Duration-dependent effects of high dose of phthalate exposure on semen quality in adult male rats.

OBJECTIVE: To determine the length of exposure to high doses of phthalate that might affect sperm quality in adult male Wistar rats. METHODS: Forty-two (42) adult male Wistar rats (weighing 150-200 g) were randomly assigned into six groups (n=7): Group A received 0.5 mL of distilled water - placebo - and served as controls; groups B, C, D, E and F received Phthalate (750 mg/kgbw) for 1, 3, 5, 7 and 9 weeks, respectively. The data obtained from the study was expressed as Mean ± SEM with a p-value <0.05 considered significant. The data was analyzed with one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test using GraphPad Prism, version 8. RESULTS: The results showed a statistically significant (p<0.05) decrease in testicular weight in the rats exposed to 750 mg/kg of phthalate for 3, 5, 7 and 9 weeks when compared with the controls. Sperm count, motility and viability were also significantly (p<0.05) reduced, while sperm cells with abnormal morphology had increased counts in the groups exposed for 3, 5, 7 and 9 weeks when compared with controls. Serum zinc and magnesium were also significantly reduced (p<0.05) in the subjects treated for 1, 3, 5, 7 and 9 weeks when compared with controls. CONCLUSIONS: The dosage of phthalate adopted in this study was deleterious to testicular function when rats were exposed to it for as short a period as three weeks.

Bull sperm selection by attachment to hyaluronic acid semi-interpenetrated hydrogels.

We report the development of a hydrogel-based approach to select bull spermatozoa, a crucial step for successful assisted reproductive techniques (ARTs). Hyaluronic acid (HA) semi-interpenetrated N-isopropylacrylamide (PNIPAM) co-20% N-Tris (hydroxymethyl) methyl acrylamide (HMA) hydrogels were synthetized on glass surfaces and cultured in presence of frozen-thawed bull spermatozoa. A fraction of motile bull spermatozoa population strongly attached to hydrogels and was partially released by treatment with hyaluronidase. Fifty-nine (59 ± 7.24) per cent of sperm cells attached to PNIPAM-HMA-HA hydrogels and 31.16 ± 4.81% of them were released upon treatment with medium containing hyaluronidase. This attached-released sperm fraction has acceptable characteristics of progressive motility (50.0 ± 5.0%), vigour (4), high viability (58.7 ± 11.7%) and low percentage of acrosome reacted spermatozoa (23.36 ± 4.1%). Our findings indicate that PNIPAM-HMA-HA hydrogels are non-toxic and allow the selection of high-quality sperm cells for ART.

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability.

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-ß-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.

Cinética e integridade de espermatozoides caprinos criopreservados utilizando Red Cushion® como estratégia de proteção espermática durante a centrifugação do sêmen / Kinetics and integrity of cryopreserved goat sperm using Red Cushion® as a strategy to sperm protection during semen centrifugation

Objetivou-se avaliar o efeito do gradiente de Iodixanol (Red Cushion®, RC) como método deproteção espermática durante a centrifugação (CE) do sêmen caprino destinado à criopreservação. Foramrealizadas colheitas de sêmen de cinco reprodutores caprinos da raça Anglo Nubiana (N= cincoejaculados/bode). Os ejaculados foram divididos em três grupos experimentais: GC (Controle); G1 (CEutilizado 1ml de RC) e G2 (foi utilizado 0,5mL de RC, misturado ao pellet espermático após a CE). Asamostras foram centrifugadas, diluídas, envasadas e criopreservadas. A cinética espermática foi avaliada porsistema computadorizado de análise (CASA) após a descongelação e pós-teste de termorresistência (TTR), efoi avaliada a integridade de membrana plasmática e acrossomal (MPAI, %). Não foram observadasdiferenças para a motilidade total (p=0,1849) avaliada pós-descongelação (GC=45,1%, G1=51,86% eG2=55,28%) ou pós-TTR (GC=10,32%, G1=11,44% e G2=13,16%), bem como para nenhum outroparâmetro cinético avaliado. Também não foram encontradas diferenças para a MPAI (GC=43,70% ±16,60; G1=36,68 ± 17,40; G2=41,72 ± 14,76; p=0,2523). Conclui-se que a utilização de Red Cushion®não promove nenhum benefício à manutenção da cinética e integridade de espermatozoides caprinossubmetidos a centrifugação previamente ao processo de criopreservação do sêmen.

