In Vitro Embryo Production

Author Title Number of pages Date Olli-Pekka Lehtinen Modifying Wheat Bran for Food Applications Effect of Wet Milling and Enzymatic Treatment 63 pages + 9 appendices 20 May 2012 Degree Bachelor of Engineering Degree Programme Biotechnology and Food Engineering Specialisation option Product Development and Quality Management Instructors Juhani Sibakov, Research Scientist, Project Manager (VTT) Mikko Halsas, Senior Lecturer (Metropolia University of Applied Sciences) The aim of this study was to develop sustainable methods to modify the structure of wheat bran used in food applications. The methods of interest were wet milling and enzymatic treatment. The effect of these methods on a product quality was tested in a pilot scale baking process. Wheat bran is composed of many nutritionally valuable components, such as dietary fibre, antioxidants and proteins. Due to its relatively low price, wheat bran holds a great potential as an ingredient to be added into food products. However, mos...

Manual dish preparation for IVF in human fertility clinics or animal laboratories heavily relies on embryologists' experience, which can lead to occupational illness due to long-term and monotonous operation. Therefore, introducing an automated technique to replace traditional methods is crucial for improving working efficiency and reducing work burden for embryologists. In the current study in the mouse, both manual and automated methods were used to prepare IVF or embryo culture dishes. A one-way analysis of variance was conducted to compare several factors, including preparation time, qualified rates, media osmolality of dishes, fertilization rates, and embryonic development to assess the efficiency and potential of automated preparation. The results showed that automation system significantly reduced the required time and increased the efficiencies and qualified rates of dish preparation, especially for embryo culture dishes, without significantly altering medium osmolalities. There were no significant differences between two preparations in fertilization rates and embryo development in mice. Thus, automated dish preparation can improve working efficiency and qualified rates while maintaining fertilization rates and subsequent embryonic development without compromising osmolality stability of medium. It presents a superior alternative to manual preparation, reducing the workload of embryologists and facilitating the standardization of operational procedures.

Background: Controlling soil-borne fungal diseases is challenging due to the fungi's ability to endure in the soil for extended time frames. Tomato plants are one of the crops that suffer from root rot disease caused by Rhizoctonia solani. Biocontrol agents, such as rhizobacteria are a new trend to control this pathogen as alternative to chemical fungicides. Methods: The morphological and physiological traits of four rhizobacterial isolates of Leclercia adecarboxylata DKS3, Bacillus halotolerans DMC8, Bacillus subtilis NAS1 and Paenibacillus polymyxa TRS4 were examined in vitro and their efficacy in managing tomato root rot disease caused by R. solani was assessed under greenhouse conditions, along with their impact on plant growth metrics and enzyme activities. Result: The isolate DMC8 significantly enhanced the germination percentage of tomato seedlings, whilst the other isolates showed no difference compared to the positive control treatment. The isolates DMC8, NAS1 and TRS4 demonstrated a substantial reduction in disease incidence, ranging from 50% to 55%, in contrast to the positive control, which recorded 85% incidence. All isolates, however, demonstrated a notable reduction in disease severity, ranging from 30% to 49%, in contrast to the positive control, which exhibited a severity rate of 56%. All bacterial isolates exhibited a notable enhancement in growth metrics in the absence of the pathogen, whereas in the presence of the pathogen, isolates DMC8, NAS1 and TRS4 demonstrated a considerable improvement in growth markers. An elevation in the activities of peroxidase and phenylalanine ammonium lyase enzymes was observed, signifying the development of resistance in the plant by the rhizobacterial isolates.

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.

T helper (h) lymphocytes in pathogenic immune response at mucosal effector site play a key role in IgA nephropathy (IgAN). We evaluated the impact of some Th1/Th2/Th3/T R -type, and of monocyte/ macrophage cytokines on IgAN susceptibility with a family-based association study including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFNc intron-1 CA repeat at position 1349 -1373; IL-13 À1055C/T; TGFb þ 915G/C; IL-10 5 0 -proximal and distal microsatellites; TNFa À308G/A, À238G/A. The FBAT multi-allelic analysis showed an association between IFNc polymorphism and susceptibility to IgAN (P ¼ 0.03). The bi-allelic analysis evidenced that the 13-CA repeat allele was preferentially transmitted to the affected individuals (P ¼ 0.006; Bonferroni P-value ¼ 0.04). The direct sequencing of IFNc amplicons showed a strict association between the 13-CA repeat allele and the A variant of the þ 874T/A single nucleotide polymorphism (SNP rs2430561) directly adjacent to the 5 0 end of the microsatellite. The in vitro production of IFNc evaluated in peripheral blood mononuclear cells from 10 genotyped patients demonstrated a correlation between the þ 874A allele and a lower production of IFNc (P ¼ 0.028 Mann -Whitney test). This SNP affects IFNc production lying within a binding site for the transcription factor NF-jB. No significant difference was observed in the 15 years renal survival between IgAN patients carrying different IFNc gene polymorphisms. This first family-based association study demonstrates that the þ 874A allele, strictly associated with IFNc 13-CA repeat allele, confers susceptibility to IgAN, without influencing renal survival.

