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	<front>
		<journal-meta>
			<journal-id journal-id-type="publisher-id">abaagrof</journal-id>
			<journal-title-group>
				<journal-title>Abanico agroforestal</journal-title>
				<abbrev-journal-title abbrev-type="publisher">Abanico agrofor.</abbrev-journal-title>
			</journal-title-group>
			<issn pub-type="epub">2594-1992</issn>
			<publisher>
				<publisher-name>Sergio Martínez González</publisher-name>
			</publisher>
		</journal-meta>
		<article-meta>
			<article-id pub-id-type="doi">10.37114/abaagrof/2020.2</article-id>
			<article-id pub-id-type="other">00101</article-id>
			<article-categories>
				<subj-group subj-group-type="heading">
					<subject>Artículos originales</subject>
				</subj-group>
			</article-categories>
			<title-group>
				<article-title>Determinación de minerales en hígado y en sangre de ovejas alimentadas con altas dosis de pollinaza por periodos prolongados</article-title>
			</title-group>
			<contrib-group>
				<contrib contrib-type="author">
					<contrib-id content-type="orcid">0000-0002-5551-9411</contrib-id>
					<name>
						<surname>Peña-Parra</surname>
						<given-names>Bladimir</given-names>
					</name>
					<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0002-8461-2797</contrib-id>
					<name>
						<surname>Duran-Puga</surname>
						<given-names>Noe</given-names>
					</name>
					<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0003-2441-9116</contrib-id>
					<name>
						<surname>Alejo -Santiago</surname>
						<given-names>Gelacio</given-names>
					</name>
					<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0002-3792-2733</contrib-id>
					<name>
						<surname>Escalera-Valente</surname>
						<given-names>Francisco</given-names>
					</name>
					<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0001-9655-1867</contrib-id>
					<name>
						<surname>Herrera-Corredor</surname>
						<given-names>Alejandra</given-names>
					</name>
					<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0003-3356-1499</contrib-id>
					<name>
						<surname>Rivas-Jacobo</surname>
						<given-names>Marco</given-names>
					</name>
					<xref ref-type="aff" rid="aff4"><sup>4</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0002-4916-0967</contrib-id>
					<name>
						<surname>Martínez-González</surname>
						<given-names>Sergio</given-names>
					</name>
					<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<contrib-id contrib-id-type="orcid">0000-0002-7766-6682</contrib-id>
					<name>
						<surname>Ávíla-Ramos</surname>
						<given-names>Fidel</given-names>
					</name>
					<xref ref-type="aff" rid="aff5"><sup>5</sup></xref>
					<xref ref-type="corresp" rid="c1"><sup>*</sup></xref>
				</contrib>
			</contrib-group>
			<aff id="aff1">
				<label>1</label>
				<institution content-type="original">Universidad Autónoma de Nayarit, Unidad Académica de Medicina Veterinaria y Zootecnia. Nayarit, México.</institution>
				<institution content-type="normalized">Universidad Autónoma de Nayarit</institution>
				<institution content-type="orgname">Universidad Autónoma de Nayarit</institution>
				<institution content-type="orgdiv1">Unidad Académica de Medicina Veterinaria y Zootecnia</institution>
				<addr-line>
					<city>Nayarit</city>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<aff id="aff2">
				<label>2</label>
				<institution content-type="original">Centro Universitario de Ciencias Biológicas y Agropecuarias. Universidad de Guadalajara. México.</institution>
				<institution content-type="normalized">Universidad de Guadalajara</institution>
				<institution content-type="orgdiv1">Centro Universitario de Ciencias Biológicas y Agropecuarias</institution>
				<institution content-type="orgname">Universidad de Guadalajara</institution>
				<country country="MX">Mexico</country>
			</aff>
			<aff id="aff3">
				<label>3</label>
				<institution content-type="original">Universidad Autónoma de Nayarit, Unidad Académica de Agricultura. Nayarit, México.</institution>
				<institution content-type="normalized">Universidad Autónoma de Nayarit</institution>
				<institution content-type="orgname">Universidad Autónoma de Nayarit</institution>
				<institution content-type="orgdiv1">Unidad Académica de Agricultura</institution>
				<addr-line>
					<city>Nayarit</city>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<aff id="aff4">
				<label>4</label>
				<institution content-type="original">Universidad Autónoma de San Luis Potosí, Facultad de Agronomía y Medicina Veterinaria. San Luis Potosí, México.</institution>
				<institution content-type="orgname">Universidad Autónoma de San Luis Potosí</institution>
				<institution content-type="orgdiv1">Facultad de Agronomía y Medicina Veterinaria</institution>
				<addr-line>
					<city>San Luis Potosí</city>
				</addr-line>
				<country country="MX">México</country>
			</aff>
			<aff id="aff5">
				<label>5</label>
				<institution content-type="original">Universidad de Guanajuato, Medicina Veterinaria y Zootecnia. Guanajuato. México.</institution>
				<institution content-type="normalized">Universidad de Guanajuato</institution>
				<institution content-type="orgname">Universidad de Guanajuato</institution>
				<institution content-type="orgdiv1">Medicina Veterinaria y Zootecnia</institution>
				<addr-line>
					<city>Guanajuato</city>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<author-notes>
				<corresp id="c1">
					<label>*</label>Autor de correspondencia: Universidad de Guanajuato, División de Ciencias de la Vida, Medicina Veterinaria y Zootecnia. Ex Hacienda El Copal, km. 9 Carretera Irapuato-Silao, CP. 36500, Irapuato, Guanajuato. México.<email>bladiuan73@gmail.com</email>,<email>noeduranpuga@yahoo.com.mx</email>, <email>gelacioalejo@hotmail.com</email>, <email>franes08@hotmail.com</email>, <email>alejandra.herrera@uaslp.mx</email>, <email>marco.rivas@uaslp.mx</email>, <email>sergiotepic@hotmail.com</email>, <email>ledif@hotmail.com</email>.</corresp>
			</author-notes>
			<pub-date date-type="pub" publication-format="electronic">
				<day>31</day>
				<month>07</month>
				<year>2020</year>
			</pub-date>
			<pub-date date-type="collection" publication-format="electronic">
				<year>2020</year>
			</pub-date>
			<volume>2</volume>
			<elocation-id>202</elocation-id>
			<history>
				<date date-type="received">
					<day>16</day>
					<month>06</month>
					<year>2019</year>
				</date>
				<date date-type="accepted">
					<day>15</day>
					<month>01</month>
					<year>2020</year>
				</date>
				<date date-type="pub">
					<day>15</day>
					<month>02</month>
					<year>2020</year>
				</date>
			</history>
			<permissions>
				<license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by-nc/4.0/" xml:lang="es">
					<license-p>Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons</license-p>
				</license>
			</permissions>
			<abstract>
				<title>RESUMEN</title>
