Funziona questo è stato sostenuto attraverso sovvenzioni dal scienze naturali e ingegneria Research Council del Canada (RGPIN 238382-2011) e Canadian Institutes of Health Research (MOP115076). Heather Paul è stato sostenuto da una borsa di studio post-laurea scienze naturali e ingegneria Research Council del Canada e una borsa di studio Alberta innova Health Solutions. Megan Hallam è stato sostenuto da un scienze naturali e ingegneria Research Council Postgraduate borse di studio, una borsa di studio Frederick Banting e Charles Best Canada laureato e premio un bambini Alberta Hospital Research Training Institute in Genetica, Child Development, e la salute.

Questo protocollo è stato approvato dalla University of Calgary Comitato cura degli animali e conforme alla Guida per la cura e l&#39;uso di animali da laboratorio. 1. Diga separato da Offspring <ol><li> Separare la diga dalla sua prole per un minimo di 5 minuti prima mungitura 12. NOTA: La diga può essere munta fino a 5-6 ore dopo la separazione 1,6,13, ma periodi di separazione più lunghi di 4 ore possono alterare la composizione del latte 14. Mentre tempo di separazione non sembra impattare latte volume di raccolta 12, è consigliabile che un tempo di separazione coerente essere mantenuta per tutto lo studio. Composizione del latte può cambiare tutta la lattazione 15, quindi i tentativi dovrebbe essere fatto per mantenere il giorno di raccolta del latte coerente. La produzione massima di latte si consiglia di verificare in lattazione giorno 14 12. </li><li> Usando una camera di riscaldamento, verificare i cuccioli sono in grado di mantenere la corretta tempera corpori senza la presenza della madre per la durata della procedura di mungitura. NOTA: Nello studio presentato di seguito, la mungitura è stata eseguita allo svezzamento, quando le dighe erano circa 22 settimane di età e le prole 21 giorni di età, quindi nessuna camera di riscaldamento è stato utilizzato. </li></ol> 2. Set-up e preparazione <ol><li> Raccogliere tutti i materiali necessari per la procedura di mungitura. NOTA: Tutti i materiali possono essere trovati nella tabella materiali ed apparecchiature. </li><li> Posizionare una piastra elettrica sulla panca dove la mungitura avrà luogo e coprire il pad con un banco assorbenti sotto-pad. </li><li> Impostare il sistema anestetico. Assicurarsi che il sistema ha ossigeno e isoflurano prima dell&#39;inizio sufficiente. Fissare la maschera anestetico che verrà utilizzato per l&#39;induzione di anestesia iniziale alla macchina. Posizionare la maschera che verrà utilizzata per l&#39;anestesia di mantenimento vicino se diversa dalla maschera anestetico iniziale. </li><li> Collegare un ago da 25 G per una siringa da 1 ml per ossitocinainiezione con tecnica asettica. </li><li> Accendere il rilievo di riscaldamento in modo che la temperatura del corpo della madre è mantenuta durante la procedura di mungitura. NOTA: Monitorare la temperatura della piastra elettrica per assicurare il pad non diventi troppo calda e causare ustioni. In alternativa, utilizzare una fonte di calore, ad esempio un tavolo operatorio riscaldato, che può essere impostato ad una temperatura specifica. </li></ol> 3. Anestetizzare la diga di Utilizzo Isoflurane <ol><li> Aprire il serbatoio di ossigeno e ruotare il flusso a 1 L (1.000 cc) per min. Accendere il flusso di isoflurano e impostare al 5%. ATTENZIONE: Evitare l&#39;inalazione diretta di anestetico e prevenire l&#39;accumulo di vapori anestetici. </li><li> Anestetizzare la diga. </li><li> Passare alla maschera di manutenzione, se necessario, ponendo la supina diga sul tampone assorbente panca. Confermare anestesia dalla mancanza di pedale reflex. </li><li> Ridurre il flusso di isoflurano al 2-3% per il mantenimento dell&#39;anestesia. Monitorare continuamente diga durante tutta la procedura per garantire la depressione di respirazione non si verifica. NOTA: Una volta sotto anestesia, gli occhi della diga devono essere protetti con un lubrificante occhio sterile per evitare che gli occhi si secchi o di diventare graffiato. </li></ol> 4. L&#39;ossitocina Iniezione <ol><li> Assicurarsi che il ossitocina (20 USP Unità / ml) non ha superato la data di scadenza. Disinfettare la fiala di ossitocina con un alcool sterile wipe / alcol detergente pad. </li><li> Utilizzando una tecnica asettica, elaborare 2 UI (0,1 ml) di ossitocina nella siringa. Usare un nuovo ago e la siringa per ogni diga che verranno munte. NOTA: L&#39;ossitocina dosi variano generalmente da singole iniezioni di 1-5 UI 1,6,12,16. In alternativa, una dose di 4 UI / kg di peso corporeo può essere utilizzato 12. Una singola dose di 2 UI può essere ripetuta una volta se si incontra difficoltà di mungitura. </li><li> Iniettare l&#39;ossitocina per via intraperitoneale. Inserire l&#39;ago nel quadrante in basso a destra dell&#39;addome con l&#39;ago rivolto verso la testa, con un angolodi 15-30 °, circa 0,5 cm di profondità. </li><li> Tirare indietro lo stantuffo per garantire pressione negativa prima dell&#39;iniezione. Se il liquido (sangue, urine, contenuto intestinale, ecc) è aspirato nella siringa, estragga l&#39;ago e tentare l&#39;iniezione con un nuovo ago e la siringa. Se nessun fluido viene aspirato iniettare l&#39;ossitocina e scartare il ago e la siringa immediatamente in un contenitore a rischio biologico. </li><li> Attendere circa 5-15 min per il ossitocina per stimolare il latte delusione. </li></ol> 5. Preparazione di siti mungitura <ol><li> Scegli i siti / mammelle da cui verrà raccolto il latte. Il latte può essere raccolto da ogni capezzolo 12. </li><li> Rimuovere delicatamente il pelo intorno ai capezzoli da mungere con i trimmer, come la pelliccia può causare difficoltà nella raccolta dei campioni a causa dell&#39;umidità del latte. Essere gentile - la pelle