<i>In vitro</i>equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems

Alonso, A.; Castex, C. Baca; Ferrante, Alejandro; Pinto, Marcelo R.; Castañeira, C.; Trasorras, Virginia Luz; Gambarotta, M.; Losinno, Luis; Miragaya, Marcelo

doi:10.1111/and.12273

Cited by 12 publications

(9 citation statements)

References 29 publications

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“…Similar results have been reported by Kim, Lee, Jun, Lee, and Chung () who obtained the initial stages of embryo development after using non‐viable porcine sperm for ICSI (they injected isolated sperm heads) and by Alonso et al. () who used air‐dried equine sperm for ICSI. All these studies would support the suggestion made by García Roselló et al.…”

Section: Discussionsupporting

confidence: 87%

“…Studies have been carried out using sucrose in human (Isachenko et al, 2008;Schulz, Muñoz, Risopatrón, & Sánchez, 2006), canine (Sánchez et al, 2011), rabbit (Rosato & Iaffaldano, 2013) In this study, not only were we able to corroborate that vitrification of porcine spermatozoa allows conservation of cells with intact and condensed chromatin (97.6 ± 1.3% and 91.6 ± 2.3% respectively), but the percentage of PN obtained shows that it also permits conservation of cells with the capacity to produce zygotes using ICSI. Similar results have been reported by Kim, Lee, Jun, Lee, and Chung (1998) who obtained the initial stages of embryo development after using non-viable porcine sperm for ICSI (they injected isolated sperm heads) and by Alonso et al (2014) who used air-dried equine sperm for ICSI.…”

Section: Discussionsupporting

confidence: 85%

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In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

Arraztoa

Castex

Álvarez

et al. 2017

Reprod Domestic Animals

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The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO and tri-gas. A group of porcine oocytes maturated in vitro were injected with vitrified-warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO or tri-gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified-warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO or under tri-gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO and tri-gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 10 spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.

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Section: Discussionsupporting

confidence: 87%

Section: Discussionsupporting

confidence: 85%

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

Arraztoa

Castex

Álvarez

et al. 2017

Reprod Domestic Animals

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“…Conventional in vitro fertilization (IVF) and embryo production has species specific problems. For instance, equine oocytes cannot be fertilized in vitro unless intracytoplasmic sperm injection (ICSI) is used, which requires expensive and dedicated technology and is labour intensive 3,65,102. For cattle, it is known that IVP embryos are of lower quality and have reduced cryosurvival compared to embryos flushed from the uterus 85,110,119.…”

Section: Improvements In Oviduct Modelling Via Bioengineeringmentioning

confidence: 99%

Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production

et al. 2016

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The oviduct was long considered a largely passive conduit for gametes and embryos. However, an increasing number of studies into oviduct physiology have demonstrated that it specifically and significantly influences gamete interaction, fertilization and early embryo development. While oviduct epithelial cell (OEC) function has been examined during maintenance in conventional tissue culture dishes, cells seeded into these two-dimensional (2-D) conditions suffer a rapid loss of differentiated OEC characteristics, such as ciliation and secretory activity. Recently, three-dimensional (3-D) cell culture systems have been developed that make use of cell inserts to create basolateral and apical medium compartments with a confluent epithelial cell layer at the interface. Using such 3-D culture systems, OECs can be triggered to redevelop typical differentiated cell properties and levels of tissue organization can be developed that are not possible in a 2-D culture. 3-D culture systems can be further refined using new micro-engineering techniques (including microfluidics and 3-D printing) which can be used to produce ‘organs-on-chips’, i.e. live 3-D cultures that bio-mimic the oviduct. In this review, concepts for designing bio-mimic 3-D oviduct cultures are presented. The increased possibilities and concomitant challenges when trying to more closely investigate oviduct physiology, gamete activation, fertilization and embryo production are discussed.

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“…Alternative preservation methods such as dehydration or airdrying sperm have been explored in different species [man: (8); mice: (9); and horses: (10,11)]. These non-conventional techniques have the disadvantage that sperm become immotile and have impaired membranes, making it necessary to recur to intracytoplasmic sperm injection (ICSI) to obtain embryos.…”

Section: Introductionmentioning

confidence: 99%

Air-Drying Llama Sperm Affects DNA Integrity

et al. 2020

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The objective of this study was to evaluate the effects of air-drying preservation on llama sperm DNA. Semen collections were carried out using electroejaculation under general anesthesia. A total of 16 ejaculates were processed from 4 males (n = 4, r = 4). Each sample was diluted 4:1 in a collagenase solution in TALP media, then incubated and centrifuged at 800 g for 8 min. The pellet was re-suspended to a concentration of 20 million sperm/ml in TALP. Then the samples were placed onto sterile slides forming lines and were left to dry under laminar flow for 15 min. After this, the slides were placed into Falcon centrifuge tubes and kept at 5°C. Sperm characteristics (motility, membrane function, viability and morphology) were evaluated in raw semen and in the air-dried samples kept at 5°C for 30 min. DNA evaluation (integrity and degree of chromatin condensation) was carried out in raw semen and in the air-dried samples after 30 min, 7, 14, 21, 30, and 60 days after preservation. To compare raw semen to the air-dried samples, a Wilcoxon test was used for all sperm characteristics except for DNA, where a paired Student t-test was applied. A split plot design was used to compare chromatin condensation between the different periods of preservation and a Kruskal Wallis test was used to compare DNA integrity. Motility, membrane function, viability and sperm with intact DNA decreased in the air-dried samples (p < 0.05), while morphology and chromatin condensation were not affected (p > 0.05). No significant differences were observed in the percentage of sperm with condensed chromatin between the different periods of preservation (p > 0.05). On the other hand, a significant decrease in the percentage of sperm with intact DNA was observed as from day 7 of preservation (p < 0.05). In conclusion the air-drying process has a negative effect on llama sperm DNA, hence the media used will need to be improved to protect DNA and be able to implement this technique in this species.

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In vitroequine embryo production using air-dried spermatozoa, with different activation protocols and culture systems

Cited by 12 publications

References 29 publications

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

Designing 3-Dimensional In Vitro Oviduct Culture Systems to Study Mammalian Fertilization and Embryo Production

Air-Drying Llama Sperm Affects DNA Integrity

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