The protocol of in vitro production (IVP) of bovine embryos is one of the critical factors determining embryo viability after cryopreservation. In this study were used two differents protocols to produce IVP bovine embryos, with variations in protein source, oocyte/zygote density per media volume, with the aim to determine the in vitro and in vivo embryo survival after vitrification using hand-pulled glass micropipettes. Expanded blastocysts (D7) were morphologically selected by size (≥180 µm) and osmotic behavior before they were randomly allocated to sub-groups by protocol: non-vitrified embryos (control; C) and vitrified embryos (V). For the evaluation of the in vitro survival, control embryos and a group of warmed vitrified embryos were in vitro-cultured (IVC) for 72 h. Re-expansion rates of warmed embryos at 24 h of IVC were 94.8% and 93.2% for Protocols 1 and 2, respectively. Hatching rates at 72 h of IVC of embryos from Protocol 1 (C=80% and V=75.8%) tended to be higher (P=0.0561, Chi 2 test) than those from Protocol 2 (C=67.2% and V=59.3%). For the evaluation of in vivo survival, 21 vitrified embryos per protocol were singly non-surgically transferred to synchronized recipients (n=42) after the in-straw cryoprotectant dilution, resulting in 4 (19%) pregnancies per group on Day 60 of gestation. In conclusion, despite a lower variation on in vitro embryo development between both IVP protocols, the use of different protocols under the same laboratory conditions did not afect the in vitro and in vivo embryo viability after vitrification into hand-pulled glass micropipettes.

Aspergillus fumigatas (Af) a frequently isolated fungus from the environment is involved in a number of disorders in humans including allergic bronchopulmonary aspergillosis (ABPA). Immunodiagnosis of ABPA depends on the demonstration of specific IgE and IgG antibodies to antigens of Af in the sera of patients. Purified well characterized antigens may lead to a more reliable diagnosis of ABPA. In order to obtain standardized and reproducible antigens, we have raised moneclonsl antibodies against culture filtrate antigens of Af. A meneclonal antibody immunoaffinity column was used to purify a diagnostically relevant antigen of Af. This purified antigen was evaluated using ELISA and RAST assays for specific IgE antibodies in sera from ABPA(n=25), allergic asthmatics (n=10) and normal controls (n= 10). Results indicate a strong correlation of IgE levels in ABPA between these two methods. The mean IgE ELISA absorbance (OD) and RAST (units) for ABPA was 1.01 and 3.78 respectively as compared to 0.10 and 0.79 for normals (p<0.001). The correlation coefficients of RAST and ELISA using purified and crude antigens were all greater than 0.96. Using RAST and ELISA the purified antigen demonstrated high IgE levels in over 90% of the sera from ABPA patients. From these observations this purified antigen appears to be a major allergen of Af and may be useful for immunodiagnosis of ABPA.

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Resumen: La producción de embriones in vitro actualmente ha cobrado importancia en el sector ganadero por ser una técnica que ayuda a la mejora genética y eficiente producción. Sin embargo, para llegar a crear embriones se necesita un protocolo que proporcione metodologías que ayuden a obtener mejores resultados, es por eso que el objetivo de este estudio fue implementar un protocolo de fertilización in vitro en bovinos tomando como base el protocolo realizado por el Dr.

Enhanced renal leukotriene production in murine lupus: Role of lipoxygenase metabolites. To investigate the potential role of leukotrienes in murine lupus, we measured renal hemodynamics and renal leukotriene production in MRL-lprllpr mice at 12 and 20 weeks of age. Over this age range, these animals develop overt manifestations of autoimmune disease with nephritis similar to human SLE. In the current study, we demonstrated that glomerular filtration rate (GFR) and PAH clearance (CPAH) deteriorated with age in MRL-lpr/lpr mice, but not in MRL-+/+ controls. Impaired renal hemodynamic function in MRL-lprllpr mice was associated with enhanced ionophore-stimulated production of both leukotriene B4 (LTB4) and leukotriene C4 (LTC4) by preparations of renal cortex. There was a significant inverse correlation between GFR and in vitro production of both LTC4 and LTB4 in kidneys from MRL-lprllpr mice, but not in control animals. In addition, in vitro LTC4 production was correlated with the severity of renal histomorphologic abnormalities. Administration of the specific peptidoleukotriene receptor antagonist SKF 104353 to 20 week old MRL-lprllpr mice significantly improved both GFR and CPAH, whereas this agent had no effect of renal hemodynamics in MRL-+/+ controls. These results suggest that renal production of LTC4 and LTB4 is increased in MRL-lprllpr mice with nephritis, and that enhanced production of peptidoleukotrienes causes reversible renal dysfunction. Increased leukotriene production within the kidney may therefore be important in the pathogenesis of lupus nephritis. Glomerulonephritis with progressive renal failure continues to be an important cause of morbidity and mortality in patients with SLE [1-31. Although the mechanisms promoting abnormal immune function and autoantibody production in SLE have received much attention 12-51, the specific effector systems which produce renal injury in lupus have not been as well characterized. Previous studies have suggested that alterations in arachidonic acid metabolism may be important in the pathogenesis of lupus nephritis 16-91. Enhanced production of cyclooxygenase metabolites has been documented in murine lupus as well as in humans with SLE . However, little is known about the potential role of altered lipoxygenase metabolism in renal disease associated with lupus. Leukotrienes are 5-lipoxygenase products of arachidonic acid that have a number of biologic actions which may be specifically relevant to the pathogenesis of renal disease . For

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

This field study aimed to investigate the relationships between the urinary zearalenone (ZEN) concentration, which reflects dietary ZEN intake, and the numbers of total and transferable embryos in superovulated cattle. A total of 38 cows (Japanese Black, n=16; Holstein, n=22) were superovulated for commercial embryo production. Urine samples were collected from all cows at the time of embryo flushing and the urinary ZEN concentration was measured. The ZEN concentration was corrected for the creatinine (Crea) concentration as follows: ZEN (pg/mL)/Crea (mg/dL); the corrected ZEN concentration was expressed in pg/mg Crea. The cows were divided into two groups according to whether the urinary ZEN level was less than (group 1) or more than (group 2) the mean value for each breed (Japanese Black: 97.4 pg/mg Crea; range 44.5-91.3 pg/mg Crea; Holstein: 155.5 pg/mg Crea; range 32.7-146.9 pg/mg Crea). The embryo flushing results were compared between the two groups within each breed. Overall, the total number of embryos collected and the number of transferable embryos did not differ significantly between the groups. These results suggest that natural ZEN contamination resulting in urine levels below the threshold value (i.e. below the maximal permissible urinary ZEN concentration) does not affect embryo production in Japanese Black and Holstein cows undergoing superovulation.

