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BACKGROUND: Using sulfated polysaccharides (SP) in fish sperm freezing medium promotes cell maintenance. OBJECTIVE: To evaluate the effect of different SP concentrations, extracted from two seaweeds (Gracilaria domingensis and Ulva fasciata), as a supplement to the sperm freezing medium of Prochilodus brevis. MATERIALS AND METHODS: Five semen pools were diluted in a solution composed of 5% glucose, 10 % dimethyl sulfoxide (DMSO) and different SP concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5 or 3.0 mg/mL). The samples were cryopreserved and, after 7 days, rewarmed and analyzed for morphology, plasma membrane integrity, DNA integrity, mitochondrial activity and sperm kinetics [total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), and wobble (WOB)]. RESULTS: There was no interaction between seaweed and SP concentrations. Similar effects were observed with SP extracted from the two seaweeds, regardless of concentration. When comparing the SP concentrations, regardless of the seaweed, 1.0 mg/mL SP showed better results for VCL and VSL. For VAP and WOB, 1.0 mg/mL SP showed better results, but differed from 3.0 mg/mL. LIN followed the same pattern, but differed from SP at 2.5 and 3.0 mg/mL. For progressive motility, 1.0 mg/mL G. domingensis showed superior results compared to the control. For mitochondrial activity, G. domingensis was superior to U. fasciata, regardless of concentration. The lowest concentrations (0.5 and 1.0 mg/mL) showed the best results, regardless of the seaweed. However, the control was superior to all treatments tested. CONCLUSION: G. domingensis SP at the lowest concentrations might be a potential supplement to the P. brevis freezing medium. doi.org/10.54680/fr22210110412.

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BACKGROUND: Sulfated polysaccharides from the skin of Nile tilapia (Oreochromis niloticus), added to the tambaqui (Colossoma macropomum) semen diluting medium, can be potential antioxidants and promote the maintenance of sperm quality. OBJECTIVE: To evaluate the use of different concentrations of glycosaminoglycans (GAGs) from the skin of Nile tilapia as a supplement in two cryogenic media for tambaqui semen. MATERIALS AND METHODS: Tambaqui males received a single dose of pituitary carp extract. The semen was collected for pool analysis and, later, cryopreserved in liquid nitrogen. The pools were diluted and frozen in a solution containing fish-specific powdered coconut water (ACP-104) and 10% DMSO or 5% Glucose and 10% DMSO and supplemented with different concentrations of GAGs. The controls had no GAGs addition. After 45 days, the samples were thawed by immersion in a water bath and evaluated for membrane and DNA integrity, morphology and sperm kinetics. RESULTS: The parameters of linearity (LIN), straightness (STR) and DNA integrity of sperm frozen in 5% Glucose showed better results than ACP-104. For membrane integrity, concentrations of 0 and 1.0 mg/mL were better than 5 mg/mL. Semen motility in 5% Glucose showed superior results at concentrations lower than 5 mg/mL of GAGs. For VCL and VAP, in ACP-104, 3.0 mg/mL exceeded the other treatments. In 5% Glucose, for VCL, 4.0 mg/mL showed the lowest results compared to concentrations of <3.5 mg/mL and, for VAP, it also differed from 4.5 mg/mL CONCLUSION: Therefore, the skin of Nile tilapia has GAGs, in low concentrations, capable of improving the post-thawed sperm quality of tambaqui, especially in 5% Glucose medium.

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Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

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Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

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Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000 g·mol-1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2 mL·min-1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.

