages for procedures for use in FTAI, identifying the method most indicated by several authors, the current state of biotechnics, its future merits and possibilities.(AU)

Impact of quercetin, carnosine, and ozone in the cryopreservation on Nellore (Bos indicus) semen

The objective of this study was to reduce the effects of cryoinjury caused in bovine semen by cryopreservation. Ejaculates were collected from Nellore bulls and subjected to freezing in C (control), ozone (15, 30, and 60 µg mL-1 of ozone), quercetin (25, 50, and 100 µg mL-1 of quercetin), and carnosine groups (100, 200, and 300 ng mL-1 of carnosine). Samples were evaluated post-thaw (M0) and post-rapid thermoresistance test (M30) for sperm kinetics (total motility, progressive motility, curvilinear speed, linearity and amplitude of lateral head displacement) and cell structure viability (plasma membrane integrity, acrosomal integrity, mitochondrial potential, membrane fluidity, and lipid peroxidation). There were no differences (P > 0.05) between the control, quercetin, and carnosine-treated groups for the parameters evaluated at M0 and M30. In turn, supplementation with ozone resulted in lower values for sperm kinetics (P < 0.05) and lower mitochondrial potential at M30 (P < 0.05). Quercetin and carnosine at the concentrations used did not promote significant gains in frozen semen, nor did they demonstrate cytotoxicity. We expected to obtain positive results with the use of ozone. Nonetheless, the addition was harmful to the parameters of sperm kinetics, and its effect was not observed as a possible pro-antioxidant. We believe that the fact that the gas did not harm the sperm structure opens avenues for tests with lower dosages, since, by reducing its concentration, we could minimize the damage to sperm kinetics.(AU0

Association of Palm gene mutations with structure and function of paralemmin proteins in Lori-Bakhtiari and Zel sheep Breeds

The aim of current study was to survey genetic variability of PALM gene'sexon 3 and 4 by PCR-SSCP and DNA sequencing in Zel and Lori Bakhtiari sheep breeds. TheSIFT(Sorting Intolerant from Tolerant) and PHyre2 program were used to predict the possible impact of amino acid substitutions on performance and structure of the paralemmin protein. A total of 140 animal's from 2 Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Zel sheep breeds were considered. The results showed that there are two polymorphic sites including a nonsynonymous substitution and an insertion mutation (49bp). Non-synonymous mutation deduced Thr20Ala amino acid exchange and ensuing two different structures for paralemmin protein that could be potentially affect protein structure and function during the interaction with glutamate in the cytosolic surface of plasma membrane. PALMgene, according to evolutionary path, is classified into two separate categories. In first covey, Gallus gallusand in second one, other species in several branches, so that the sequence of cow and sheep is placed in a sub-branch which forms a clade beside goat. Comparison of illustrated coding region sequences, PALMgene among different species, is of orthologous which are derived from a common ancestor.(AU)

Antifungal effect of (R) and (S)-citronellal enantiomers and their predictive mechanism of action on Candida albicans from voriconazole-resistant onychomycoses / Efeito antifúngico dos enantiômeros (R) e (S)-citronelal e seu mecanismo de ação preditivo sobre Candida albicans de onicomicoses resistentes ao voriconazol

Onychomycosis is the most common disease affecting the nail unit and accounts for at least 50% of all nail diseases. In addition, Candida albicans is responsible for approximately 70% of onychomycoses caused by yeasts. This study investigated the antifungal effect of (R) and (S)-citronellal enantiomers, as well as its predictive mechanism of action on C. albicans from voriconazole-resistant onychomycoses. For this purpose, in vitro broth microdilution and molecular docking techniques were applied in a predictive and complementary manner to the mechanisms of action. The main results of this study indicate that C. albicans was resistant to voriconazole and sensitive to the enantiomers (R) and (S)-citronellal at a dose of 256 and 32 µg/mL respectively. In addition, there was an increase in the minimum inhibitory concentration (MIC) of the enantiomers in the presence of sorbitol and ergosterol, indicating that these molecules possibly affect the integrity of the cell wall and cell membrane of C. albicans. Molecular docking with key biosynthesis proteins and maintenance of the fungal cell wall and plasma membrane demonstrated the possibility of (R) and (S)-citronellal interacting with two important enzymes: 1,3-ß-glucan synthase and lanosterol 14α-demethylase. Therefore, the findings of this study indicate that the (R) and (S)-citronellal enantiomers are fungicidal on C. albicans from onychomycoses and probably these substances cause damage to the cell wall and cell membrane of these micro-organisms possibly by interacting with enzymes in the biosynthesis of these fungal structures.

