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Digital, The effect of L-165041 (PPARδ-agonist) on decreasing apoptosis and intracellular lipid content was assessed in fresh and vitrified–warmed in vitro-produced bovine embryos. It was hypothesised that the addition of L-165041 to the culture medium enhances development and cryopreservation. Oocytes were allocated to one of two treatments: control-standard culture medium, or L-165041 added to the medium on day 1 with no media change. Ultrastructure, cleavage, and blastocyst rates were evaluated in fresh, and in post-vitrification cultured embryos by optical and electronic microscopy. A subset of fresh embryos were fixed for TUNEL assay and for Sudan-Black-B histochemical staining. Vitrified–warmed embryos were assessed using MALDI-MS technique. Cleavage and blastocyst rates (control 49.4 ± 5.2, L-165041 51.8 ± 4.3) were not influenced by L-165041. The proportion of inner cell mass cells (ICM) was higher in fresh embryos, and the rate of total and ICM apoptosis was lower in L-165041. In warmed-embryos, total and ICM apoptosis was lower in L-165041. The overall hatching rate was higher in L-165041 (66.62 ± 2.83% vs 53.19 ± 2.90%). There was less lipid accumulation in fresh L-165041-embryos. In conclusion, the use of L-165041 is recommended to improve the viability of in vitro-derived bovine embryos., Ciencias Agropecuarias

Embryonic morphofunctional competence features regulating post-cryopreservation resumption of development are still poorly understood. In this study, we investigated the correlation between embryonic viability and the speed and ability to resume post-cryopreservation development. Thus, in vitro produced blastocysts were vitrified by the Cryotop method using standard protocols. Subsequently, the embryos were warmed, re-cultured, and classified into groups according to their speed and ability to resume post-cryopreservation development: embryos not re-expanded at 12h (NE12); embryos re-expanded at 12h and hatched at 24h (E12H24); embryos re-expanded at 12h and hatched at 48h (E12H48); embryos re-expanded at 12h and not hatched at 48h (E12NH48). Subsequently, the embryos were subjected to monitoring of total cell number and apoptosis. We identified that the blastocoel's ability to re-expand was negatively affected by the significant higher percentage of apoptotic cells observed in the NE12 group than in the other groups. A greater (P 0.05) number of total cells, found in groups E12H24 and E12H48, seems to have a positive influence on the hatching capacity of blastocysts after cryopreservation. In conclusion, the total number of cells and apoptotic index correlated with the speed and ability to resume post-cryopreservation development. Apoptosis was a determinant for embryonic re-expansion, and the total cell number was crucial for blastocyst hatching.

Made available in DSpace on 2021-06-25T10:17:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development. Center for Natural and Human Sciences Federal University of ABC School of Veterinary Medicine Federal University of Pampa In vitro Brazil – IVB ABS Pecplan Department of Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology Institute of Biosciences University of São Paulo State (Unesp) Department of Genetics and Evolutionary Biology University of São Paulo Department of Biological Science School of Sciences and Languages University of São Paulo State Department of Genetics and Evolution Federal University of São Carlos Department of Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology Institute of Biosciences University of São Paulo State (Unesp)

The effect of L-165041 (PPARδ-agonist) on decreasing apoptosis and intracellular lipid content was assessed in fresh and vitrified-warmed in vitro -produced bovine embryos. It was hypothesised that the addition of L-165041 to the culture medium enhances development and cryopreservation. Oocytes were allocated to one of two treatments: control-standard culture medium, or L-165041 added to the medium on day1 with no media change. Ultrastructure, cleavage, and blastocyst rates were evaluated in fresh, and in post-vitrification cultured embryos by optical and electronic microscopy. A subset of fresh embryos were fixed for TUNEL assay and for Sudan-Black-B histochemical staining. Vitrified-warmed embryos were assessed using MALDI-MS technique. Cleavage and blastocyst rates (control 49.4±5.2, L-165041 51.8±4.3) were not influenced by L-165041. The proportion of inner cell mass cells (ICM) was higher in fresh embryos, and the rate of total and ICM apoptosis was lower in L-165041. In warmed-embryos, total and ICM apoptosis was lower in L-165041. The overall hatching rate was higher in L-165041 (66.62±2.83% vs 53.19±2.90%). There was less lipid accumulation in fresh L-165041-embryos. In conclusion, the use of L-165041 is recommended to improve the viability of in vitro -derived bovine embryos.

