The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.(AU)

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Citometria de Fluxo em andrologia: da pesquisa básica para a rotina clínica / Flow citometry in andrology: from basic research to clinical routine

Os primeiros estudos de espermatozoides com citometria de fluxo com espermatozoides iniciaram no final da década de 1970. Com os avanços tecnológicos, hoje contamos com equipamentos com alta sensibilidade e eficiência que, em conjunto com amplo catálogo de sondas fluorescentes, podemos mensurar com alta precisão características celulares. Como exemplo, destacam-se dano em membrana plasmática, atividade mitocondrial, produção de espécies reativas de oxigênio, dano ao DNA espermático e muito mais. Na presente revisão, as potencialidades e limitação para a implementação da citometria de fluxo na análise seminal de espécies domésticas são exploradas e comentadas.

The first flow cytometry studies with spermatozoa were published in the late 1970s. With the technological advances in the following years, today we have equipments with high sensitivity and efficiency that, together with a large number of commercially available fluorescent probes, allow us to measure different cell characteristics with high accuracy. Some of the most evaluated characteristics are plasma membrane damage, mitochondrial activity, production of reactive oxygen species, sperm DNA damage, and much more. In this review, the potentials and limitations for the implementation of flow cytometry in the seminal analysis of domestic species are explored and commented.

Efeito da adição do ácido palmítico e da vitamina E ao diluidor Tris-gema na criopreservação de sêmen canino / Effect of addition of palmitic acid and Vitamin E to the Tris-egg yolk diluter in canine semen cryopreservation

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. 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. 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 100µM 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.(AU)

Chlorophyll a fluorescence and leaf temperature are early indicators of oil palm diseases

Bud rot (BR) caused by Phytophthora palmivora and lethal wilt (LW) whose causal agent is unknown, are two diseases currently posing a threat to the oil palm ( Elaeis guineensis . Jacq) industry. BR, first reported in 1964, has destroyed more than 85,000 ha. LW, first reported in 1994 in the Llanos Orientales in Colombia, has destroyed more than 5,000 ha. Chlorophyll a fluorescence is useful as a provider of information about the efficiency of the photosynthetic process when plants are subjected to biotic or abiotic stresses. Oil palms affected by BR and LW showed anomalies in the photosynthetic system, manifested by reductions in Fv / F M and PSII. Changes in PSII, variable fluorescence yield ( Fv ) and maximum fluorescence in light-adapted leaves ( F M ) were observed from the start of BR infection. The most sensitive and early indicators of LW disease were leaf temperature and basal fluorescence ( F 0 ). Fv/F 0 significantly changed in diseased palms, indicating problems with movement of electrons through the electron transport chain. Leaf temperature changed in response to both diseases, but variation was greater in LW. We concluded that damage to the photochemical system caused by the diseases affected the processes by which the plant captures and transports energy, causing a physiological imbalance in the plant reflected in the observed variations in chlorophyll a fluorescence and leaf temperature. The two parameters began to change early in the onset of BR and before visual symptoms appeared in LW, which is very important to the management of both diseases, the foundation of which is early detection.

Chlorophyll a fluorescence and leaf temperature are early indicators of oil palm diseases

Bud rot (BR) caused by Phytophthora palmivora and lethal wilt (LW) whose causal agent is unknown, are two diseases currently posing a threat to the oil palm ( Elaeis guineensis . Jacq) industry. BR, first reported in 1964, has destroyed more than 85,000 ha. LW, first reported in 1994 in the Llanos Orientales in Colombia, has destroyed more than 5,000 ha. Chlorophyll a fluorescence is useful as a provider of information about the efficiency of the photosynthetic process when plants are subjected to biotic or abiotic stresses. Oil palms affected by BR and LW showed anomalies in the photosynthetic system, manifested by reductions in Fv / F M and PSII. Changes in PSII, variable fluorescence yield ( Fv ) and maximum fluorescence in light-adapted leaves ( F M ) were observed from the start of BR infection. The most sensitive and early indicators of LW disease were leaf temperature and basal fluorescence ( F 0 ). Fv/F 0 significantly changed in diseased palms, indicating problems with movement of electrons through the electron transport chain. Leaf temperature changed in response to both diseases, but variation was greater in LW. We concluded that damage to the photochemical system caused by the diseases affected the processes by which the plant captures and transports energy, causing a physiological imbalance in the plant reflected in the observed variations in chlorophyll a fluorescence and leaf temperature. The two parameters began to change early in the onset of BR and before visual symptoms appeared in LW, which is very important to the management of both diseases, the foundation of which is early detection.(AU)