IL-6 in vitro production, as well as the serum/plasma concentration of the cytokine, increase with age. In the present investigation, a total of 62 individuals (31 males and 31 females), aged from 29 to 93 years of age (mean age of males: 60.4 years; mean age of females: 59.4 years) were assessed for IL-6 plasma concentration, and for IL-6 in vitro production, using supernatants of 4 h cultured adherent peripheral blood mononuclear cells (aPBMC). The subjects were examined for a C to G transition at nucleotide 2174 of the IL-6 gene promoter (2174 C/G locus), and were classi®ed as C allele carriers (C 1 ) or non-carriers (C 2 ). We found that: (i) aPBMC from C 1 individuals produced smaller amounts of IL-6 in vitro than C 2 individuals; (ii) IL-6 production by aPBMC increased with age in C 1 but not in C 2 subjects; (iii) there was no correlation between IL-6 plasma levels and in vitro IL-6 production by aPBMC; (iv) IL-6 C 1 individuals had lower plasma levels than C 2 individuals, and this phenomenon was signi®cant only in men. On the whole our data indicate that the production of IL-6 is genetically controlled and age-and gender-dependent.

Background: Microarray studies can supplement QTL studies by suggesting potential candidate genes in the QTL regions, which by themselves are too large to provide a limited selection of candidate genes. Here we provide a case study where we explore ways to integrate QTL data and microarray data for the pig, which has only a partial genome sequence. We outline various procedures to localize differentially expressed genes on the pig genome and link this with information on published QTL. The starting point is a set of 237 differentially expressed cDNA clones in adrenal tissue from two pig breeds, before and after treatment with adrenocorticotropic hormone (ACTH). Results: Different approaches to localize the differentially expressed (DE) genes to the pig genome showed different levels of success and a clear lack of concordance for some genes between the various approaches. For a focused analysis on 12 genes, overlapping QTL from the public domain were presented. Also, differentially expressed genes underlying QTL for ACTH response were described. Using the latest version of the draft sequence, the differentially expressed genes were mapped to the pig genome. This enabled co-location of DE genes and previously studied QTL regions, but the draft genome sequence is still incomplete and will contain many errors. A further step to explore links between DE genes and QTL at the pathway level was largely unsuccessful due to the lack of annotation of the pig genome. This could be improved by further comparative mapping analyses but this would be time consuming.

In vitro embryo production (IVEP) is a promising tool with many applications in producing calves from genetically superior animals desired for propagation and in the conservation and revival of endangered species. The techniques of IVEP were adopted from cattle and refined to suit the water buffalo requirements. From the collection of ovaries from a local abattoir and the collection of oocytes by ovum pick up from live animals, gamete storage, collection techniques, handling of ovaries and oocytes to keep the viability and developmental competence, selection of oocytes to the type of culture media and in vitro culture condition, and treatment of the sperm cells for in vitro fertilization are all-important components of the process that requires careful and precise action to ensure success. Trials on intracytoplasmic injection, the use of sex-sorted sperm cells as a tool for producing sex-predetermined embryos, and the somatic cell nuclear transfer are methods that can be used to pro...

Artificial insemination is a reproductive technology commonly employed in the genetic improvement program on water buffaloes in the Philippines. However, conception rate with artificial insemination is still hampered due to low reproductive efficiency of these animals. In this study, estrus cornification index was investigated in three bubaline breeds namely, Native Philippine Carabao (NPC), Bulgarian Murrah (BuM) and Brazilian Murrah (BrM) to develop a method that will increase heat detection and synchronize administration of artificial insemination in field conditions. A conception rate of 100% after artificial insemination was observed on each of the breeds inseminated with cornification index within the range 46% to 66%, 39% to 64%, and 55% to 67% in NPC, BuM, and BrM, respectively. A cornification index within the specified range reinforced with the natural

Field trials were carried out on the use of swamp buffaloes (2n = 48) as recipients of in vitro-derived vitrified river buffalo (2n = 50) embryos. River buffalo embryos were produced through a total in vitro production system using frozen semen from a top progeny-tested bull and slaughter-house-derived ovaries and cryopreserved by a vitrification technique. Recipient swamp buffaloes were Philippine carabaos selected on body condition, parity, and reproductive status and owned by village farmers. Eighty postwarmed riverine embryos were transferred non-surgically to 40 swamp buffalo recipients, of which three healthy riverine calves were born by normal delivery and one stillbirth, representing a 5.0% (4/80) full-term development and 10% (4/40) calving rate with 75% (3/4) normal and 25% (1/4) stillbirth. The results demonstrate that, under field conditions and despite the difference in chromosome number between the embryo and recipient, swamp buffaloes are potential surrogate mothers of imported, genetically superior river buffalo frozen embryos. Thus, embryo IVP-vitrification and transfer technology is a potential tool in the propagation of genetically superior buffaloes. Precautionary measures, however, are recommended to avoid dystocia and stillbirth.

A tecnica do TUNEL tem se mostrado eficiente como indicadora de qualidade embrionaria em embrioes bovinos e de camundongos. Entretanto, em ratos, a tecnica nao e amplamente utilizada. Objetivou-se avaliar a qualidade do desenvolvimento de embrioes de ratas Wistar e o uso da tecnica de TUNEL. Animais (n=8) foram superovulados atraves da injecao intraperitoneal de 150 UI/kg de peso via intraperitoneal (IP) de PMSG (Pregnant Mare Gonadotropin) e de 75 UI/kg de hCG 48h apos. Femeas superovuladas foram colocadas para acasalar. 107 embrioes de 4-8 celulas foram coletados 72 horas apos hCG e cultivados em meio KSOM com 5% albumina serica humana (BSA), em incubadora com 5% de CO2, 95% de umidade a 37,0oC, por 48 horas. Apos o cultivo, foi calculada a taxa de blastocisto e realizada a tecnica do TUNEL. A taxa de blastocisto foi de 83,17% (89/107), com 19,62% (21/107) de blastocisto inicial, 14,01% (15/107) de blastocisto, e 49,53% (53/107) de blastocisto expandido. O numero total de blastome...