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This study evaluated the effect of glucose orBeltsville Thawing Solution (BTS™) combined withdimethyl sulfoxide (DMSO) or methylglycol (MG)under two different freezing protocols on the kineticsand morphology of cryopreserved Prochilodus brevissperm. The semen samples were diluted using one of fourdifferent treatments (glucose+DMSO, glucose+MG,BTS™+DMSO, and BTS™+MG), loaded into 0.25-mlstraws and subjected to two different freezing processes(programmed freezing machine and dry shipper). After10 days, the semen samples were thawed, and the spermmorphology and kinetics were evaluated. Thephysicochemical parameters of the semen in naturawere similar to those observed in other studies ofCharaciformes, indicating the feasibility of semencryopreservation. Glucose, when used as a diluentwith the cryoprotectant MG (glucose+MG), yieldedhigher percentages of mobile spermatozoa afterfreezing in a dry shipper (76.88 ± 4.84%) and in aprogrammed freezing machine (70.95 ± 1.76%)compared with the combination of glucose and DMSO.Moreover, the glucose+MG treatment yielded a highersperm velocity (curvilinear velocity: 79.52 ± 2.88 µm s-1; straight-line velocity: 45.46 ± 3.01 µm s-1; averagepath velocity: 67.92 ± 3.08 µm s-1) than the otherstudied treatments, and a higher amount of normalsperm (74.56 ± 0.77%) was observed in the semensamples cryopreserved using a programmed freezingmachine. The sperm abnormalities observed included abent tail morphology. Therefore, the use ofglucose+MG in combination with either a dry shipper ora programmed freezing machine is recommended forthe cryopreservation of P. brevis sperm because thesemethods yielded high numbers of motile andmorphologically normal spermatozoa.

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This study evaluated the effect of glucose orBeltsville Thawing Solution (BTS™) combined withdimethyl sulfoxide (DMSO) or methylglycol (MG)under two different freezing protocols on the kineticsand morphology of cryopreserved Prochilodus brevissperm. The semen samples were diluted using one of fourdifferent treatments (glucose+DMSO, glucose+MG,BTS™+DMSO, and BTS™+MG), loaded into 0.25-mlstraws and subjected to two different freezing processes(programmed freezing machine and dry shipper). After10 days, the semen samples were thawed, and the spermmorphology and kinetics were evaluated. Thephysicochemical parameters of the semen in naturawere similar to those observed in other studies ofCharaciformes, indicating the feasibility of semencryopreservation. Glucose, when used as a diluentwith the cryoprotectant MG (glucose+MG), yieldedhigher percentages of mobile spermatozoa afterfreezing in a dry shipper (76.88 ± 4.84%) and in aprogrammed freezing machine (70.95 ± 1.76%)compared with the combination of glucose and DMSO.Moreover, the glucose+MG treatment yielded a highersperm velocity (curvilinear velocity: 79.52 ± 2.88 µm s-1; straight-line velocity: 45.46 ± 3.01 µm s-1; averagepath velocity: 67.92 ± 3.08 µm s-1) than the otherstudied treatments, and a higher amount of normalsperm (74.56 ± 0.77%) was observed in the semensamples cryopreserved using a programmed freezingmachine. The sperm abnormalities observed included abent tail morphology. Therefore, the use ofglucose+MG in combination with either a dry shipper ora programmed freezing machine is recommended forthe cryopreservation of P. brevis sperm because thesemethods yielded high numbers of motile andmorphologically normal spermatozoa.(AU)

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O objetivo deste trabalho foi comparar a influência da administração, na fase pré-púbere, de diferentesdietas alimentares e formas de esterilização da ração sobre o número de embriões de duas células e a quantidadede estruturas coletadas de camundongos fêmeas superovuladas. Foram utilizados animais B6.129P2-Nos2,divididos em quatro grupos, conforme a ração comercial ingerida: G1- comum autoclavada; G2- comum nãoautoclavada; G3- para fins de reprodução e G4- comum irradiada. Os animais foram pesados semanalmente ealimentados com 184g da ração do grupo correspondente. Após duas semanas, as fêmeas foram superovuladas eacasaladas com machos férteis. Aquelas positivas tiveram sua tuba lavada com meio M2, e as estruturasembrionárias coletadas e classificadas. Os resultados foram analisados por análise de variância e teste de Tukey.Fêmeas do G3 apresentaram maior ganho de peso do que as do G1 e do G4, mas semelhante às do G2. Já emrelação à recuperação de embriões de duas células, as do G2 e do G4 obtiveram os melhores resultados, semvariação entre si. O grupo que apresentou as menores taxas de recuperação de embriões de duas células foi o G3.Conclui-se que o uso de ração comercial para reprodução na fase pré-púbere aumentou o ganho de peso dosanimais, mas não o número de embriões de duas células em fêmeas superovuladas.