Made available in DSpace on 2020-12-12T02:26:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ± 3.7) and Control (883 ± 5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos. University of Londrina (UEL) Laboratory of Animal Reproduction São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology Center for Natural and Human Sciences University ABC Federal São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science Laboratório de Virologia Animal Departamento de Medicina Veterinária Preventiva Universidade Estadual de Londrina Department of Chemistry and Center for Analytical Instrumentation Development Purdue University Instituto de Ciências Biomédicas Universidade de São Paulo Centro de Ciências Naturais e Humanas Universidade Federal Do ABC São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science CNPq: 403862/2016-7

SummaryHuman embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8–16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

Embryonic lipids are crucial for the formation of cellular membranes and dynamically participate in metabolic pathways. Cells can synthesize simple fatty acids, and the elongation of fatty acids facilitates the formation of complex lipids. The aim of this work was to investigate the involvement of the elongation of very long chain fatty acid enzyme 5 (ELOVL5) in embryonic development and lipid determination. Bovine embryos were produced in vitro using a standard protocol and randomly divided to receive one of three treatments at Day 4: morpholino (Mo) gene expression knockdown assay for ELOVL5 (ELOVL5-Mo), Mo antisense oligonucleotides for the thalassemic β-globulin human mRNA (technical control Mo), and placebo (biological control). The phenotypes of embryonic development, cell number, ELOVL5 protein abundance, lipid droplet deposits, and lipid fingerprint were investigated. No detrimental effects (p &gt, 0.05) were observed on embryo development in terms of cleavage (59.4 ± 3.5%, 63.6 ± 4.1%, and 65.4 ± 2.2%), blastocyst production (31.3 ± 4.2%, 28.1 ± 4.9%, and 36.1 ± 2.1%), and blastocyst cell number (99.6 ± 7.7, 100.2 ± 6.2, 86.8 ± 5.6), respectively, for biological control, technical control Mo, and ELOVL5-Mo. ELOVL5 protein abundance and cytoplasmic lipid droplet deposition were increased (p &lt, 0.05) in ELOVL5-Mo–derived blastocysts compared with the controls. However, seven lipid species, including phosphatidylcholines, phosphatidylethanolamines, and triacylglycerol, were downregulated in the ELOVL5-Mo–derived blastocysts compared with the biological control. Therefore, ELOVL5 is involved in the determination of embryonic lipid content and composition. Transient translational blockage of ELOVL5 reduced the expression of specific lipid species and promoted increased cytoplasmic lipid droplet deposition, but with no apparent deleterious effect on embryonic development and blastocyst cell number.

The expansion of the use of in vitro production techniques has revolutionized the bovine embryo market. In the last decade, we have seen the number of in vitro produced (IVP) embryos surpass the number of in vivo-derived (IVD) embryos obtained worldwide. Concomitantly, other biotechnologies were also improved, following the global trend. Embryo cryopreservation has received special attention, as it is one of the tools capable of disseminating in vitro production. Currently, two protocols are available: slow freezing and vitrification. Both have advantages and disadvantages regarding their application and, many aspects need to be considered before their use. In this review, we discuss in vitro production market trends, cellular and molecular features involved in embryo response to cryopreservation, and addressed cryo-storage period and embryonic developmental stage on cryosurvival. In addition, we also presented an overview of some aspects that impact the pregnancy rate following transfer of fresh and cryopreserved IVP embryos.

Contents It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin-like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy-associated plasma protein-A (PAPPA) in the cumulus–oocyte complexes (COCs) of Gir, 1/2 Holstein × 1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein × 1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at −80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real-time RT-PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8-16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

Made available in DSpace on 2018-12-11T17:29:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-01. Added 1 bitstream(s) on 2021-07-15T14:34:10Z : No. of bitstreams: 1 S0102-09352016000400958.pdf: 303959 bytes, checksum: e14f48cc607089919e5fbb51a24f98aa (MD5) Autonomic nervous system activity can be checked by heart rate variability (HRV), method that quantifies and thus infers on cardiac autonomic modulation, reflecting the functioning of the autonomic nervous system. During the neonatal period significant differences as regards the autonomic maturation, are described in various species. Although the sheep come being used as experimental model of several protocols in neonatology and cardiology in humans, descriptive studies about the VFC using healthy animals and not sedated are scarce in the literature. The objective of this study was to describe the HRV in the Bergamasca race lambs during the first 35 days of age. Electrocardiographic examinations were performed in 20 lambs of the Bergamasca race from birth, in the 7th, 14th, 21st, 28th, and 35th days of age. The HRV was analyzed by normal RR interval (sinus rate), heart rate, index of vagal tone, and the cardiovascular manifestations (square root of the average of the square of the differences between normal RR intervals adjacent). All parameters have changed over the weeks. The average heart rate increased between the birth and the first seven days of age, with decrease in the subsequent four weeks, being the smallest value found at 35 days of age (145.63±37.80bpm). Between 21, 28 and 35 days of age, the iTVV increased significantly, the RMSM from the 28 day, and 35 days, the cardiovascular manifestations, reflex parasympathetic activation, exhibited differences in relation to subsequent times. The beginning of parasympathetic dominance, reflected in the HRV indices, particularly the iTVV, occurs at 21 days of age. From the 35 days of age, the RMSM indices and RMSSD can be used as reliable markers of change in sympathetic and parasympathetic effect on the heart of lambs. Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Unesp Universidade Estadual Paulista Faculdade de Medicina Unesp Universidade Federal do Pampa Unipampa Universidade de São Paulo Instituto de Pesquisas Energéticas e Nucleares USP Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Unesp Universidade Estadual Paulista Faculdade de Medicina Unesp