Insulina e IGF-1 no meio extensor de criopreservação seminal bovina / Insulin and IGF-I in extender medium for bovine semen cryopreservation

Objetivou-se avaliar a condição metabólica e estrutural das células espermáticas bovinas após congelação, com adição prévia de IGF-I e insulina no meio diluidor seminal. Os ejaculados de seis touros Nelore foram submetidos a quatro tratamentos: controle; insulina (100µUI/mL); IGF-I (150ng/mL) e insulina + IGF-I (50µUI/mL e 75ng/mL, respectivamente). Após a congelação, realizaram-se os testes de termorresistência rápida, coloração pelo corante azul de tripan e Giemsa, além da análise computadorizada da motilidade espermática, da integridade das membranas plasmática e acrossomal, e da peça intermediária por meio de sondas fluorescentes. O teste de termorresistência rápida apresentou efeito dentro do tempo de cada tratamento, mas não entre os tratamentos. Na análise computadorizada da motilidade espermática, foram observados movimento, motilidade e velocidade espermáticos; não houve efeitos dos tratamentos sobre qualquer uma dessas variáveis. Respostas iguais foram obtidas com as sondas fluorescentes e o corante azul de tripan/Giemsa. A adição de insulina e IGF-I, de forma isolada ou combinada, ao meio diluidor para congelação de sêmen não produziu efeitos sobre as condições metabólica e estrutural das células espermáticas.(AU)

This study aimed to evaluate the metabolic and structural condition of the spermatic bovine cells after the freezing, with addition, previously, of IGF-I and Insulin in the seminal thinner medium. The semen of 6 Nellore bulls were submitted to four treatments: Control, Insulin (100µUI/mL); IGF-I (150ng/mL) and Insulin + IGF-I (50µUI/mL and 75ng/mL, respectively). After freezing, rapid resistance tests, Tripan and Giemsa Blue staining, and computerized analysis of sperm motility and integrity of the plasma and acrosomal membranes and the intermediate part were performed by fluorescent probes. The term rapid resistance test had effect within the time of each treatment, but not between treatments. In the computer analysis of sperm motility, sperm movement, motility and velocity no effects of treatments were observed on any of these variables. The same results were obtained with the fluorescent probes and the Blue dye Trypan / Giemsa. The addition of Insulin and IGF-I, alone or in combination, to the semen freezing dilution medium had no effect on the metabolic and structural condition of sperm cells.(AU)

Insulina e IGF-1 no meio extensor de criopreservação seminal bovina / Insulin and IGF-I in extender medium for bovine semen cryopreservation

Objetivou-se avaliar a condição metabólica e estrutural das células espermáticas bovinas após congelação, com adição prévia de IGF-I e insulina no meio diluidor seminal. Os ejaculados de seis touros Nelore foram submetidos a quatro tratamentos: controle; insulina (100µUI/mL); IGF-I (150ng/mL) e insulina + IGF-I (50µUI/mL e 75ng/mL, respectivamente). Após a congelação, realizaram-se os testes de termorresistência rápida, coloração pelo corante azul de tripan e Giemsa, além da análise computadorizada da motilidade espermática, da integridade das membranas plasmática e acrossomal, e da peça intermediária por meio de sondas fluorescentes. O teste de termorresistência rápida apresentou efeito dentro do tempo de cada tratamento, mas não entre os tratamentos. Na análise computadorizada da motilidade espermática, foram observados movimento, motilidade e velocidade espermáticos; não houve efeitos dos tratamentos sobre qualquer uma dessas variáveis. Respostas iguais foram obtidas com as sondas fluorescentes e o corante azul de tripan/Giemsa. A adição de insulina e IGF-I, de forma isolada ou combinada, ao meio diluidor para congelação de sêmen não produziu efeitos sobre as condições metabólica e estrutural das células espermáticas.(AU)