A onicomicose é a doença mais comum que afeta a unidade ungueal e representa pelo menos 50% de todas as doenças ungueais. Além disso, a Candida albicans é responsável por aproximadamente 70% das onicomicoses causadas por leveduras. Nesse estudo, foi investigado o efeito antifúngico dos enantiômeros (R) e (S)-citronelal, bem como seu mecanismo de ação preditivo sobre C. albicans de onicomicoses resistentes ao voriconazol. Para este propósito, foram aplicadas técnicas in vitro de microdiluição em caldo e docking molecular de forma preditiva e complementar para os mecanismos de ação. Os principais resultados deste estudo indicam que C. albicans foi resistente ao voriconazol e sensível aos enantiômeros (R) e (S)-citronelal na dose de 256 e 32 µg/mL respectivamente. Além disso, houve aumento da concentração inibitória mínima (CIM) dos enantiômeros na presença do sorbitol e do ergosterol, indicando que estas moléculas possivelmente afetem a integridade da parede e da membrana celular de C. albicans. O docking molecular com proteínas chave da biossíntese e manutenção da parede celular e da membrana plasmática fúngica, demonstraram a possibilidade do (R) e (S)-citronelal interagir com duas importantes enzimas: 1,3-ß-glucan sintase e lanosterol 14α-demetilase. Portanto, os achados desse estudo indicam que os enantiômeros (R) e (S)-citronelal são fungicidas sobre C. albicans de onicomicoses e provavelmente essas substâncias causem danos a parede e a membrana celular desses microrganismos possivelmente por interagir com as enzimas da biossíntese destas estruturas fúngicas.

In vitro antibacterial activities of silver nanoparticles synthesised using the seed extracts of three varieties of Phoenix dactylifera

Abstract Green synthesis of silver nanoparticles (AgNPs) is an ecofriendly, cost-effective and promising approach for discovery of novel therapeutics. The aim of the current work was to biogenic synthesize, characterize AgNPs using seed extracts of three economically important varieties of date palm (Iklas, Irziz and Shishi), and assess their anti-pathogenic bacterial activities. AgNPs were synthesised then characterised using electron microscopy and Fourier transform infrared analyses. The bactericidal activities of AgNPs against five different bacterial pathogens, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae, were determined in vitro. In particular, changes in membrane integrity of virulent bacterial strains in response to AgNPs were investigated. Results of lactate dehydrogenase, alkaline phosphatase activity assays, and measurement of membrane potential revealed that the cytotoxic effects of the AgNPs were mainly centred on the plasma membrane of bacterial cells, leading to loss of its integrity and eventually cell death. In conclusion, green synthesis of AgNPs is an efficient, cost-effective and promising strategy to combat virulent antibiotic-resistant strains.

Resumo A síntese verde de nanopartículas de prata (AgNPs) é uma abordagem ecologicamente correta, econômica e promissora para a descoberta de novas terapêuticas. O objetivo do presente trabalho foi sintetizar biogênica, caracterizar AgNPs usando extratos de sementes de três variedades economicamente importantes de tamareira (Iklas, Irziz e Shishi) e avaliar suas atividades bacterianas antipatogênicas. AgNPs foram sintetizados e caracterizados usando microscopia eletrônica e análise de infravermelho por transformada de Fourier. As atividades bactericidas de AgNPs contra cinco diferentes patógenos bacterianos, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus resistente à meticilina e Streptococcus pneumoniae, foram determinadas in vitro. Em particular, foram investigadas alterações na integridade da membrana de cepas bacterianas virulentas em resposta a AgNPs. Os resultados da lactato desidrogenase, dos ensaios da atividade da fosfatase alcalina e da medição do potencial de membrana revelaram que os efeitos citotóxicos dos AgNPs estavam principalmente centrados na membrana plasmática das células bacterianas, levando à perda de sua integridade e, eventualmente, à morte celular. A síntese verde de AgNPs é uma estratégia eficiente, econômica e promissora para combater cepas virulentas resistentes a antibióticos.

