• View in gallery

    Micrographs of testicular structures of chronically Trypanosoma cruzi-infected dogs. (A) Seminiferous tubules (H & E × 400), (B) epididymal tubes filled with sperm (H & E × 100). Four hundred fields per dog were analyzed.

  • 1.

    OPS/OMS, 2005. Plan Estratégico Nacional de Chagas 2003–2007. Honduras: Enfermedad de Chagas: Modelo de la Armonización de la Cooperación Interagencial, 535.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization, 2012. Global polio eradication initiative: 7th meeting of the Independent Monitoring Board. Wkly Epidemiol Rec 87: 509526.

    • Search Google Scholar
    • Export Citation
  • 3.

    Sosa-Jurado F, Zumaquero-Rios JL, Reyes PA, Cruz-Garcia A, Guzman-Bracho C, Monteón VM, 2004. Factores bióticos y abióticos que determinan la seroprevalencia de anticuerpos contra Trypanosom acruzi en el municipio de Palmar de Bravo, Puebla, México. Salud Publica Mex 46: 3948.

    • Search Google Scholar
    • Export Citation
  • 4.

    Estrada-Franco JG, Bhatia V, Diaz-Albiter H, Ochoa-Garcia L, Barbabosa A, Vazquez-Chagoyan JC, Martinez-Perez MA, Guzman-Bracho C, Garg N, 2006. Human Trypanosoma cruzi infection and seropositivity in dogs, Mexico. Emerg Infect Dis 12: 624630.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jiménez-Coello M, Ortega-Pacheco A, Guzmán-Marin E, Guiris-Andrade DM, Martínez-Figueroa L, Acosta-Viana KY, 2010. Stray dogs as reservoirs of the zoonotic agents Leptospira interrogans, Trypanosoma cruzi and Aspergillus spp. in an urban area of Chiapas in southern México. Vector Borne Zoonotic Dis 10: 135141.

    • Search Google Scholar
    • Export Citation
  • 6.

    Balán LU, Yerbes IM, Piña MA, Balmes J, Pascual A, Hernández O, López R, Monteón V, 2011. Higher seroprevalence of Trypanosoma cruzi infection in dogs than in humans in an urban area of Campeche, México. Vector Borne Zoonotic Dis 11: 483484.

    • Search Google Scholar
    • Export Citation
  • 7.

    Ikede BO, 1979. Genital lesions in experimental chronic Trypanosoma brucei infection in rams. Res Vet Sci 26: 145151.

  • 8.

    Anosa VO, Isoun TT, 1980. Further observations on the testicular pathology in Trypanosoma vivax infection of sheep and goats. Res Vet Sci 28: 151160.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ikede BO, Akpavie SO, 1982. Delay in resolution of trypanosome-induced genital lesions in male rabbits infected with Trypanosoma brucei and treated with diminazene aceturate. Res Vet Sci 32: 374376.

    • Search Google Scholar
    • Export Citation
  • 10.

    Anosa VO, Kaneko JJ, 1984. Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus). Light and electron microscopic study of testicular lesions. Rev Vet Pathol 21: 238246.

    • Search Google Scholar
    • Export Citation
  • 11.

    Lamano-Carvalho TL, Ribeiro RD, Lopes RA, 1991. The male reproductive organs in experimental Chagas' disease. I. Morphometric study of the vas deferens in the acute phase of the disease. Exp Pathol 41: 203214.

    • Search Google Scholar
    • Export Citation
  • 12.

    Boly H, Humblot P, Tillet Y, Thibier M, 1994. Effects of Trypanosoma congolense infection on the pituitary gland of Baoulé bulls: immunohistochemistry of LH- and FSH-secreting cells and response of plasma LH and testosterone to combined dexamethasone and GnRH treatment. J Reprod Fertil 100: 157162.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lenzi HL, Castelo-Branco MT, Pelajo-Machado M, Oliveira DN, Gattass CR, 1998. Trypanosoma cruzi: compromise of reproductive system in acute murine infection. Acta Trop 71: 117129.