This study aimed to evaluate the metabolic and structural condition of the spermatic bovine cells after the freezing, with addition, previously, of IGF-I and Insulin in the seminal thinner medium. The semen of 6 Nellore bulls were submitted to four treatments: Control, Insulin (100µUI/mL); IGF-I (150ng/mL) and Insulin + IGF-I (50µUI/mL and 75ng/mL, respectively). After freezing, rapid resistance tests, Tripan and Giemsa Blue staining, and computerized analysis of sperm motility and integrity of the plasma and acrosomal membranes and the intermediate part were performed by fluorescent probes. The term rapid resistance test had effect within the time of each treatment, but not between treatments. In the computer analysis of sperm motility, sperm movement, motility and velocity no effects of treatments were observed on any of these variables. The same results were obtained with the fluorescent probes and the Blue dye Trypan / Giemsa. The addition of Insulin and IGF-I, alone or in combination, to the semen freezing dilution medium had no effect on the metabolic and structural condition of sperm cells.(AU)

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.

Dimethylacetamide alone or in combination with glycerol can be used for cryopreservation of ovine semen

Dimethylacetamide has been included in different extenders for the cryopreservation of semen from species with promising results. The objective of this study was to evaluate the use of dimethylacetamide (DMA) in different concentrations, associated or not with glycerol (GLY), for the cryopreservation of ovine semen, and its effects on in vitro sperm parameters and post-thaw in vivo fertility. Five semen samples of five adult Santa Ines sheep (n=25) were used. The collected ejaculates were divided among the seven treatments for subsequent cryopreservation. The treatments presented different concentrations of DMA and GLY, being divided as G1: GLY 6%; G2: DMA 3%; G3: GLY 5% + DMA 1%; G4: GLY 4% + DMA 2%; G5: GLY 3% + DMA 3%; G6: GLY 2% + DMA 4%; G7: GLY 1% + DMA 5%. %. Post-thawing of the straws, aliquots were evaluated for computerized sperm kinetics (CASA) and plasma membrane integrity, using fluorescent probes and flow cytometry. After the in vitro evaluation of the sperm parameters, in vivo testing was performed by laparoscopic artificial insemination of 72 females. The post-thaw total motility (%) evaluated by CASA were 51.4, 51.4, 50.1, 53.6, 52.3, 52.8 and 46.9, respectively, for the seven groups. And the plasma membrane integrity (%) were 19.7, 28.4, 22.3, 29.4, 24.3, 17.9 and 16.9, respectively. There were no differences (P> 0.05) between the treatments for the parameters of spermatic kinetics and membrane integrity. For females inseminated with semen from the control group (G1, GLY6%), the percentage of pregnant females was 36.1%, a result similar to that obtained with G3 treatment (GLY5% + DMA1%). In conclusion, dimethylacetamide, either alone or in combination with glycerol, can be used for cryopreservation of ovine semen.(AU)

Diluente Tris suplementado com óleo de Mauritia flexuoxa sobre a qualidade do sêmen caprino após a criopreservação / Tris diluent supplemented with Mauritia flexuoxa oil on the quality of goat semen after cryopreservation

