Supplementation of sulfate polysaccharides in the seminal cooling medium of common curimata (Prochilodus brevis).

BACKGROUND: The use of sulfated polysaccharides (PS) in seminal cooling is known to improve seminal quality. OBJECTIVE: To evaluate the effect of different concentrations of PS, extracted from the macroalgae Gracilaria domigensis as a supplement to the seminal cooling medium of the reophilic fish Prochilodus brevis (common curimatã). MATERIALS AND METHODS: Five semen pools were diluted in ACP-104 (treatment T1), in BTS® (T2) and in BTS® with different concentrations of PS (0.5 [T3]; 1.0 [T4] and 1.5 [T5]). The samples were cooled for different times (0, 6, 24, 48, 72, 96 and 120 h) and after each hour they were analyzed for: morphology, membrane integrity, DNA integrity and sperm kinetics. RESULTS: There were no significant differences between the treatments containing different concentrations of sulfated polysaccharides. Regarding the different cooling times, it was possible to observe that after hour 96, there was a reduction in the parameters of sperm kinetics. For DNA integrity there was no significant difference in relation to the treatments nor in relation to the hours. For membrane integrity, a reduction was noted as of hour 96, but there was no influence of polysaccharides. For the sperm morphology, there was no statistical difference between the hours, however the BTS was better than the ACP-104. CONCLUSION: It is concluded that the use of polysaccharides in seminal cooling has no negative effect on sperm parameters and proves that seminal cooling keeps the material viable for up to 72 hours. Doi: 10.54680/fr23410110512.

Sulfated polysaccharides from seaweed as a supplement to Prochilodus brevis sperm freezing medium.

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.

The effect of glycosaminoglycans, extracted from the skin of tilapia, in the sperm freezing medium of Colossoma macropomum.

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.

Efeito de vitaminas adicionadas ao diluente ACP-104 sobre a qualidade do sêmen criopreservado de carpa comum (Cyprinus carpio) / Effect of vitamins added to the ACP-104 extender on the quality of cryopreserved semen of common carp (Cyprinus carpio)

O objetivo da presente pesquisa foi alcançado com a divisão da pesquisa em dois experimentos: (1) aperfeiçoar o protocolo de congelação utilizando água de coco em pó (ACP-104) como diluente para a criopreservação seminal de carpa comum; (2) avaliar o efeito da suplementação das vitaminas C (ácido ascórbico) ou E (α-tocoferol) sobre os melhores diluidores testados no experimento 1 na qualidade do sêmen pós-descongelado da espécie. Para o experimento 1, foram formados oito pools de sêmen, provenientes de 14 machos selecionados. As amostras seminais coletadas foram avaliadas quanto à motilidade total, à velocidade, ao percentual de espermatozoides normais e à vitalidade espermática antes e depois da criopreservação seminal. Esta foi realizada em meio ACP-104 acrescido de dimetilsulfóxido (DMSO), ou etilenoglicol (EG), ou glicerol, ou metanol, todos à concentração de 10%, diluídos em 1:3 (sêmen:diluidor). As amostras foram, então, congeladas em vapor de nitrogênio líquido em dry shipper e estocadas em nitrogênio líquido (-196°C). Para o experimento 2, foram formados oito pools provenientes da coleta de sêmen de 15 machos. As amostras seminais foram avaliadas seguindo as mesmas análises do experimento 1, acrescentando-se a duração da motilidade total. A criopreservação seminal utilizou-se do meio ACP-104 acrescido de DMSO ou EG, suplementado ou não com vitamina C ou E. Os melhores resultados encontrados no experimento 1 foram obtidos com o DMSO e o EG. Estes não diferiram significativamente entre si para a motilidade total (24% e 28%; P>0,05) e a normalidade espermática (32% e 26%; P>0,05), respectivamente. Para o experimento 2, o EG suplementado com vitamina E produziu significativamente resultados superiores de motilidade total, normalidade espermática e duração da motilidade em relação ao DMSO, concluindo-se que o EG deve ser, portanto, o crioprotetor de escolha a ser utilizado com o ACP-104 suplementado ou não com vitamina E.(AU)

