RESUMO
In this study, it was aimed to determine the effect of sulforaphane (SFN) on rabbit semen cryopreservation. Semen collected from animals was divided into 5 equal volumes as Control, SFN 5 µM, SFN 10 µM, SFN 25 µM and SFN 50 µM groups. Afterwards, semen analyzes were performed. According to our results, there was no statistical difference between the groups at 4°C. However after freezing thawing, the highest total motility, progressive motility and rapid spermatozoa rate was seen in the 10 µM SFN group, while the lowest was observed in the 50 µM SFN group (P<0.05). Static sperm ratio was highest in the 50 µM group, while the lowest was observed in the 10 µM SFN group. When flow cytometry results examined the rate of acrosomal damaged and dead sperm was the lowest in the 10 µM SFN group, a statistical difference was observed between the control group (P<0.05). The highest rate of sperm with high mitochondrial membrane potential was seen in the 5 µM SFN and 10 µM SFN groups. Apoptosis and ROS rates were found to be lower in the experimental groups compared to the control groups (P<0.05). As a result, SFN supplementation at a dose of 10 µM increased the quality of sperm in the freezing and thawing processes of rabbit semen. In conclusion, 10 µM SFN improved the quality of cryopreservation of rabbit semen.(AU)
Assuntos
Animais , Coelhos , Preservação do Sêmen/efeitos adversos , Isotiocianatos/efeitos adversos , Criopreservação , Apoptose/fisiologia , Estresse Oxidativo/efeitos dos fármacosRESUMO
Effects were assessed of the dilutants TRIS and ACP - 101c® with the addition of different guinea fowl (Numida meleagris) egg yolk concentrations. Fifteen ejaculates were collected from five goats of the Anglo Nubian breed. The ejaculates were pooled and then divided into 12 groups, two control groups (GC1 TRIS, with 2.5% Gallus gallus domesticus hen egg yolk GOGD), (GC2 Control Group ACP - 101c®, with the addition of 2.5% Gallus gallus domesticus hen egg yolk GOGD) and ten experimental groups (EG), containing TRIS and ACP added with different concentrations of egg yolk from guinea hen (Numida meleagris) (TRIS 2,5% GONM; TRIS 5% GONM; TRIS 10% GONM; TRIS 15% GONM; TRIS 20% GONM; ACP® 2,5% GONM; ACP® 5% GONM; ACP® 10% GONM; ACP® 15% GONM; ACP® 20% GONM). Then cryopreservation was carried out and the samples stored in liquid nitrogen (-196 °C). After seven days, the samples were thawed and assessed for spermatic kinetics, immunofluorescence and sperm morphology. Analysis of GOMN by the CASA system showed that the various parameters were similar to those of GOGD (P>0.05). The membrane integrity, mitochondrial potential and the acrosome were not influenced by the treatment (P>0.05) nor by the dilutant used for cryopreservation (P>0.05). The spermatic morphology was also preserved by the different GOGD and GONM concentrations in the ACP® and TRIS dilutants, with no statistically significant differences (P<0.05). It was concluded that Numida meleagris egg yolk, as external membrane cryoproctant added to the dilutants ACP-101c® and TRIS, improved goat semen quality.(AU)
Assuntos
Animais , Masculino , Preservação do Sêmen/efeitos adversos , Ruminantes/fisiologia , Criopreservação/veterinária , Gema de Ovo/química , Alimentos de Coco , Crioprotetores/administração & dosagem , GalliformesRESUMO
BACKGROUND: The sperm vitrification developed by this group is based on the ultrarapid freezing of a vitrification solution composed of a non-permeable cryoprotectant (saccharides and protein), in which previously selected spermatozoa are resuspended, free of seminal plasma, and then plunged directly into liquid nitrogen. Compared to traditional sperm freezing, vitrification does not cause chemical or physical damage to the intracellular structures and reduces the damage to the plasma membrane because no ice crystals form, thus preserving motility and DNA integrity. OBJECTIVES: This manuscript is a review of the vitrification methodology developed by the authors' research group, including studies showing the application in human reproduction therapy. MATERIALS AND METHODS: The authors perform a review of the work initiated more than a decade ago by this research group, on the implementation of sperm vitrification, a more effective technique for cryopreservation of human spermatozoa, discussing the results obtained by other authors and the projection of this technique. RESULTS AND DISCUSSION: The vitrification technique has been developed in selected spermatozoa free of seminal plasma supplemented with saccharides such as sucrose, trehalose, and dextran, together with albumin, providing a high motility rate and protective structures of the cytoskeleton. In patients, it can be used to preserve their fertility for oncological reasons, genetics, inflammatory diseases, or reproductive medicine techniques. The possibility that vitrified spermatozoa can be preserved at temperatures of -80°C can simplify sample storage, optimizing the space and time as well as operator safety. CONCLUSION: Vitrification techniques have demonstrated the preservation of selected spermatozoa without seminal plasma and with non-permeable cryoprotectants and protein. Currently, it is one of the most effective ways to maintain sperm function and has been used in in vitro fertilization or intrauterine insemination in humans, achieving healthy live births.