Objetivou-se verificar os efeitos, nos parâmetros espermáticos, na integridade mitocondrial, acrossomal e de membrana em células espermáticas, desencadeados pelo uso do Tris (Tris hidroximetil aminometano) suplementado com óleo de Mauritia flexuoxacomo diluente para criopreservação de sêmen caprino. Quatro caprinos clinicamente saudáveis foram utilizados. Os animais eram alimentados diariamente com volumoso (Pennisetum purpureum, Schum.), concentrado (ração peletizada com teor de 20% proteína, 300 g/animal/dia) e sal mineral específico para Caprinos (Caprinofós®), à vontade. Dois ensaios foram realizados: I ­ Teste de toxicidade; II ­ Criopreservação do sêmen com concentrações ideais. No teste de toxicidade as concentrações avaliadas foram: 5%, 10%, 15% e 20% de diluente a base de óleo de Mauritia flexuoxa. Após o teste de toxicidade, foi escolhido a concentraçãoque apresentou o melhor resultado (5%). Logo após, foram realizadas mais 32 coletas, que foram diluídas em Tris-gema-glicerol (grupo controle) ou diluente contendo óleo vegetal (Mauritia flexuoxa). As amostras foram criopreservadas com auxílio do aparelho Tk3000®. Após o período mínimo de uma semana as palhetas foram descongeladas em banho-maria a 37 °C por 30 segundos, acondicionadas em microtubos de centrifugação e homogeneizadas para a análise imediata de motilidade, vigor espermático e morfologia. Em seguida, por meio de sondas fluorescentes foram avaliadas a integridade de acrossomo, membrana plasmática (Diacetato de Carboxifluresceína e Iodeto de Propídeo) e função mitocondrial sob microscopia de epifluorescência. Quanto a motilidade e vigor, integridade mitocondrial e acrossomal, o grupo buriti foi inferior ao grupo controle. O Tris suplementado com óleo de Mauritia flexuoxa na concentração de 5% não influenciou significativamente a qualidade espermática, porém, observou-se morfologia e integridade de membrana favoráveis. Dessa forma, sendo uma alternativa para substituição de diluentes a base de produtos de origem animal.

The objective was to verify the effects, sperm parameters, mitochondrial, acrosomal and membrane integrity in sperm cells, triggered by the use of Tris (Tris hydroxymethyl aminomethane) supplemented with Mauritia flexuoxa oil as a diluent for cryopreservation of goat semen. Four goats clinically healthy were used. The animals were fed daily with bulky (Pennisetum purpureum, Schum.), concentrate (pelleted feed with 20% protein content, 300 g / animal / day) and mineral salt Specific for Goats (Caprinofós®), ad libitum. Two tests were carried out: I - Toxicity test; II - Semen cryopreservation with ideal concentrations. In the toxicity test as selected were: 5%, 10%, 15% and 20% of Mauritia flexuoxa oil-based diluent. After the toxicity test, the concentration that showed the best result (5%) was chosen. Soon after, a further 32 samples were obtained, which were diluted in Tris-glycerol (control group) or diluent containing vegetable oil (Mauritia flexuoxa). The samples were cryopreserved using the Tk3000® machine. After a minimum of one week, the samples were thawed in a 37 ° C water bath for 30 seconds, packed in centrifugation microtubes and homogenized for immediate analysis of motility, sperm vigor and morphology. Then, by means of fluorescent probes, the integrity of the acrosome, plasma membrane (Carboxyflurescein diacetate and Propidium Iodide) and mitochondrial function under epifluorescence microscopy were evaluated. As for motility and vigor, mitochondrial and acrosomal integrity, the buriti group was inferior to the control group. Tris supplemented with Mauritia flexuoxa oil at a concentration of 5% did not significantly influence sperm quality, however, favorable motility, morphology and membrane integrity were observed. Thus, being an alternative to replace diluents based on products of animal origin.

