Effect of protein and lipids levels in a growth diet on adult whitebait Galaxias maculatus (Jenyns 1842) / Efeito dos níveis de proteína e lipídio em uma dieta de crescimento em whitebait Galaxias maculatus adulto (Jenyns 1842)

Abstract Galaxias maculatus aquaculture objectives is to produce millions of eggs. Wild females are small (2 g), have quick sexual maturity and low mean fecundity (500 eggs/female), requiring larger fishes with higher fecundity. This study aim is to evaluate experimentally the effect of the levels of protein, lipid and dietary energy on weight increases in adults. Five independent experiments were performed at different sequential time periods at the UCT hatchery, Chile. Specimens were obtained from a) Crystalline sea return specimen catches in the Tolten estuary (4 -6 cm, 0.3-0.4 g.). b) Hatchery cultured fish. Fish were fed by hand ad libitum. In experiments 1 to 4, pelleted diets were prepared with 3 to 5 levels of protein (treatments 27 up to 57%), crumble size, three 100 L fibre ponds replicates. In experiment 5 the effect of two lipid levels (8 and 21%) was evaluated with commercial extruded Salmon Nutra Starter isoproteic crumble 1 diet at 63%, replicated in 4 ponds. The results show: A tendency to increased weight in all sizes with an increased protein level in the pelleted diet.A maximal adult growth is obtained with a diet containing a minimum of 37% crude protein, with 40% the optimal value. A higher % protein in the diet or growth in weight lower feed conversion ratio. The feed conversion ratio in the extruded diet reaches up to 0.5 and in the pelleted vary from 0.7 to 1.5. Fish 0.6 g fed with 63% protein, extruded commercial diet with two different lipid levels (8 and 21%, 20.40 and 23.84 MJ kg-1, PE/TE 0.62 and 0.71) increased weight the first month 67 and 105% each. It has been established that high-energy diets with optimal levels of protein and lipid are a good short-term solution to obtain G. maculatus of higher weight.

Resumo O objetivo da aquicultura de Galaxias maculatus é produzir milhões de ovos. As fêmeas selvagens são pequenas (2 g) e têm maturidade sexual rápida e fecundidade média baixa (500 ovos/fêmea), necessitando de peixes maiores e com fecundidade superior. O objetivo deste estudo é avaliar experimentalmente o efeito dos níveis de proteínas, lipídios e energia da dieta sobre o aumento de peso em adultos. Cinco experimentos independentes foram realizados em diferentes períodos sequenciais de tempo no incubatório UCT, Chile. Os espécimes foram obtidos a partir de: a) capturas de espécimes de retorno do mar cristalino no estuário de Tolten (4-6 cm, 0,3-0,4 g); b) peixes de cultura em incubatório. Os peixes foram alimentados à mão ad libitum. Nos experimentos de 1 a 4, dietas peletizadas foram preparadas com três a cinco níveis de proteína (tratamentos 27 a 57%), tamanho do crumble, três repetições de tanques de fibra de 100 L. No experimento 5, o efeito de dois níveis de lipídios (8 e 21%) foi avaliado com dieta comercial isoproteica crumble 1 de Salmon Nutra Starter extrusada a 63%, replicada em quatro tanques. Os resultados mostram: uma tendência ao aumento de peso em todos os tamanhos, com um aumento do nível de proteína na dieta peletizada; um crescimento adulto máximo com uma dieta contendo um mínimo de 37% de proteína bruta, com 40% do valor ideal; uma porcentagem maior de proteína na dieta ou crescimento em peso com menor taxa de conversão alimentar. A taxa de conversão alimentar na dieta extrusada chega a 0,5, e na peletizada varia de 0,7 a 1,5. Peixes de 0,6 g alimentados com 63% de proteína e dieta comercial extrusada com dois níveis lipídicos diferentes (8 e 21%; 20,40 e 23,84 MJ kg-1; PE / TE 0,62 e 0,71) aumentaram de peso no primeiro mês em 67 e 105% cada, respectivamente. Foi estabelecido que dietas de alta energia com níveis ótimos de proteínas e lipídios são uma boa solução de curto prazo para obter G. maculatus de peso mais alto.