The objective was achieved by dividing the research into two experiments: (1) improving the freezing protocol using powdered coconut water (ACP-104) as a diluent for the cryopreservation seminal of common carp; (2) evaluating the effect of supplementation of vitamins C (ascorbic acid) or vitamin E (α-tocoferol) with the best extenders tested in experiment 1 on the quality of post-thawed. For experiment 1, semen pools from 14 selected males were formed. Seminal samples were evaluated for total motility, velocity, percentage of normal sperm and sperm vitality before and after the seminal cryopreservation. This was done in ACP-104 extender plus dimethyl sulfoxide (DMSO), or ethylene glycol (EG), or glycerol or methanol all at concentration 10% diluted in 1:3 (semen:extender). The samples were frozen in vapors of nitrogen into dry shippers and stored in liquid nitrogen (-196 °C). For experiment 2, eight pools were formed from the 15 males. The semen samples were evaluated following the same analysis of experiment 1 adding duration of total motility. The sperm cryopreservation was performed in extenders ACP-104 plus DMSO or EG supplemented or not with vitamin C or E. The best results found in Experiment 1 were obtained with DMSO and EG. They do not differ significantly for total motility (24% and 28%; P>0.05) and normal sperm (32% and 26%; P>0.05) respectively. For experiment 2, EG supplemented with vitamin E, produced significantly better results overall motility, sperm normality and duration of motility relative to DMSO. In conclusion, EG should be the cryoprotectant of choice for use with the ACP-104 supplemented or not with vitamin E.(AU)

Cinética e morfologia de espermatozoides de carpa comum criopreservados em água de coco em pó acp-104 / Kinetics and morphology of common carp sperm cryopreserved in powdered coconut water acp-104

O objetivo deste estudo foi avaliar o efeito de diferentes taxas de diluição seminal sobre a motilidade, velocidade e presença de anormalidades espermáticas no sêmen descongelado de carpa comum (Cyprinus carpio). Utilizaram-se 20 machos sexualmente maduros, pertencentes ao Departamento Nacional de Obras Contra as Secas (DNOCS). A partir do sêmen coletado, foi formado um total de nove pools. O sêmen de cada pool foi avaliado quanto à motilidade, velocidade e morfologia espermática antes e depois da criopreservação. A criopreservação seminal foi realizada em meio ACP-104 + DMSO 10% diluído em 1:1 ou 1:3 (sêmen:diluidor). As amostras foram envasadas em palhetas de 0,25mL, congeladas em vapor de nitrogênio líquido (caixa de poliestireno) e estocadas em nitrogênio líquido (-196 °C). Não houve diferença significativa (P>0,05) entre as taxas de diluições sobre os parâmetros cinéticos e morfológicos de sêmen descongelado de carpa comum. Entretanto, esses parâmetros apresentaram diferença significativa (P<0,05) em relação ao sêmen fresco (controle). Baixos valores de velocidade foram registrados no sêmen descongelado (VCL 42,6-46,5μm/s; VSL 33,2-37,1μm/s; VAP 38,9-43,2μm/s); contudo, observaram-se motilidades acima de 59% (59,8-67,8%) e baixos índices de anormalidades espermáticas (19,3-19,5%), sugerindo que o ACP pode ser usado como um meio favorável à criopreservação do sêmen de carpa comum em qualquer uma das taxas de diluição testadas.

The aim of this study was to determine the effects of various dilution ratios on thaw sperm motility, velocity and presence of spermatic abnormalities in the common carp. 20 males with three years of age, raised at the Departamento Nacional de Obras Contra as Secas (DNOCS) were used. From the collected semen a total of nine pools were formed. The semen from each pool was evaluated for motility, velocity and spermatic morphology before and after cryopreservation. The sperm cryopreservation was performed in medium ACP-104 + DMSO 10% diluted in 1:1 or 1:3 (sperm:extender). The samples were loaded into 0.25mL straws, frozen in liquid nitrogen vapor (Styrofoam) and stored in liquid nitrogen (-196 °C). There was no significant difference (P>0.05) between the ratios of dilution on the kinetic and morphological parameters of thawed semen from common carp. However, these parameters were significantly different (P<0.05) compared to fresh semen (control). Low velocity values were recorded in thawed semen (VCL 42.6-46.5μm/s; VSL 33.2-37.1μm/s; VAP 38.9-43.2μm/s), however, observed motility above 59% (59.8-67.8%), and low levels of spermatic abnormalities (19.3-19.5%) suggested that the ACP-104 can be used as a suitable medium for cryopreservation of common carp semen in any of the tested dilution ratios.