The aim of this study was to evaluate the efficacy of iodixanol gradient centrifugation (RedCushion®, RC) as a method of protection of goat semen submitted to centrifugation previously tocryopreservation process. Semen were collected from five Anglo Nubian goats (n = five ejaculates/goat).The ejaculates were divided into three experimental groups: GC (Control; conventional centrifugation);G1 (centrifugation using 1ml RC) and G2 (centrifugation using 0.5 mL RC, mixed to sperm pellet beforethe cryopreservation). Samples were centrifuged (CE) at 600xg for 10 minutes, diluted, packed andcryopreserved. Sperm kinetics were evaluated using a computer assisted semen analysis (CASA) afterthawing and after thermoresistance test (TTR), and the integrity of the plasma and acrosomal membranewas evaluated (MPAI, %). No differences were observed for total motility (P =0.1849) assessed afterthawing (GC=45.1%, G1=51.86% and G2=55.28%) or post-TTR (GC=10.32%, G1=11.44% andG2=13.16%), as well as for the other kinetic parameters analyzed. Furthermore, no differences for MPAI(CG =43.70% ± 16.60; G1=36.68 ± 17.40; G2=41.72 ± 14.76; P=0.2523) were found comparing thedifferent groups. In conclusion, Red Cushion® was not able to improve the kinetics and integrity of thegoat semen submitted to the cryopreservation process.

Cinética e integridade de espermatozoides caprinos criopreservados utilizando Red Cushion® como estratégia de proteção espermática durante a centrifugação do sêmen / Kinetics and integrity of cryopreserved goat sperm using Red Cushion® as a strategy to sperm protection during semen centrifugation

Objetivou-se avaliar o efeito do gradiente de Iodixanol (Red Cushion®, RC) como método deproteção espermática durante a centrifugação (CE) do sêmen caprino destinado à criopreservação. Foramrealizadas colheitas de sêmen de cinco reprodutores caprinos da raça Anglo Nubiana (N= cincoejaculados/bode). Os ejaculados foram divididos em três grupos experimentais: GC (Controle); G1 (CEutilizado 1ml de RC) e G2 (foi utilizado 0,5mL de RC, misturado ao pellet espermático após a CE). Asamostras foram centrifugadas, diluídas, envasadas e criopreservadas. A cinética espermática foi avaliada porsistema computadorizado de análise (CASA) após a descongelação e pós-teste de termorresistência (TTR), efoi avaliada a integridade de membrana plasmática e acrossomal (MPAI, %). Não foram observadasdiferenças para a motilidade total (p=0,1849) avaliada pós-descongelação (GC=45,1%, G1=51,86% eG2=55,28%) ou pós-TTR (GC=10,32%, G1=11,44% e G2=13,16%), bem como para nenhum outroparâmetro cinético avaliado. Também não foram encontradas diferenças para a MPAI (GC=43,70% ±16,60; G1=36,68 ± 17,40; G2=41,72 ± 14,76; p=0,2523). Conclui-se que a utilização de Red Cushion®não promove nenhum benefício à manutenção da cinética e integridade de espermatozoides caprinossubmetidos a centrifugação previamente ao processo de criopreservação do sêmen.(AU)

The aim of this study was to evaluate the efficacy of iodixanol gradient centrifugation (RedCushion®, RC) as a method of protection of goat semen submitted to centrifugation previously tocryopreservation process. Semen were collected from five Anglo Nubian goats (n = five ejaculates/goat).The ejaculates were divided into three experimental groups: GC (Control; conventional centrifugation);G1 (centrifugation using 1ml RC) and G2 (centrifugation using 0.5 mL RC, mixed to sperm pellet beforethe cryopreservation). Samples were centrifuged (CE) at 600xg for 10 minutes, diluted, packed andcryopreserved. Sperm kinetics were evaluated using a computer assisted semen analysis (CASA) afterthawing and after thermoresistance test (TTR), and the integrity of the plasma and acrosomal membranewas evaluated (MPAI, %). No differences were observed for total motility (P =0.1849) assessed afterthawing (GC=45.1%, G1=51.86% and G2=55.28%) or post-TTR (GC=10.32%, G1=11.44% andG2=13.16%), as well as for the other kinetic parameters analyzed. Furthermore, no differences for MPAI(CG =43.70% ± 16.60; G1=36.68 ± 17.40; G2=41.72 ± 14.76; P=0.2523) were found comparing thedifferent groups. In conclusion, Red Cushion® was not able to improve the kinetics and integrity of thegoat semen submitted to the cryopreservation process.(AU)