Eficiência de duas técnicas de recuperação de espermatozoides epididimários de cães e avaliação seminal pós-criopreservação / [Efficiency of two techniques for recovering epididymal spermatozoids from dogs and seminal evaluation post cryopreservation]

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.(AU)

The objective of this work was to evaluate the recovery of epididymal spermatozoa from castrated dogs using retrograde flow (FL) and flotation (FL) techniques in Tris-egg yolk diluent, before and after cryopreservation. Thirty testicle-epididymal complexes (CTE) were collected, 15 for FR and 15 for FL and soon after spermatozoid recovery, morphological changes in these spermatic cells were analyzed. After addition of the diluent, the parameters of total motility (MOT) and vigor (V) were evaluated. The post-cryopreserved semen was submitted to thermoresistance (TTR) test at T0, T30, T60 and T90 minutes, as well as the plasma and acrosomal membrane evaluation by fluorescent probes. There was no statistically significant difference between techniques tested for MOT and vigor in the diluted semen (FR-MOT: 82.3% and V: 3.4, FL-MOT: 79.6% and V: 3.2) and post-cryopreserved (FR-MOT: 34% and V: 2.8, FL-MOT: 30% and V: 2.7). From the T30 there was a significant difference regarding MOT and vigor in the used techniques, and the time also damaged the spermatic acrosome from the T30. It is concluded that the epididymal spermatozoa recovering techniques from castrated dogs, tested in this study, can be used for semen refrigeration and cryopreservation.(AU)

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.(AU)

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.(AU)

Diluente Tris suplementado com óleo de Mauritia flexuoxa sobre a qualidade do sêmen caprino após a criopreservação / Tris diluent supplemented with Mauritia flexuoxa oil on the quality of goat semen after cryopreservation

Objetivou-se verificar os efeitos, nos parâmetros espermáticos, na integridade mitocondrial, acrossomal e de membrana em células espermáticas, desencadeados pelo uso do Tris (Tris hidroximetil aminometano) suplementado com óleo de Mauritia flexuoxa como diluente para criopreservação de sêmen caprino. Quatro caprinos clinicamente saudáveis foram utilizados. Os animais eram alimentados diariamente com volumoso (Pennisetum purpureum, Schum.), concentrado (ração peletizada com teor de 20% proteína, 300 g/animal/dia) e sal mineral específico para Caprinos (Caprinofós®), à vontade. Dois ensaios foram realizados: I Teste de toxicidade; II Criopreservação do sêmen com concentrações ideais. No teste de toxicidade as concentrações avaliadas foram: 5%, 10%, 15% e 20% de diluente a base de óleo de Mauritia flexuoxa. Após o teste de toxicidade, foi escolhido a concentração que apresentou o melhor resultado (5%). Logo após, foram realizadas mais 32 coletas, que foram diluídas em Tris-gema-glicerol (grupo controle) ou diluente contendo óleo vegetal (Mauritia flexuoxa). As amostras foram criopreservadas com auxílio do aparelho Tk3000®. Após o período mínimo de uma semana as palhetas foram descongeladas em banho-maria a 37 °C por 30 segundos, acondicionadas em microtubos de centrifugação e homogeneizadas para a análise imediata de motilidade, vigor espermático e morfologia. Em seguida, por meio de sondas fluorescentes foram avaliadas a integridade de acrossomo, membrana plasmática (Diacetato de Carboxifluresceína e Iodeto de Propídeo) e função mitocondrial sob microscopia de epifluorescência. Quanto a motilidade e vigor, integridade mitocondrial e acrossomal, o grupo buriti foi inferior ao grupo controle. O Tris suplementado com óleo de Mauritia flexuoxa na concentração de 5% não influenciou significativamente a qualidade espermática, porém, observouse morfologia e integridade de membrana favoráveis. Dessa forma, sendo uma alternativa para substituição de diluentes a base de produtos de origem animal.(AU)

