Estudio Histológico en Embriones de Cobayo (Cavia porcellus) y su Utilidad como Modelo para la Comprensión del Desarrollo Embrionario Humano / Histological Study in Guinea Pig Embryos (Cavia porcellus) and its Usefulness as a Model for Understanding Human Embryonic Development

El cobayo es un modelo animal ampliamente utilizado en la investigación biomédica debido a sus similitudes biológicas con los seres humanos. El objetivo de nuestro estudio es proporcionar sustento morfológico para utilizar preparados histológicos de embriones de cobayo como modelo de estudio para comprender los procesos del desarrollo embrionario humano. Nuestros resultados muestran que los embriones de cobayo presentan características morfológicas similares a las observadas en los embriones humanos, lo que sugiere que pueden utilizarse como un modelo efectivo para estudiar el desarrollo embrionario humano. Este hallazgo tiene importantes implicancias para la investigación y la docencia utilizando este modelo animal. Se analizaron preparados histológicos de embriones de cobayo teñidos con hematoxilina eosina, adquiridos por la Universidad Autónoma de Chile. Se tomaron microfotografías de preparados histológicos de cobayo en diferentes estadios del desarrollo y se seleccionaron las mejores imágenes para la descripción de estructuras y establecer estimados de la embriogénesis. Del análisis de los preparados se desprende que órganos como esófago, médula espinal y corazón presentan similitudes anatómicas e histológicas que hacen posible compararlas con el desarrollo embrionario humano y la edad de gestación en etapas tempranas. El uso de preparados de embriones de cobayo y su análisis desde un aspecto histológico resulta ser una estrategia metodológica factible debido a las similitudes en la embriogénesis de los mamíferos y las concordancias morfológicas con el desarrollo de los órganos entre humanos y roedores. Esto permite implementar este modelo animal como una herramienta para comprender el desarrollo embrionario humano.

SUMMARY: The guinea pig is an animal model widely used in biomedical research due to its biological similarities with humans. The objective of our study is to provide morphological support to use histological preparations of guinea pig embryos as a study model to understand the processes of human embryonic development. Our results show that guinea pig embryos present morphological characteristics similar to those observed in human embryos, suggesting that they can be used as an effective model to study human embryonic development. This finding has important implications for research and teaching using this animal model. Histological preparations of guinea pig embryos stained with hematoxylin eosin, acquired by the Autonomous University of Chile, were analyzed. Photomicrographs of histological preparations of guinea pigs at different stages of development were taken and the best images were selected to describe structures and establish estimates of embryogenesis. From the analysis of the preparations it is clear that organs such as the esophagus, spinal cord and heart present anatomical and histological similarities that make it possible to compare them with human embryonic development and gestation age in early stages. The use of guinea pig embryo preparations and their analysis from a histological aspect turns out to be a feasible methodological strategy due to the similarities in mammalian embryogenesis and the morphological concordances with the development of organs between humans and rodents. This allows this animal model to be implemented as a tool to understand human embryonic development.

Effect of umbilical cord length on early fetal biomechanics.

The umbilical cord suspends the fetus within the amniotic cavity, where fetal dynamics is one of its many functions. Hence, the umbilical cord is a viable index in determining fetal activity. Fetal movements result in mechanical loads that are fundamental for fetal growth. At present, mechanical environment during early human fetal development is still largely unknown. To determine early fetal movement dynamics at given physiological (0.060 m) and pathological umbilical cord lengths (0.030 m, 0.020 m, 0.017 m and 0.014 m) a 2D computational model was created to simulate dynamic movement conditions. Main findings of this computational model revealed the shortest umbilical cord length (0.014 m) with a 6(10-6)N, twitch force amplitude had a two-fold increase on linear velocity (0.12 m/s) in comparison with other lengths (0.05m/s). Moreover, umbilical cord length effect presented an increasing exponential tension on the fetus body wall from longest to shortest, from 0 N in the control length to 0.05 N for the shortest umbilical cord. Last, tension was always present over a period of time for the shortest cord (0.03 N to 0.08 N). Collectively, for all variables evaluated the shortest umbilical cord (0.014 m) presented remarkable differences with other lengths in particular with the second shortest umbilical cord (0.017 m), suggesting a 0.003 m difference represents a greater biomechanical effect. In conclusion, this computational model brings new insights required by clinicians, where the magnitude of these loads could be associated with different pathologies found in the clinic.

