Efeitos da melatonina exógena na viabilidade embrionária e no ambiente uterino de ovelhas / Effects of exogenous melatonin on embryo viability and uterine environment of ewes

O objetivo deste artigo é revisar o efeito da melatonina na viabilidade embrionária e ambiente uterino em ovinos, tanto in vivo quanto in vitro, considerando as vias pelas quais esse hormônio pode ter influência no embrião ou o mecanismo de reconhecimento materno e estabelecimento de gravidez em ovelhas.(AU)

The aim of this article is to review the effect of melatonin on embryo viability and uterine environment in sheep, both in vivo and in vitro, considering the pathways by which this hormone could have influences at the embryo or the mechanism of maternal recognition and establishment of pregnancy in sheep.(AU)

Effect of temperature on embryonic development and first exogenous feeding of goldfish Carassius auratus (Linnaeus, 1758) / Efeito da temperatura no desenvolvimento embrionário e na primeira alimentação exógena do goldfish Carassius auratus (Linnaeus, 1758)

Goldfish or Kinguio is a widely marketed species worldwide due to the ornamental market. There is some lack of acknowledgment of the production of the species under specific climatic conditions. To evaluate the effect of temperature on embryonic development and the first exogenous feeding of goldfish, an experiment was proposed. Fifteen incubators, organized in five treatments (18, 22, 26, 30, and 34 °C) with three replications each, were used to keep the fertilized goldfish eggs until the first exogenous feeding of the larvae. The main development events were observed to understand the possible effects of these temperatures on embryos and larvae of the species. Temperature influences embryo development and the time of first exogenous feeding of goldfish. The temperature of 34 °C was lethal to the species causing 100% of anomalies in the embryos and larvae. The experiment data allow us to conclude that the species presents a maximum thermal limit during embryogenesis, and these data are important to the aquaculture industry and to understand the effect of climate changes on goldfish. The data obtained in this experiment will assist in the management of invasive species and production of the species (aquaculture).

O peixe dourado ou Kinguio é uma espécie amplamente comercializada em todo o mundo devido ao mercado ornamental. Existe alguma falta de conhecimento da produção da espécie em condições climáticas específicas. Para avaliar o efeito da temperatura no desenvolvimento embrionário e na primeira alimentação exógena do Kinguio, um experimento foi proposto. Quinze incubadoras, organizadas em cinco tratamentos (18, 22, 26, 30 e 34 °C) com três repetições cada, foram utilizadas para manter os ovos fertilizados de Kinguio até a primeira alimentação exógena das larvas. Os principais eventos do desenvolvimento foram observados para entender os possíveis efeitos dessas temperaturas em embriões e larvas da espécie. A temperatura influencia o desenvolvimento do embrião e o tempo da primeira alimentação exógena do Kinguio. A temperatura de 34°C foi letal para a espécie causando 100% de anomalias nos embriões e larvas. Os dados do experimento permitem concluir que a espécie apresenta um limite térmico máximo durante a embriogênese, sendo esses dados importantes para a indústria da aquicultura e para entender o efeito das mudanças climáticas no Kinguio. Os dados obtidos neste experimento auxiliarão no manejo de espécies invasoras e na produção da espécie (aquicultura).

A preliminary approach to embryonic development of Austrolebias wolterstorffi, an endangered neotropical annual fish species / Desenvolvimento embrionário de Austrolebias wolterstorffi, uma espécie de peixe anual neotropical criticamente ameaçada de extinção

Annual fish live exclusively in temporary wetlands and are among the shortest-lived vertebrates in the world. These fish persist in these habitats due to drought-resistant eggs, that which, through diapauses are able to detect stimuli from the environment to start the development processes. They are also able to direct their embryonic development in different trajectories with different development times. Our objective in this paper was to describe the different stages of embryonic development of Austrolebias wolterstorffi, a critically endangered annual fish. A total of 27 stages of embryonic development were identified for the two observed developmental pathways (direct and diapause 2). Of these 27 developmental stages observed, 24 were identical between the two types of trajectories and three different. A total of 90% of the embryos that completed their development proceeded normally, without pauses. One embryo had a different development from the others, entering diapause 2, with a pause in development for 48 h. Although the embryonic development of A. wolterstorffi is similar to that of other Neotropical annual fish species, the diapause 2 occurs when the embryo has a large body size and a well-developed brain, indicating that the lack of embryonic information of the genus Austrolebias may hide characteristics still undescribed or even different survival strategies than what has been observed for other annual fish.

