Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.

Are specialized sperm function tests clinically useful in planning assisted reproductive technology?

CASE STUDY 40-year-old male patient and 32-year-old female partner, with a history of primary infertility of two years duration. The workup revealed idiopathic mild oligoasthenotheratozoospermia, and no apparent female infertility factors. The couple has failed three intrauterine insemination (IUI) cycles, planning more IUI cycles but also considering in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).

Increasing the chances of natural conception: opinion statement from the the brazilian federation of gynecology and obstetrics associations - FEBRASGO committee of gynecological endocrinology / Aumentando as chances de concepção natural: parecer da comissão nacional de ginecologia endócrina da Federação Brasileira das Associações de Ginecologia e Obstetrícia

Abstract Considering that myths and misconceptions regarding natural procreation spread rapidly in the era of easy access to information and to social networks, adequate counseling about natural fertility and spontaneous conception should be encouraged in any kind of health assistance. Despite the fact that there is no strong-powered evidence about any of the aspects related to natural fertility, literature on how to increase the chances of a spontaneous pregnancy is available. In the present article, the Brazilian Federation of Gynecology and Obstetrics Associations (FEBRASGO, in the Portuguese acronym) Committee on Endocrine Gynecology provides suggestions to optimize counseling for non-infertile people attempting spontaneous conception.

Resumo Uma vez que mitos e equívocos sobre a procriação natural se espalham rapidamente na era do fácil acesso à informação e às redes sociais, o aconselhamento adequado sobre a fertilidade natural e a concepção espontânea deve ser encorajado em qualquer tipo de assistência à saúde. Apesar do fato de não haver evidências fortes sobre qualquer dos aspectos relacionados à fertilidade natural, existe literatura sobre como aumentar as chances de uma gravidez espontânea. No presente artigo, a Comissão Nacional de Ginecologia Endócrina da Federação Brasileira das Associações de Ginecologia e Obstetrícia (FEBRASGO) oferece sugestões para otimizar o aconselhamento a pessoas que tentam a concepção espontânea, na ausência do diagnóstico de infertilidade.

Teaching blind women about the anatomy and physiology of the female reproductive system through educational manual / Aprendizado de cegas sobre anatomia e fisiologia do sistema reprodutor feminino mediante manual educativo

Abstract Objectives: to evaluate blind women's learning on the anatomy and physiology of the female reproductive system through the use of an educational material. Methods: methodological development study performed with blind women. The participants responded the pre-test to evaluate their knowledge on sexual and reproductive health and received a manual on anatomy and the reproduction of the physiology After an individual reading, in a period ranging from three to fifteen days, they responded the post-test. Results: there was an increase of correct answers in the post-test in all the items in relation to the categories of The Woman's Body and How One Gets Pregnant becoming significant in the following knowledge of "clitoris increases with the woman excited" (p=0.009), "the function of the vagina in a sexual intercourse "(p<0.001), "How does fertilization occurs"(p<0.001) and "the ovulating period" (p<0.001). Conclusions: the manual enabled the participants to learn about the female anatomy and the physiology of fertilization after educational assistive technology.

Resumo Objetivos: avaliar o aprendizado de cegas sobre anatomia e fisiologia do sistema reprodutor feminino mediante o uso de manual educativo. Métodos: estudo de desenvolvimento metodológico, realizado com mulheres cegas. As participantes responderam o pré-teste para avaliação do conhecimento sobre saúde sexual e reprodutiva, e receberam o manual sobre anatomia e fisiologia da reprodução. Após leitura individual, em prazo que variou entre três e quinze dias, responderam o pós-teste. Resultados: houve aumento de respostas corretas no pós-teste em todos os itens com relação às categorias O Corpo da Mulher e Como se Engravida, sendo significativo os seguintes conhecimentos "clitóris aumenta com a mulher excitada" (p=0,009), "função da vagina na relação sexual" (p<0,001), "como ocorre a fecundação" (p<0,001) e "período da ovulação" (p<0,001). Conclusões: o manual permitiu aprendizado das participantes quanto à anatomia feminina e à fisiologia da fecundação após utilização da tecnologia educativa assistiva.