Aloe vera na criopreservação do sêmen de tambaqui (Colossoma macropomum) / Aloe vera in the cryopreservation of tambaqui (Colossoma macropomum) sperm

This study aimed to evaluate the extract of Aloe vera (AV) associated or not with 10% Dimethylsulfoxide (DMSO) in cryopreservation of tambaqui semen. For the formation of the pools (n= 14), 30 males were hormonally induced twice. Each pool had the objective motility, curvilinear velocity, straight-line velocity, average path velocity and morphology analyzed before and after cryopreservation of semen. The means for cryopreservation were constituted of Powder Coconut Water-104 diluent added DMSO and/or AV (5 or 10%). After cryopreservation, motility, velocities and morphology were reduced significantly when compared to fresh semen. For sperm motility the best treatment was that using only DMSO (20,86±8,31) and DMSO + 5% AV (15.71±9.77). For the velocities, the worse treatment was DMSO+10% AV. Treatment with only the addition of DMSO had a significantly higher effect than others on percentage of morphologically normal sperm. The mean correlation found was between motilityand the rate of morphologically normal sperm (r = 0.687). In conclusion, the addition of AV does not provide greater protection for spermatozoa during cryopreservation.

Determinação da dose inseminante e embriogênese na fertilização artificial de tambaqui (Colossoma macropomum) / Determination of insemination dose and embryonic development in the artificial fertilization of tambaqui (Colossoma macropomum)

Determinou-se a dose inseminante para fertilização artificial e descreveu-se o desenvolvimento embrionário de tambaqui (Colossoma macropomum). Os gametas foram coletados de reprodutores induzidos hormonalmente. Foi realizada fertilização artificial nas proporções de espermatozoides/ovócito de D1-50.666; D2-75.999; D3-101.332; D4-126.665; D5-151.998. O desenvolvimento embrionário foi acompanhado por meio de observações periódicas em estereoscópio até a eclosão dos ovos. Na fase de fechamento do blastóporo foi calculada a taxa de fertilização nas diferentes doses inseminantes. A porcentagem de fertilização aumentou de forma linear segundo a equação Ŷ =0,050 + 0,00000773X (R²=97,5), atingindo um platô em 84% na proporção de 102.486 espermatozoides/ovócito. Os embriões apresentaram segmentação meroblástica discoidal, típica de ovos telolécitos, com eclosão ocorrendo aos 357 horas-grau após a fertilização. Conclui-se que o desenvolvimento embrionário de tambaqui obedece ao esperado para peixes com ovos telolécitos e recomenda-se o uso da dose inseminante de aproximadamente 100.000 espermatozoides/ovócito na rotina de fertilização artificial dessa espécie.

The objective of this research was to determine the insemination dose for artificial fertilization and describe the embryonic development of tambaqui (Colossoma macropomun). The gametes were collected from induced breeding hormonally. An artificial fertilization was performed with different sperm/oocyte ratios of D1-50666, D2-75999, D3-101 332, 126 665-D4, D5-151 998 sperm/oocyte. Embryonic development was monitored through periodic stereoscopic observations until hatching. When embryos reached the blastopore closure stage, the rate of fertilization in different insemination doses was calculated. A regression equation was estimated to determine the ideal proportion of the gametes. The fertilization rate increased linearly according to the equation Ŷ = 0.050 + 0.00000773 X (R² = 97.5), up to the proportion of 102.486 spermatozoa/oocyte, and, from this point, the fertilization rate was maintained at 84%. The embryonic development of tambaqui was meroblastic discoidal, as expected from telolecithal eggs and we recommend the use of the insemination dose of approximately 100.000 sperm/oocyte in the artificial fertilization of tambaqui.

Preservaçäo do sistema genital de cabras para a avaliaçäo em microscopia de luz / Preservation of goat genital system for microscopic evaluation purpose

The preservation of the goat genital organs with a fixed solution of paraformaldehyde 10 per cent and a saturated solution of Bouin during 4, 8, 12 and 24 hours of fixation, included in paraffin and stained with hematoxylin-eosin was evaluated. The Bouin solution with four hours of fixation showed to be the best protocol of fixation for the ovary, oviduct, uterus and vagina, resulting in little tissue retraction and better cellular integration when compared to the other fixing times (8, 12 and 24 hours). The fixation with paraformaldehyde showed significant alteration in the tissue architecture of the oviduct and vagina. Althouh paraformaldehyde is not the most adequate solution for the goat genital system preservation, its use can be considered in the absence of Bouin solution