The aim of this study was to observe the influence, in prepuberal phase, of different diets andsterilization types on number of 2-cells embryos/ total quantity of structures recovered from superovulatedfemale mice. B6.129P2-Nos2 animals were divided into 4 groups according to the commercial diet: G1-commonautoclaved; G2- common not-autoclaved; G3-reproduction and G4-common irradiated. Animals were weightedweekly and fed with 184 g of corresponding diet. After two weeks, females were superovulated and mated withfertile males. Those positives had their tuba washed with M2 medium and the embryonic structures collected andclassified. The results were analyzed by Variance Analysis and Tukey Test. G3 females had higher weight gainthan G1 and G4, but similar to G2. In relation to recovery of 2 cells embryos, G2 and G4 had obtained the bestresults, without variation among themselves. The group that presented the smallest recovery rates of 2 cellsembryos was G3. It was concluded that the use of reproduction diet in prepuberal phase increased animals’weight gain, but not number of 2-cells embryos in superovulated female mice.

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O objetivo deste trabalho foi comparar a influência da administração, na fase pré-púbere, de diferentesdietas alimentares e formas de esterilização da ração sobre o número de embriões de duas células e a quantidadede estruturas coletadas de camundongos fêmeas superovuladas. Foram utilizados animais B6.129P2-Nos2,divididos em quatro grupos, conforme a ração comercial ingerida: G1- comum autoclavada; G2- comum nãoautoclavada; G3- para fins de reprodução e G4- comum irradiada. Os animais foram pesados semanalmente ealimentados com 184g da ração do grupo correspondente. Após duas semanas, as fêmeas foram superovuladas eacasaladas com machos férteis. Aquelas positivas tiveram sua tuba lavada com meio M2, e as estruturasembrionárias coletadas e classificadas. Os resultados foram analisados por análise de variância e teste de Tukey.Fêmeas do G3 apresentaram maior ganho de peso do que as do G1 e do G4, mas semelhante às do G2. Já emrelação à recuperação de embriões de duas células, as do G2 e do G4 obtiveram os melhores resultados, semvariação entre si. O grupo que apresentou as menores taxas de recuperação de embriões de duas células foi o G3.Conclui-se que o uso de ração comercial para reprodução na fase pré-púbere aumentou o ganho de peso dosanimais, mas não o número de embriões de duas células em fêmeas superovuladas.(AU)

The aim of this study was to observe the influence, in prepuberal phase, of different diets andsterilization types on number of 2-cells embryos/ total quantity of structures recovered from superovulatedfemale mice. B6.129P2-Nos2 animals were divided into 4 groups according to the commercial diet: G1-commonautoclaved; G2- common not-autoclaved; G3-reproduction and G4-common irradiated. Animals were weightedweekly and fed with 184 g of corresponding diet. After two weeks, females were superovulated and mated withfertile males. Those positives had their tuba washed with M2 medium and the embryonic structures collected andclassified. The results were analyzed by Variance Analysis and Tukey Test. G3 females had higher weight gainthan G1 and G4, but similar to G2. In relation to recovery of 2 cells embryos, G2 and G4 had obtained the bestresults, without variation among themselves. The group that presented the smallest recovery rates of 2 cellsembryos was G3. It was concluded that the use of reproduction diet in prepuberal phase increased animalsweight gain, but not number of 2-cells embryos in superovulated female mice.(AU)

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Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

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