The objective was to verify the effects, sperm parameters, mitochondrial, acrosomal and membrane integrity in sperm cells, triggered by the use of Tris (Tris hydroxymethyl aminomethane) supplemented with Mauritia flexuoxa oil as a diluent for cryopreservation of goat semen. Four goats clinically healthy were used. The animals were fed daily with bulky (Pennisetum purpureum, Schum.), concentrate (pelleted feed with 20% protein content, 300 g / animal / day) and mineral salt Specific for Goats (Caprinofós®), ad libitum. Two tests were carried out: I - Toxicity test; II - Semen cryopreservation with ideal concentrations. In the toxicity test as selected were: 5%, 10%, 15% and 20% of Mauritia flexuoxa oil-based diluent. After the toxicity test, the concentration that showed the best result (5%) was chosen. Soon after, a further 32 samples were obtained, which were diluted in Tris-glycerol (control group) or diluent containing vegetable oil (Mauritia flexuoxa). The samples were cryopreserved using the Tk3000® machine. After a minimum of one week, the samples were thawed in a 37 ° C water bath for 30 seconds, packed in centrifugation microtubes and homogenized for immediate analysis of motility, sperm vigor and morphology. Then, by means of fluorescent probes, the integrity of the acrosome, plasma membrane (Carboxyflurescein diacetate and Propidium Iodide) and mitochondrial function under epifluorescence microscopy were evaluated. As for motility and vigor, mitochondrial and acrosomal integrity, the buriti group was inferior to the control group. Tris supplemented with Mauritia flexuoxa oil at a concentration of 5% did not significantly influence sperm quality, however, favorable motility, morphology and membrane integrity were observed. Thus, being an alternative to replace diluents based on products of animal origin.(AU)

Diluente Tris suplementado com óleo de Mauritia flexuoxa sobre a qualidade do sêmen caprino após a criopreservação / Tris diluent supplemented with Mauritia flexuoxa oil on the quality of goat semen after cryopreservation

Objetivou-se verificar os efeitos, nos parâmetros espermáticos, na integridade mitocondrial, acrossomal e de membrana em células espermáticas, desencadeados pelo uso do Tris (Tris hidroximetil aminometano) suplementado com óleo de Mauritia flexuoxa como diluente para criopreservação de sêmen caprino. Quatro caprinos clinicamente saudáveis foram utilizados. Os animais eram alimentados diariamente com volumoso (Pennisetum purpureum, Schum.), concentrado (ração peletizada com teor de 20% proteína, 300 g/animal/dia) e sal mineral específico para Caprinos (Caprinofós®), à vontade. Dois ensaios foram realizados: I – Teste de toxicidade; II – Criopreservação do sêmen com concentrações ideais. No teste de toxicidade as concentrações avaliadas foram: 5%, 10%, 15% e 20% de diluente a base de óleo de Mauritia flexuoxa. Após o teste de toxicidade, foi escolhido a concentração que apresentou o melhor resultado (5%). Logo após, foram realizadas mais 32 coletas, que foram diluídas em Tris-gema-glicerol (grupo controle) ou diluente contendo óleo vegetal (Mauritia flexuoxa). As amostras foram criopreservadas com auxílio do aparelho Tk3000®. Após o período mínimo de uma semana as palhetas foram descongeladas em banho-maria a 37 °C por 30 segundos, acondicionadas em microtubos de centrifugação e homogeneizadas para a análise imediata de motilidade, vigor espermático e morfologia. Em seguida, por meio de sondas fluorescentes foram avaliadas a integridade de acrossomo, membrana plasmática (Diacetato de Carboxifluresceína e Iodeto de Propídeo) e função mitocondrial sob microscopia de epifluorescência. Quanto a motilidade e vigor, integridade mitocondrial e acrossomal, o grupo buriti foi inferior ao grupo controle. O Tris suplementado com óleo de Mauritia flexuoxa na concentração de 5% não influenciou significativamente a qualidade espermática, porém, observouse morfologia e integridade de membrana favoráveis. Dessa forma, sendo uma alternativa para substituição de diluentes a base de produtos de origem animal.