intorno i capezzoli è estremamente sensibile e può essere secco e come tale è soggetta a graffi e rotture. </li><li> Sterilizzazione della tettarella iNon s necessario, ma opzionalmente, pulire il capezzolo con acqua tiepida dopo il pelo viene rimosso. Preparare almeno due siti come più di un sito può essere richiesta per la raccolta del latte. NOTA: Se l&#39;analisi del latte comprende profiling microbica, la zona del capezzolo può richiedere la sterilizzazione con iodio 5. </li></ol> 6. Latte Collection <ol><li> Premere delicatamente la base del capezzolo, espellendo manualmente il latte per la raccolta. NOTA: Se l&#39;analisi del latte comprende profiling microbica, le prime gocce di latte devono essere eliminate. </li><li> Raccogliere le gocce di latte in un tubo capillare, riempiendo il tubo capillare. Tubi capillari che possono ospitare volumi maggiori (ad esempio, 50 microlitri) facilitare il processo. NOTA: Se si riscontra difficoltà nella raccolta del latte, una seconda dose di ossitocina può essere somministrata. Si raccomanda di non superare la dose di 4 UI totale. </li><li> Erogare il latte dal tubo capillare in una provetta steriletoccare l&#39;estremità del tubo che è stato utilizzato per disegnare il latte dal capezzolo al lato della provetta - osservare il latte viene estratto dal tubo capillare tramite azione capillare. NOTA: &#39;Soffiare&#39; il latte che non aspirata nel tubo con un ago 18 G attaccato ad una siringa da 1 ml. </li><li> Monitorare continuamente la diga per segni di dolore o di depressione respiratoria e regolare il flusso di isoflurano conseguenza. </li><li> Continuare a raccogliere il latte, come descritto in questa sezione fino a quando il latte sufficiente sono stati raccolti per l&#39;analisi del latte prescelto. Per la creamatocrit e proteine ​​determinazione della concentrazione descritto di seguito, raccogliere 0,25 ml di latte. NOTA: utilizzare un sito di mungitura diverso se il latte delusione rallenta o il sito scelto non espelle il latte a sufficienza. Un massimo di circa 2,5 ml di latte per animale può essere raccolto 17, o fino a 0,5 ml per tettarella. Gli autori raccomandano che il tempo totale in anestesia essere limitato a circa 45-60 minuti, or 45 min di mungitura. </li><li> Raccogliere latte in una provetta per la misura creamatocrit microematocrito da campioni di latte fresco, e sigillare l&#39;estremità del tubo con argilla sigillante. Etichettare la provetta microematocrito con l&#39;ID diga e conservare il campione di latte in posizione verticale. </li><li> Quando la mungitura è completata, spegnere il flusso di isoflurano e ossigeno. Rimuovere la maschera dalla diga e continuare a monitorare diga fino sveglio. Si raccomanda che se non pienamente cosciente, la diga deve essere collocato su un tampone assorbente panchina durante il periodo di recupero, anziché direttamente sulla biancheria gabbia, per evitare che la biancheria venga aspirato o graffiare gli occhi della diga durante il recupero. NOTA: Non lasciare incustodita la diga fino a che non ha ripreso conoscenza sufficiente a mantenere recumbancy sternale. </li><li> Se non sono necessarie ulteriori analisi, congelare il latte a -80 ° C. Altri hanno suggerito che il latte può essere conservato fino a 3 ore a 4 ° C o 5 mesi a -20 ° C 18. </li></ol> 7. Creamatocrit misura <ol><li> Stimare il contenuto di grasso nel latte calcolando la creamatocrit latte (la percentuale di crema nel campione di latte) 19. NOTA: Le misurazioni con latte umano hanno dimostrato che il latte fresco o congelato può essere utilizzato per una misurazione creamatocrit, tuttavia latte fresco è più strettamente correlata (r = 0,92 rispetto r = 0,90) con concentrazione di lipidi 11. L&#39;uso di latte fresco o congelato per misurazioni creamatocrit deve essere mantenuto costante attraverso lo studio, come il latte scongelato è associato ad una piccola diminuzione creamatocrit valori 11. </li><li> Per il latte fresco, raccogliere un campione di latte dal capezzolo in un tubo microematocrito; riempire almeno ¾ (circa 15-20 ml). In alternativa, disegnare il latte fresco dal campione raccolto nel tubo capillare dopo mescolando bene. Sigillare la fine con l&#39;argilla sigillante. </li><li> Posizionare il tubo capillare nella filatore ematocrito, con ilestremità sigillata orientate verso l&#39;esterno, assicurando la centrifuga è equilibrata. </li><li> Iniziare lo spin ematocrito (120 sec a 13.700 xg). NOTA: il tempo di rotazione o la velocità possono cambiare a seconda del modello di centrifuga utilizzata. </li><li> Rimuovere il tubo dalla centrifuga dopo lo spin è completa ed eseguire le misurazioni per calcolare il creamatocrit. Osservare separazione del campione in uno strato di crema e uno strato trasparente. </li><li> Misurare e registrare la lunghezza totale del fluido nel tubo e la lunghezza del grasso (crema) strato utilizzando pinze o un righello. NOTA: Il creamatocrit è espresso come percentuale dello strato di crema all&#39;interno del campione del latte 19, calcolato come (lunghezza dello strato di crema / lunghezza totale della colonna latte) x 100 (Figura 1A). Calcolare valori di concentrazione e di energia grasso da valutazione creamatocrit come segue: concentrazione di grasso (g / L) = (creamatocrit (%) - 0,59) /0.146 (Figura 1B) 19; Valore energetico (kcal / L) = 290 + (66,8 * creamatocrit (%) (Figura 1C) 19. </li></ol> 8. Proteine ​​Concentrazione Determinazione <ol><li> Utilizzando Bovine Serum Albumin come standard proteine, determinare la concentrazione totale di proteine ​​del latte usando un test standard di proteine, come una proteina test Lowry 6. NOTA: La diluizione del latte può essere necessario per le misurazioni della proteina del latte a cadere all&#39;interno della curva standard del dosaggio. </li></ol>

