ABSTRACT: didactic models are a very useful tool for pedagogy in Agrarian Science careers (i.e. Veterinary
Medicine and Animal Science). Objective: the aim of this work was to create a didactic model of the chicken
embryo development using modified Dawson´s diaphanization and staining technique, which allows the centers
of ossification to be viewed. Methods: chick embryos from day 5 to day 21 were diaphanized with KOH, stained
with Alizarin Red, and stored in glycerol. Results: growth of primary ossification centers during embryonic
development was easily visualized. Conclusion: to our knowledge, this is the first literature report showing an
anatomical model of all embryonic stages of chicken development. Impact of this model in pedagogy must be
evaluated in future works.RESUMEN: los modelos didácticos son una muy buena herramienta para el proceso de enseñanzaaprendizaje
en los cursos de Ciencias Agrarias (Medicina Veterinaria y Zootecnia, entre otros). Objetivo:hacer un modelo didáctico del desarrollo embrionario del pollo usando la técnica modificada de Dawson para
transparentación y tinción, que permite visualizar los centros de osificación. Métodos: embriones de pollo
del día 5 al 21 fueron transparentados con KOH, teñidos con Rojo de Alizarina y almacenados en glicerina.
Resultados: se pudo visualizar el crecimiento de los centros primarios de osificación durante el desarrollo
embrionario. Conclusión: este es el primer reporte en literatura que muestra un modelo anatómico de todas
las etapas del desarrollo embrionario del pollo. En futuras investigaciones se debe evaluar el impacto que
tiene este modelo en el proceso enseñanza-aprendizaje

Cano Valderrama, Ana I.

Cuartas Martínez, Brayian A.

Lenis Sanin, Yasser Y.

Mejía Giraldo, Carlos A.

Suárez Avendaño, Paula A.

Tamayo Arango, Lynda Jhailú

Yepes Ciro, Sergio A.