				<p>El objetivo fue medir los niveles de minerales en hígado y en sangre de ovinos alimentados con altos niveles de pollinaza. Se consideraron dos grupos experimentales, un grupo tratado (vientres ovinos que consumieron pollinaza por al menos tres años) y un grupo testigo (corderos en engorda que no consumieron pollinaza). Se sacrificaron al azar seis animales de cada grupo (n=6), previo al sacrificio se colectaron las muestras sanguíneas, y posteriormente se tomaron muestras de tejido hepático. Los minerales analizados fueron cobre (Cu), hierro (Fe) y zinc (Zn). En el grupo tratado, las concentraciones en hígado de Cu, Fe y Zn fueron de 176.23, 9.58, y 72.63 mg/L, respectivamente; mientras que en el grupo testigo fueron de 85.35,13.41 y 112.0 mg/L, respectivamente. Las determinaciones de Cu del grupo tratado fueron superiores tanto en hígado como sangre (p&lt;0.05). Sin embargo, a pesar del tiempo de exposición al consumo de pollinaza, los animales no presentaron signos de intoxicación por cobre, lo que pudo estar condicionado por la baja concentración del mineral en las excretas avícolas.</p>
			</abstract>
			<kwd-group xml:lang="es">
				<title>Palabras clave:</title>
				<kwd>ovinos</kwd>
				<kwd>minerales salud</kwd>
				<kwd>cobre</kwd>
				<kwd>hierro</kwd>
				<kwd>zinc y pollinaza</kwd>
			</kwd-group>
			<counts>
				<fig-count count="0"/>
				<table-count count="6"/>
				<equation-count count="0"/>
				<ref-count count="36"/>
				</counts>
		</article-meta>
	</front>
	<body>
		<sec sec-type="intro">
			<title>INTRODUCCIÓN</title>
			<p>En México, la producción de ovinos se ve limitada por los altos costos de los alimentos; además la ovinocultura aún es de traspatio; es decir, como actividad económica secundaria (<xref ref-type="bibr" rid="B18">Martínez <italic>et al.,</italic> 2011</xref>). Este tipo de explotación da lugar a la utilización de desechos industriales, tal como la pollinaza en la alimentación animal. México está consolidado como el quinto productor de pollo y huevo a nivel mundial (<xref ref-type="bibr" rid="B32">SAGARPA, 2016</xref>), por lo que la producción de pollinaza es muy alta, lo que la convierte en un residuo potencial para la alimentación ovina.</p>
			<p>El uso de la pollinaza, como suplemento alimenticio, se debe a la facilidad de adquisición y costo moderado. Entre sus ventajas es el aporte importante de proteína y de minerales para los animales, sobre todo de fósforo disponible. Entre las desventajas se encuentra su elevado contenido de cobre, mismo que puede provocar intoxicación (<xref ref-type="bibr" rid="B5">Castellanos, 2007</xref>). Existen recomendaciones de no utilizar la pollinaza por tiempos prolongados ni en altas concentraciones (<xref ref-type="bibr" rid="B29">Ríos <italic>et al.,</italic> 2005</xref>), para evitar la presentación de intoxicaciones; sin embargo, el productor abusa del empleo de la pollinaza, incluyendo altos niveles en la dieta; pensando en alimentar mejor y más barato a su ganado, lo que resulta contraproducente <xref ref-type="bibr" rid="B5">Castellanos, 2007</xref>).</p>
			<p>El cobre (Cu) es un elemento traza esencial para la mayoría de los procesos biológicos de plantas y animales. Es útil para el metabolismo normal de hierro, síntesis de elastina y colágeno, producción de melanina y para la integridad del sistema nervioso central (<xref ref-type="bibr" rid="B16">Kimberling, 1998</xref>); también es un cofactor de muchas cuproenzimas, pero es extremadamente tóxico en exceso (<xref ref-type="bibr" rid="B12">Horn and Turner, 1999</xref>; <xref ref-type="bibr" rid="B21">Mercer, 2001</xref>).</p>
			<p>Todos los organismos vivos han desarrollado mecanismos homeostáticos altamente especializados para reclutar, trasladar y eliminar el cobre; así como para neutralizar su efecto tóxico (<xref ref-type="bibr" rid="B6">Dameron and Harrison, 1998</xref>; Harris, 2000; <xref ref-type="bibr" rid="B21">Mercer, 2001</xref>). Varias especies de animales muestran variación en su tolerancia a los incrementos de los niveles de cobre en la dieta (<xref ref-type="bibr" rid="B13">Howell y Gooneratne, 1987</xref>).</p>
			<p>Sin duda, los ovinos son los más susceptibles a la toxicidad crónica de cobre; sin embargo, en cuanto a la resistencia a la toxicidad por cobre, existen diferencias entre razas, pues algunos autores comentan que la toxicidad por cobre, ocurre más frecuentemente en la raza Dorper que en la Merino (<xref ref-type="bibr" rid="B2">Bath, 1979</xref>; <xref ref-type="bibr" rid="B10">Harrison <italic>et al.,</italic> 1987</xref>).</p>
			<p>Por otro lado, está la cuestión de la inocuidad de los alimentos, algunos autores (<xref ref-type="bibr" rid="B29">Ríos <italic>al.,</italic> 2005</xref>) comentan la importancia de la salud animal, sobre todo cuando se evalúan la cama de pollo y la gallinaza, como suplemento en la alimentación animal; pues los productos que se generan a partir de esos animales serán consumidos por el hombre. Por lo tanto, el objetivo del presente trabajo fue determinar las concentraciones de cobre, hierro y zinc en hígado y sangre; así como valorar la salud de los animales en una granja que suplementaba a sus ovejas con alto contenido de pollinaza en la dieta.</p>
		</sec>
		<sec sec-type="materials|methods">
			<title>MATERIAL Y MÉTODOS</title>
			<p>Para la realización de este trabajo y del cuidado de los animales, se tomaron en cuenta los lineamientos de la Norma Oficial Mexicana (<xref ref-type="bibr" rid="B25">NOM-062-ZOO-1995</xref>): Especificaciones técnicas para el cuidado y uso de animales de laboratorio, explotaciones ganaderas, granjas, centros de producción, reproducción y cría.</p>
			<p>El trabajo se realizó en la granja “El Refugio” la cual se encuentra localizada en Lo de García, Tepic, Nayarit. La región cuenta con clima cálido sub húmedo, lluvias en verano, temperatura promedio anual de 21.3 °C, precipitación promedio de 1152.3 mm y una altitud de 915 m sobre el nivel del mar (<xref ref-type="bibr" rid="B29">Ríos <italic>et al.,</italic> 2005</xref>). Se contaba con 200 vientres ovinos (grupo tratado, suplementados con pollinaza), y 30 corderos en engorda (grupo testigo, alimentados sin pollinaza).</p>
			<p>La pollinaza fue adquirida en una empresa avícola de la región, a dicha pollinaza se le realizó análisis de los minerales de Cu, Fe y Zn. El manejo de las excretas se realizó siguiendo las indicaciones de la Norma Oficial Mexicana (<xref ref-type="bibr" rid="B24">NOM-044-ZOO-1995</xref>).</p>
			<sec>
				<title>Grupos experimentales</title>
				<p>Grupo tratado: la raza de estas hembras era Pelibuey/Katahdin; las edades oscilaban entre los 10 meses y los 5 años. La condición corporal se situaba entre 2 y 3.5 siguiendo la clasificación de De Lucas (2007). El manejo de los vientres (durante los últimos tres años) era en un sistema de alimentación semiestabulado, con pastoreo y acceso <italic>ad libitum</italic> al suplemento. Dicho suplemento contenía el 60 % pollinaza, 10 % de grano de maíz, 28 % de melaza y 2 % de mezcla comercial de minerales para ovinos. Cada kilogramo de esta mezcla mineral contenía: calcio (Ca) 130 g, fósforo (P) 50 g, sodio (Na) 109 g, Cloro (Cl) 200 g, Hierro (Fe) 4.3 g, Magnesio (Mg) 10 g, Manganeso (Mn) 3.3 g, minerales traza y el resto salvado de trigo, melaza de caña y saborizante vegetal.</p>
				<p>Grupo testigo: la edad promedio de estos animales era de seis meses, de raza Pelibuey/Katahdin. En estos animales el sistema de producción era totalmente estabulada; de esta manera, la alimentación fue de forma continua y consumían una dieta comercial a base de maíz grano, rastrojo de maíz, sorgo grano, soya, melaza, cañóla y 2 % de la mezcla comercial de minerales para ovinos ya descrita.</p>
			</sec>
			<sec>
				<title>Sacrificio de animales</title>