Os peixes anuais vivem exclusivamente em áreas úmidas temporárias e estão entre os vertebrados de vida mais curta do mundo. Esses peixes persistem nesses habitats devido a ovos resistentes à seca, que, por meio de diapausas, são capazes de detectar estímulos do ambiente para iniciar os processos de desenvolvimento embrionário. Eles também são capazes de direcionar seu desenvolvimento embrionário em diferentes trajetórias com diferentes tempos de desenvolvimento. O objetivo deste trabalho foi descrever os diferentes estágios do desenvolvimento embrionário de Austrolebias wolterstorffi, um peixe anual criticamente ameaçado. Um total de 27 estágios de desenvolvimento embrionário foram identificados para as duas trajetórias de desenvolvimento observadas (direto e diapausa 2). Desses 27 estágios de desenvolvimento observados, 24 eram idênticos entre os dois tipos de trajetórias e três diferentes. Um total de 90% dos embriões que completaram seu desenvolvimento seguiu normalmente, sem pausas. Um embrião teve um desenvolvimento diferente dos demais, entrando na diapausa 2, com pausa no desenvolvimento de 48h. Embora o desenvolvimento embrionário de A. wolterstorffi seja semelhante ao de outras espécies de peixes anuais neotropicais, a diapausa 2 ocorre quando o embrião tem um corpo grande e um cérebro bem desenvolvido, indicando que a falta de informação embrionária do gênero Austrolebias pode escondem características ainda não descritas ou mesmo estratégias de sobrevivência diferentes das observadas para outros peixes anuais.

The interaction between the environment and embryo development in assisted reproduction

It can be assumed that the natural processes of selection and developmental condition in the animal provide the best prerequisites for embryogenesis resulting in pregnancy and subsequent birth of a healthy neonate. In contrast, circumventing the natural selection mechanisms and all developmental conditions in a healthy animal harbors the risk of counteracting, preventing or reducing the formation of embryos or substantially restricting their genesis. Considering these facts, it seems to be obvious that assisted reproductive techniques focusing on early embryonic stages serve an expanded and unselected germ cell pool of oocytes and sperm cells, and include the culture of embryos outside their natural habitat during and after fertilization for manipulation and diagnostic purposes, and for storage. A significant influence on the early embryonic development is seen in the extracorporeal culture of bovine embryos (in vitro) or stress on the animal organism (in vivo). The in vitro production per se and metabolic as well as endocrine changes in the natural environment of embryos represent adequate models and serve for a better understanding. The purpose of this review is to give a brief presentation of recent techniques aimed at focusing more on the complex processes in the Fallopian tube to contrast in vivo and in vitro prerequisites and abnormalities in early embryonic development and serve to identify potential new ways to make the use of ARTs more feasible.(AU)

Beneficial Impact of Hypercapnic Conditions During Early Incubation on Broiler Hatchability. Embryo Mortality and Postnatal Performance

We assessed the extent to which CO2 levels altered different hatching and chick parameters. In Experiments 1 and 2, a total of 16,184 eggs from Cobb 500 breeders were incubated in single stage incubators under three different conditions: (a) standard ventilated incubator (CON, Exp.1 and 2); (b) increasing CO2 levels during the first 10 days of incubation until 0.7% (V7000, Exp. 1) and (c) until 0.8% (V8000, Exp. 2). High levels of CO2 improved hatchability, possibly due to lower embryo mortality from ED18 to ED21. Internal and external pipping in experiment V8000 started later than in CON; nevertheless, the hatch still occurred before in V8000 as a result of the shorter durations of external pipping and hatch. In Experiment 3, a total of 12,138 eggs from Cobb 500 were incubated in single stage incubators under three different conditions: (a) standard ventilated incubator (CON); (b) increasing CO2 levels until 1.0% with ventilation (V10000); and (c) increasing CO2 levels until 1.0% without ventilation (NV10000). Hypercapnic conditions led to better hatchability and lower embryo mortality from ED18 to ED21. Internal pipping started earlier in NV10000, but only V10000 differed from CON in terms of the average time for hatch. Hypercapnic groups also showed shorter durations of external pipping and hatch when compared to CON. Post-hatch analysis revealed no differences among incubation conditions in terms of body weight gain, feed conversion ratio, mortality by sudden death syndrome, and production factor. Nevertheless, V10000 showed a lower mortality by ascites and a better viability when compared to CON, while NV10000 presented a higher mortality by other causes. Altogether, our findings indicate that in addition to not being detrimental to embryo survival, high CO2 levels reduce embryonic mortality at 18-21 days of incubation and increase hatchability.(AU)

