efeitos da exposição ao chumbo no sistema reprodutivo de
TRANSCRIPT
UNIVERSIDADE FEDERAL DO RIO GRANDE - FURG
INSTITUTO DE CIÊNCIAS BIOLÓGICAS
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FISIOLÓGICAS: FISIOLOGIA
ANIMAL COMPARADA
Efeitos da exposição ao chumbo no sistema reprodutivo
de Chrysomus ruficapillus (AVES: Icteridae)
Danusa Leidens
Orientadora Profª Dra. Carine Dahl Corcini
Co-orientadora Profª Dra. Cecilia Perez Calabuig
Março de 2013
Rio Grande
Dissertação defendida no âmbito do
Programa de Pós Graduação em
Ciências Fisiológicas - Fisiologia
Animal Comparada como parte dos
requisitos para obtenção do título de
MESTRE em Fisiologia Animal
Comparada
1
SUMÁRIO
Agradecimentos ............................................................................................................ 2
Resumo ......................................................................................................................... 3
Introdução .................................................................................................................... 4
Justificativa ................................................................................................................. 12
Objetivo ...................................................................................................................... 13
Capítulo 1 ................................................................................................................... 14
Abstract ..................................................................................................................... 16
Introduction ............................................................................................................... 17
Materials and Methods ............................................................................................... 18
Results ....................................................................................................................... 25
Discussion ................................................................................................................. 27
Acknowledgements .................................................................................................... 32
References ................................................................................................................. 32
Table .......................................................................................................................... 37
Legend to Figures ...................................................................................................... 38
Figures ....................................................................................................................... 40
Conclusão e considerações gerais ............................................................................... 44
Referências ................................................................................................................ 45
2
I. AGRADECIMENTOS
Primeiramente, gostaria de agradecer aos meus pais e meu irmão pelo carinho e
apoio incondicional em todos os momentos da minha vida.
A minha orientadora Prof. Dra. Carine Dahl Corcini e a minha co-orientadora
Prof. Dra. Cecilia Perez Calabuig pela oportunidade, confiança e pelas inúmeras ajudas
no trabalho de campo.
A Granjas Quatro Irmãos pela oportunidade e disponibilidade de trabalho dentro
de sua propriedade.
Aos professores Dr. Antonio Sergio Varela Junior e Dr. Carlos Eduardo Rosa
pela contribuição e dedicação nos laboratórios.
Aos colegas de campo que foram ajudar nas coletas.
Ao Márcio Alberto Geihs pela amizade e apoio ao meu trabalho.
A Roberta Socoowski Britto, minha sempre colega, que além de colega virou
uma grande amiga, que sempre esteve no meu lado, apoiando na amizade e nas dúvidas
e ajudas no laboratório
As minhas amigas Daiane Sena Kaffer, Jéssica Borges Cantos, Beatriz Sena,
pelo apoio e os momentos de amizade e diversão proporcionados.
A Cynthia Harayashiki, companheira de sempre no laboratório, sempre apoiando
e ajudando nas conversas e trabalho.
Aos amigos da Pós-graduação de Ciências Fisiológicas – Fisiologia Animal
Comparada pela convivência e amizade.
A Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo
auxilio a pesquisa.
Ao Programa de Pós-graduação Ciências Fisiológicas – Fisiologia Animal
Comparada e a FURG, pela disponibilização de todos os meios necessários para
execução desse trabalho.
Obrigada a todos
3
RESUMO
O chumbo é um metal pesado que constitui um dos grandes problemas
ambientais em termos de poluição atmosférica, aquática e terrestre. O impacto da
exposição ao chumbo tem consequências nas características morfológicas e bioquímicas
deem aves, porém são escassos os estudos sobre os efeitos no sistema reprodutivo das
aves. O objetivo deste trabalho é avaliar os efeitos do acetato de chumbo em parâmetros
de integridade, histopatologia e bioquímica emnas células espermáticas. Foram
coletadas 36 aves silvestres (Chrysomus ruficapillus) adultos, machos e expostas em
gaiolas. Foi administrada uma dose única de 50 e 100 mg/kg de acetato de chumbo
através de uma injeção intraperitoneal e o grupo controle recebeu uma injeção de
solução salina. Após sete dias da administração das doses, foi realizada a coleta dos
ductos deferentes e testículos para as análises nas células espermáticas. Os resultados
mostraram que houve deterioração na integridade da membrana e DNA, e diminuição
da funcionalidade mitocondrial nos testículos das aves expostas ao acetato de chumbo
nas duas doses do estudo (P<0,05). Na histopatologia foi observada diminuição na
quantidade de células dos estágios de desenvolvimento da espermatogênese, além de
patologias nas mesmas. Observou-se danos oxidativos nas aves tratadas com 100mg/kg
e um aumento da peroxidação lipídica nos testículos. Portanto, o acetato de chumbo
causou efeitos negativos no aparelho reprodutivo de Chrysomus ruficapillius.
Palavras-chave: acetato de chumbo, ave silvestre, sistema reprodutor
4
II. INTRODUÇÃO
Os metais são componentes naturais do meio ambiente e, muitos deles, são
micronutrientes essenciais para os organismos vivos, podendo ser encontrados em
sistemas terrestres, aquáticos e também no ar. Podem se concentrar através de redes de
alimentos, de onde as espécies, no topo da cadeia alimentar, podem acumular níveis
elevados de metais (Hernandez et al. 1999, Niecke et al. 1999). Ao contrário de muitos
compostos orgânicos, os metais não podem ser facilmente metabolizados em compostos
menos tóxicos, portanto metais têm longo tempo de permanência no solo podendo
causar efeitos nocivos muito tempo depois de haver ocorrido a poluição pelo próprio
metal (Dauwe et al. 2004, Berglund et al. 2009).
Os seres vivos necessitam de pequenas quantidades de metais, incluindo:
cobalto, cobre, manganês, molibdênio, vanádio, estrôncio e zinco para a realização de
funções vitais no organismo. Porém, níveis excessivos desses elementos podem ser
extremamente tóxicos por serem elementos químicos altamente reativos e os
organismos têm dificuldade em eliminá-los. Outros metais pesados como o mercúrio,
chumbo e cádmio não possuem nenhuma função dentro de organismos vivos e sua baixa
ou alta acumulação pode provocar graves doenças (Pereira & Ebecken 2009). Nos
últimos anos, efeitos tóxicos de metais pesados em organismos vivos, principalmente
como resultado da sua contínua mobilização antropogênica no ambiente, têm atraído
considerável atenção mundial (Schmitt-Jansen et al. 2008, Seebaugh et al. 2005).