Made available in DSpace on 2018-12-11T16:43:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-06-01. Added 1 bitstream(s) on 2019-10-09T18:28:52Z : No. of bitstreams: 1 S0100-736X2016000600551.pdf: 720307 bytes, checksum: 76cec92b8f78c83f71cbcef62a7529b6 (MD5) The veterinary cardiology has growing importance in equine medicine. There are studies of standardization of electrocardiographic parameters of many races, according to their stature and ability. However, no studies are in the literature with the American Miniature Horse. To evaluate the electrocardiogram (ECG) tracing configuration of this breed at rest and to verify the influence of age and sex on ECG parameters, 203 horses including 143 females and 60 males were divided into four age groups (foals, yearlings, adults and elderly). Electrocardiographic parameters were performed by computerized electrocardiogram (TEB), and the parameters were evaluated in six leads of frontal plane (Lead I, II, III, aVR, aVL and aVF) and base-apex (BA). Heart rates (HR) decreased with increasing age were higher in males than in females. Sinus tachycardia followed by sinus arrhythmia was dominant in both sexes. The cardiac axis was higher in males and ranged between 120° and 150° for foals, 30° and 60° for yearlings and adults, and 60° and 90° for the elderly. The P wave was bifid in several animals. The P-wave amplitude and T-wave duration from lead II and BA were larger in males than in females. The majority of the animals exhibited ST segment depression and a negative T-wave. The most common QRS complex morphology was Qr. Differences were observed between the electrocardiographic tracings of males and females, and age influenced the ECG parameters. Therefore, this study established the ECG patterns for the American Miniature Horse breed and could be used to determine the influence of age and sex on several of the studied variables. Faculdade de Medicina Veterinária e Zootecnia Universidade Estadual Paulista (Unesp), Rubião Jr s/n. Laboratório de Genética e Zootecnia Faculade de Medicia Veterinária Universidade Federal do Pampa (Unipampa), Av. General Osório 900 Faculdade de Medicina Veterinária e Zootecnia Universidade Estadual Paulista (Unesp), Rubião Jr s/n.

There are studies on electrocardiogram (ECG) in several breeds of horses, which highlights the growing importance of cardiology in this species. But few authors have addressed the influence of pregnancy on the cardiac physiology of the mare, and specifically, there are no studies on the Mini-horse breed, as well as no correlation of body weight (BW) and heart rate (HR) with electrocardiographic parameters in this breed. The aims of this study were to determine the effects of pregnancy on ECG parameters and to examine the relationships between these variables and body weight (BW) and heart rate (HR). A total of 203 animals were used, including 143 females (66 pregnant) and 60 males. Electrocardiographic examinations were performed by computerized electrocardiogram (TEB), and the parameters were evaluated in six leads in the frontal plane (Lead I, II, III, aVR, aVL and aVF) and base-apex (BA). BW was inversely proportional to HR, which in turn showed an inverse relationship with the duration of the P-wave and the PR and QT intervals. The P-wave amplitude (lead II) was higher in pregnant animals than in non-pregnant animals. The effect of reproductive status should be monitored by ECG throughout the entire gestational period. The 66 pregnant mares in this study were examined during the first third of their pregnancies, thus, few differences were observed between pregnant and non-pregnant animals. RESUMO Existem estudos sobre eletrocardiograma (ECG) em diversas raças de equinos, o que ressalta a crescente relevância da cardiologia nessa espécie. Porém, poucos autores abordaram a influência da gestação na fisiologia cardíaca da égua, e, especificamente, não há trabalhos sobre a raça Mini-horse, assim como não há correlação do peso vivo (PV) e frequência cardíaca (FC) com os parâmetros eletrocardiográficos nessa raça. O objetivo deste estudo foi estudar a influência da prenhez sobre os parâmetros eletrocardiográficos, além de verificar a correlação do PV e FC com essas variáveis. Utilizaram-se 203 animais dessa raça, hígidos, sendo 143 fêmeas (66 prenhes) e 60 machos. Os exames eletrocardiográficos foram realizados por eletrocardiograma computadorizado (TEB), e os parâmetros foram avaliados em seis derivações do plano frontal (I, II, III, aVR, aVL e aVF) e base-ápice. O PV foi inversamente proporcional à FC, que também mostrou relação inversa com a duração da onda P e os intervalos PR e QT. Na derivação II, a amplitude da onda P foi maior nos animais prenhes do que vazios. No entanto, sugere-se que a influência do estado reprodutivo seja estudada de forma progressiva pelo acompanhamento de todo o período gestacional, visto que as 66 éguas estudadas se apresentavam no terço inicial da prenhez, portanto poucas diferenças foram observadas entre animais prenhes e vazios.

