MORPHOMETRY AND STEREOLOGY OF THE INTERSTITIAL COMPARTMENT
IN THE WILD RODENT HYLAEAMYS MEGACEPHALUS (RODENTIA:
CRICETIDAE) FROM CENTRAL BRAZIL
Fabiana Cristina Silveira Alves de Melo1, Tatiane Pires de Sousa2; Kyvia Lugate
Cardoso Costa3; Sérgio Luis Pinto da Matta3; Fabiano Rodrigues de Melo1
1
Biological Sciences, Federal University of Goiás, Campus Jataí, Jataí, Brazil
([email protected]);
2
Ecology and Conservation Program, State University of Mato Grosso, Campus Nova
Xavantina, Nova Xavantina, Brazil;
3
Department of General Biology, Federal University of Viçosa, Viçosa, Brazil.
Recebido em: 31/03/2015 – Aprovado em: 15/05/2015 – Publicado em: 01/06/2015
ABSTRACT
Little is known about the testicular components in wild rodents, particularly the
interstitial compartment. The aim of this study was to describe the testis morphometry
and stereology of Hylaeamys megacephalus, with emphasis in the interstitial
compartment. In this study were used five animals captured in forest fragments in
southwestern Goiás. The testes were removed, fixed in Karnovsky, dehydrated,
embedded in methacrylate, sectioned with two-micrometer-thick, and stained with 1%
toluidine blue/sodium borate. The histological sections of testes were evaluated using
light microscopy. The interstitial compartment volume density was 5.5%, consisting
predominantly of Leydig cells (3.21%), followed by blood vessels (0.97%), connective
tissue (0.83%) and lymphatic space (0.53%). The Leydigossomatic index was
0.015%. The interstitial pattern in H. megacephalus was similar to pattern I of
Fawcett’s classification as was found in capybara. The Leydig cell average volume in
H. megacephalus was 350.80 µm3, close to the lowest values observed in several
domestic, wild and laboratory species. The number of Leydig cells per testis and per
gram of testis was higher than observed in other small rodents.
KEYWORDS: Leydig cell, lymphatic space, connective tissue, testes, blood vessel.
MORFOMETRIA E ESTEREOLOGIA DO COMPARTIMENTO INTERSTICIAL NO
ROEDOR SILVESTRE HYLAEAMYS MEGACEPHALUS (RODENTIA:
CRICETIDAE) DO BRASIL CENTRAL
RESUMO
Pouco se sabe sobre os componentes testiculares em roedores silvestres,
particularmente o compartimento intersticial. O objetivo deste estudo foi descrever a
morfometria e estereologia testicular de Hylaeamys megacephalus, com ênfase no
compartimento intersticial. Neste estudo foram utilizados cinco animais capturados
em fragmentos florestais no sudoeste de Goiás. Os testículos foram fixados em
Karnovsky, desidratados, embebidos em metacrilato, seccionados com dois
micrômetros de espessura e corados com azul de toluidina-borato de sódio 1%. Os
cortes histológicos dos testículos foram avaliados ao microscópio de luz. A
densidade volumétrica do compartimento intersticial foi de 5,5%, consistindo
predominantemente de células de Leydig (3,21%), seguido por vasos sanguíneos
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2331
2015
(0,97%), tecido conjuntivo (0,83%) e espaço linfático (0,53%). O índice
Leydigossomático foi de 0,015%. O padrão de organização do tecido intersticial em
H. megacephalus foi similar ao padrão I da classificação de FAWCETT et al. (1973),
como foi encontrado em capivaras. O volume médio da célula de Leydig em H.
megacephalus foi 350,80 µm3, perto dos menores valores observados em diversas
espécies domésticas, silvestres e de laboratório. O número de células de Leydig por
testículo e por grama de testículo foi maior do que o observado em outros pequenos
roedores.
PALAVRAS-CHAVE: célula de Leydig, espaço linfático, tecido conjuntivo, testículos,
vaso sanguíneo.