O Chumbo (Pb) é um metal não essencial, tóxico, encontrado em todos os
compartimentos da biosfera e em diversas formas químicas. Suas principais fontes
naturais são as emissões vulcânicas e o intemperismo. Entre as fontes antropogênicas
encontram-se as fábricas de baterias de automóveis, as ligas metálicas, os pigmentos de
5
tinta, munição, mineração, fundição e a gasolina (ATSDR 2007, Fisher et al. 2006),
constituindo-se assim um dos grandes problemas em termos de poluição atmosférica. O
chumbo é o segundo na lista das 275 substâncias perigosas - Lista Prioritária da Lei de
Responsabilidade, Compensação e Resposta Ambiental (ATSDR 2007).
Durante alguns séculos o Pb foi amplamente utilizado em diversas indústrias,
por ter propriedades de baixo ponto de fusão, durabilidade, ductibilidade e facilidade em
formar ligas metálicas, (Florea & Busselberg 2006). Desde meados do século XX, as
quantidades de Pb liberadaos no meio ambiente têm aumentado, devido ao acelerado
crescimento populacional que levou ao desenvolvimento industrial, urbanização e
aumento na oferta de transportes (ATSDR 2007). Até aproximadamente 1970, quase
toda a gasolina utilizada no mundo continha chumbo como um de seus aditivos (UNEP
1999), e mesmo com a eliminação do chumbo acrescentado à gasolina, o mesmo
constitui-se um grave problema de saúde ocupacional e ambiental (ATSDR 2007).
Outra fonte de contaminação por chumbo no meio ambiente é a tinta a base de chumbo,
o envenenamento com a tinta ainda é um problema no interior de muitas cidades e em
ambientes aquáticos em geral (Tajkarimi et al. 2008).
Vanz et al. (2003), analisando o chumbo proveniente das precipitações sólidas
atmosféricas na cidade do Rio Grande e São José do Norte, constataram que as maiores
concentrações de chumbo encontravam-se nas áreas próximas a cidade do Rio Grande e
na região estuarina ao redor. Isto ocorre devido a poeira do ar da cidade do Rio Grande
conter chumbo que pode variar de 4,0 à 1.165,0 mg.m3 dependendo da área da cidade.
A maior concentração da deposição atmosférica seca, nas margens do estuário da área
industrial da cidade, tem como fontes a área de permanência de pescadores (devido às
chumbadas), a parte da cidade antiga (tintas a base de chumbo) e a zona industrial
(deposição atmosférica) (Mirlean et al. 2005).
6
A absorção do chumbo por parte dos seres vivos é determinada por suas
propriedades físico-químicas, pela dose, frequência, duração e via da exposição,
podendo ser influenciada por fatores como idade, sexo, estilo de vida, estado fisiológico
e nutricional e ainda pela susceptibilidade individual do organismo exposto (Alexander
et al. 1998). O chumbo é distribuído de acordo com sua afinidade pelos tecidos, em
casos de exposição crônica, cerca de 95% da carga corpórea de chumbo acumula-se nos
ossos, o tecido ósseo constitui o principal sítio de estocagem de longa vida do chumbo
(Smith et al. 1995). A quantidade de chumbo no esqueleto não é distribuída
homogeneamente, pois ele também se distribui e acumula em outros órgãos, tais como
fígado, rim e cérebro (Pain, 1995).
O chumbo (Pb) após entrar na cadeia alimentar através do ar, água e do solo,
pode ter uma bioacumulação em todos os organismos que estão no topo da cadeia
alimentar. É bem conhecido que o Pb tem efeito nocivo para os organismos vivos, com
efeitos adversos para a saúde, causando danos fisiológicos e comportamentais e,
potencialmente, a morte.
Estudos mostraram os efeitos da exposição por chumbo aguda e crônica
associados a danos graves neurocomportamentais (Burger & Gochfeld 2005),
hematológicos (Pain et al. 2007), nefrotóxicos (Cory-Slechta & Schaumburg 2000) e
reprodutivos (Telišman et al. 2007) em seres humanos e outros animais. No modelo
animal, uma série de estudos tem avaliado o impacto da exposição ao chumbo com
consequências nas características morfológicas, bioquímicas e nos hormônios
reprodutivos.
A interação do organismo com o chumbo pode levar a consequências graves
entre os componentes do eixo reprodutivo, o que pode ocasionar anomalias
reprodutivas. Os efeitos adversos do chumbo sobre a fertilidade masculina em animais
7
podem ocorrer nos órgãos reprodutivos e/ou no sistema endócrino, incluindo alterações
na motilidade dos espermatozoides, presença de espermatozoides imaturos, diminuição
da espermatogênese, redução da fertilidade, e outras funções dependentes da integridade
do sistema reprodutor masculino (Garu et al. 2011). O acetato de chumbo em altas
dosagens reduziu a percentagem de espermatozoides móveis e aumentou a percentagem
de espermatozoides anormais em camundongos (Wadi & Ahmad 1999). Outros
trabalhos descreveram alterações histológicas nos tubos seminíferos, incluindo
desorganização da espermatogênese, danos na membrana e presença de vacúolos nas
células de ratos tratados com chumbo (50 e 200 mg/kg) durante três meses (Batra et al.
2001). Lesões histopatológicas são importantes, pois recolhem dados pertinentes ao
estado de saúde, dados gerados que são uteis em fornecer informações complementares
para apoiar os marcadores bioquímicos e celulares baseados, como aqueles usados em
programas de monitoramento ambiental (Shaw et al. 2011).
Os efeitos dos metais pesados sobre a reprodução podem estar relacionados com
a agressão causada às membranas celulares dos gametas e/ou ovos embrionados
(Brooks 1997). Almeida (2001) sugere que metais pesados podem apresentar tal efeito
pelo seu grande potencial de oxirredução, em função da liberação de radicais livres que
contém oxigênio, conhecidos como espécies reativas de oxigênio (ROS). Os organismos
desenvolveram mecanismos para se proteger contra compostos orgânicos e inorgânicos,
como os metais. Um dos mecanismos mais importantes contra compostos tóxicos é de
defesa antioxidante, o qual é capaz de desintoxicar e remover tóxicos prejudiciais do
corpo. Tem sido sugerido que os efeitos tóxicos de metais são parcialmente devido ao
metal induzida por stress oxidativo (Ercal et al. 2001). Processos oxidativos são normais
para o metabolismo de defesa imunológica do organismo todos os dias e são
compensados por uma variedade de sistemas antioxidantes (Isaksson et al. 2005).
8
Estresse oxidativo é uma condição em que há um desequilíbrio entre a defesa
antioxidante e a produção de espécies reativas de oxigénio, de modo que a defesa é
superado pela formação de radicais causando danos oxidativos em biomoléculas
(Halliwell & Gutteridge 2007). O dano oxidativo está geralmente relacionado com a
produção de espécies reativas de oxigénio (ROS) por metais, e, portanto a defesa
antioxidante tem um papel importante na proteção de organismos contra o stress
oxidativo induzido. Com a contaminação do metal, a manutenção de uma elevada
capacidade antioxidante em células pode aumentar a tolerância contra os diferentes
tipos de stress ambiental (Thomas et al. 1999 ). Quando a produção de ROS ocorre em
grandes quantidades podem ser prejudiciais para biomoléculas, causando dano
oxidativo ao DNA, proteínas e lipídios de membrana, bem como alterações nas enzimas
antioxidantes (Finkel & Holbrook 2000, Valavanidis et al. 2006).