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.

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.

Effect of protein and lipids levels in a growth diet on adult whitebait Galaxias maculatus (Jenyns 1842) / Efeito dos níveis de proteína e lipídio em uma dieta de crescimento em whitebait Galaxias maculatus adulto (Jenyns 1842)

Galaxias maculatus aquaculture objectives is to produce millions of eggs. Wild females are small (2 g), have quick sexual maturity and low mean fecundity (500 eggs/female), requiring larger fishes with higher fecundity. This study aim is to evaluate experimentally the effect of the levels of protein, lipid and dietary energy on weight increases in adults. Five independent experiments were performed at different sequential time periods at the UCT hatchery, Chile. Specimens were obtained from a) Crystalline sea return specimen catches in the Tolten estuary (4 -6 cm, 0.3-0.4 g.). b) Hatchery cultured fish. Fish were fed by hand ad libitum. In experiments 1 to 4, pelleted diets were prepared with 3 to 5 levels of protein (treatments 27 up to 57%), crumble size, three 100 L fibre ponds replicates. In experiment 5 the effect of two lipid levels (8 and 21%) was evaluated with commercial extruded Salmon Nutra Starter isoproteic crumble 1 diet at 63%, replicated in 4 ponds. The results show: A tendency to increased weight in all sizes with an increased protein level in the pelleted diet.A maximal adult growth is obtained with a diet containing a minimum of 37% crude protein, with 40% the optimal value. A higher % protein in the diet or growth in weight lower feed conversion ratio. The feed conversion ratio in the extruded diet reaches up to 0.5 and in the pelleted vary from 0.7 to 1.5. Fish 0.6 g fed with 63% protein, extruded commercial diet with two different lipid levels (8 and 21%, 20.40 and 23.84 MJ kg-1, PE/TE 0.62 and 0.71) increased weight the first month 67 and 105% each. It has been established that high-energy diets with optimal levels of protein and lipid are a good short-term solution to obtain G. maculatus of higher weight.

O objetivo da aquicultura de Galaxias maculatus é produzir milhões de ovos. As fêmeas selvagens são pequenas (2 g) e têm maturidade sexual rápida e fecundidade média baixa (500 ovos/fêmea), necessitando de peixes maiores e com fecundidade superior. O objetivo deste estudo é avaliar experimentalmente o efeito dos níveis de proteínas, lipídios e energia da dieta sobre o aumento de peso em adultos. Cinco experimentos independentes foram realizados em diferentes períodos sequenciais de tempo no incubatório UCT, Chile. Os espécimes foram obtidos a partir de: a) capturas de espécimes de retorno do mar cristalino no estuário de Tolten (4-6 cm, 0,3-0,4 g); b) peixes de cultura em incubatório. Os peixes foram alimentados à mão ad libitum. Nos experimentos de 1 a 4, dietas peletizadas foram preparadas com três a cinco níveis de proteína (tratamentos 27 a 57%), tamanho do crumble, três repetições de tanques de fibra de 100 L. No experimento 5, o efeito de dois níveis de lipídios (8 e 21%) foi avaliado com dieta comercial isoproteica crumble 1 de Salmon Nutra Starter extrusada a 63%, replicada em quatro tanques. Os resultados mostram: uma tendência ao aumento de peso em todos os tamanhos, com um aumento do nível de proteína na dieta peletizada; um crescimento adulto máximo com uma dieta contendo um mínimo de 37% de proteína bruta, com 40% do valor ideal; uma porcentagem maior de proteína na dieta ou crescimento em peso com menor taxa de conversão alimentar. A taxa de conversão alimentar na dieta extrusada chega a 0,5, e na peletizada varia de 0,7 a 1,5. Peixes de 0,6 g alimentados com 63% de proteína e dieta comercial extrusada com dois níveis lipídicos diferentes (8 e 21%; 20,40 e 23,84 MJ kg-1; PE / TE 0,62 e 0,71) aumentaram de peso no primeiro mês em 67 e 105% cada, respectivamente. Foi estabelecido que dietas de alta energia com níveis ótimos de proteínas e lipídios são uma boa solução de curto prazo para obter G. maculatus de peso mais alto.

