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Rhinoplasty is one of the most complex and challenging procedures in plastic surgery, even for experienced surgeons. Among the steps leading to an aesthetic and functional nose, there is the nasal tip improvement. Today's approach to nasal tip is the product of different techniques shifting through time, mainly from a resection tendency, to preservation and suture use to reshape cartilages. Addressing the lateral crura is vital to an aesthetic nasal tip and it is frequently obtained by adequate suture techniques. The alar-spanning suture described by Perkins is one of such. Regardless of its importance, it was not able to improve convex crura in some cases. The inverted alar-spanning suture (ISS) is an adaptation designed to treat those cases with the suture alone. ISS is a novel technique that can lead to better results treating the convex lateral crura by distributing the force vector in a more effective way. New techniques in rhinoplasty have multiplied, bringing this procedure to a new level and keeping up with the updated notion of restoration instead of excision the ISS is a new, precise, approach to an old problem.

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The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil.

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The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

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Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time.

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This experiment was designed to study mechanisms affecting growth of in vivo-derived (IVD) and in vitro-produced (IVP) fetuses of cattle. Day-7 IVD or IVP cattle blastocysts were transferred to recipients, with pregnant females being slaughtered on Days 90 or 180 of gestation or allowed to undergo parturition. Uteri and contents were dissected and physically measured, and maternal and fetal plasma and amniotic and allantoic fluids were collected for IGF-1 and IGF-2 determinations, and IGFBP profile characterization. Transcripts for IGF-1 and IGF-2 mRNA in placental and fetal tissues, and IGF-1r and IGF-2r in placentomes were determined. There was a greater fetal weight in the IVP group, which was associated with greater IGF-1 and IGF-2 concentrations in maternal circulation, and changes in IGFBP profiles within fetal fluids. Day-90 IVP-derived fetuses were longer, had greater organ weights, larger placentomes, less placentome IGF-2r mRNA transcript, and greater maternal IGF-1 and IGF-2 concentrations than controls. On Day 180 and at parturition tissues from IVP-derived fetuses/calves were from larger uteri, with larger placentomes/fetal membranes, fetuses/calves weighed more, had greater fetal hepatic IGF-2 mRNA transcript, had less fetal plasma IGF-1 and greater allantoic IGF-2 concentrations, greater and lesser IGFBP activities in the allantoic and amniotic fluids, respectively, and greater glucose and fructose accumulation in fetal fluids. Components of the IGF system were differentially regulated not only according to the gestation period (Days 90 or 180) and fluid type (maternal or fetal plasma, amniotic or allantoic fluids), but also based on conceptus origin (IVP or IVD) in cattle.

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Introduction The predictability of nasal tip projection and rotation after aesthetic surgery is a challenge. Tongue-in-groove (TIG) is an effective technique to control tip projection and rotation, but there may be a small loss of projection and rotation of the tip lobe due to lack of support between the anterior septal angle and the domus, since this region is sustained by medial crusts suture-linked and interdomus sutures. Objective To describe a new surgery technique in an attempt to correct the lack of support for the nasal tip after lowering the nasal dorsum. Methods The horn technique consists in preserving a square of cartilage during the removal of the nasal dorsum and septum excess in patients with long and projected nose. This piece will give greater support to the TIG technique and greater predictability of the rotation and projection of the nasal tip. Results Between 2016 and 2018, 50 patients with long and projected noses were submitted to the "horn technique" surgery. They were submitted to the TIG technique associated to the horn technique. A retrospective review of the preoperative and postoperative photographs (3 months to 1 year) of these patients treated with the horn technique were analyzed and showed better support of the nasal tip. Conclusion The horn technique provides greater support to the projection and rotation of rhinoplasties in patients with long and projected nose.