Assuntos
Criopreservação , Crioprotetores/uso terapêutico , Preservação da Fertilidade , Infertilidade/terapia , Preservação do Sêmen , Espermatozoides/efeitos dos fármacos , Crioprotetores/efeitos adversos , Difusão de Inovações , Feminino , Fertilidade , Preservação da Fertilidade/efeitos adversos , Humanos , Infertilidade/diagnóstico , Infertilidade/fisiopatologia , Masculino , Gravidez , Fatores de Risco , Preservação do Sêmen/efeitos adversos , Espermatozoides/patologia , Resultado do Tratamento , VitrificaçãoRESUMO
Our aim was to evaluate the effects of three thermal environments over time on kinetics, functionality and in vitro fertility of cryopreserved bovine spermatozoa. Four ejaculates from five bulls (n = 20) were cryopreserved. After thawing, semen was evaluated (0 hr), incubated for 4 hr in T36.0 (36.0°C), T38.0 (38.0°C) and T39.5 (39.5°C), and analysed every hour (1 hr, 2 hr, 3 hr, 4 hr). In vitro production of embryos was performed at 0 hr and 4 hr. Sperm motility and cell kinetics (Computer-Assisted Sperm Analysis) were impaired after 2 hr at T38.0 and T39.5 (p < 0.05). Flow cytometry revealed an increase in the cells with injured plasma membrane to 39.5°C and a general reduction in the mitochondrial potential over time (p < 0.05). In vitro fertility was impaired in all temperatures after 4 hr, but there was no difference between 36.0°C and 38.0°C. Our results suggest that the ex situ resilience of semen at 36.0°C after thawing with no major damage to the quality is limited to 3 hr. In normothermia or in thermal stress, sperm cells present a gradual reduction of movement and functionality, which were more significant after 1 hr of incubation. The in vitro production of embryos is impaired when the semen is kept in a thermal environment ≥36.0°C for 4 hr.
Assuntos
Criopreservação/veterinária , Fertilidade/fisiologia , Temperatura Alta/efeitos adversos , Preservação do Sêmen/efeitos adversos , Espermatozoides/fisiologia , Animais , Bovinos , Feminino , Fertilização in vitro/veterinária , Inseminação Artificial/veterinária , Masculino , Ovário , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/fisiologia , Fatores de TempoRESUMO
Pre-treatment of boar semen with a red light photostimulation procedure increases its "in vivo" fertilising ability. However, "in vitro" conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short-term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620-630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca2+ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca2+ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short-term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.
Assuntos
Temperatura Baixa/efeitos adversos , Inseminação Artificial/veterinária , Luz , Sêmen/efeitos da radiação , Estresse Fisiológico/efeitos da radiação , Criação de Animais Domésticos/métodos , Animais , Sobrevivência Celular/efeitos da radiação , Inseminação Artificial/métodos , Masculino , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sêmen/metabolismo , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides/efeitos da radiação , Suínos , Fatores de TempoRESUMO
The aim of this study was to evaluate the effect of four methods of sperm selection, on the integrity and stability of the plasma membrane, integrity of the acrosomal membrane and spermatic morphology in frozen/thawed ovine semen. Two types of colloidal silica: colloidal silica-silane and colloidal silica-polyvinylpyrrolidone (PVP), and two aliquots: 1 and 4 ml, were used for sperm selection. Probes FITC-PSA and PI were used to measure the integrity of the plasma and acrosomal membranes. Plasma membrane stability was measured, using fluorescent probes M540 and YOPRO1. Effective reduction in the incidence of spermatozoa with acrosomal pathologies was only achieved using 1 ml colloidal silica-silane. All methods were efficient in select viable and unreacted spermatozoa. Only methods using 1 ml of silica were efficient in decrease spermatozoa stained by PI (death). Methods using silica colloidal-silane were more efficient to decrease apoptotic cells after selection when compared to silica colloidal-PVP. In conclusion, sperm selection in colloidal silica-silane and colloidal silica-PVP improved sperm quality when compared to the controls. The method using 1 ml of colloidal silica-silane is the preferred method because its effectiveness and lower cost.
Assuntos
Membrana Celular/metabolismo , Coloides/química , Criopreservação/métodos , Preservação do Sêmen/métodos , Sêmen/metabolismo , Acrossomo/metabolismo , Acrossomo/patologia , Animais , Sobrevivência Celular , Criopreservação/economia , Criopreservação/veterinária , Citometria de Fluxo , Corantes Fluorescentes , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/economia , Preservação do Sêmen/veterinária , Ovinos , Carneiro Doméstico/fisiologia , Motilidade dos Espermatozoides/fisiologiaRESUMO
Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3) and CRISP3c.+716A>G (SNP4). CRISP-3 protein content in seminal plasma was determined by enzyme-linked immunosorbent assay. Semen from 30 stallions was cryopreserved and post-thaw motility, kinetics, abnormal morphology (AM), sperm vitality (SV) and membrane integrity (MI) were evaluated. Generalized linear models were fitted and means were compared using Tukey's test. Correlation and regression analyses were performed. For SNP1 and SNP3, the AA genotype had the highest results for motility and MI; for SNP2, the best results for motility and AM were obtained with the CC genotype. For SNP4, the GG genotype had the lowest results, except for MI. A high level of CRISP-3 protein in seminal plasma had the best results for motility, kinetics, SV and AM. In conclusion, there was a relationship between CRISP-3 genotype and seminal plasma protein and post-thawing semen quality in stallions.