<ol>
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Gli autori non hanno conflitti di interesse da dichiarare. Tutti gli esperimenti sugli animali sono stati condotti secondo protocolli approvati CCAC.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The method requires no specialized vacuum equipment and can be performed by a single person. 
While this protocol is intended for use at a single time point during lactation, others have performed serial milk collections throughout their studies12,15. However, as serial milking may affect milk composition17, it is advised that researchers determine the frequency and number of milk collections that best suit their study, based on the outcome of interest. Additionally, while others have used injectable anesthetics during milking procedures in the rat14,17, this protocol involves anesthetization of the rat using isoflurane. Isoflurane is an inhalable anesthetic, and its advantages include rapid induction and recovery rates20 and the ability to easily adjust the time the animal is anesthetized. However, in mice, isoflurane may result in decreased milk yield in comparison to injectable anesthetics10, although this does not seem to have been tested in rats. Furthermore, protocols involving vacuum suction may not require anesthetic, and may result in higher milk yield depending on the efficiency of the suction apparatus12. However, if proper suction does not occur, milk yield will decrease. 
It is possible for the researcher to encounter difficulty in manual expression of milk, which can prolong time under anesthesia or decrease the milk yield. If difficulty in milking is encountered, some possibilities are: 1) sufficient time has not passed for the oxytocin to take effect; 2) improper or inexperienced milking technique; 3) physical differences in the nipple; 4) incorrect intraperitoneal injection of the oxytocin. The researcher may therefore wait a few more minutes prior to attempting to milk again; if this does not result in increased milk yield, the researcher may choose to move to a different milking site. Using a vacuum, each set of teats (upper thoracic, lower thoracic, upper abdominal, and lower abdominal) was equally successful in expelling milk when the milker is proficient, and approximately 0.5 ml can be obtained from each teat12. Therefore, if one milking site is proving difficult, moving to another site may help. The authors are not aware of differences in milk composition between milking sites. Finally, a second dose of oxytocin may be administered to further stimulate milk letdown. Diluting the oxytocin to a standard dose volume/kg body weight can also be attempted, for example 1 ml/kg body weight12, however be aware of maximum intraperitoneal injection volumes. 
It is also possible that other factors, including but not limited to, rodent strain, health status, parity, litter number, level of stress, diet, time of day, and food intake may affect the outcome of the milk collection. While the discussion of all these variables is beyond the scope of this protocol, it is encouraged that the investigators consider the multitude of factors that may influence the efficiency of milk collection in their experiments.
Finally, a simple method of measuring the proportion of cream in the milk, the creamatocrit, which is an accurate estimation of the amount of lipid in the samples11, is described herein. The creamatocrit can then be used to estimate the fat concentration and energy value of the milk. Though there were no differences in creamatocrit, fat concentration, energy value, or protein concentrations between the maternal dietary groups in the results presented above, others have found differences in creamatocrit values based on maternal characteristics such as gestational age21 and duration of lactation22. Differences in oligosaccharide content in maternal milk has also been demonstrated with maternal dietary manipulation6. 
In summary, milk contains a plethora of bioactive compounds whose specific roles in early life development of offspring, and their subsequent effect on disease susceptibility throughout life, remain to be clarified. The protocol reported here allows milk to be collected from lactating rats by a single individual, requires no specialized vacuum apparatus, and provides sufficient milk for subsequent analysis. Mastering this technique will provide the researcher with the opportunity to contribute significant insight into the processes involved in early-life development and establishment of health.