In dentistry, a variety of potentially allergenic metals are used, such as mercury, palladium, nickel, gold, chromium, cobalt and other metals. This paper describes a diagnostic approach from a dentist&#39;s point of view, which enables analysis of metals in a patient&#39;s oral cavity. If metal allergy is suspected, a micro analysis can be used to determine which metals are present in the restorations. When the exact composition of the dental materials is known, the patient can be tested in vivo (patch test) and/or in vitro (lymphocyte proliferation test) to reveal sensitization. Two patients with nickel allergy are described where removal of nickel-containing materials (bridge and orthodontic wire) resulted in the marked alleviation of symptoms and improvement of health. Finally, if allergy to specific metals has been established, the restorations containing the implicated metals should be removed to discontinue the exposure and thus facilitate the patient&#39;s health.

Effects of adrenal corticosteroids on reproductive and endocrine functions of the gonads are well known, but reciprocal effects of gonadal hormones on the hypothalamo-pituitary-adrenal (HPA) axis and on adrenocortical steroidogenesis in particular have received much less attention. We investigated effects of gonadectomy and testosterone (T) replacement on adrenocortical cell function in a year-long field study of male Sceloporus undulatus (Eastern Fence Lizard) and in a shorter term laboratory study with male Sceloporus jarrovii (Yarrow's Spiny Lizard). We also compared females to males in Sceloporus virgatus (Striped Plateau Lizard) and investigated effects of gonadectomy in short-term laboratory experiment on females of this species. As measured by in vitro production of progesterone (P 4 ), corticosterone (B), and aldosterone (ALDO), sensitivity of adrenocortical cells to corticotrophin (ACTH) was lower in control males than females of S. virgatus. In S. jarrovii males, cellular sensitivity to ACTH was reduced by orchiectomy but was not restored to levels of intact controls by T replacement. By contrast, in S. undulatus, cellular sensitivity to ACTH was not affected by orchiectomy alone but was reduced by T replacement in orchiectomized males. Maximal rates of steroid production were less consistently affected by experimental treatments, but were lower in males than in females of S. virgatus and were dramatically reduced by T replacement in orchiectomized S. undulatus males. Overall, our experiments clearly demonstrate two distinct sources of variation in functional capacities of dispersed adrenocortical cells isolated from Sceloporus lizards: (1) naturally occurring differences between males and females , and (2) species-dependent changes in response to surgical gonadectomy with or without exogenous testosterone. Sex differences and functional lability in adrenocortical cells are probably widespread among vertebrates and may be an important component of variation in output of the HPA.

Cholinesterases are targets for organophosphorus compounds which are used as insecticides, chemical warfare agents and drugs for the treatment of disease such as glaucoma, or parasitic infections. The widespread use of these chemicals explains the growing of this area of research and the ever increasing number of sequences, structures, or biochemical data available. Future advances will depend upon effective management of existing information as well as upon creation of new knowledge. The ESTHER database goal is to facilitate retrieval and comparison of data about structure and function of proteins presenting the a/b hydrolase fold. Protein engineering and in vitro production of enzymes allow direct comparison of biochemical parameters. Kinetic parameters of enzymatic reactions are now included in the database. These parameters can be searched and compared with a table construction tool. ESTHER can be reached through internet (). The full database or the specialised X-window Client-server system can be downloaded from our ftp server (). Forms can be used to send updates or corrections directly from the web.

A pecuária brasileira é, em parte, sustentada pela utilização e aperfeiçoamento de biotecnologias da reprodução. Pesquisas que buscam solucionar problemas como a distância existente entre os laboratórios e as grandes fazendas produtoras de gado são necessárias. O objetivo deste trabalho foi avaliar a eficácia de um sistema de cultivo durante períodos longos de transporte de embriões bovinos produzidos in vitro. Os oócitos, obtidos de ovários de vacas de abatedouro, passaram pelos procedimentos de produção de embriões in vitro, e no quinto dia do cultivo, foram alocados no sistema de transporte para compor os tratamentos: Tcontroleembriões controle mantidos na incubadora; T48embriões retirados do meio de cultivo no 5º dia e simulado o transporte por 48 horas; T24 -embriões retirados do meio de cultivo no 6º dia e simulado o transporte por 24 horas. Após os tratamentos, os embriões foram avaliados quanto à taxa de blastocistos no 7º dia e a taxa eclosão no 9º dia. O delineamento experimental foi inteiramente casualizado com três tratamentos e cinco repetições cada. Não foram observadas diferenças (P> 0,05) na taxa de blastocisto (31, 33 e 35%) e na taxa de eclosão (74, 78 e 80%), respectivamente para os tratamentos Tcontrole, T48 e T24. Concluiu-se que o sistema de cultivo durante o transporte, por até 48 horas, foi eficiente quanto à viabilidade embrionária inferindo que o ambiente de transporte simulou as condições de atmosfera e de temperatura do laboratório de forma satisfatória. Biotecnologia da reprodução. Blastocistos. Produção in vitro de embriões. The Brazilian livestock is based on the use and in the improvement of reproductive biotechnologies. Researches that aims to solve problems such as the distance between laboratories and the large cattle producing farms are necessary. The objective of this study was to evaluate the effectiveness of a culture system during long periods of transportation on bovine embryos produced in vitro.. The oocytes obtained from slaughterhouse cows ovaries, passed through in vitro production of embryos procedures such as: maturation, fertilization and embryo culture and subsequently were allocated into the transportation system to compose the treatments: Tcontrol -control embryos kept in the incubator; T48embryos removed from the culture medium on the 5th day and transportation simulated for 48 hours; T24 -embryos removed from the culture medium on the 6th day and transportation simulated for 24 hours. After the treatments, the embryos were evaluated for blastocyst on the 7th day and hatching rate on the 9th day. The experimental design was completely randomized. No differences were found (P> 0.05) for blastocyst rate (31, 33 and 35%) and hatching rate (74, 78 and 80%), respectively, for Tcontrol, T48 and T24 treatments. It was concluded that the culture system during transport, up to 48 hours, was efficient when considering the embryonic viability, inferring that the transportation environment simulated atmosphere and temperature conditions of the laboratory in a satisfactory manner.