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The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

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The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen-thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

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O programa de Esporte Unificado, da Special Olympics, possibilita práticas esportivas para pessoas com e sem deficiência intelectual conjuntamente, visando a promoção da inclusão social. Fundamentado neste programa, foi realizada uma pesquisa-ação objetivando analisar a relação entre atletas com e sem deficiência intelectual em aulas de ginástica rítmica fundamentadas no Esporte Unificado. Para isso, 18 meninas, 12 com deficiência intelectual e 6 sem essa condição, realizaram aulas de ginástica rítmica, conjuntamente, por 12 semanas, com 2 sessões semanais de 2 horas cada, em uma quadra de esportes de Guaratinguetá, São Paulo, Brasil. Ao final do programa, foi empregada uma entrevista semiestruturada com as meninas sem deficiência, apreciando os dados por análise de conteúdo. Os resultados apontaram que a interação entre atletas com e sem deficiência promoveu melhoras na performance das atletas e possibilitou a construção de conhecimento sobre as diversidades, alcançando atitudes de aceitação e respeito pelas diferenças.

The Special Olympics Unified Sport program enables sports practices for people with and without intellectual disabilities to work together to promote social inclusion. Based on this program, an action research was carried out aiming to verify possible implications achieved through the implementation of a rhythmic gymnastics project based on the Unified Sport, from the perspective of non-disabled participants. To that end, 18 girls, 12 with intellectual disabilities and 6 without this condition, performed rhythmic gymnastics classes together for 12 weeks, with 2 weekly sessions of 2 hours each, in a sports court in Guaratinguetá, São Paulo, Brazil. At the end of the program, a semi-structured interview with non-disabled girls was used, assessing the data by content analysis. The results showed improvements in the performance of athletes, stimulation of the interactions between athletes with and without disabilities and the construction of knowledge about the diversities, reaching attitudes of acceptance and respect for differences.

El programa de deportes unificados de Olimpiadas Especiales permite prácticas deportivas para personas con y sin discapacidad intelectual conjuntamente, con vistas a la promoción de la inclusión social. Fundamentado en este programa, se realizó una investigación-acción objetivando verificar posibles implicaciones alcanzadas por medio de la implantación de un proyecto de gimnasia rítmica fundamentado en el Deporte Unificado, desde la perspectiva de los partícipes sin discapacidad. Para ello, 18 niñas, 12 con discapacidad intelectual y 6 sin esa condición, realizaron clases de gimnasia rítmica, conjuntamente, por 12 semanas, con 2 sesiones semanales de 2 horas cada una, en una cuadra de deportes de Guaratinguetá, São Paulo, Brasil. Al final del programa, se empleó una entrevista semiestructurada con las niñas sin discapacidad, apreciando los datos por análisis de contenido. Los resultados apuntaron mejoras en el desempeño de las atletas, estímulo a las interacciones entre atletas con y sin discapacidad ya la construcción de conocimiento sobre las diversidades, alcanzando actitudes de aceptación y respeto por las diferencias.

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Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/µl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/µl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/µl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

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Nuclear reprogramming in somatic cell cloning is one of the key factors for proper development, with variations in the protocol appearing to improve cloning efficiency. This study aimed to determine the effects of two fusion-activation intervals and the aggregation of bovine cloned embryos on subsequent in vitro and in vivo development. Zygotes produced by handmade cloning were exposed to two fusion-activation intervals (2 h or 4 h), and then cultured in microwells either individually (1 × 100%) or after aggregation of two structures (2 × 100%). Zona-intact oocytes and zona-free oocytes and hemi-oocytes were used as parthenote controls under the same fusion-activation intervals. Day-7 cloned blastocysts were transferred to synchronous recipients. Cleavage (Day 2), blastocyst (Day 7) and pregnancy (Day 30) rates were compared by the χ2 test (P < .05). Extending fusion-activation interval from 2 to 4 h reduced cleavage (91.0 vs. 74.4%) but not blastocyst (34.8 vs. 42.0%) rates. On a microwell basis, cloned embryo aggregation (2 × 100%) increased cleavage (91.5% vs. 74.4%) and blastocyst (46.0% vs. 31.3%) rates compared to controls (1 × 100%), but did not improve the overall embryo production efficiency on Day 7 (23.0% vs. 31.3%), on a per reconstructed embryo basis, respectively. Treatments had no effects on in vitro developmental kinetics, embryo quality, and in vivo development. In summary, the fusion-activation interval and/or the aggregation of cloned bovine embryos did not affect cloning efficiency based on the in vitro development to the blastocyst stage and on pregnancy outcome.