A patogenia da intoxicação por chumbo nos espermatozoides não está
totalmente elucidada e múltiplos mecanismos de ação são fornecidos para explicar este
efeito. Estudos recentes sugerem que a contaminação por chumbo perturba o equilíbrio
pró-oxidante / antioxidante e pode contribuir, pelo menos em parte, a este elemento de
toxicidade, afetando os sistemas de defesa antioxidante diminuindo a integridade das
membranas e de DNA espermático (Hsu & Guo 2002). De fato, um aumento de
espécies reativas de oxigénio (ROS) foi observada após exposição ao chumbo no
espermatozoide e nos órgãos do sistema reprodutivo, em ratos (Acharya et al.
2003, Marchlewicz et al. 2007) e em humanos (Kasperczyk et al. 2008). Este aumento
foi associado a diminuição da concentração e a queda da motilidade espermática
(Kasperczyk et al. 2008), além de ter elevado o percentual de patologias nos
espermatozoides (Acharya et al. 2003).
Na natureza, os animais podem desempenhar um papel de bioindicadores por
9
fornecer aviso de potenciais efeitos adversos para os organismos em si, para os
organismos que atacam sobre eles, e como indicadores da exposição e os efeitos para os
seres humanos (Burger & Gochfeld 2004). Dessa forma, torna-se cada vez mais
importante desenvolver modelos através de animais selvagens para entender os
potenciais riscos ecológicos como indicadores de riscos para a saúde humana.
A contaminação e o posterior envenenamento de aves pelo chumbo estão bem
documentados em várias situações e por diferentes meios. Há estudos que detectaram
contaminação de aves nos órgãos ou em penas, que estavam em locais próximos a
fundições e minas, depósitos de resíduos e áreas industriais (García-Fernández et al.
1995, Berglund 2010). Ainda, a contaminação de diferentes espécies de aves tem sido
relacionada com a contaminação do sedimento em regiões com história de caça
(Guillemain et al. 2007 ) ou antigas minas (Blus et al. 1995).
Aves terrestres e aquáticas, podem contaminar-se pela ingestão de munições de
chumbo ou pelos fragmentos de bala alojados em seus corpos (Fisher et al.
2006 ). Fragmentos de munições com chumbo foram encontradas no estômago e no
fígado de algumas aves mortas (Blus et al. 1995 e Beyer et al. 1998), a morte por
envenenamento de aves de rapina têm se atribuído à ingestão de presas mortas
jácontaminadas (Kendall et al. 1996). Resíduos de tintas a base de chumbo (lascas)
foram encontradas em aves envenenadas, tanto de cativeiro como a Grus
canadensis (Kennedy et al. 1977), como em aves silvestres Phoebastria immutabilis
(Finkelstein et al. 2003). O depósito de lodo proveniente de estações de tratamento de
esgoto em terras agrícolas pode ser uma fonte de chumbo que consequentemente causa
a contaminação de organismos que usam esses locais (Pain et al. 1995).
10
A probabilidade de se tornar uma ave contaminada está relacionada com o
tempo, frequência e histórico de exposição ao chumbo, e fatores como o estado
nutricional e estresse ambiental ao qual essa ave está exposta (Pattee & Pain 2003).
Apesar da detecção de níveis maiores de chumbo principalmente nos ossos e fígado,
muito estudos têm demonstrando que o chumbo causa efeitos em vários órgãos,
particularmente nos testículos, em seres humanos e animais selvagens (Fair & Ricklefs
2002, Snoeijs et al. 2004). A maioria dos estudos sobre os efeitos do chumbo na
fisiologia reprodutiva foram desenvolvidos em mamíferos em laboratório e são poucos
os estudos relacionando os efeitos do chumbo no sistema reprodutivo de aves (Partyka
et al. 2012).
O Chrysomus ruficapillus é uma espécie de ave silvestre amplamente distribuída
na América do Sul. Esta espécie está presente em uma variedade de habitats, incluindo
pântanos naturais, canaviais, valas na estrada, estações de tratamento de esgoto,
pequenas plantações de eucaliptos e campos agrícolas (Jaramillo & Burke 1999). Pode
ser encontrada em grandes bandos durante praticamente todo o ano. Durante a
reprodução, entre o período de agosto a março, separa-se em pequenos grupos, isso
ocorre em banhados naturais ou nas lavouras de arroz (Fallavena 1988).
Nas plantações de arroz, o Garibaldi (C.ruficapillus) é, muitas vezes,
considerado uma praga (Silva et al. 1997a). Pelo menos no Rio Grande do Sul, seu
alcance e abundância aumentaram ao longo do século XX com o aumento do cultivo de
arroz irrigado (Belton 1994). Grandes bandos dessa espécie são observados após a
colheita em arrozais e comendo os grãos de arroz que vão sendo perdidos durante o
transporte da colheita (Silva et al. 1997b). O crescimento populacional da espécie pode
ser devido ao aumento da oferta alimentar que permite uma alta taxa de sobrevivência
para jovens e adultos (Silva et al. 1997a). Dessa forma, têm entrado em processo de
11
expansão populacional ao encontrar nas lavouras de arroz as condições propícias de
abrigo, alimentação e reprodução (Cirne & López-Iborra 2005).
Apesar do conhecimento do uso de pesticidas contendo chumbo, da fabricação
de tintas a base de chumbo durante o século XX e da ampla cultura de caça que o Rio
Grande do Sul possui, são escassos os estudos sobre os efeitos do chumbo associado às
aves silvestres que frequentam ambientes expostos à contaminação com chumbo. Dessa
forma, a proposta do estudo é entender os efeitos do chumbo sobre o sistema
reprodutivo de aves usando o Garibaldi como modelo.
12
III. JUSTIFICATIVA
As aves vêm sendo usadas como indicadores da condição ambiental porque são
particularmente sensíveis a mudanças de origem antrópica (Primack & Rodrigues
2002). Este estudo escolheu uma espécie silvestre e de fácil acesso para estudar o efeito
do chumbo, que possui uma elevada importância devido ao seu alto nível de toxicidade,
que pode causar uma série de problemas nos organismos dos animais afetados.
O estudo pretendeu verificar os efeitos sobre os órgãos reprodutivos das aves
testadas e compará-las com aves capturadas paralelamente sem administração de acetato
de chumbo (aves controle). Este estudo proporcionou conhecimento sobre as mudanças
fisiológicas de aves que podem sofrer expostas a doses de chumbo e identificou se o
chumbo interfere no seu aparelho reprodutivo.