				<p>Se sacrificaron 6 animales (elegidos al azar) de cada grupo; aunque en el grupo tratado se procuró que los animales tuvieran más de cuatro años de edad, con la finalidad de asegurar que habían consumido pollinaza durante tres años.</p>
				<p>Para llevar a cabo este trabajo y el cuidado de los animales, se tomaron en cuenta las pautas de la Norma Oficial Mexicana (<xref ref-type="bibr" rid="B25">NOM-062-ZOO-1995</xref>): Especificaciones técnicas para el cuidado y uso de animales de laboratorio, granjas ganaderas y granjas, centros de producción, reproducción y cría. Los animales fueron sacrificados siguiendo las recomendaciones de la Norma Oficial Mexicana (<xref ref-type="bibr" rid="B23">NOM-033-ZOO-1995</xref>): sacrificio humanitario de animales domésticos y salvajes.</p>
				<p>La toma de muestras sanguíneas se realizó antes del sacrificio de los animales, mediante venopunción yugular, con aguja vacutainer de 21G; la sangre obtenida se colocó en tubos heparinizados de 3 cc. Una vez muertos los animales, se tomaron muestras de tejido hepático del lóbulo izquierdo. Se determinaron las concentraciones de Cu, Fe y Zn; en sangre, hígado y pollinaza, mediante espectrofotometría de absorción atómica con ayuda de un equipo Spectra AA de Marca Varían.</p>
				<p>Las muestras de sangre se analizaron con una dilución 1:1 (vol:vol), con agua destilada, e inmediatamente se procedió a realizar la lectura en el equipo de absorción atómica, con la lámpara respectiva para cada elemento. En el caso de muestras de hígado y de pollinaza se requirió de un proceso de digestión gradual en una plancha de digestión, iniciando en 50 °C, hasta llegar a 250 °C, para la destrucción total del tejido. Una vez que terminó el proceso de digestión, se obtuvo una muestra transparente, y posteriormente se realizó una dilución de la muestra en matraces volumétricos de 25 mi, con agua destilada, y se procedió a la lectura de los elementos, en el equipo de absorción atómica. Los ácidos utilizados fueron, ácido nítrico con ácido perclórico y grado reactivo, en proporción 2:1 (vokvol) (<xref ref-type="bibr" rid="B1">Alcántar y Sandoval, 1999</xref>). Las recomendaciones de MediCalc® (<xref ref-type="bibr" rid="B20">MediCalc®, 2017</xref>) se utilizaron para la conversión a unidades internacionales (de pg / di a pmol / L).</p>
			</sec>
			<sec>
				<title>Análisis estadístico</title>
				<p>Se realizó un análisis descriptivo que incluyó el valor medio, la desviación estándar, mínimo y máximo. El contraste de hipótesis se realizó mediante una prueba de t-Student para muestras independientes; estos análisis fueron realizados con el programa estadístico SPSS Versión 20.0 (<xref ref-type="bibr" rid="B14">IBM, 2011</xref>).</p>
			</sec>
		</sec>
		<sec sec-type="results|discussion">
			<title>RESULTADOS Y DISCUSIÓN</title>
			<sec>
				<title>Valores de pollinaza</title>
				<p>De las muestras de pollinaza analizadas, el hierro fue el que se encontró en mayor concentración, seguido del zinc y por último el cobre (<xref ref-type="table" rid="t1">tabla 1</xref>). Los valores de los minerales analizados siguen la misma tendencia de presentación que la descrita por<xref ref-type="bibr" rid="B28">Pacheco <italic>etal.</italic> (2003)</xref>, es decir las concentraciones de hierro son las más altas, seguidas de las de zinc y por último el cobre.</p>
				<p>
					<table-wrap id="t1">
						<label>Tabla 1</label>
						<caption>
							<title>Niveles de minerales en pollinaza (ppm, base fresca)</title>
						</caption>
						<table style="border-collapse:separate;">
							<colgroup>
								<col/>
								<col/>
								<col/>
								<col/>
								<col/>
							</colgroup>
							<tbody>
								<tr>
									<td style="border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none; text-align: center;"></td>
									<td style="border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none; text-align: center;">Promedio</td>
									<td style="border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none; text-align: center;">Desviación estándar</td>
									<td style="border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none; text-align: center;">Mínimo</td>
									<td style="border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none; text-align: center;">Máximo</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">Cobre mg/L</td>
									<td style="text-align: center; border:0;">2.06</td>
									<td style="text-align: center; border:0;">2.75</td>
									<td style="text-align: center; border:0;">0</td>
									<td style="text-align: center; border:0;">6.6</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">Hierro mg/L</td>
									<td style="text-align: center; border:0;">182.68</td>
									<td style="text-align: center; border:0;">166.46</td>
									<td style="text-align: center; border:0;">35.2</td>
									<td style="text-align: center; border:0;">638.2</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">Zinc mg/L</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">41.38</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">9.142</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">27.7</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">56.6</td>
								</tr>
							</tbody>
						</table>
					</table-wrap>
				</p>
				<p>Los valores de cobre en pollinaza (3.096 ppm) son inferiores a los publicados por <xref ref-type="bibr" rid="B28">Pacheco <italic>etal.</italic> (2003)</xref>, quienes reportaron un promedio general de 82 ppm. <xref ref-type="bibr" rid="B8">Deshck <italic>etal.</italic>(1998)</xref>; encontraron valores de cobre en pollinaza, en un rango de 29.0-122.7, con un promedio de 51.4; valores que son superiores a los encontrados en este estudio.</p>
				<p>Es sabido que las dietas en ovinos deben contener cobre; sin embargo, existe controversia en cuanto a las concentraciones ideales. Tomando en cuenta las tablas de alimentación, éstas mencionan que los ovinos toleran hasta 25 ppm (<xref ref-type="bibr" rid="B26">NRC, 1985</xref>);<xref ref-type="bibr" rid="B11">Hartmans (1975)</xref> comentó que las dieta para ovinos con más de 15 ppm (15 mg/kg) de cobre, puede causar envenenamiento por este mineral; mientras que <xref ref-type="bibr" rid="B30">Plumlee (2004)</xref>comenta que la dosis tóxica comienza con 30 ppm, y como rangos normales menciona de 10-20 ppm. El nivel de cobre administrado en la presente investigación fue de 1.85 ppm (1.85 mg/kg), lo que está muy por debajo de los considerados como aconsejables.</p>
				<p>Si bien es cierto que la concentración de cobre fue baja, se debe tener en cuenta tanto el alto porcentaje de inclusión de pollinaza en la dieta (60 %), así como el tiempo prolongado de exposición (tres años); lo que pudiera provocar la acumulación de cobre en hígado.</p>
				<p>En cuanto al porcentaje de inclusión de pollinaza, existen trabajos en los que se ha incluido niveles desde un 50 % (<xref ref-type="bibr" rid="B29">Pérez, 2004</xref>), 60 % (<xref ref-type="bibr" rid="B35">Vivas, 2002</xref>) y hasta 85 % (<xref ref-type="bibr" rid="B19">Mavimbela <italic>et al.,</italic> 2000</xref>), con un periodo de exposición máximo de 4 meses.</p>