Influência de diferentes sistemas e curvas de congelamento na congelabilidade e fertilidade do sêmen equino / Influence of systems and freezing curve in equine semen freezability and fertility

Avaliou-se o efeito de curvas de congelação nos parâmetros espermáticos e na fertilidade, usando sêmen de alta e baixa congelabilidade. Experimento 1 - utilizou-se sêmen de quatro garanhões resistentes à congelação: grupo 1, palhetas refrigeradas até 5°C e congeladas com curva de -8°C/min; grupos 2 e 3, palhetas refrigeradas até 5°C (0,5°C/min.) e congeladas com curvas de -20°C/min e -10°C/min, respectivamente. Experimentos 2 e 3 - utilizaram-se cinco garanhões (Mangalarga Marchador), respectivamente, de alta e baixa congelabilidade: grupo 4, a mesma metodologia descrita no grupo 1; grupos 5 e 6, palhetas refrigeradas até 5°C (0,5°C/min) e congeladas com curva de -20°C/min, entre 5°C e -60°C, e -10°C/min, entre -60°C e -100ºC (grupo 5), e -25°C/min, de 5°C até -100°C (grupo 6). O sêmen foi avaliado após descongelamento pelo método computadorizado. No experimento 1, não houve diferença nos parâmetros avaliados. No experimento 2, os parâmetros motilidade total (MT) e motilidade progressiva foram superiores aos do grupo 6 em relação ao grupo 4. No experimento 3, a MT foi superior no grupo 6 em relação ao grupo 4. As curvas de congelação mais rápidas apresentaram melhores parâmetros de cinética espermática, após a descongelação, para o sêmen de garanhões da raça Mangalarga Marchador.(AU)

The effect of freezing curves on sperm parameters and fertility, using resistant and sensitive semen to cryopreservation, was evaluated. In experiment 1, Semen from 4 stallions resistant to freezing was used: Group 1, straws were cooled to 5°C and frozen with a curve of - 8°C/min; Groups 2 and 3, straws were cooled to 5°C (0.5°C/min) and frozen with curves of - 20°C / min and - 10°C/min, respectively. In experiments 2 and 3, 5 stallions (Mangalarga Marchador) presenting respectively resistant and sensitive sperm to cryopreservation were used: Group 4, same methodology described for Group 1 was performed; Groups 5 and 6, straws were cooled to 5°C (0.5°C/min) and frozen with a curve of - 20°C/min. between 5°C and - 60°C and -10°C/min. between - 60°C and - 100°C (Group 5) and - 25°C/min. 5°C to - 100°C (Group 6). Thawed-semen was evaluated by the computerized method CASA. In Experiment 1, there was no difference in the evaluated parameters. In Experiment 2, total motility (MT) and progressive motility (PM) were higher in Group 6 compared to Group 4. In Experiment 3, TM was higher in Group 6 than Group 4. The faster freezing curves showed better parameters of sperm kinetics after thawing, for the Mangalarga Marchador stallion semen.(AU)

Influência de diferentes sistemas e curvas de congelamento na congelabilidade e fertilidade do sêmen equino / Influence of systems and freezing curve in equine semen freezability and fertility

Avaliou-se o efeito de curvas de congelação nos parâmetros espermáticos e na fertilidade, usando sêmen de alta e baixa congelabilidade. Experimento 1 - utilizou-se sêmen de quatro garanhões resistentes à congelação: grupo 1, palhetas refrigeradas até 5°C e congeladas com curva de -8°C/min; grupos 2 e 3, palhetas refrigeradas até 5°C (0,5°C/min.) e congeladas com curvas de -20°C/min e -10°C/min, respectivamente. Experimentos 2 e 3 - utilizaram-se cinco garanhões (Mangalarga Marchador), respectivamente, de alta e baixa congelabilidade: grupo 4, a mesma metodologia descrita no grupo 1; grupos 5 e 6, palhetas refrigeradas até 5°C (0,5°C/min) e congeladas com curva de -20°C/min, entre 5°C e -60°C, e -10°C/min, entre -60°C e -100ºC (grupo 5), e -25°C/min, de 5°C até -100°C (grupo 6). O sêmen foi avaliado após descongelamento pelo método computadorizado. No experimento 1, não houve diferença nos parâmetros avaliados. No experimento 2, os parâmetros motilidade total (MT) e motilidade progressiva foram superiores aos do grupo 6 em relação ao grupo 4. No experimento 3, a MT foi superior no grupo 6 em relação ao grupo 4. As curvas de congelação mais rápidas apresentaram melhores parâmetros de cinética espermática, após a descongelação, para o sêmen de garanhões da raça Mangalarga Marchador.(AU)