The differential expression of many productive and reproductive traits has already been associated with the genotype effect observed between Bos taurus indicus (Bos indicus) and Bos taurus taurus (Bos taurus) breeds. Lipids have a crucial role during oocyte development. The aim of this work was to characterize the lipid profiles of oocytes recovered from dairy breeds: Gir (Bos indicus), Holstein (Bos taurus) and their crossbred (Gir x Holstein). Cumulus-oocyte complexes were collected by ovum pick-up from Gir (n=21), Holstein (n=36), and crossbreed (n=33) cows and submitted to MALDI-MS lipid fingerprint analysis. Multi- and univariate analyses were used to characterize lipid profiles of oocytes derived from each breed. Third lipid profiles (Gir n=7; Holstein n=12; and crossbred n=11) were evaluated and allowed characterization of oocyte's lipids among breeds. The lipid profiles from Gir and Holstein were observed to be the most distinct, whereas Gir and crossbred oocytes presented greater similarities. Overall, a wide-range oocyte lipid profile characterization from distinctive bovine dairy breeds recovered by OPU were provided. We have identified two distinguishable lipid profile patterns among groups and revealed that phospholipids and triacylglycerol were the predominantly lipid assigned in Gir (Bos indicus) and Holstein (Bos taurus) oocytes, respectively.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

SummaryTemporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus–oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

SummaryThe inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. Thus, the objective of this work was to evaluate the effect of blocking oocyte meiosis with the addition of forskolin, an efficient inhibitor of nuclear maturation, in in vitro maturation (IVM) medium. Forskolin was added to the IVM medium for 6 h at concentrations of 0.1 mM, 0.05 mM or 0.025 mM, then the oocytes were allowed to mature in drug-free medium for 18 h. The oocytes were assessed for the stage of nuclear maturation, the activity and distribution of mitochondria, oocyte ultrastructure, the number of viable cells and the apoptosis rate. After forskolin treatment, the oocytes were fertilized in vitro and cultured for 7 days. On day 7, the blastocyst rate, the ultrastructure, the number of intact cells and the apoptosis rate of the blastocysts were measured. No differences were observed for the stage of nuclear maturation of the oocyte, the mitochondrial activity and distribution, the blastocyst rate or total number of intact cells. However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (P < 0.05). We conclude that all the experimental groups reached the MII stage after the addition of forskolin and that the highest concentration of forskolin caused cellular degeneration without harming embryo production on the 7th day.

Made available in DSpace on 2022-04-29T08:45:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Mammalian oocytes resume meiosis spontaneously after removal from the ovarian follicle. We tested the effects of a 2-h prematuration treatment (Pre-IVM) with forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) in bovine cumulus-oocyte complexes (COCs) on the lipid content of oocytes and blastocysts, on the membrane lipid composition of blastocysts and on the transcriptional profiling of cumulus cells and blastocysts in a high-throughput platform. Embryonic development rates to the morula (mean 56.1%) or blastocyst (mean 26.3%) stages were unaffected by treatment. Lipid content was not affected after Pre-IVM, but was increased after IVM in treated oocytes. Conversely, the lipid content was reduced in Pre-IVM blastocysts. Pre-IVM COCs generated blastocysts containing blastomeres with more unsaturated lipids in their membranes. Pre-IVM also altered the relative abundance of 31 gene transcripts after 2 h and 16 transcripts after 24 h in cumulus cells, while seven transcripts were altered in blastocysts. Our results suggest that the Pre-IVM treatment affected the lipid composition and transcriptional profiles of COCs and blastocysts. Therefore, Pre-IVM with FSK and IBMX could be used either to prevent spontaneous meiotic resumption during IVM or to modulate lipid composition in the membrane and cytoplasm of blastocysts, potentially improving bovine embryos. Nove de Julho University, Rua Nicolau Assis, 15 School of Veterinary Medicine Federal University of Pampa, Caixa Postal 118 Department of Pharmacology Institute of Bioscience University of São Paulo State, Distrito de Rubião Junior s/n ThoMSon Mass Spectrometry Laboratory Institute of Chemistry University of Campinas-UNICAMP, Rua Sérgio Buarque de Holanda s/n University of Western São Paulo Rodovia Raposo Tavares Km 572 - Bairro Limoeiro Department of Biological Sciences Faculty of Sciences and Letters University of São Paulo State, Avenuenida Dom Antonio, 2100