				<p>Los resultados, en cuanto al tiempo de exposición al Cu necesario para producir intoxicación en ovinos, son diversos (<xref ref-type="bibr" rid="B4">Cantón <italic>et al.,</italic> 1994</xref>), pues estos investigadores no registraron mortalidad dentro de los 91 días de alimentación con 87 ppm de Cu; mientras que otros grupos de trabajo describen mortalidad desde los 67 días, e incluso desde los 30 días de exposición al cobre, con niveles más bajos de este mineral en la dieta (67 ppm y 60 ppm, respectivamente) (<xref ref-type="bibr" rid="B3">Bostwick, 1982</xref>; <xref ref-type="bibr" rid="B36">Zervas 1990</xref>).</p>
			</sec>
			<sec>
				<title>Valores en hígado</title>
				<p>Los resultados de las concentraciones de cobre, hierro y zinc, determinadas en hígado, se describen en la <xref ref-type="table" rid="t2">tabla 2</xref>. En esta tabla se observa que el cobre e hierro, fueron superiores en el grupo tratado; aunque sólo el cobre alcanzó diferencia estadística significativa (p&lt;0.05). Por otro lado, el zinc fue superior en el grupo testigo, aunque sin significación estadística.</p>
				<p>
					<table-wrap id="t2">
						<label>Tabla 2</label>
						<caption>
							<title>Concentraciones medias de minerales en hígado de ovejas (ppm, base fresca) que consumieron pollinaza</title>
						</caption>
						<table style="border-collapse:separate;">
							<colgroup>
								<col span="2"/>
								<col/>
								<col/>
								<col/>
								<col/>
								<col/>
							</colgroup>
							<tbody>
								<tr>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;" colspan="2"> </td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">N</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Promedio</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Desviación estándar</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Mínimo</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Máximo</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;" rowspan="2">Cobre mg/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">176.2<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">59.2</td>
									<td style="text-align: center; border:0;">104.9</td>
									<td style="text-align: center; border:0;">243.7</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">0 % (Control)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">85.4<xref ref-type="table-fn" rid="TFN6"><sup>b</sup></xref></td>
									<td style="text-align: center; border:0;">13.7</td>
									<td style="text-align: center; border:0;">65.0</td>
									<td style="text-align: center; border:0;">99.8</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;" rowspan="2">Hierro mg/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">72.6<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">30.9</td>
									<td style="text-align: center; border:0;">26.2</td>
									<td style="text-align: center; border:0;">111.0</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">0 % (Control)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">112.0<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">70.7</td>
									<td style="text-align: center; border:0;">48.8</td>
									<td style="text-align: center; border:0;">239.2</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;" rowspan="2">Zinc mg/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">9.6<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">3.5</td>
									<td style="text-align: center; border:0;">3.1</td>
									<td style="text-align: center; border:0;">13.1</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0 % (Control)</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">6</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">13.4<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">2.9</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">8.5</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">16.1</td>
								</tr>
							</tbody>
						</table>
						<table-wrap-foot>
							<fn id="TFN6">
								<label>a,b</label>
								<p>Distintas literales indican diferencia estadística significativa entre los tratamientos (p&lt;0.05).</p>
							</fn>
						</table-wrap-foot>
					</table-wrap>
				</p>
				<p>El rango de valores de cobre hepático publicado por <xref ref-type="bibr" rid="B34">Underwood y Suttle (1999)</xref>, es de 33.3-100 pmol/l, por lo que los valores de cobre hepático encontrado en el presente trabajo, en el grupo tratado y testigo, pueden considerarse como elevados (2819.2 y 1365.6 pmol/l respectivamente).</p>
				<p>
					<xref ref-type="bibr" rid="B30">Plumlee (2004)</xref>, comenta que en la mayoría de los rumiantes la acumulación de más de 250 ppm de cobre en hígado es considerada como tóxica; este valor es más alto del encontrado en hígado del grupo tratado (176.2 ppm), y casi el triple del valor del grupo testigo (85.3 ppm).</p>
				<p>Los corderos muestreados por <xref ref-type="bibr" rid="B33">Sivertsen and Løverg (2014)</xref>, en su primer año de estudio (noviembre, marzo y junio) promediaron un valor de 110 mg/kg en base húmeda; mientras que en el segundo año promediaron un total de 114 mg/kg. Estos valores son superiores a los descritos en la presente investigación en corderos de 5 meses de edad (85.35 mg/kg); esta diferencia puede estar dada por el consumo de pastos que contenían niveles altos de cobre.</p>
				<p>
					<xref ref-type="bibr" rid="B27">Oruc <italic>et al.</italic> (2009)</xref> describieron valores de 302 mg/kg en base húmeda, estas muestras provenían de tres ovejas que habían muerto y que presentaban signos de intoxicación por cobre, estos valores son casi el doble de los de las borregas que consumieron pollinaza (176.2 mg/kg).</p>
				<p>Otro factor que pudo contribuir es la resistencia de la raza, pues <xref ref-type="bibr" rid="B10">Harrison <italic>et al.</italic> (1987)</xref> comentaron que, en cuanto al metabolismo del cobre, existen diferencias genéticas entre las razas ovinas. En ese sentido <xref ref-type="bibr" rid="B17">Lewis <italic>et al.</italic> (1997)</xref> menciona que la raza Suffolk está particularmente en riesgo; mientras que <xref ref-type="bibr" rid="B2">Bath (1979)</xref> encontró que la toxicidad por cobre ocurre más frecuentemente en la raza Dorper, que en la raza Merino.</p>
			</sec>
			<sec>
				<title>Valores en sangre</title>
				<p>Los niveles de cobre, hierro y zinc, analizados en suero sanguíneo, fueron superiores en el grupo tratado con respecto al grupo testigo: aunque sólo el cobre mostró diferencias estadísticas significativas (p&lt;0.05) (<xref ref-type="table" rid="t3">tabla 3</xref>).</p>
				<p>
					<table-wrap id="t3">
						<label>Tabla 3</label>
						<caption>
							<title>Valor medio de minerales en sangre de ovinos (ppm, base fresca) que consumieron pollinaza</title>
						</caption>
						<table>
							<colgroup>
								<col span="2"/>
								<col/>
								<col/>
								<col/>
								<col/>
								<col/>
							</colgroup>
							<tbody>
								<tr><td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;" colspan="2"> </td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">N</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Promedio</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Desviación estándar</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Mínimo</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Máximo</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;" rowspan="2">Cobre mq/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">0.35<xref ref-type="table-fn" rid="TFN2"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">0.17</td>