The effect of freezing curves on sperm parameters and fertility, using resistant and sensitive semen to cryopreservation, was evaluated. In experiment 1, Semen from 4 stallions resistant to freezing was used: Group 1, straws were cooled to 5°C and frozen with a curve of - 8°C/min; Groups 2 and 3, straws were cooled to 5°C (0.5°C/min) and frozen with curves of - 20°C / min and - 10°C/min, respectively. In experiments 2 and 3, 5 stallions (Mangalarga Marchador) presenting respectively resistant and sensitive sperm to cryopreservation were used: Group 4, same methodology described for Group 1 was performed; Groups 5 and 6, straws were cooled to 5°C (0.5°C/min) and frozen with a curve of - 20°C/min. between 5°C and - 60°C and -10°C/min. between - 60°C and - 100°C (Group 5) and - 25°C/min. 5°C to - 100°C (Group 6). Thawed-semen was evaluated by the computerized method CASA. In Experiment 1, there was no difference in the evaluated parameters. In Experiment 2, total motility (MT) and progressive motility (PM) were higher in Group 6 compared to Group 4. In Experiment 3, TM was higher in Group 6 than Group 4. The faster freezing curves showed better parameters of sperm kinetics after thawing, for the Mangalarga Marchador stallion semen.(AU)

Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications.

SummaryThe aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 µs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Cryopreservation of boar semen in 0. 5 mL straws at low spermatozoa concentration is better than high concentration to maintain sperm viability / A manutenção da viabilidade do sêmen suíno criopreservado em palhetas de 0, 5 mL em baixas concentrações espermáticas é melhor do que em altas concentrações

To date, no studies have been performed evaluating the effect of boar spermatozoa concentration in 0.5mL freezing straws, leading us to examine this question. Each sperm-rich fraction of the ejaculate (n=25) was diluted at five different sperm concentrations (100, 200, 300, 600 and 800 x 106 spermatozoa/mL), packaged in 0.5mL straws, and subsequently frozen. After thawing, the sperm from all of treatment groups were analyzed to determine motility characteristics using a sperm class analyzer (SCA-CASA), and their plasma and acrosomal membrane integrity, mitochondrial membrane potential, sperm membrane lipid peroxidation and fluidity were analyzed by flow cytometry. An increase in spermatozoa concentration above 300x106 spermatozoa/mL in a 0.5mL straw impaired (p<0.05) the total and progressive motility, curvilinear velocity, straight-line velocity, linearity and beat cross frequency. However, the plasma and acrosomal membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation and fluidity were not influenced (p>0.05) by high spermatozoa concentrations at freezing. Therefore, to increase spermatozoa survival and total and progressive motility after thawing, boar spermatozoa should be frozen at concentrations up to 300x106 spermatozoa/mL.(AU)

Até o momento, não foram realizados estudos que avaliassem o efeito da concentração de espermatozoides/mL em palhetas (0,5mL) para a criopreservação, levando-nos a analisar esta questão. Cada fração-rica do ejaculado (n=25) foi diluída em cinco diferentes concentrações de espermatozoides (100, 200, 300, 600 e 800x106 espermatozoides/mL), envasadas em palhetas de 0,5mL e posteriormente congeladas. Após a descongelação, os espermatozoides de todos os tratamentos foram avaliados a fim de determinar as características de motilidade usando um sistema de análise computadorizada dos espermatozoides (SCA-CASA). A integridade das membranas plasmática e acrosomal, o potencial de membrana mitocondrial, a peroxidação lipídica e a fluidez da membrana foram analisadas por citometria de fluxo. O aumento na concentração de espermatozoides acima de 300x106 espermatozoides/mL diminuiu (p<0,05) a motilidade total e progressiva, velocidade curvilínea, velocidade linear, linearidade e frequência de batimento. No entanto, a integridade da membrana plasmática e acrosomal, potencial de membrana mitocondrial, peroxidação lipídica e fluidez de membrana não foram influenciados (p>0,05) por altas concentrações de espermatozoides durante a criopreservação. Portanto, a fim de melhorar a sobrevivência dos espermatozoides suínos e a motilidade total e progressiva após a descongelação, os espermatozoides suínos devem ser congelados a concentrações não superiores a 300x106 espermatozoides/mL.(AU)

Evaluation of cooling and freezing systems of bovine semen.