Assuntos
Criopreservação/veterinária , Cavalos/genética , Polimorfismo de Nucleotídeo Único , Proteínas e Peptídeos Salivares/genética , Preservação do Sêmen/veterinária , Proteínas de Plasma Seminal/genética , Espermatozoides/química , Animais , Sobrevivência Celular , Frequência do Gene , Heterozigoto , Homozigoto , Humanos , Cinética , Modelos Lineares , Masculino , Fenótipo , Fatores de Risco , Proteínas e Peptídeos Salivares/análise , Preservação do Sêmen/efeitos adversos , Proteínas de Plasma Seminal/análise , Motilidade dos Espermatozoides , Espermatozoides/patologiaRESUMO
This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalos semen due to its nutritive and protective properties.
Assuntos
Animais , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Mel , Mel/análise , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/veterinária , Búfalos , Clivagem do DNA , Estudos de ViabilidadeRESUMO
This study testified the effect of honey supplementation (0.5-4.0%) in milk on the quality of chilled and frozen buffalo spermatozoa. Semen was chilled with/without honey and examined for motility, viability, plasma membranes integrity by hypo-osmotic swelling test (HOS) at 0, 1, 2 and 4 h. Frozen-thawed semen was examined for the same criteria beside the viability index and in vitro cleavage rate. The motility, livability and HOS of chilled semen upsurge with honey supplementation 1.0-2.0%. The normality of chilled spermatozoa was improved in the presence of 2.0-4.0% of honey at 4 h. Tail abnormalities decreased with milk honey 0.5, 1.0 and 2.0% at 2, 1 and 4 h, respectively. Incorporation of honey in milk extenders at levels of 0.5-2.0% was associated with an enhanced post equilibration motility. The post-thawing motility showed a steady increase with honey levels. The viability index increased (P < 0.001) with milk-honey 2.0% (109.00 ± 9.91) and 4.0% (112.00 ± 14.41). In vitro cleavage rate was clearly (P 0.001) enhanced in the co-existence of milk-honey 2.0% compared with control (74.00 vs. 45.83). In the meantime, a reasonable high cleavage rate (67.00%) was encountered with milk honey 0.5%. In conclusion, incorporation of honey in skim milk extenders is promising to enhance the characteristics and fertilizing potential of stored buffalos semen due to its nutritive and protective properties.(AU)
Assuntos
Animais , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/veterinária , Mel/análise , Mel , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Búfalos , Estudos de Viabilidade , Clivagem do DNARESUMO
O objetivo foi avaliar o efeito da adição de diferentes concentrações de taurina ao diluente de resfriamento de sêmen ovino. Foram utilizados neste experimento 40 ejaculados de oito carneiros, diluídos nostratamentos: controle Tris-gema (T1) adicionado de taurina nas concentrações de 1 µM (T2), 2 µM (T3) e 3 µM (T4), sob refrigeração a 5°C por 48 h e avaliados através da análise de parâmetros de qualidade espermática de integridade de membrana, DNA e acrossoma, funcionalidade de mitocôndria e motilidade espermática. Observou-se que a motilidade espermática nas 48 h foi inferior no T2 (29,3%) em relação ao T4 (37,6%) (P < 0,05). Quanto aos parâmetros de integridade de membrana e DNA não se verificou diferença estatística entre os tratamentos.Para integridade de acrossoma, em amostras de sêmen fresco, encontrou-se 59.0%, e após 48 h de refrigeração, foram observadas taxas integridade nos grupos adicionados de taurina de 50.7% (T2), 51,3% (T3) e 51.6% (T4),que não diferiram do sêmen fresco. Conclui-se que a taurina nas concentrações testadas foi eficiente para manter a integridade de acrossoma no sêmen ovino refrigerado a 5°C.
The objective of this work was to assess the effect of different concentrations of taurine added to ramsperm cooling extender. We used in this experiment 40 ejaculates from eight rams, diluted according to the following treatments: Tris-yolk control (T1) taurine-added at the concentrations of 1 µM (T2), 2 µM (T3) and 3 µM (T4), under 5º C refrigeration for 48 hours and assessed through sperm quality parameters of membrane integrity, DNA and acrosome, mitochondrial functionality and sperm motility. We observed that sperm motilitywithin 48 hours was lower in T2 (29.3%) when compared to T4 (37.6%) (P < 0.05). As for the parameters membrane integrity and DNA we did not observe statistical differences among the treatments. As for acrosome integrity, in fresh semen samples, we obtained 59.0%, and after 48 hours refrigeration we observed integrity rates in the taurine-added groups of 50.7% (T2), 51,3% (T3) and 51.6% (T4), that did not differ from fresh semen. In conclusion, the concentrations of taurine we tested was efficient to keep acrosome integrity within cooled ram sperm at 5ºC.