13
IV. OBJETIVOS
2.1 Geral
- Determinar as alterações sobre o aparelho reprodutivo de Chrysomus ruficapillus
(Garibaldi) após sua exposição a doses não letais de chumbo.
2.2 Específicos
a) Avaliar a integridade e funcionalidade da célula espermática de Garibaldi dosados
com chumbo com as de Garibaldi do grupo controle;
b) Avaliar a histopatologia dos testículos de Garibaldi expostos a doses de chumbo com
as do grupo controle;
c) Verificar a capacidade antioxidante total contra peroxi-radicais nos testículos de aves
expostas as doses de chumbo;
d) Avaliar as espécies reativas de oxigênio nos testículos de aves expostas as doses de
chumbo;
e) Verificar dano oxidativo em termos de peroxidação lipídica nos testículos de aves
expostas as doses de chumbo.
14
V. CAPITULO 1
Lead accumulation and effects in testis of the fowl Chrysomus ruficapillus (Fowl:
Icteridae)
Submetido à revista Reproductive Toxicology
15
Lead accumulation and effects in testis of the fowl Chrysomus ruficapillus (Fowl:
Icteridae)
Danusa Leidensa,b
, Adalto Bianchinia, Antonio Sergio Varela Junior
b, Carlos Eduardo
Rosaa, Cecilia Perez Calabuig
c, Carine Dahl Corcini
a,d*
aPrograma de Pós-Graduação em Ciências Fisiológicas - Fisiologia Animal Comparada
Instituto de Ciências Biológicas, Universidade Federal do Rio Grande, Rio Grande, RS,
Brazil. ([email protected]; [email protected]; [email protected] )
bReprodução Animal Comparada (RAC), Instituto de Ciências Biológicas, Universidade
Federal do Rio Grande, Rio Grande, RS, Brazil. ([email protected] )
cDepartamento de Ciências Animais, Universidade Federal Rural do Semiárido,
Mossoró, RN, Brazil. ([email protected] )
dReproPel – Reprodução Animal, Departamento de Patologia, Faculdade de Veterinária,
Universidade Federal de Pelotas, Pelotas, RS, Brazil ([email protected] )
* Corresponding author: Carine Dahl Corcini
Universidade Federal do Rio Grande - FURG
Instituto de Ciências Biológicas (ICB)
Av Itália km 8 s/n - Caixa Postal 474
CEP 96200-970
Rio Grande, RS, BRAZIL
Phone: 55 5332759167
E-mail: [email protected]
16
Abstract
In the present study, lead (Pb) effects on sperm quality parameters, as well as testis
histology and biochemistry were evaluated in male adult fowls (Chrysomus
ruficapillus). Wild fowls were captured, maintained in captivity and treated with a
single intraperitoneal injection of saline solution (control) or saline solution containing
Pb acetate (50 or 100 mg Pb/Kg). Seven days after injection, samples were collected
from the ductus deferens and testes for analyses. Increased in both blood and testis of
fowls injected with both doses of Pb acetate. Histological analysis revealed the presence
of pathological alterations. In sperm cells, Pb exposure induced loss of membrane
integrity, mitochondrial functionality, and DNA integrity. Also, oxidative damage was
observed in testes of fowls injected with 100 mg Pb/Kg. Taken altogether, these
findings indicate that Pb is highly accumulated in testis of the fowl C. ruficapillius,
inducing severe morphological and biochemical damages that could compromise the
reproductive performance of male fowls.
Key words: histological, fowl, lead toxicity, oxidative damage, reproduction, spermatic
cells.
17
1. Introduction
Lead (Pb) is a nonessential and toxic metal that can be found in all compartments of
the biosphere under different chemical forms. The main natural sources of Pb are
volcanic emissions and weathering. Anthropogenic sources include car batteries, metal
alloy, paint pigments, ammunition, mining, casting and gasoline [1] and [2].
Uncontrolled discharge of Pb represents a global environmental issue, concerning
atmospheric, aquatic and terrestrial ecosystems. In fact, Pb is the second chemical listed
among the 275 most dangerous substances in the Priority List of Hazardous Substances
[2].
Adverse effects of Pb exposure on male fertility can be a result of its direct
interaction with the reproductive organs and/or the endocrine system. These effects
include altered sperm motility and maturation, reduced spermatogenesis and
testosterone level, as well as disturbances of other functions depending on the integrity
of the male reproductive system [3] and [4]. For example, studies with administration of
Pb (as Pb acetate) in captive fowls have reported neurobehavioural effects on growth
[5], [6] and [7]. [8] evaluated the effects of the oral administration of Pb (4-110 mg as
ammunition) on fertility of adult male turtle doves (Streptophelia risoria). In this case,
significant testicular degeneration and absence of sperm in the seminiferous tubules
were observed.
Fowls have been used as biomonitors of environmental contamination with organic
pollutants and heavy metals [7] and [9]. In fact, they are top predators sensitive to
environmental changes, being exposed to many sources of environmental pollutants
[10]. Therefore, it is important to evaluate the effects of environmental contaminants on
18
this wild bird species to better understand the potential risks to the ecosystem and
human health.
The fowl Chrysomus ruficapillus is widely distributed in South America. This bird
species is present in a variety of habitats, including natural marshes, reed beds, roadside
ditches, sewage treatment site, small eucalyptus plantations and farmland [11]. In rice
fields, they enter into the process of population expansion because they find safe shelter,
food and optimal conditions for reproduction [12].
Despite of the widespread use of Pb-containing pesticides and Pb-based paints
during the twentieth century, as well as the increased hunting practices in some
countries, there are only few studies on Pb effects on wild fowls inhabiting areas
subjected to these sources of contamination. In light of this, the aim of the present study
was to evaluate the effects of Pb exposure on reproductive parameters in the male fowl
C. ruficapillus.
2. Materials and Methods
2.1. Fowls capture and maintenance
Experiments performed in the present study were approved by the Ethics
Committee of the Federal University of Rio Grande (license #23116.006225/2011-39).
Adult male fowls C. ruficapillus (body mass: 36.1 ± 2.79 g; n = 50) were captured
in the wild during the reproductive period (October/2012) using mist nets (SISBIO,
capture license #30228-1). Fowls were randomly divided into two groups: (1) birds
sampled immediately after capture and transfer to the laboratory, as described below
(field group: n = 5); and (2) birds kept in captivity, treated, and sampled, as described
below (n = 45).
19
Fowls were kept in cages (2 birds/m2) with covered floor to avoid environmental
contamination. Cages were provided with shrubs and trees, with shade, water mirror and
feeder following the criteria established by the Federal Normative Instruction 04/2002
issued by the Brazilian Institute of Environment (IBAMA). Food and water were
completely renewed every day over the whole experimental period (7 days).