A Correlative Study of Oocytes Morphology with Fertilization, Cleavage, Embryo Quality and Implantation Rates after Intra Cytoplasmic Sperm Injection.

ntroduction: Non-invasive selection of developmentally human oocytes may increase the overall effi ciency of human assisted reproduction. Morphologic abnormalities in the oocyte are relevant for determining its developmental fate. Th e objective is to evaluate the infl uence of MII oocyte morphology on intra cytoplasmic sperm injection (ICSI) outcomes. Material and Methods: 132 patients undergoing ICSI cycles and having female factors of infertility and unexplained infertility. Couples having male factors of infertility were excluded. A total of 1200 oocytes were retrieved from 132 ICSI cycles, of which 1056 MII oocytes were evaluated. Th e criteria for morphological evaluations were: (i) Normal MII oocytes showing clear cytoplasm with uniform texture and homogenous fi ne granularity, a round or ovoid fi rst polar body with a smooth surface, and perivitelline space of normal size. (ii) MII oocytes with extra cytoplasmic abnormalities (fi rst polar body and perivitelline space abnormalities). (iii) MII oocytes with cytoplasmic abnormalities (dark cytoplasm, granular cytoplasm, inclusion body and presents of vacuoles). (iv) MII oocytes with combined abnormalities. Result: From 1056 MII oocytes, 180 (17.04%) had normal morphology while 876 (82.95%) had at least one demonstrable morphological abnormality. Cytoplasmic abnormalities were observed in 516 (58.9%) of the oocytes. Extra cytoplasmic abnormalities were observed in 104 (11.87%) while combined abnormalities were responsible for the remaining 256 (29.22%). Th ere were no signifi cant diff erences in fertilization, cleavage, and embryo quality between the groups but there was a highly signifi cant diff erence in implantation rate which was higher in the group of normal oocytes morphology than abnormal oocytes morphology, oocytes with cytoplasmic, extracytoplasmic and combined abnormality 11.11%, 7.33%, 9.03%, 2.3%, and 4.34% respectively. Conclusion: MII oocyte morphology did not aff ect fertilization, cleavage, and embryo quality, but aff ecting implantation rate.

Viabilidade de embriões vitrificados oriundos da fertilização in vitro de oócitos de vacas suplementadas com canola / Viability of vitrified embryos obtained from the in vitro fertilization of oocytes from cows supplemented with canola

Avaliou-se a produção de oócitos e embriões de vacas Nelore in vitro e a resistência à vitrificação. Foram utilizadas 12 vacas Nelore, distribuídas aleatoriamente em dois tratamentos: T1-tratados com canola em grão (2,0kg/animal/dia) e T2-controle. Cada animal foi aspirado quatro vezes para obtenção de óocitos para fecundação in vitro. Os oócitos foram quantificados e classificados em viáveis ou inviáveis. Os zigotos foram cultivados in vitro e, sete dias após, os embriões foram avaliados quanto à qualidade e grau de desenvolvimento e vitrificados em hastes próprias. Na sequência, foram descongelados e cultivados em 6, 12 e 24 horas, observando-se a taxa de expansão e eclosão. Não houve diferenças (P>0,05) no número total de oócitos viáveis, T1=12,7% e T2=11,0%, na taxa de clivagem, T1=60,6% e T2=61,4%, e taxa de blastocistos, T1=23,7% e T2=27,0%, em função do tratamento. Também não houve influência na taxa de re-expansão, T1=70,5 e T2=59,6%, após a vitrificação e descongelamento. Todavia houve diferença (P<0,06) para a taxa de eclosão, T1=69,2 e T2=35,7. Conclui-se que a adição de canola na dieta de vacas não alterou a produção de embriões; entretanto, os embriões resultantes de oócitos coletados de vacas alimentadas com canola são mais resistentes à vitrificação.