Early and natural embryonic death in Lagostomus maximus: Association with the uterine glands, vasculature, and musculature.

The uterus is an organ with great plasticity due to the morphological and physiological changes it experiences during the estrous cycle and pregnancy. In mammals, pregnancy requires diverse sex hormones, growth factors and cytokines, among others, for promoting uterine remodeling to favor implantation, placentation, and embryo/fetus survival and growth. The hystricognathi rodent Lagostomus maximus (plains viscacha) has a high rate of embryonic resorption. The cranial and middle implants are reabsorbed 25-35 days after intercourse while the caudal embryos continue with their development until two precocial offspring are born. So far, no uterine studies of non-pregnant L. maximus females were performed to determine the possible existence of variations in the organ that could be related to the differential survival of the implants. We used ultrasonography, as well as morphological, morphometric, histochemical, lectinhistochemical, and immunohistochemical methods to study differences in the uterine glands (area), vasculature (area), and musculature (thickness) along the uterine horns in non-pregnant females. Along the uterus, all these structures were in more advanced developmental condition in the caudal region as compared to more anterior positions. These regional variations could be decisive in explaining the reason why only caudal implantations come to term. In contrast, no differences in the in the luminal and glandular epithelial cells, nor in the degree of cell proliferation and apoptosis, and hormonal receptor staining were found. These parameters could be related to implantation along the uterine horns, but not to the differential survival of the implants.

The embryo of the silky shrew opossum, Caenolestes fuliginosus (Tomes, 1863): First description of the embryo of Paucituberculata.

The development of caenolestid marsupials (order Paucituberculata) is virtually unknown. We provide here the first description of Caenolestes fuliginosus embryos collected in the Colombian Central Andes. Our sample of four embryos comes from a single female caught during a fieldtrip at Río Blanco (Manizales, Caldas), in 2014. The sample was processed for macroscopic description using a Standard Event System and for histological descriptions (sectioning and staining). The grade of development of the lumbar flexure and coelomic closure differed between embryos, two of them being more advanced than the others (similar to McCrady's stages 30 and 29, respectively). The pericardial and peritoneal cavities were present, the hepatic anlage was organized in hepatic cords, the heart was in its final position, and the mesonephros was functional. Compared to other Neotropical marsupials, an early appearance of the frontonasal-maxillary fusion and the cervical growth (thickness) was observed; however, absorption of the pharyngeal arches into the body and lung development was delayed. Besides these differences, embryos were similar to equivalent stages in Didelphis virginiana and Monodelphis domestica. Previous proposals of litter size of four for C. fuliginosus are supported.

Key characteristics of the ovary and uterus for reproduction with particular reference to poly ovulation in the plains viscacha (Lagostomus maximus, chinchillidae).

One of the dogmas of mammalian reproduction states is that primordial germ cells in females are restricted to the intrauterine phase, and that only a small portion of oocytes is available for ovulation during the adult life. Among the rare exceptions to this rule is the plains viscacha. This specie polyovulates up to 800 oocytes per cycle, from which 10 to 12 are implanted, but only 1-2 conceptuses survive. To better understand the key mechanisms of this pattern of embryonic to uterine interactions, we analyzed 19 female genital systems by means of gross morphology, histology, stereology and immunohistochemistry. Data showed that a specialized, highly convoluted structure of the ovarian cortex developed during the intrauterine phase as a prerequisite for the massive super-ovulation, likely associated with the inhibition of apoptosis and continued proliferation of germ cells, as well as maintenance of several corpora lutea during the adult life. In addition, specializations of uterine vasculature and musculature were demonstrated. Altogether, these key morphological characteristics evolved in order to contribute as compensatory or controlling mechanism for polyovulation and polyimplantation that led these species into becoming an unique enigma in reproductive biology, and a potential animal model to provide explanations regarding to developmental specializations.

Sexual determination and differentiation during embryonic and fetal development of New Zealand rabbit females / Determinación y diferenciación sexual durante el desarrollo embrionario y fetal de conejos hembras de Nueva Zelanda

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.

El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.

Morphology of alpaca (Vicugna pacos) embryos in the first third of pregnancy.

