Je tiens à remercier Daniel Van Dyk du Centre d&#39;étude intégrative du comportement animal (CISAB) pour une grande aide dans la conception du modèle et l&#39;accès aux archives vidéo. Christopher Evans (CISAB) fourni un appui technique, Daniel Warner (Université de Sydney) a fourni le modèle empaillés, et Richard Peters (Australian National University) a fourni les caractéristiques de conception tôt.

Conception d&#39;animation Objet de numérisation Acquérir lézards empaillés. Analyser le modèle avec un Konica Minolta VI-9i. Il produit un objet 3D (*. LWO et les fichiers *. obj) dans un maillage unique de 50 000 polygones. LightWave ® v8.3 a deux programmes utilisés pour l&#39;animation 3D: Modeleur et mise en page. LightWave ® conçoit Modeller et manipule l&#39;objet. Programme de mise en page Lightwave ® crée des scènes d&#39;animation. LightWave ® Modeller et mise en page ont trois dimensions de positionnement, l&#39;orientation et la rotation: rubrique (Y), hauteur (X), et la banque (Z). Acquisition Texture Photographie d&#39;un lézard en direct en utilisant une 12,8 mégapixels Canon EOS 5D Appareil photo numérique à partir de deux angles (de face et orthogonaux), trois positions (frontale, orthogonales, ventrale et dorsale), et trois régions du corps les parties (tête, corps, la queue, et des membres) sur une feuille de papier blanc à blanc pour la couleur équilibrée standardisée. Fusible photographies ainsi que dans Adobe ® Photoshop ® Elements 3.0. Créer une carte UV Atlas dans LightWave ® Modeller. La carte UV rompt l&#39;objet dans des chaînes de connexion polygones. Capturer l&#39;image JPEG de l&#39;Atlas UV carte avec Grab V1.2 pour créer une image distincte JPEG. Incruster le calque de fond dans Adobe ® Photoshop ® Elements 3.0. Dans le modeleur LightWave ®, les polygones en évidence sur la carte UV Atlas pour identifier les domaines spécifiques sur le lézard. Superficie de la culture et de superposer sur le fond l&#39;Atlas UV JPEG carte. Lorsque tous les fragments photographiques sont posés sur le JPEG Atlas UV carte, enlever le fond et créer un seul fichier TIFF. Importer le fichier TIFF dans LightWave ® Modeller et attribuer des coordonnées UV. Skelegons implantation et Bones Dans LightWave ® Modeller, skelegons ont été conçues et incorporées. Skelegons agi comme des espaces réservés pour les os. Créer 61 os. Créer skelegons pour imiter le nombre de vertèbres dans un dragon Jacky, avec une grande skelegon utilisé pour simuler la tête. Créer une colonne vertébrale virtuels à partir du cou jusqu&#39;à la fin de la queue. Fusible les membres antérieurs, qui se composent de quatre skelegons chacun, à la colonne vertébrale. Lorsque tous skelegons sont créés, envoyer l&#39;objet entier à LightWave ® Mise en page, où l&#39;skelegons doivent être transformé en os. Ajout d&#39;ombrage Poids Cartes de poids indépendants ont une gamme de valeur de -100% à +100% pour répartir uniformément le mouvement. Désignés les membres, la queue et la tête avec positif (+) des valeurs Plan du poids. Donner le corps un effet négatif - la valeur de poids map (). Capturez des vidéos d&#39;archives de stimulation Images d&#39;archives de lézard a été montré à l&#39;aide de Sony MiniDV enregistreur vidéo numérique PAL joueur sur un moniteur Sony Trinitron à un lézard vivant a tenu dans son enceinte (pour l&#39;acquisition vidéo initiale, voir Ord et Evans, 2002; Ord, Peters, Evans et Taylor, 2002; Van Dyk et Evans, 2007). Réponses des lézards ont été enregistrés en utilisant un Canon (MV650i) caméscope numérique placé sur le mètre environ de l&#39;enceinte. Taille Lizard a été ajustée à la taille représentative de la perche sur le moniteur. Exporter en tant individuelles des images JPEG séquentiel dans Apple QuickTime ™ v7.0. Rotoscopie pour la réplication de stimulation «Rotoscopie» est une technique où l&#39;animation d&#39;objets se superposent trame par trame dans le plan de la séquence de fond (Gatsey, Middleton, Jenkins, &amp; Shubin, 1999). Envoyer l&#39;animation à LightWave ® Mise en page, où la lumière, caméra, objet, et caractéristiques de fond peut être contrôlée pour la scène. Sélectionnez schémas moteurs de la vidéo numérique d&#39;archives et de l&#39;exportation comme une séquence d&#39;images (JPEG) avec Apple QuickTime Player ™ 7.0. L&#39;objet doit être en vue de la caméra. Importer le premier fichier JPEG dans la séquence d&#39;image dans le fond. Manipuler l&#39;objet en utilisant les os et les superposer dans la position actuelle qui reflète l&#39;image de fond. Chaque cadre est d&#39;être «images clés, tels que la position de l&#39;objet est sauvegardé. Remplacez l&#39;image de fond avec l&#39;image suivante consécutifs et de manipuler l&#39;objet dans la nouvelle position. Répétez le processus jusqu&#39;à ce que la séquence est terminée. Rendu la séquence pour l&#39;achèvement Plans de séquence Large ou traitement par lots sont rendues avec Render Farm commandant v2.9. Rendu Ferme commandant (RFC) relie des ordinateurs pour produire une série de processeurs à l&#39;aide de rendu plus rapide. Quatre Apple Mac G5 bi-processeurs (huit fils) ont été utilisés pour distribuer le rendu, réduisant ainsi le temps de quatre heures. Toutes les séquences ont été maintenus à PAL-DV standard (720 576 pixels Résolution ¥; compression 5:1; résolution horizontale 575 lignes, 25 trames S-1).