It was evaluated the production of oocytes and embryos from Nellore cows in vitro, as well as its resistance to vitrification, when the animals were supplemented with canola grains. Twelve Nellore cows were randomly divided into two treatments: T1-treated with Canola grains (2.0kg/animal/day) and T2-control. Each animal was submitted to other four aspirations, to obtain oocytes for the in vitro fertilization. The oocytes were quantified and classified as viable or not viable. The embryos were cultivated in vitro, seven days after the quality and the level of development of embryos was evaluated and they were vitrified in vitrification straws. Then, the embryos were thawed and grown during 6, 12 and 24 hours, and the rate of expansion and hatching were recorded. There were no differences (P> 0.05) in the whole number of viable oocytes T1: 12.7±1.71 and T2: 11.0±1.77, cleavage rate T1= 60.6±4,72 and T2= 61.4±4.88 or blastocysts rate T1=23,7±5.12 and T2=27,0±5.30 due to the treatment. The treatments did not influence the rate of re-expansion T1= 70,5±6.99 and T2: 59.6±7.09 after vitrification and thawing. However, there was a significant difference (P <0.06) in the hatching rate (T1: 69.17±7.43; T2: 35.66±6.86). Thus, we conclude that supplementation with canola grains did not change embryos production, but the embryos yielded from oocytes of cows fed canola grains are more resistant to vitrification.

Qualidade do corpo lúteo e do embrião de ovelhas Santa Inês superovuladas com FSH associado à rbST / Quality of corpus luteum and embryo of Santa Inês ewes superovulated with FSH associated with rbST

Avaliou-se o efeito da somatotropina bovina recombinante (rbST) sobre a qualidade do corpo lúteo e do embrião e sobre a concentração de progesterona (P4), em ovelhas da raça Santa Inês doadoras de embriões. Foram utilizadas 13 doadoras, distribuídas aleatoriamente em dois grupos, G1 (n=7) e G2 (n=6). Em ambos os grupos, as doadoras foram sincronizadas com implante vaginal por 14 dias, receberam 100µg de PGF2α (Ciosin®) no sétimo dia e foram superovuladas, a partir do 12º dia, com 250UI de FSH (Folltopin-V®) em seis doses decrescentes. No dia da retirada do implante, as doadoras do G2 receberam 125mg de rbST (Boostin®) e foram cobertas por macho Santa Inês a cada 12 horas até o final do estro. No sexto dia após a primeira cobrição, foram avaliadas, por laparoscopia, a resposta superovulatória e a quantidade e a qualidade dos corpos lúteos. Após as avaliações, as colheitas embrionárias foram realizadas por laparotomia. A administração da rbST no protocolo de superovulação promoveu aumento significativo (P<0,05) no número de corpos lúteos do tipo I (9,00 x 5,28) e na qualidade embrionária (4,33 x 2,00).

We evaluate the effect of recombinant bovine somatotropin (rbST) on the quality of the corpus luteum and embryo and on the concentration of progesterone (P4) in donors embryo sheep Santa Ines. 13 donors were used, randomly assigned to two groups, G1 (n=7) and G2 (n=6). In both groups, the donors were synchronized with vaginal implant for 14 days and received 100µg of PGF2α (Ciosin®) on the seventh day and were superovulated, from the 12th day, with 250IU of FSH (Folltopin-V®) in six decreasing doses. On the day of implant removal the donor G2 received 125mg of rbST (Boostin®), and were covered by Santa Inês male every 12 hours until the end of estrus. In the sixth day after the first mating were evaluated by laparoscopy, superovulatory response and the amount and quality of the corpus luteum. After the evaluations, the embryonic harvests were performed by laparotomy. The administration of rbST in superovulation protocol promoted a significant increase (P <0.05) in the number of corpus luteum of type I(9.00 x 5.28) and embryo quality (4.33 x 2.00).

Avaliação ultrassonográfica da involução das estruturas umbilicais extra e intracavitárias em bezerros sadios da raça Nelore concebidos naturalmente e produtos de fertilização in vitro / Ultrasound evaluation of extra- and intra-abdominal umbilical structures involution in healthy Nelore calves products of natural conception or in vitro fertilization