									<td style="text-align: center; border:0;">0.23</td>
									<td style="text-align: center; border:0;">0.62</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">0 % (Control)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">0.12<xref ref-type="table-fn" rid="TFN6"><sup>b</sup></xref></td>
									<td style="text-align: center; border:0;">0.03</td>
									<td style="text-align: center; border:0;">0.06</td>
									<td style="text-align: center; border:0;">0.14</td>
								</tr>
								<tr>
									<td style="text-align: left;" rowspan="2">Hierro mq/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">36.25<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">30.91</td>
									<td style="text-align: center; border:0;">8.90</td>
									<td style="text-align: center; border:0;">95.20</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">0 % (Control)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">17.40<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">9.88</td>
									<td style="text-align: center; border:0;">6.50</td>
									<td style="text-align: center; border:0;">31.40</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;" rowspan="2">Zinc mq/L</td>
									<td style="text-align: center; border:0;">60 % (Tratado)</td>
									<td style="text-align: center; border:0;">6</td>
									<td style="text-align: center; border:0;">0.98<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">0.84</td>
									<td style="text-align: center; border:0;">0.02</td>
									<td style="text-align: center; border:0;">2.06</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0 % (Control)</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">6</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.93<xref ref-type="table-fn" rid="TFN6"><sup>a</sup></xref></td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.36</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.40</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">1.37</td>
								</tr>
							</tbody>
						</table>
						<table-wrap-foot>
							<fn id="TFN2">
								<label>a,b</label>
								<p> Distintas literales indican diferencia estadística entre los tratamientos (p&lt;0.05).</p>
							</fn>
						</table-wrap-foot>
					</table-wrap>
				</p>
				<p>Los resultados de ovejas que consumieron pollinaza, son ligeramente inferiores a los publicados por <xref ref-type="bibr" rid="B22">Mohammed <italic>et al.</italic> (2014)</xref>, independientemente del estado fisiológico y de la estación; esto puede deberse a la raza (<xref ref-type="bibr" rid="B2">Bath, 1979</xref>; <xref ref-type="bibr" rid="B17">Lewis <italic>et al.,</italic> 1997</xref>), aunque haría falta más estudios para apoyar la afirmación de que la raza Pelibuey es más resistente a la intoxicación por cobre.</p>
			</sec>
		</sec>
		<sec sec-type="conclusions">
			<title>CONCLUSIÓN</title>
			<p>Los niveles de hierro y de zinc analizados en suero sanguíneo y en hígado del grupo tratado con respecto al grupo testigo, no mostraron diferencia estadística. Las determinaciones de cobre del grupo tratado fueron superiores, tanto en sangre como en hígado; sin embargo, ningún animal manifestó síntomas de intoxicación.</p>
		</sec>
	</body>
	<back>
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	<sub-article article-type="translation" id="s1" xml:lang="en">
		<front-stub>
			<article-categories>
				<subj-group subj-group-type="heading">
					<subject>Original Articles</subject>
				</subj-group>
			</article-categories>
			<title-group>
				<article-title>Determination of minerals in liver and blood of sheep fed with high poultry manure doses for prolonged periods</article-title>
			</title-group>
			<abstract>
				<title>ABSTRACT</title>
				<p>The objective was to measure the levels of minerals in the liver and blood of sheep, fed with high levels of poultry manure. Two experimental groups were considered: a treated group (ovine bellies that consumed poultry manure for at least three years) and a control group (lambs in fattening that did not consume poultry manure). Six animals (n=6) from each group were randomly sacrificed, blood samples were collected prior to slaughter, and hepatic tissue samples were subsequently taken. The minerals analyzed were copper (Cu), iron (Fe) and zinc (Zn). In the treated group, concentrations of Cu, Fe andZn were 176.23, 9.58, and 72.63 mg/L, respectively. While in the treated group they were 85.35, 13.41 and 112.0 mg/L, respectively. The Cu determinations of the treated group were higher in both liver and blood (p &lt;0.05). H owever, in spite of the time of exposure to poultry manure consumption, the animals showed no signs of copper intoxication, which could be conditioned by the low concentration of the mineral in the poultry excreta.</p>
			</abstract>
			<kwd-group xml:lang="en">
				<title>Keywords:</title>
				<kwd>ovines</kwd>
				<kwd>health</kwd>
				<kwd>copper</kwd>
				<kwd>iron</kwd>
				<kwd>zinc</kwd>
				<kwd>poultry manure</kwd>
			</kwd-group>
		</front-stub>
		<body>
			<sec sec-type="intro">
				<title>INTRODUCTION</title>
				<p>In Mexico, sheep production is limited (<xref ref-type="bibr" rid="B18">Martinez <italic>etal.,</italic> 2011</xref>), among other things, due to food issues, since the costs are very high, and sheep production in Mexico is still from backyard, as a secondary activity; which leads to the use of industrial waste such as poultry manure in animal feeding. Mexico is consolidated as the fifth largest producer of chicken and egg in the world (<xref ref-type="bibr" rid="B32">SAGARPA, 2016</xref>), so the production of poultry manure is very high, which makes it a potential residue for sheep feeding.</p>
				<p>The use of poultry manure as a dietary supplement is due to its ease of acquisition and its moderate cost. Among the advantages is that it represents an important contribution of protein and minerals for animals, especially available phosphorus. Among the disadvantages is its high copper content, which may lead to intoxication (<xref ref-type="bibr" rid="B5">Castellanos, 2007</xref>). This happens, despite the recommendations of not using the poultry manure for prolonged periods or high levels (<xref ref-type="bibr" rid="B31">Rios <italic>etal.,</italic> 2005</xref>), when the producer abuses the poultry manure use, including high levels in the diet, thinking of feeding better and cheaper to their cattle, which can be counterproductive (<xref ref-type="bibr" rid="B5">Castellanos, 2007</xref>).</p>
				<p>Copper (Cu) is an essential trace element for most biological processes in plants and animals. It is useful for normal iron metabolism, elastin and collagen synthesis, melanin production, and central nervous system integrity (<xref ref-type="bibr" rid="B16">Kimberling, 1998</xref>). It is also a cofactor of many cuproenzymes, but it is extremely toxic in excess (<xref ref-type="bibr" rid="B12">Horn and Turner, 1999</xref>;<xref ref-type="bibr" rid="B21">Mercer, 2001</xref>).</p>
