Daniel Salamone - Academia.edu (original) (raw)

Papers by Daniel Salamone

Figure S1. Expression of NANOG and SOX2 in day 8 bovine embryos. Figure S2. Expression of NANOG, ... more Figure S1. Expression of NANOG and SOX2 in day 8 bovine embryos. Figure S2. Expression of NANOG, SOX2, GATA4, GATA6 in day 8 and 10 bovine embryos. Figure S3. Expression of NANOG (N=4), SOX2 (N=2) and SOX17 (N=2) in bovine embryos with ZP. Figure S4. A representative negative control of the immunofluorescence protocol. (DOCX 2405 kb)

L'invention concerne un outil de maintenance (9) pour une chaudiere a condensation, caracteri... more L'invention concerne un outil de maintenance (9) pour une chaudiere a condensation, caracterise en ce qu'il comprend - une lame (21) de nettoyage mecanique, - une tige de maintien (23) de la lame de nettoyage, la lame (21) de nettoyage etant fixee a la tige de maintien (23) avec une orientation perpendiculaire a l'axe de la tige de maintien (23), et - un actionneur (11) de mise en mouvement oscillant de la lame (21) de nettoyage selon un mouvement oscillant autour de l'axe de la tige de maintien (23).

Theriogenology, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reproduction, Fertility and Development, 2014

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived ... more Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of ...

Cellular Reprogramming, 2010

Theriogenology, 2010

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICS... more In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.

Reproductive Biology and Endocrinology, 2011

Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Nogg... more Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100...

Reproduction, Fertility and Development, 2011

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the... more Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cel...

Methods in Molecular Biology

Reproduction, Fertility and Development, 2014

Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning p... more Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning procedure, and it has been shown that different methods affect embryo development. The goal of the present study was to determine the effect of serum starvation in combination with growth to confluence of the somatic donor cells, on in vitro embryo development and quality of aggregated cloned equine embryos. Oocyte collection, maturation, cloning, and activation procedures were performed as described previously by (Gambini et al. 2012 Biol. Reprod. 87, 15). Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of 2 horses. They were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. After cell multiplication, they were cryopreserved and stored in liquid nitrogen. After thawing, 2 groups of cell synchronization were established. Group I: growth to confluence for 3 to 5 days before NT followed by serum ...

Zygote, 2012

SummaryThis study was designed to evaluate the quality and viability of bovine embryos produced b... more SummaryThis study was designed to evaluate the quality and viability of bovine embryos produced byin vitrofertilization (IVF), after intracytoplasmic injection of pCX–EGFP–liposome complexes or pBCKIP2.8–liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX–EGFP–liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culturein vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP exp...

Reproduction, Fertility and Development, 2011

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the ... more Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measur...

Reproduction, Fertility and Development, 2011

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogen... more Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P < 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) an...

Reproduction, Fertility and Development, 2011

Assisted reproduction techniques in the domestic cat could be successfully applied in endangered ... more Assisted reproduction techniques in the domestic cat could be successfully applied in endangered wild felids for sustaining genetic biodiversity. One technique with great potential is intracytoplasmic sperm injection (ICSI). The objective of this study was to evaluate the development of domestic cat embryos after ICSI exposed to ionomycin activation, using a free-radical reducer (insulin-transferrin-selenium, ITS) in oocyte maturation and low oxygen tension in culture. Domestic cat ovaries were recovered from cats subjected to ovariectomy and transported to the laboratory within 2 h. They were washed in TALP-H and the oocytes were released from the follicle by repeatedly puncturing and scraping the ovaries. The cumulus–oocyte-complexes selected were in vitro matured in TCM 199 containing 1 IU mL–1 HCG, 10 ng mL–1 ECG, 2.2 mM calcium lactate, 0.3 mM pyruvate, 0.3% BSA, and 3% antibiotic-antimycotic. Matured oocytes were denuded of cumulus cells after 22 h of culture and individually ...

Reproduction, Fertility and Development, 2013

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may af... more Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the we...

Reproduction, Fertility and Development, 2014

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) co... more Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups...

Reproduction, Fertility and Development, 2012

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilize... more Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO2 in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transf...

Figure S1. Expression of NANOG and SOX2 in day 8 bovine embryos. Figure S2. Expression of NANOG, ... more Figure S1. Expression of NANOG and SOX2 in day 8 bovine embryos. Figure S2. Expression of NANOG, SOX2, GATA4, GATA6 in day 8 and 10 bovine embryos. Figure S3. Expression of NANOG (N=4), SOX2 (N=2) and SOX17 (N=2) in bovine embryos with ZP. Figure S4. A representative negative control of the immunofluorescence protocol. (DOCX 2405 kb)

L'invention concerne un outil de maintenance (9) pour une chaudiere a condensation, caracteri... more L'invention concerne un outil de maintenance (9) pour une chaudiere a condensation, caracterise en ce qu'il comprend - une lame (21) de nettoyage mecanique, - une tige de maintien (23) de la lame de nettoyage, la lame (21) de nettoyage etant fixee a la tige de maintien (23) avec une orientation perpendiculaire a l'axe de la tige de maintien (23), et - un actionneur (11) de mise en mouvement oscillant de la lame (21) de nettoyage selon un mouvement oscillant autour de l'axe de la tige de maintien (23).

Theriogenology, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reproduction, Fertility and Development, 2014

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived ... more Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of ...

Cellular Reprogramming, 2010

Theriogenology, 2010

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICS... more In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.