<ol>
	<li>Gatesy, S. M., Middleton, K. M., Jenkins, F. A., Shubin, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+preservation+of+foot+movements+in+Triassic+theropod+dinosaurs.">Three-dimensional preservation of foot movements in Triassic theropod dinosaurs.</a> Nature. 399, 141-144 (1999).</li><li>Newtek, Inc, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lightwave+[8]+Reference+Manual.">Lightwave [8] Reference Manual.</a> , (2004).</li><li>Ord, T. J., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactive+video+playback+and+opponent+assessment+in+lizards.">Interactive video playback and opponent assessment in lizards.</a> Behav. Process. 59, 55-65 (2002).</li><li>Ord, T. J., Peters, R. A., Evans, C. S., Taylor, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+video+playback+and+visual+communication+in+lizards.">Digital video playback and visual communication in lizards.</a> Anim. Behav. 63, 879-890 (2002).</li><li>Rosenthal, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+and+techniques+for+constructing+video+stimuli.">Design considerations and techniques for constructing video stimuli.</a> Acta Ethol. 3, 49-54 (2000).</li><li>Van Dyk, D. A., Evans, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familiar-unfamiliar+discrimination+based+on+visual+cues+in+the+Jacky+dragon,+Amphibolurus+muricatus.">Familiar-unfamiliar discrimination based on visual cues in the Jacky dragon, Amphibolurus muricatus.</a> Anim. Behav. 74, 33-44 (2007).</li></ol>

Le financement a été soutenue par le Prix d&#39;études supérieures et de CISAB Département de psychologie de l&#39;Université Macquarie, et l&#39;Australian Research Council. Des recherches ont été menées conformément aux directives éthiques de l&#39;Université Macquarie Comité d&#39;éthique animale (2003/014) et le New South Wales National Parks et Wildlife Services (S11024).

Animations générées par ordinateur sont de plus en plus un outil populaire dans lequel pour enquêter sur des questions concernant la communication dans le comportement animal. En utilisant des techniques d&#39;animation, les chercheurs peuvent se concentrer sur des aspects spécifiques de conception des signaux, et de manipuler des fonctions qui étaient auparavant incapables d&#39;être étudiés grâce à la technologie limitée. Bien que mis en scène des animaux conventionnels se heurte encore à démontrer les principes théoriques...

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<td>New Item</td>
<td>Digital camera</td>
<td>Konica Minolta Holdings, Inc. </td>
<td>VI-9i</td>
<td>Used to acquire 3D mesh (done by New Dawn&#174; - Bexley North, NSW Australia)
 
</td>
</tr>
<tr>
<td>Lightwave&#174; 3D</td>
<td>Software program</td>
<td>NewTek Inc.</td>
<td>v8.3</td>
<td>To create animation</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camera</td>
<td>Canon, Inc.</td>
<td>EOS 5D</td>
<td>12.8 megapixels</td>
</tr>
<tr>
<td>Grab</td>
<td>Software program</td>
<td>Apple Computer, Inc.</td>
<td>v1.2</td>
<td>Extract texture from photographs and UV map</td>
</tr>
<tr>
<td>Adobe&#174; Photoshop&#174; Elements</td>
<td>Software program</td>
<td>Adobe Systems Inc.</td>
<td>v3.0</td>
<td>Extract texture from photographs</td>
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<td>Render Farm Commander</td>
<td>Software program</td>
<td>Bruce Rayne&#169;</td>
<td>v2.9</td>
<td>Mass rendering across local area network