Esse trabalho foi desenvolvido com o objetivo de caracterizar a involução das estruturas umbilicais em bezerros sadios da raça Nelore ao longo dos primeiros 35 dias de vida, e de comparar esse processo em bezerros concebidos por métodos naturais ou por fertilização in vitro (FIV). Quarenta bezerros foram distribuídos em dois grupos (n=20) de acordo com o método de concepção (natural ou FIV) e cada grupo foi composto por dez machos e dez fêmeas. A ultrassonografia (transdutor microconvexo de 7,5 MHz) foi empregada para examinar o conjunto das estruturas remanescentes do cordão umbilical que compõem o umbigo externo e as estruturas abdominais (veia umbilical, artéria umbilical esquerda e ducto alantóide), mensurando-se os seus diâmetros em locais definidos. Os exames foram realizados entre 24 e 36 horas de vida e aos 7, 14, 21, 28 e 35 dias de idade. Testaram-se os efeitos do sexo, da idade e do método de concepção por meio da análise de variâncias de medidas repetidas. O exame ultrassonográfico provou-se adequado para a avaliação das estruturas umbilicais extra e intracavitárias permitindo a caracterização do processo fisiológico de involução das mesmas. No umbigo externo, as veias umbilicais foram observadas como imagem individualizada até os 14 dias de vida e um conjunto de estruturas em processo de atrofia era visualizado após essa idade. No abdômen, a veia e a artéria umbilicais foram visualizadas até os 35 dias de idade e o ducto alantóide somente durante a primeira semana de vida. Essas estruturas apresentaram-se com parede hiperecóica regular e contínua e lúmen homogeneamente anecóico. O diâmetro de todas as estruturas umbilicais estudadas se reduziu continuamente ao longo do primeiro mês de vida (p<0,05), sem efeito do sexo (p>0,05). Comparados aos bezerros concebidos por métodos naturais, os produtos de FIV nasceram com os vasos umbilicais e o ducto alantóide um pouco mais calibrosos (diâmetros 1 a 3 mm maiores). Distintamente dos valores ...

This study was carried out to characterize the involution of the umbilical cord structures in healthy Nelore calves during their first 35 days of life, and to compare this process in calves conceived by natural methods or by in vitro fertilization (IVF). Forty calves were separated in two groups (n=20) according to their conception method (natural or IVF) and each group consisted of ten male and ten female calves. The ultrasound (7.5 MHz micro convex transducer) was used to examine all the remaining structures of the umbilical cord that make the external navel and the abdominal structures (umbilical vein, left umbilical artery and allantoic duct), and their diameters were measured in distinct locations. The examinations were performed between 24 and 36 hours of life and at 7, 14, 21, 28 and 35 days of age. The effects of sex, age and method of conception were tested by repeated measures ANOVA. The ultrasound examination was suitable for evaluation of extra- and intra-abdominal umbilical structures and characterization of its involutive physiological process. Both veins were visualized in the external umbilicus up to 14 days of life and set of structures in process of atrophy were seen after this age. In the abdomen, the artery and the vein could be examined up to 35 days of age, and the allantoic duct only during the first week of life. These structures showed a regular and consistent hyperechoic wall and a homogeneous anechoic lumen. The diameter of all studied structures decreased throughout the first month of life (p<0.05) without any sex effect (p>0.05). The umbilical vessels and the allantoic duct were slightly wider (diameter 1-3 mm larger) in calves conceived by IVF. Differently from the highest values previously demonstrated for Bos taurus calves, we can disclose that in healthy newborn Nelore calves the thickness of the structures which make the external navel should not exceed 2 cm, the diameter of the umbilical vein and artery ...

Unexplained Male infertility: diagnosis and Management

Unexplained male infertility is a diagnosis reserved for men in whom routine semen analyses results are within normal values and physical as well as endocrine abnormalities were ruled out. In addition to erectile problems and coital factors, immunologic causes and sperm dysfunction may contribute to such condition. New etiologies of unexplained male infertility include low level leukocytospermia and mitochondrial DNA polymerase gene polymorphism. Contemporary andrology may reveal cellular and sub-cellular sperm dysfunctions which may explain subfertility in such cases, thus aiding the clinician to direct the further work-up, diagnosis and counseling of the infertile male. The objective of this article is to highlight the concept of unexplained male infertility and focuses on the diagnosis and treatment of this condition in the era of modern andrology and assisted reproductive techniques. Extensive literature review was performed using the search engines: Pubmed, Science-direct, Ovid and Scopus.