INTRODUCTION
The Rodentia order is the most numerous of the Mammalia class and it is
present in all continents except the Antarctic. This order has a long evolutionary
history, great diversity and varied habits and trophic strategies (BONVICINO et al.,
2008). The Cricetidae is the richest family in species diversity in Brazil and all
members belong to the Neotropical subfamily Sigmodontinae. The wild rodent
Hylaeamys megacephalus has terrestrial habits, inhabiting forested and open
vegetation areas in the main Biomes of Brazil, including Amazon Rainforest, Atlantic
Forest and Cerrado. In Brazil, this species is founded in Amazonas, Roraima, Pará,
Western Amapá, Mato Grosso, Maranhão, Tocantins, Goiás, Distrito Federal, Mato
Grosso do Sul, Western Minas Gerais, and Northeastern São Paulo
(CARMIGNOTTO et al., 2012).
The mammalian testis can be divided into tubular or spermatogenic
compartment and interstitial or intertubular compartment. The tubular compartment
contains seminiferous tubules and the interstitial compartment contains blood and
lymphatic vessels, nerves, a variable population of cells in the connective tissue, and
Leydig cells (MORAIS et al., 2013a). The percentage of the interstitial components is
variable in different species (FAWCETT et al., 1973, FRANÇA & RUSSELL, 1998),
and the component that has the greater percentage variation is the Leydig cell
(COSTA et al., 2010).
FAWCETT et al., (1973) described three patterns of interstitial tissue
organization according to the arrangement and the proportion of the elements in this
compartment. The first pattern has small volume of Leydig cells, scarce connective
tissue, and great part of this compartment is occupied by the lymphatic vessels. In
the second pattern the clusters of Leydig cells are widely scattered in a very loose
connective tissue stroma drained by conspicuous lymphatic vessels. The third
pattern has the Leydig cells occupying almost all the interstitial area, with little
connective tissue and few lymphatic vessels. Although little is known about the
physiological implications of these variations, they are probably related to the ability
of lymphatic vessels in move out of the testes materials vascularly secreted and
maintain adequate concentrations of androgens in the testis and blood vessels
(FAWCETT et al., 1973).
Little is known about the testicular components in wild rodents, particularly the
interstitial compartment. The assessment of histological parameters of gonads and
other reproductive organs becomes an important tool in the analysis of the
reproductive conditions of an animal in its natural habitat. The aim of this study was
to describe the testicular morphology, morphometry and stereology of the interstitial
compartment in H. megacephalus.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2332
2015
MATERIAL AND METHODS
Animal capture and histological processing
The animals were captured in forest fragments in southwestern Goiás through
traditional methods using live animal traps, from April to August 2009 (IBAMA
License number 11621-1 and Ethics Committee Protocol 233/10 CEP/UFG).
Five adult animals were anesthetized by intraperitoneal injection of xylazine
(1mg/kg) and ketamine (15mg/kg) and killed with an overdose of the same
anesthetic. After euthanasia, the testes were removed and fixed in Karnovsky
solution, dehydrated and embedded in methacrylate. Two-micrometer sections from
each testis were stained with 1% toluidine blue/sodium borate. Images of the
testicular parenchyma were obtained using the photomicroscope Olympus AX-70
and the morphometric analyses were carried out using the Image Pro-Plus software.
Testis morphometry and stereology
The albuginea from one of the testes was removed and weighed and it was
subtracted of the testicular weight to calculate the weight of testicular parenchyma.
Since the testicular density is near 1, the weight was considered equal to that
testicular volume (COSTA et al., 2010).
The volume density of the tubular and interstitial compartments was estimated
by counting 2,500 points projected onto images captured in histological sections per
animal. In the interstitial compartment were considered points on nuclei and
cytoplasm of the Leydig cells, blood vessels, lymphatic space and connective tissue
(Figure 1).
The volume (mL) occupied by the tubular and interstitial compartments was
estimated considering the percentage of each compartment multiplied by the
testicular parenchyma volume. Choosing only spherical nuclei and evident nucleoli,
the average diameter of the Leydig cell nucleus was obtained from the assessment
of 50 cells per animal.