This study evaluated the effect of hormonal ovarian stimulation regimes on the quantity and quality of the cumulus-oocyte complexes (COCs) recovered by laparoscopy and their subsequent in vitro maturation (IVM). Eighteen cyclic Canindé goats received a vaginal sponge with 60 mg medroxyprogesterone acetate for 11 days, together with an injection of 50 μg d-cloprostenol on the 8 day, along with additional treatment regimens, as follows: i) five doses (5D), 120 mg of NIH-FSH-P1 in five injections at 12 h intervals; ii) three doses (3D), 120 mg of NIH-FSH-P1 in three injections at 24 h intervals; iii) single dose (1D), 70 mg of NIH-FSH-P1 and 200 IU of eCG at 36 h prior to sponge removal. Three sessions of hormonal treatment/oocyte recovery were performed and goats (n = 6/group) were allocated to different groups. The oocytes were collected by laparoscopy at the time of sponge removal and the IVM of the oocytes was monitored. A total of 14.8 ± 0.8 follicles were aspirated per animal wit...

Leptin is a circulating hormone which plays an important role in the regulation of energy balance, haemopoiesis and reproduction. Leptin and its receptor (leptin-R) are localized in human placental tissue but their function is not known. In this study we have investigated the expression of leptin and leptin-R in the human placenta with particular attention to extravillous cytotrophoblastic cell islands and cell columns which play a pivotal role in trophoblast invasion and placental growth. We demonstrate that leptin-R immunoreactivity shows a strong expression in the distal extravillous cytotrophoblastic cells of cell columns invading the basal plate, whereas leptin expression is homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located extravillous cytotrophoblast of cell columns is known to be regulated by a variety of proteases and some extracellular matrix molecules, we tested the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by cytotrophoblastic cells. We demonstrate that leptin increases, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN and enhances the activity of MMP-9 in cultured cytotrophoblastic cells. Our results suggest that leptin and leptin-R could have a role in the invasive processes of the extravillous cytotrophoblastic cells by modulating the expression of MMPs. In addition, these results provide a foundation for studying pathological conditions characterized by insufficient or excessive trophoblast invasion.

Twenty-nine Fusarium isolates, representing three new taxa originated by Nirenberg from F. sambucinum Fuckel sensu lato, namely: F. sambucinum Fuckel sensu stricto, F. venenotum Nirenb., and F. torulosum (Berk. & Curt.) Nirenb., were tested for in vitro production of toxic secondary metabolites on autoclaved corn kernels. F. sambucinum sensu stricto was able to produce type A trichothecenes and enniatin B (EB). In particular, amongst the 14 isolates tested, 5 produced only diacetoxyscirpenol (DAS) (up to 700 pg/g); 1 produced only neosolaniol (NEOS) (250 pg/g); 2 produced T-2 toxin (T-2) + NEOS (up to 175 and 150 #g/g, respectively); 1 produced NEOS + DAS (300 and 100 #g/g, respectively); and 5 produced DAS + EB (up to 500 and 140 #g/g, respectively). All six isolates of F. venenotum were able to produce only DAS (up to 100 #g/g). F. torulosum produced no trichothecenes, but four out of nine tested isolates were able to produce EB (up to 140/.tg/g). Zearalenones and type B trichothecenes were not found. The toxicity of the culture extracts towards Artemia salina L. was correlated in general with the occurrence of the above toxins, except for some F. torulosum strains. However, the lack of correlation between the amounts of toxins recovered and toxic activity observed in the Geotrichum candidum Link ex Pers. and A. salina assays suggested the presence of unknown toxic compounds.

O objetivo do presente trabalho, foi o estudo dos efeitos da idade e da fase do ciclo estral dos bovinos na quantidade e qualidade de ovócitos e de embriões produzidos in vitro. Para isso, foram utilizados ovócitos de 63 vacas da raça Holstein-Friesian abatidas em matadouro, com idades compreendidas entre 1 e 15 anos, as quais foram divididas em 5 lotes de acordo com a idade. Cada lote foi subdividido de acordo com a fase do ciclo estral em que o animal se encontrava no dia do abate, pela presença ou não de corpo lúteo funcional na superfície do ovário. Os lotes referentes à idade foram: ;8 anos. Após punção dos folículos, os ovócitos foram divididos nas classes A, B, C e D, tendo em conta o seu aspecto morfológico, em que apenas os de classe A e B foram usados para a FIV. Observou-se que o número de ovócitos produzidos foi diretamente proporcional à idade das vacas (R=0,99 - P&lt;0,001). Quanto à fase do ciclo estral, as vacas em fase luteínica, produziram maior número de ovócitos ...