A light- and electron microscopic study of primordial germ cells in the zebra fish (Danio rerio)

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. This study explores the origin of primordial germ cells (PGCs) of the zebra fish and examines their morphology during early development (1st day-15th day). PGCs were selectively stained by the alkaline phosphatase histochemical reaction and viewed by light and electron microscopy from the time they are first detectable in the yolk sac endoderm. PGCs occurred in the subendodermal space on the syncytial periblast; differing from the surrounding endodermal cells. Later the PGCs moved to between the blastoderm and yolk sac and transferred to the dorsal mesentery where they formed gonadal anlage with mesoderm cells. PGCs were easily distinguished from somatic cells by their morphology and low electron density of their nuclei. Under light microscopy, PCGs were rounded with a distinct cytoplasmic membrane.

The opportunistic feeding and reproduction strategies of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes: Rivulidae) inhabiting ephemeral habitats on southern Brazil

A maioria dos peixes da família Rivulidae são popularmente conhecidos como anuais por completarem todo seu ciclo biológico em pequenos corpos de água temporários que secam obrigatoriamente em determinados períodos do ano causando a morte dos indivíduos adultos. Possuem características biológicas peculiares como pequeno porte, maturação sexual precoce, reprodução contínua, um elaborado padrão de corte e uma grande capacidade reprodutiva entre os peixes. Os rivulídeos se encontram amplamente distribuídos nas Américas do Norte, Central e Sul. Este trabalho analisou a dieta e a biologia reprodutiva de Cynopoecilus melanotaenia. Foram coletados 263 exemplares e a análise de 233 conteúdos gastrointestinais revelou uma dieta invertívora composta principalmente por microcrustáceos (Cladocera, Amphipoda e Ostracoda) e insetos imaturos (Chaoboridae, Culicidae, Syrphidae, mas principalmente larvas de Chironomidae). Foi registrada a lepidofagia na dieta dos machos. A fecundidade foi estimada pela análise de 59 pares de ovários maduros e variou entre 2 a 157 ovócitos (média, 19 ± 26[DP]). A espécie apresentou desova parcelada, uma estratégia para aumentar a chance de sobrevivência a depleções prolongadas. Este trabalho apresenta a primeira investigação sobre a biologia reprodutiva de C. melanotaenia. Os resultados encontrados sugerem a estratégia reprodutiva oportunista de C. melanotaenia e fornecem novas informações biológicas que podem contribuir para pesquisas de conservação e manutenção da espécie em seu ambiente natural.

Most Rivulidae fishes are popularly known as annual fishes which live in ephemeral environments such as pools, that obligatorily dry out seasonally causing the death of adult individuals. They have unique biological characteristics such as small body size, early sexual maturation, continuous reproduction, an elaborated courtship behavior, and a great reproductive capacity among fishes. The rivulids are widely distributed in North, Central and South America. In this study, the diet and reproductive biology of Cynopoecilus melanotaenia was analyzed. A total of 263 specimens were collected and the analysis of 233 gastrointestinal contents revealed an invertivorous diet composed mainly of small crustaceans (Cladocera, Amphipoda, and Ostracoda) and immature insects (Chaoboridae, Culicidae, Syrphidae, but mainly Chironomidae larvae). Lepidophagy on male’s diet was also registered. Fecundity was estimated by analyzing 59 pairs of mature ovaries and ranged from 2 to 157 oocytes (mean, 19 ± 26[SD]). The species has fractional spawning, a strategy to increase the chance of survival to prolonged depletions. This study is the first to investigate the reproductive biology of C. melanotaenia. The results confirmed the opportunistic character of the rivulid C. melanotaenia and provided unreported reproductive information that may aid conservation of the species.

Análise do desenvolvimento e das glicoproteínas presentes nos folículos ovarianos de poecilia vivipara (cyprinodontiformes, poeciliidae) / Analyses of the development and glycoproteins present in the ovarian follicles of poecilia vivipara (cyprinodontiformes, poeciliidae)

