Morphometry of testis and seminiferous tubules of the adult crab-eating

Os parâmetros de biometria corporal e testicular são de grande importância no estabelecimento dos padrões reprodutivos e, consequentemente, no desenvolvimento de protocolos para a reprodução assistida nas diferentes espécies. Uma correlação direta entre o peso do testículo e a população espermática é observada nas diferentes espécies já estudadas, uma vez que o tamanho do testículo reflete principalmente o volume total do túbulo seminífero, seu principal componente. O presente trabalho tem como objetivos definir parâmetros volumétricos testiculares e correlacionar os dados de morfometria testicular e do túbulo seminífero com a massa corporal em seis cachorros-domato adultos. O peso corporal médio dos cachorros-do-mato foi de 6,53 Kg, dos quais cerca de 0,068% são alocados em massa testicular e 0,042% especificamente em túbulos seminíferos, que representam 87,5% do parênquima testicular. A albugínea representa 12,5% da massa testicular. O diâmetro médio dos túbulos seminíferos em cachorro-do-mato foi de 236 μm, e a espessura média do epitélio seminífero foi de 62,9 μm, sendo encontrados em média 18 metros de túbulo seminífero por grama de testículo. Os valores para os parâmetros tubulares indicam uma produtividade espermática próxima àquelas observadas nos carnívoros já estudados.


INTRODUCTION
The crab-eating fox (Cerdocyon thous Linnaeus, 1766), also known as the forest fox, common fox and the common zorro, and, in Brazil, as cachorro-do-mato, raposacaranguejeira, lobinho, and graxaim-do-mato, is relatively common in Colombia, Venezuela, Guyana, Surinam, eastern Peru, eastern Bolivia, Paraguay, Uruguay, northern Argentina and in most habitats of Brazil (Fowler, 2001).The species occupies mainly the biomes Cerrado, Caatinga, Pantanal, Atlantic and Southern Plains, using forest edges, and altered areas inhabited by humans (Fonseca et al., 1996).The abundance of the crab-eating fox is due in part to its eating habit.It is an omnivorous species whose diet includes everything from insects, rodents, reptiles, birds, river crabs, eggs and even fruit (Bisbal & Ojasti, 1980).Thanks to the high consumption of fruits, this species can act as seed dispersers (Rocha et al., 2004).
The crab-eating fox is listed in Appendix II of the Conservation on International Trade in Endangered Species (CITES, 2007).It has a high occurrence in nature and is assigned at the Least Concern category regarding the extinction risk.However, according to Vieira (1996), death by road accidents of crab-eating fox individuals occurs in a considerable amount and may contribute to large impacts on populations of this species.
With regard to reproductive characteristics, a positive correlation has been reported between testicular weight and sperm production (Amann, 1970;Olar et al., 1983;França & Russell, 1998).However, testicular size is proportional to body size, i.e., somatic investment in gonadal mass is greater in small animals compared with larger ones (Kenagy & Trombulak, 1986).The amount of sperm produced is always much larger than the number required for fertilization, and the fact that the testis size is not, necessarily, proportional to the body size contributes to energy saving, since large animals would produce excessive high amounts of sperm (Paula, 1999).
According to Kenagy & Trombulak (1986), the relative testis size can provide information on the reproductive physiology and even on the mating system.Thus, testicular size is directly related to reproductive behavior, as monogamous and polygamous animals have lower gonadosomatic index (GSI) compared with species with promiscuous or polyandrous mating.In the first case, the small testicular investment reflects the formation of pairs or harems where one male is responsible for copulations.Among promiscuous or polyandrous animals, several males have access to copulation with the same female, being better able to paternity the animals with high sperm production and, hence, increased testicular size.
Seminiferous tubules are usually the most abundant components of the testicular parenchyma.All quantitative parameters related to seminiferous tubules, such as tubular diameter, thickness of the seminiferous epithelium and the total length per gram of testis have a positive relationship with spermatogenic activity (França & Russell, 1998;Paula, 1999).Tubular measurement is, thus, the approach traditionally used as indicator of spermatogenic activity in investigations of testicular function (Navarro et al., 2004;Souza et al., 2005;Silva Jr et al., 2006;Mascarenhas et al., 2006).
The study of testicular morphophysiology and body biometry in wild animals allows the establishment of physiological and behavioral patterns important to understand aspects of the reproductive biology of different species, which make it possible to develop protocols for assisted reproduction.In this context, the objective of this work was to determine testicular volume parameters such as gonadosomatic index and volume of testicular parenchyma, compare the GSI with the tubulossomatic index (STI) between monogamous or polygamous species and promiscuous or polyandrous species, as well as to evaluate the seminiferous tubule diameter, the thickness of the seminiferous epithelium, seminiferous tubule length per gram of testis and the total length of seminiferous tubule in six adult crab-eating foxes .