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.(AU)

Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality / Efeito da inclusão de quercetina ou butil-hidroxitolueno na qualidade do sêmen de carneiro resfriado ou congelado

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 µg/mL; Q10, 10 µg/mL; Q15, 15 µg/mL) or DMSO alone (DMSO1, 0.055 µL DMSO per mL; DMSO2, 0.165 µL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 µg/mL; BHT2, 1 µg/mL; BHT3, 1.5 µg/mL) or DMSO alone (DMSO3, 0.375 µL DMSO per mL; DMSO4, 1.125 µL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.(AU)

O resfriamento e o congelamento causam danos físicos e químicos aos espermatozoides por choque térmico e estresse oxidativo. Portanto, este estudo teve como objetivo avaliar o efeito da inclusão de dois antioxidantes em um diluente à base de gema de ovo sobre os parâmetros espermáticos do sêmen ovino resfriado e congelado. Trinta carneiros tiveram o sêmen coletado e processado em dois experimentos consecutivos para testar a inclusão de diferentes concentrações de quercetina e hidroxitolueno butilado (BHT) em diluente de sêmen à base de gema de ovo. O DMSO foi adicionado como solvente ao diluente de sêmen em uma proporção de 1 mL de DMSO parra 90 mg de quercetina e 1 Ml de DMSO para 880 mg de BHT. Após a coleta, o sêmen foi diluído a 200 × 106 espermatozoides móveis/mL (Controle) e dividido em diferentes grupos em cada experimento. Experimento 1, Quercetina (Q5, 5 µg / mL; Q10, 10 µg / mL; Q15, 15 µg / mL) ou DMSO (DMSO1, 0,055 µL de DMSO por ml; DMSO2, 0,165 µL de DMSO / mL) foram adicionados ao extensor. Experimento 2, BHT (BHT1, 0,5 µg / mL; BHT2, 1 µg / mL; BHT3, 1,5 µg / mL) ou DMSO (DMSO3, 0,375 µL de DMSO por ml; DMSO4, 1,125 µL de DMSO / mL) foram adicionados à o extensor. Após a diluição, o sêmen foi dividido em duas alíquotas. O primeiro foi resfriado a 5 ° C por 24h, enquanto o segundo foi congelado. Os parâmetros de motilidade espermática e integridade da membrana plasmática (PMI) foram avaliados, imediatamente após a diluição (0h) e 24h após o resfriamento e nas amostras congeladas, pelo CASA e microscopia de epifluorescência, respectivamente. A inclusão de quercetina ou BHT não afetou os parâmetros de motilidade espermática e PMI de espermatozoides frescos, resfriados ou congelados (P < 0,05). Portanto, a inclusão de quercetina e BHT não beneficiou os parâmetros espermáticos do sêmen ovino submetido a armazenamento líquido a 5 ° C por 24h ou protocolo de congelamento no presente estudo. No entanto, mais estudos são necessários para testar o efeito desses antioxidantes na fertilidade do sêmen ovino criopreservado.(AU)

Supplementation of cryodiluent medium with sulfated polysaccharides from green seaweeds in the freezing of Colossoma macropomum semen / Suplementação do meio criodiluidor com polissacarídeos sulfatados de macroalgas verdes na congelação do sêmen de Colossoma macropomum