Os aspectos morfofuncionais da oogênese do Poecilia vivipara foram estudados nesse trabalho. Esse estudo contribuiu para o aprimoramento das informações sobre a biologia reprodutiva e do desenvolvimento de espécies com fecundação interna, em especial aquelas pertencentes à família Poecilidae. Para tanto, caracterizou-se os estágios de maturação gonadal e desenvolvimento folicular através de análises mesoscópicas, histológicas, histoquímicas e citoquímicas com lectinas. O estudo mesoscópico permitiu a classificação do desenvolvimento ovariano em seis estágios: imaturo, em maturação I, em maturação II, maturo I, maturo II e pós-desova. O exame microscópico dos ovários permitiu a identificação dos oócitos pré-vitelogênicos (OPVt) e vitelogênicos (OVt). Na fase pré-vitelogênese, verificou-se os oócitos tipo I (OI) e tipo II (OII), e na fase vitelogênese, foram encontrados oócitos tipo III (OIII), tipo IV (OIV) e os pós-fertilização (OV). Com o avanço da oogênese, o citosol dos oócitos aumentaram em volume, com crescente acúmulo de grânulos citoplasmáticos, caracterizando a vitelogênese. Além disso, a zona radiata (ZR) aumentou de espessura e alterou suas características histocitoquímicas, e o epitélio folicular (EF), inicialmente delgado e constituído por células pavimentosas, nos FOs na fase III tornaram-se cúbico simples. As análises histoquímicas e citoquímicas permitiram diagnosticar alterações na composição molecular das estruturas que formam os FOs ao longo do desenvolvimento gonadal. O presente estudo indica diferenças no sistema reprodutor feminino entre espécies de peixes com fecundação interna e aquelas com fecundação externa e sugere que o P. vivipara pode ser usado como modelo experimental em testes de toxicidade ambiental.

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

The effect of cigarette smoke on fertilization and pre-implantation development: assessment using animal models, clinical data, and stem cells

Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time.

The beginning of life of a new human being from the scientific biological perspective and its bioethical implications

The issue of when the human life begins is a very important subject since it has a significant impact on the decisions that we have to take in relation to human beings in development, particularly human embryos. In this article we discuss some of the more relevant biological evidence supporting the fact that beginning human life begins unquestionably at fertilization and the bioethical consequences.

Bull spermatozoa express receptors for platelet-activating factor

Platelet-activating factor (PAF; 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a ubiquitous phospholipid that is implicated in the mediation of a wide variety of reproductive processes. To better understand the role of PAF in bovine reproduction, it was designed experiments to: (a) determine whether bull spermatozoa express receptors for PAF and (b) study the effect of exogenous PAF on in vitro sperm physiology (i.e., capacitation, acrosome reaction, motility, and fertilizing ability). Bull sperm express PAF receptor as determined by two approaches: RT-PCR and immunofluorescence. However, exposure of spermatozoa to different concentrations of exogenous PAF (10-11-10-6 M) did not affect capacitation, acrosome reaction or motility. Consistent with these findings, coculture of gametes in medium containing increasing concentrations of PAF (1 x 10-8-8 x 10-6 M) did not improve in vitro fertilization outcome as measured by percentage of inseminated oocytes reaching 2-cell stage 48 h after fertilization. In contrast, PAF at 8 x 10-6 M concentration significantly inhibited IVF. In conclusion, although bull sperm have PAF receptors, exposure of bull spermatozoa to exogenous PAF failed to enhance the sperm function parameters measured in this study. Additional studies are warranted to elucidate the biological role of PAF on bull spermatozoa.

El factor activador de plaquetas (PAF; del inglés Platelete Activating Factor; 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) es un fosfolípido ampliamente distribuido que participa como mensajero mediador en diferentes procesos reproductivos. Para comprender mejor la participación del PAF en la fisiología espermática bovina se diseñaron experimentos para: (a) determinar si los espermatozoides de toro expresan receptores para PAF y (b) estudiar el efecto del PAF sobre el comportamiento de los espermatozoides bovinos in vitro (capacitación, reacción acrosomal y capacidad fertilizante). De acuerdo a los resultados obtenidos por RT-PCR e inmunofluorescencia, los espermatozoides de toro expresan receptores para PAF. Sin embargo, la exposición de los espermatozoides a concentraciones crecientes de PAF exógeno (10-11-10-6 M) no afectó la capacitación, reacción de acrosoma ni la motilidad. En concordancia con estos hallazgos, el cocultivo de gametas (ovocitos y espermatozoides) en medio al cual se le había adicionado PAF (1 x 10-8-8 x 10-6 M) no mejoró la tasa de fertilización medida como el porcentaje de ovocitos inseminados que alcanzaron el estadio de 2 células 48 hs después de la inseminación. Por el contrario, PAF a una concentración de 8 x 10-6 M inhibió significativamente la tasa de fertilización. En conclusión, a pesar de que los espermatozoides bovinos poseen receptores para PAF, el agregado de PAF al medio de cultivo no mejora las funciones espermáticas examinadas en el presente trabajo. Otros estudios serán necesarios para dilucidar la participación del PAF en la fisiología espermática del toro.