MATERIAL AND METHODS
Testicular fragments were collected from six adult crabeating foxes (Cerdocyon thous Linnaeus, 1766).These animals are routinely found hit by motor vehicles on highways in the vicinities of Viçosa (state of Minas Gerais, Brazil, 20º45' S, 42º51' W, 693 m.a.s.l.) and, some are taken to the Screening Center for Wild Animals, Federal University of Viçosa (UFV-CETAS).The animals who survive the trauma of being hit are rehabilitated and released, when possible, near the areas of occurrence.The procedures adopted in the methodology of this study were approved by the Ethics Committee of the Veterinary Department, Federal University of Viçosa-UFV, process no.65/2007.
As we are dealing with animals of the Brazilian fauna, the experiment was conducted under permission of the Brazilian Institute of Environment and Natural Resources (IBAMA) number 11373-1.
Two animals were anesthetized using darts containing 10 mg/kg tiletamine-zolazepam.The whole procedure was monitored by assessing animals' temperature, respiratory movements and heart rate every 10 minutes.The animals were weighed and it was taken the measures of width, thickness and length of the testis using a digital caliper.
The gonadosomatic index (GSI) was calculated using of the weight of both testis as a proportion of the total body weight, which represents the percentage of body mass allocated to the testis.
After trichotomy and local antisepsis, the scrotal skin was incised using a 4mm diameter circular scalpel and a fragment was excised from the mid-region of the right or left testicle.The incision was sutured in separate layers with absorbable synthetic thread, using buried intradermal suture on the skin.Each animal received antiphlogistic agents and antibiotics in the postoperative treatment.
The animals that died during hospitalization (n = 4) were weighed and had their testis removed.The excised testis were measured following the previously described methodology and fragments were collected from their midregion.
The samples were immediately fixed in Karnovsky's fixative (4% paraformaldehyde and 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) at room temperature for at least two hours, and subsequently stored in the same buffer under refrigeration.
For light microscope studies, fragments were dehydrated in an ethanol series of increasing concentration (70, 80, 90 and 100% GL) and then embedded in plastic resin (Leica Historesin®).The fragments were positioned to allow cross sections of the testicular albuginea.Sequential histological sections (3-μm thick) were obtained using a rotary microtome (Leica RM2155®) with a glass knife and stained with 1% toluidine blue/ sodium borate, for one minute.
The mean thickness of the testicular albuginea was determined in 20 cross sections of each animal, using a light microscope in combination with an ocular micrometer (20-fold magnification).The volume of the testicular albuginea was estimated by subtracting the volume of testicular parenchyma from the total testicular volume.The volume of testicular parenchyma was also calculated using the volume formula for an ellipse, by subtracting the testicular size from the thickness of the albuginea.When possible, the testicular albuginea was dissected and its thickness measured, using a digital caliper with resolution of 0.01mm.The data showed no significant difference from values obtained by a light microscope.
Ten random histological images obtained by photomicroscope (200-fold magnification) were used to calculate the volume proportion occupied by seminiferous tubules and the intertubular space.A graticule with 400 intersections was superimposed on the images.The intersections overlying the seminiferous tubule and intertubular space were counted.Using the volume proportion of the seminiferous tubule and intertubular space, we calculated the total volume of these testicular components.
The mean diameter of the seminiferous tubules was estimated by measuring 20 tubule cross sections, made as circular as possible, of each animal.Measurements were taken using an ocular micrometer in a light microscope and a 20X objective lens.In the same sections, we also measured the height of the seminiferous epithelium, from the basal membrane to the luminal edge.The length of the seminiferous tubule was calculated using the volume formula for a cylinder, where the length is the volume divided by the base area.Thus, the total volume of seminiferous tubules divided by the area of the cross section corresponds to the total length of seminiferous tubules per testis, which can also be converted to meters per gram of testis.
The tubulossomatic index (ITS) represents the percentage of body weight allocated to the seminiferous tubules.It was calculated by inferring to the gonadosomatic index (GSI) the percentage of the testicular parenchyma occupied by seminiferous tubules.
Data were analyzed by descriptive statistics, where mean, standard deviation and coefficient of variation were calculated by using the statistical functions of Microsoft Office Excel® 2003.