</td>
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<td>Mac Pro</td>
<td>Computer</td>
<td>Apple Computer, Inc.</td>
<td>&nbsp;</td>
<td>Four dual processors (eight threads); Create and render animation</td>
</tr>
<tr>
<td>Sony MiniDV</td>
<td>Digital video recorder</td>
<td>Sony Corporation</td>
<td>GV-D300E</td>
<td>Playback stimuli during stimulus acquisition</td>
</tr>
<tr>
<td>Sony Trintron</td>
<td>Colour viewfinder</td>
<td>Sony Corporation</td>
<td>PVM-14M2A</td>
<td>One monitor for stimulus acquisition</td>
</tr>
<tr>
<td>Canon</td>
<td>Digital camcorder</td>
<td>Cannon Inc.</td>
<td>MV650i</td>
<td>Records behavior during stimulus acquisition</td>
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computer-generated animal model stimuli

La communication entre les animaux est diversifié et complexe. Les animaux peuvent communiquer en utilisant auditif, sismiques, chimiosensoriels, les signaux électriques, ou visuelle. En particulier, la compréhension des contraintes sur la conception des signaux visuels pour la communication a été d&#39;un grand intérêt. Les méthodes traditionnelles d&#39;enquête sur les interactions des animaux ont utilisé les techniques de base d&#39;observation, mise en scène des rencontres, ou de la manipulation physique de la morphologie. Des méthodes moins intrusives ont essayé de simuler en utilisant des outils de lecture congénères brut, comme des miroirs, des images fixes, ou des modèles. Comme la technologie est devenue plus avancée, la lecture vidéo est apparue comme un autre outil dans lequel d&#39;examiner la communication visuelle (Rosenthal, 2000). Cependant, pour aller encore plus loin, l&#39;application de l&#39;ordinateur de l&#39;animation permet maintenant aux chercheurs d&#39;isoler spécifiquement les composants critiques nécessaires pour obtenir des réponses sociales à partir des congénères, et de manipuler ces fonctions pour contrôler les interactions. Ici, je fournir des détails sur la façon de créer une animation à l&#39;aide du dragon Jacky comme un modèle, mais ce processus peut être adapté pour d&#39;autres espèces. Dans la construction de l&#39;animation, j&#39;ai choisi d&#39;utiliser Lightwave 3D pour modifier la morphologie objet, ajouter de la texture, installer les os, et fournir de l&#39;ombre de poids comparable qui empêche tout mouvement exagéré. L&#39;animation est ensuite apparié pour sélectionner les modèles à reproduire les caractéristiques du moteur mouvement critique. Enfin, la séquence doit rendu dans un clip de présentation individuelle. Bien qu&#39;il existe d&#39;autres techniques adaptables, cette méthode particulière a été démontré pour être efficace dans obtenir des réponses à la fois visible et mise en scène sociale dans les interactions.

Watch this Scientific Journal Video about Computer-Generated Animal Model Stimuli at JoVE.com

Computer-Generated Animal Model Stimuli

Générées par ordinateur en utilisant des stimuli du dragon Jacky comme un modèle.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Research

JoVE Journal

Biology

10.9K vues.  Macquarie University. Générées par ordinateur en utilisant des stimuli du dragon Jacky comme un modèle.

Vidéo: Computer-Generated Animal Model Stimuli

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 &#x2013; 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6&deg;C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5&deg;C/min, enabling further dehydration, to a temperature of -34&deg;C before being plunged into liquid nitrogen (-196&deg;C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37&deg;C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage.

Cryoconservation d&#39;embryons pré-implantatoire de bovins, ovins et caprins

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage.  Preimplantation embryos consist predominantly ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

Le génotypage des échantillons de plantes et d&#39;animaux sans purification d&#39;ADN Avant

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

Utéro-tubaire transfert d&#39;embryons et la vasectomie dans le modèle de souris

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.

This protocol provides a convenient set of methods, which enables extremely fast, easy, non-invasive, reliable and low-cost, molecular sex determination of birds and their non-invasive, quick, safe and easily recognizable marking shortly after hatching. Only limited handling of chicks is required. This convenient toolbox of methods complies entirely with the RRR-guidelines.

Qui est qui? Méthodes non-invasives pour Individuellement sexe et Mark nidicoles poussins

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Un protocole pour l’utilisation de gènes série d’analyses d’enrichissement pour identifier le modèle Animal approprié de recherche translationnelle

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Un modèle de Culture tissulaire d’oestrogène-production primaire Bovine Granulosa cellules

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Techniques de biopsie des glandes mammaires bovine

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Analyse de spectrométrie de masse pour identifier l’ubiquité de cenp-A marqué par EYFP (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

Scrunching planaire comme une lecture comportementale quantitative pour la détection de stimuli nocifs

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...