Revista Colombiana de Ciencias Pecuarias

English

Biblioteca Digital del Sistema de Bibliotecas de la Universidad de Antioquia

Tamayo LJ et al. Didactic model of the chicken embryo620Rev Colomb Cienc Pecu 2012; 25:620-624Didactic model of the chicken embryo development using modified Dawson’s diaphanization and staining technique¤Modelo didáctico del desarrollo embrionario del pollo usando la técnica modificada de Dawson para transparentación y tinciónModelo didático do desenvolvimento embrionário do frango usando a técnica modificada de Dawson para diafanização e coloraçãoLynda J Tamayo Arango1,*, MV, Dr Sci Agr; Paula A Suárez Avendaño2, Est Zoot; Ana I Cano Valderrama2, Est Zoot; Brayian A Cuartas Martínez2, Est Zoot; Sergio A Yepes Ciro2, Est Zoot; Carlos A Mejía Giraldo3, Biol, MSc; Yasser Y Lenis Sanin4, MVZ, Esp, MSc1Faculty of Agrarian Sciences, University of Antioquia, BIOGENESIS, Carrera 75 N° 65-87. AA 1226. Medellín, Colombia.2 Faculty of Agrarian Sciences, University of Antioquia. Carrera 75 N° 65-87. AA 1226. Medellín, Colombia.3 Faculty of Medicine, University of Antioquia. Carrera 51d N° 62-29. Medellín, Colombia.4Faculty of Agrarian Sciences, University of Antioquia, CENTAURO, Carrera 75 N° 65-87. AA 1226. Medellín, Colombia.(Received: 4 may, 2012; accepted 19 august, 2012)SummaryBackground: didactic models are a very useful tool for pedagogy in Agrarian Science careers (i.e. Veterinary Medicine and Animal Science). Objective: the aim of this work was to create a didactic model of the chicken embryo development using modified Dawson´s diaphanization and staining technique, which allows the centers of ossification to be viewed. Methods: chick embryos from day 5 to day 21 were diaphanized with KOH, stained with Alizarin Red, and stored in glycerol. Results: growth of primary ossification centers during embryonic development was easily visualized. Conclusion: to our knowledge, this is the first literature report showing an anatomical model of all embryonic stages of chicken development. Impact of this model in pedagogy must be evaluated in future works. Key words: anatomy, cartilage, embryology, organic system, pedagogy.ResumenAntecedentes: los modelos didácticos son una muy buena herramienta para el proceso de enseñanza-aprendizaje en los cursos de Ciencias Agrarias (Medicina Veterinaria y Zootecnia, entre otros). Objetivo: ¤	 To	cite	this	article:	Tamayo	LJ,	Suárez	PA,	Cano	AI,	Cuartas	BA,	Yepes	SA,	Mejía	CA,	Lenis	YY.	Didactic	model	of	the	chicken	embryo	development	using	modified	Dawson´s	diaphanization	and	staining	technique.	Rev	Colomb	Cienc	Pecu	2012;	25:620-624.*	 Corresponding	author:	Lynda	J	Tamayo	Arango.	University	of	Antioquia,	Faculty	of	Agrarian	Sciences,	BIOGENESIS.	Carrera	75	N°	65-87.	AA	1226.	Medellín,	Colombia.	Email:	lyndatamayo@gmail.comRevista Colombiana de Ciencias Pecuarias621Tamayo LJ et al. Didactic model of the chicken embryoRev Colomb Cienc Pecu 2012; 25:620-624hacer un modelo didáctico del desarrollo embrionario del pollo usando la técnica modificada de Dawson para transparentación y tinción, que permite visualizar los centros de osificación. Métodos: embriones de pollo del día 5 al 21 fueron transparentados con KOH, teñidos con Rojo de Alizarina y almacenados en glicerina. Resultados: se pudo visualizar el crecimiento de los centros primarios de osificación durante el desarrollo embrionario. Conclusión: este es el primer reporte en literatura que muestra un modelo anatómico de todas las etapas del desarrollo embrionario del pollo. En futuras investigaciones se debe evaluar el impacto que tiene este modelo en el proceso enseñanza-aprendizaje.Palabras clave: anatomía, cartílago, embriología, pedagogía, sistemas orgánicos.ResumoAntecedentes:	os	modelos	didáticos	são	uma	boa	ferramenta	para	o	processo	ensino-aprendizado	nos	cursos	de Ciências Agrárias (Medicina Veterinária, Zootecnia, entre outros). Objetivo: fazer um modelo didático do desenvolvimento	do	frango	usando	a	técnica	modificada	de	Dawson	para	diafanização	e	coloração,	que	permite	visualizar	os	centros	de	ossificação.	