ABSTRACT
Since bull fertility prediction remains challenging, the identification of potential fertility markers is important considering the economic benefits to the livestock industry. The main goal of this study was to determine the Na/K-ATPase activity and expression in thawed sperm of high (HF)- and low-fertility (LF) Angus bulls. Samples from three different batches/bulls with HF (n = 4) and LF (n = 4) were used. The Na/K-ATPase activity was determined after thawing, whereas sperm kinematics, membrane integrity, and expression of Na/K-ATPase on sperm surface were evaluated immediately post-thaw and after 120 minutes of incubation. Within the same incubation time, there was no difference on sperm membrane integrity, kinematics, and the expression of Na/K-ATPase on the sperm surface between HF and LF bulls. Kinematic parameters of LIN and VCL were not influenced by incubation time in samples from HF and LF, respectively. A tendency (P = 0.06) of higher Na/K-ATPase enzymatic activity for sperm of HF bulls compared to LF bulls was observed (0.49 ± 0.07 and 0.32 ± 0.06, respectively). In conclusion, Na/K-ATPase activity and expression in thawed sperm from Angus bulls are not related to the fertility index after fixed-time artificial insemination. However, sperm kinematics related to hyperactivation might indicate higher sperm cryotolerance for HF bulls.
ABSTRACT
A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.
Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.
Subject(s)
Male , Animals , Galactose , Sheep , Semen Preservation/veterinary , Vitrification/drug effectsABSTRACT
A vitrificação de espermatozoides é uma técnica que apresenta grande potencial para criopreservação de material genético, e sua eficácia tem sido superior aos métodos convencionais em algumas espécies. No entanto, existem poucos estudos sobre sua eficiência com sêmen ejaculado de carneiros e o uso da galactose como crioprotetor extracelular durante a vitrificação. Objetivou-se com este estudo avaliar o efeito da galactose (0,01 M), associada ou não ao glicerol (3% e 7%), em meio comercial (Steridyl® - controle), na criopreservação de espermatozoides de carneiros pelo método de palhetas, comparando o método clássico de congelação e a vitrificação. Ejaculados de seis carneiros da raça Dorper em idade reprodutiva foram coletados com vagina artificial, aliquotados, diluídos individualmente (100 × 106 espermatozoides/mL) nos meios testados, envasados em palhetas de 0,25 mL e submetidos à congelação clássica ou vitrificação. Foram analisadas a cinemática, morfologia, morfometria, viabilidade, integridade física e funcional da membrana espermática. A congelação clássica obteve melhores resultados de motilidade total e progressiva do que a vitrificação nos quatro extensores testados, uma vez que as amostras vitrificadas não apresentaram motilidade pós-reaquecimento (p < 0,05). A adição de galactose ou glicerol ao meio comercial não trouxe efeito benéfico tanto para a vitrificação quanto congelação clássica.
Sperm vitrification is a technique with great potential for cryopreservation of genetic material, with superior effectiveness compared to conventional methods in some species. However, few studies have shown its efficiency with ejaculated sperm of rams and the use of galactose as an extracellular cryoprotectant during vitrification. This study aimed to evaluate the effect of galactose (0.01 M), with or without glycerol addition (3% and 7%) in a commercial extender (Steridyl® - control) for ram sperm cryopreservation in straws, comparing the classic freezing method and vitrification. Ejaculates from six breeding soundness Dorper rams were collected with an artificial vagina, aliquoted, individually diluted (100 × 106 sperm/mL) on extenders tested, loaded into 0.25 mL straws, and subjected to a classic freezing method or vitrification. Sperm kinematics, morphology, morphometry, viability, and physical and functional integrity of the sperm membrane were evaluated. The classic freezing method resulted in higher total and progressive motility than vitrification, as no motility was detected in vitrified samples after rewarming (p<0.05). The addition of galactose or glycerol to the commercial medium did not benefit both vitrification and the classic freezing method.
Subject(s)
Animals , Male , Semen , Sheep , Cryopreservation/methods , Cryopreservation/veterinary , Galactose , Cryoprotective Agents , GlycerolABSTRACT
Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.
ABSTRACT
Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.(AU)
Subject(s)
Animals , Ouabain/analysis , Ouabain/chemistry , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/immunology , Blastocyst/chemistry , HorsesABSTRACT
Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.
Subject(s)
Animals , Sodium-Potassium-Exchanging ATPase/analysis , Sodium-Potassium-Exchanging ATPase/immunology , Blastocyst/chemistry , Horses , Ouabain/analysis , Ouabain/chemistryABSTRACT
As afecções dentárias são de alta incidência em equinos, muitas vezes evoluindo para os alvéolos dentários causando periostites e sinusites. Quando afetam molares e pré-molares a exodontia faz-se imprescindível, sendo que duas técnicas mais citadas: trepanação óssea e repulsão de molares sob anestesia geral e extração intraoral sob neuroleptoanalgesia. Uma égua de 18 anos, raça Campolina, com sinais clínicos de inapetência e perda de peso foi atendida no ambulatório do GRUPEQUI-UFAL. Ao exame radiográfico lateral obliquo, apresentou imagens sugestivas de reabsorção do dente 109 e alvéolo-periostite do 108, além de área de radiopacidade sobre as raízes, confirmando diagnóstico de sinusite secundária. A opção cirúrgica foi a trepanação óssea e repulsão de molares sob neuroleptoanalgesia, em posição quadrupedal, pouco citada na literatura. A sedação foi realizada com detomidina (0,6 ucg/kg/IV), o bloqueio anestésico foi efetuado de maneira local subcutânea na linha de incisão e perineural infiltrativa no nervo maxilar, com Lidocaina 2% sem vasoconstritor, com volumes respectivos de 10 e 20 mL. Para o bloqueio do nervo maxilar, foi utilizada a técnica de acesso ventral ao processo zigomático e dorsal aos vasos faciais transversos. Conseguiu-se a extração dos dentes 108 e 109 em 45 minutos. Após a extração, inseriu-se uma sonda de Foley, para limpeza e drenagem nos primeiros 4 dias pós-operatório. A sutura foi de Cushing no espaço subcutâneo com Poliglactina 910 nº0 e interrompida simples com Nylon n°0 na pele. O pós cirúrgico foi de rotina com Penicilina benzatina (40000 UI/Kg/IMq48 horas/3 aplicações), Metronidazol (10 mg/kg/V0/12hs/5 dias) e Flunixin Meglumine intramuscular (1,1 mg Kg/IM/24 hs/5 dias). As lavagens da boca com mangueira foram realizadas quatro vezes por dia, após as alimentações, além de utilização de Líquido de Dakin no alvéolo periostal. Os curativos externos eram realizados com povidine e spray repelente. Os pontos foram retirados com 15 dias. A trepanação óssea e extração de molares com o animal sob neuroleptoanalgesia, utilizando detomidina e bloqueio perineural maxilar, apresentou-se como técnica satisfatória para a extração de molares em equinos idosos.