Analysis of physical traits, clinical parameters, and energy metabolism of in vivo- and in vitro-derived Flemish newborn calves during the first day of life.

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.

Physiological impact of the environment on the welfare of transgenic goats raised in a tropical climate.

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.

Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.

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.

Expression of a recombinant bacterial L-asparaginase in human cells.

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.

Developmental Outcome and Related Abnormalities in Goats: Comparison Between Somatic Cell Nuclear Transfer- and In Vivo-Derived Concepti During Pregnancy Through Term.

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.

Microbiological and functional evaluation of an alternative device (OB®) for estrous synchronization in ewes / Avaliação microbiológica e funcional de um dispositivo vaginal alternativo (OB®) para sincronização de cio em ovelhas

The use of synthetic progestagens released by vaginal devices is an important tool to overcome the reproductive seasonality in sheep, but cost and/or subsequent vaginitis are limiting factors for their use. To identify economic, simple and innocuous alternative vaginal devices for estrous synchronization/induction protocols in sheep, this study aimed to evaluate the microbiological and functional viability of the human vaginal tampons (OB®) impregnated with medroxyprogesterone acetate (MAP) on reproductive performance of ewes. The study compared them with commercial vaginal inserts (CIDR®) and polyurethane sponges impregnated with MAP. In Experiment 1, the device loss rate, the degree of vaginitis during the device removal, the count and identification of bacterial colonies at the device insertion and removal, and efficiency in estrous synchronization and estrus temporal distribution were evaluated. Pubertal ewes at the beginning of the breeding season were randomly allocated to three experimental groups: CIDR®, PSP (polyurethane sponge) and OB®. No device losses occurred in any group, but the use of OB® caused milder signs of vaginitis than polyurethane sponges, with a similar vaginal bacterial growth and microbiota than the CIDR group. The estrus distribution was more disperse in the CIDR than PSP or OB groups. In Experiment 2, pregnancy rates using CIDR® or OB® devices were compared, with estrus manifestation (85.4 percent and 89.8 percent) and pregnancy rates (58.3 percent and 49.0 percent) being similar between groups (P>0.05), respectively. In conclusion, the use of human intra-vaginal tampons (OB®) impregnated with MAP was proven highly hygienic, practical and effective as a low-cost alternative for estrous synchronization and AI in sheep.

O uso de progestágeno sintético liberado por pessários vaginais é uma importante ferramenta para suplantar a sazonalidade reprodutiva em ovelhas. Todavia, seu uso é limitado pelo custo ou pelas subsequentes vaginites. Na busca de uma alternativa simples e de baixo custo para sincronizar estro em ovelhas, este estudo avaliou o tampão vaginal humano (OB®) impregnado com MAP, na performance reprodutiva de ovelhas, comparando com o CIDR® e as esponjas de poliuretano, estas também impregnadas com MAP. No experimento 1 foram avaliados a taxa de perdas; o grau das vaginites no momento da remoção do pessário; a contagem e identificação das colônias bacterianas; bem como a eficiência da sincronização e a distribuição temporal dos cios. As ovelhas foram aleatoriamente distribuídas em um de três grupos experimentais: CIDR, Esponjas e OB, no inicio da estação reprodutiva. Não ocorreram perdas de pessários em qualquer grupo, porém o OB causou menor grau de vaginite em relação às esponjas, com um crescimento bacteriano e microbiota similares ao grupo CIDR. A distribuição dos cios foi mais dispersa no grupo CIDR do que nos grupos Esponja ou OB. No experimento 2, foram comparados o CIDR e OB em relação à manifestação de cio (85,4 por cento e 89,8 por cento) e taxa de prenhez (58,3 por cento e 49,0 por cento), que foram similares (P<0,05). Conclui-se que o pessário OB impregnado com MAP é higiênico, de baixo custo, prático e efetivo como para a sincronização de cios e IA em ovelhas.