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Studies investigating physiological deviations from normality in newborn calves derived from in vitro fertilization procedures remain important for the understanding of factors that reduce calf survival after birth. The aim of this study was to investigate parameters affecting health and welfare of newborn Flemish calves derived from in vitro embryo production (IVP) in the first hours of life in comparison to in vivo-derived calves. Physical traits of newborn calves and fetal membranes (FM) were recorded soon after birth. Newborn venous blood samples were collected at several time points within the first 24 h of life for analyses of energy substrates, electrolytes, blood gases, acid-base balance, blood chemistry, and haematology. A liver biopsy was taken within the first hour after birth for analysis of gene expression of key enzymes of the fructolytic and glycolytic pathways. Newborn IVP calves were heavier and larger at birth, which was associated with heavier FM. At several time points during the first 24 h of life, IVP-derived calves had altered rectal temperature, blood gases, electrolyte concentrations, blood parameters for liver, kidney and muscle function, and acid-base balance, plasma lipid metabolism, and hemogram parameters. The relative mRNA abundances for triokinase and lactate dehydrogenase-B were greater in IVP calves. In summary, IVP-derived newborn calves were at higher risk of clinical problems after birth, which was markedly greater in heavier and larger calves. Such animals take longer to adapt to extrauterine life and should receive a special attention during the immediate neonatal period.

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The present study evaluated the general welfare state of two strains of transgenic goats bred in a region with a hot and humid tropical climate. Nine females were used, being three transgenic for human lysozyme (hLZ group), three transgenic for human glucocerebrosidase (hGCase group), and three non-transgenic (control group). The temperature and humidity index (THI) were recorded during the morning, afternoon, and evening. The physiological parameters measured were respiratory rate, heart rate, and rectal and vaginal temperatures. Venous blood samples were collected using Vacutainer® tubes containing 10% ethylenediaminetetraacetic acid (EDTA). Also, analysis of erythrogram, leukogram, and some biochemical parameters of serum was performed. It was observed that the afternoon shift presented the largest THI, being potentially more impactful on the physiology of animals. In general, respiratory and heart rates were higher in transgenic animals, especially in the hLZ group compared to the control group (P < 0.05). Regarding the hematological parameters, the quantification of red blood cells, hemoglobin, and hematocrit was significantly lower (P < 0.05) in the hGCase group compared to that in the hLZ and control. The leukocyte count was considerably lower (P < 0.05) in the hLZ group compared to that in the hGCase and control. Correlation analysis showed that the increase in THI was associated with a change in physiological parameters normally used as indicators of thermal stress. Despite the differences found among the experimental groups, all the physiological parameters remained within the normal limits recommended for the goat species. Further studies involving a larger number of animals from different categories should be carried out to elucidate the impacts that transgenesis can have on animal welfare under different THI conditions.

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Os equinos são animais muito utilizados em eventos esportivos, sendo, desta forma, propensos a sofrer acidentes que podem levar a lesões articulares e ósseas. Na rotina clínica, artifícios como o uso de raio X e ultrassonografia são de grande importância para que o correto diagnóstico seja feito e a conduta clínica possa ser melhor planejada. Além disso, alguns biomateriais, como o plasma rico em plaquetas (PRP) e células-tronco mesenquimais (CTMs), têm se inserido nos protocolos terapêuticos, demonstrando grande efetividade no tratamento desse tipo de lesão, devido à grande quantidade de fatores de crescimento presentes em ambos os biomateriais. O PRP é um derivado sanguíneo, caracterizado pela alta concentração plaquetária. Foi produzido a partir de duas centrifugações, a primeira para separar o plasma das hemácias e a segunda para concentrar as plaquetas. As CTMs foram isoladas de tecido adiposo, cultivadas, transportadas e aplicadas de forma autóloga, assim como o PRP. No presente relato, um equino, fêmea, Brasileiro de Hipismo com 8 anos, foi diagnosticado com desmopatia nos ligamentos colaterais da articulação interfalângica distal e foi tratado com PRP e CTMs, de forma associada e sequencial. Foi encontrada uma melhora do quadro clínico, significativa, em comparação aos dados encontrados na literatura, demonstrando grande potencialidade do uso associado de PRP e CTMs no tratamento de lesões ligamentares.