    • Search Google Scholar
    • Export Citation
  • 14.

    Herrera L, Urdaneta-Morales S, 2001. Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice. Mem Inst Oswaldo Cruz 96: 713717.

    • Search Google Scholar
    • Export Citation
  • 15.

    Diniz SA, Melo MS, Borges AM, Bueno R, Reis BP, Tafuri WL, Nascimento EF, Santos RL, 2005. Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs. Vet Pathol 42: 650658.

    • Search Google Scholar
    • Export Citation
  • 16.

    Adamu S, Fatihu MY, Useh NM, Mamman M, Sekoni VO, Esievo KA, 2007. Sequential testicular and epididymal damage in Zebu bulls experimentally infected with Trypanosoma vivax. Vet Parasitol 143: 2934.

    • Search Google Scholar
    • Export Citation
  • 17.

    Carvalho LO, Abreu-Silva AL, Hardoim D de J, Tedesco RC, Mendes VG, da Costa SC, Calabrese KS, 2009. Trypanosoma cruzi and myoid cells from seminiferous tubules: interaction and relation with fibrous components of extracellular matrix in experimental Chagas' disease. Int J Exp Pathol 90: 5257.

    • Search Google Scholar
    • Export Citation
  • 18.

    Raheem AK, Fayemi EO, Leigh OO, Ameen SA, 2009. Selected fertility parameters of West African dwarf bucks experimentally infected with Trypanosoma congolense. Folia Vet 53: 6871.

    • Search Google Scholar
    • Export Citation
  • 19.

    Silva FL, Oliveira RG, Silva TM, Xavier MN, Nascimento EF, Santos RL, 2009. Venereal transmission of canine visceral leishmaniasis. Vet Parasitol 160: 5559.

    • Search Google Scholar
    • Export Citation
  • 20.

    Assis VP, Ribeiro VM, Rachid MA, Castro AC, Valle GR, 2010. Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy. Anim Reprod Sci 117: 183186.

    • Search Google Scholar
    • Export Citation
  • 21.

    Leigh OO, Fayemi OE, 2010. Ejaculate characteristics of rabbits infected with Trypanosoma congolense and changes caused offer treatment with Diminazene aceturate (Diminaveto). Rev Int J Morphol 28: 471475.

    • Search Google Scholar
    • Export Citation
  • 22.

    Mbaya AW, Nwosu CO, Kumshe HA, 2011. Genital lesions in male red fronted gazelles (Gazella rufifrons) experimentally infected with Trypanosoma brucei and effect of melarsamine hydrochloride (Cymelarsan) and diminazene aceturate (Berenil) in its treatment. Theriogenology 76: 721728.

    • Search Google Scholar
    • Export Citation
  • 23.

    Manna L, Paciello O, Morte RD, Gravino AE, 2012. Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report. Parasites & Vectors 5: 216.

    • Search Google Scholar
    • Export Citation
  • 24.

    Kegler K, Habierski A, Hahn K, Amarilla SP, Seehusen F, Baumgärtner, 2013. Vaginal canine transmissible venereal tumor associated with intra-tumoral Leishmania spp. amastigotes in an asymptomatic female dog. J Comp Path 149: 156161.

    • Search Google Scholar
    • Export Citation
  • 25.

    Monteón VM, Furuzawa-Caballeda J, Alejandre-Aguilar R, Aranda-Fraustro A, Rosales Encina JL, Reyes PA, 1996. American trypanosomosis: in situ and generalized features of parasitism and inflammation kinetics in a murine model. Exp Parasitol 83: 267274.

    • Search Google Scholar
    • Export Citation
  • 26.

    Espinoza B, Solorzano-Domínguez N, Vizcaino-Castillo A, Martínez I, Elias-López AL, Rodríguez-Martínez JA, 2011. Gastrointestinal infection with Mexican TcI Trypanosoma cruzi strains: different degrees of colonization and diverse immune responses. Int J Biol Sci 7: 13571370.

    • Search Google Scholar
    • Export Citation
  • 27.