Assuntos
Animais , Antioxidantes/síntese química , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Taurina/administração & dosagem , Taurina/efeitos adversos , OvinosRESUMO
O objetivo foi avaliar o efeito da adição de diferentes concentrações de taurina ao diluente de resfriamento de sêmen ovino. Foram utilizados neste experimento 40 ejaculados de oito carneiros, diluídos nostratamentos: controle Tris-gema (T1) adicionado de taurina nas concentrações de 1 µM (T2), 2 µM (T3) e 3 µM (T4), sob refrigeração a 5°C por 48 h e avaliados através da análise de parâmetros de qualidade espermática de integridade de membrana, DNA e acrossoma, funcionalidade de mitocôndria e motilidade espermática. Observou-se que a motilidade espermática nas 48 h foi inferior no T2 (29,3%) em relação ao T4 (37,6%) (P < 0,05). Quanto aos parâmetros de integridade de membrana e DNA não se verificou diferença estatística entre os tratamentos.Para integridade de acrossoma, em amostras de sêmen fresco, encontrou-se 59.0%, e após 48 h de refrigeração, foram observadas taxas integridade nos grupos adicionados de taurina de 50.7% (T2), 51,3% (T3) e 51.6% (T4),que não diferiram do sêmen fresco. Conclui-se que a taurina nas concentrações testadas foi eficiente para manter a integridade de acrossoma no sêmen ovino refrigerado a 5°C.(AU)
The objective of this work was to assess the effect of different concentrations of taurine added to ramsperm cooling extender. We used in this experiment 40 ejaculates from eight rams, diluted according to the following treatments: Tris-yolk control (T1) taurine-added at the concentrations of 1 µM (T2), 2 µM (T3) and 3 µM (T4), under 5º C refrigeration for 48 hours and assessed through sperm quality parameters of membrane integrity, DNA and acrosome, mitochondrial functionality and sperm motility. We observed that sperm motilitywithin 48 hours was lower in T2 (29.3%) when compared to T4 (37.6%) (P < 0.05). As for the parameters membrane integrity and DNA we did not observe statistical differences among the treatments. As for acrosome integrity, in fresh semen samples, we obtained 59.0%, and after 48 hours refrigeration we observed integrity rates in the taurine-added groups of 50.7% (T2), 51,3% (T3) and 51.6% (T4), that did not differ from fresh semen. In conclusion, the concentrations of taurine we tested was efficient to keep acrosome integrity within cooled ram sperm at 5ºC.(AU)
Assuntos
Animais , Taurina/administração & dosagem , Taurina/efeitos adversos , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Antioxidantes/síntese química , OvinosRESUMO
OBJECTIVE: The main objective of this study was to assess semen characteristics of patients with testicular cancer before cryopreservation and after thawing, to evaluate the consequences of this technique on sperm quality in patients with testicular cancer. METHODS: Five hundred eighty-nine samples from 543 patients with testicular cancer were cryopreserved between 1995 and 2015, one aliquot per patient was used for a thawing test to assess the impact of cryopreservation on sperm motility; semen analysis was performed before cryo preservation and after thawing, the result interpretation was carried out using the 2010 World Health Organization (WHO) Laboratory Manual, and consent forms were signed by the patients for freezing and when sperm was used for reproductive purposes. RESULTS: Hypospermia was observed in 28.7% of samples, the median sperm concentration was 18 million/mL with 35% oligozoospermia; twenty-two patients (4.1%) had azoospermia and 12.7% had severe oligozoospermia, the median sperm count was 31.3 million and 261 semen samples (44.3%) were normal in all parameters according to the WHO; total motile sperm count before cryopreservation and after thawing was 12 (0-412.2) and 7 (0-303.9) million sperm, respectively (p < 0.00001, 95% CI 5.48-14.91), which represents a 32% reduction; concerning the utilization of cryopreserved semen samples, only twelve patients (2.2%) used their frozen sperm for reproductive purposes. CONCLUSIONS: An impairment in semen quality was found in almost half of the samples from patients with testicular cancer, only few patients had azoospermia or severe oligozoospermia; sperm cryopreservation significantly reduces sperm motility and total motile sperm count and very few patients use their frozen sperm for reproductive purposes.
Assuntos
Criopreservação , Análise do Sêmen , Preservação do Sêmen/efeitos adversos , Neoplasias Testiculares/patologia , Adulto , Humanos , Masculino , Contagem de EspermatozoidesRESUMO
The aim of this work was to submit sperm cells to different laboratory challenges and to compare in vitro results with in vivo semen fertility. Four different batches from the same Brangus bull were used in a timed-AI program of 332 Brangus cows. Each batch (B) was submitted to the following procedure: semen sample was thawed at 36°C for 30 seconds (control). Sperm motility parameters, plasma membrane integrity, sperm morphology, and concentration were assessed. Then, an aliquot of thawed sample was incubated in a water bath at 45°C for 40 min (thermal challenge group; TCG) and another aliquot was centrifuged at 500 xg (Percoll gradient 45%/90%) for 15 min (centrifugation challenge group; CCG). Centrifuged semen was also submitted to another thermal challenge, being incubated (water bath) at 45°C for 40 min (centrifugation + thermal challenge group; CTCG). At the end of each challenge (CCG, TCG, and