The breeding of South American camelids is the main economic activity of the high Andean region of South America and it, is potentially, the most profitable resource in of the Puna environmental conditions of the Puna. The duration of the gestation in alpaca is 339.7 ± 12 days. The objective of the present work was to macroscopically and microscopically describe the ontogenic development of the splanchnic cavities of the alpaca and to determine the gestational time in which the post-cranial ossification centers are observed in the embryos/fetuses of this species, from day 21 to 107 of gestation. The documentation of normal ontogenic development, which is vacant for this period, is of the utmost importance to understand the consequences of the alterations at the different gestational times, as well as for the estimation of the gestational age in the case of abortions. Forty-seven alpaca specimens of both sexes, at different times of their gestational development, collected during slaughter at local slaughterhouses of the Department of Huancavelica, Peru, were evaluated. Specimens were assigned to seven groups according to their morphological characteristics. The embryogenesis in the alpaca was characterized by a series of changes comparable to those occurring in other mammals with similar gestational periods. Despite these similarities, species differences were found in some organs as stomach, which are observed too in adult individuals.

Macroscopic and microscopic analysis of 2 embryos and 1 foetus derived from a sheep (Ovis aries) without breed / Análise macroscópica e microscópica de 2 embriões e 1 feto derivados de ovelha (Ovis aries) sem raça

The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.(AU)

O interesse em Embriologia, a ciência do desenvolvimento de um zigoto em um feto completamente desenvolvido, tem aumentado consideravelmente nos últimos anos devido a uma série de estudos envolvendo células-tronco pluripotentes embrionárias e induzidas. Além disso, o desenvolvimento de técnicas como a clonagem tem ajudado a compreender os eventos críticos que ocorrem durante o desenvolvimento embrionário. Neste estudo, descrevemos a morfologia de dois embriões de ovinos e um feto utilizando técnicas macroscópicas e microscópicas. Obtivemos ovelhas sem raça definida com 24, 32 e 50 dias de gestação (estimado pelo método de Crown-Rump, CR). Os conceptos foram mensurados, pesados e caracterizados a olho nu. Macroscopicamente, observamos o desenvolvimento dos embriões E1 (24 dias), apresentando globo ocular sem pigmentação de retina e broto do membro torácico e pélvico. Já o E2 (32 dias), apresentava globo ocular com pigmentação na retina e os membros torácicos e pélvicos mais desenvolvidos. O F1 apresentou olhos cobertos com uma membrana e membros torácicos e pélvicos mais desenvolvidos. Enquanto isso, microscopicamente observamos no E1 somitos, ventrículo, átrio e cavidade oral ainda em desenvolvimento. Porém, no F1 já era possível observar ossificação da coluna espinhal, coração com estruturas mais complexas, como ventrículo, átrio, septo interventricular e saco pericárdio. Além disso, na cavidade oral observamos a formação da língua. Este trabalho fornece informações precisas e detalhadas sobre as características morfológicas dos principais órgãos dos sistemas (nervoso, circulatório, respiratório, digestivo e urinário) em cada fase embrionária e fetal analisadas.(AU)

Macroscopic and microscopic analysis of 2 embryos and 1 foetus derived from a sheep (Ovis aries) without breed / Análise macroscópica e microscópica de 2 embriões e 1 feto derivados de ovelha (Ovis aries) sem raça

The interest in embryology, the science of the development of a zygote into a completely developed foetus, has increased greatly in recent years due to a number of studies involving embryonic and induced pluripotent stem cells. In addition, the development of techniques such as cloning has aided to understand the critical events that occur during embryonic development. In this study, we describe the morphology of two sheep embryos and one foetus using macroscopic and microscopic techniques. We investigated sheep without defined breed on days 24, 32, and 50 of gestation (estimated by crown-rump length [CR]). Macroscopically, we observed the development of E1 (24 days), with visible optic vesicle, but without retinal pigmentation and the forelimbs bud in development. In the E2 (32 days), we noticed the presence of optic retinal pigmentation and forelimbs more developed in comparison with E1. As expected, F1 revealed an eyeball already covered and the forelimbs developed. Meanwhile, microscopic analysis revealed somite, ventricle, atrium, and oral cavity in development in E1. However, in F1 we were able to identify more complex structures, such as ossification in the spine, ventricle, atrium, intraventricular septum, pericardial sac, and oral cavity with tongue. This work brings more precise and detailed data on the morphological characteristics of the major organ systems (nervous, circulatory, respiratory, digestive, and urinary) at each embryonic and foetal stage analysed.(AU)