    WHO Technical Report Series, 2002. Control of Chagas Disease. Second report of the WHO expert committee. Geneva: World Health Organization, 1109.

    • Search Google Scholar
    • Export Citation
  • 28.

    Rodríguez-Morales O, Ballinas-Verdugo MA, Alejandre-Aguilar R, Reyes-López PA, Arce-Fonseca M, 2011. Trypanosoma cruzi connatal transmission in dogs with Chagas disease. Experimental case report. Vector Borne Zoonotic Dis 11: 13651370.

    • Search Google Scholar
    • Export Citation
  • 29.

    Norma Oficial Mexicana NOM-0062-ZOO-1999, 1999. Especificaciones Técnicas para el Cuidado y Uso de Animales de Laboratorio. Mexico: Diario Oficial de la Federación, 158.

    • Search Google Scholar
    • Export Citation
  • 30.

    Linde-Forsberg C, 1991. Achieving canine pregnancy using frozen or chilled extended semen. Vet Clin North Am Small Anim Pract 21: 467485.

    • Search Google Scholar
    • Export Citation
  • 31.

    Dostal LA, Juneau P, Rothwell CE, 2001. Repeated analysis of semen parameters in Beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin. Toxicol Sci 61: 128134.

    • Search Google Scholar
    • Export Citation
  • 32.

    Gutiérrez Y, Hernández J, Fernández F, Cruz H, 2004. Congelación de espermatozoides obtenidos de colas de epidídimos de bovinos. Rev Salud Anim 26: 192196.

    • Search Google Scholar
    • Export Citation
  • 33.

    Rodríguez-Morales O, Pérez-Leyva MM, Ballinas-Verdugo MA, Carrillo-Sánchez SC, Rosales-Encina JL, Alejandre-Aguilar R, Reyes PA, Arce-Fonseca M, 2012. Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model. Vet Res 43: 79.

    • Search Google Scholar
    • Export Citation
  • 34.

    Rodríguez-Morales O, Carrillo-Sánchez SC, García-Mendoza H, Aranda-Fraustro A, Ballinas-Verdugo MA, Alejandre-Aguilar R, Rosales-Encina JL, Vallejo M, Arce-Fonseca M, 2013. Effect of the plasmid-DNA vaccination on macroscopic and microscopic damage caused by the experimental chronic Trypanosoma cruzi infection in the canine model. Biomed Res Int 2013: 826570.

    • Search Google Scholar
    • Export Citation
  • 35.

    Olar TT, Amann RP, Pickett BW, 1983. Relationships among testicular size, daily production and output of spermatozoa, and extragonadal spermatozoa reserves of the dog. Biol Reprod 29: 11141120.

    • Search Google Scholar
    • Export Citation
  • 36.

    Souza FF, Leme DP, Uechi E, Trinca LA, Lopes MD, 2004. Evaluation testicular fine needle aspiration cytology and serum testosterone levels in dogs. Braz J Vet Res Anim Sci 41: 98105.

    • Search Google Scholar
    • Export Citation
  • 37.

    Reyes LA, Segovia FJ, Cerpa RE, 2011. Volumen testicular: el tamaño sí importa. (Testicular volumen: size does matter). Rev Colomb Radiol 22: 33483351.

    • Search Google Scholar
    • Export Citation
  • 38.

    England GC, 1991. Relationship between ultrasonographic appearance, testicular size, spermatozoal output and testicular lesions in the dog. J Small Anim Pract 32: 306311.

    • Search Google Scholar
    • Export Citation
  • 39.

    Cortez AA, Aquino-Cortez A, Silva AR, Cardoso Rc S, Silva LD, 2002. Relação entre perímetro escrotal e concentração espermática emcães, clinicamentenormais, da raça Pastor Alemão. Arq Bras Med Vet Zootec 54: 549550.

    • Search Google Scholar
    • Export Citation
  • 40.

    Corti L, 2003. Evaluación de la capacidad fecundante del semen congelado del perro (Canis familiaris), en ova recuperadas de perras de celo inducido. Bachelor Thesis for Doctor in Veterinary Medicine. Facultad de Ciencias Veterinarias de Chile, 171.