CTCG), the same laboratory tests used for control group were repeated. The following conception rates (CR) were observed for each batch: B1 = 48.9% (44/90); B2 = 44.2% (23/52); B3 = 55.5% (40/72); B4 = 43.2% (51/118); (p < 0.10). In the lab, B3 presented higher (p ≤ 0.05) progressive motility (PM) than B4 after thawing (control group) and after all sperm challenges (TCG, CCG, and CTCG). However, despite B3 and B4 having demonstrated a similar percentage of plasma membrane integrity (PMI) to the control group (B3 = 66.7 ± 1.3 and B4 = 65.2 ± 3.3), B3 demonstrated higher (P ≤ 0.05) percentage of PMI (37.2 ± 2.5) than B4 (26.7 ± 3.3) after passing through the most stressing in vitro challenge (CTCG). The semen batch presenting the highest resistance to in vitro challenges was the one that presented a trend for higher in vivo fertility, suggesting that submitting semen samples to laboratory challenges may be an interesting alternative for selecting batches with greater field fertility.(AU)
O objetivo deste estudo foi estressar células espermáticas em diferentes desafios laboratoriais e comparar os resultados in vitro com a fertilidade in vivo do sêmen. Quatro partidas de um mesmo touro Brangus foram utilizadas em um programa de IATF de 332 vacas Brangus. Cada partida foi submetida ao seguinte procedimento: a amostra de sêmen foi descongelada a 36°C por 30 segundos (grupo controle). Foram avaliados parâmetros de motilidade espermática (CASA), integridade da membrana plasmática (PMI), morfologia e concentração espermática. Em seguida, uma alíquota da amostra descongelada foi incubada em banho-maria a 45°C durante 40 minutos (grupo de desafio térmico, TCG) e outra alíquota foi centrifugada a 500 xg (gradiente de Percoll 45%/90%) durante 15 min (grupo desafio de centrifugação, CCG). Uma aliquota do sêmen centrifugado foi ainda submetida ao desafio térmico, sendo incubado a 45°C durante 40 min (grupo de desafio térmico + centrifugação, CTCG). No final de cada desafio (CCG, TCG e CTCG), os mesmos testes laboratoriais utilizados para o grupo de controle foram realizados. A seguinte taxa de concepção (CR) foi observada para cada partida (B): B1 = 48,9% (44/90), B2 = 44,2% (23/52), B3 = 55,5% (40/72) e B4 = 43,2% (51/118); (P < 0,10). No laboratório, B3 apresentou maior (P ≤ 0,05) motilidade progressiva (PM) do que B4 logo após o descongelamento (grupo controle) e após todos os desafios laboratoriais (TCG, CCG e CTCG). Porém, apesar de B3 e B4 demonstrarem similar porcentagem de PMI no grupo controle (B3 = 66,7 ± 1,3 e B4 = 65,2 ± 3,3), B3 apresentou maior (P ≤ 0,05) PMI (37,2 ± 2,5%) do que B4 (26,7 ± 3,3%) após passar pelo maior desafio laboratorial (CTCG). A partida seminal que in vitro apresentou maior resistência aos desafios laboratoriais foi a mesma que apresentou tendência para maior fertilidade in vivo. Assim, sugere-se que submeter amostras seminais a desafios laboratoriais pode ser uma alternativa interessante para selecionar partidas com maior fertilidade a campo.(AU)
Assuntos
Animais , Bovinos , Fertilização in vitro/veterinária , Inseminação Artificial/veterinária , Técnicas de Reprodução Assistida/veterinária , Preservação do Sêmen/efeitos adversosRESUMO
The aim of this work was to submit sperm cells to different laboratory challenges and to compare in vitro results with in vivo semen fertility. Four different batches from the same Brangus bull were used in a timed-AI program of 332 Brangus cows. Each batch (B) was submitted to the following procedure: semen sample was thawed at 36°C for 30 seconds (control). Sperm motility parameters, plasma membrane integrity, sperm morphology, and concentration were assessed. Then, an aliquot of thawed sample was incubated in a water bath at 45°C for 40 min (thermal challenge group; TCG) and another aliquot was centrifuged at 500 xg (Percoll gradient 45%/90%) for 15 min (centrifugation challenge group; CCG). Centrifuged semen was also submitted to another thermal challenge, being incubated (water bath) at 45°C for 40 min (centrifugation + thermal challenge group; CTCG). At the end of each challenge (CCG, TCG, and CTCG), the same laboratory tests used for control group were repeated. The following conception rates (CR) were observed for each batch: B1 = 48.9% (44/90); B2 = 44.2% (23/52); B3 = 55.5% (40/72); B4 = 43.2% (51/118); (p < 0.10). In the lab, B3 presented higher (p ≤ 0.05) progressive motility (PM) than B4 after thawing (control group) and after all sperm challenges (TCG, CCG, and CTCG). However, despite B3 and B4 having demonstrated a similar percentage of plasma membrane integrity (PMI) to the control group (B3 = 66.7 ± 1.3 and B4 = 65.2 ± 3.3), B3 demonstrated higher (P ≤ 0.05) percentage of PMI (37.2 ± 2.5) than B4 (26.7 ± 3.3) after passing through the most stressing in vitro challenge (CTCG). The semen batch presenting the highest resistance to in vitro challenges was the one that presented a trend for higher in vivo fertility, suggesting that submitting semen samples to laboratory challenges may be an interesting alternative for selecting batches with greater field fertility.(AU)