O interesse em Embriologia, a ciência do desenvolvimento de um zigoto em um feto completamente desenvolvido, tem aumentado consideravelmente nos últimos anos devido a uma série de estudos envolvendo células-tronco pluripotentes embrionárias e induzidas. Além disso, o desenvolvimento de técnicas como a clonagem tem ajudado a compreender os eventos críticos que ocorrem durante o desenvolvimento embrionário. Neste estudo, descrevemos a morfologia de dois embriões de ovinos e um feto utilizando técnicas macroscópicas e microscópicas. Obtivemos ovelhas sem raça definida com 24, 32 e 50 dias de gestação (estimado pelo método de Crown-Rump, CR). Os conceptos foram mensurados, pesados e caracterizados a olho nu. Macroscopicamente, observamos o desenvolvimento dos embriões E1 (24 dias), apresentando globo ocular sem pigmentação de retina e broto do membro torácico e pélvico. Já o E2 (32 dias), apresentava globo ocular com pigmentação na retina e os membros torácicos e pélvicos mais desenvolvidos. O F1 apresentou olhos cobertos com uma membrana e membros torácicos e pélvicos mais desenvolvidos. Enquanto isso, microscopicamente observamos no E1 somitos, ventrículo, átrio e cavidade oral ainda em desenvolvimento. Porém, no F1 já era possível observar ossificação da coluna espinhal, coração com estruturas mais complexas, como ventrículo, átrio, septo interventricular e saco pericárdio. Além disso, na cavidade oral observamos a formação da língua. Este trabalho fornece informações precisas e detalhadas sobre as características morfológicas dos principais órgãos dos sistemas (nervoso, circulatório, respiratório, digestivo e urinário) em cada fase embrionária e fetal analisadas.(AU)

Association between Number of Formed Embryos, Embryo Morphology and Clinical Pregnancy Rate after Intracytoplasmic Sperm Injection.

Introduction Infertility has a high prevalence in the general population, affecting ∼ 5 to 15% of couples in reproductive age. The assisted reproduction techniques (ART) include in vitro manipulation of gametes and embryos and are an important treatment indicated to these couples. It is well accepted that the implantation rate is positively influenced by the morphology of transferred embryos. However, we question if, apart from the assessment of embryo morphology, the number of produced embryos per cycle is also related to pregnancy rates in the first fresh transfer cycle. Purpose To evaluate the clinical pregnancy rate according to the number of formed embryos and the transfer of top quality embryos (TQEs). Methods In a retrospective cohort study, between January 2011 and December 2012, we evaluated women who underwent intracytoplasmic sperm injection (ICSI), aged < 40 years, and with at least 1 formed embryo fresh transferred in cleavage stage. These women were stratified into 3 groups according to the number of formed embryos (1 embryo, 2-3 and ≥ 4 embryos). Each group was divided into 2 subgroups according to the presence or not of at least 1 transferred TQE (1 with TQE; 1 without TQE; 2-3 with TQE, 2-3 without TQE; ≥ 4 with TQE; ≥ 4 without TQE). The clinical pregnancy rates were compared in each subgroup based on the presence or absence of at least one transferred TQE. Results During the study period, 636 women had at least one embryo to be transferred in the first fresh cycle (17.8% had 1 formed embryo [32.7% with TQE versus 67.3% without TQE], 42.1% of women had 2-3 formed embryos [55.6% with TQE versus 44.4% without TQE], and 40.1% of patients had ≥ 4 formed embryos [73.7% with TQE versus 26.3% without TQE]). The clinical pregnancy rate was significantly higher in the subgroup with ≥ 4 formed embryos with at least 1 transfered TQE (45.2%) compared with the subgroup without TQE (28.4%). Conclusions Having at least two available embryos and at least one TQE for embryo transfer are predictors of the pregnancy rates.

Association between Number of Formed Embryos, Embryo Morphology and Clinical Pregnancy Rate after Intracytoplasmic Sperm Injection / Associação entre número de embriões formados, morfologia embrionária e taxa de gravidez clínica após injeção intracitoplasmática de espermatozoides