Métodos:	Embriões	de	frango	do	dia	5	ao	21	foram	transparentados	com	KOH, corados com Vermelho de Alizarina e armazenados em glicerina. Resultados: foi possível visualizar o crescimento	dos	centros	primários	de	ossificação	durante	o	desenvolvimento	embrionário.	Conclusão: este é o	primeiro	reporte	na	literatura	de	um	modelo	anatômico	do	desenvolvimento	embrionário	do	frango.	Futuras	pesquisas devem ser focadas no impacto que tem este modelo no processo ensino-aprendizado. Palavras chave: anatomia, cartilagem, embriologia, pedagogia, sistemas orgânicos.IntroductionDidactic models are tools used to improve analytic and argumentative capacity, imagination, and innovation skills in students in order to enrich their learning process in agrarian careers. The integrative course in Veterinary Medicine and Animal Science at	the	Faculty	of	Agrarian	Sciences	of	the	University	of Antioquia is titled Organic Systems. Aiming to improve the integration of anatomy, histology and physiology concepts, students have to develop a didactic model and present it in an exhibition during this	course	(Lenis	and	Tamayo,	2011).	Avian embryo development is of great economic importance because it is during this time that the genetic basis of the line or breed to be produced is consolidated.	Nowadays,	 it	 is	 very	 common	 to	 use	animal didactic models for improving pedagogy in agrarian science careers (i.e. Veterinary Medicine and Animal Science). Dawson´s technique is an anatomical technique which diaphanizes soft tissues and stains embryo bones	 (Dawson,	 1926).	 Several	 modifications	have	 been	 made	 since	 the	 first	 description	 of	 the	technique	 (Randle,	 1969;	 Tipton	 and	 Burt,	 1977;	Sanchez et al.,	 1996;	 Staples	 and	 Schnell,	 1964;	Jensh	 and	 Brent,	 1966;	 Kawamura	 et al.,	 1990),	and it is considered as a very useful technique for studying the embryological development of the skeletal system. Essentially, the method consists of macerating the fetus with alkali, followed by staining	it	with	a	dye,	which	has	a	strong	affinity	for	calcium salts, such as Alizarin Red (a dye derived from anthraquinone). The resulting specimens allow the visualization of the stained red bone beneath the transparent soft tissues.  It is necessary for Animal Science and Veterinary Medicine students to develop and have access to didactic models for studying skeletal system ossification.	 The	 study	 of	 this	 important	 process	allows the students to better understand some of the skeletal problems that animals can encounter, especially those derived from developmental impairments. The aim of this work was to describe the diaphanization and stain of the skeletal system of chicken embryos aged 5 to 21 days for its use as a didactic model for teaching purposes. Tamayo LJ et al. Didactic model of the chicken embryo622Rev Colomb Cienc Pecu 2012; 25:620-624Material and methodsAnimalsChicken embryos provided by Marruecos poultry farm (Medellín, Colombia) from 5 to 21 days of incubation were used. Day 5 was chosen because skeletal system development (endochondral and intramembranous	 ossification)	 starts	 at	 that	 day,	with	 small	 ossification	 centers	 in	 the	 skull,	 limb	buds and prevertebrae (Sawad et al.,	 2009).	 Each	embryo was separated from the attached structures of	the	egg	and	placed	in	individual	250	mL	bottles.	Specimen processingIn order to avoid excessive manipulation and damage the embryos were not transferred from the bottles. Taking into account the small size of the specimens, it was not considered necessary to eviscerate	or	 skin	 the	embryos.	They	were	fixed	 in	10%	 formalin	 (Protokimica,	 Medellín,	 Colombia)	for	 eight	 days	 to	 attain	 an	 adequate	 fixation,	 then	dehydrated	 in	 70%	 ethyl	 alcohol	 (Protokimica,	Medellín, Colombia) mixed with molecular iodine	 (Carlo	 Erba,	 Val-de-Reuil,	 France)	 for	 two	days. Molecular iodine was used as a mordant to potentiate	fixation	of	Alizarin	Red	in	bone	calcium.	