RESULTS AND DISCUSSION
The mean body weight of the studied animals was 6.53 kg (Table 1), within the range considered normal for the species (5 to 8 kg) (Brady, 1979).The testicular weight was estimated from the testis dimensions, once for this work samples were taken by testicular biopsy.Testicular biopsy is the elective procedure to study reproductive morphophysiology in wild animals (Azevedo et al., 2006;Guião-Leite et al., 2006;Sarti, 2006), as it is a conservative method that maintains the reproductive capacity, showing no harmful effects on sperm production in the long term (Lopate et al., 1989;Threlfall & Lopate, 1993;Attia et al., 2000).Unilateral testicular biopsy in dogs of undetermined breed (mongrel dog) showed no changes in the cell population, the intrinsic efficiency of the spermatogenic process and the Sertoli cell index, either in the biopsied testis or in the contralateral testis, after a period of 60 days (Mascarenhas et al., 2006).
According Kenagy & Trombulak (1986), the largest allocation to testicular mass is found in smaller animals, compared with animals of large body mass.This explains the close similarity of GSI of crab-eating fox (0.068%, Table 1) and domestic cats (0.07%, Godinho, 1999), which are higher than GSI of larger animals such as the maned wolf (0.04%, Bittencourt et al., 2007), the jaguar (0.034%, Azevedo et al., 2006), the African lion (0.015%, Barros, 2005) and the puma (0.03%, Guião-Leite & Paula, 2003).Kenagy & Trombulak (1986) also discussed that the relative testis size reflects the mating system of a given species, i.e., the testis size is directly related to the reproductive behavior.Monogamous and polygamous animals have lower GSI in relation to promiscuous or polyandrous species.In the former case (monogamy and polygamy), the male competition for the production of genetic offspring is by access to females, whereas for promiscuous and polyandrous species, the male competition for production of offspring occurs within the female genital tract and, thus, males with higher sperm production and consequently, with larger relative testis size are better able to paternity.The crab-eating fox is a monogamous species in which mating can occur at any time of the year (Cheida et al., 2006).Hence, it is expected that the GSI of crab-eating fox is lower than species of polyandrous or promiscuous behavior, as is the case of the domestic dog, which although showing mean body size higher than the crab-eating fox, it has higher IGS (0.1%, Mascarenhas et al., 2006).This also seems to be the case observed in ocelots that, even having twice the body weight recorded for the crab-eating fox, it shows a much higher GSI (0.12%, Sarti, 2006).However, further ethological studies on this species are needed to validate this statement.
In most domestic species, the volume proportion of the albuginea and mediastinum testis is usually around 10% (França & Russell, 1998).The animals evalueted in this study had mean volume proportion of testicular albuginea of 12.5% (Table 2), very close to that recorded for maned wolves (11%, Bittencourt, 2003).But other studied carnivores show a higher proportion of testicular albuginea, domestic cats, for instance, show values near 18% (Godinho, 1999).Mascarenhas et al. (2006) reported similar values for dogs of undetermined breed and Barros (2005) for African lions, whereas the ocelot has the highest proportion of albuginea among carnivore studied species, with an average of 23.42% (Sarti, 2006).