Made available in DSpace on 2018-11-26T17:29:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of super stimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 degrees C until extraction. The lipid screening was performed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development. (C) 2017 Published by Elsevier Inc. Univ Estadual Paulista, Inst Biociencias, Dept Farmacol, Botucatu, SP, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Assis, SP, Brazil Univ Estadual Campinas, UNICAMP, Lab Espectrometria Massa, Campinas, SP, Brazil Univ Estadual Campinas, UNICAMP, Thomson, Campinas, SP, Brazil Univ Fed Pampa, Curso Med Vet, Uruguaiana, RS, Brazil Univ Oeste Paulista, Presidente Prudente, SP, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Farmacol, Botucatu, SP, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Assis, SP, Brazil FAPESP: 2011/50593-2 FAPESP: 2013/11480-3 FAPESP: 2012/07206-0

Background Diabetic pregnancy have increased rates of congenital malformation and neonatal mortality. In vitro studies suggest hyperglycemia associated with diabetes impair embryogenesis but in vivo investigations on maternal hyperglycemic insult and early embryo development are scarce. We evaluated the embryofetal development on experimental diabetes models to assess whether hyperglycemia at preimplantation period impairs the progression of pregnancy. Methods Different hyperglycemic intensities were obtained by two experimental diabetes models. Female Sprague Dawley rats received streptozotocin at birth (mild diabetes) or at day 90 of life (severe diabetes). For both diabetic groups hyperglycemia was confirmed 5 days after diabetes induction and the mating was performed around 100 day of life. For preimplantation analysis, embryos were recovered at D4 of pregnancy. Another group of animals was submitted to laparotomy at D21 to assess contents of the uterus and fetal viability. Results Mild (i) and Severe (ii) diabetes modified the early development. Degenerating embryos percentage was higher compared to control (11%) (i) 30.7%, (ii) 37.3%. Cell number mean dropped according to hyperglycemic intensity (control 30.57, (i) 21.42, (ii) 13.42). Pre and post-implantation loss rates were higher in diabetic groups. The fetal viability also decreased from 96% in the control group to (i) 78.7% and (ii) 80.6%. Conclusion Our results show that during diabetic pregnancy, preimplantation embryos present decreased cell number due to higher apoptosis rates, which are dependent of the hyperglycemic intensity. Moreover, fetal viability was also decreased suggesting that the quality of these embryos at long-term may be questioned. Copyright © 2014 John Wiley & Sons, Ltd.

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:51:08Z No. of bitstreams: 1 S0100-736X2013000400019.pdf: 1564211 bytes, checksum: 70ecbfccb45b5b58cf39232c91b792cc (MD5) Made available in DSpace on 2013-08-22T18:51:08Z (GMT). No. of bitstreams: 1 S0100-736X2013000400019.pdf: 1564211 bytes, checksum: 70ecbfccb45b5b58cf39232c91b792cc (MD5) Previous issue date: 2013-04-01 Made available in DSpace on 2013-09-30T18:18:05Z (GMT). No. of bitstreams: 2 S0100-736X2013000400019.pdf: 1564211 bytes, checksum: 70ecbfccb45b5b58cf39232c91b792cc (MD5) S0100-736X2013000400019.pdf.txt: 40775 bytes, checksum: 74ba842d82e89ec17bf04502faa46af5 (MD5) Previous issue date: 2013-04-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:41:56Z No. of bitstreams: 2 S0100-736X2013000400019.pdf: 1564211 bytes, checksum: 70ecbfccb45b5b58cf39232c91b792cc (MD5) S0100-736X2013000400019.pdf.txt: 40775 bytes, checksum: 74ba842d82e89ec17bf04502faa46af5 (MD5) Made available in DSpace on 2014-05-20T13:41:56Z (GMT). No. of bitstreams: 2 S0100-736X2013000400019.pdf: 1564211 bytes, checksum: 70ecbfccb45b5b58cf39232c91b792cc (MD5) S0100-736X2013000400019.pdf.txt: 40775 bytes, checksum: 74ba842d82e89ec17bf04502faa46af5 (MD5) Previous issue date: 2013-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura. The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature. Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia Veterinária Unesp FMVZ Departamento de Clínica Veterinária Universidade Federal de Pelotas Faculdade de Medicina Veterinária Departamento de Clínicas Veterinárias Universidade Estadual Paulista Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia Veterinária Unesp FMVZ Departamento de Clínica Veterinária