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Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.

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Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.(AU)

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.(AU)

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The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.(AU)

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The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.

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The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.

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Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.(AU)

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Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.

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Cloning by somatic cell nuclear transfer (SCNT) is characterized by low efficiency and the occurrence of developmental abnormalities, which are rather poorly studied phenomena in goats. This study aimed at comparing overall SCNT efficiency in goats by using in vitro-matured (IVM) or in vivo-matured oocytes and fibroblast donor cells (mock transfected, transgenic, or wild type), also characterizing symptoms of the Abnormal Offspring Syndrome (AOS) in development, comparing results with pregnancies produced by artificial insemination (AI) and in vivo-derived (IVD) embryos. The SCNT group had lower pregnancy rate (18.3%, 11/60), total number of concepti (20.0%, 12/60), term births (3.3%, 2/60), and live births (1.7%, 1/60) than both the IVD (77.8%, 7/9; 155.5%, 14/9; 122.2%, 11/9; 88.8%, 8/9) and the AI (71.4%, 10/14; 121.4%, 17/14; 100%, 14/14; 78.5%, 11/14) groups, respectively (p < 0.05). No SCNT pregnancies reached term using IVM oocytes, but in vivo-matured oocytes resulted in two term transgenic cloned kids. The proportion fetal membrane (FM) weight/birth weight reflected an increase in FM size and cotyledonary enlargement in clones, for disproportionally bigger newborns in relation to cotyledonary numbers. Overall, goat cloning showed losses and abnormality patterns similar to the AOS in cloned cattle and sheep, which have not been previously well recognized in goats.

Assuntos

RESUMO

Background: Modified embryo in vitro culture (IVC) systems have been devised for the culture of individual embryos, suchas in microdroplets, capillary tubes, or co-cultures systems. However, the development of the Well-of-the-Well (WOW)system takes advantage of both the individual and the grouped culture systems, as embryos are cultured into microwellsdisposed into a larger well. The aim of this study was to evaluate the in vitro and in vivo developmental potential of handmade cloning (HMC)-derived cloned bovine embryos after the IVC in three microwell (WOW) systems.Material, Methods & Results: Adipose tissue-derived mesenchymal stem cells (MSCs) isolated after subcutaneous biopsy from a Nellore adult female were used as nucleus donor cells for cloning. Collected adipose tissue was incubated in0.075% collagenase for 60 min at 39oC. After digestion, cell suspension was centrifuged at 80 g for 10 min, supernatantwas discarded, and 2 mL of Red Blood Cells Lysis Buffer (RBC) was added to the pellet, remaining for 2 min. The RBCbuffer was diluted with 20 mL PBS, with cell suspension spun at 80 g for 5 min. Supernatant was discarded and the pelletre-suspended in DMEM culture medium + 10% fetal calf serum (FCS). Following cell counting, 5 x 105 cells were seededin 25-cm² bottles containing 6 mL DMEM + 10% FCS, and cultured at 38.3°C, 5% CO2 in air and saturated humidity.After IVM, oocytes were denuded and incubated in demecolcine, followed by zona pellucida removal, oocyte bisection,embryo reconstruction, electrofusion and chemical activation. Cloned embryos were allocated to one of three IVC groups:cWOW: conventional microwells (250 µm, round); mWOW: modified microwells (130 µm, conical); and WOW-PDMS:microwells in polydimethylsiloxane chips (170 µm, cylindrical, with interconnecting microchannels); IVF embryos wereused as controls. Cleavage (D2), blastocyst (D7) and pregnancy (D30) rates were analyzed by the χ2 test, for P < 0.05...(AU)

Assuntos