Abstract Introduction Infertility has a high prevalence in the general population, affecting 5 to 15% of couples in reproductive age. The assisted reproduction techniques ( ART ) include in vitro manipulation of gametes and embryos and are an important treatment indicated to these couples. It is well accepted that the implantation rate is positively influenced by the morphology of transferred embryos. However, we question if, apart from the assessment of embryo morphology, the number of produced embryos per cycle is also related to pregnancy rates in the first fresh transfer cycle. Purpose To evaluate the clinical pregnancy rate according to the number of formed embryos and the transfer of top quality embryos ( TQEs ). Methods In a retrospective cohort study, between January 2011 and December 2012 , we evaluated women who underwent intracytoplasmic sperm injection (ICSI), aged < 40 years, andwith at least 1 formed embryo fresh transferred in cleavagestage. These women were stratified into 3 groups according to the number of formed embryos (1 embryo, 2-3 and ≥ 4 embryos). Each group was divided into 2 subgroups according to the presence or not of at least 1 transferred TQE (1 with TQE; 1 without TQE; 2-3 with TQE, 2-3 without TQE; ≥4with TQE; ≥4withoutTQE). The clinicalpregnancy rateswerecomparedineach subgroup based on the presence or absence of at least one transferred TQE. Results During the study period, 636 women had at least one embryo to be transferred in thefirst fresh cycle (17.8% had 1 formed embryo [32.7% with TQEversus 67.3% without TQE], 42.1% of women had 2-3 formed embryos [55.6% with TQE versus 44.4% without TQE], and 40.1%ofpatientshad ≥4 formedembryos[73.7%withTQEversus26.3%withoutTQE]).The clinical pregnancy rate was significantly higher in the subgroup with ≥4 formed embryos with at least 1 transfered TQE (45.2%) compared with the subgroup without TQE (28.4%). Conclusions Having at least two available embryos and at least one TQE for embryo transfer are predictors of the pregnancy rates.

Resumo Introdução A infertilidade tem uma alta prevalência na população geral, afetando 5 a 15% dos casais em idade reprodutiva. As técnicas de reprodução assistida ( TRA ) incluem a manipulação in vitro de gametas e embriões e são um importante tratamento indicado para esses casais. Sabe-se que a taxa de implantação é positivamente influenciada pela morfologia dos embriões transferidos. No entanto, questiona-se, se além da avaliação da morfologia do embrião, o número de embriões produzidos por ciclo também está relacionado com as taxas de gravidez do primeiro ciclo de transferência fresco. Objetivo Avaliar a taxa de gravidez clínica de acordo com o número de embriões formados e a transferência de embrião com ótima morfologia ( EOM ). Métodos Em um estudo de coorte retrospectivo, entre janeiro de 2011 e dezembro de 2012, avaliamos mulheres submetidas a ICSI com idade < 40 anos e com pelo menos um embrião formado e transferido a fresco em estágio de clivagem. Estas mulheres foram estratificadas em 3 grupos de acordo com o número de embriões formados (1 embrião, 2-3 e ≥ 4 embriões). Cada grupo foi subdividido em 2 subgrupos de acordo com a presença ou não de EOM transferido (1 com EOM; 1 sem EOM; 2-3 com EOM; 2-3 sem EOM; 4 com EOM; ≥4 sem EOM). As taxas de gravidez clínica foram comparadas em cada subgrupo segundo a presença ou não de pelo menos um EOM transferido. Resultados Durante o período do estudo, 636 mulheres tiveram pelo menos 1 embrião para ser transferido no primeiro ciclo a fresco (17,8% possuíram 1 embrião formado [32,7% com EOM versus 67,3% sem EOM], 42,1% das mulheres apresentaram 2-3 embriões formados [55,6% com EOM versus 44,4% sem EOM], e 40,1% das pacientes formaram ≥4 embriões [73,7% com EOM versus 26,3% sem EOM]). A taxa de gravidez clínica foi significativamente maior no subgrupo com ≥4 embriões formados com transferência de pelo menos 1 EOM (45,2%) comparando-se ao subgrupo sem EOM (28,4 % ). Conclusões Ter pelo menos dois embriões e pelo menos um EOM para transferência são fatores preditivos da taxa de gravidez.

Development of the Liver in Alpaca (Vicugna pacos): A Microscopic and Macroscopic Description.

South American camelids have several biological, morphological and behavioural adaptations that allow them to live in geographical areas dominated by high altitudes. The liver has hematopoietic functions during the prenatal life, which could be modified in response to the unfavorable habitat. However, there are no previous data on the prenatal development of the liver in these species. In the present work, a study on the macroscopic and microscopic morphology of the liver of the alpaca during ontogeny was performed. Forty-one animals ranging in age from 20 days of embryonic development to adults were studied. Macroscopic and microscopic observations were performed on samples subjected to different techniques. Less than 7-g specimens were studied with stereoscopic magnifying glass. The general characteristics of the prenatal liver are similar to those of other mammals, and the structures related to hematopoietic function follow an ontogenic pattern similar to that of previously studied precocial species. However, there are differences in morphology when compared to descriptions for the Old World camelids, including the absence of relation between the caudate lobe and the right kidney and the lack of interlobular connective tissue.