O objetivo deste estudo foi estressar células espermáticas em diferentes desafios laboratoriais e comparar os resultados in vitro com a fertilidade in vivo do sêmen. Quatro partidas de um mesmo touro Brangus foram utilizadas em um programa de IATF de 332 vacas Brangus. Cada partida foi submetida ao seguinte procedimento: a amostra de sêmen foi descongelada a 36°C por 30 segundos (grupo controle). Foram avaliados parâmetros de motilidade espermática (CASA), integridade da membrana plasmática (PMI), morfologia e concentração espermática. Em seguida, uma alíquota da amostra descongelada foi incubada em banho-maria a 45°C durante 40 minutos (grupo de desafio térmico, TCG) e outra alíquota foi centrifugada a 500 xg (gradiente de Percoll 45%/90%) durante 15 min (grupo desafio de centrifugação, CCG). Uma aliquota do sêmen centrifugado foi ainda submetida ao desafio térmico, sendo incubado a 45°C durante 40 min (grupo de desafio térmico + centrifugação, CTCG). No final de cada desafio (CCG, TCG e CTCG), os mesmos testes laboratoriais utilizados para o grupo de controle foram realizados. A seguinte taxa de concepção (CR) foi observada para cada partida (B): B1 = 48,9% (44/90), B2 = 44,2% (23/52), B3 = 55,5% (40/72) e B4 = 43,2% (51/118); (P < 0,10). No laboratório, B3 apresentou maior (P ≤ 0,05) motilidade progressiva (PM) do que B4 logo após o descongelamento (grupo controle) e após todos os desafios laboratoriais (TCG, CCG e CTCG). Porém, apesar de B3 e B4 demonstrarem similar porcentagem de PMI no grupo controle (B3 = 66,7 ± 1,3 e B4 = 65,2 ± 3,3), B3 apresentou maior (P ≤ 0,05) PMI (37,2 ± 2,5%) do que B4 (26,7 ± 3,3%) após passar pelo maior desafio laboratorial (CTCG). A partida seminal que in vitro apresentou maior resistência aos desafios laboratoriais foi a mesma que apresentou tendência para maior fertilidade in vivo. Assim, sugere-se que submeter amostras seminais a desafios laboratoriais pode ser uma alternativa interessante para selecionar partidas com maior fertilidade a campo.(AU)
Assuntos
Animais , Bovinos , Inseminação Artificial/veterinária , Fertilização in vitro/veterinária , Técnicas de Reprodução Assistida/veterinária , Preservação do Sêmen/efeitos adversosRESUMO
Two different extenders were compared for their effects on preservation of semen from Indigenous rams and on pregnancy rate (PR) in Indigenous ewes. Semen was collected from nine Indigenous rams (Ovis aries) once a week using an artificial vagina. Each ejaculate was divided into 2 aliquots, diluted with either commercial (Triladyl®) or locally manufactured (tris, fructose, citric acid, egg yolk: TFE, prepared in own laboratory) extenders and kept at 4°C for 48 h. Motility, viability, functional integrity and morphological changes were evaluated at 0, 24 and 48 h. Synchronized oestrus ewes inseminated transcervically with 24 and 48 h of preserved chilled semen diluted with Triladyl and TFE extenders separately. Semen preserved in Triladyl had better motility, viability, and functional integrity at 24 and 48 h (P < 0.001) than did in TFE. The morphologically normal spermatozoa up to 48 h of preservation did not differ between extenders. However, in abnormalities studied, Triladyl had detrimental effect on sperm acrosome and TFE on sperm tail (P < 0.001) at 24 and 48 h of preservation. But, midpiece was not affected by any extender (P > 0.05) over the entire period of preservation. The quality of semen decreased (P < 0.001) with increasing preservation time for both extenders. The extenders did not differ (P > 0.05) the overall PR after transcervical AI (TCAI) in ewes. Increased preservation time (48 h) negatively affected the PR in TFE extended semen compared with than that of Triladyl. The results suggest that the quality of chilled semen (motility, viability, and functional integrity) is more improved when preserved in Triladyl than if extended with a TFE. PR may higher when TCAI is performed with chilled semen preserved in Triladyl for a longer time than TFE. However, TFE extender may be used to dilute the semen for chilling and used in TCAI to get similar PR of Triladyl up to 24 h of preservation.(AU)
Assuntos
Animais , Feminino , Ovinos/embriologia , Ovinos/genética , Taxa de Gravidez , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/veterinária , Análise do Sêmen/veterináriaRESUMO
Two different extenders were compared for their effects on preservation of semen from Indigenous rams and on pregnancy rate (PR) in Indigenous ewes. Semen was collected from nine Indigenous rams (Ovis aries) once a week using an artificial vagina. Each ejaculate was divided into 2 aliquots, diluted with either commercial (Triladyl®) or locally manufactured (tris, fructose, citric acid, egg yolk: TFE, prepared in own laboratory) extenders and kept at 4°C for 48 h. Motility, viability, functional integrity and morphological changes were evaluated at 0, 24 and 48 h. Synchronized oestrus ewes inseminated transcervically with 24 and 48 h of preserved chilled semen diluted with Triladyl and TFE extenders separately. Semen preserved in Triladyl had better motility, viability, and functional integrity at 24 and 48 h (P 0.05) over the entire period of preservation. The quality of semen decreased (P 0.05) the overall PR after transcervical AI (TCAI) in ewes. Increased preservation time (48 h) negatively affected the PR in TFE extended semen compared with than that of Triladyl. The results suggest that the quality of chilled semen (motility, viability, and functional integrity) is more improved when preserved in Triladyl than if extended with a TFE. PR may higher when TCAI is performed with chilled semen preserved in Triladyl for a longer time than TFE. However, TFE extender may be used to dilute the semen for chilling and used in TCAI to get similar PR of Triladyl up to 24 h of preservation.