Horses are animals widely used in sporting events and are therefore prone to accidents that can lead to joint and bone injuries. In the clinical routine, devices such as the use of X-ray and ultrasound are of great importance for the correct diagnosis to be made and the clinical conduct can be better planned. In addition, some biomaterials such as platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) have been included in the therapeutic protocols, demonstrating great effectiveness in the treatment of this type of injury due to the large amount of growth factors present in both biomaterials. PRP is a blood derivative, characterized by high platelet concentration. It is produced from two centrifugations, the first to separate plasma from red blood cells and the second to concentrate platelets. MSCs were isolated from adipose tissue, cultured, transported and applied autologously, as well as PRP. In the present report, an 8-year-old female Brazilian equestrian horse was diagnosed with desmopathy in the collateral ligaments of the distal interphalangeal joint and was treated with PRP and MSCs in an associated and sequential manner. A significant improvement in the clinical picture was found in comparison to the data found in the literature, demonstrating great potential of the associated use of PRP and MSCs in the treatment of ligament injuries.

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The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

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OBJECTIVE: L-Asparaginase (ASNase) is an enzyme used in the treatment of acute lymphoblastic leukemia (ALL). As the therapeutic ASNases has bacterial origin, severe side effects are associated with its use, among them hypersensitivity and inactivation of the enzyme. In this context, the objective of this work was to produce a recombinant ASNase of bacterial origin in human cells in order to determine the presence and consequences of potential post-translational modifications on the enzyme. RESULTS: Recombinant ASNase was expressed in human cells with a molecular weight of 60 kDa, larger than in Escherichia coli, which is 35 kDa. N-glycosylation analysis demonstrated that the increased molecular weight resulted from the addition of glycans to the protein by mammalian cells. The glycosylated ASNase presented in vitro activity at physiological pH and temperature. Given that glycosylation can act to reduce antigenicity by masking protein epitopes, our data may contribute to the development of an alternative ASNase in the treatment of ALL in patients who demonstrate side effects to currently marketed enzymes.

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A maturação in vitro de oócitos submetidos ao processo de criopreservação, ainda compreende um desafio para o sucesso da reprodução assistida na medicina veterinária. Devido a isso, estudos são desenvolvidos a fim de identificar, amenizar e superar as limitações encontradas. Nesse sentido, objetivou-se realizar a avaliação da maturação in vitro de oócitos ovinos após criopreservação pelo método de congelação lenta. Para tanto, foram colhidos 204 ovários oriundos de 102 ovelhas púberes (SPRD) pertencentes a abatedouros localizados no município de Teresina, Piauí. Os ovários foram transportados para o laboratório e, posteriormente, foram aspirados por meio de um aspirador cirúrgicoadaptado. Um total de 180 oócitos foram desnudados e, então, submetidos à congelação lenta em sistema automatizado (TK 3000®). Posteriormente foram descongelados, e submetidos à maturação in vitro(MIV). Em seguida, procedeu-se a avaliação da maturação nuclear. Os resultados foram avaliados por meio do teste de Qui-quadrado de Pearson (P ≤ 0,05). Após descongelação, 22,8% dos oócitos na avaliação em estereomicroscópio (45x) apresentavam lesões de zona pelúcida e de oolema. Dos 139 oócitos submetidos a MIV, oito maturaram (5,75%). Conclui-se que a congelação lenta de oócitos ovinos pode influenciar a maturação in vitro, devido a lesões de membrana plasmática e zona pelúcida.(AU)

The in vitro maturation of oocytes submitted to the cryopreservation process, still comprises a challenge for the success of assisted reproduction in veterinary medicine. Due to this, studies are developed in order to identify, ameliorate and overcome the limitations found. The objective of this study was to evaluate the in vitro maturation of ovine oocytes after cryopreservation by the slow freezing method. For that, 204 ovaries from 102 pubertal sheep (SPRD) belonging to slaughterhouses located in the city of Teresina, Piauí, were collected. The ovaries were transported to the laboratory and subsequently aspirated by means of an adapted surgical aspirator. A total of 180 CCO's were obtained, which were stripped and then subjected to slow freezing in an automated system (TK 3000®). Later they were thawed and submitted to in vitro maturation (IVM). Next, the nuclear maturation was evaluated. Results were evaluated using Pearson's chi-square test (P ≤ 0.05). After thawing, 22.8% of the oocytes in the stereomicroscope (45x) evaluation presented lesions of the zona pellucida and oolema. Of the 139 oocytes submitted to IVM, eight maturated (5.75%). It is concluded that slow freezing of sheep oocytesmay influence in vitro maturation due to plasma membrane and zona pellucida lesions.(AU)