Made available in DSpace on 2022-04-29T08:45:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The presence of fetal calf serum in culture medium influences embryo quality, causing a reduction in postcryopreservation survival. Forskolin has been used to induce lipolysis and increase cryotolerance, functioning as an activator of adenylate cyclase and elevating cAMP levels. In the present experiment, bovine zygotes were cultured in synthetic oviduct fluid with amino acid plus 2.5% fetal calf serum for 6 days, when forskolin was added in three concentrations: 2.5, 5, and 10 μM. Treatment with forskolin lasted for 24 hours. Blastocyst formation rate, quantification of lipid granules, total cell numbers, and apoptosis rate were evaluated. In a second assessment, embryos were vitrified, and warming, re-expansion rate, total cell numbers, and apoptosis rate were also evaluated. There was no difference due to forskolin in blastocyst formation or re-expansion rates after vitrification. However, lipid measurements were lower (control: 136.8 and F 2.5 μM: 128.5; P < 0.05), and number of cells per embryo higher (control: 140.1 and F 2.5 μM: 173.5; P < 0.05) than controls for 2.5 μM forskolin but not for higher forskolin concentrations. The number of intact cells per embryo was higher, and the rate of apoptosis was lower in fresh than in vitrified embryos (number of cells of warmed embryos, control: 104.1, F 2.5 μM: 101.3, F 5 μM: 115.4, F 10 μM: 95.1; apoptotic of fresh cells, control: 12.1%, F 2.5 μM: 16.7%, F 5 μM: 11.1%, F 10 μM: 14.2%; and apoptotic warmed embryos, control: 22.3%, F 2.5 μM: 37.3%, F 5 μM: 33.2%, F 10 μM: 30.3%; P < 0.05). It was concluded that forskolin is an effective lipolytic agent even at low concentrations, leading to formation of blastocysts with a comparatively larger number of cells. Departmento de Medicina Veterinária Faculdade de Zootecnia e Engenharia de Alimentos Universidade de São Paulo Universidade Federal do Pampa Faculdade de Medicina Veterinária Departmento de Reprodução Animal Faculdade de Medicina Veterinária e Zootecnia Universidade do Estado de São Paulo Departmento de Clínica Cirurgia e Reprodução Animal Faculdade de Medicina Veterinária Universidade do Estado de São Paulo FAPESP: 2010/50410-2 FAPESP: 2011/08926-4 FAPESP: 2014/21289-1

Made available in DSpace on 2018-12-11T17:14:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress. Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Dr. Walter Mauricio Correra Street Department of Animal Sciences Luiz de Queiroz College of Agriculture (ESALQ) University of São Paulo (USP), Pádua Dias Avenue 11 Department of Animal Reproduction School of Veterinary Medicine and Animal Science (FMVZ) University of São Paulo (USP), Orlando Marques de Paiva Street 87 Institute of Biosciences Chemistry and Pharmacology Federal University of Saõ Paulo (UNIFESP), Prof. Artur Riedel Street 275 Department of Pharmacology Institute of Biosciences São Paulo State University (UNESP) Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science (FMVZ) São Paulo State University (UNESP), Prof. Dr. Walter Mauricio Correra Street

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:27:29Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:27:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-01 It is believed the temporary meiosis arrest with roscovitine or cycloheximide may improve the in vitro developmental competence of oocytes in different animal species. However, little is known about the effects of these inhibitors on ultrastructure of ovines cumulus-oocyte complexes (COCs). The aim of this study was to evaluate the progression of cytoplasmic maturation and the ultrastructural changes in sheep COCs exposed to roscovitine or cycloheximide, at acceptable concentrations. COCs were in vitro cultured for 24. h in maturation medium (control group) containing 100 μM roscovitine or 1 μg/mL cycloheximide (treatment groups). After this time, some COCs were cultured for further 22. h in inhibitor-free medium. The ultrastructure organization of COCs was evaluated by transmission electron microscopy before (immature group) and after in vitro culture for 24 and 46. h. As expected, signs of immaturity and maturity were observed in immature and control groups, respectively. In treatment with roscovitine, there were cumulus cells degeneration, swelling of mitochondrias, few cortical granules and many vesicles with electron-dense material. However, in cycloheximide treatment there were not signs of degeneration or cellular senescence. Metabolic units and mitochondrial pleomorphism were found in all experimental groups. These evidences demonstrate that roscovitine promoted irreversible ultrastructural changes while cycloheximide did not affect the cytoplasmic maturation. However, the implications on embryo development are still unclear. © 2012 Elsevier B.V. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University, UNESP, Distrito de Rubião Junior s/no, 18603-970, Botucatu, SP Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University, UNESP, Distrito de Rubião Junior s/no, 18603-970, Botucatu, SP

Made available in DSpace on 2018-12-11T17:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 The objective of the study was to assess clinical alterations, electrocardiographic, hematological, biochemical, hemogasometric, electrolytic, and hormone plasma concentrations in bitches with eutocia and dystocia. Overall, 28 bitches (dystocia, n = 22 and eutocia, n = 6) were assessed. The evaluations were performed at 2 time points, M1 (1 hour prepartum—eutocia group and cesarean or clinical intervention—dystocia group) and M2 (postpartum—eutocia or dystocia group and anesthetic recovery—dystocia group). The main clinical finding was the hypothermia (mean: 36.9°C dystocia vs. 36.8°C eutocia). Sinus arrhythmia and tachycardia were the electrocardiographic parameters predominant in eutocia and sinus rhythm in dystocia group. The P wave amplitude, heart rate, creatinine concentration, hematocrit, and hemoglobin were increased in M1 (P