Congenital rubella syndrome after rubella vaccination in 1-4 weeks periconceptional period.

OBJECTIVE: To examine weather exposure to rubella vaccine during 1-4 wk periconceptional period can cause congenital rubella syndrome (CRS). METHODS: This prospective study was performed in 60 pregnant women who received rubella vaccine inadvertently 1-4 wk pre or post conception. Time of conception was determined by last menstrual period (LMP) and first trimester sonography. In addition to gathering mother's obstetric and demographic information, all neonates were evaluated for CRS signs by systemic physical examination and anti rubella IgG and IgM antibody titers in cord blood samples. RESULTS: A total of 60 pregnant women with the median gestational age of 38 weeks were studied. The mean maternal age was 22 years and 58.3% of pregnancies were unintended. In 90% of mothers there were no post vaccination side effects (fever, lymphadenopathy, arthritis, arthralgia). None of the mothers had a history of drug abuse, smoking or teratogenic exposures. Mean neonatal weight was 3100grs and 6.7% of them were premature. No signs of CRS were found in the neonates based on systemic physical exam at birth and one month later. Mean value of cord blood anti rubella IgG titere was 148/28+/-67/26 lu/ml. cord blood anti rubella IgM was negative in all of the neonates. CONCLUSION: In this study inadvertent rubella vaccination 1-4 wk before and after conception did not cause CRS in neonates and according to all reasearches pregnancy termination is not indicated in these cases.

Ultrastructural changes of the egg apparatus associated with fertilisation of natural tetraploid Trifolium pratense L. (Fabaceae)

The aim of this study is to describe the ultrastructural changes of the egg apparatus associated with fertilisation of the natural tetraploid Trifolium pratense. The pollen tube enters one of the synergids through the filliform apparatus from the micropyle. Before the entry of the pollen tube into the embryo sac, one of the synergids begins to degenerate, as indicated by increased electron density and a loss of volume. This cell serves as the site of entry for the pollen tube. Following fertilization, the vacuolar organisation in the zygote changes; in addition to the large micropylar vacuole, there are several small vacuoles of varying size. Ribosomal concentration increases significantly after fertilisation. In T. pratense, ultrastructural changes between the egg cell and zygote stages are noticeable. Several marked changes occur in the egg cell because of fertilisation. The zygote cell contains ribosomes has many mitochondria, plastids, lipids, vacuoles. After fertilization, most of the food reserves are located in the integument in the form of starch. The zygote shows ultrastructural changes when compared to the egg cell and appears to be metabolically active.

Avaliação de protocolos para indução de inatividade ovariana em gatas domésticas / Evaluation of protocols for induction of inactivity ovarian in domestic cats

Os índices de prenhez após inseminação artificial em felídeos selvagens não são satisfatórios devido ao variável ambiente endócrino após a estimulação com gonadotropinas. O objetivo deste estudo consistiuem aumentar a taxa de sucesso em programas de inseminação artificial em gatas domésticas (animal modelo). As fêmeas (n=9) foram divididas em três grupos, cada um com três animais, sendo: 1) controle(C), somente 200 UI eCG/ 100 UI hCG ; 2) levonorgestrel oral (L)(0,075 mg) durante 37 dias + eCG/hCG; 3) etonogestrel (E), implante subdérmico durante 37 dias + eCG/hCG. Foram submetidas ao exame laparoscópico 29-39 horas após a administração de hCG para verificação da resposta ovariana e realização de esfregaço vaginal para monitoração da fase do ciclo estral. Foram coletadas amostras de fezes 60 dias antes e 60 dias após o tratamento com gonadotropinas para dosagem hormonal de estrógenos. Os resultados foram avaliados através do Teste ANOVA. Os níveis de significância mostraram que o Grupo E, em contraste com o Grupo C e o Grupo L, apresentou inibição satisfatória das concentrações de estrógenos durante a sua utilização. O grupo L não apresentou inibição ovariana durante o tratamento e diferença significativa em relação ao Grupo C. No exame laparoscópico todas as fêmeas dos grupos C, L e E apresentaram folículos e 77% das fêmeas apresentaram corpo lúteo. Também apresentaram células epiteliais superficiais anucleadas e nucleadas características de estro. Concluiu-se que a utilização de implantes de etonogestrel em gatas domésticas mostrou-se eficaz, possibilitando asua utilização prévia aos programas de inseminação artificial, aspiração folicular e também para a contracepção.