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.

Effect of protein and lipids levels in a growth diet on adult whitebait Galaxias maculatus (Jenyns 1842)

Abstract Galaxias maculatus aquaculture objectives is to produce millions of eggs. Wild females are small (2 g), have quick sexual maturity and low mean fecundity (500 eggs/female), requiring larger fishes with higher fecundity. This study aim is to evaluate experimentally the effect of the levels of protein, lipid and dietary energy on weight increases in adults. Five independent experiments were performed at different sequential time periods at the UCT hatchery, Chile. Specimens were obtained from a) Crystalline sea return specimen catches in the Tolten estuary (4 -6 cm, 0.3-0.4 g.). b) Hatchery cultured fish. Fish were fed by hand ad libitum. In experiments 1 to 4, pelleted diets were prepared with 3 to 5 levels of protein (treatments 27 up to 57%), crumble size, three 100 L fibre ponds replicates. In experiment 5 the effect of two lipid levels (8 and 21%) was evaluated with commercial extruded Salmon Nutra Starter isoproteic crumble 1 diet at 63%, replicated in 4 ponds. The results show: A tendency to increased weight in all sizes with an increased protein level in the pelleted diet.A maximal adult growth is obtained with a diet containing a minimum of 37% crude protein, with 40% the optimal value. A higher % protein in the diet or growth in weight lower feed conversion ratio. The feed conversion ratio in the extruded diet reaches up to 0.5 and in the pelleted vary from 0.7 to 1.5. Fish 0.6 g fed with 63% protein, extruded commercial diet with two different lipid levels (8 and 21%, 20.40 and 23.84 MJ kg-1, PE/TE 0.62 and 0.71) increased weight the first month 67 and 105% each. It has been established that high-energy diets with optimal levels of protein and lipid are a good short-term solution to obtain G. maculatus of higher weight.

Resumo O objetivo da aquicultura de Galaxias maculatus é produzir milhões de ovos. As fêmeas selvagens são pequenas (2 g) e têm maturidade sexual rápida e fecundidade média baixa (500 ovos/fêmea), necessitando de peixes maiores e com fecundidade superior. O objetivo deste estudo é avaliar experimentalmente o efeito dos níveis de proteínas, lipídios e energia da dieta sobre o aumento de peso em adultos. Cinco experimentos independentes foram realizados em diferentes períodos sequenciais de tempo no incubatório UCT, Chile. Os espécimes foram obtidos a partir de: a) capturas de espécimes de retorno do mar cristalino no estuário de Tolten (4-6 cm, 0,3-0,4 g); b) peixes de cultura em incubatório. Os peixes foram alimentados à mão ad libitum. Nos experimentos de 1 a 4, dietas peletizadas foram preparadas com três a cinco níveis de proteína (tratamentos 27 a 57%), tamanho do crumble, três repetições de tanques de fibra de 100 L. No experimento 5, o efeito de dois níveis de lipídios (8 e 21%) foi avaliado com dieta comercial isoproteica crumble 1 de Salmon Nutra Starter extrusada a 63%, replicada em quatro tanques. Os resultados mostram: uma tendência ao aumento de peso em todos os tamanhos, com um aumento do nível de proteína na dieta peletizada; um crescimento adulto máximo com uma dieta contendo um mínimo de 37% de proteína bruta, com 40% do valor ideal; uma porcentagem maior de proteína na dieta ou crescimento em peso com menor taxa de conversão alimentar. A taxa de conversão alimentar na dieta extrusada chega a 0,5, e na peletizada varia de 0,7 a 1,5. Peixes de 0,6 g alimentados com 63% de proteína e dieta comercial extrusada com dois níveis lipídicos diferentes (8 e 21%; 20,40 e 23,84 MJ kg-1; PE / TE 0,62 e 0,71) aumentaram de peso no primeiro mês em 67 e 105% cada, respectivamente. Foi estabelecido que dietas de alta energia com níveis ótimos de proteínas e lipídios são uma boa solução de curto prazo para obter G. maculatus de peso mais alto.