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:52:53Z No. of bitstreams: 1 S0100-879X2012001200008.pdf: 2235930 bytes, checksum: 352d1b6b4e48528e2848748d816a6f74 (MD5) Made available in DSpace on 2013-08-22T18:52:53Z (GMT). No. of bitstreams: 1 S0100-879X2012001200008.pdf: 2235930 bytes, checksum: 352d1b6b4e48528e2848748d816a6f74 (MD5) Previous issue date: 2012-12-01 Made available in DSpace on 2013-09-30T18:15:43Z (GMT). No. of bitstreams: 2 S0100-879X2012001200008.pdf: 2235930 bytes, checksum: 352d1b6b4e48528e2848748d816a6f74 (MD5) S0100-879X2012001200008.pdf.txt: 26410 bytes, checksum: 5cf09de1533d8848bd1ceda2b458c8e9 (MD5) Previous issue date: 2012-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:41:56Z No. of bitstreams: 2 S0100-879X2012001200008.pdf: 2235930 bytes, checksum: 352d1b6b4e48528e2848748d816a6f74 (MD5) S0100-879X2012001200008.pdf.txt: 26410 bytes, checksum: 5cf09de1533d8848bd1ceda2b458c8e9 (MD5) Made available in DSpace on 2014-05-20T13:41:56Z (GMT). No. of bitstreams: 2 S0100-879X2012001200008.pdf: 2235930 bytes, checksum: 352d1b6b4e48528e2848748d816a6f74 (MD5) S0100-879X2012001200008.pdf.txt: 26410 bytes, checksum: 5cf09de1533d8848bd1ceda2b458c8e9 (MD5) Previous issue date: 2012-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium. Universidade Estadual de São Paulo Faculdade de Medicina Veterinária e Zootecnia Departamento de Reprodução Animal e Radiologia Veterinária Universidade Estadual de São Paulo Faculdade de Medicina Veterinária e Zootecnia Departamento de Cirurgia e Anestesiologia Veterinária Universidade Estadual de São Paulo Faculdade de Medicina Veterinária e Zootecnia Departamento de Patologia Veterinária Universidade de São Paulo Instituto de Química de São Carlos UNESP

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos producedin vitrowith or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos producedin vivowere used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared within vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P< 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

SummaryThe objective of the present study was to correlate some parameters (cleavage, blastocyst production, quality degree score, total cell number, fresh apoptosis and lipid content) with embryo survival after cryopreservation. A total of 1727 in vitro-produced bovine blastocysts were used to establish the parameters (mean ± standard error of the mean (SEM)) for cleavage (85.6 ± 0.8), blastocyst production (39.9 ± 1.4), quality degree score (1.6 ± 0.1), total cell number (140.1 ± 2.9), fresh apoptosis (20.8 ± 1.1) and lipid content (21.3 ± 0.8 droplets). On the same way 1316 blastocysts were vitrified for the determination of post-cryopreservation embryo survival (49.4 ± 1.9). Fresh apoptosis rate and total lipid droplets value were correlated (P < 0.05) with embryo survival after cryopreservation (r = 0.91 and r = 0.59; respectively). However, cleavage, blastocyst production, quality degree score and total cell number were not correlated (P > 0.05) with embryo cryotolerance (r = 0.23, r = 0.38, r = 0.22 and r = 0.28; respectively). Therefore, the increased lipid content was moderately correlated with apoptosis in vitrified blastocysts. On the other hand, increased apoptosis in fresh blastocysts was strongly correlated with apoptosis in vitrified blastocysts, which indicated that the apoptosis rate in fresh embryos was a better parameter than the lipid content to predict post-vitrification embryo survival.

Made available in DSpace on 2013-08-12T19:22:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-01 Made available in DSpace on 2013-09-30T18:25:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:41:58Z No. of bitstreams: 1 S0100-736X2012000500013.pdf: 2425689 bytes, checksum: 89632b3737cdeb5449d034c3f81d4d92 (MD5) Made available in DSpace on 2014-05-20T13:41:58Z (GMT). No. of bitstreams: 1 S0100-736X2012000500013.pdf: 2425689 bytes, checksum: 89632b3737cdeb5449d034c3f81d4d92 (MD5) Previous issue date: 2012-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P

The objective of this study was to evaluate the potential of bovine IVF blastocysts at different stages of embryonic development in establishing ESC-like. Furthermore, blastocysts cultured in medium containing (10%) and (2.5%) fetal calf serum (FCS) were compared to determine if the serum concentration during in vitro culture alters the blastocyst's potential to establish ESC-like culture. It was observed that only ICM's from expanded blastocysts adhered to the monolayer (n=160) independent of the concentration of serum used during IVF culture. There were no observable differences in potential to establish ESC-like in embryos cultured with 2,5% or 10% FCS . The bFGF didn´t seems to be required for maintenance of bovine ESC-like regardless of culture conditions. Blastocysts and colonies in primary culture and after the first passage were positive for Oct4, Nanog, SSEA-3 and TRA-1-81. Expanded blastocysts gave rise to ESC-like colonies, and the addition of LIF was sufficient to maintain cells undifferentiated in culture.