Reproductive Biology and Endocrinology, 2011

Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Nogg... more Background BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine. Methods For m experiments, COCs were collected from slaughterhouse ovaries and in vitro matured in TCM with 100 ng/ml of either BMP4 or Noggin. After 24 h, the nuclear stage of the oocytes was determined by staining with Hoechst 33342. In addition, RT-qPCR was performed on MII oocytes to study the relative concentration of ZAR1, GDF9, BAX, MATER and HSP70 transcripts. Treated oocytes were submitted to parthenogenic activation (PA) or in vitro fertilization (IVF) and cultured in CR2. For c experiments, non-treated matured oocytes were submitted to PA or IVF to generate embryos that were exposed to 100...

Reproduction, Fertility and Development, 2011

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the... more Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cel...

Methods in Molecular Biology

Reproduction, Fertility and Development, 2014

Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning p... more Donor cell synchronization for nuclear transfer (NT) is one of the crucial steps in the cloning procedure, and it has been shown that different methods affect embryo development. The goal of the present study was to determine the effect of serum starvation in combination with growth to confluence of the somatic donor cells, on in vitro embryo development and quality of aggregated cloned equine embryos. Oocyte collection, maturation, cloning, and activation procedures were performed as described previously by (Gambini et al. 2012 Biol. Reprod. 87, 15). Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of 2 horses. They were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. After cell multiplication, they were cryopreserved and stored in liquid nitrogen. After thawing, 2 groups of cell synchronization were established. Group I: growth to confluence for 3 to 5 days before NT followed by serum ...

Zygote, 2012

SummaryThis study was designed to evaluate the quality and viability of bovine embryos produced b... more SummaryThis study was designed to evaluate the quality and viability of bovine embryos produced byin vitrofertilization (IVF), after intracytoplasmic injection of pCX–EGFP–liposome complexes or pBCKIP2.8–liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX–EGFP–liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culturein vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP exp...

Reproduction, Fertility and Development, 2011

Development of cloned equine embryo is still inefficient. The aim of our study was to assess the ... more Development of cloned equine embryo is still inefficient. The aim of our study was to assess the aggregation of zona-free genetically identical cloned embryos as a strategy to improve in vitro and in vivo development. Oocyte collection, maturation, cloning, and activation procedures were performed as described by (Lagutina et al. 2007 Theriogenology 67, 90–98). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 with 5% of FBS in the well of well system in 3 different groups: I, only one RE per well; II, two RE per well; and III, three RE per well. Cleavage and blastocyst formation (7 to 8 days) of all experimental groups was assessed. At day 8, some embryos of each group were either fixed to determine Oct-4 expression by immunocytochemistry or transferred transcervically to a synchronized mare. Pregnancies were assessed by ultrasound from 7 days after embryo transfer until day 45 to 50 of pregnancy every 7 to 10 days, and sizes of vesicles and embryos were measur...

Reproduction, Fertility and Development, 2011

Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogen... more Oocyte genome cloning refers to obtaining haploid maternal embryos in such a way that parthenogenetic haploid blastomeres (PHB) from these embryos should be considered as a clone of the original gamete. Our main objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Moreover, we generated biparental homogeneous transgene-expressing embryos using PHB that express a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation in 5 μM ionomycin for 4 min followed by culture in SOF for 3 h to permit second polar body extrusion and subsequently treated with 1.9 mM DMAP for 3 h. To generate transgene-expressing PHB, parthenogenetic embryos were injected 3 h post-activation with pCX-EGFP–liposome complexes. These treatments were analysed by Fisher test (P < 0.05). The cleavage rate of the haploid and diploid parthenogenetic embryos control was 87.3% (103/118) an...

Reproduction, Fertility and Development, 2011

Assisted reproduction techniques in the domestic cat could be successfully applied in endangered ... more Assisted reproduction techniques in the domestic cat could be successfully applied in endangered wild felids for sustaining genetic biodiversity. One technique with great potential is intracytoplasmic sperm injection (ICSI). The objective of this study was to evaluate the development of domestic cat embryos after ICSI exposed to ionomycin activation, using a free-radical reducer (insulin-transferrin-selenium, ITS) in oocyte maturation and low oxygen tension in culture. Domestic cat ovaries were recovered from cats subjected to ovariectomy and transported to the laboratory within 2 h. They were washed in TALP-H and the oocytes were released from the follicle by repeatedly puncturing and scraping the ovaries. The cumulus–oocyte-complexes selected were in vitro matured in TCM 199 containing 1 IU mL–1 HCG, 10 ng mL–1 ECG, 2.2 mM calcium lactate, 0.3 mM pyruvate, 0.3% BSA, and 3% antibiotic-antimycotic. Matured oocytes were denuded of cumulus cells after 22 h of culture and individually ...

Reproduction, Fertility and Development, 2013

Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may af... more Somatic donor cells play a major role during the NT procedure. In vitro culture conditions may affect the capability of these cells to be reprogrammed and to allow embryo development. The aim of this study was to evaluate the effect of in vitro culture at low (5%) or atmospheric (20%) oxygen tension in somatic donor cells for cloned equine embryo production. Adult fibroblasts were obtained through culture of minced tissue from neck biopsies of one horse skin. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics in 2 groups: (1) 5% CO2 and (2) 5% CO2 and 5% O2, both groups in humidified air at 39°C. Quiescence of donor cells was induced by growth to confluency for 3 to 5 days prior to NT. Oocyte collection, maturation, cloning, and activation procedures were performed as described by Gambini et al. (2012 Biol. Reprod. 87, 1–9.). After activation, reconstructed embryos (RE) were cultured in DMEM/F12 supplemented with 5% FBS in the well of the we...

Reproduction, Fertility and Development, 2014

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) co... more Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups...

Reproduction, Fertility and Development, 2012

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilize... more Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO2 in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transf...