FIGURE 1 – Cross section of H. megacephalus testis (ST=
seminiferous tubules; IT= interstitial tissue; LC=
Leydig cell (cluster); LS= lymphatic space). Bar:
16µm.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2333
2015
The nuclear volume (µm3) was obtained from the knowledge of the mean
nuclear diameter, and the 4/3R3 formula was used (R=nuclear diameter/2). The
cytoplasmatic volume (µm3) was estimated multiplying the percentage of cytoplasm
by the nuclear volume, divided by the nuclear percentage. The single cell volume
(µm3) was estimated by the sum of the nuclear and cytoplasmatic volumes.
The number of Leydig cells per testis was calculated from the Leydig cell
individual volume and the total volume occupied by Leydig cells in the testicular
parenchyma. To obtain the number of Leydig cells per gram of testis, the number of
Leydig cells was divided by the gonadal weight. The Leydigosomatic index (LSI) was
estimated by dividing the volume of Leydig cells in the parenchyma by body weight
and multiplying the quotient by 100. This index quantifies the investment in Leydig
cells related to the mass of the body.
All data are reported as mean and standard deviation.
RESULTS AND DISCUSSION
The testicular morphometry, volume density and volume of the testicular
parenchyma components in H. megacephalus are presented in Table 1.
TABLE 1 - Morphometry and stereology of the testicular parenchyma
components in H. megacephalus (mean + standard deviation;
n=5).
Parameters
Body weight (g)
Mean + SD
47.84+8.7
Testicular weight (g)
0.195+0.089
Albuginea weight (g)
Parenchyma weight (g)
Volume density of seminiferous tubule (%)
0.006+0.001
0.189+0.087
94.46+1.74
Volume density of interstitial tissue (%)
Volume of seminiferous tubule (mL)
5.54+1.74
0.178+0.081
Volume of interstitial tissue (mL)
0.011+0.007
Volume density in testicular parenchyma (%)
Leydig cells
Blood vessel
3.21+2.04
0.97+0.46
Connective tissue
Lymphatic space
0.83+0.29
0.53+0.96
Volume in testicular parenchyma (mL)
Leydig Cells
0.006+ 0.006
Connective tissue
Blood vessel
0.002+ 0.001
0.002+ 0.001
Lymphatic space
0.001+ 0.001
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2334
2015
The organization of the testis in H. megacephalus was similar to that described
for most mammalian studied species (FRANÇA & RUSSELL, 1998). The proportion
between the tubular and interstitial compartments is variable, and constitutes one of
the main factors responsible for the differenciated efficiency in sperm production
observed in several species (BALARINI, 2013).
Approximately 5% of the testicular parenchyma of H. megacephalus is
constituted by interstitial tissue, which is a smaller percentage than observed in other
rodents like Cuniculus paca 9% (CARRETA JÚNIOR, 2012); Dasyprocta aguti
15.25% (ASSIS NETO et al., 2003); Hydrochoerus hydrochaeris 50% (PAULA et al.,
2002), and closer to the value reported for small rodent Necromys lasiurus 7%
(SILVA et al., 2010).
The interstitial compartment is composed of Leydig cells, blood and lymphatic
vessels, nerves, and a variable population of connective tissue (MORAIS et al.,
2013b). The interstitial compartment in H. megacephalus showed Leydig cells
grouped in clusters and also found blood vessels, as well as lymphatic space and
connective tissue (Figure 1).
The interstitial compartment presents great variation in volume and proportion
of its components in different species (FAWCETT et al., 1973, FRANÇA &
RUSSELL, 1998, COSTA et al., 2010, MORAIS et al., 2014), but usually the Leydig
cell is the most frequent component. In H. megacephalus, the Leydig cell was the
most abundant component in the interstitial compartment, followed by blood vessels,
connective tissue and lymphatic space (Table 1). This pattern was observed in other
wild rodents Akodon cursor, Akodon montensis, Necromys lasiurus and
Oligoryzomys nigripes (CORDEIRO JÚNIOR, 2009).
The patterns described by FAWCETT et al., (1973) indicated a great variation
related the amount of the Leydig cells and the disposition of the lymphatic vessels.
The rodents, in general, present the first pattern described by FAWCETT et al.,
(1973) where a small amount of Leydig cells and blood vessels are separated from
the seminiferous epithelium by a developed lymphatic space. In relation to
arrangements of the interstitial tissue components, the distribution in H.
megacephalus is similar to that observed in the first pattern described by Fawcett to
guinea-pigs and chinchillas, except for the amount of Leydig cells, making possible
the inclusion of this species into one more variation of the type I classification like
observed in capybara (PAULA et al., 2007).