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A maturação in vitro de oócitos submetidos ao processo de criopreservação, ainda compreende um desafio para o sucesso da reprodução assistida na medicina veterinária. Devido a isso, estudos são desenvolvidos a fim de identificar, amenizar e superar as limitações encontradas. Nesse sentido, objetivou-se realizar a avaliação da maturação in vitro de oócitos ovinos após criopreservação pelo método de congelação lenta. Para tanto, foram colhidos 204 ovários oriundos de 102 ovelhas púberes (SPRD) pertencentes a abatedouros localizados no município de Teresina, Piauí. Os ovários foram transportados para o laboratório e, posteriormente, foram aspirados por meio de um aspirador cirúrgicoadaptado. Um total de 180 oócitos foram desnudados e, então, submetidos à congelação lenta em sistema automatizado (TK 3000®). Posteriormente foram descongelados, e submetidos à maturação in vitro(MIV). Em seguida, procedeu-se a avaliação da maturação nuclear. Os resultados foram avaliados por meio do teste de Qui-quadrado de Pearson (P ≤ 0,05). Após descongelação, 22,8% dos oócitos na avaliação em estereomicroscópio (45x) apresentavam lesões de zona pelúcida e de oolema. Dos 139 oócitos submetidos a MIV, oito maturaram (5,75%). Conclui-se que a congelação lenta de oócitos ovinos pode influenciar a maturação in vitro, devido a lesões de membrana plasmática e zona pelúcida.

The in vitro maturation of oocytes submitted to the cryopreservation process, still comprises a challenge for the success of assisted reproduction in veterinary medicine. Due to this, studies are developed in order to identify, ameliorate and overcome the limitations found. The objective of this study was to evaluate the in vitro maturation of ovine oocytes after cryopreservation by the slow freezing method. For that, 204 ovaries from 102 pubertal sheep (SPRD) belonging to slaughterhouses located in the city of Teresina, Piauí, were collected. The ovaries were transported to the laboratory and subsequently aspirated by means of an adapted surgical aspirator. A total of 180 CCO's were obtained, which were stripped and then subjected to slow freezing in an automated system (TK 3000®). Later they were thawed and submitted to in vitro maturation (IVM). Next, the nuclear maturation was evaluated. Results were evaluated using Pearson's chi-square test (P ≤ 0.05). After thawing, 22.8% of the oocytes in the stereomicroscope (45x) evaluation presented lesions of the zona pellucida and oolema. Of the 139 oocytes submitted to IVM, eight maturated (5.75%). It is concluded that slow freezing of sheep oocytesmay influence in vitro maturation due to plasma membrane and zona pellucida lesions.

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

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Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...]

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Background: The production of transgenic animals has been envisioned as a viable strategy to improve food quality, animal yield, and for the production of bioproducts that can be used for the benefit of the human and animal population. Transgenic animals have been used to improve production traits, to add value to animal products, to minimize the impact on the environment, to promote disease resistance, and most notably, to produce recombinant proteins in natural fluids, such as milk, that can be collected, purified and used as biomedical products (biopharming). This review aims to discuss past and recent technological advances in animal transgenesis, and the perspective for biopharming in Brazil.Review: Since the production of recombinant human insulin from Escherichia coli in the 1970s, continuous development of new platforms has allowed a significant expansion in the biopharmaceutical market. The animal platform has been shown to be highly competitive by adding value as low cost implementation, production and scale up, as well as high productivity of synthesized proteins. The expression of recombinant proteins in milk represents the most developed system for production of biopharmaceutical drugs in animals, with two approved biopharmaceuticals for human use: Atryn®, a recombinant antithrombin produced in the milk of goats, approved in 2006 by European Medicines Agency (EMA) and in 2009 by US Food and Drug Administration (FDA), and more recently, Ruconest®, a recombinant human C1 esterase inhibitor protein (C1INH) produced in the milk of rabbits, first approved by EMA in 2012, followed by the FDA approval in 2014. Transgenic animals have been produced by many strategies that have gradually evolved over the decades, including the use of embryo microinjection, viral vectors and transposable elements, sperm-mediated gene transfer, and cloning by somatic cell nuclear transfer (SCNT).[...](AU)

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