Brilliant cresyl blue (BCB) staining is a method used for selection of developmentally competent porcine oocytes, in addition to the standard morphological classification. The aim of the current study was to assess the relationship between BCB staining in porcine oocytes with high and low morphological characteristics and its maturation rate. In the first part of the study, cumulus-oocytes complexes (COCs) (n=434) were aspirated from 60 ovaries. According to the morphological classification and BCB staining, they were divided in 4 groups: COC+/BCB+, COC−/BCB+, COC+/BCB−, and COC−/BCB−. In the second experiment, COCs (n=445) were categorized into 3 groups: control, BCB+, and BCB−. Significant differences in the maturation rate were observed between COC+/BCB+ and COC−/BCB− (66% vs. 23%), and between COC+/BCB+ and COC−/BCB+ (66% vs. 38%) (p&lt;0.0001). Significant differences were also registered between COC−/BCB+ and COC−/BCB-(38% vs. 23%) and between COC+/BCB− and COC−/BCB− (53% vs. ...

Mice were selected in this study as a model to induce E.coli mastitis and sttudying the role of purified LPS in performed the protection against induce E.coli infection of mammary glands in mice. After LD50 of purified antigen were determined (250 µg/ml of purified LPS). The experimental study was included 56 mouse which divided into two groups and the LPS group was included 26 mouse, while the control group was included 30 mouse. The mouse in LPS group was immunized by: 2.5 µg of LPS /gm of mice and by sterile PBS (pH=7.2) in control group. In all groups, the route of inoculation was subcutaneous, and these doses repeated at 15 days, 27 days after first inoculation as boostered doses. At 34 days post the first inoculation the humoral and cellular immune response were assesement by many particular tests to determine the activity of their antigen in provided protection against challenge dose by virulent E.coli O111:B4 which gave intramammary, and histopathogical study was carried out...

Cytokines appear to be involved in the pathogenesis of Alzheimer's Disease (AD). Their modulation by treatment has been investigated only in a few studies. The aim of our study was to evaluate the effect of acetylcholinesterase inhibitors (AChEI) on Interleukin-4 (IL-4) production in AD patients. IL-4 levels were measured by ELISA on peripheral blood mononuclear cell cultures in the presence or absence of Concanavalin A or Phytohaemagglutinin. Linear regression analysis shows that patients who have been treated, have higher levels of IL-4 independently from age, gender and comorbidity. The increased production of IL-4 in AChEI treated patients might represent an additional mechanism through which AChEI act on AD progression.

Five species of meat-borne, coagulase-negative staphylococci were screened for their in vitro production of 3-methyl-1-butanol. The highest production level was encountered for Staphylococcus sciuri αSG2, despite its poor growth. With respect to Staphylococcus species that are generally applied in sausage starter cultures, production of 3-methyl-1-butanol was higher with Staphylococcus xylosus 3PA6 than with Staphylococcus carnosus 833. Mathematical modelling was used to link the kinetics of 3-methyl-1-butanol production by S. xylosus 3PA6 and S. carnosus 833 in meat simulation medium to bacterial growth and environmental factors, in casu temperature and pH. The specific production rate of 3-methyl-1-butanol was about ten times higher for S. xylosus 3PA6 than for S. carnosus 833, indicating a higher production rate per amount of biomass. This explains the higher concentrations of 3-methyl-1-butanol in the medium with S. xylosus 3PA6, despite its poorer growth.

Five species of meat-borne, coagulase-negative staphylococci were screened for their in vitro production of 3-methyl-1-butanol. The highest production level was encountered for Staphylococcus sciuri αSG2, despite its poor growth. With respect to Staphylococcus species that are generally applied in sausage starter cultures, production of 3-methyl-1-butanol was higher with Staphylococcus xylosus 3PA6 than with Staphylococcus carnosus 833. Mathematical modelling was used to link the kinetics of 3-methyl-1-butanol production by S. xylosus 3PA6 and S. carnosus 833 in meat simulation medium to bacterial growth and environmental factors, in casu temperature and pH. The specific production rate of 3-methyl-1-butanol was about ten times higher for S. xylosus 3PA6 than for S. carnosus 833, indicating a higher production rate per amount of biomass. This explains the higher concentrations of 3-methyl-1-butanol in the medium with S. xylosus 3PA6, despite its poorer growth.

The continuous search for natural preservatives that improve the microbial quality, physicochemical characteristics, shelf life, and sensory attributes of food products has become an issue of interest to researchers and consumers alike. Lactic acid bacteria, especially Lactobacillus plantarum, have been frequently used as a starter culture for fermented sausage production. Water Kefir is another valuable product that causes sausage fermentation and enhances its flavor, quality, and shelf life. Therefore, this study was conducted to compare Lactobacillus plantarum, which acts as a control group, and different concentrations of Kefir 1%, 3%, and 5% in the fermentation process of oriental sausages. The results demonstrated that including water Kefir notably improved the sausages’ quality. Specifically, water Kefir 3% and 5% concentration groups showed overall acceptability till the 15th and 17th days. In comparison, water Kefir 1% and the control group showed acceptability till the 11th and 7th day, respectively. In addition, a significant difference (P<0.05) in levels of total bacterial, psychrotrophic, and yeasts and molds counts, especially on the 7th day between the control group (7.78±.01a, 4.38±.01a and 4.96±.01a), water Kefir 1% (6.83±.01b, 3.73±.01band 4.66±.01b), 3% (5.69±.01c, 3.54±.01c and4.53 ±.01c) and 5% (5.60±.03d, 3.41±.01d and 4.50±.01d), respectively. It was observed that water Kefir 3% and 5% had significant differences (P<0.05) effects on color intensity, optimum acidity levels, increased water holding capacity, and reduced drip and cooking losses compared to 1% water Kefir and the control one. In conclusion, Water Kefir was preferred to be used in the fermented sausage as it not only enriched the flavor and texture of the sausages but also decreased total bacterial, total psychrotrophic, and total mycotic counts, enhanced the physicochemical characteristics, sensory qualities, and overall standard of the sausages.