The Leydig cells represent the endocrine portion of the testes. These cells are
responsible for the synthesis of testosterone which is involved in the the
spermatogenesis, development and maintenance of the secondary sex
characteristics (GILBERT, 2010). Testosterone is still related to the reproductive
behavior of the animals, such as libido, demarcation of territory and the status of a
male in a colony. The morphometry of the Leydig cell is shown in Table 2.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2335
2015
TABLE 2 - Morphometry of the Leydig cell in H. megacephalus (mean +
standard deviation; n=5).
Parameters
Mean + SD
Nuclear diameter (µm)
5.94+0.45
Nuclear percentage (%)
37.06+11.64
Cytoplasmatic percentage (%)
62.94+11.64
Nuclear volume (µm3)
111.18+23.66
Cytoplasmatic volume (µm3)
239.61+187.55
Leydig cell volume (µm3)
350.80+206.56
Number of Leydig cells per testis (x106)
9.73+8.65
Number of Leydig cells per gram of testis (x106)
89.58+46.77
Leydigosomatic index (%)
0.015+0.01
The Leydig cell average volume in H. megacephalus was 350.80 µm3, close to
the lowest values observed in several domestic, wild and laboratory species (3502.580 µm3;)(FRANÇA & RUSSELL, 1998, COSTA & PAULA, 2006, AZEVEDO et al.,
2010). Variations in the synthesis and secretion of testosterone depend more on the
individual capacity of this cell than on their total volume in the testes (EWING et al.,
1979). According to ZIRKIN et al. (1980), the productive capacity of the Leydig cell is
closely related to the amount of smooth endoplasmic reticulum in this cell.
The Leydig cell number per testis is converted to the Leydig cell number per
gram of testis to compare the different species independently of the size of the
testes. The numbers of Leydig cells per testis and per gram of testis in H.
megacephalus (9.73x106 and 89.58x106, respectively) were higher than observed in
other small rodents (5.2 and 19.2x106 A. cursor; 3.1 and 14.6 x106 A. montensis; 4.0
and 17.1x106 N. lasiurus; 1.9 and 33.2x106 O. nigripes;)(CORDEIRO JÚNIOR,
2009). The number of Leydig cells per gram of testis in H. megacephalus has one of
the largest values observed in domestic species, which varies from 20x106 to 87x106
cells (FRANÇA & RUSSELL, 1998).
Many factors can influence amount of Leydig cell per animal, such as the
number of LH receptors per cell, amount of LH available, amount of testosterone
secreted by the Leydig cell, the rate at which the testosterone leaves the testis, and
the testosterone metabolism rate (YE et al., 2011). The amount of Leydig cells varies
greatly between individuals and between mammalian species, and this variation was
not found for other endocrine glands (FAWCETT et al., 1973).
The Leydigosomatic index (ILS) is the percentage of body mass allocated in
Leydig cells, allowing comparison of individuals with different body sizes. The ILS in
the present study was 0.015%. This value was two times greater than observed in
Cuniculus paca (0.007%;)(CARRETA JÚNIOR, 2008) and three times greater than
observed in N. lasiurus (0.005%;)(SILVA & MELO, 2010), but lower than the values
reported for H. hydrochaeris (0.037%; COSTA et al., 2006), guinea pig, mice and rats
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2336
2015
(0.044, 0.030 and 0.055, respectively;)(RUSSELL, 1996).
According to EWING et al., (1979) the steroid production to regulate tissue
physiology and animal behavior in each species is more related to the individual
ability of Leydig cell to secrete such hormones than to the proportions, volume, and
population differences of these cells in testis.
CONCLUSIONS
This study showed that several morphometric parameters of interstitial tissue
evaluated in H. megacephalus are within the values described for most mammals.
H. megacephalus has one of the lowest values of Leydig cell volume than
observed for domestic, wild and laboratory species.
The number of Leydig cells per gram of testis in H. megacephalus has one of
the largest values observed in domestic species and in other small rodents.