The objective was to verify the effects, sperm parameters, mitochondrial, acrosomal and membrane integrity in sperm cells, triggered by the use of Tris (Tris hydroxymethyl aminomethane) supplemented with Mauritia flexuoxa oil as a diluent for cryopreservation of goat semen. Four goats clinically healthy were used. The animals were fed daily with bulky (Pennisetum purpureum, Schum.), concentrate (pelleted feed with 20% protein content, 300 g / animal / day) and mineral salt Specific for Goats (Caprinofós®), ad libitum. Two tests were carried out: I - Toxicity test; II - Semen cryopreservation with ideal concentrations. In the toxicity test as selected were: 5%, 10%, 15% and 20% of Mauritia flexuoxa oil-based diluent. After the toxicity test, the concentration that showed the best result (5%) was chosen. Soon after, a further 32 samples were obtained, which were diluted in Tris-glycerol (control group) or diluent containing vegetable oil (Mauritia flexuoxa). The samples were cryopreserved using the Tk3000® machine. After a minimum of one week, the samples were thawed in a 37 ° C water bath for 30 seconds, packed in centrifugation microtubes and homogenized for immediate analysis of motility, sperm vigor and morphology. Then, by means of fluorescent probes, the integrity of the acrosome, plasma membrane (Carboxyflurescein diacetate and Propidium Iodide) and mitochondrial function under epifluorescence microscopy were evaluated. As for motility and vigor, mitochondrial and acrosomal integrity, the buriti group was inferior to the control group. Tris supplemented with Mauritia flexuoxa oil at a concentration of 5% did not significantly influence sperm quality, however, favorable motility, morphology and membrane integrity were observed. Thus, being an alternative to replace diluents based on products of animal origin.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106 ), G2 (2*106 ) and G3 (4*106 ) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chisquare (P0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0,05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106 ), G2 (2*106 ) e G3 (4*106 ) espermatozoides/mL. Às 18 h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o ComputerAssisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Correlation between insemination dose and bovine sperm traits on the in vitro embryo production outcomes / Correlação da dose inseminante e características do sêmen bovino no sucesso da produção in vitro de embriões

In the present study we aimed to test the best insemination dose for in vitro embryo production (IVEP) and to correlate sperm traits in bovine. In vitro matured oocytes were inseminated with three different sperm concentrations of the same bull: G1 (1*106), G2 (2*106) and G3 (4*106) sperm/mL. At 18 h post-insemination (hpi), presumptive zygotes [G1 (n=114), G2 (n=139) and G3 (n=136)] were stained to evaluate the pronuclei numbers, or continued to in vitro culture [G1 (n=102), G2 (n=111) and G3 (n=106)]. Sperm kinetics were analyzed using Computer-Assisted Semen Analysis (CASA). Sperm plasma membrane, acrosome integrity and mitochondrial activity were analyzed using fluorescent probes. In vitro fertilization (IVF) and IVEP data were compared using chi-square (P<0.05) and correlated with CASA and fluorescence data using Person Correlation (P<0.05). The IVF efficiency, cleavage and total blastocyst rates did not show any significant difference (P>0.05) among the groups. In G3, the polyspermy rate was the highest (7.4%; P<0.05) without difference (P>0.05) between G1 (0%) and G2 (0%). In G1, the early blastocyst rate was the highest (7.8%; P<0.05), without significant difference (P>0.05) between G2 (1.8%) and G3 (0.9%). The IVF efficiency and total blastocyst rates were positively correlated with curvilinear velocity (VCL) (r≃+1; P<0.05). We concluded that the reduction of insemination dose may negatively affect embryo development and VCL may be used as a parameter to improve the IVEP outcomes.