This study proposes to investigate the addition of sulfated polysaccharides (SP) extracted from two species of green seaweeds, Ulva lactuca and Caulerpa racemosa, to Colossoma macropomum semen cryodiluent medium. Four concentrations of SP (1.0, 2.0, 3.0, or 4.0 mg mL-1) of each seaweed were evaluated. Semen was collected during the month of September in Fortaleza - CE, Brazil. Fresh semen samples were analyzed for the parameters of total sperm motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), sperm morphology, membrane integrity, and DNA integrity. Then, the samples were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO) + 5% glucose, which was supplemented with different concentrations of SP. An unsupplemented treatment was used as control. After 15 days, they were thawed in a water bath at 45 ºC for eight seconds and the same analyses of fresh semen were performed. Statistical analysis revealed that there were no significant differences (p > 0.05) between the different tested concentrations of SP for any of the evaluated parameters. Compared with the control, there was no difference in concentrations (p > 0.05) for total motility; however, for VCL, VSL, and VAP, the U. lactuca concentrations of 3.0 and 4.0 mg mL-1 were detrimental (p < 0.05). The same was observed with 4.0 mg mL-1 of C. racemosa for VSL and VAP. In terms of morphology, 1.0 and 4.0 mg mL-1 of C. racemosa reduced normal sperm (p < 0.05), whereas for the other concentrations there was no difference (p > 0.05). All concentrations of both seaweeds maintained plasma membrane integrity (p > 0.05). As for DNA integrity, only 4.0 mg mL-1 of U. lactuca produced lower results than the control (p < 0.05), whereas the other concentrations maintained the number of spermatozoa with intact DNA (p > 0.05). Based on the results, higher concentrations of SP are harmful to tambaqui sperm in the freezing medium, whereas lower concentrations maintain sperm parameters. Further research is warranted to better investigate the antioxidant potential of these polymers in cryodiluent medium for C. macropomum as well as other fish species.

O estudo teve como objetivo avaliar a adição de polissacarídeos sulfatados (PS) extraídos de duas espécies de macroalgas verdes, Ulva lactuca e Caulerpa racemosa, no meio criodiluidor do sêmen de Colossoma macropomum. Para isso, foram avaliadas quatro concentrações de PS (1,0; 2,0; 3,0 ou 4,0 mg mL-1), de cada macroalga. A coleta de sêmen foi realizada durante o mês de setembro, em Fortaleza, Ceará, Brasil. As amostras de sêmen fresco foram analisadas quanto aos parâmetros de motilidade total dos espermatozoides, velocidade curvilinear (VCL), velocidade em linha reta (VSL), velocidade média do trajeto (VAP), morfologia espermática, integridade de membrana e integridade de DNA. Em seguida, foram criopreservadas em meio de congelação contendo dimetilsulfóxido (DMSO) 10% + glicose 5%, e suplementadas com as diferentes concentrações de PS, tendo ainda um tratamento não suplementado como controle. Após 15 dias, foram descongeladas em banho-maria a 45 ºC por oito segundos, e as mesmas análises do sêmen fresco foram realizadas. Através da análise estatística, os resultados mostraram que não houveram diferenças significativas (p > 0,05) entre as diferentes concentrações de PS testadas para nenhum dos parâmetros avaliados. Já em relação ao controle, não houve diferença nas concentrações (p > 0,05) para a motilidade total, no entanto, para VCL, VSL e VAP, as concentrações de 3,0 e 4,0 mg mL-1 de U. lactuca foram prejudiciais (p < 0,05). O mesmo foi observado em 4,0 mg mL-1 de C. racemosa para VSL e VAP. Para a morfologia, 1,0 e 4,0 mg mL-1 de C. racemosa reduziram os espermatozoides normais (p < 0,05), enquanto para as demais concentrações não houve diferença (p > 0,05). Para todas as concentrações de ambas as macroalgas, a integridade de membrana plasmática foi mantida (p > 0,05). Quanto à integridade do DNA, apenas 4,0 mg mL-1 de U. lactuca foi inferior ao controle (p < 0,05), enquanto as demais concentrações mantiveram o número de espermatozoides com DNA íntegro (p > 0,05). De acordo com os resultados obtidos, concentrações mais elevadas de PS são prejudiciais aos espermatozoides de tambaqui no meio de congelação, enquanto concentrações mais baixas mantiveram parâmetros espermáticos. Estudos posteriores são indicados para melhor avaliar o potencial antioxidante destes polímeros no meio criodiluidor do sêmen de C. macropomum, bem como de outras espécies de peixes.