ACKNOWLEDGMENTS
We thank the Biological Sciences students from the Federal University of Goiás
– Campus Jataí for the collection of animals, the Structural Biology Laboratory at the
Federal University of Viçosa for processing the biological material, and the
Foundation for Research Support from the State of Goiás (FAPEG) for the financial
support.
REFERENCES
ASSIS NETO, A. C.; MELO, M. I. V.; CARVALHO, M. A. M.; MIGLINO, M. A.;
OLIVEIRA, M. F.; MENEZES, D. J. A.; PAPA, P. C.; KFOURY-JÚNIOR, J. R. Análise
quantitativa do estabelecimento da espermatogênese em cutias (Dasyprocta aguti)
criadas em cativeiros. Brazilian Journal of Veterinary Research and Animal
Science, v.40, n.3, p. 175-179, 2003.
AZEVEDO, M. H. F.; PAULA, T. A. R.; FONSECA, S. L. P.; COSTA, C. C.; COSTA,
E. P.; PEIXOTO, J. V. Cell population indexes of spermatogenic yield and testicular
sperm reserves in adult jaguars (Panthera onca). Animal Reproduction Science. v.
118, p.83-88, 2010.
BALARINI, M. K. Avaliação morfofuncional do testículo e do processo
espermatogênico dos roedores silvestres Akodon cursor e Oligoryzomys
nigripes (RODENTIA: Cricetidae). Ph.D. Thesis. Universidade Federal de Viçosa.
81 p., 2013.
BONVINCINO, C. R.; OLIVEIRA, J. A.; D’ANDREA, P. S. Guia dos roedores do
Brasil, com chaves para gêneros baseadas em caracteres externos. Centro
Pan-Americano de Febre Aftosa - OPAS/OMS, Rio de Janeiro, Brasil, 2008.
CARMIGNOTTO, A. P.; VIVO, M.; LANGGUTH, A. Mammals of the Cerrado and
Caatinga: distribution patterns of the tropical open biomes of Central South
America. In: PATTERSON, B. D.; COSTA, L. P. (Eds.), Bones, Clones, and Biomes:
The history and Geography of Recent Neotropical Mammals v.1, University of
Chicago Press; Chicago, p. 307-350, 2012.
CARRETTA JÚNIOR, M. Estudo comparativo do processo espermatogênico e
duração do ciclo do epitélio seminífero através da técnica de
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2337
2015
imunohistoquímica com bromodeoxiuridina de três diferentes espécies de
roedores da subordem Hystricomorpha: cutia (Dasyprocta leporina), paca
(Cuniculus paca) e capivara (Hydrochoerus hydrochaeris). Ph.D. Thesis.
Universidade Federal de Viçosa. 131 p., 2012.
CORDEIRO JÚNIOR, D. A. Estrutura e função testiculares em Roedores
Silvestres das Famílias Echimyidae (Trinomys moojeni) e Cricetidae (Akodon
cursor, Akodon montensis, Necromys lasiurus e Oligoryzomys migripes), da
Reserva Particular do Patrimônio Natural do Caraça – MG. Ph.D. Thesis.
Universidade Federal de Minas Gerais. 109 p., 2009.
COSTA, D. S.; PAULA, T. A. R. 2006. Testosterone level, nasal gland volume and
Leydig cell morphometry in capybaras (Hydrochoerus hydrochaeris). Arquivo
Brasileiro de Medicina Veterinária e Zootecnia. v.58, n.6, p.1086-1091, 2006.
COSTA, G. M.; LEAL, M. C.; FERREIRA, A. C.; GUIMARÃES, D. A.; FRANÇA, L. R.
Duration of spermatogenesis and spermatogenic efficiency in two large neotropical
rodent species: the agouti (Dasyprocta leporina) and paca (Agouti paca). Journal of
Andrology, v.31, n.5, p. 489–499, 2010.
EWING, L. L.; ZIRKIN, B .R.; COCHRAN, R. C.; KROMANN, N.; PETERS, C.; RUIZBRAVO, N. Testosterone secretion by rat, rabbit, guinea pig, dog, and hamster
testes perfused in vitro: correlation with Leydig cell mass. Endocrinology, v.105, p.