				<p>All living organisms have developed highly specialized homeostatic mechanisms to recruit, remove and remove copper, and neutralize its toxic effect (<xref ref-type="bibr" rid="B6">Dameron and Harrison, 1998</xref>; Harris, 2000; <xref ref-type="bibr" rid="B21">Mercer, 2001</xref>). Several species of animals show variation in their tolerance to increases in copper levels in the diet (<xref ref-type="bibr" rid="B13">Howell and Gooneratne, 1987</xref>).</p>
				<p>Undoubtedly, sheeps are the most susceptible to chronic copper toxicity; however, in terms of resistance to copper toxicity, there are differences among breeds, as some authors comment that copper toxicity occurs more frequently in the Dorper breed than in Merino one (<xref ref-type="bibr" rid="B2">Bath, 1979</xref>; <xref ref-type="bibr" rid="B10">Harrison <italic>etal.,</italic> 1987</xref>).</p>
				<p>On the other hand, there is the question of food safety, some authors (<xref ref-type="bibr" rid="B31">Rios <italic>et al.,</italic> 2005</xref>) comment on the importance of animal health, especially when evaluating the chicken bed and chicken dung as a supplement in animal feed, because the products that are generated from these animals will be consumed by the man. Therefore, the objective of the present study was to determine the concentrations of hepatic and blood copper and to evaluate the health of the animals in a farm that supplemented their sheep with high content of poultry manure in the diet.</p>
			</sec>
			<sec sec-type="materials|methods">
				<title>MATERIAL AND METHODS</title>
				<p>The work was carried out in the farm &quot;El Refugio&quot; which is located in Lo de Garcia, Tepic, Nayarit. The region has a warm subhumid climate with rainfall in summer with an average annual temperature of 21.3 °C, with an average rainfall of 1152.3 mm, with an altitude of 915 meters above sea level (m.a.s.l) (INEGI, 2006) with 200 ovine bellies (Treated group, supplemented with poultry manure), and 30 lambs in fattening (control group, fed without poultry manure).</p>
				<p>The poultry manure was acquired in a poultry company of the region; to this poultry manure was realized analysis of the minerals of Cu, Fe and Zn. The excreta management was carried out following the indications of Official Mexican Standard (<xref ref-type="bibr" rid="B24">NOM-044-ZOO-1995</xref>).</p>
				<sec>
					<title>Experimental groups</title>
					<p>Treated group: the breed of these females was Pelibuey/Katahdin; the ages ranged from 10 months to 5 years. The body condition was between 2 and 3.5 following the classification of De Lucas (2007). The management of the bellies (during the last three years) was in a semi-frozen feeding system, with grazing and ad libitum access to the supplement. This supplement contained 60% poultry manure, 10% maize grain, 28% molasses and 2% commercial mixture of minerals for sheep. Each kilogram of this mineral mixture contained: Calcium (Ca) 130g, Phosphorus (P) 50g, Sodium (Na) 109g, Chlorine (Cl) 200g, Iron (Fe) 4.3g, Magnesium (Mg) 10g, Manganese (Mn) 3.3g, trace minerals and wheat bran residue, cane molasses and vegetable flavoring).</p>
					<p>Control group: the average age of these animals was six months, Pelibuey / Katahdin breed. In these animals, the operating system was fully stabbed. In this way, the diet was continuous and consumed a commercial diet based on maize grain, corn stubble, grain sorghum, soybean, molasses, canola and 2% of the commercial mixture of minerals for sheep already described.</p>
				</sec>
				<sec>
					<title>Sacrifice of animals</title>
					<p>Six animals (randomly chosen) from each group were sacrificed. Although the treated group tried to ensure that the animals were more than four years old, in order to ensure that they had consumed poultry manure for three years.</p>
					<p>In order to carry out this work and the care of the animals, the guidelines of the Official Mexican Standard (NOM-062-ZOO-1995) were taken into account: Technical specifications for the care and use of laboratory animals, livestock farms, and farms, centers of production, reproduction and breeding. The animals were sacrificed following the recommendations of Official Mexican Standard (<xref ref-type="bibr" rid="B23">NOM-033-ZOO-1995</xref>): Humanitarian slaughter of domestic and wild animals.</p>
					<p>Blood sampling was performed before the animals were slaughtered by jugular venipuncture with a 21G Vacutainer needle, the blood obtained was placed in 3 cc heparinized tubes. Once the animals were dead, liver tissue samples were taken from the left lobe. Concentrations of Cu, Fe and Zn were determined; in blood, liver and poultry, by means of atomic absorption spectrophotometry with the aid of Varían Mark Spectra AA equipment. The blood samples were analyzed with a 1:1 (vokvol) dilution with distilled water and immediately read in the atomic absorption equipment with the respective lamp for each element. In the case of liver and poultry manure samples, a gradual digestion process was required in a digestion plate, starting at 50 °C until reaching 250 °C, for the total destruction of the tissue. After the digestion process was completed, a clear sample was obtained, and the sample was then diluted into 25 ml volumetric flasks with distilled water and the elements were read in the atomic absorption equipment. The acids used were nitric acid with perchloric acid, reactive grade, in a ratio of 2: 1 (vol: vol) (<xref ref-type="bibr" rid="B1">Alcántar and Sandoval, 1999</xref>). MediCalc® recommendations (<xref ref-type="bibr" rid="B20">MediCalc®, 2017</xref>) were used for the conversion to international units (from pg/dl to pmol/L).</p>
				</sec>
				<sec>
					<title>Statistical analysis</title>
					<p>A descriptive analysis was performed that included the mean value, the standard deviation, minimum and maximum. The hypothesis test was performed using a t-Student test for independent samples. These analyzes were performed with the statistical software SPSS Version 20.0 (<xref ref-type="bibr" rid="B14">IBM, 2011</xref>).</p>
				</sec>
			</sec>
			<sec sec-type="results|discussion">
				<title>RESULTS AND DISCUSSION</title>
			</sec>
			<sec>
				<title>Values in poultry manure</title>
				<p>From the samples of poultry manure analyzed the iron was the one that was found in greater concentration, followed by the zinc and finally the copper (<xref ref-type="table" rid="t4">Table 1</xref>), being there statistical difference among the three. The values of the analyzed minerals follow the same trend of presentation as that described by <xref ref-type="bibr" rid="B28">Pacheco (2003)</xref>, that is concentrations are highest followed by zinc and finally copper.</p>
				<p>
					<table-wrap id="t4">
						<label>Table 1</label>
						<caption>
							<title>Mineral levels in poultry manure (ppm, fresh basis).</title>
						</caption>
						<table>
							<colgroup>
								<col/>
								<col/>
								<col/>
								<col/>
							</colgroup>
							<tbody>
								<tr>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;"></td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Mean</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Typical deviation</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Minimum</td>
									<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Maximum</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">Cu mg L<sup>-1</sup></td>
									<td style="text-align: center; border:0;">2.06<xref ref-type="table-fn" rid="TFN3"><sup>a</sup></xref></td>
