Genetic variation (mtDNA) of the European conger eel, Conger conger, was compared across five locations in the north-eastern Atlantic (Madeira, Azores, South Portugal, North Portugal and Ireland) and one location in the western Mediterranean (Mallorca). Genetic diversity of conger eel was high, and differentiation among regions was not significant. Additionally, comparisons of element:Ca ratios (Sr:Ca, Ba:Ca, Mn:Ca and Mg:Ca) in otolith cores (larval phase) and edges (3 months prior to capture) among the Azores, North Portugal, Madeira and Mallorca regions for 2 years indicated that variation among regions were greater for edges than cores. Therefore, while benthic conger may display residency at regional scales, recruitment may not necessarily be derived from local spawning and larval retention. Furthermore, data from otoliths suggest a separated replenishment source for western Mediterranean and NE Atlantic stocks. The combination of genetics and otolith chemistry suggests a population model for conger eel involving a broad-scale dispersal of larvae, with limited connectivity for benthic juvenile life stages at large spatial scales, although the existence of one or multiple spawning grounds for the species remains uncertain. Communicated by T. Reusch. Alberto T. Correia and Rita Castilho contributed equally to this work.
Resumo. O processo de criopreservação acarreta estresse oxidativo à célula espermática e a adição de antioxidantes aos meios de refrigeração e congelação de sêmen pode auxiliar na proteção dos espermatozoides contra o dano induzido pelas espécies reativas de oxigênio (ROS), incluindo perda da motilidade de forma irreversível, peroxidação lipídica e fragmentação do DNA, interferindo na capacidade de fertilização do espermatozoide. Objetivou-se com este trabalho avaliar o efeito da adição dos antioxidantes Hidroxi butiltolueno (BHT) e extrato etanólico da casca do caule do mufumbo (Cumbretum leprosum) ao diluidor Botucrio ® para criopreservação de sêmen equino. Foram coletados quatro ejaculados de quatro garanhões da raça Quarto de Milha, por meio de vagina artificial. Após a descongelação das amostras em banho Maria a 37º C foi estimada a taxa de peroxidação lipídica dos espermatozoides pela medida do nível de malonaldeído. O delineamento experimental foi em bloco ao acaso. As variáveis de volume seminal, motilidade espermática, vigor espermático e HOST foram submetidas a análise de variância (ANOVA) utilizando-se o procedimento modelos lineares gerais (Proc GLM) e para comparação de média foi utilizado o teste Tukey, na probabilidade de 5%. A quantificação malonaldeído foi submetida à análise de variância (ANOVA), seguido de Tukey como post hoc teste, na probabilidade de 5%. Não houve diferença, pois o acréscimo de BHT nas concentrações 5, 1,0 e 2,0 mM de BHT e de extrato de mufumbo nas doses de 30, 60 e 120 mg ao diluidor Botucrio não melhorou a qualidade espermática. Desta forma, os antioxidantes não foram determinantes em promover a diminuição formação de radicais livres e, consequentemente, não melhoraram a qualidade do sêmen após a crioinjúria provocando danos à membrana dos espermatozoides. A suplementação nas doses 0,5, 1,0 e 2,0 mM de BHT e de extrato de mufumbo nas doses de 30, 60 e 120 mg ao diluidor botucrio ® não influenciou na qualidade seminal pós criopreservação.
Background: Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal’s species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing.Materials, Methods & Results: Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog’s semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn´t exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2.Discussion: The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100 µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.Keywords: dog, semen, antioxidants, cryopreservation.Descritores: cão, sêmen, antioxidantes, criopreservação.
RESUMO O objetivo deste trabalho foi avaliar a recuperação de espermatozoides epididimários de cães castrados, utilizando as técnicas de fluxo retrógrado (FR) e flutuação (FL) em diluidor Tris-gema, antes e após a criopreservação. Foram coletados 30 complexos testículo-epididímos (CTE), sendo 15 para FR e 15 para FL, e, logo após a recuperação dos espermatozoides, foram analisadas as alterações morfológicas nessas células espermáticas. Após a adição do diluidor, foram avaliados os parâmetros de motilidade total (MOT) e vigor (V) espermáticos. O sêmen pós-criopreservado foi submetido ao teste de termorresistência nos tempos T0, T30, T60 e T90 minutos, além da avaliação das membranas plasmática e acrossomal por sondas fluorescentes. Não houve diferença estatística entre as técnicas quanto à MOT e ao vigor no sêmen diluído (FR-MOT: 82,3% e V: 3,4; FL-MOT: 79,6% e V: 3,2) e pós-criopreservado (FR-MOT: 34% e V: 2,8; FL-MOT: 30% e V: 2,7). A partir do T30, houve diferença significativa quanto à MOT e ao vigor nas técnicas utilizadas, e o tempo também prejudicou o acrossoma espermático a partir do T30. Conclui-se que as técnicas de recuperação de espermatozoides epididimários de cães castrados, testadas neste trabalho, podem ser utilizadas para refrigeração e criopreservação de sêmen.
Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.
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