1135-1142, 1979.
FAWCETT, D. W.; NEAVES, W. B.; FLORES, M. N. Comparative observations on
intertubular lymphatic and the organization of the interstitial tissue of the mammalian
testis. Biology of Reproduction, v. 9, p. 500-532, 1973.
FRANÇA, L. R.; RUSSELL, L. D. The testis of domestic animals. In: MARTINEZGARCIA, F.; REGADERA, J. (Eds.), Male Reproduction: A Multidisciplinary
Overview, Churchill Livingstone; Madrid, p. 197-219, 1998.
GILBERT, S.F. Developmental Biology. Massachusetts: Sinauer Associates. 711
p., 2010.
MORAIS, D. B.; CUPERTINO, M. C.; GOULART, L. S.; FREITAS, K. M.; FREITAS,
M. B. D.; PAULA, T. A. R.; MATTA, S. L. P. Histomorphometric evaluation of the
Molossus molossus (Chiroptera, Molossidae) testis: The tubular compartment and
indices of sperm production. Animal Reproduction Science, v. 140, p. 268–278,
2013a.
MORAIS, D. B.; OLIVEIRA, L. C.; CUPERTINO, M. C.; FREITAS, K. M.; FREITAS,
M. B. D.; PAULA, T. A. R.; MATTA, S. L. P. Organization and seasonal quantification
of the intertubular compartment in the bat Molossus molossus (Pallas, 1776) testis.
Microscopy Research and Technique, v. 76, n. 1, p. 94-101, 2013b.
MORAIS, D. B.; BARROS, M. S.; FREITAS, M. B. D.; PAULA, T. A. R.; MATTA, S. L.
P. Histomorphometric characterization of the intertubular compartment in the testes
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2338
2015
of the bat Sturnira lilium. Animal Reproduction Science, v. 147, p. 180-186, 2014.
PAULA, T. A. R.; COSTA, D. S.; MATTA, S. L. P. Avaliação histológica quantitativa
do testículo de capivaras (Hydrochoerus hydrochaeris) adultas. Bioscience Journal,
v. 18, p. 121–136, 2002.
PAULA, T. A. R.; MATTA, S. L. P.; SILVA JR, V. A.; COSTA, D. S.; FONSECA, C.
C.; NEVES, M. T. D. Intertubular space characterization in adult capybara
(Hydrochoerus hydrochaeris) testis. Brazilian Archives of Biology and
Technology, v. 50, n. 2, p. 289-297, 2007.
RUSSELL, L. D. Mammalian Leydig cell structure. In: PAYNE, A. H.; HARDY, M.
P.; RUSSELL, L. D. (Eds.), The Leydig Cell, Cache River Press, Vienna, p. 43-96,
1996.
SILVA, K. C. S.; MELO, F. C. S. A. Morfologia e morfometria testicular de
Necromys lasiurus (Cricetidae, Sigmodontinae) em área de Cerrado no
Sudoeste goiano. In: Congresso de Pesquisa, Ensino e Extensão da Universidade
Federal de Goiás, Goiânia, GO, 2010.
SILVA, K. C. S.; SOUSA, T. P.; MILEN, L. C.; MELO, F. C. S. A.; MATTA, S. L. P.;
MELO, F. R.; SANTA RITA, R. M.; OLIVEIRA, F. C. G. Testicular morphometry of
the wild Necromys lasiurus (Rodentia: Cricetidae). In: Meeting of the Brazilian
Society for Cell Biology, São Paulo, SP, 2010.
ZIRKIN, B. R.; EWING, L. L.; KROMANN, N.; COCHRAN, R. C. Testosterone
secretion by rat, rabbit, guinea pig, dog, and hamster testes perfused in vitro:
correlation with Leydig cell ultrastructure. Endocrinology, v. 107, p. 1867-1874,
1980.
YE, L.; SU, Z. J.; GE, R. S. Inhibitors of testosterone biosynthetic and metabolic
activation enzymes. Molecules, v. 16, n. 12, p. 9983-10001, 2011.
ENCICLOPÉDIA BIOSFERA, Centro Científico Conhecer - Goiânia, v.11 n.21; p. 2339
2015