2.2. Laboratory procedures
Fowls from the field group were weighed (body mass) and measured (total culmen,
bill height, bill width, tarsus, tail and wings). Blood samples were collected by puncture
of the tarsal vein using disposable syringes and needles. Fowls were then euthanized by
cervical dislocation (Report of the AVMA Panel on Euthanasia, 2007) and had their
testes collected through laparotomy. Whole-blood and testis samples were immediately
stored in Eppendorf-type tubes and frozen (-80oC) until Pb content determination, as
described below
Fowls kept in captivity were weighed (body mass), measured (total culmen, bill
height, bill width, tarsus, tail and wings), tagged with leg rings, and randomly divided
into three groups: (1) fowls treated with a single intraperitoneal injection (1 mL) of
saline (0.9% NaCl) solution (control group; n = 12); (2) fowls treated with a single
intraperitoneal injection (1 mL) of saline (0.9% NaCl) solution containing Pb acetate
(50 mg Pb/Kg group; n = 15); and (3) fowls treated with a single intraperitoneal
injection (1 mL) of saline (0.9% NaCl) solution containing Pb acetate (100 mg Pb/Kg
group: n = 18).
Seven days after treatment, whole-body samples were collected and fowls were
euthanized by cervical dislocation, as described above for the field group. Ductus
deferens and testes were collected through laparotomy. Isolated ductus deferens were
20
sectioned longitudinally, individually immersed in saline solution, and kept at room
temperature (~20oC) for 10 min to allow sperm migration to the incubation medium.
Seminal quality parameters were evaluated immediately after semen dilution, as
described below. Regarding testes, one testis was fixed in 4% paraformaldehyde for
histological analysis, as described below, while the other was immediately divided into
three subsamples. One subsample was kept on ice (2-4ºC) for reactive oxygen species
(ROS) and antioxidant capacity against peroxyl radicals (ACAP) analysis, as described
below. The other two subsamples were stored in ultra-freezer (-80°C) for further
analyses of lipid peroxidation (LPO) and Pb content, as described below.
2.3. Semen quality analysis
The following quality parameters were evaluated in sperm cells: membrane
integrity, mitochondrial functionality and DNA integrity. A total of 200 sperm cells
were assessed in each sample slide.
Plasma membrane integrity was evaluated using the fluorescent probes
carboxyfluorescein diacetate (CFDA) (Sigma, St. Louis, MO, USA) and propidium
iodate (PI) (Sigma, St. Louis, MO, USA), as described by [13]. Samples were evaluated
under epifluorescence microscope (400x magnification) (Olympus BX 51, América,
São Paulo, SP) equipped with WU filter with excitation between 450-490 nm and
emission at 516 (CFDA) or 617 nm (PI). Green-stained cells were considered viable
whereas red and red-green stained cells were considered damaged. Data were expressed
as percentage of cells showing membrane integrity.
Mitochondrial functionality was assessed by rhodamine 123 (Rh123) staining
(Sigma, St. Louis, MO, USA) according to [14]. Semen aliquots (100 μL) were placed
in 1.5 mL microtubes and 5 μL of formaldehyde (1.7 mM), 5 μL of PI (7.3 mM) and 5
21
μL of Rh123 (0.2 mM) were then added. Cells were evaluated under the epifluorescence
microscope (400x magnification; excitation: 450-490 nm; emission: 516–617 nm). Cells
with intense green fluorescence were considered as containing functional mitochondria
whereas those not showing intense green staining were considered as containing non-
functional mitochondria. Data were expressed as percentage of cells showing
mitochondrial functionality.
Sperm DNA integrity was assessed using acridine orange (Sigma, St. Louis,
MO, USA) staining in dried slides. Analysis was performed under the epifluorescence
microscope (400x magnification; excitation: 525 nm). Sperm cells exhibiting green
fluorescence were considered normal (bicatenary DNA) whereas red- or yellow-stained
sperm cells were considered abnormal, with denatured DNA (monocatenary DNA).
Data were expressed as percentage of sperm cells showing DNA integrity.
2.4. Histological analysis
Testes were fixed in 4% paraformaldehyde for 2 h. They were then processed in
an automated vacuum processor (ASP 200, Leica, Germany) according to standard
histological techniques. Samples were impregnated and embedded in ParaplastXtra®
(Sigma, St. Louis, MO, USA). Glass slides with tissue sections (7-μm thickness) were
stained with hematoxylin and eosin [15]. Histological analysis was performed under
bright-field light microscope BX51 equipped with a DP73 camera (Olympus, Japan).
For all samples, histological alterations were quantified using blind analysis. The
abundance or absence of the different cell types of the seminiferous epithelium was
scored.
22
2.5. Reactive oxygen species (ROS) determination
Testis samples freshly collected were weighed and homogenized (1:20 w/v) in
phosphate buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 1.15 g/L
Na2HPO4.7H2O; pH 7.6). Homogenized samples were centrifuged (10,000 xg) at 4°C
for 20 min. The supernatant was collected for ROS determination [16]. Protein
concentration in the supernatant was determined using a commercial reagent kit (Doles,
Goiânia, GO, Brazil) based on the Biuret method. ROS measurement was performed
using 2',7'–dichlorofluorescein diacetate (H2DCF-DA, Molecular Probes, USA). The
H2DCF-DA cleavage by cellular esterases from the homogenized supernatant in the
presence of ROS generates a fluorochrome, which fluorescence can be detected at
excitation and emission wavelengths of 488 and 525 nm, respectively. The assay was
performed using white bottom microplates. Fluorescence reading was done every 5 min
up to 60 min using a fluorescence microplate reader (Victor2 Perkin-Elmer,
USA). Fluorescence area was calculated integrating fluorescence units (FU) over time,
adjusting FU data to a second order polynomial function. ROS content was expressed as
FU per milligram protein in the homogenized supernatant.
2.6. Antioxidant capacity against peroxyl radicals (ACAP)
Supernatants obtained during ROS determination, as described above, were used
for ACAP determination, which was assessed following procedures described by [17].
Briefly, an aliquot of 10 µL from each homogenized supernatant was transferred to a
white-bottom 96-wells microplate. Each sample was tested in six different wells. The
reaction buffer (127.5 µL) containing 30 mM HEPES (pH 7.2), 200 mM KCl and 1 mM
MgCl2 was added to the wells with samples. In three wells of each sample, 7.5 mL of
23
2,2 '-azo-bis-di-hidrocloretomethylpropionamidine2 (ABAP, 4 mM; Aldrich, USA)
were added. The same volume of ultrapure water was then added to the remaining three
wells of the corresponding sample. The fluorescence of the reaction mixture was read
using the fluorescence microplate reader at 37°C to determine the sample background.