Pontos críticos na produção de embriões equinos com enfoque na doadora / Critical points in the production of equine embryos with a donor approach

Inúmeras são as vantagens da utilização da técnica de transferência de embriões (TE) em equinos. No entanto, alguns fatores podem afetar a taxa de recuperação embrionária: dia da colheita do embrião; condição uterina e idade da doadora; manejos reprodutivo, sanitário e nutricional; garanhão/qualidade do sêmen; habilidade do técnico; condições climáticas, número de ovulações e adaptação da doadora. O objetivo deste artigo é revisar os fatores que podem exercer influência sobre a taxa de recuperação embrionária tendo impacto direto nos índices reprodutivos de um programa de TE.(AU)

There are countless advantages of using the embryo transfer (ET) technique in horses. However, some factors may affect the embryonic recovery rate: day of embryo collection; uterine condition and donor age; reproductive, health and nutritional management; stallion/semen quality; skill of the technician; climatic conditions, number of ovulations and adaptation of the donor. The objective of this article is to review those factors that can influence the embryonic recovery rate, having a direct impact on the reproductive rates of an ET program.(AU)

Efeitos imediatos, tardios e transgeracionais do estresse térmico em gametas / Immediate, late and transgenerational effects of heat stress on gametes

As perdas de produtividade e fertilidade animal associadas ao estresse térmico durante os meses mais quentes do ano é um dos maiores desafios do setor pecuário. Na indústria de leite as perdas econômicas causadas pelo estresse térmico foram estimadas em mais de 1,5 bilhões de dólares por ano. No que tange a reprodução, já foi demonstrado que o estresse térmico exerce múltiplos efeitos deletérios, causando disfunções endócrinas e alterando a sequência orquestrada de eventos importantes para a gametogênese e para o desenvolvimento embrionário inicial. Estudos recentes têm esclarecido o padrão temporal no qual os danos são estabelecidos e carreados dependendo da intensidade do estresse. Enquanto os efeitos imediatos do estresse térmico nos gametas já são bem caracterizados, existem evidências de que alguns danos podem ser carreados de forma tardia e possivelmente entre gerações. Além disso, dados emergentes indicam que o estresse térmico compromete a reprogramação da metilação do DNA que ocorre durante a gametogênese e a programação do desenvolvimento in utero. Dessa forma, esse artigo visa explorar os efeitos imediatos, tardios e transgeracionais do estresse térmico nos gametas.(AU)

The drop on animal productivity and fertility associated with heat stress during the hot months of the year is one of the biggest challenges for the livestock sector. For the dairy industry the economic losses caused by heat stress have been estimated over 1.5 billion dollars per year. It has already been demonstrated that heat stress exerts multiple deleterious effects on reproductive function, causing endocrine dysfunctions as well as changes in the sequence of events required for gametogenesis and early embryonic development. Recent studies have shed a light in the temporal pattern in which heat-induced damage is established and carried forward depending on the intensity of stress. While the immediate effects of heat stress on gametes are well characterized, there is evidence that some damage can be carried over for longer periods and even across generations. Furthermore, emerging data indicate that heat stress compromises DNA methylation reprogramming that occurs during gametogenesis and developmental programming in utero. Thus, this paper aims to explore the immediate, late and transgenerational effects of heat stress on gametes.(AU)

Impacto da qualidade seminal e dos fatores relativos ao macho na produção in vitro de embriões bovinos / Impact of semen quality and other male-related factors on in vitro embryo production in cattle

A importância da qualidade do sêmen no processo comercial de produção in vitro de embriões (PIVE) é bem conhecida, ainda que não devidamente relatada na literatura. Existe não apenas uma significativa diferença entre touros nas taxas de clivagem e de blastocistos, mas também nas taxas de prenhezes subsequentes. Adicionalmente, há evidências de interação entre touro e tecnologia de processamento do sêmen (particularmente na separação de espermatozoides por sexo), entre touro e protocolo de preparação do sêmen para fertilização in vitro, e ainda entre touro e doadora. Controlar estes efeitos em uma rotina comercial tem sido um desafio crescente para os laboratórios, particularmente com a alta oferta de novos touros decorrente da recente adoção da seleção genômica. O presente trabalho aborda algumas destas questões, com base na experiência da Bio Biotecnologia da Reprodução Animal nesta área.(AU)

