In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation

Macedo, M.C.; Lucia, Thomaz; Rambo, Gissele; Filho, E.B. Ferreira; Rosa, Armelle; Fabiane, C.; Cabral, Marcelo Araújo; Deschamps, João Carlos

doi:10.1016/j.anireprosci.2009.05.013

Cited by 8 publications

(10 citation statements)

References 23 publications

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“…For production animals, conventional methods of semen quality evaluation, such as sperm motility, morphology, and concentration, are routinely used to estimate male reproductive potential, although their correlations with actual fertility are unsatisfactory (Amann and Hammerstedt ; Love, ). Potential male fertility can be also estimated by the capacity of spermatozoa to penetrate homologous and heterologous oocytes in vitro (Zaho et al, ; Macedo et al, , ). For boar sperm, the in vitropenetration (IVP) assay using homologous oocytes as substrates detected a decline in sperm penetrating capacity after only 24 hr of storage, which was undetected by conventional semen quality evaluations after storage for 72 hr (Macedo et al, ).…”

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confidence: 99%

“…Nonetheless, the IVP assay required freshly recovered oocytes and a CO 2 incubator for gametes coincubation. Advances in the IVP assay were achieved using swine oocytes frozen in cryotubes as substrates and glass flasks and bag systems for gametes coincubation without a CO 2 incubator (Macedo et al, ), but such assay still needs further reduction in cost and labor and is sensitive to individual variation in sperm‐penetrating capacity among sperm donors (Popwell and Flowers, ; Macedo et al, , ).…”

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confidence: 99%

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In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species

Corcini

Stephan²,

Colares³

et al. 2011

J. Exp. Zool.

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Small vesper mice (Calomys laucha) may be considered as an animal model for in vitro fertilization studies, but limited data about in vitro evaluations of their sperm quality and fertility are available. The in vitro penetration (IVP) assay is used to estimate potential sperm fertility for many mammal species, but it still requires reduction in cost and labor. This study tested improvements in the IVP assay for C. laucha sperm using swine oocytes and perivitelline layers (PVL) of chicken eggs as substrates, and evaluated associations among C. laucha sperm quality, IVP, and in vivo fertility after natural mating. In the IVP assay, gametes coincubation was carried out flat-bottomed wells with M2, in water bath at 37°C for 2 hr. C. laucha sperm presented motility, normal morphology, membrane integrity, and acrosome integrity equal to 90.6 ± 5.6, 90.2 ± 6.6, 88.7 ± 9.6, and 90.5 ± 11.5%, respectively. The IVP rate was 39.8% in swine oocytes and 87.5% in the inner PVL. Considering in vivo fertility as the gold standard, the IVP assay in swine oocytes presented a sensitivity of 16.0% and specificity of 83.3%. The sensitivity of the IVP assay in the inner PVL was 84.0%, but the specificity was not determined because there were no true negative results. Sperm membrane integrity was correlated with parturition after natural mating (r = 0.38, P<0.01) and litter size (r = 0.54; P<0.0002).The IVP assay using swine oocytes as substrates can be performed in nearly 2 hr without gametes' coincubation in CO(2).

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confidence: 99%

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confidence: 99%

In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species

Corcini

Stephan²,

Colares³

et al. 2011

J. Exp. Zool.

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“…para que houvesse a sedimentação, após sendo feito busca em lupa estéreo-microscópica em placa de Petri contendo PBS 1X. O processamento dos ovócitos foi realizado como descrito por Macedo et al (2006;2010). No teste de adesão in vitro utilizou-se em cada tratamento para cada macho pools de 30 ovócitos, conforme Maleszewski et al (1995).…”

Section: Methodsunclassified

“…Após a co-incubação, os ovócitos foram recuperados, lavados, corados com Hoechst 33342 (10 mg/mL), e avaliados a 400X em microscópio de epi-fluorescência (Olympus BX 51, América INC, São Paulo, SP). Os ovócitos foram considerados aderidos quando sua ZP continha pelo menos um espermatozoide no espaço perivitelíneo ou citoplasma (MACEDO et al, 2006;2010). Para cada macho, o número de ovócitos aderidos e de espermatozoides por ovócitos foram contados, e a taxa de adesão in vitro calculada.…”

Section: Methodsunclassified

Teste De Adesão Espermática Heterólogo in Vitro Modificado

et al. 2016

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O teste de adesão espermática heterólogo (espécies diferentes doadoras de gameta masculino e feminino) in vitro, apesar de ser sensível para determinação da fertilidade, conta com meios e sistemas de incubação onerosos. Assim, o objetivo deste trabalho foi simplificar o teste de adesão espermática heterólogo in vitro, avaliando a viabilidade de utilização do Meio M2 em banho maria a 39 oC frente ao meio KRB em estufa de CO2 a 5% (método original). Os ovócitos foram obtidos de leitoas pré-púberes, com diâmetro folicular entre 3-6 mm, selecionados e desnudados por sucessivas pipetagens. Os espermatozoides foram obtidos através do corte da cauda epididimária de ratos Wistar adultos. A concentração final no meio de fertilização foi de 1x106 espermatozoides/mL. Em cada tratamento foram utilizados pools de 30 ovócitos para cada macho. O grupo controle foi incubado em meio de Krebs-Ringer bicarbonato (KRB) e em incubadora de CO2 a 5%, e no grupo tratado (T1) o meio foi M2 (Sigma, M7167) com HEPES e albumina sérica bovina (BSA) e incubado em banho-maria a 39 °C. A co-incubação (espermatozoides/ovócito) foi realizada por 2 h a 37 °C. A adesão foi avaliada por exposição dos ovócitos a uma solução de Hoescht 33342 em PBS e incubados a 37 °C/15 min, seguido de avaliação em microscópio de epi-fluorescência em aumento de 400X. A taxa de adesão de espermatozoides no ovócito, no grupo controle e T1, não diferiram estatisticamente, foram de 23,5±3,9% e 24,7±4,2%, respectivamente. Também não foi observada diferença significativa entre os tratamentos para o número de espermatozoides/ovócito (Controle = 0,9±0,1 e T1 = 0,8±0,1) (p>0,05). Estes resultados demonstraram que o método simplificado (T1) não interfere nos resultados do teste de adesão espermática. Desse modo, o teste de adesão espermática pode ser simplificado com a utilização do banho-maria, sem que ocorram alterações nas taxas de adesão e polispermia.

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“…In horses, conventional methods of semen quality evaluation, such as sperm motility, morphology, viability and concentration, are routinely used to rapidly estimate reproductive potential, but the results obtained do not correlate well with the final fertility rates achieved (Amann and Hammerstedt, 2002;Kuisma et al, 2006;Love, 2011;Rodríguez-Martínez, 2003). A better alternative is to examine the capacity of spermatozoa to penetrate homologous or heterologous oocytes in vitro (Cox et al, 1994;García-Álvarez et al, 2009;Macedo et al, 2006Macedo et al, , 2010Slavík and Fulka, 1992;Zhao et al, 2002). This has provided excellent results in donkeys (Taberner et al, 2010).…”

Section: Introductionmentioning

confidence: 94%

Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen–thawed, capacitated stallion sperm

Morató

Soares

Orero

et al. 2013

Animal Reproduction Science

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In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation

Cited by 8 publications

References 23 publications

In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species

In vitro assays for vesper mice (Calomys laucha) sperm using heterologous substrates from nonrodent species

Teste De Adesão Espermática Heterólogo in Vitro Modificado

Pre-selection by double layer density gradient centrifugation improves the fertilising capacity of frozen–thawed, capacitated stallion sperm

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