Assuntos
Feminino , Animais , Análise do Sêmen/veterinária , Ovinos/embriologia , Ovinos/genética , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/veterinária , Taxa de GravidezRESUMO
The effects of semen extenders containing coconut (Cocos nucifera) water alone or combined with egg yolk on viability of spermatozoa during cryopreservation of semen obtained from West African Dwarf (WAD) bucks were studied. Coconut water (5, 10, 15 and 20 ml/100 ml) was diluted with pooled semen samples in study I, while in study II, the pooled semen samples were diluted with egg yolk and coconut water (EYCW) at 5:10, 10:10 and 10:5 in Tris-based extenders. The control consisted of Tris-based extenders plus sodium citrate. The diluted semen samples were cryopreserved for 30 days and evaluated for sperm viability parameters. Following cryopreservation, acrosome reaction and capacitation of spermatozoa were induced in vitro. The results showed that motility was higher (P < 0.05) in coconut water extenders compared to the control and improvement in this parameter was best at 10% coconut water. More spermatozoa cryopreserved with coconut water underwent induced acrosome reaction and capacitation compared to the control and 10% coconut water had the highest values (P < 0.05). The results showed that motility was better preserved in EYCW5:10 and EYCW10:10 extenders (P < 0.05). While more spermatozoa underwent induced acrosome reaction and capacitation with coconut water and egg yolk extenders at different combinations compared to the control (P < 0.05), the highest values of acrosome integrity and membrane integrity were observed in EYCW5: 10 extenders. The results revealed that 10% coconut water and EYCW5:10 gave better improvement of sperm viability parameters. The findings indicated that coconut water extender improved the quality of cryopreserved spermatozoa of WAD bucks.(AU)
Assuntos
Alimentos de Coco , Proteínas do Ovo/administração & dosagem , Criopreservação , Criopreservação/veterinária , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodosRESUMO
The effects of semen extenders containing coconut (Cocos nucifera) water alone or combined with egg yolk on viability of spermatozoa during cryopreservation of semen obtained from West African Dwarf (WAD) bucks were studied. Coconut water (5, 10, 15 and 20 ml/100 ml) was diluted with pooled semen samples in study I, while in study II, the pooled semen samples were diluted with egg yolk and coconut water (EYCW) at 5:10, 10:10 and 10:5 in Tris-based extenders. The control consisted of Tris-based extenders plus sodium citrate. The diluted semen samples were cryopreserved for 30 days and evaluated for sperm viability parameters. Following cryopreservation, acrosome reaction and capacitation of spermatozoa were induced in vitro. The results showed that motility was higher (P < 0.05) in coconut water extenders compared to the control and improvement in this parameter was best at 10% coconut water. More spermatozoa cryopreserved with coconut water underwent induced acrosome reaction and capacitation compared to the control and 10% coconut water had the highest values (P < 0.05). The results showed that motility was better preserved in EYCW5:10 and EYCW10:10 extenders (P < 0.05). While more spermatozoa underwent induced acrosome reaction and capacitation with coconut water and egg yolk extenders at different combinations compared to the control (P < 0.05), the highest values of acrosome integrity and membrane integrity were observed in EYCW5: 10 extenders. The results revealed that 10% coconut water and EYCW5:10 gave better improvement of sperm viability parameters. The findings indicated that coconut water extender improved the quality of cryopreserved spermatozoa of WAD bucks.
Assuntos
Alimentos de Coco , Criopreservação , Criopreservação/veterinária , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/métodos , Proteínas do Ovo/administração & dosagemRESUMO
Este estudo descreveu as características seminais, da membrana plasmática e do acrossoma de espermatozoide congelado/descongelado de 19 ejaculados de garanhões da raça Nordestina. Os aspectos analisados incluíram os parâmetros físicos do sêmen fresco; a motilidade e a longevidade do sêmen diluído e descongelado; a morfologia espermática, integridade funcional e estrutural da membrana plasmática do espermatozoide e a habilidade de ligação do espermatozoide à membrana perivitelina da gema do ovo de galinha do sêmen descongelado. As variáveis foram avaliadas pela ANOVA com post hoc teste de Student Newman-Keuls (P<0,05). A MT e a MP foram maiores (P<0,05) no sêmen diluído do que no descongelado. A percentagem média de defeitos maiores, menores e totais foi muito inferior ao limite recomendado pelo CBRA. A porcentagem de reativos ao HOST foi de 14,21±1,12% e a porcentagem média de membranas íntegras detectadas pelo teste supravital de 62,22±9,06% e pela sonda SYBR-14 de 81,47±26,90. O número médio de espermatozoides ligados à MPV após a descongelação do sêmen foi de 230,39±57,09. A MT e MP no tempo 0 min do TTR foi superior (P<0,05) em relação a 150 min, não diferindo nos tempos 10 min e 30 min. Os resultados demonstram que a utilização dos testes laboratoriais adicionais ajudam no processo de avaliação das amostras, possibilitando a obtenção de informações mais confiáveis e precisas. Embora a criopreservação tenha provocado queda na motilidade seminal, o uso de diluidor contendo amidas minimizou os danos osmóticos nas células espermáticas e manteve a integridade morfológica, funcional e estrutural da membrana plasmática do espermatozoide. Estes resultados são um referencial em estudos futuros uma vez que, inexistem dados comparativos nesta raça...