    • Search Google Scholar
    • Export Citation
  • 41.

    Kawakami E, Hori T, Tsutsui T, 1998. Changes in semen quality and in vitro sperm capacitation during various frequencies of semen collection in dogs with both asthenozoospermia and teratozoospermia. J Vet Med Sci 60: 607614.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Sperm Morphological Features Associated with Chronic Chagas Disease in the Semen of Experimentally Infected Dogs

View More View Less
  • Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez,” Mexico City, Mexico; Department of Agricultural and Animal Production, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma Metropolitana Unidad Xochimilco, Mexico City, Mexico; Department of Parasitology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico; Department of Pathological Anatomy, Instituto Nacional de Cardiología “Ignacio Chávez,” Mexico City, Mexico

The presence of trypanosomatids in the reproductive systems of different mammals (causing genital lesions in the acute stage of the disease) may predispose the animals to low semen quality. However, there are no studies examining the alterations in the sperm morphological features in the chronic stage of Trypanosoma cruzi infection. Knowledge of these aspects is important to understand the other ways of transmission of the Chagas disease. Progressive motility, mass motility, concentration, and sperm morphology of 84 ejaculates of dogs that were chronically infected with T. cruzi were evaluated. Most of the findings were consistent with the reference values and with those obtained from healthy control dogs. The scrotal circumference was not correlated with spermatozoa concentration in the infected animals. In conclusion, the T. cruzi Ninoa (MHOM/MX/1994/Ninoa) strain does not cause significant alterations in the semen quality of dogs experiencing chronic Chagas disease (at concentrations of 5 × 104 to 1 × 106 parasites per animal).

Introduction

Chagas disease (CD) is caused by Trypanosoma cruzi (T. cruzi), a hemoflagellate protozoan. Although CD was a Latin America-endemic disease at first, in recent decades, this disease has been observed most often in the United States, Canada, many European countries, and others in the western Pacific. This spread is observed mainly because of population mobility between Latin America and the rest of the world. The CD is now considered to be the sixth leading cause of death among Latin American adults. Treatment is usually successful when the infection is detected in the acute stage of the disease or when the patient suffering from chronic CD is < 15 years of age. The 30% of the people who are in the chronic stage develop cardiac damage and die.1 Globally, ~10 million people are infected, and more than 25 million are at risk of acquiring the disease.2 A wide variety of mammalian hosts can also be infected and/or act as wild or domestic reservoirs. In Mexico, the seroprevalence in the dog population ranges between 1.6% and 21% depending on the geographical region of the country. Estado de México, Puebla, Chiapas, Morelos, Campeche, and Yucatán are the only states (6 of 32) where dogs infected with T. cruzi have been reported.36 Therefore, a notably large number of dogs act as reservoirs and as sources of bug blood meals, thereby creating a public health problem.

Trypanosoma cruzi is characterized by chronic chagasic cardiomyopathy and digestive and nervous manifestations. Although sexual transmission has been suggested since the discovery of Chagas disease, it has not been shown to date. The presence of trypanosomatids, such as Trypanosoma vivax, Trypanosoma brucei, Trypanosoma congolense, T. cruzi, and Leishmania sp., has been reported in the reproductive systems of a wide variety of mammals. Many authors have shown inflammation and parasite colonization in the genital tissues of such animals. These researchers have also reported the shedding of parasites in the semen accompanied by several reproductive disorders, such as low semen quality, reduced libido, testicular degeneration, impaired spermatogenesis, and alterations in the extracellular matrix of the lamina propria of the seminiferous tubules.724 The presence of trypanosomatids in the semen of infected dogs indicates the possibility of venereal transmission of CD. Therefore, the use of chagasic dogs as sires should be avoided. The studies of semen quality or damage in the reproductive organs of individuals with CD will help to evaluate the hypothesis that CD affects sexual transmission. This can provide important epidemiological data to increase our knowledge about the natural history of the disease. The aim of this study was to evaluate the semen quality of chronically T. cruzi infected dogs.