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage / Conceptos bovinos (Bos indicus) produzidos por transferência nuclear de células somáticas e partenogênese apresentam variações morfológicas desde o estágio de blastocisto

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas.

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage / Conceptos bovinos (Bos indicus) produzidos por transferência nuclear de células somáticas e partenogênese apresentam variações morfológicas desde o estágio de blastocisto

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles(AU)

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas(AU)

Development of the Superaltricial Monk Parakeet (Aves, Psittaciformes): Embryo Staging, Growth, and Heterochronies.

Knowledge about the embryonic stages of birds is important in answering many questions about development and evolution. We give the first description of 41 embryological stages of the monk parakeet (Myiopsitta monachus) on the basis of external morphology and comparison with the chicken. We also provide measurements of some external morphological characters (i.e. body mass, crown-rump, beak, forelimb, and third toe lengths) and perform comparisons with other precocial and altricial birds with the aim of identifying heterochronous developmental features. The following differences in the development of characters in the monk parakeet when compared with other birds were found: (1) delay of the feathers primordia, (2) wing buds initially greater than leg buds, (3) forelimbs and hindlimbs with similar relative size, (4) retroversion of the toe IV, (5) ventral curvature of the upper jaw, (6) positive regressions between stages and beak length with acceleration and higher values and III toe lengths with deceleration and lower values in the monk parakeet compared to the chicken. The growth pattern of the monk paraket Myiopsitta monachus could be influenced by some heterochronic processes like post-displacement, acceleration and/or deceleration. Results of this research allow the standard identification of stages in different species of parrots, recognize similarities and differences between precocial (the chicken) and altricial species (Myiopsitta), and provide planning data for future studies.

Dynamic changes in the spatio-temporal expression of the ß-defensin SPAG11C in the developing rat epididymis and its regulation by androgens.

Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the ß-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of ß-defensins in the epididymis.

Postnatal ontogeny of limb proportions and functional indices in the subterranean rodent Ctenomys talarum (Rodentia: Ctenomyidae).

Burrow construction in the subterranean Ctenomys talarum (Rodentia: Ctenomyidae) primarily occurs by scratch-digging. In this study, we compared the limbs of an ontogenetic series of C. talarum to identify variation in bony elements related to fossorial habits using a morphometrical and biomechanical approach. Diameters and functional lengths of long bones were measured and 10 functional indices were constructed. We found that limb proportions of C. talarum undergo significant changes throughout postnatal ontogeny, and no significant differences between sexes were observed. Five of six forelimb indices and two of four hindlimb indices showed differences between ages. According to discriminant analysis, the indices that contributed most to discrimination among age groups were robustness of the humerus and ulna, relative epicondylar width, crural and brachial indices, and index of fossorial ability (IFA). Particularly, pups could be differentiated from juveniles and adults by more robust humeri and ulnae, wider epicondyles, longer middle limb elements, and a proportionally shorter olecranon. Greater robustness indicated a possible compensation for lower bone stiffness while wider epicondyles may be associated to improved effective forces in those muscles that originate onto them, compensating the lower muscular development. The gradual increase in the IFA suggested a gradual enhancement in the scratch-digging performance due to an improvement in the mechanical advantage of forearm extensors. Middle limb indices were higher in pups than in juveniles-adults, reflecting relatively more gracile limbs in their middle segments, which is in accordance with their incipient fossorial ability. In sum, our results show that in C. talarum some scratch-digging adaptations are already present during early postnatal ontogeny, which suggests that they are prenatally shaped, and other traits develop progressively. The role of early digging behavior as a factor influencing on morphology development is discussed.