Effect of protein and lipids levels in a growth diet on adult whitebait Galaxias maculatus (Jenyns 1842).

Galaxias maculatus aquaculture objectives is to produce millions of eggs. Wild females are small (2 g), have quick sexual maturity and low mean fecundity (500 eggs/female), requiring larger fishes with higher fecundity. This study aim is to evaluate experimentally the effect of the levels of protein, lipid and dietary energy on weight increases in adults. Five independent experiments were performed at different sequential time periods at the UCT hatchery, Chile. Specimens were obtained from a) Crystalline sea return specimen catches in the Tolten estuary (4 -6 cm, 0.3-0.4 g.). b) Hatchery cultured fish. Fish were fed by hand ad libitum. In experiments 1 to 4, pelleted diets were prepared with 3 to 5 levels of protein (treatments 27 up to 57%), crumble size, three 100 L fibre ponds replicates. In experiment 5 the effect of two lipid levels (8 and 21%) was evaluated with commercial extruded Salmon Nutra Starter isoproteic crumble 1 diet at 63%, replicated in 4 ponds. The results show: A tendency to increased weight in all sizes with an increased protein level in the pelleted diet.A maximal adult growth is obtained with a diet containing a minimum of 37% crude protein, with 40% the optimal value. A higher % protein in the diet or growth in weight lower feed conversion ratio. The feed conversion ratio in the extruded diet reaches up to 0.5 and in the pelleted vary from 0.7 to 1.5. Fish 0.6 g fed with 63% protein, extruded commercial diet with two different lipid levels (8 and 21%, 20.40 and 23.84 MJ kg-1, PE/TE 0.62 and 0.71) increased weight the first month 67 and 105% each. It has been established that high-energy diets with optimal levels of protein and lipid are a good short-term solution to obtain G. maculatus of higher weight.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (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.

Sperm characteristics of wild and captive lebranche mullet Mugil liza (Valenciennes, 1836), subjected to sperm activation in different pH and salinity conditions.

In this article we describe basic aspects of the sperm biology of lebranche mullet (Mugil liza) in the wild and in captivity, in particular assessing the effects of salinity (0, 10, 20, 30, 35, 40, 50 and 60 g L-1) and pH (6, 7, 8, 9 and 10) on sperm motility. Our results indicate that the highest percentage of motility was recorded with salinity 34.6 g L-1 (95 ±â€¯10%) and the longest motility time was obtained with a salinity of 34.8 g L-1 (189 ±â€¯15 s). Variations in the salinity between 30 and 35 g L-1 did not produce any significant alterations in sperm motility; however salinities of 20 and 50 g L-1 produced a significant loss of sperm motility. The highest percentage of motility was obtained at pH 8.5 (93 ±â€¯12%), and the longest motility period at pH 8.7 (218 ±â€¯13 s), while pH lower than or equal to 7 and equal to 10 both produced a significant loss in sperm motility. A positive correlation was found between pH/salinity and the motility percentage (R2 = 0.94 and R2 = 0.97) and motility time (R2 = 0.86 and R2 = 0.98). In seminal and morphometric parameters, statistically significant differences were observed in semen volume, sperm density, plasma membrane integrity and sperm morphometry between the groups studied, showing that the characteristics of the fish have a direct influence on sperm quality. The information generated in this research will be useful for developing biotechnology tools for the effective management of Mugil liza gametes.

Effect of the age of broodstock males on sperm function during cold storage in the trout (Oncorhynchus mykiss).