Afterwards, 10 µl of H2DCF-DA were added to each microplate well at a final
concentration of 40 μM. Thermal decomposition of ABAP at 37ºC produces peroxy
radicals which in contact with fluorochromes generates fluorescence. Fluorescence
emission was determined using the fluorescence microplate reader (excitation: 488 nm;
emission: 525 nm). Readings were performed every 5 min up to 60 min. FU were
integrated over time after adjusting data to a second order polynomial function. Results
were calculated as the difference in FU area per min in the same sample with and
without ABAP, and then normalized to ROS area without ABAP (background area).
The relative difference between ROS area with and without ABAP was considered a
measurement of antioxidant capacity. Therefore, antioxidant capacity decreases as the
relative area increases.
2.7. Lipid peroxidation (LPO)
Lipid peroxidation in testis samples of the wild fowl C. ruficapillus was
determined using the FOX method [18]. This method is based on the Fe2+
oxidation by
lipid hydroperoxides under acid pH in the presence of xylenol orange (Sigma, St. Louis,
MO, USA) which forms a complex with Fe3+
. Testis samples were weighed,
homogenized (1:20 w/v) in 100% methanol (4°C) and centrifuged (1,000 xg) at 4°C, for
5 min. The supernatant was used for the spectrophotometric assay (580 nm). Cumene
hydroperoxide (CHP; Sigma, St. Louis, MO, USA) was used as standard. Results were
expressed as nmoles CHP per gram of wet tissue.
24
2.8. Tissue Pb content
Pb content in the whole-blood and testis of fowls from the 4 groups (field,
control, 50 mg Pb/Kg, and 100 mg Pb/Kg) was determined by absorption atomic
spectrophotometry (AAS-932 Plus, GBC, Australia), following the procedures and
quality assurance controls as described by [19]. Briefly, blood and testis samples were
thawed at room temperature, dried at 60oC for 72 h, weighed (dry weight), and
completely digested with concentrated nitric acid (65% HNO3, SupraPur, Merck, São
Paulo, SP, Brazil). After complete digestion, samples were diluted (1:2) with Milli-Q
water and Pb concentration was determined by AAS. Measurement accuracy and
standard curves were obtained using a standard Pb solution (Standard Reference
Material 3114; National Institute of Standards & Technology, Gaithersburg, MD, USA).
Percentages of metal recovery based on standard reference material (European
Reference Material ERM-CE278, Geel, Belgium) prepared as described for tissue
samples was 98.3%. Results were expressed as mg Pb/g dry weight.
2.9. Statistical analysis
Continuous data were tested for normal distribution using the Shapiro-Wilk test.
All dependent variables with normal distribution were subjected to analysis of variance
(ANOVA) followed by the Tukey multiple comparison test. Treatment was considered
as the independent variable while biometric, membrane integrity, mitochondrial
functionality, DNA integrity, ROS content, LPO, ACAP, and Pb content data were
considered as dependent variables. Data from histological examination were compared
using the chi-square test. All analyses were performed using the software Statistix 9.0
(Analitical Software, Tallahassee, FL, USA).
25
3. Results
3.1. Mortality
Regarding fowls kept in captivity, no mortality was observed in those from the
control and 50 mg Pb/Kg groups. However, two days after treatment, three fowls from
the 100 mg Pb/Kg group were found dead.
3.2. Biometry
No significant difference in body weight, total culmen, bill height, bill width,
tarsus, and wing and tail length was observed among the fowl groups (P>0.05) (Table
1).
3.3. Tissue Pb content
No detectable level of Pb was observed in the whole-blood of fowls from the
field and control groups. However, high levels of accumulated Pb were observed in
blood and testis of fowls injected with 50 and 100 mg Pb/Kg. In fact, a ~5-6-fold
increase in Pb accumulation was observed in both whole-blood and testis of these fowls.
Pb content was ~10-fold higher in testis than in blood of Pb-exposed fowls (Fig. 1).
3.4. Histological analysis
Moderate to severe testicular atrophy was observed in fowls injected with 50 mg
Pb/Kg (67% atrophy or with 100 mg Pb/Kg (89% atrophy). Also, vacuoles were
observed in Pb-exposed fowls (50 mg Pb/Kg: 98%; 100 mg Pb/Kg: 67%). Moderate to
severe decrease in the number of germinal cells was observed. Results for fowls injected
26
with 50 and 100 mg Pb/Kg were 25 and 44% for spermatogonia; 83 and 77% for
spermatocytes; 75 and 100% for spermatids; 83 and 100% for spermatozoa; and 16 and
33% for Leydig cells (Fig. 2).
3.5. Semen quality analysis
It was observed a significant decrease (P<0.05) in membrane integrity and
mitochondrial functionality in Pb-exposed fowls compared to those from the control
group. However, no significant difference was observed between fowls injected with 50
and 100 mg Pb/Kg. Regarding DNA integrity of spermatic cells, a significant decrease
(P<0.05) was observed in fowls injected with 100 mg Pb/Kg when compared to those
from the control group or those injected with 50 mg Pb/Kg. No significant difference
was observed between control fowls and those injected with 50 mg Pb/Kg (P>0.05)
(Fig.3).
3.6. Biochemical analysis
No significant difference in ROS content was observed between control and Pb-
exposed fowls (P>0.05). However, a significantly lower (P<0.05) ROS content was
observed in fowls injected with 100 mg Pb/Kg than in those injected with 50 mg Pb/Kg.
ACAP was significantly reduced (P<0.05) in fowls injected with 100 mg Pb/Kg when
compared to those from the control group. No significant difference was observed
between control fowls and those injected with 50 mg Pb/Kg. Also, no significant
difference was observed between fowls injected with Pb (50 and 100 mg Pb/Kg
groups). A significantly higher (P<0.05) lipid peroxidation level was observed in fowls
injected with 100 mg Pb/Kg when compared to those from the control and 50 mg Pb/Kg
groups. No significant difference (P>0.05) was observed between control fowls and
27
those injected with 50 mg Pb/Kg (Fig. 4).
4. Discussion
Data on Pb content showed non-detectable levels of the metal in the blood of field
and control fowls. However, Pb was found to accumulate in testis of fowls from both
groups. In fact, it is known that after entering the systemic circulation, Pb acetate
crosses the blood-testis barrier and accumulates in the testis of animals and humans [20]
and [21]. It is important to note that similar Pb content was found in testis of field and
control fowls. These findings indicate that no additional Pb contamination was
introduced through water or food provided during fowl maintenance in captivity over
the experimental period.
In the fowl C. ruficapillus kept and treated with Pb in captivity, we also observed
that Pb (as Pb acetate) has effectively reached the blood after being injected into the
peritoneum. As mentioned above, no detectable levels of Pb were observed in the
whole-blood of field and captive fowls non-exposed to Pb. In contrast, Pb was
significantly accumulated in the whole-blood of fowls seven days after being treated
with a single injection of Pb acetate. Fowls injected with a single dose of 100 mg Pb/Kg
reached a mean value as high as 0.68 mg Pb/g dw in the blood. A similar result was
observed in fowls injected with a single dose of 50 mg Pb/Kg. As expected, after
reaching the blood, Pb crossed the blood testis-barrier and accumulated in the testis of
the fowl C. ruficapillus. Again, similar results were observed with both Pb doses tested
(50 and 100 mg Pb/Kg). These findings indicate that tissue (blood and testis) Pb level
was already saturated with the lower dose applied.