American-Eurasian Journal of Sustainable Agriculture, 3(3): 328-331, 2009 ISSN 1995-0748 © 2009, American Eurasian Network for Scientific Information This is a refereed journal and all articles are professionally screened and reviewed ... Corresponding Author: Aman ...

This field study aimed to investigate the relationships between the urinary zearalenone (ZEN) concentration, which reflects dietary ZEN intake, and the numbers of total and transferable embryos in superovulated cattle. A total of 38 cows (Japanese Black, n=16; Holstein, n=22) were superovulated for commercial embryo production. Urine samples were collected from all cows at the time of embryo flushing and the urinary ZEN concentration was measured. The ZEN concentration was corrected for the creatinine (Crea) concentration as follows: ZEN (pg/mL)/Crea (mg/dL); the corrected ZEN concentration was expressed in pg/mg Crea. The cows were divided into two groups according to whether the urinary ZEN level was less than (group 1) or more than (group 2) the mean value for each breed (Japanese Black: 97.4 pg/mg Crea; range 44.5-91.3 pg/mg Crea; Holstein: 155.5 pg/mg Crea; range 32.7-146.9 pg/mg Crea). The embryo flushing results were compared between the two groups within each breed. Overall, the total number of embryos collected and the number of transferable embryos did not differ significantly between the groups. These results suggest that natural ZEN contamination resulting in urine levels below the threshold value (i.e. below the maximal permissible urinary ZEN concentration) does not affect embryo production in Japanese Black and Holstein cows undergoing superovulation.

RESUMO -O objetivo deste trabalho foi avaliar a influência do diluidor do sêmen no desenvolvimento in vitro de ovócitos bovinos após a maturação e fecundação in vitro. Ejaculado de um reprodutor foi fracionado e submetido a três diluidores: Lactose/gema de ovo (LG), Citrato/gema de ovo (CG) e Tris/gema de ovo (TG). Amostras deste material foram envasadas, congeladas e estocadas em N 2 e, posteriormente, descongeladas; a fração móvel foi separada por gradiente descontínuo de Percoll. A concentração espermática foi ajustada para 10 x 10 6 /mL e a capacitação espermática, induzida com 10 µg/mL de heparina. Após 24 horas de cultura para maturação in vitro, os ovócitos, aspirados de folículos ovarianos, foram inseminados com sêmen diluído em meio TALP e, após 48 horas de cultura, os zigotos foram transferidos para gotas de meio TCM 199, com 5% de soro fetal bovino, 5% de soro de vaca em estro e suspensão de células epiteliais do oviduto bovino, cobertas com óleo de silicone, e mantidos em cultura por nove dias. Todas as culturas foram realizadas a 38,5 o C em atmosfera com 5% de CO 2 . Os dados foram analisados pelo teste do qui-quadrado e houve diferença com relação à taxa de clivagem (TC), sendo as médias de 66,0; 69,3; e 54,4% para LG, CG e TG, respectivamente. Não houve diferença entre tratamentos com relação às taxas de mórulas/blastocistos ou de eclosão. O diluidor do sêmen não teve efeito sobre o desenvolvimento in vitro de embriões bovinos, embora a TC tenha sido afetada. Palavras-chave: bovinos, diluidor do sêmen, embrião, fecundação in vitro, taxa de clivagem ABSTRACT -This study investigated the effect of semen extender on in vitro development of bovine oocytes after in vitro maturation and fertilization. The ejaculate from one bull was divided in three parts which were diluted with three different extenders: Egg yolk/Lactose (EYL), Egg yolk/Citrate (EYC) and Egg yolk/Tris (EYT). Samples of semen diluted with these extenders were frozen in liquid nitrogen and, after thawing, were filtered by discontinuous Percoll gradient. The sperm concentration was adjusted to 10 x 106 cells/mL and the sperm capacitation was induced with 10 µg/mL of heparin. After 24 hours of culture for in vitro maturation, the oocytes, aspirated from ovarian follicles, were inseminated with semen diluted in TALP medium and 48 h after the zigotes were transferred into droplets prepared with TCM-199 medium containing 5% of bovine fetal serum, 5% of estrous cow serum and bovine oviduct epithelial cells in suspension under silicon oil and were further cultured of 9 days. All the cultures were performed at 38.5ºC in 5% CO 2 in air. Data were analyzed by chi-square analysis. There was a significant difference in terms of cleavage rate (CR) with means of 66.0, 69.3 and 54.4% for EYL, EYC and EYT, respectively. There were no differences in terms of the morulae/blastocysts and hatching rates. These results suggest that the semen extender had no effect on in vitro development of bovine, even the CR have been affected.

Background and Aim: Heat stress (HS) can negatively impact oocytes by disrupting mitochondrial activity, increasing the production of reactive oxygen species, and decreasing antioxidant levels. This study investigated the impact of orange peel (OP) exposure on zebrafish oocytes (ZOs) diameter, survival rate, and germinal vesicle breakdown (GVBD) during HS.

Materials and Methods: We investigated the antioxidant effect of flavonoids (concentration = 328.58 ppm) derived from OP (Citrus sinensis) added to in vitro maturation (IVM) media of ZOs (Danio rerio) under non-heat stress (NHS) and HS conditions to mimic in vivo HS conditions due to the global warming phenomenon on females. ZO in stage 3 (n = 1080) was treated with 4 μL of OP extract (not treated/control) under HS: 32°C (Heat stress 32°C solution/Heat stress 32°C orange peel [HS32S/HS32O]) and 34°C (Heat stress 34°C solution/Heat stress 34°C orange peel [HS34S/HS34O]); and NHS: 28°C (Non-heat stress solution/Non-heat stress orange peel [NHSS/NHSO]), during maturation. After 24 h of maturation, we observed the oocyte diameter, survival rate, and GVBD rate. The data were analyzed with IBM Statistics 23 software using two-way analysis of variance and Kruskal–Wallis (p < 0.05).