In vitro antibacterial activities of silver nanoparticles synthesised using the seed extracts of three varieties of Phoenix dactylifera / Atividades antibacterianas in vitro de nanopartículas de prata sintetizadas usando extratos de sementes de três variedades de Phoenix dactylifera

Green synthesis of silver nanoparticles (AgNPs) is an ecofriendly, cost-effective and promising approach for discovery of novel therapeutics. The aim of the current work was to biogenic synthesize, characterize AgNPs using seed extracts of three economically important varieties of date palm (Iklas, Irziz and Shishi), and assess their anti-pathogenic bacterial activities. AgNPs were synthesised then characterised using electron microscopy and Fourier transform infrared analyses. The bactericidal activities of AgNPs against five different bacterial pathogens, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae, were determined in vitro. In particular, changes in membrane integrity of virulent bacterial strains in response to AgNPs were investigated. Results of lactate dehydrogenase, alkaline phosphatase activity assays, and measurement of membrane potential revealed that the cytotoxic effects of the AgNPs were mainly centred on the plasma membrane of bacterial cells, leading to loss of its integrity and eventually cell death. In conclusion, green synthesis of AgNPs is an efficient, cost-effective and promising strategy to combat virulent antibiotic-resistant strains.

A síntese verde de nanopartículas de prata (AgNPs) é uma abordagem ecologicamente correta, econômica e promissora para a descoberta de novas terapêuticas. O objetivo do presente trabalho foi sintetizar biogênica, caracterizar AgNPs usando extratos de sementes de três variedades economicamente importantes de tamareira (Iklas, Irziz e Shishi) e avaliar suas atividades bacterianas antipatogênicas. AgNPs foram sintetizados e caracterizados usando microscopia eletrônica e análise de infravermelho por transformada de Fourier. As atividades bactericidas de AgNPs contra cinco diferentes patógenos bacterianos, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus resistente à meticilina e Streptococcus pneumoniae, foram determinadas in vitro. Em particular, foram investigadas alterações na integridade da membrana de cepas bacterianas virulentas em resposta a AgNPs. Os resultados da lactato desidrogenase, dos ensaios da atividade da fosfatase alcalina e da medição do potencial de membrana revelaram que os efeitos citotóxicos dos AgNPs estavam principalmente centrados na membrana plasmática das células bacterianas, levando à perda de sua integridade e, eventualmente, à morte celular. A síntese verde de AgNPs é uma estratégia eficiente, econômica e promissora para combater cepas virulentas resistentes a antibióticos.

Sperm cell capacitation status of ram semen after cooling

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

Erythrogram and oxidative stress in confined cattle fed with Brachiaria sp hay and supplemented with antioxidants / Eritrograma e estresse oxidativo em bovinos confinados alimentados com feno de Brachiaria sp. e suplementados com antioxidantes

Brachiaria sp contains sporidesmin that can be oxidized by lipoperoxidation and cause oxidative stress. In the present study we evaluated the effects of different antioxidants on lipoperoxidation of erythrocytes from Nelore cattle fed with Brachiaria sp hay. The experimental design was entirely randomized, in which 40 whole male cattle were divided into five treatments (G1: control - no supplementation; G2: selenium and vitamin E supplementation; G3: zinc supplementation; G4: selenium supplementation and G5: vitamin E supplementation) and allocated in feedlot pens for 105 days. The samples heparinized and withethylenediaminetetraacetic acid (EDTA) were obtained every 28 days for hematological and oxidative stress evaluation (0, 28 56, 84 and 105 days). In the erythrogram total erythrocyte count, hemoglobin, and hematocrit (Ht) were measured. For the evaluation of oxidative stress, in order to analyze the characteristics of the erythrocyte membrane, the thiobarbituric acid reactive substances (TBARS), total glutathione (GSH-T), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were determined. The results showed that regardless of the treatment there was no oxidative stress during the experimental confinement period and that the joint association of selenium and vitamin E in the bovine diet provided a lower incidence of deleterious alterations on erythrocytes.