The importance of semen quality in a commercial in vitro embryo production (IVEP) routine is well-known, although underreported in the literature. There is not only a significant difference among sires on cleavage and blastocyst rates, but also on subsequent pregnancy rates. Moreover, there are evidences of interaction between sire and sperm processing technology (particularly in the case of sex-sorted semen), between sire and the protocol for sperm preparation for in vitro fertilization, and between sire and donor. Controlling such effects in a commercial routine has been a growing challenge for the laboratories, especially due to the high turnover of sires caused by the recent adoption of genomic selection in most breeds. The current study discusses some of these aspects, from the perspective of the experience of Bio Biotecnologia da Reprodução Animal in this field.(AU)

In vivo embryo development in bitches inseminated laparoscopically after ovulation time estimated based on a single progesterone determination

Logistic and economical limitations are often the causes of dog owners not accurately monitoring the estrous cycle and the optimal insemination time. The aim of this study was to evaluate in vivo early embryonic development in bitches, after the analysis of sequential vaginal cytologies associated to single progesterone measurement and single laparoscopic insemination with high quality semen (fresh and with high spermatozoa concentration) or low-quality semen (frozen/thawed and with low spermatozoa concentration) at 48 h post- ovulation time predicted on a single progesterone measurement. Ten bitches were inseminated with 250 x 106 fresh spermatozoa (80% motility), and ten with 80 x 106 frozen/thawed spermatozoa (60% motility) in the cranial part of each uterine horn. Seven days later, ovariohysterectomy was performed and the oviducts and uterine horns and body were flushed to recover embryos and unfertilized oocytes. In 80% of the bitches inseminated with fresh and 50% of bitches inseminated with frozen/thawed semen, embryos at 2 to 8 cells stage were recovered mostly from the, oviducts. This study indicates that pregnancies can be obtained with a single laparoscopic intrauterine insemination after single serum progesterone measurement, although with a low number of embryos. This result should be taken into account in case economic or logistic restrictions that affect the possibility of owners to plan an accurate monitoring of the optimal breeding time using fresh and frozen semen.(AU)

Opportunities involving microfluidics and 3D culture systems to the in vitro embryo production

Traditional methods of gamete handling, fertilization, and embryo culture often face limitations in efficiency, consistency, and the ability to closely mimic in vivo conditions. This review explores the opportunities presented by microfluidic and 3D culture systems in overcoming these challenges and enhancing in vitro embryo production. We discuss the basic principles of microfluidics, emphasizing their inherent advantages such as precise control of fluid flow, reduced reagent consumption, and high-throughput capabilities. Furthermore, we delve into microfluidic devices designed for gamete manipulation, in vitro fertilization, and embryo culture, highlighting innovations such as droplet-based microfluidics and on-chip monitoring. Next, we explore the integration of 3D culture systems, including the use of biomimetic scaffolds and organ-on-a-chip platforms, with a particular focus on the oviduct-on-a-chip. Finally, we discuss the potential of these advanced systems to improve embryo production outcomes and advance our understanding of early embryo development. By leveraging the unique capabilities of microfluidics and 3D culture systems, we foresee significant advancements in the efficiency, effectiveness, and clinical success of in vitro embryo production.(AU)

Lab partners: oocytes, embryos and company. A personal view on aspects of oocyte maturation and the development of monozygotic twins