Next,	 specimens	 were	 transferred	 to	 a	 2%	 KOH	solution (Protokimica, Medellín, Colombia) added to	 1%	 Alizarin	 Red	 (Merck,	 Parkville,	 Australia)	for	24	hours.	The	embryos	were	transferred	to	a	2%	KOH solution for eight days for clearing. At this time, embryos were completely stained by Alizarin Red, and no difference between muscle and bone was	observed.	For	an	additional	clearing,	a	1:1:1	2%	KOH solution, ammonia (Protokimica, Medellín, Colombia) and glycerol (Protokimica, Medellín, Colombia) was	used	for	eight	days.	Finally,	glycerol	was used to store the specimens. Results Embryos were successfully diaphanized, yielding	the	ossification	centers	in	red	(Figure	1).	To	attain the main objective of this work—that is, the process of forming a didactic model—all the bottles were placed in a specially designed shelf including photos of the fresh specimen in all the stages to enhance the steps of the embryologic development process,	as	shown	in	figure	2.Figure 1. 16-day-old chick embryo. Primary centers of ossification are clearly seen in red at bone diaphysis, while cartilage is seen in white at bone extremities. Figure 2. Final aspect of the didactic model displaying chicken development stages from days 5 to 21. Fetuses were placed in individual bottles. Photographs were placed in the central panel for additional explanations. 623Tamayo LJ et al. Didactic model of the chicken embryoRev Colomb Cienc Pecu 2012; 25:620-624The resulting didactic embryo model was as the	 final	 project	 of	 the	 Organic	 Systems	 course	at	 the	 Faculty	 of	 Agrarian	 Sciences-University	 of	Antioquia	 (Colombia),	 exhibited	 during	 the	 First	Undergrad	 Students	 Exhibition	 of	Animal	Didactic	Models	(Lenis	and	Tamayo,	2011).DiscussionTo	 our	 knowledge,	 this	 is	 the	 first	 literature	report showing an anatomical model of all the embryonic stages of chicken development. The modified	 Dawson´s	 technique	 stained	 the	 calcified	bone and allowed the visualization of the primary ossification	center	development.	Given multiple factors such as species, development stage, size, and evisceration, experience is needed to optimize diaphanization and	 staining	 of	 ossification	 processes.	 Several	modifications	 of	 Dawson´s	 technique	 have	been proposed to optimize the diaphanization process. Sanchez et al.	 (1996)	 used	 a	 higher	KOH	concentration	 (15%)	 to	 decrease	 the	 time	 spent	in diaphanization from one month to one week in	 human	 fetuses.	 Betti	 (2005)	 used	 2-4%	 KOH	to	 diaphanize	 fish	 larvae	 but	 only	 for	 10	 minutes,	while Maiolino et al.	 (2009)	used	6%	KOH,	under	an	 unspecified	 period	 of	 time.	 Care	must	 be	 taken	when using higher KOH concentrations due to the risk of tissue disintegration. When pigmentation is present, H202	 10	V	 solution	must	 be	 used	 for	 3	 to	5 minutes to clarify the specimen (Maiolino et al., 2009).	Sanchez et al.	 (1996)	 changed	 the	 Alizarin	Red	 to	 20%	 alcoholic	 carmine,	 with	 good	 results.	Rodrigues	(2005)	recommends	0.05%	methyl	green	in	 70%	 ethylic	 alcohol	 for	 additional	 cartilage	staining	(Lundwall	method)	(Da	Rocha,	2005).	This	can also be achieved using Alcian blue (Maiolino, 2009;	Sawad	et al.,	2009;	Whitaker	and	Dix,	1979;	McLeod,	 1980;	 Webb	 and	 Byrd,	 1994;	 Depew,	2000;	Narotsky	and	Rogers,	2000).	