Materials and Methods

In this study, eight male dogs (5 Beagle and 3 mongrel), aged 2.5 (± 0.35) years, weighing 13.3 (± 6.2) kg were used. The animals were inoculated with a well-characterized Mexican T. cruzi Ninoa strain25,26 (MHOM/MX/1994/Ninoa [T. cruzi I])27 by intraperitoneal injection at different times and with different doses of infection (Table 1). The establishment of acute infection was evaluated by examining freshly isolated blood samples that were collected every third day. Two hundred to 400 parasites/mL were observed (as the limits of detection) between day 21 and day 50 post infection only in those animals that were infected with doses of 2 × 105, 5 × 105, and 1 × 106 parasites per animal (Table 2). All of the dogs were monitored clinically with general physical examinations and electrocardiographic studies. The infected animals were serologically positive for T. cruzi based on standardized enzyme-linked immunosorbent assays and indirect immunofluorescence that were performed using methods described previously.28

Table 1

Physical features, times, and doses of infection of chronic chagasic experimental dogs

Dog no.BreedAge (months)Weight (kg)Time of infection (months)Metacyclic trypomastigotes* by intraperitoneal injection (mt/IP)
1Beagle3011.0Control healthy
2Beagle2412.6Control healthy
3Beagle2615.0241 × 106
4Beagle2615.0245 × 105
5Beagle2610.0245 × 105
6Mongrel3427.0322 × 105
7Mongrel347.6325 × 104
8Mongrel348.0325 × 104

The Trypanosoma cruzi Ninoa (MHOM/MX/1994/Ninoa) strain that was used was maintained by serial passage in reduviid vectors. Metacyclic trypomastigotes were obtained from urine and feces of triatomines and resuspended in physiological saline solution.

Table 2

Parasitemia detection in experimentally infected dogs with Trypanosoma cruzi

Dog no.Days after inoculation
10–1516–2021–2526–3031–3536–4041–4546–50
1 (Beagle)        
2 (Beagle)        
3 (Beagle)  ++     
4 (Beagle)   + +++++
5 (Mongrel)    +++  
6 (Mongrel)    ++++
7 (Mongrel)   NDNDNDNDND
8 (Mongrel)   NDNDNDNDND

Detection of 1 or 2 parasites per blood sample of fresh drop examination.

ND = not determined.

The animal handling followed the established guidelines of the International Guiding Principles for Biomedical Research involving Animals and the Norma Oficial Mexicana: Technical Specifications for the Care and Use of Laboratory Animals.29 The experimental protocol was approved by the Bioethics Committee of the Instituto Nacional de Cardiología, Ignacio Chávez.

General physical examinations evaluating the mental state and respiratory patterns of the dogs were performed. Vital signs, including rectal temperature, capillary refill time, beats per minute, and exploration for enlarged lymph nodes were recorded.

The male reproductive tract was examined by inspection and palpation of the scrotum, epididymis, testes, penis, prepuce, and prostate gland. The size, symmetry, content, consistency, skin thickness, lesions, anatomic abnormalities, and cryptorchidism were evaluated.

Testicular measurements were made while each dog was restrained in a standing position, and the measurements were obtained by a single person (E.P-M). The testes were aligned side by side and pushed down into the distal part of the scrotum at the level of the spermatic cord. The measurement was recorded at the widest point with the help of a flexible measuring tape.

The male dogs were trained for a month before the start of the experiment such that their semen could be collected by digital manipulation. Twelve ejaculates were collected from the control and the infected dogs using a routine protocol with intervals ranging from 7 to 12 days.30