As Brachiaria sp contêm esporidesminas que podem ser oxidadas por lipoperoxidação e ocasionar estresse oxidativo. No presente estudo foram avaliados os efeitos de diferentes antioxidantes na lipoperoxidação dos eritrócitos de bovinos da raça Nelore, alimentados com feno de Brachiaria sp. O delineamento experimental foi inteiramente casualizado, em que 40 bovinos machos, inteiros, foram divididos, em cinco tratamentos (G1: controle - sem suplementação; G2: suplementação de selênio e vitamina E; G3: suplementação de zinco; G4: suplementação de selênio e G5: suplementação de vitamina E) e alocados em baias de confinamento, por 105 dias. As amostras de plasma heparinizado ou com ácido etilenodiamino tetra-acético (EDTA) foram obtidas a cada 28 dias para avaliação hematológica e de estresse oxidativo (0, 28 56, 84 e 105 dias). No eritrograma foi mensurado a contagem total de eritrócitos, a hemoglobina e o volume globular (VG). Para a avaliação do estresse oxidativo, com o objetivo de analisar as características da membrana do eritrócito foram determinadas as substâncias reativas ao ácido tiobarbitúrico (TBARS), glutationa total (GSH-T), glutationa peroxidase (GSH-Px), catalase (CAT) e superóxido dismutase (SOD). Os resultados demonstraram que independente do tratamento não houve estresse oxidativo durante o período do confinamento experimental e que a associação conjunta de selênio e vitamina E na dieta dos bovinos proporcionaram menor incidência de alterações deletérias sobre os eritrócitos.

Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.(AU)

In vitro antibacterial activities of silver nanoparticles synthesised using the seed extracts of three varieties of Phoenix dactylifera / Atividades antibacterianas in vitro de nanopartículas de prata sintetizadas usando extratos de sementes de três variedades de Phoenix dactylifera

Green synthesis of silver nanoparticles (AgNPs) is an ecofriendly, cost-effective and promising approach for discovery of novel therapeutics. The aim of the current work was to biogenic synthesize, characterize AgNPs using seed extracts of three economically important varieties of date palm (Iklas, Irziz and Shishi), and assess their anti-pathogenic bacterial activities. AgNPs were synthesised then characterised using electron microscopy and Fourier transform infrared analyses. The bactericidal activities of AgNPs against five different bacterial pathogens, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae, were determined in vitro. In particular, changes in membrane integrity of virulent bacterial strains in response to AgNPs were investigated. Results of lactate dehydrogenase, alkaline phosphatase activity assays, and measurement of membrane potential revealed that the cytotoxic effects of the AgNPs were mainly centred on the plasma membrane of bacterial cells, leading to loss of its integrity and eventually cell death. In conclusion, green synthesis of AgNPs is an efficient, cost-effective and promising strategy to combat virulent antibiotic-resistant strains.

A síntese verde de nanopartículas de prata (AgNPs) é uma abordagem ecologicamente correta, econômica e promissora para a descoberta de novas terapêuticas. O objetivo do presente trabalho foi sintetizar biogênica, caracterizar AgNPs usando extratos de sementes de três variedades economicamente importantes de tamareira (Iklas, Irziz e Shishi) e avaliar suas atividades bacterianas antipatogênicas. AgNPs foram sintetizados e caracterizados usando microscopia eletrônica e análise de infravermelho por transformada de Fourier. As atividades bactericidas de AgNPs contra cinco diferentes patógenos bacterianos, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus resistente à meticilina e Streptococcus pneumoniae, foram determinadas in vitro. Em particular, foram investigadas alterações na integridade da membrana de cepas bacterianas virulentas em resposta a AgNPs. Os resultados da lactato desidrogenase, dos ensaios da atividade da fosfatase alcalina e da medição do potencial de membrana revelaram que os efeitos citotóxicos dos AgNPs estavam principalmente centrados na membrana plasmática das células bacterianas, levando à perda de sua integridade e, eventualmente, à morte celular. A síntese verde de AgNPs é uma estratégia eficiente, econômica e promissora para combater cepas virulentas resistentes a antibióticos.