This paper describes the seminal characteristics of the plasma membrane of frozen-thawed sperm. Nineteen ejaculates of Nordestino horse breed. The aspects analyzed in the physical parameters of fresh semen were total and progressive motility and your longevity after dilution or thawed; sperm morphology, functional and structural integrity of the plasma membrane of the sperm and the sperm-binding ability to the perivitelline membrane of the yolk (MPV) after thawed. The variables were assessed by ANOVA with post hoc test of Student Newman-Keuls test (P<0.05). The total and progressive motility were higher in diluted semen than thawed (P<0.05). The average percentage of the major, minor and total defects was lower than the limit recommended by the CBRA. The percentage of reactive to hypo-osmotic swelling test was 14.21±1.12%, the intact membrane detected by supravitally test was 62.22±9.06% and the SYBR-14 was 81.47±26.9. The ability of sperm to bind to the MPV after thawing semen was 230.39±57.09. The total and progressive motility at time 0 min of termo resistance test was higher than to 150 minutes (P<0.05), and no difference was observed in the times 10 and 30 minutes. The results demonstrate that the use of additional laboratory tests help in the process of evaluation of samples, making possible to obtain more reliable and accurate information. Although cryopreservation has caused decrease in sperm motility and was used diluents with amides to diluted and cryopreservation protocol and this minimized the osmotic damage to sperm cells and maintained the morphological, functional and structural integrity of the plasma membrane of the sperm. These results are a reference for future studies since there are no comparative data on this breed...
Assuntos
Animais , Masculino , Cavalos/fisiologia , Espermatozoides/fisiologia , Análise do Sêmen/veterinária , Criopreservação/veterinária , Motilidade dos Espermatozoides/fisiologia , Preservação do Sêmen/efeitos adversos , Preservação do Sêmen/veterináriaRESUMO
O objetivo desta revisão foi apresentar e discutir os efeitos da sazonalidade nas características seminais de touros Bos taurus e Bos indicus antes e após o congelação, a fim de possibilitar maior rentabilidade econômica. Apesar da reprodução da espécie bovina não se caracterizar pela sazonalidade, estudos mostram a interferência das estações do ano na qualidade do sêmen de touros. O aumento da temperatura ambiente pode levar a elevação da temperatura testicular, das taxas metabólicas e das exigências de oxigênio, ocasionando alterações na espermatogênese e, por consequência, a um impacto negativo na reprodução. Touros Bos indicus obtêm melhor desempenho na estação mais quente do ano apresentando uma qualidade de sêmen satisfatória em relação a touros Bos taurus. Isso se deve à maior resistência dos zebuínos às altas temperaturas e a peroxidação lipídica causada pelo calor resultando em menor produção de espécies reativas ao oxigênio e menor aumento nos defeitos nas células espermáticas. Desse modo, touros de origem europeia não apresentam um bom desempenho durante alguns períodos mais quentes do ano o que leva a questionar se a relação custo-benefício de aproveitamento do sêmen para criopreservação justifica o processamento do mesmo pelas Centrais de Coleta e Processamento de Sêmen nesta época. Esse fato tem levado algumas empresas a suspender atividades em determinados meses do ano.(AU)
The objective of this review was to present and discuss the effects of seasonality in the semen characteristics of Bos taurus and Bos indicus bulls before and after freezing in order to enable greater economic profitability. Even though the bovine reproduction is not characterized by seasonality, studies have shown the interference of the season on semen quality in bulls. The increase of temperature may lead to increase in testicular temperature, metabolic rates and oxygen requirements. Both thermal and oxidative stress will cause changes in the normal development of spermatogenesis, changing volume, sperm concentration, progressive motility, vigor and morphology of sperm cells, resulting in an economically negative impact on reproduction. Bos indicus bulls presented better performance in the hottest season of the year, showing a satisfactory quality of semen when compared to Bos taurus bulls. This is due to the greater resistance to high temperatures of zebu bulls and lipid peroxidation caused by the heat resulting in lower production of species reactive to oxygen and smaller increases in defects in sperm cells. Thus, European bulls, such as Bos Taurus, do not present a good performance in some of the hottest periods of the year, which leads to questioning if the cost-effective use of semen cryopreservation justifies its processing by the Semen Collection and Processing Centers during these periods. This fact has led some national centers for semen suspend activities in certain months of the year.(AU)
El objetivo de esta revisión ha sido presentar y discutir los efectos de la estacionalidad en las características del semen de toros Bos taurus y Bos indicus pre y post congelación, con el fin de proporcionar mayor rentabilidad económica. A pesar de la reproducción vacuna no caracterizarse por la estacionalidad, estudios muestran la interferencia de las estaciones del año en la calidad del semen de toros El aumento de la temperatura ambiente puede conducir a elevada temperatura testicular, de las tasas metabólicas y de las exigencias de oxígeno, ocasionando alteraciones en la espermatogénesis y, por lo tanto, a un impacto negativo en la reproducción. Toros Bos indicus presentaron mejor rendimiento en la temporada más calurosa del año, presentando calidad de semen satisfactoria en comparación con toros Bos taurus. Esto es debido a mayor resistencia del ganado cebú a las altas temperaturas y la peroxidación de lípidos causada por el calor, que resulta en menor producción de especies reactivas al oxígeno y menos defectos en las células espermáticas. Así, toros de origen europea no presentan buen desempeño durante algunos períodos más calurosos del año, lo que lleva a cuestionar si la relación costo beneficio de aprovechamiento del semen para criopreservación justifica la tramitación del mismo por las Centrales de Recogida y Procesamiento de Semen en este periodo. Este hecho ha llevado a algunas empresas a suspender las actividades en ciertos meses del año.(AU)