The present review addresses the oocyte and the preimplantation embryo, and is intended to highlight the underlying principle of the "nature versus/and nurture" question. Given the diversity in mammalian oocyte maturation, this review will not be comprehensive but instead will focus on the porcine oocyte. Historically, oogenesis was seen as the development of a passive cell nursed and determined by its somatic compartment. Currently, the advanced analysis of the cross-talk between the maternal environment and the oocyte shows a more balanced relationship: Granulosa cells nurse the oocyte, whereas the latter secretes diffusible factors that regulate proliferation and differentiation of the granulosa cells. Signal molecules of the granulosa cells either prevent the precocious initiation of meiotic maturation or enable oocyte maturation following hormonal stimulation. A similar question emerges in research on monozygotic twins or multiples: In Greek and medieval times, twins were not seen as the result of the common course of nature but were classified as faults. This seems still valid today for the rare and until now mainly unknown genesis of facultative monozygotic twins in mammals. Monozygotic twins are unique subjects for studies of the conceptus-maternal dialogue, the intra-pair similarity and dissimilarity, and the elucidation of the interplay between nature and nurture. In the course of in vivo collections of preimplantation sheep embryos and experiments on embryo splitting and other microsurgical interventions we recorded observations on double blastocysts within a single zona pellucida, double inner cell masses in zona-enclosed blastocysts and double germinal discs in elongating embryos. On the basis of these observations we add some pieces to the puzzle of the post-zygotic genesis of monozygotic twins and on maternal influences on the developing conceptus.(AU)

Interação da sazonalidade e nutrição na viabilidade embrionária ovina / Interaction between seasonality and nutrition on embrio viability of ovines

A sazonalidade reprodutiva e a condição nutricional são os principais fatores que influenciam o desempenho reprodutivo em ovinos, principalmente em áreas onde a disponibilidade de alimentos é altamente sazonal. A desnutrição pode comprometer a competência folículo-oocitária, a função lútea e o desenvolvimento embrionário. Por outro lado, o tratamento com melatonina é um método eficaz para induzir ciclicidade, aumentando a ovulação e melhorando a viabilidade do embrião durante o anestro. Portanto, levantamos a hipótese de que a melatonina pode substituir os efeitos prejudiciais da desnutrição para melhorar o desempenho reprodutivo em ovelhas. A este respeito, esta revisão enfoca os efeitos da interação entre melatonina e nutrição na viabilidade do embrião e resume a informação disponível sobre o efeito da melatonina exógena na viabilidade do embrião em ovelhas desnutridas.(AU)

Reproductive seasonality and nutritional condition are the main factors that influence reproductive performance in sheep, particularly in the areas where the availability of food is highly seasonal. Undernutrition can compromise follicle-oocyte competence, luteal function and embryo development. On the other hand, melatonin treatment is an effective method for inducing estrous cycles, increasing ovulation and improving embryo viability during anestrus. Therefore, we hypothesized that melatonin can override the detrimental effects of undernutrition to improve reproductive performance in ewes. In this regard, this review focuses on the effects of the interaction between melatonin and nutrition on embryo viability and summarizes the available information about the effect of exogenous melatonin on embryo viability in undernourished ewes.(au)

The embryo non-invasive pre-implantation diagnosis era: how far are we?

Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters.(AU)

Maternal contributions to pregnancy success: from gamete quality to uterine environment

The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle.(AU)

Mecanismos epigenéticos nos espermatozoides e o impacto sobre a fertilidade masculina / Epigenetic mechanisms on sperm cells and the impact on male fertility

Por ser uma célula altamente especializada, o espermatozoide apresenta diferentes mecanismos epigenéticos, sendo os principais as metilações do DNA, o código de histonas, os ncRNAs (RNAs não codificadores), e a alta condensação da cromatina pela presença das protaminas. Estes mecanismos interagem entre si, contribuindo para a formação do epigenoma espermático, que modela a carga molecular espermática, que, por sua vez, pode impactar sobre as características do desenvolvimento embrionário e da progênie. Dessa forma, atualmente é consenso que o papel do espermatozoide ultrapassa a entrega de DNA de qualidade para o oócito no momento da fecundação. Pesquisas recentes de diversos grupos, incluindo o nosso, mostram que além da contribuição com DNA de qualidade, o espermatozoide entrega moléculas ao oócito no momento da fecundação que influenciam o desenvolvimento do embrião. Recentemente, essas moléculas de origem espermática (Em inglês: sperm-borne) também são associadas com alterações metabólicas e cognitivas da progênie. Embora ainda pouco se entenda como esses mecanismos podem persistir mesmo com o ciclo de reprogramação celular que ocorre logo após a fecundação, é evidente que estes podem impactar as características da progênie. Nesta revisão abordaremos sobre a modulação do epigenoma espermático e seus efeitos no desenvolvimento embrionário.(AU)