In vitro antibacterial activities of silver nanoparticles synthesised using the seed extracts of three varieties of Phoenix dactylifera / Atividades antibacterianas in vitro de nanopartículas de prata sintetizadas usando extratos de sementes de três variedades de Phoenix dactylifera

Green synthesis of silver nanoparticles (AgNPs) is an ecofriendly, cost-effective and promising approach for discovery of novel therapeutics. The aim of the current work was to biogenic synthesize, characterize AgNPs using seed extracts of three economically important varieties of date palm (Iklas, Irziz and Shishi), and assess their anti-pathogenic bacterial activities. AgNPs were synthesised then characterised using electron microscopy and Fourier transform infrared analyses. The bactericidal activities of AgNPs against five different bacterial pathogens, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae, were determined in vitro. In particular, changes in membrane integrity of virulent bacterial strains in response to AgNPs were investigated. Results of lactate dehydrogenase, alkaline phosphatase activity assays, and measurement of membrane potential revealed that the cytotoxic effects of the AgNPs were mainly centred on the plasma membrane of bacterial cells, leading to loss of its integrity and eventually cell death. In conclusion, green synthesis of AgNPs is an efficient, cost-effective and promising strategy to combat virulent antibiotic-resistant strains.(AU)

A síntese verde de nanopartículas de prata (AgNPs) é uma abordagem ecologicamente correta, econômica e promissora para a descoberta de novas terapêuticas. O objetivo do presente trabalho foi sintetizar biogênica, caracterizar AgNPs usando extratos de sementes de três variedades economicamente importantes de tamareira (Iklas, Irziz e Shishi) e avaliar suas atividades bacterianas antipatogênicas. AgNPs foram sintetizados e caracterizados usando microscopia eletrônica e análise de infravermelho por transformada de Fourier. As atividades bactericidas de AgNPs contra cinco diferentes patógenos bacterianos, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Staphylococcus aureus resistente à meticilina e Streptococcus pneumoniae, foram determinadas in vitro. Em particular, foram investigadas alterações na integridade da membrana de cepas bacterianas virulentas em resposta a AgNPs. Os resultados da lactato desidrogenase, dos ensaios da atividade da fosfatase alcalina e da medição do potencial de membrana revelaram que os efeitos citotóxicos dos AgNPs estavam principalmente centrados na membrana plasmática das células bacterianas, levando à perda de sua integridade e, eventualmente, à morte celular. A síntese verde de AgNPs é uma estratégia eficiente, econômica e promissora para combater cepas virulentas resistentes a antibióticos.(AU)

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen / Efeito do diluente à base de gema de ovo e remoção do plasma seminal na viabilidade espermática do sêmen refrigerado de jumento

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.(AU)

O desenvolvimento de protocolos de sêmen resfriado eficazes é essencial para aumentar as taxas de prenhez e eficiência reprodutiva em jumentos. O objetivo desse estudo foi avaliar o efeito do diluente à base de proteínas do leite com 1 ou 2% de gema de ovo sobre os parâmetros cinéticos do sêmen e integridade da membrana em sêmen resfriado de jumento, com ou sem a remoção do plasma seminal. Vinte e quatro ejaculados de seis jumentos foram coletados. Cada ejaculado foi dividido em quatro alíquotas e diluído em diluente com 1% (EY1) ou 2% (EY2) de gema de ovo. Uma amostra por grupo foi centrifugada e o plasma seminal removido (grupos CEY1 e CEY2, respectivamente). Os pellets foram novamente ressuspendidos nas mesmas concentrações e diluentes. Em seguida, as quatro alíquotas foram refrigeradas a 5°C por 24 horas. Amostras de sêmen fresco e refrigerado foram avaliadas quanto à motilidade espermática e integridade da membrana plasmática. Motilidade total, motilidade progressiva, parâmetros de cinética espermática ou células espermáticas vivas não apresentaram diferença significativa quando o sêmen foi resfriado com diluente suplementado com 1% ou 2% de gema de ovo. A remoção do plasma seminal não afetou a motilidade total ou os parâmetros de cinética espermática; entretanto, a motilidade progressiva diminuiu (P<0,05) quando o sêmen foi diluído com 2% de gema de ovo e o plasma seminal removido. Nas amostras centrifugadas, a integridade da membrana foi afetada (P<0,05). Em conclusão, os resultados sugerem que não há diferença na cinética espermática e na integridade da membrana quando 1% ou 2% de gema de ovo são adicionados ao diluente Equiplus e a remoção do plasma seminal por centrifugação não teve nenhum efeito benéfico no resfriamento de sêmen de jumento. Mais estudos são necessários para relacionar esses resultados com testes de fertilidade in vivo com sêmen resfriado em jumentos.(AU)