Reproductive success in endangered captive small felids species is veryl ow. Due to great variability in endocrine environment post gonadotropin treatment, after artificial insemination pregnancy rates are very low. Nowadays, ovarian activity controll improves the AI success in many species. In this study, new protocols were compared to improve the fertilization rates in artificial insemination programs in domestic cat. Female domestic cats were divided in three treatments: 1) control (eCG/hCG); 2) levonorgestrel (0.075 mg) orally during 37days + eCG/hCG; 3) etonogestrel subdermal implant during 37days + eCG/hCG: Laparoscopies were done 29-39 hours post hCG treatment to verify ovarian activity. Vaginal swabs were collected at laparoscopic procedures. Fecal samples were colected 60 days before, during and 60 days after the gonadotropin treatment for estradiol assay. Means comparisons were done by ANOVA test. Results demonstrated that etonogestrel (implant) and not oral levonorgestrel successfully suppressed ovarian activity. The levonorgestrel group didnot show ovarian inactivity during the administration, presenting oestradiol peaks and without significative diference comparing to control group. All females presented anuclear and nuclear superficial vaginal epithelial cells at laparoscopies. In conclusion, the etonogestrel implant used in the domestic cat was efficient and can be used previous to gonadotropin protocol in artificial insemination programs, follicular aspiration and contraception

Efeitos de diferentes fontes de energia sobre taxa ovulatória, fertilidade e sobrevivência embrionária em marrãs cíclicas / Effects of different energy sources on ovulation rate, pregnancy rate and embryo survival in cyclic gilts

Cinqüenta e quatro marrãs cíclicas, uniformizadas quanto à linhagem, família, ganho de peso, espessura de toucinho, peso, precocidade sexual, número de cios e escore clínico, foram alocadas em dois grupos experimentais com dietas isocalóricas, isoprotéicas e isolisínicas. Duas fontes de energia foram testadas: amido de milho (T1) e óleo de soja (T2). Sincronizou-se o segundo estro com allyl-trenbolone, para inseminação no terceiro estro. Foi realizada cateterização não cirúrgica em 21 marrãs, submetidas a dois ciclos de coleta para dosagem de glicose e insulina, aos 14 e 21 dias do ciclo. Todas as marrãs foram abatidas aos 28,6 dias de gestação média, para análises biométricas do trato reprodutivo. Marrãs do T1 apresentaram maior taxa ovulatória em relação às do T2 (16,52 vs 14,70, P<0,01). Não houve diferença entre os tratamentos nas taxas de prenhez e sobrevivência embrionária. É possível alterar a eficiência reprodutiva por intermédio de manipulação dietética, mesmo em marrãs em estado anabólico. O uso do amido de milho na fase pré-cobertua melhorou a eficiência reprodutiva dos animais avaliados.

Fifty four cyclic gilts were randomly selected and uniformized according to genetic background, litter of origin, weight gain, backfat, number of cycles and clinical score. Gilts were alloted to one of two groups fed isocaloric, isoproteic and isolysinic diets. Two energy sources were tested: corn starch (T1) and soybean oil (T2). Second estrus was synchronized with oral allyl-trenbolone, so that insemination was carried out at third estrus. Indweeling catheters were implanted by non-surgical technic in 21 gilts, which were submitted to consecutive blood samplings for glicose and insulin determination. Timing of ovulation was estimated by means of ultrasonography. All gilts were slaughtered at an average gestation lenght of 28.6 days. Starch-fed gilts (T1) showed higher ovulation rates than T2 gilts (16.52 vs 14.70; P<0.01). There was no effect of treatments on pregnancy rate and embryo survival. Results indicate it is possible to manipulate reproductive efficiency through diet even in anabolic experimental models like cyclic gilts. Feeding starch as main energy source during pre-mating flushing phase improved reproductive efficiency of cyclic gilts.