Semen cryopreservation comprises different steps, among them are the cooling and freezing rates which significantly influence the quality of thawed sperm. Different systems with variable freezing rates are used for freezing bull semen in the field, with a consequence of variable success rates. The objective of this study was to compare different systems for freezing bull semen in the field. Five cooling methods of semen and two methods for the subsequent freezing phase (5 × 2 factorial scheme) were used. Two to four ejaculates were collected from 12 bulls with an electroejaculator. The ejaculates were diluted in BotuBov® to a concentration of 50 × 106 spermatozoa/mL in 0.5-mL straws. After dilution, the straws were cooled to 5 °C in five cooling systems: TK 4000® at a cooling rate of -0.25 °C/min (R1); TK 4000® at a rate of -0.5 °C/min (R2); Minitube® refrigerator at a rate of -2.8 °C/min (R3); Botutainer® at a rate of -0.65 °C (R4), and domestic refrigerator at a rate of -2.0 °C/min (R5). After stabilization at 5 °C for 4 h, these straws were then submitted to two freezing systems: TK 4000® at a freezing rate of -15 °C/min (C1) and Styrofoam box with liquid nitrogen at a rate of -19 °C/min (C2). Sperm kinetics were evaluated by computer-assisted sperm analysis at four time points: in fresh semen, after cooling, post-thawing, and after the rapid thermal resistance test (TRT). In addition, plasma and acrosomal membrane integrity, mitochondrial potential and intracellular H2O2 were analyzed after thawing by flow cytometry. The R1, R2 and R4 cooling systems were the most efficient in preserving sperm viability, membrane integrity and intracellular H2O2. Samples frozen in the C1 system exhibited better post-thaw and post-TRT kinetics than C2 samples. In conclusion, slower cooling curves in conjunction with a constant freezing rate obtained with the programmable unit were more efficient for freezing bull semen in the field.

Fertility in male rats: Disentangling adverse effects of arsenic compounds.

Arsenic impairs male reproductive functions. However, it is not clear whether different arsenic compounds similarly affect fertility. In this study, we compared the impact of sodium arsenite and arsenate on sperm quality and fertility. After 56 d exposure, male Wistar rats were mated and pregnant females were evaluated by fertility indexes. Clearly, exposure to 10 mg/L arsenite reduced daily sperm production via H2O2 overproduction and germ cells loss. Animals from this group also showed a decrease in epididymal sperm counts and percentage of sperm with intact membranes. Moreover, they presented low fertility potential and high preimplantation loss. In contrast, 10 mg/L arsenate caused oxidative stress in testis, mineral imbalance in epididymis, and sperm membranes damage, with no effects on fertility. Both arsenic compounds at 0.01 mg/L altered reproductive parameters. We concluded that arsenite is more harmful than arsenate to sperm quality and male fertility, with negative influences in early pregnancy.

Could cryopreserved human semen samples be stored at -80°C?

OBJECTIVE: To evaluate storage time effects in cryopreserved human semen samples, kept in the freezer at a controlled temperature of -80°C, on sperm viability after thawing. METHODS: We used 20 semen samples. Each sample was cryopreserved in 10 fingers, which were divided into five groups: one group was kept in cryogenic canisters throughout the experiment(control), and four groups were kept in a VIP Ultra Low MDF-U76V- PE freezer, with the temperature set at -80°C, for 24, 48, 72 and 96 hours, respectively. After the exposure time, the samples were stored in cryogenic canisters after being thawed. The analyzed parameters were: motility, vitality and mitochondrial activity. RESULTS: After thawing, we noticed decreased sperm motility, vitality and mitochondrial activity, when comparing the tested groups with the control group, as well as a progressive reduction in the analyzed parameters between the times evaluated. CONCLUSIONS: Cryopreservation of semen samples at -80°C is potentially harmful to sperm viability, causing damage when submitted to longer exposure times.