Made available in DSpace on 2013-09-27T14:54:19Z (GMT). No. of bitstreams: 1 WOS000296681000016.pdf: 301005 bytes, checksum: 32ff2ce42c38d877b706dc2611af743b (MD5) Previous issue date: 2011-11-01 Made available in DSpace on 2013-09-30T18:29:07Z (GMT). No. of bitstreams: 1 WOS000296681000016.pdf: 301005 bytes, checksum: 32ff2ce42c38d877b706dc2611af743b (MD5) Previous issue date: 2011-11-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:42:17Z No. of bitstreams: 1 WOS000296681000016.pdf: 301005 bytes, checksum: 32ff2ce42c38d877b706dc2611af743b (MD5) Made available in DSpace on 2014-05-20T13:42:17Z (GMT). No. of bitstreams: 1 WOS000296681000016.pdf: 301005 bytes, checksum: 32ff2ce42c38d877b706dc2611af743b (MD5) Previous issue date: 2011-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) This study assessed the reuse of norgestomet implants in Nelore cows that were superstimulated with eCG. In a crossover design trial, eight cows were randomly divided into two experimental groups and twice superstimulated: Group 1 - half of the cows received a new norgestomet implant and 2 mg estradiol benzoate (EB) on Day 0; Group 2 - remaining cows received two once-used norgestomet implants and 2 mg EB also on Day 0. on Day 4 all cows received a single dose of 2000 IU eCG, and on Day 6 cows were treated with two doses of PGF(2 alpha), 12 h apart. Ovulation was induced with 12.5 mg pLH 12 h after implant withdrawal (on Day 7), and fixed-time artificial inseminations were carried out 12 and 24 h later. Seven days after pLH injection embryos were recovered and blood samples were taken to determine circulating progesterone. Ultrasound examinations were performed at pLFI administration and at embryo recovery. The number of large follicles (>= 8 mm) was greater (P0.05) were found between treatments (respectively, for Group 1 and Group 2 cows) in the follicle diameter (10.5 +/- 1.6 and 102 +/- 1.7 mm), corpus luteum (CL) diameter (15.7 +/- 1.8 and 14.4 +/- 1.7 mm), CL volume (2232 1356 and 1893 +/- 1828 mm(3)), ova/embryos recovered (6.3 +/- 1.1 and 4.0 +/- 1.9), transferable embryos (4.0 +/- 1.8 and 2.5 +/- 1.0) and plasma progesterone concentration (36.1 +/- 83 and 35.1 +/- 6.9 ng/mL). In conclusion, the stimulatory effects on the ovaries provided by the use of two once-used implants were less intense than the ones verified after using a new implant. (C) 2011 Elsevier B.V. All rights reserved. São Paulo State Univ UNESP, Sch Vet Med & Anim Sci FMVZ, Dept Anim Reprod & Vet Radiol, BR-18618970 Botucatu, SP, Brazil Univ São Paulo USP, Dept Anim Sci, BR-13418900 Piracicaba, SP, Brazil Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) SE Cattle, BR-13560970 São Carlos, SP, Brazil São Paulo State Univ UNESP, Sch Vet Med & Anim Sci FMVZ, Dept Anim Reprod & Vet Radiol, BR-18618970 Botucatu, SP, Brazil FAPESP: 06/55199-2

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:26:16Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:36:30Z : No. of bitstreams: 1 2-s2.0-84859947498.pdf: 1188327 bytes, checksum: 2810311ca1a614402555d3020f4254f9 (MD5) Made available in DSpace on 2014-05-27T11:26:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-01 The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP. © 2011 Mateus José Sudano et al. Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University, 18618-000 Botucatu, SP Department of Surgical Clinics and Animal Reproduction School of Odontology São Paulo State University, 16050-680 Araçatuba, SP Department of Statistics São Carlos Federal University, 13565-905 São Carlos, SP Embrapa Pecúaria Sudeste, 13560-970 São Carlos, SP Department of Animal Reproduction and Veterinary Radiology School of Veterinary Medicine and Animal Science São Paulo State University, 18618-000 Botucatu, SP Department of Surgical Clinics and Animal Reproduction School of Odontology São Paulo State University, 16050-680 Araçatuba, SP

Neonatal veterinarians still observe higher mortality rates among their patients than those observed among humans. Establishment of a neonatal assessment protocol is fundamental to the identification of the medical status of the neonate and the need for medical intervention. The neonatal Apgar score evaluation, which is commonly used in clinical practice, should be complemented by other methods of analysis. This study proposes, in addition to an Apgar score analysis, the evaluation of laboratory parameters and weight. We believe that knowledge of these reference values is essential for diagnosing at-risk neonates and for establishing suitable treatments.