Il latte è l&#39;unica fonte di nutrimento per i mammiferi neonati, fornendo energia e nutrienti per la crescita infantile e lo sviluppo 1,2. Mentre il latte è composto principalmente da cellule, lipidi e proteine ​​1, contiene anche una pletora di composti bioattivi che modulano lo sviluppo primi anni di vita della prole compresi gli enzimi, carboidrati, ormoni, anticorpi, fattori di crescita, citochine, esosomi, microvescicole, e piccoli RNA tali come microRNA 1,2. Il ruolo fondamentale del latte materno nello stabilimento della prole immunitario e la salute intestinale 3, insieme con la prova che allattati al seno i neonati sono meno suscettibili alle malattie 2, sottolinea l&#39;importanza di identificare i componenti del latte associati ai processi di malattia nella prima infanzia ed i meccanismi molecolari coinvolti nelle loro azioni. Il ratto di sviluppo è un modello popolare per studiare l&#39;effetto di vari nutrizionale, fisiologico, e interventi chimici sulle precocesviluppo -life 4. L&#39;analisi del latte di ratto può quindi fornire romanzo spaccato la salute materna e prole. Progressi scientifici attuali forniscono ora aumentare le opportunità per le approfondite indagini sugli effetti di specifici componenti del latte sulla salute e la malattia. Ad esempio, il sequenziamento di latte profili batterici ha chiarito il loro ruolo nella prima colonizzazione intestinale dell&#39;intestino infantile 5, analisi di spettrometria di massa di oligosaccaridi del latte hanno fornito spaccato l&#39;alterazione dei profili latte oligosaccaridi via dieta materna 6, e sequenziamento profondo di microRNA secreto in i globuli di grasso del latte materno evidenzia possibili ruoli nella trascrizione genica, il metabolismo, e la funzione immunitaria 7. Modelli di ratto rappresentano uno dei più popolari organismi modello usati in studi materni 8,9. Un vantaggio è la loro gestazione e allattamento periodi brevi, che durano solo approximately 21 giorni ciascuno; il tempo totale dall&#39;inizio della gravidanza per allattamento rappresenta un breve periodo di tempo in cui i dati importanti possono essere generati. Le maggiori dimensioni dei ratti rispetto ai topi, nel contesto di raccolta del latte, può fornire un vantaggio significativo rispetto al volume di latte e facilità di raccolta del latte; produzione di latte nel topo, per esempio, sembra essere dipende dal peso corporeo totale con topi pesanti producono più latte 10. Qui, è fornita una descrizione generale per la raccolta manuale del latte di ratti che allattano. Questo protocollo richiede attrezzatura minima, non è invasiva, poco costoso, e può essere utilizzato per raccogliere adeguati volumi di latte per ulteriori analisi a valle. In breve, la diga è anestetizzato con isoflurano, latte letdown è stimolata da ossitocina, e latte viene raccolto in provette capillari tramite spremitura manuale del latte. Infine, come due componenti principali di latte sono grassi e proteine, una breve description di stimare latte tenore di materia grassa utilizzando misurazioni creamatocrit 11 e la quantificazione della concentrazione proteica totale usando un test standard di proteine ​​è presentato.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