									<td style="text-align: center; border:0;">2.75</td>
									<td style="text-align: center; border:0;">0</td>
									<td style="text-align: center; border:0;">6.6</td>
								</tr>
								<tr>
									<td style="text-align: left; border:0;">Fe mg L<sup>-1</sup></td>
									<td style="text-align: center; border:0;">182.68<xref ref-type="table-fn" rid="TFN3"><sup>b</sup></xref></td>
									<td style="text-align: center; border:0;">166.46</td>
									<td style="text-align: center; border:0;">35.2</td>
									<td style="text-align: center; border:0;">638.2</td>
								</tr>
								<tr>
									<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">Zn mg L<sup>-1</sup></td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">41.38<xref ref-type="table-fn" rid="TFN3"><sup>c</sup></xref></td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">9.142</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">27.7</td>
									<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">56.6</td>
								</tr>
							</tbody>
						</table>
						<table-wrap-foot>
							<fn id="TFN3">
								<label>a, b c</label>
								<p> Different literals indicate significant differences between minerals (p&lt;0.05).</p>
							</fn>
						</table-wrap-foot>
					</table-wrap>
				</p>
				<p>The values of copper in poultry manure (3.096 ppm) are lower than those published by <xref ref-type="bibr" rid="B28">Pacheco <italic>et at.</italic> (2003)</xref>, who reported an overall average of 82 ppm. <xref ref-type="bibr" rid="B8">Deshck <italic>et at.</italic> (1998)</xref>found values of copper in poultry manure in a range of 29.0-122.7 with an average of 51.4, values that are higher than those found by us.</p>
				<p>It is known that diets in sheep must contain copper; however, controversy exists as to ideal concentrations. Taking into account feeding tables, they mention that sheep tolerate up to 25 ppm (<xref ref-type="bibr" rid="B26">NRC, 1985</xref>), <xref ref-type="bibr" rid="B11">Hartmans (1975)</xref> comments that diet for sheep with more than 15 ppm (15 mg/kg) of copper can cause Copper, and whereas <xref ref-type="bibr" rid="B30">Plumlee (2004)</xref>comments that the toxic dose begins with 30 ppm and as normal ranges mentions of 10-20 ppm. The level of copper administered in the present investigation was 1.85 ppm (1.85 mg/kg), which is well below those considered advisable.</p>
				<p>Although copper concentration was low, both the high inclusion rate of poultry manure in the diet (60%) and the long exposure time (three years) should be taken into account, which could lead to accumulation of copper in liver.</p>
				<p>As for the inclusion percentage of poultry manure, there are studies that have included levels from 50% (<xref ref-type="bibr" rid="B29">Pérez, 2004</xref>), 60% (<xref ref-type="bibr" rid="B35">Vivas, 2002</xref>) and up to 85% (<xref ref-type="bibr" rid="B19">Mavimbela <italic>etal.,</italic> 2000</xref>) with a period of exposure of maximum 4 months.</p>
				<p>As for exposure time to Cu, necessary to produce intoxication in sheep, the opinions are diverse (<xref ref-type="bibr" rid="B4">Cantón <italic>et al.,</italic> 1994</xref>), as these investigators did not register mortality within 91 days of feeding with 87 ppm of Cu, while other working groups describe mortality from 67 days, and even from 30 days, exposure to copper, with lower levels of this mineral in the diet (67 ppm and 60 ppm, respectively) (<xref ref-type="bibr" rid="B3">Bostwick, 1982</xref>; <xref ref-type="bibr" rid="B36">Zervas <italic>etal.,</italic> 1990</xref>).</p>
				<sec>
					<title>Values in liver</title>
					<p>The results of the concentrations of copper, iron and zinc, determined in liver, are described in <xref ref-type="table" rid="t5">Table 2</xref>. In this table can observe that copper and iron were higher in the treated group, although only the Copper reached significant statistical difference (p &lt;0.05). On the other hand, zinc was higher in the control group, although without statistical significance.</p>
					<p>
						<table-wrap id="t5">
							<label>Table 2</label>
							<caption>
								<title>Mean concentrations (standard deviation) of minerals in sheep liver (ppm, fresh basis).</title>
							</caption>
							<table>
								<colgroup>
									<col span="2"/>
									<col/>
									<col/>
									<col/>
									<col/>
									<col/>
								</colgroup>
								<tbody>
									<tr>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;" colspan="2"> </td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">N</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Mean</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Typical deviation</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Minimum</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Maximum</td>
									</tr>
									<tr>
										<td style="text-align: left; border:0;" rowspan="2">Cu mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">176.2<xref ref-type="table-fn" rid="TFN4"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">59.2</td>
										<td style="text-align: center; border:0;">104.9</td>
										<td style="text-align: center; border:0;">243.7</td>
									</tr>
									<tr>
										<td style="text-align: left; border:0;">0% (Control)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">85.4<xref ref-type="table-fn" rid="TFN4"><sup>b</sup></xref></td>
										<td style="text-align: center; border:0;">13.7</td>
										<td style="text-align: center; border:0;">65.0</td>
										<td style="text-align: center; border:0;">99.8</td>
									</tr>
									<tr>
										<td style="text-align: left; border:0;" rowspan="2">Fe mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">72.6<xref ref-type="table-fn" rid="TFN4"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">30.9</td>
										<td style="text-align: center; border:0;">26.2</td>
										<td style="text-align: center; border:0;">111.0</td>
									</tr>
									<tr>
										<td style="text-align: left; border:0;">0% (Control)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">112.0<xref ref-type="table-fn" rid="TFN4"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">70.7</td>
										<td style="text-align: center; border:0;">48.8</td>
										<td style="text-align: center; border:0;">239.2</td>
									</tr>
									<tr>
										<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;" rowspan="2">Zn mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">9.6<xref ref-type="table-fn" rid="TFN4"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">3.5</td>
										<td style="text-align: center; border:0;">3.1</td>
										<td style="text-align: center; border:0;">13.1</td>
									</tr>
									<tr>
										<td style="text-align: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0% (Control)</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">6</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">13.4<xref ref-type="table-fn" rid="TFN4"><sup>a</sup></xref></td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">2.9</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">8.5</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">16.1</td>
									</tr>
								</tbody>
							</table>
							<table-wrap-foot>
								<fn id="TFN4">
									<label>a, b</label>