We want to highlight the particularities of our method, which are the use of molecular iodine and ammonia. Molecular iodine acts as a mordant, avoiding discoloration of the specimen with time and guaranteeing that the specimen will withstand time while remaining in good condition. The addition of ammonia makes specimens more transparent than only KOH and has the advantage of being less corrosive to the delicate tissues of the fetus. Although diaphanization protocols are widely used for research, they are poorly used as didactic models for the explanation of basic science topics in agrarian careers. With this model we obtained a useful tool for the pedagogy of avian embryology.Prospective future works include constructing didactic embryological models for other species and evaluating the impact that these models have upon pedagogy. We want to stain not only the bone but also the cartilage using Alcian blue in chickens and other animal species to study the musculoskeletal system at earlier stages. AcknowledgmentsWe wish to thank Marruecos poultry farm, owned by Super Pollo Paisa company, for supplying the fertilized eggs required for all the development stages. ReferencesBetti P. Desarrollo larval de merluza (Merluccius hubbsi) considerando aspectos ecomorfológicos. Tesis de Licenciatura en	 Ciencias	 Marinas.	 Universidad	 de	 Buenos	 Aires	2005;	 [April	 19	 2012].	 URL:	 http://www.oceandocs.org/bitstream/1834/2599/1/Betti_2005.pdfDa Rocha JM. Staining of adult cartilage by Lundwall’s methods.	The	Anatomical	Record	2005;	13:447-449.Dawson A. A note on the staining of the skeleton of cleared specimens	 with	 Alizarin	 Red	 S.	 Biotech	 &	 Histochem	 1926;	1:123-124.Depew MJ. Analysis of skeletal ontogenesis through differential staining	of	bone	and	cartilage.	Meth	Mol	Biol2000;	461:37-45.	Jensh RP, Brent RL. Rapid schedules for KOH clearing and Alizarin Red S staining of fetal rat bone. Biotech & Histochem 1966;	41:179-183.Kawamura S, Hirohashi A, Kato T, Yasuda M. Bone-staining technique for fetal rat specimens without skinning and removing adipose tissue. Cong	Anom	1990;	30:93-95.	Tamayo LJ et al. Didactic model of the chicken embryo624Rev Colomb Cienc Pecu 2012; 25:620-624Lenis Y, Tamayo LJ. Modelos didáticos como iniciativa para o	ensino	da	anatomia	e	fisiologia	animal.	 J	Morphol	Sci	2011;	28:34.	 [October	 22,	 2012].	 URL:	 http://jms.org.br/PDF/v28nea34.pdfMaiolino	CV,	Mikos	JD,	Kirschnik	PG,	Rios	FSA.	Method	for	diafanization	 and	 staining	 of	 skeleton	 adapted	 to	 young	 fish	forms. Acta	 Microsc	 2009;18.	 [September	 3rd,	 2011]	 URL:	http://www.actamicroscopica.org/uploads/Suplementos/Vol_18_Supp_B_2009_Memorias_SBMM_2009/files/pdf/b06502.pdfMcLeod MJ. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratol 1980;	22:299-301.	Narotsky	MG,	 Rogers	 JM.	 Examination	 of	 the	 axial	 skeleton	of	 fetal	 rodents.	 In:	Tuan	RS,	Lo	CW,	 editors.	Developmental	biology	protocols.	Vol	1.	New	Jersey:	Hum	Press	Inc;	2000.	p.	139-50.	Randle AD. Automation of skeletal staining in the evaluation of teratogenicity. Lab	Anim	1969;	3:197-205.Rodrigues	 H.	 Técnicas	 anatómicas.	 3ª	 ed.	 Espíritu	 Santo:	Universidade	Federal	do	Espíritu	Santo;	2005.	Sanchez I, Terreros V, Zevallos J, Carpio J, Suero S. Visualización del desarrollo óseo en fetos humanos por técnica modificada	 de	Dawson.	 La	 revista	Médica	 del	 CIEM	 1996;	 1	[October	 20,	 2012]	 URL:	 http://www.ucsm.edu.pe/ciemucsm/larev/desos.htmSawad	AA,	 Hana	 BA,	Al-Silawi	AN.	Morphological study of the skeleton development in chick embryo (Gallus domesticus). Int	J	Poul	Sci	2009; 8:710-714.Staples	RE,	Schnell	VL.	Refinements	 in	 rapid	clearing	 technic	in the KOH-Alizarin Red S Method for foetal bone. Stain Tech 1964; 39:61-63.Tipton PW, Burt ME. A method for mechanised staining of rat and mouse foetuses for teratological examination. Lab Anim 1977;	11:265-267.	Webb	 GN,	 Byrd	 RA.	 Simultaneous	 differential	 staining	 of	cartilage	 and	 bone	 in	 rodent	 fetuses:	 an	 Alcian	 Blue	 and	Alizarin Red S procedure without glacial acetic acid. Biotech & Histochem	1994;	69:181-185.	Whitaker J, Dix KM. Double staining technique for rat foetus skeletons	in	teratological	studies.	Lab	Anim	1979;	13:309-310.

Modelo didáctico del desarrollo embrionario del pollo usando la técnica modificada de Dawson para transparentación y tinción

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Modelo didáctico del desarrollo embrionario del pollo usando la técnica modificada de Dawson para transparentación y tinción

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