Bovine sperm cells viability during incubation with or without exogenous DNA

Feitosa, Weber Beringui; Milazzotto, Marcella Pécora; Simões, Rafael Plana; Rovegno, M.; Nicacio, Alessandra Corallo; Nascimento, A. B.; Gonçalves, J. S. A.; Visintin, José Antônio; Assumpção, Mayra Elena Ortiz D’Ávila

doi:10.1017/s0967199409005449

Cited by 17 publications

(8 citation statements)

References 22 publications

(33 reference statements)

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“…Using flow cytometry, our results showed that incubation with exogenous DNA induced a slightly negative effect on sperm viability. In contrast, recently Feitosa et al (2009) did not report any DNA effect regarding viability using microscopy examination. The negative effect regarding motility and viability, which was higher after a long incubation time, could partially explain the frequent failure of IVF and AI with exogenous DNA reported by some authors.…”

Section: Discussionmentioning

confidence: 83%

“…A putative negative effect of DNA binding on motility could explain the failure of SMGT reported by numerous authors. Previous studies reported opposing results regarding a DNA effect: one group of works described a negative effect of the exogenous DNA on the motility of bovine spermatozoa (Schellander et al, 1995; Anzar and Buhr, 2006; Feitosa et al, 2009), while others did not report any effect (Rieth et al, 2000; Alderson et al, 2006). This could be partially explained by the variations between observers during subjective assessments.…”

Section: Discussionmentioning

confidence: 98%

“…SMGT is a technique for producing transgenic animals with huge potential in different fields, including modeling human disease and agricultural applications (revised by Lavitrano et al, 2006; Robl et al, 2007; Niu and Liang, 2008). In the case of bovine SMGT, binding of exogenous DNA to spermatozoa was first showed in 1990 (Castro et al, 1990) with subsequent reports elucidating additional aspects of the process (Atkinson et al, 1991; Gagne et al, 1991; Camaioni et al, 1992; Schellander et al, 1995; Sperandio et al, 1996; Alderson et al, 2006; Hoelker et al, 2007; Feitosa et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

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Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Cánovas

Gutiérrez‐Adán²,

Gadea

2010

Molecular Reproduction Devel

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Sperm mediated gene transfer (SMGT) could provide the opportunity to carry out transgenesis on a mass scale using spermatozoa as vectors for exogenous DNA. However, the efficiency of sperm-mediated DNA transfer is still questionable, and the mode of transmission to the egg has not yet been well understood. Our aim was to investigate the capacity of bovine spermatozoa to carry exogenous DNA and its relationship to sperm functionality. We studied these parameters using flow cytometry to measure viability (necrosis and apoptosis) and capacitation status, computer-assisted semen analysis (CASA) to measure motility parameters and in vitro fertilization (IVF) to assess fertilizing capacity. Furthermore, we studied the effect of capacitation status on interaction with exogenous DNA, and the role of heparin supplementation in this process. Bull spermatozoa showed a high capacity to bind DNA quickly and reached a maximum after 30 min, with approximately half of the DNA-bound spermatozoa being viable. Incubation with exogenous DNA induced a decrease in sperm viability and motility and increased the proportion of apoptotic cells, but did not affect the cleavage rate in IVF assay. Heparin increased high-lipid disorder and the number of sperm with DNA bound (viable and dead). In conclusion, this study shows that live spermatozoa can bind exogenous DNA with a slight negative effect in some parameters of sperm function that in our opinion, would not drastically compromise fertility.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

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Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Cánovas

Gutiérrez‐Adán²,

Gadea

2010

Molecular Reproduction Devel

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“…In a previous work, our group described that incubation period was a factor in decreased sperm viability, causing acrosome membrane disruption during greater incubation periods (Feitosa et al, 2009). This result suggests that not only length of the incubation period, but also CaI concentration is crucial for maintenance of sperm viability.…”

Section: Discussionmentioning

confidence: 85%

Sperm-mediated gene transfer: effect on bovine in vitro embryo production

Simões

Nicacio

Binelli

et al. 2012

Zygote

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The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozenthawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 F capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.

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“…We can speculate that exogenous DNA concentration was not able to trigger endonuclease activity; hence, embryo development rates from all experimental groups were not diff erent (p > 0.05) from control group which suggests that endonuclease activity is not a barrier for SMGT in this species. Feitosa et al (2009), reported that incubation time could reduce sperm viability causing acrosome membrane disruption during increased incubation periods. Our data is in accordance with Canovas, Gutierrez-Adan and Gadea (2010) that showed that aft er 5 min of incubation, more than 20% of bovine spermatozoa bind to exogenous DNA.…”

Section: Discussionmentioning

confidence: 99%

Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

Simões

Feitosa

Milazzotto

et al. 2015

Braz. J. Vet. Res. Anim. Sci.

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Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.Keywords: Spermatozoa. Cattle. Animal transgenesis. In vitro fertilization. Embryos. ResumoApesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal propósito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência gênica, na qual a célula espermática tem habilidade espontânea de se ligar à molécula de DNA e internalizá-la. Dado o potencial da transferência gênica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência gênica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões po...

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Bovine sperm cells viability during incubation with or without exogenous DNA

Cited by 17 publications

References 22 publications

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Effect of exogenous DNA on bovine sperm functionality using the sperm mediated gene transfer (SMGT) technique

Sperm-mediated gene transfer: effect on bovine in vitro embryo production

Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

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