28
Regarding the biological effects, findings from the present study clearly
demonstrate that acute exposure to Pb induced severe damage to the male reproductive
tract in the wild fowl C. ruficapillus. This statement is based on the fact that fowls
showed reduced semen quality seven days after being treated with a single
intraperitoneal injection of saline solution containing Pb acetate (50 and 100 mg
Pb/Kg).Reduced sperm quality was evidenced by a lower frequency of cells showing
membrane integrity, mitochondrial functionality, and DNA integrity. These findings are
in complete agreement with those reported by [22]. These authors reported an increased
percentage of sperm pathologies and a reduced percentage of motile sperm in rats
intraperitoneally injected with 100 mg Pb/Kg, one of the doses tested in the present
study. It is important to note that similar results of membrane integrity and
mitochondrial functionality were observed with a lower dose (50 mg Pb/Kg) of Pb in
the fowl C. ruficapillus, which is in complete agreeing with data on Pb accumulation in
the fowl testis.
Sperm parameters evaluated in the present study are very important for the whole
reproductive process in vertebrates, since the observed decrease in membrane integrity
reduces sperm viability [13]. Furthermore, membrane lesions induced by Pb exposure
would facilitate the metal access into the cells and its interaction with organelles and
nucleus, bringing them more susceptible to Pb-induced damages. In fact, a significant
reduction in mitochondrial functionality and DNA integrity was observed in Pb-exposed
fowls. It is important to stress that a reduced mitochondrial functionality would decrease
the amount of energy available for sperm motility. Regarding DNA, [23] also observed
a high frequency (60%) of sperm cells with DNA lesion after treating mice with a high
dose of Pb acetate. In fact, these authors demonstrated that Pb can be incorporated into
29
sperm nuclei during development in the testis or during maturation in the epididymis.
Therefore, Pb effects on sperm quality (membrane integrity, mitochondrial functionality
and DNA integrity) in the fowl C. ruficapillus seems to result from a direct interaction
of the metal with the testis and sperm nuclei, as previously suggested by [20] and [21].
Considering the Pb effects described above on sperm quality, a lower reproductive
potential would be expected in male fowls acutely exposed to Pb. In fact, male fowls
exposed to both doses of Pb acetate showed a reduced number of spermatogonia,
spermatocytes, spermatids and spermatozoa. Therefore, it is clear that Pb exposure is
negatively interfering on fowl spermatogenesis, leading to a reduced availability of all
spermatogenic cells types. Furthermore, Leydig cells responsible for testosterone
synthesis were also reduced in fowls exposed to both doses of Pb. A similar result was
observed in mice treated with Pb acetate [24]. This finding suggests that a lower level of
circulating testosterone would be occurring in Pb-exposed fowls, thus affecting germ
cells.
The observed decreased number and function of Leydig in mice treated with
different doses of Pb acetate was suggested to be resulting from LPO induced by an
increased oxidative stress. It is known that increased ROS production without a
corresponding increase in tissue total antioxidant capacity or a reduced amount of non-
enzymatic antioxidant agents and/or lower activity of the enzymes involved in the
antioxidant defence system can lead to the oxidative stress condition and LPO [17]. For
example, increased ROS levels were observed after injection of Pb acetate (40 mg
Pb/Kg) and associated with testicular germ cells destruction, membrane damage and
significant decrease in sperm count in mice [25].
In the present study, a dose-dependent increase in LPO was observed in testis of the
fowl C. ruficapillus exposed to a single dose o Pb acetate. Indeed, testis of fowls
30
injected with 100 mg Pb/Kg showed a significantly higher LPO than those from the
control group. This finding clearly point out the Pb-induced oxidative stress as a
reasonable explanation for damages observed in the testis of the fowl C. ruficapillus,
especially that associated with the membrane integrity of sperm cells. In fact, the sperm
membrane of birds is rich in poly-unsaturated fatty acids (PUFAs), thus being subject to
LPO in the presence of ROS [26]. Peroxidation of PUFAs in sperm cell membrane is an
autocatalytic, self-propagating reaction that can cause cell dysfunction with loss of
membrane integrity, leading to a decrease in sperm fertilizing capacity [27]. Actually,
the oxidative degradation of PUFAs has been considered as the main cause of
membrane fluidity and permeability loss, inducing damage to germ cells and sperm
[28]. Therefore, the higher LPO level observed in testis of fowls exposed to the higher
dose tested (100 mg Pb/Kg) can be a reasonable explanation for the lower integrity of
cell and nuclear membrane, as well as the consequently reduced sperm quality observed
in fowls subjected to this experimental condition.
Although an increased LPO level was observed in Pb-exposed fowls, no significant
change in ROS content was observed in testis of these fowls. This lack of change in
ROS content could be explained by considering a Pb-induced increase in the capacity of
the antioxidant system to scavenge the ROS generated. In fact, a dose-dependent
decrease in ACAP was observed in fowls exposed to Pb, being significantly higher in
fowls treated with 100 mg Pb/Kg than in those from the control group. Therefore, the
lack of change in ROS content paralleled with the reduced ACAP observed after fowl
treatment with Pb acetate can easily explain the damages reported for sperm cells and
discussed above. Also, it can be at the basis of the structural disorder and atrophy
observed in testis of fowls exposed to Pb, as discussed below.
31
In a broad view, damages observed in sperm cells were also reflected at a higher
level, i.e., cellular structural disorder and testis atrophy. Actually, vacuole formation
was observed in more than 50% of testis from Pb-exposed fowls analyzed. These
finding is in complete agreement with data reported for rats. Exposure of adult rats to a
low dose of Pb acetate (20 mg Pb/Kg) caused testicular lesion while exposure to higher
doses induced lesions that included spermatogenesis disorder and disruption with
accumulation of immature cells in the tubular lumen, as well as lesions such as atrophy
and vacuoles [29]. Similar findings were also reported in adult male of the quail
Coturnix coturnix exposed to Pb acetate in drinking water (0.1, 0.25, 0.5 and 1%), for 1
to 6 months [30]. In this case, chronic exposure induced histopathological alterations
including spermatic cells hyperplasia, Leydig cells degeneration, decreased
spermatocyte number and tubular atrophy. Taken altogether, these findings suggest that
damages observed in testis of the fowl C. ruficapillus are certainly related to the
observed loss of sperm function and viability discussed above.