Results: The highest oocyte diameter data were in NHS treated with OP extract (NHSO) group (0.759 ± 0.01; mean ± standard error) compared with HS group using and without OP extract (HS32S [0.583 ± 0.02]; HS32O [0.689 ± 0.02]; HS34S [0.554 ± 0.02]; and HS34O [0.604 ± 0.02]). The survival rate of OP treated group, namely, NHSO (93% ± 3%), HS32O (85% ± 2%), and HS34O (80% ± 2%) was higher than that of the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]). ZO treated with OP extract (NHSO [93% ± 3%], HS32O [85% ± 2%], and HS34O [80% ± 2%]) showed a higher GVBD rate than the group without treatment (NHSS [83% ± 3%], HS32S [71% ± 6%], and HS34S [63% ± 3%]).

Conclusion: It revealed that OP can enhance the oocyte diameter, survival rate, and GVBD rate of ZO under NHS and HS. Further investigation should be conducted to determine the effect of OP extract (C. sinensis) on in vivo conditions in females as an alternative treatment to face global warming.

Keywords: heat stress, orange peel extract, Zebrafish oocytes.

A simple high density cell culture system is described which demonstrates many of the features of commercially available hollow-fibre systems, but without the need to invest in a dedicated system. The system has been shown to achieve product concentrations of up to 40-fold greater than that obtained in batch culture making gram production of MAbs possible with considerable saving on serum costs. List of symbols ELISA FBS IgG IgM MAb pH pO2 RPMI1640 enzyme-linked immunosorbent assay foetal bovine serum immunoglobulin G immunoglobulin M monoclonal antibody negative common logarithm of the hydrogen ion concentration dissolved oxygen concentration Roswell Park Memorial Institute medium 1640

We studied the role of three glycosidases (αmannosidase-α-ΜΑΝ, β-N-acetyloglucosaminidase-NAGASE and β-galactosidase-β-GAL) in follicular fluid (FF) and in fertilization medium (FM) of bovine oocytes. Oocytes were allocated into 3 groups according to the follicular size (controls-CF: 2-8 mm, small follicle group-SF: 2-5 mm, large follicle group-LF: >5-8 mm). Bovine embryos were produced in vitro either in groups (experiment 1, n = 2099 oocytes) or individually (experiment 2, n = 79 oocytes). In both experiments, the activity of all glycosidases in the FF of large follicles was significantly lower than in the FF of small follicles group. In the FM of LF-group oocytes, α-MAN and NAGASE were significantly higher compared to SF-and CF-group oocytes (experiment 1) and β-GAL was significantly higher in SF-compared to CF-group oocytes (experiment 2). Cleavage rate was similar among all groups in both experiments; however significantly higher blastocyst formation was noted in CF-group compared to LF-(days 7, 8, 9) and SF-(days 8, 9) groups (experiment 1). In follicular fluid of small follicle group, β-GAL was associated positively with degenerating oocytes' number and negatively with blastocyst rate at days 7, 8 (P = 0.065) and 9 (experiment 1). In fertilization medium of control group, α-MAN related negatively to cleavage rate (P < 0.05) and β-GAL to blastocyst rate at day 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1). During fertilization, in experiment 1 all oocytes consumed β-GAL and only control or small follicle oocytes consumed α-MAN; in experiment 1, only large follicle oocytes released NAGASE, whereas all oocytes released all three glycosidases in experiment 2. In conclusion, glycosidases affect the developmental competence of oocytes collected from different sized follicles during in vitro fertilization, performed either in groups or individually; their role in follicular fluid is different from that in fertilization medium.

In the course of a preliminary clarification of the mechanisms of the leaf mixture extract of Aloe buettneri, Justicia insularis, Dicliptera verticillata and Hibiscus macranthus, locally used to regulate the menstrual cycle and to treat dysmenorrhea or cases of infertility in women, pieces of proestrus rat ovary were incubated in the presence of increasing concentration of the plant extract and/or human Chorionic Gonadotropin (hCG). The in vitro production of estradiol and progesterone by ovarian cells of proestrus rat was significantly increased in the presence of various concentration of hCG (P < 0.05). The different concentration of the plant extract increased the production of estradiol by twofold. In addition, the in vitro production of estradiol by ovarian cells increased by 13-fold when they were incubated with hCG (0.1 IU/ml) and a concentration of 130 g/ml of the plant extract. These results clearly attest the direct effects of some chemical components of the leaf mixture of the plants on ovarian steroidogenesis.

The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical. Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT. The AKI conditions involve biphasic cultures. In phase 1 the organism is grown in a still tube for 4 h. In phase 2 the medium is poured into a flask to continue growth with shaking. Virtually no expression of CT occurs if this protocol is not followed. Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere. The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

In the present investigation we analysed Interleukin 6 (IL-6) in vitro production by Epstein Á/Barr virus (EBV)-immortalized B lymphocytes established from 43 subjects, 15 young people and 28 elderly people, including 18 centenarians, after 3, 6, 9, 24, 48 and 72 h of culture. The subjects were genotypized for the C to G transition at nucleotide (/174 of IL-6 gene promoter ((/174 C/G) and were classified as C allele carriers (C'/) and non-carriers (C(/). We found that: (i) the interindividual difference in in vitro IL-6 production was wider in elderly individuals in respect to young individuals, leading to different coefficient of variation in the two groups; (ii) the (/174 C/G polymorphism had an age-related effect on IL-6 in vitro production. Only among C(/ people, cells from elderly subjects produced significant higher level of IL-6 than cells from young subjects. These data are consistent with our previous results regarding the IL-6 serum levels in a large group of people of different age, including centenarians. Thus, the EBVimmortalized B lymphocytes can be considered a useful in vitro model for studying the genetic control of IL-6 production and its changes with age.