Diagnóstico genético pré-implantacional de embriões bovinos utilizando plataformas de genotipagem de marcadores moleculares do tipo SNP / Preimplantation genetic diagnosis of bovine embryos using genotyping platforms of SNP molecular markers

Apesar do grande desenvolvimento das biotecnologias da reprodução animal, como a produção in vitro de embriões (PIV), o diagnóstico genético pré-implantacional (PGD) ainda é aplicado com restrição na rotina da transferência de embriões em animais. Os recentes avanços da genômica têm permitido associar características fenotípicas com informações moleculares, possibilitando a realização da seleção assistida por marcadores moleculares. O objetivo do presente estudo foi de realizar o PGD de embriões bovinos em plataforma de alta densidade de SNP (BeadChip/6.909 SNP). A pequena quantidade de DNA genômico (gDNA) obtida na biópsia embrionária é a principal limitação para a análise de grande número de SNP. Dessa forma, a metodologia de amplificação total do genoma (WGA) (Repli-g Mini Kit, Qiagen, Hilden, Alemanha) foi utilizada para aumentar a quantidade de gDNA da biópsia e permitir a análise de milhares de SNP simultaneamente. Foram utilizados 88 embriões bovinos PIV, submetidos à micromanipulação pela técnica de microaspiração, possibilitando a formação de 3 grupos com diferentes números de células biopsiadas: G1) 5-10 (n=28); G2) 10-20 (n=37); G3) >100 - blastocisto eclodido (n=23). Todas as amostras foram submetidas ao mesmo protocolo de WGA e 4µL de cada amostra foram utilizados para a genotipagem em plataforma iScan/Illumina. A qualidade dos genótipos foi avaliada pela análise do Call Rate (CR), 10o percentil do GCScore (GC10), Alelle Drop In (ADI) e Alelle Drop Out (ADO). O teste de Kruskal-Wallis foi utilizado para investigar diferenças na distribuição das variáveis entre os grupos. O coeficiente de correlação de postos de Spearman revelou correlação significativa entre todas as variáveis. Os resultados apresentaram correlação positiva entre o CR e GC10 (0,99/P<0,001), enquanto as taxas de ADI e ADO apresentaram correlação negativa com o CR e CG10 (ADI/CR: - 0,87; ADI/GC10:-0,88; ADO/CR:-0,87; ADO/GC10:-0,86), com P<0,001 para todas as variáveis. O teste de Kruskal-Wallis apontou para diferença significativa em todas as variáveis analisadas (CR, GC10, ADI e ADO) entre os 3 grupos de biópsias (G1, G2 e G3). O CR médio foi de 59,26%, 78,47% e 95,97%, para G1, G2 e G3, respectivamente. Foi desenvolvido programa computacional (mendelFix) baseado na Lei da Segregação, para inferência dos genótipos não determinados baseado no genótipo do progenitores, aumentando o CR dos grupos 1, 2 e 3 para 79,69%, 88,20% e 97,28%, respectivamente. Os resultados do presente estudo permitem concluir que é possível realizar a genotipagem de embriões bovinos em plataforma de alta densidade de SNP com amostras submetidas ao protocolo WGA/MDA, mas o número de células obtidas na biópsia embrionária afeta a qualidade da genotipagem. A associação das biotecnologias descritas no presente trabalho permite a aplicação da seleção assistida por marcadores moleculares em embriões bovinos, contribuindo para acelerar ainda mais o processo de melhoramento genético animal.

Despite the great development of animal reproduction biotechnology, such as embryo in vitro production (IVP), preimplantation genetic diagnosis (PGD) is still applied with restraint in animals embryo transfer. Recent advances in genomics have associated phenotypic characteristics with molecular information, allowing the development of marker assisted selection. The aim of this study was to perform PGD in bovine embryos using high-throughput SNP platform (BeadChip/6,909 SNP). The small amount of genomic DNA (gDNA) obtained from embryo biopsy is the main limitation for the high-density SNP analysis. Thus, the Whole Genome Amplification (WGA) (Repli-g Mini Kit, Qiagen, Hilden, Germany) was used to increase the amount of gDNA from embryo biopsy and allow the analysis of thousands SNP simultaneously. Eighty-eight IVP bovine embryos were subjected to micromanipulation by microaspiration, allowing the formation of three groups with different numbers of biopsied cells: G1) 5-10 (n=28); G2) 10-20 (n=37); G3) > 100 - hatched blastocyst (n = 23). All samples were subjected to the same WGA protocol, and 4µL of each sample were used for genotyping on iScan/Illumina platform. The genotyping quality was assessed using the Call Rate (CR), 10th percentile GCScore (GC10), Alelle Drop In (ADI) and Alelle Drop Out (ADO). Kruskal-Wallis test was applied to investigate differences in the distribution of variables among groups. Spearman`s rank correlation coefficient revealed a significant correlation between all variables. The results showed a positive correlation between CR and GC10 (0.99/P <0.001), while ADI and ADO rates were negatively correlated with CR and CG10 (ADI/CR: -0.87; ADI/GC10: - 0.88; ADO/CR: -0.87; ADO/GC10: -0.86), P<0.001 for all variables. Kruskal Wallis pointed to significant differences in all variables (CR, GC10, ADO and ADI) among the 3 groups of biopsies (G1, G2 and G3). The CR average was 59.26%, 78.47% and 95.97% for G1, G2 and G3, respectively. Was developed a script (mendellFix) based on the /"Law of Segregation/", for inference of not determined genotypes based on the parents genotypes, thus increasing the CR of group 1, 2 and 3 to 79.69%, 88.20% and 97 28%, respectively. The results of this study show that it is possible to perform the genotyping of bovine embryos in highthroughput SNP platform with samples subjected to WGA/MDA protocol, but the number of cells obtained by embryo biopsy affects the quality of genotyping. The association of biotechnologies described in this work allows the application of marker-assisted selection in bovine embryo, contributing to further accelerate the process of animal breeding.