Since it is a highly specialized cell, the spermatozoa display different epigenetic mechanisms; the main ones are DNA methylation, histone code, ncRNAs (non-coding RNAs), and high chromatin condensation by the presence of protamines. These mechanisms act in synergy contributing to forming the sperm epigenome, which modulates the spermatic molecular cargo, and, may impact embryo and offspring development features. Thus, it is currently a consensus that the role of spermatozoa goes beyond delivering quality DNA to the oocyte at fertilization. Relevant findings from several research groups, including ours, have shown that sperm delivers several molecules to the oocyte at fertilization, beyond the contribution to DNA, which influences the development of the embryo. Recently, these sperm-borne molecules have also been associated with metabolic and cognitive changes in the offspring. Although the mechanism by which these changes can persist even after embryo reprogramming is not completely understood, evidence shows that sperm cell molecular content impacts embryo and offspring development. This review will mainly focus on the modulation of the sperm epigenome and its effects on embryo development.(AU)

Effect of GnRH administration on pregnancy-associated glycoproteins in dairy sheep with different reproductive status

Background: Measurement of pregnancy-associated glycoproteins (PAGs) by radioimmunoassay or enzyme-linked immunosorbent assay has been commonly used for early pregnancy diagnosis in ruminants. However, an accurately pregnancy detection depends on test antibody, breed and number of embryos. Only few studies have conducted to detect or predict animals at risk of late embrionic mortality (LEM) and to use hormonal interventions for embryo losses reducing, but this area is still open in sheep. The current study aimed to evaluate the effect of gonadotropin releasing hormone (GnRH) administration on Day 4 after artificial insemination on the pregnancy-associated glycoproteins in dairy sheep with different reproductive status in relation to pregnancy and late embrionic mortality detection. Materials, Methods & Results: Sixty-five East Friesian sheep were divided in 2 groups - I (Control group, n = 35) and II (GnRH group, n = 30) and sublected to estrus synchronization and artificial insemination (AI). Group I was not treated and Group II received 50 µg GnRH on Day 4 after AI. PAGs in blood serum were measured by Alertys Ruminant Pregnancy test on Days 4, 12, 20, 25 and 35 after AI and ultrasound pregnancy test was conducted on Days 20, 25, 35 and 60. Reproductive status (non-pregnant, pregnant and animals with LEM) was determined by ultrasound and the results between different groups were compared. The PAGs mean values according to reproductive status and Day after AI were analysed. Accuracy, sensitivity and specificity of the PAGs test for pregnancy diagnosis on Day 35 were calculated. On Day 20 after AI the pregnat sheep (83.3%) in group I tended to be higher than those (77.1%) in group II with decrease of 25.7% and 20% for the same groups on Day 25. On day 25 LEM was recorded in 33.3% and 24% in group I and II, respectively. The ultrasound exams on Day 60 confirmed the results from Day 25 after AI. A total value of non-pegnant, pregnant animals and LEM was 20%, 56.9% and 28.8%, respectively. The mean values of PAGs in animals with the same reproductive status in group I and II no differed statistically between Days 4 and 35 after AI. On Day 25 the PAGs values in LEM groups (0.126 ± 0.072 and 0.179 ± 0.029) were higher than those (0.062 ± 0.038 and 0.083 ± 0.023) in the non-pregnant groups, but no significan difference was deternined. On Day 35 after AI the mean values of PAGs (0.414 ± 0.125 and 0.421 ± 0.121) for the pregnant groups were significantly (P < 0.05) higher than those (0.078 ± 0.053 and 0.093 ± 0.034) for the non-pregnant groups. The values of PAGs in LEM groups on Day 25 (0.099 ± 0.062 and 0.113 ± 0.058) were decreased and close to the mean values in non-pregnant sheep. A significant effect of the day after AI on the PAGs values was evidenced in pregnant animals only (control r = 0.97 and GnRH r = 0.98; P < 0.05). The PAGs started to rise rapidly between Days 25 and 35 after AI. On Day 35 the accuracy (98%), the sensitivity (97.3%) and the specificity (100%) of the PAG test for pregnancy diagnosis were similar to the values (100%) for ultrasound method. Discussion: The gonadotropin treatment on Day 4 after AI tended to improve the reproductive performance in the sheep, but no significant effect of GnRH on the PAGs values in the animals with different reproductive status was found. The PAGs profiles in non-pregnant and LEM sheep were close and distinguishing between non-pregnant and LEM ewes on Day 35 was impossible. The PAGs values in pregnant sheep showed significantly (P < 0.05) increase between Days 25 and 35 after AI with higher mean values in pregnant than non-pregnant groups on Day 35 (P < 0.05). Alertys Ruminant Pregnancy test was a reliable for pregnancy detection in sheep on Day 35 after artificial insemination.

Pancreatic cancer orthotopic graft in a murine model

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with increasing incidence. Even if progress have been made, the five-year overall survival remains lower than 10%. There is a desperate need in therapeutic improvements. In the last two decades, new in-vitro models have been developed and improved, including tridimensional-culture spheroids and organoids. However, animal studies remain mandatory in the upscaling before clinical studies. Orthotopic and syngeneic grafting is a robust model to test a drug efficiency in a tumor and its microenvironment. Methods: We described a method for orthotopic and syngeneic graft of KRAS mutated, p53 wildtype, 8305 cells in a C57BL/6J mouse model. Results: With this microsurgical method, 30 mice were grafted, 24 by a junior and six by a senior, resulting in 95,8 and 100% of (partial and total) successful tumoral implantation, respectively. Twenty mice underwent ultrasound follow-up. It was an efficient method for the tumoral growth evaluation. At day 16 after grafting, 85% of the tumors were detectable by ultrasound, and at day 22 all tumors were detected. Conclusions: The presented method appears to be a robust and reliable method for pre-clinical studies. A junior master student can provide positive results using this technique, which can be improved with training.

Embryo biopsies for genomic selection in tropical dairy cattle

Genomic selection has transformed the livestock industry, enabling early-life selection of animals. Biopsy sampling of pre-implantation embryos has been described since 1968. However, it was only after 2010, with the advancement of molecular biology techniques such as whole genomic amplification and SNP Chips, that next-generation sequencing became commercially available for bovine embryos. It is now possible to make decisions about which embryos to transfer not only based on recipients' availability or embryo morphology but also on genomic estimates. This technology can be implemented for a wide spectrum of applications in livestock. In this review, we discuss the use of embryo biopsy for genomic selection and share our experience with Gir and Girolando Brazilian breeding programs, as well as future goals for implementing it in Brazilian bovine in vitro embryo production practices.(AU)

Utilização de filamentos de ouro em pontos de acupuntura em lobo-guará (Chrysocyon brachyurus) com espondilose / Use of gold filaments in acupuncture points in maned wolf (Chrysocyon brachyurus) with spondylosis / Implantación de filamentos de oro en puntos de acupuntura en aguará guazú (Chrysocyon brachyurus) com espondilosis

Um lobo-guará (Chrysocyon brachyurus) macho com 15 anos de idade apresentou dificuldade ao se levantar e deitar, relutância ao caminhar e aspecto arqueado da coluna vertebral. Após contenção química, o paciente foi examinado e submetido a exames radiográficos e colheita de material biológico para análises laboratoriais. Os resultados dos exames apontaram um animal saudável, porém visualizaram-se espondiloses em coluna vertebral na região cervical e torácica. Foi iniciado protocolo para controle de dor, inflamação e uso de regenerador articular, a fim de estabilizar o quadro do paciente. Novo manejo foi agendado para implante de filamentos de ouro em pontos de acupuntura. Após essa terapia, o paciente foi acompanhado pela equipe veterinária, e alguns dias após o tratamento houve melhora do quadro clínico o animal deixou de apresentar dificuldade para caminhar, deitar, levantar e ficar em estação. O paciente retornou ao recinto, dividindo o local com a fêmea, e até o momento está assintomático.(AU)

A 15-year-old male maned wolf (Chrysocyon brachyurus) showed difficulty getting up and lying down, reluctance to walk and an arched aspect of the spine. After chemical containment, the patient underwent physical examination and was submitted to radiographic examens and collection of biological material for laboratory analysis. The test results indicated a healthy animal, but spondylosis was seen in the cervical and thoracic spine. A protocol was started to pain control, inflammation, and use of condroprotective to stabilize the patient's condition. A new management was scheduled for the implantation of gold filaments in acupuncture points. After this therapy, the patient was followed up by the veterinary team, and days after the treatment, there was an improvement in the clinical condition- the animal had no more difficulty walking, lying down, getting up and standing still. The patient returned to the enclosure, sharing the place with the female, and so far, the clinical signs presented are absent and/or reduced.(AU)