In summary, data reported in the present study clearly show that Pb can accumulate
in testis of the fowl C. ruficapillus after a single intraperitoneal injection of Pb acetate
and induce toxicity. Toxic effects on the reproductive tract of male fowls included loss
of membrane integrity, mitochondrial functionality and DNA integrity in sperm cells, as
wells as cellular structural disorder and testis atrophy. The observed effects were quite
severe and likely resulting from an oxidative stress condition induced by Pb exposure.
These effects indicate that Pb can negatively affect testis function and sperm quality in
adult male fowl C. ruficapillus, being a potential threat to reproduction of these wild
birds.
32
Acknowledgements - The present study was financially supported by the Brazilian
“Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)”. A.
Bianchini is a research fellow from the Brazilian CNPq (Proc. # 304430/2009-9) and
supported by the International Canada Research Chair Program from IDRC. Authors are
grateful to “Granjas Quatro Irmãos” for all the assistance provided
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37
Table 1. Biometric parameter (mm) of wild fowls (Chrysomus ruficapillus) injected
with 1 mL of saline solution (control) or with 1 mL of saline solution containing Pb
acetate (50 or 100 mg Pb/Kg). Data are expressed as mean ± standard error. No
significant difference was observed among groups for all parameters analyzed (P<0.05).
Group
Parameter Field
(n = 5)
Control
(n = 12)
50 mg Pb/Kg
(n = 15)
100 mg Pb/Kg
(n = 15)
Body weight 38.18 ± 3.22 36.15 ± 2.79 36.94 ± 2.41 37.07 ± 2.33
Total culmen 17.06 ± 0.68 17.33 ± 1.21 17.65 ± 1.23 17.48 ± 0.85
Bill height 9.57 ± 0.26 9.35 ± 0.25 9.23 ± 0.55 9.37 ± 0.43
Bill width 8.71 ± 0.13 8.74 ± 0.31 8.71 ± 0.25 8.73 ± 0.43
Tarsus 34.83 ± 1.73 34.56 ± 1.25 34.91 ± 2.05 34.65 ± 2.15
Tail 69.43 ± 2.88 73.00 ± 2.86 71.56 ± 3.43 73.03 ± 3.39
Wing 92.00 ± 3.00 94.36 ± 3.80 94.50 ± 3.49 93.30 ± 3.42
38
Legend to Figures
Fig.1. Pb content in whole-blood and testis of the fowl Chrysomus ruficapillus. Data are
expressed as mean ± standard error (n = 5 for each group). Pb content in the blood of
fowls from the field and control groups was below the detection limit of the technique
employed. Different letters indicate significant difference (P<0.05) among experimental
groups for each tissue.
Fig. 2. Histological analysis in testis of the fowl Chrysomus ruficapillus. (A) Testis of
control fowls showing normal organization of seminiferous tubules and all stages of
spermatogenic and Leydig cells (HE; 100x magnification). (B) Testis of fowls from the
50 mg Pb/Kg group showing severe atrophy, low sperm cell density and vacuolization
(HE; 100x magnification). (C) Testis of fowls from the 100 mg Pb/Kg group showing
total disorganization of seminiferous tubules with many vacuoles, low sperm cell
density, cell disorganization and few Leydig cells (HE; 100x magnification). -
spermatogonia, - spermatocyte, - spermatozoa, - Leydig cell and - vacuole.
Fig. 3. Sperm quality parameters in the fowl Chrysomus ruficapillus seven days after
injection of 1 mL of saline solution (control group) or 1 mL of saline solution
containing Pb acetate (50 or 100 mg Pb/Kg groups). Data are expressed as mean ±
standard error (control group: n = 12; 50 and 100 mg/Kg groups: n = 15). MI:
membrane integrity; MF: mitocondrial functionality; DI: DNA integrity. Different
letters indicate significant difference (P<0.05) among experimental groups for each
parameter.
39
Fig. 4. Reactive oxygen species (ROS), total antioxidant capacity (ACAP), and lipid
peroxidation (LPO) in testis of the wild fowl Chrysomus ruficapillus seven days after
injection of 1 mL of saline solution (control group) or 1 mL of saline solution
containing Pb acetate (50 or 100 mg Pb/Kg). Data are expressed as mean ± standard
error (control group: n = 12; 50 and 100 mg/Kg groups: n = 15). Different letters
indicate significant different mean values among experimental groups (P<0.05) for each
parameter.
40
Figure 1
Blood T estis
Blo
od
Pb
co
nte
nt
(mg
/g d
w)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Te
sti
s P
b c
on
ten
t (m
g/g
dw
)
0
2
4
6
8
10
12
F ie ld
C ontro l
Pb-exposed (50 m g Pb/Kg)
Pb-exposed (100 m g Pb/Kg)
a a
b
aa
b
b
b
41
Figure 2
42
Figure 3
M I M F D I
Fre
qu
en
cy
(%
)
0
20
40
60
80
100
120
C ontro l
Pb-exposed (50 m g Pb/Kg)
Pb-exposed (100 m g Pb/Kg)
a
b bbb
a a
a
b
43
Figure 4
R O S AC AP LPO
RO
S c
on
ten
t (f
luo
res
ce
nc
e u
nit
s/m
g p
rote
in)
0
10 5
2x10 5
3x10 5
4x10 5
5x10 5
6x10 5
AC
AP
(re
lati
ve
are
a)
an
d L
PO
(n
mo
l C
HP
/g w
w)
0
1
2
3
4
5
6
150
175
200
225
250
C ontro l
Pb-exposed (50 m g Pb/Kg)
Pb-exposed (100 m g Pb/Kg)
ab
b
a
b
ab
a
a
ab
b
44
VI. CONCLUSÃO E CONSIDERAÇÕES GERAIS
Concluiu-se que a exposição crônica ao chumbo causa efeitos nas células
espermáticas e no aparelho reprodutivo masculino em Chrysomus ruficapillus adultos
que afetará o desempenho reprodutivo dessas aves silvestres. O chumbo é um dos
contaminantes ambientais mais comuns, com alta toxicidade para homens e animais,
que pode causar mortalidade ou interferir nas populações através de seus efeitos tóxicos.
Estudos relacionando a exposição de metais e seus compostos com o trato reprodutivo
têm sido associados a um aumento de anormalidades morfológicas, baixa motilidade
espermática e infertilidade masculina. Aves terrestres selvagens estão expostas a
inúmeras formas de contaminação, frequentam vários ambientes e tem uma diversidade
alimentar, sendo animais mais sensíveis e mais susceptíveis a doenças, por isso, são
consideradas bioindicadores ambientais. Porém, estudos com aves terrestres ainda são
escassos devido a manutenção e custos de manter elas em laboratório, e pela
manipulação para conseguir os tecidos. Torna-se necessário aprofundar conhecimentos
no que diz a respeito à influência do acetato de chumbo no sistema reprodutivo e como
esse interage na fisiologia reprodutiva, os mecanismos de ação e que danos causam,
pois esses conhecimentos podem fornecer informações para a saúde pública e
ambiental.
45
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