Effect of different mini-volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer-assisted semen analyzer.

Straws of sex-sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106  sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex-sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane-coated silica colloid dilutions and layering configurations during centrifugation of sex-sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex-sorted sperm samples were centrifuged using mini-volume single-layer (40%, 60% and 80%) and mini-volume two-layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm® . A single layer of 40% PureSperm® recovered significantly more sex-sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two-layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single-layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini-volume single layer of 80% PureSperm® was determined to be an effective alternative to a two-layer centrifugation configuration for sex-sorted sperm selection. In addition, single-layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.

Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability.

The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains limited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evaluated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motility, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation.

QUALIDADE ESPERMÁTICA DURANTE A CURVA DE RESFRIAMENTO DO SÊMEN SUÍNO DILUÍDO EM ÁGUA DE COCO EM PÓ VISANDO SUA CRIOPRESERVAÇÃO

Abstract Semen cryopreservation is associated with low productivity results. This study aimed to test the Coconut Water Powder (ACP®-103) as a ressuspension diluent after thawing semen, also evaluate sperm quality during the cooling curve until thawing of the semen. For this, the semen was collected from 15 boars once a week, incubated at 30 ºC for 15 minutes, and afterwards, the samples were diluted in the diluent Beltsville Thawing Solution - BTS (Control) or ACP-103®, and subjected to a slow cooling curve, where the force and the motility were analyzed in each step. The thawed semen was ressuspended in its respective solvents and analyzed in the characteristics of vigor, motility, vitality, acrosome integrity and functionality of the membrane. During the analysis of vigor and motility that make up the cooling curve, and thawing, for analysis of vitality and intact acrosomal membrane, it was observed that there was no significant difference between treatments. Besides, after thawing, the BTS showed better results of sperm vigor, sperm motility and membrane functionality. However, the cooling curve and coconut water powder can be used in the protocol for cryopreservation of boar semen, as both ensured quality of sperm viability that may be used in artificial insemination.

Resumo A criopreservação seminal apresenta baixos resultados produtivos. Objetivou-se testar a Água de Coco em Pó (ACP-103®) como diluente de ressuspensão após a descongelação seminal e avaliar a qualidade espermática durante a curva de resfriamento até a descongelação do sêmen. Para isso, o sêmen de 15 reprodutores foi coletado uma vez por semana, incubado a 30 ºC por 15 minutos, e em seguida diluído em Beltsville Thawing Solution - BTS (controle) ou em ACP-103®, e submetidos a uma curva de resfriamento lenta, onde foram feitas análises de vigor e motilidade em cada passo. O sêmen descongelado foi ressuspenso em seus respectivos diluentes e analisado quanto às características: vigor, motilidade, vitalidade, integridade acrossomal e funcionalidade da membrana. Durante as análises de vigor e motilidade que compõem a curva de resfriamento, e na descongelação, para as análises de vitalidade e membrana acrossomal intacta, observou-se que não houve diferença significativa entre os tratamentos. Já após a descongelação, o BTS apresentou melhores resultados de vigor, motilidade espermática e funcionalidade da membrana. No entanto, a curva de resfriamento e o ACP-103® podem ser utilizadas no protocolo de criopreservação do sêmen suíno, visto que ambas asseguraram qualidade da viabilidade espermática.

Diluentes seminais para pequenos ruminantes / Seminal extenders for small ruminants

Visando prolongar a conservação e o uso seminal, muitos diluentes vêm sendo utilizados em pequenos ruminantes, como os convencionais e os alternativos à base de substâncias vegetais, tendo como funções principais a proteção das membranas, a nutrição e a manutenção da pressão osmótica, bem como a multiplicação das doses inseminantes. Desta forma, este artigo tem como objetivo levantar os principais diluentes convencionais e alternativos utilizados na criopreservação seminal em pequenos ruminantes, a fim de maximizar seu uso e promover uma melhor qualidade espermática.

Looking to prolong preservation and seminal use, diluent muts are used in small ruminants, such as conventional ones and alternative ones based on vegetais substitions, tending as main functions to membrane protection, to nutrição e manutenção da pressão osmótica, bem as a multiplicação You give two inseminants. This way, this article aims to raise the main conventional and alternative diluents used in seminal cryopreservation, in small ruminants, in order to maximize its use and promote a better sperm quality.