The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4 years old). Sperm samples of each reproductive age were stored in Storfish® during 10 days at 4°C, and then, motility, viability, mitochondrial function (MMP), superoxide anion (O2-) level and DNA fragmentation (DNAfrag ) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNAfrag and O2- level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3 years old were superior to those of 4 years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level O2- during short-term storage. This information must be considered for optimum utilization of broodstock males in aquaculture.

Porphyromonas gingivalis and its lipopolysaccharide differently modulate epidermal growth factor-dependent signaling in human gingival epithelial cells.

Periodontitis is an inflammatory disease induced by pathogenic bacteria such as Porphyromonas gingivalis. Little is known about epidermal growth factor (EGF) signals in human gingival epithelial cells (HGEC), which are major targets of P. gingivalis, and how the expression of proteins participating in EGF signaling-that is, EGF-receptor (EGFR), suppressor of cytokine signaling-3 (SOCS-3), interferon regulatory factor-1 (IRF-1), and signal transducers and activators of transcription (STAT-3)-are modified. This study aimed to assess the effects of P. gingivalis and its purified lipopolysaccharide (LPS-Pg) on EGF signaling. HGEC were infected for 2 h in a dose-dependent manner with P. gingivalis and with heat-killed P. gingivalis, and activated for 2 and 24 h by 1 µg/mL of purified LPS-Pg. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to measure mRNA and protein levels for SOCS-3, IRF-1 EGF, EGFR, and STAT-3. The tyrosine-phosphorylation status of STAT-3 was also examined. The results showed that infection of HGEC cells with P. gingivalis, but not with heat-killed P. gingivalis, led to significant reductions in expression levels of mRNAs and proteins for SOCS-3, IRF-1, and EGFR, while LPS-Pg over time significantly increased the expression of these mRNAs and proteins. Tyrosine-phosphorylation of STAT-3 was significantly increased during infection with P. gingivalis and activation by LPS-Pg but not modified during infection with heat-killed P. gingivalis. This study highlights that P. gingivalis and its purified LPS differentially modulated the expression of proteins (SOCS-3, IRF-1, EGFR, and STAT-3) interfering with EGF signaling.

Short-term storage of salmonids semen in a sodium alginate-based extender.

Short-term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate-based extender on sperm function in the short-term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish® (Ext-C) and Storfish® supplemented with sodium alginate (Ext-A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O2- ) level and DNA fragmentation (DNA Frag) were assessed. Ext-A had positive effect in preservation of sperm motility, viability, ΔΨmit, O2- level and DNA integrity in the three species analysed compared to control samples. In Ext-A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O2- level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate-based extender is effective for protecting sperm quality during 10 days of short-term storage.

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology.

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N2 L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (ΔΨMMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10(7) spermatozoa oocyte(-1) , by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d. DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (VCL ) 61·2 ± 17·4 µm s(-1) ; average-path velocity (VAP ) 50·1 ± 17·3 µm s(-1) ; straight-line velocity (VSL ) 59·1 ± 18·4 µm s(-1) ; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, VCL , VAP and VSL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, VCL and VSL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with VCL and VSL (r = 0·59 and r = 0·55, respectively).

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions.

Effect of short-term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry.

The short-term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short-term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short-term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning - undiluted (SSD) and diluted (SD) (Storfish(®) 1 : 2v/v; IMV AI solutions, France) - were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short-term storage evaluated by flow cytometry.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification.

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-µL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).

Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report.

The aims of this investigation were to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland(®) medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: CM + 1% BSA + 40% seminal plasma + 0.125 m sucrose. For cooling, 20-µl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM + 1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR-14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM + 1% BSA + 40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA + 40% seminal plasma.

Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness.

The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland(®)-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125 M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125 M sucrose. For cooling, 20 µL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P(1,2,3,4,5)<0.001; P(2,3,4,5) <0.01)(P(3-5)>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters.