Abstract in English:Abstract Due to the peculiarities of the reproductive cycle of the female dog, which makes it difficult to accurately ascertain the date of conception, it may be challenging to precisely estimate the gestational age in bitches. Using fetal measurements obtained by ultrasound, it is possible to estimate the gestational age in dogs; however, due to the differences in size of the various breeds, such estimates may have a significant standard deviation, which represents less accuracy when specifying the date of birth. The purpose of this study was to evaluate pregnant female Chihuahuas, establishing relations between the fetal dimensions measured by ultrasound and the remaining time until delivery. Using 13 pregnant female Chihuahuas, weekly ultrasound assessments and measurements were performed, of the inner chorionic cavity, cranial-caudal length, biparietal diameter, abdominal diameter and thoracic diameter. Such parameters were retroactively correlated to the date of delivery, and linear regressions were established between the gestational measurements and remaining days until delivery. The fetal measurement presenting the highest correlation (r = 0.99; P<0.0001) and reliability (R2 = 0.98, P<0.0001) with the probable date of delivery was the biparietal diameter, a measurement that can be easily and safely obtained and, when applying a specific formula (Y = -15.46X1 + 38.72), can accurately predict the date of delivery in Chihuahua female dogs.
Abstract in English:Abstract Coenzyme Q9 (COQ9), a coenzyme Q (CoQ) precursor, is an essential component of the mitochondrial electron transport chain that drives adenosine triphosphate production. COQ9 polymorphism 18:25527339 is characterized by substitution of guanine (allele G) for adenine (allele A), which modifies the function of the protein encoded by the gene. In Holsteins, allele A has been associated with better reproductive performance in terms of the conception rate, number of services per conception (SPC) and days open (DO). The signal transducer and activator of transcription (STAT) protein is a transcription factor activated in the presence of cytokines and growth factors. STAT5A polymorphism 19:42407732 in exon 8 has been associated with higher fertility and embryonic survival rates. The objective of this study was to determine the relationship of COQ9 and STAT5A polymorphisms with reproductive parameters [calving to first heat interval (CFHI), DO and SPC]. Blood samples were taken from 112 lactating Holstein from a herd in México for allele genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). To estimate the association between reproductive parameters and genotypes, a linear mixed-effect model was performed. The COQ9 AG genotype was associated significantly with lower SPC (P<0.05) but not with DO or CFHI. No significant association with any reproductive parameter was found for STAT5A. Our findings suggest that the COQ9 18:25527339 polymorphism is a useful molecular marker for improvement of reproductive performance in dairy herds.
Abstract in English:Abstract Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.
Abstract in English:Abstract The objective of this study was to investigate the influence of long-term temperature stress during the in vitro maturation (IVM) of oocytes on the in vitro embryo production (IVP) and the abundance of HSP70 and HSP90 in zebu cattle. Viable cumulus-oocyte complexes (COCs) were incubated for 24 h at 37 °C, 38.5 °C, or 40 °C for the low-, physiological, and high-temperature stress treatments, respectively. Thereafter, they were subjected to in vitro fertilization and culture. Temperature did not affect the polar body extrusion. However, IVP was adversely affected when IVM took place at 37 °C and 40 °C. The highest abundance of HSP70 was observed in cumulus cells after maturation of COCs at 40 °C. In contrast, HSP70 was more abundant in oocytes at both 37 °C and 40 °C; however, at 40 °C, the difference to the control group (38.5 °C) was not significant. In contrast, the highest abundance of HSP90 was observed in oocytes and cumulus cells at 37 °C. It appears that HSP70 and HSP90 respond to cold and heat stress in different ways. In conclusion, moderately high (40 °C) and low (37 °C) thermal stress for 24 h during IVM is detrimental to the developmental competence of oocyte and is accompanied by changes in the abundances of HSP70 and HSP90, especially in cumulus cells.
Abstract in English:Abstract The objective of the present study was to determine the effect of follicle size on recovery rate, quality, and in-vitro developmental competence of oocytes in Bos indicus cows. The ovaries (n = 507) of Bos indicus cows having age of 5-8 years, with mixed parity, BCS 2.75 ± 0.25, and clinically normal reproductive tracts were collected from the local abattoir. The follicles on the ovaries were divided into two groups based upon their size; 1) ≥6 mm diameter, and 2) <6 mm diameter. After initial evaluation of quality of the oocytes, the COCs were in vitro matured, fertilized, and cultured to determine the in vitro developmental competence. The oocyte recovery, quality, maturation, cleavage, 4-cell, 8-cell, and 16-cell stages were analyzed using PROC GLIMMIX procedure of SAS. However, the number of oocytes recovered per ovary was analyzed using MIXED procedure of SAS. Results revealed that the recovery of oocytes (LSM ± SEM) derived from the follicles having size <6 mm per ovary was greater (1.02 vs. 3.14 ± 0.13; P < 0. 0001). However, the percentage (n/n) recovery [69.8 (474/679) vs. 62.7% (1454/2320); P = 0.01] and grade I_+_II oocytes [68.4 (324/474) vs. 57.9% (842/1454); P < 0.0001] was greater in ≥6 mm as compared with <6 mm group, respectively. However, maturation rate did not differ [92.9 (288/310) vs. 92.2% (296/321); P = 0.98] between the groups. In contrast, cleavage rate [58.1 (180/310) vs. 47.4% (152/321); P = 0.01], the 4-cell [34.5 (107/310) vs. 18.7% (60/321); P = 0.0003], 8-cell [15.5 (48/310) vs. 7.8% (25/321); P = 0.008], and 16-cell [8.7 (27/310) vs. 2.1% (7/321); P = 0.004] stage embryos were greater in ≥6 mm group. It can be concluded that oocytes derived from follicle ≥6 mm have better in vitro developmental competence based on embryonic conversion in Bos indicus cows.
Abstract in English:Abstract The creation of a genetic resource bank of avian species aims to prevent the decline and fragmentation of wild bird populations, which in turn lead to the loss of genetic diversity and, in more serious cases, the extinction of the most threatened species. In order for the collected genetic material to be stored in a bank and useful when necessary, it is essential to improve the technique ensuring its effectiveness. Thus, our study used feather follicle cells from the domestic gallus species to standardize the technique of cell culture and subsequent cryopreservation. This study aimed to establish a protocol, in vitro, of isolation and primary culture of somatic cells derived from the feather follicle, with the purpose of establishing a cell lineage, and evaluate its viability for the biobank formation. Developing feathers of gallus domesticus were collected at 12, 21 and 34 days of age. The feathers were morphologically analyzed and then we selected the region of the calamus due to the presence of pulp for cell culture and cryopreservation. The results showed that it is possible to find cells with distinct morphology; cells in elliptical shape with central nucleus also in elliptical shape, cells with shape and round nucleus, cells compatible with the fibers of the barbules, cell agglomerates and cells adhered to the bottom of the plate with fibroblastatoid shape. After 24 hours of culture there was the presence of primary culture with 80% of confluence and after cryopreservation the average viability after freezing was 68.8%, with cellular morphologies being maintained. Therefore, we proved the isolation of somatic cells from the follicle of bird’s feathers, suggesting that this is a source of great value, viable and effective for obtaining biological material for the elaboration of a biobank.
Abstract in English:Abstract Seminal plasma (SP) supports not only sperm function but also the ability of spermatozoa to withstand biotechnological procedures as artificial insemination, freezing or sex sorting. Moreover, evidence has been provided that SP contains identifiable molecules which can act as fertility biomarkers, and even improve the output of assisted reproductive technologies by acting as modulators of endometrial and embryonic changes of gene expression, thus affecting embryo development and fertility beyond the sperm horizon. In this overview, we discuss current knowledge of the composition of SP, mainly proteins and cytokines, and their influence on semen basic procedures, such as liquid storage or cryopreservation. The role of SP as modulator of endometrial and embryonic molecular changes that lead to successful pregnancy will also be discussed.
Abstract in English:Abstract Understanding preimplantation embryonic development is crucial for the improvement of assisted reproductive technologies and animal production. To achieve this goal, it is important to consider that gametes and embryos are highly susceptible to environmental changes. Beyond the metabolic adaptation, the dynamic status imposed during follicular growth and early embryogenesis may create marks that will guide the molecular regulation during prenatal development, and consequently impact the offspring phenotype. In this context, metaboloepigenetics has gained attention, as it investigates the crosstalk between metabolism and molecular control, i.e., how substrates generated by metabolic pathways may also act as players of epigenetic modifications. In this review, we present the main metabolic and epigenetic events of pre-implantation development, and how these systems connect to open possibilities for targeted manipulation of reproductive technologies and animal production systems.
Abstract in English:Abstract Assisted reproductive technologies such as artificial insemination have delivered significant benefits for farm animal reproduction. However, as with humans, assisted reproduction in livestock requires the manipulation of the gametes and preimplantation embryo. The significance of this ‘periconception’ period is that it represents the transition from parental genome regulation to that of the newly formed embryo. Environmental perturbations during these early developmental stages can result in persistent changes in embryonic gene expression, fetal organ development and ultimately the long-term health of the offspring. While associations between maternal health and offspring wellbeing are well-defined, the significance of paternal health for the quality of his semen and the post-conception development of his offspring have largely been overlooked. Human and animal model studies have identified sperm epigenetic status (DNA methylation levels, histone modifications and RNA profiles) and seminal plasma-mediated maternal uterine immunological, inflammatory and vascular responses as the two central mechanisms capable of linking paternal health and post-fertilisation development. However, there is a significant knowledge gap about the father’s contribution to the long-term health of his offspring, especially with regard to farm animals. Such insights are essential to ensure the safety of widely used assisted reproductive practices and to gain better understanding of the role of paternal health for the well-being of his offspring. In this article, we will outline the impact of male health on semen quality (both sperm and seminal plasma), reproductive fitness and post-fertilisation offspring development and explore the mechanisms underlying the paternal programming of offspring health in farm animals.
Abstract in English:Abstract The use of in-vitro produced (IVP) embryo transfer (ET) in dairy herds is growing fast. Much of this growth is on dairy farms where the focus is on milk production and not on selling breeding stock. The value of implementing IVP-ET in a dairy herd arises from a higher genetic merit of the IVP-embryo, but the cost to produce a pregnancy with an IVP embryo is greater than the cost of artificial insemination (AI). The first objective of this study was to review estimates of the net benefit of using IVP-ET over AI in dairy herds using existing literature. Another objective was to show how much IVP-ET use in a herd is optimal. Most of the literature is based on simulation modeling, including our own work that focuses on the dairy industry in the USA. We found that the most profitable use of AI and IVP-ET is often a combination of the two. More IVP-ET should be used when the value of surplus calves is greater and the cost of IVP-ET is lower, among many other factors. In the future, use of IVP-ET will be further improved by more accurately identifying superior donors and recipients, reducing the generation interval, and achieving greater efficiency in embryo production.
Abstract in English:Abstract The environment contributes to production diseases that in turn badly affect cow performance, fertility and culling. Oestrus intensity is lower in lame cows, and in all cows 26% potential oestrus events are not expressed (to avoid getting pregnant). To understand these trade-offs, we need to know how animals react to their environment and how the environment influences hypothalamus-pituitary-adrenal axis (HPA) interactions with the hypothalamus-pituitary-ovarian axis (HPO). Neurotransmitters control secretion of GnRH into hypophyseal portal blood. GnRH/LH pulse amplitude and frequency drive oestradiol production, culminating in oestrus behaviour and a precisely-timed GnRH/LH surge, all of which are disrupted by poor environments. Responses to peripheral neuronal agents give clues about mechanisms, but do these drugs alter perception of stimuli, or suppress consequent responses? In vitro studies confirm some neuronal interactions between the HPA and HPO; and immuno-histochemistry clarifies the location and sequence of inter-neurone activity within the brain. In both species, exogenous corticoids, ACTH and/or CRH act at the pituitary (reduce LH release by GnRH), and hypothalamus (lower GnRH pulse frequency and delay surge release). This requires inter-neurones as GnRH cells do not have receptors for HPA compounds. There are two (simultaneous, therefore fail-safe?) pathways for CRH suppression of GnRH release via CRH-Receptors: one being the regulation of kisspeptin/dynorphin and other cell types in the hypothalamus, and the other being the direct contact between CRH and GnRH cell terminals in the median eminence. When we domesticate animals, we must provide the best possible environment otherwise animals trade-off with lower production, less intense oestrus behaviour, and impaired fertility. Avoiding life-time peri-parturient problems by managing persistent lactations in cows may be a worthy trade-off on both welfare and economic terms – better than the camouflage use of drugs/hormones/feed additives/intricate technologies? In the long term, getting animals and environment in a more harmonious balance is the ultimate strategy.
Abstract in English:Abstract Endocrine-disrupting compounds (EDCs) and foodborne contaminants are environmental pollutants that are considered reproductive toxicants due to their deleterious effects on female and male gametes. Among the EDCs, the phthalate plasticizers are of growing concern. In-vivo and in-vitro models indicate that the oocyte is highly sensitive to phthalates. This review summarizes the effects of di(2-ethylhexyl) phthalate and its major metabolite mono(2-ethyhexyl) phthalate (MEHP) on the oocyte. MEHP reduces the proportion of oocytes that fertilize, cleave and develop to the blastocyst stage. This is associated with negative effects on meiotic progression, and disruption of cortical granules, endoplasmic reticulum and mitochondrial reorganization. MEHP alters mitochondrial membrane polarity, increases reactive oxygen species levels and induces alterations in genes associated with oxidative phosphorylation. A carryover effect from the oocyte to the blastocyst is manifested by alterations in the transcriptomic profile of blastocysts developed from MEHP-treated oocytes. Among foodborne contaminants, the pesticide atrazine (ATZ) and the mycotoxin aflatoxin B1 (AFB1) are of high concern. The potential hazards associated with exposure of spermatozoa to these contaminants and their carryover effect to the blastocyst are described. AFB1 and ATZ reduce spermatozoa's viability, as reflected by a high proportion of cells with damaged plasma membrane; induce acrosome reaction, expressed as damage to the acrosomal membrane; and interfere with mitochondrial function, characterized by hyperpolarization of the membrane. ATZ and AFB1-treated spermatozoa show a high proportion of cells with fragmented DNA. Exposure of spermatozoa to AFB1 and ATZ reduces fertilization and cleavage rates, but not that of blastocyst formation. However, fertilization with AFB1- or ATZ-treated spermatozoa impairs transcript expression in the formed blastocysts, implying a carryover effect. Taken together, the review indicates the risk of exposing farm animals to environmental contaminants, and their deleterious effects on female and male gametes and the developing embryo.
Abstract in English:Abstract Researchers, veterinarians, and farmers' pursuit of a consistent diagnosis, treatment, and prevention of uterine diseases remains challenging. The diagnosis and treatment of metritis is inconsistent, a concerning situation when considered the global threat of antimicrobial resistance dissemination. Endometritis is an insidious disease absent on routine health programs in many dairy farms and from pharmaceutical therapeutics arsenal in places like the US market. Conversely, a multitude of studies advanced the understanding of how uterine diseases compromise oocyte, follicle, and embryo development, and the uterine environment having long-lasting effects on fertility. The field of uterine disease microbiome also experienced tremendous progress and created opportunities for the development of novel preventives to improve the management of uterine diseases. Activity monitors, biomarkers, genomic selection, and machine learning predictive models are other innovative developments that have been explored in recent years to help mitigate the negative impacts of uterine diseases. Albeit novel tools such as vaccines for metritis, immune modulators, probiotics, genomic selection, and selective antimicrobial therapy are promising, further research is warranted to implement these technologies in a systematic and cost-effective manner.
Abstract in English:Abstract Reproductive efficiency is critically dependent on embryo survival, establishment of a successful pregnancy and placental development. Recent advances in gene editing technology have enabled investigators to use gene knockdown and knockout approaches to better understand the role of hormone signaling in placental function and fetal growth and development. In this review, an overview of ruminant placentation will be provided, including recent data highlighting the role of histone lysine demethylase 1A and androgen signaling in ruminant placenta and pregnancy. Studies in ruminant placenta establish a role for histone lysine demethylase 1A in controlling genetic networks necessary for important cellular events such as cell proliferation and angiogenesis, as well as androgen receptor signaling during early placentation.
Abstract in English:Abstract Genomic evaluations have revolutionized dairy cattle breeding, and the demand for embryos produced from very young heifers with high genetic merit has increased over time. The combination of low oocyte recovery, young age of donors, and milk production status can make the in vitro embryo production (IVP) of Holstein cattle incredibly challenging. Several factors need to be coordinated to obtain a live calf from an IVP embryo, but the quality of the oocyte at the start of the process is one of the key factors. Aspects related to oocyte quality, laboratory quality control, embryo quality and recipient selection are addressed here, based on the measures that the RuAnn Genetics Laboratory (Riverdale, California, USA) adopted in the last 12 years, with the goal of improving production of live, healthy calves from Holstein embryos. Follicular wave synchronization and stimulation with follicular stimulating hormone (FSH) is necessary to improve oocyte quality and consequently embryo production. Laboratory quality control and the use of high-quality supplies are essential to reduce variability in production and facilitate identification of other factors that might interfere with embryo production. High pregnancy rates can be achieved with good quality embryos selected at optimal time and stage of development, transferred by an experienced embryo transfer (ET) technician, to well managed recipients 7 or 8 days after estrus. Attention to detail at every step of the process is crucial to success.
Abstract in English:Abstract The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this ‘translational gap’, we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.
Abstract in English:Abstract Reproductive failure and pregnancy loss in cattle are some of the largest economic burdens to cattle producers and one of most perplexing factors influencing management decisions. Pregnancy loss may occur at any point during gestation with the largest percentage of loss occurring in the first 30 days and, subsequently, decreasing as the pregnancy progresses. Losses may be attributed to numerous factors, predisposed issues or environmental conditions such as nutritional stressors or disease. From a research perspective, determining the exact causes of pregnancy loss or embryonic mortality in cattle have been difficult, due to limitations of accurately determining early gestation pregnancy status. Until methods that precisely determine embryo success early in gestation are available, our understanding of in vivo pregnancy loss will lack clarity necessary to develop management strategies to decrease such loss. In this review, we will briefly discuss the pivotal periods of pregnancy loss affecting beef and dairy cattle, methods and technologies to determine pregnancy status and embryo viability and potential opportunities to decrease reproductive failure.
Abstract in English:Abstract Developmental biology seeks to understand the sophisticated regulated process through which a single cell – a fertilized egg – generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.