Viabilidade espermática em sêmen in natura e refrigerado de veado-catingueiro / Sperm viability of brown brocket deer fresh and cooled sêmen

The brown brocket deer is in risk of extinction in two brazilian states. Therefore, more studies on semen evaluation may be useful to improve male reproductive capacity assessment. In this sense, this study evaluated the sperm viability using eosin-nigrosin and compared the results of fresh and cooled semen samples. Although the viability decreased in cooled semen, it maintained a satisfactory result.

Diluentes seminais para pequenos ruminantes / Seminal extenders for small ruminants

Visando prolongar a conservação e o uso seminal, muitos diluentes vêm sendo utilizados em pequenos ruminantes, como os convencionais e os alternativos à base de substâncias vegetais, tendo como funções principais a proteção das membranas, a nutrição e a manutenção da pressão osmótica, bem como a multiplicação das doses inseminantes. Desta forma, este artigo tem como objetivo levantar os principais diluentes convencionais e alternativos utilizados na criopreservação seminal em pequenos ruminantes, a fim de maximizar seu uso e promover uma melhor qualidade espermática.(AU)

Looking to prolong preservation and seminal use, diluent muts are used in small ruminants, such as conventional ones and alternative ones based on vegetais substitions, tending as main functions to membrane protection, to nutrição e manutenção da pressão osmótica, bem as a multiplicação You give two inseminants. This way, this article aims to raise the main conventional and alternative diluents used in seminal cryopreservation, in small ruminants, in order to maximize its use and promote a better sperm quality.(AU)

Qualidade espermática durante a curva de resfriamento do sêmen suíno diluído em água de coco em pó visando sua criopreservação / Sperm quality during the cooling curve of swine semen diluted in coconut water extender aiming your cryopreservation

A criopreservação seminal apresenta baixos resultados produtivos. Objetivou-se testar a Água de Coco em Pó (ACP-103®) como diluente de ressuspensão após a descongelação seminal e avaliar a qualidade espermática durante a curva de resfriamento até a descongelação do sêmen. Para isso, o sêmen de 15 reprodutores foi coletado uma vez por semana, incubado a 30 oC por 15 minutos, e em seguida diluído em Beltsville Thawing Solution BTS (controle) ou em ACP-103®, e submetidos a uma curva de resfriamento lenta, onde foram feitas análises de vigor e motilidade em cada passo. O sêmen descongelado foi ressuspenso em seus respectivos diluentes e analisado quanto às características: vigor, motilidade, vitalidade, integridade acrossomal e funcionalidade da membrana. Durante as análises de vigor e motilidade que compõem a curva de resfriamento, e na descongelação, para as análises de vitalidade e membrana acrossomal intacta, observou-se que não houve diferença significativa entre os tratamentos. Já após a descongelação, o BTS apresentou melhores resultados de vigor, motilidade espermática e funcionalidade da membrana. No entanto, a curva de resfriamento e o ACP-103® podem ser utilizadas no protocolo de criopreservação do sêmen suíno, visto que ambas asseguraram qualidade da viabilidade espermática.(AU)

Semen cryopreservation is associated with low productivity results. This study aimed to test the Coconut Water Powder (ACP®-103) as a ressuspension diluent after thawing semen, also evaluate sperm quality during the cooling curve until thawing of the semen. For this, the semen was collected from 15 boars once a week, incubated at 30 °C for 15 minutes, and afterwards, the samples were diluted in the diluent Beltsville Thawing Solution BTS (Control) or ACP-103®, and subjected to a slow cooling curve, where the force and the motility were analyzed in each step. The thawed semen was ressuspended in its respective solvents and analyzed in the characteristics of vigor, motility, vitality, acrosome integrity and functionality of the membrane. During the analysis of vigor and motility that make up the cooling curve, and thawing, for analysis of vitality and intact acrosomal membrane, it was observed that there was no significant difference between treatments. Besides, after thawing, the BTS showed better results of sperm vigor, sperm motility and membrane functionality. However, the cooling curve and coconut water powder can be used in the protocol for cryopreservation of boar semen, as both ensured quality of sperm viability that may be used in artificial insemination.(AU)