									<p> Different literals indicate significant differences among treatments (p&lt;0.05).</p>
								</fn>
							</table-wrap-foot>
						</table-wrap>
					</p>
					<p>The range of hepatic copper values published by <xref ref-type="bibr" rid="B34">Underwood and Suttle (1999)</xref> is 33.3-100 pmol/l, so the values of hepatic copper found by us in the treated and control groups can be considered as high (2819.2 and 1365.6 pmol/l respectively).</p>
					<p>
						<xref ref-type="bibr" rid="B30">Plumlee (2004)</xref> reports that in most ruminants the accumulation of more than 250 ppm of copper in the liver is considered as toxic, this value is higher than that found in the liver of the treated group (176.2 ppm) and almost triple of the control group value (85.3 ppm).</p>
					<p>The lambs sampled by <xref ref-type="bibr" rid="B33">Sivertsen and Loverg (2014)</xref> in their first year of study (November, March and June) averaged a value of 110 mg/kg on a wet basis, whereas in the second year they averaged a total of 114 mg/kg. These values are higher than those described in the present investigation in 5-month-old lambs (85.35 mg/kg), this difference may be due to the consumption of pastures containing high levels of copper.</p>
					<p>
						<xref ref-type="bibr" rid="B27">Oruc <italic>et al.</italic> (2009)</xref> reported values of 302 mg/kg on wet basis, these samples came from three sheep that had died and showed signs of copper intoxication, these values being almost twice that of lambs that consumed poultry manure 176.2 mg/kg).</p>
					<p>Another factor that could contribute is the resistance of the breed, as <xref ref-type="bibr" rid="B10">Harrison <italic>etal.</italic> (1987)</xref> states that, in terms of copper metabolism, there are genetic differences among sheep breeds. In this sense <xref ref-type="bibr" rid="B17">Lewis <italic>et al.</italic> (1997)</xref> mentions that the Suffolk breed is particularly at risk, whereas <xref ref-type="bibr" rid="B2">Bath (1979)</xref> found that copper toxicity occurs more frequently in the Dorper breed than in the Merino one. The copper determinations of the treated group were superior both in blood and in liver, however, no animal showed intoxication symptoms.</p>
				</sec>
				<sec>
					<title>Values in blood</title>
					<p>The levels of copper, iron and zinc analyzed in blood serum were higher in the treated group than in the control group, although only copper showed significant statistical differences (p &lt;0.05) (<xref ref-type="table" rid="t6">Table 3</xref>).</p>
					<p>
						<table-wrap id="t6">
							<label>Table 3</label>
							<caption>
								<title>Mean value (standard deviation) of sheep blood minerals (ppm, fresh basis).</title>
							</caption>
							<table>
								<colgroup>
									<col span="2"/>
									<col/>
									<col/>
									<col/>
									<col/>
									<col/>
								</colgroup>
								<tbody>
									<tr>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;" colspan="2"></td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">N</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Mean</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Typical deviation</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Minimum</td>
										<td style="text-align: center; border-top: 2px solid black; border-bottom: 2px solid black; border-left: none;  border-right: none;">Maximum</td>
									</tr>
									<tr>
										<td align="left" rowspan="2">Cu mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6 </td>
										<td style="text-align: center; border:0;">0.35<xref ref-type="table-fn" rid="TFN5"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">0.17 </td>
										<td style="text-align: center; border:0;">0.23 </td>
										<td style="text-align: center; border:0;">0.62</td>
									</tr>
									<tr>
										<td style="text-aling: left;">0%(Control)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">0.12<xref ref-type="table-fn" rid="TFN5"><sup>b</sup></xref></td>
										<td style="text-align: center; border:0;">0.03</td>
										<td style="text-align: center; border:0;">0.06</td>
										<td style="text-align: center; border:0;">0.14</td>
									</tr>
									<tr>
										<td style="text-aling: left;" rowspan="2">Fe mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">36.25<xref ref-type="table-fn" rid="TFN5"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">30.91</td>
										<td style="text-align: center; border:0;">8.90</td>
										<td style="text-align: center; border:0;">95.20</td>
									</tr>
									<tr>
										<td style="text-aling: left;">0% (Control)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">17.40<xref ref-type="table-fn" rid="TFN5"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">9.88</td>
										<td style="text-align: center; border:0;">6.50</td>
										<td style="text-align: center; border:0;">31.40</td>
									</tr>
									<tr>
										<td style="text-aling: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;" rowspan="2">Zn mg L<sup>-1</sup></td>
										<td style="text-align: center; border:0;">60% (Treated)</td>
										<td style="text-align: center; border:0;">6</td>
										<td style="text-align: center; border:0;">0.98<xref ref-type="table-fn" rid="TFN5"><sup>a</sup></xref></td>
										<td style="text-align: center; border:0;">0.84</td>
										<td style="text-align: center; border:0;">0.02</td>
										<td style="text-align: center; border:0;">2.06</td>
									</tr>
									<tr>
										<td style="text-aling: left; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0% (Control)</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">6</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.93<xref ref-type="table-fn" rid="TFN5"><sup>a</sup></xref></td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.36</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">0.40</td>
										<td style="text-align: center; border-top: none; border-bottom: 2px solid black; border-left: none;  border-right: none;">1.37</td>
									</tr>
								</tbody>
							</table>
							<table-wrap-foot>
								<fn id="TFN5">
									<label>a, b</label>
									<p> different literals indicate significant differences between treatments (p&lt;0.05).</p>
								</fn>
							</table-wrap-foot>
						</table-wrap>
					</p>
					<p>The results of sheep that consumed pollinaza were slightly lower than those published by <xref ref-type="bibr" rid="B22">Mohammed <italic>etal.</italic> (2014)</xref> independently of the physiological state and of the season. This may be due to race (<xref ref-type="bibr" rid="B2">Bath, 1979</xref>; <xref ref-type="bibr" rid="B17">Lewis <italic>et al.,</italic> 1997</xref>), although further studies would be needed to support the claim that the Pelibuey breed is more resistant to copper intoxication.</p>
				</sec>
			</sec>
			<sec sec-type="conclusions">
				<title>CONCLUSION</title>
				<p>The iron and zinc levels analyzed in blood serum and liver of the treated group with respect to the control group showed no statistical difference. The copper determinations of the treated group were superior both in blood and in liver, however, no animal showed intoxication symptoms.</p>
			</sec>
		</body>
	</sub-article>
</article>