Un aguará guazú (Chrysocyon brachyurus) macho de 15 años mostró dificultad para levantarse y acostarse, renuencia a caminar y un aspecto arqueado de la columna vertebral. Después de la contención química, el paciente fue examinado y sometido a exámenes radiográficos y recolección de material biológico para análisis de laboratorio. Los resultados de la prueba indicaron un animal sano, pero se observó espondilosis en la columna cervical y torácica. Se inició protocolo de control del dolor, inflamación y uso de protectores articulares, con el fin de estabilizar el estado del paciente. Con el objetivo de brindar una mejor calidad de vida, ya que se trata de un animal salvaje, incapaz de realizar ordinariamente terapias invasivas, se implantaron filamentos de oro en puntos de acupuntura. Posteriormente a esta terapia, el paciente fue acompañado por el equipo veterinario, y dias después del tratamiento hubo una mejoría en el estado clínico - el animal no tuvo dificultad para caminar, acostarse, levantarse y estar de pie. El paciente regresó al recinto, compartiendo el lugar con una hembra, y hasta el momento los signos clínicos anteriormente presentados están ausentes y/o reducidos.(AU)

Persistência do vítreo primário hiperplásico e catarata em cão / Persistence of hyperplastic primary vitreous and cataract in a dog

Background: Persistent hyperplastic primary vitreous is a rare ocular condition that is usually unilateral, formed when normal regression of the hyaloid vascular system does not occur. Diagnosis is possible through ultrasonography, by obtaining images that provide information and also serve as a differential diagnosis. Clinically the condition presents with signs of leukocoria, microphthalmia, and cataracts, and it can be further classified into 6 degrees according to the evolution. The objective of this work is to describe the case and treatment of a canine of the Pointer breed that presented opacity in both eyes, with diagnosis confirmed through ocular ultrasonography. Case: A 6-year-old male pointer dog, weighing 27 kg, was referred to the Veterinary Medical Teaching Hospital of the Federal University of Santa Maria (UFRGS), Santa Maria, RS - Brazil with a history of blindness. According to the owner, the animal's right eye has always been "white", since it was a puppy and the left eye began to present the same characteristic a short time ago. On ophthalmological examination, neither eye showed signs of ocular discomfort, the Schirmer's tear test was within the range expected for the species, fluorescein and threat tests were negative, and a positive pupillary reflex was observed in the left eye. The right eye had total corneal opacity associated with a chronic lesion and the presence of pigment cells in the medial portion of the eyeball, the opacity making it impossible to visualize the other internal structures of the eyeball. The left eye presented lens opacity. In the ultrasound of the right eye, there were several alterations, the presence of a triangular-shaped hyperechogenic structure in the vitreous chamber, a decrease in the depth of the anterior chamber, and the presence of abnormality in the development of the lens. The findings are compatible with and confirmed the diagnosis of PHPV. In the left eye, the ultrasound images indicated hyperechogenicity of the posterior and anterior poles of the lens, findings compatible with cataracts. Through the animal's history, complete ophthalmic examination, and use of ocular ultrasonography, it was possible to diagnose and differentiate the causes of the white eye in the patient. The preoperative screening was continued with an electroretinography examination, which showed a satisfactory electrical response for the retina of the left eye and an unsatisfactory electrical response for the retina of the right eye. Phacoemulsification surgery was performed to remove the cataract in the left eye with intraocular lens implantation. The positive threat reflex returned in this eye, with a return of visual acuity. Discussion: Persistent hyperplastic tunica lentis and persistent hyperplastic primary vitreous (PHTVL/PHPV) are congenital anomalies caused by a failure to regress the fetal hyaloid vasculature. These conditions are not common, but occur sporadically in dogs. The PHPV classification can be classified into degrees, based on the morphological aspect of the lesion. Following this classification scheme, the PHPV of the animal in question refers to the 6th degree of evolution. The total opacity of the cornea in the right eye was associated with the presence of a hyperplastic primary vitreous and the leukocoria in the left eye was due to the presence of a mature cataract. The conclusion of the diagnosis was only possible through ocular ultrasonography, which is very useful to differentiate the causes of ocular opacity and leukocoria, in addition to allowing complete evaluation of the intraocular structures and being considered an essential exam to confirm the diagnosis of PHPV.