Although testicular parenchyma is not an entirely appropriate term to name the testis devoid of albuginea and mediastinum, it is widely mentioned in the literature (Berndtson, 1977;Johnson et al., 1981;Russell et al., 1990a;Johnson et al., 2000).The testicular parenchyma can be divided into two compartments: tubular and intertubular (Amann, 1970;Fawcett et al., 1973;Amann & Schanbacher, 1983;Russell et al., 1990b).The intertubular compartment consists of Leydig cells, blood vessels, lymph vessels, nerves and a varied population of cells that includes fibroblasts, macrophages and mast cells (Russell, 1996).The crab-eating fox allocates 12.7% of the testicular parenchyma to the intertubular tissue (Table 3), lower than the values recorded for the ocelot (19.39%;Parameter Sarti, 2006), the maned wolf (19.97%;Bittencourt et al., 2007), the puma (21.88%;Guião-Leite et al., 2006), the jaguar (22.7%;Azevedo et al., 2006) and the African lion (24.3%;Barros, 2005).The tubular compartment is the main component of the testis in most mammals, with great influence on the testicular weight and sperm production (Amann, 1970;França & Russell, 1998;Paula et al., 2007).Except for the low values reported for the marmot and the capybara (Russell et al., 1990b;Paula et al., 2007), the majority of animals investigated had between 70 and 90% of seminiferous tubules in the testicular parenchyma (França & Russell, 1998 ).The mean volume of seminiferous tubules observed in the crab-eating fox was 3.23 mL in both testis, representing on average 87.5% of the testicular parenchyma (Table 3), which is close to 90% reported for the domestic cat (Godinho, 1999), but higher than the values reported for the ocelot (80.58%;Sarti, 2006), the maned wolf, (79.3%;Bittencourt et al., 2007), the puma (78%; Guião-Leite et al., 2006), the jaguar (77.7%;Azevedo et al., 2006) and the African lion (75.6%;Barros, 2005).Tubulossomatic Index (STI) is a parameter used to quantify the investment in seminiferous tubules in relation to body mass.This parameter allows for more discussion on the influence of reproductive behavior on the testicular morphology.Thus, species known to be monogamous or polygamous, such as the maned wolf and the large cats, show less tubulossomatic investment than promiscuous or polyandrous species, such as domestic dogs and cats (Table 4).The crab-eating fox allocates on average 0.042% of body weight to seminiferous tubules (Table 4), this value, as expected, is closer to those of species with monogamous and polygamous behavior than to promiscuous and polyandrous species (Table 4).The ocelot has the highest STI among wild carnivores (mean of 0.074%; Sarti, 2006), which is higher than the value found for the domestic cat (0.063%; Godinho, 1999).This demonstrates a greater investment in sperm production than expected for an animal of its size and points to the need for further studies on their reproductive behavior.

Table 1 .
Body weight, right testis weight, left testis weight, weight of both testis and gonadosomatic index of the crab-eating fox b Coefficient of Variation.

Table 2 .
Volume of testicular parenchyma, albuginea thickness, albuginea volume and albuginea of the adult crab-eating fox

Table 3 .
Volume proportion of seminiferous tubules, volume proportion of intertubular tissue, total volume of the seminiferous tubule and total volume of the intertubular tissue in crab-eating fox

Table 5 .
Seminiferous tubule diameter, thickness of the seminiferous epithelium, tubular length per gram of testis and total length of the seminiferous tubule in the crab-eating fox