O objetivo deste estudo foi testar a melhor dose inseminante para a produção de embriões in vitro (IVEP) e sua correlação com as características espermáticas na espécie bovina. Oócitos maturados in vitro foram inseminados com três concentrações diferentes de espermatozoides de único touro: G1 (1*106), G2 (2*106) e G3 (4*106) espermatozoides/mL. Às 18h pós-inseminação (hpi), os presumíveis zigotos [G1 (114), G2 (139) e G3 (136)] foram corados para avaliar o número de pronúcleos, ou continuaram para o cultivo in vitro [G1 (102), G2 (111) e G3 (106)]. Os parâmetros da cinética espermática foram analisados usando o Computer-Assisted Semen Analysis (CASA). A integridade de membrana plasmática espermática, acrossomal e a atividade mitocondrial foram analisadas usando sondas fluorescentes. Os dados da fertilização in vitro (FIV) e IVEP foram comparadas com qui-quadrado (P=0,05) e correlacionados com dados de CASA e Fluorescência usando Correlação de Pearson (r=±1; P<0,05). A eficiência da FIV, taxas de clivagem e blastocisto total não mostraram diferença significativa (P>0,05) entre os grupos. Em G3, a taxa de polispermia foi a maior (7,4%; P<0,05), sem diferença (P>0,05) entre G1 (0%) e G2 (0%). Em G1, a taxa de blastocisto inicial foi a maior (7,8%; P<0,05), sem apresentar diferença significativa (P>0,05) com G2 (1,8%) e G3 (0,9%). A eficiência de FIV e a taxa de blastocisto total foram positivamente correlacionadas com velocidade curvilinear (VCL) (P<0,05). Concluímos que a dose inseminante reduzida pode negativamente afetar o desenvolvimento embrionário e VCL pode ser usada como parâmetro para melhorar os resultados da PEIV.

Validation of the CellRox Deep Red ® fluorescent probe to oxidative stress assessment in equine spermatozoa

Considering the importance of ROS influence on sperm functionality and some limitations in sperm oxidative stress assessment methods, a field to studies of new techniques are still open. In this sense, the aim of this study is to validate the ROS detection technique through the CellRox Deep Red Reagent®probe in stallion sperm. Four stallions were used and the analyses were conducted on four replicates of semen samples from each of stallion (n = 16). The results of the polynomial regression presented a quadratic effect, high determination coefficient value (R2 = 0.88) and high significant P value (P < 0.0001). The CellRox Deep Red® fluorescent probe is able to detect reactive oxygen species in equine sperm, indicating accurately the occurrence of oxidative stress in stallion semen.

Validation of the CellRox Deep Red ® fluorescent probe to oxidative stress assessment in equine spermatozoa

Considering the importance of ROS influence on sperm functionality and some limitations in sperm oxidative stress assessment methods, a field to studies of new techniques are still open. In this sense, the aim of this study is to validate the ROS detection technique through the CellRox Deep Red Reagent®probe in stallion sperm. Four stallions were used and the analyses were conducted on four replicates of semen samples from each of stallion (n = 16). The results of the polynomial regression presented a quadratic effect, high determination coefficient value (R2 = 0.88) and high significant P value (P < 0.0001). The CellRox Deep Red® fluorescent probe is able to detect reactive oxygen species in equine sperm, indicating accurately the occurrence of oxidative stress in stallion semen.(AU)

Fluorescent test accurately predicts leak of ischemic colon anastomosis in rats

Purpose: To investigate if fluorescein fluorescent test can predict dehiscence in a model of ischemic colonic anastomosis in rats. Methods: This experimental controlled trial randomly assigned 55 rats to four groups. Anastomoses were performed in non-ischemic colon segments (control group) and in ischemic colon segments measuring 1, 2 or 3 cm long (groups 1, 2 and 3, respectively). Fluorescein was injected and the tissues were examined under ultraviolet light. Seven days later, a second-look surgery was performed to check for the presence or absence of anastomosis dehiscence. Results: Twenty-four rats presented anastomotic dehiscence during the second-look surgery. Reticular and nonfluorescent patterns were significantly associated with the occurrence of anastomotic dehiscence. Fluorescein fluorescence had a sensitivity of 95.8%, specificity of 89.2%, positive predictive value of 88.4%, negative predictive value of 96.2%, and accuracy of 92.3% to predict anastomotic dehiscence. Conclusion: Fluorescein fluorescent test can accurately predict leak in a model of ischemic colonic anastomosis in rats.(AU)