Embryonic and fetal development in - pigmy rice rat - Oligoryzomys sp. (Rodentia, Sigmodontinae) and its significance for being a new experimental model.

Oligoryzomys (Cricetidae, Sigmodontinae) is a common rodent genus from South America that includes a couple of very similar species. Related species have been used as experimental model for understanding several diseases for which these species are reservoirs. In order to provide a better understanding of the embryological aspects of this group, herein we showed data on the embryonic and fetal development in Oligoryzomys sp. Eight specimens of different stages of gestation were obtained from the Collection of the Zoology Museum of University of Sao Paulo, Brazil. Gestational ages were estimated by crown-rump-length according to Evans and Sack (1973). To address our analysis after examining the gross morphology, tissues from several organs were processed for light and scanning electron microscopy. Morphological data on the systems (nervous system, cardiorespiratory system, intestinal tract and urogenital system) were described in detail. Finally, the findings were compared with what is known about embryological aspects in other rodent species in order to establish similarities and differences during the organogenesis in different species.

Associação das técnicas de criopreservação e cultivo in vitro de folículos pré-antrais para a obtenção de embriões caprinos / Cryopreservation and in vitro culture of ovarian preantral follicles to obtaining goat embryos

A criopreservação tem sido de extrema importância para a preservação de material genético de machos e fêmeas para a utilização posterior em programas de reprodução assistida. Por essa razão, o emprego da criopreservação de oócitos inclusos em folículos ovarianos pré-antrais tem se mostrado como uma ferramenta complementar para as biotecnologias da reprodução que visam maximizar o potencial reprodutivo de animais de grande valor genético mesmo após a morte ou descarte programado. A aplicação da criopreservação de folículos pré-antrais, isolados ou inclusos no tecido ovariano, permitirá a implantação de bancos de germoplasma animal/humano, com o intuito de preservar o patrimônio genético e, consequentemente, a preservação e manutenção da fertilidade de fêmeas. Vários estudos em diferentes espécies, notadamente em humanos, têm demonstrado a retomada da função reprodutiva relatando inclusive o nascimento de crias saudáveis após transplante de tecido ovariano previamente criopreservado. No entanto, em pequenos ruminantes, especialmente em caprinos, os avanços ainda são pouco significativos. Portanto, conclui-se que mais estudos são requeridos com o objetivo de definir um protocolo (congelação lenta ou vitrificação) que garanta a viabilidade e função ovariana e folicular, normais após a descongelação/aquecimento.(AU)

Cryopreservation has been extremely important for the preservation of male and female genetic material for later use in assisted reproduction procedures. Thus, the use of cryopreservation of oocytes enclosed in preantral follicles has been shown as a complementary tool for the reproduction biotechnologies aimed at maximizing the reproductive potential of animals of great genetic value even after death, unexpected or not, as well as after planed elimination. The application of isolated or in situ preantral follicles cryopreservation, will allow salvage animal genetic resources in gene/banks animal / or even genetic human, and consequently to preserve and maintaining the female fertility. Several studies in different species, especially humans, have demonstrated the resumption of reproductive function including reporting the birth of healthy offspring after transplantation of cryopreserved ovarian tissue previously. However, in small ruminants, especially in goats, improvements are still not very significant. Therefore, it is concluded that further studies are required in order to define a protocol (slow freezing or vitrification) that ensures the viability and normal function of ovary and preantral follicles after thawing/warming.(AU)