Il latte è stato raccolto come descritto allo svezzamento da dighe Wistar (circa 22 settimane di età, peso 350 a 400 g) che consuma un controllo (AIN-93G, n = 5), ad alto contenuto proteico (40% di caseina peso / peso, n = 5) , o di fibre prebiotiche (21,6% peso / peso, rapporto 1: 1 di oligofruttosio e l&#39;inulina, n = 4) dieta durante la gravidanza e l&#39;allattamento. La dose di ossitocina era 2 UI. Il latte è stato raccolto in provette capillari, e un tubo è stato filato usando un filatore ematocrito per determinare creamatocrit (Figura 1A), che è stato poi utilizzato per stimare la concentrazione di grasso e valore energetico secondo: concentrazione di grasso (g / L) = (creamatocrit (% ) -0.59) /0.146 (Figura 1B); Valore energetico (kcal / L) = 290 + (66,8 * creamatocrit (%) (Figura 1C) 19. Concentrazione di proteine ​​del latte è stata determinata utilizzando il saggio di proteine ​​Bio-Rad DC (figura 2). Non vi erano differenze in creamatocrit (p = 0,674), la concentrazione di grassi (p = 0,674), valore energetico (p = 0,674), ola concentrazione di proteine ​​R (p = 0,127), in base a dieta materna (ANOVA). <img alt="Figura 1" src="/files/ftp_upload/53476/53476fig1.jpg" /> Figura 1. Il latte creamatocrit, concentrazione di grasso, e valore energetico. I campioni di latte sono stati raccolti allo svezzamento da dighe Wistar per il controllo (n = 5), High Protein (n = 5), o Alta prebiotica Fibra (n = 4) diete tutta la gravidanza e l&#39;allattamento. Misure Creamatocrit (A) sono stati utilizzati per calcolare la concentrazione grasso del latte (B) e valore energetico (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53476/53476fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53476/53476fig2.jpg" /> Figura 2. Totale concentrazione di proteine ​​del latte. I campioni di latte erano raccogliereed allo svezzamento da dighe Wistar per il controllo (n = 5), High Protein (n = 5), o High Fibre prebiotica (n = 4) diete tutta la gravidanza e l&#39;allattamento. Concentrazione totale di proteine ​​è stata determinata utilizzando il saggio di proteine ​​Bio-Rad DC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53476/53476fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Milk Collection nella Rat Utilizzando tubi capillari e stima di tenore di materia grassa per Creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Nanyang Technological University

Research

JoVE Journal

Biology

12.3K Views.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Video: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Ricerca

Biologia

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imaging del recettore dell&#39;estrogeno α in Rat Arteriole Pial utilizzando un microscopio digitale immunofluorescenza

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento dei fibroblasti Portal ratto<em&gt; In situ</em&gt; Fegato perfusione

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imaging funzionale del grasso bruno nei topi con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Raccolta, isolamento e di arricchimento di batteri naturalmente presenti magnetotactic dall&#39;ambiente

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Metodi Latte di raccolta per topi e Reeves &#39;Muntjac

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Raccolta e analisi dei<em&gt; Arabidopsis</em&gt; Floema Essudati Utilizzo del metodo EDTA-agevolata

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Raccolta dello sperma aviaria Cloacal massaggi e isolamento di DNA da sperma

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...