The second fraction of the ejaculate, corresponding to the sperm-rich fraction, was collected in a separate tube using a pre-warmed plastic funnel attached to a graduated 15-mL Falcon conical tube (Becton Dickinson, Franklin Lakes, NJ), although taking care to avoid contamination with the two other (pre-spermatic and prostatic) fractions. The sperm-rich fraction was kept at 37°C and was examined immediately after harvest to determine the appearance, pH, volume, mass motility, progressive motility, sperm concentration, percentages of viable sperm, and morphologically abnormal and immature sperm using routine methods.3032 Briefly, the pH of the semen was measured using pH test strips. The progressive motility (percentage of linear and forward movement of spermatozoa) and the mass motility (scores 0–5; 0 = absence of movement, 1 = lesser speed, and 5 = faster speed or vigorous movement) were visually evaluated by a single person (E.P-M) with an optical microscope (Leitz, Wetzler, Germany) under 400× magnification using a small drop of semen between the coverslip and a warmed slide glass. The concentration of sperm in the semen was determined by diluting 20 μL of semen in 1.0 mL of saline formaldehyde solution and by hematocytometer counts (number of sperm cells/mL of semen). The number of sperm cells/ejaculate was obtained by multiplying the number of sperm cells/mL by the ejaculate volume. The percentages of viable sperm were estimated by counting actively motile sperm on a warmed slide glass and by the eosin-nigrosin staining method. The sperm morphology (percentage of the sperm defects) was examined by the eosin-nigrosin staining method using phase-contrast microscopy (Leitz, Germany) under 1,000× magnification. Spermatozoa with a cytoplasmic droplet attached to the mid-piece were counted as immature sperm.

The dogs were euthanized using sodium pentobarbital (Barbithal, Holland Animal Health, Mexico) as a general anesthetic at a dose of 30 mg/kg applied intravenously, and a lethal dose of intravenous 15% potassium chloride was subsequently administered. Testis sections were fixed in 10% buffered formalin solution for 24 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin for histological examination. Sections (5 μm) were stained with hematoxylin and eosin and evaluated by light microscopy (Carl Zeiss, K7, Jena, Germany). Images were obtained through a Bio-Doc-It Imaging System image analyzer (UVP, LLC, Upland, CA).

Statistical analyses were performed using the SPSS 13.0 software (Statistical Package for Social Sciences, Inc., Chicago, IL). The differences between the means were analyzed statistically using analysis of variance or two-way analysis of variance followed by the Tukey test and were considered to be significant when P ≤ 0.05. Regression analysis was used to correlate the testicular size (scrotal circumference) with the sperm-concentration data from semen analysis (calculated using Excel software [Microsoft 2007, Redmond, WA]).

Electrocardiograms and echocardiograms performed on month eight post-inoculation generated data consistent with ischemia, local conduction abnormalities, hypertrophy, diminished ejection fraction, left ventricular dilation, right bundle branch blocks, or ventricular premature complexes.28,33,34 However, mortalities were not observed during the study.

Results

All of the control and experimental dogs appeared to be reproductively healthy. Only dog #5 was excluded (Table 3) because of a lack of libido, and dog #4 always needed the olfactory and visual stimulus of a teaser bitch (in any stage of the estral cycle). Both testicles and epididymis were normal on palpation. The total scrotal widths of the infected dogs averaged 15.0 ± 1.6 cm. The mean of this parameter was not different from that of healthy control dogs (P > 0.05). Rectal palpation of the prostatic glands showed no anomalies: the glands were smooth, bilobed, symmetrical, non-painful, and easily movable.

Table 3

Semen quality* from chronically Trypanosoma cruzi-infected dogs (means ± SD)

Dog no.Semen 2nd fraction volume(mL)pHMass motility (0–5)Progressive motility (%)Sperm concentration/mL (×106)Normal sperm (%)Primary abnormalities (%)Secondary abnormalities (%)Viable sperm (%)
1 (Beagle)1.3 ± 0.76.5 ± 0.32.8 ± 1.189.4 ± 5.9173.3 ± 86.289.6 ± 3.64.8 ± 3.35.6 ± 1.389.9 ± 3.5
2 (Beagle)2.0 ± 0.56.5 ± 0.44.2 ± 0.992.6 ± 3.7224.2 ± 112.292.8 ± 3.63.0 ± 2.24.3 ± 2.390.9 ± 5.6
Means ± SD1.6 ± 0.56.5 ± 0.03.5 ± 0.991.1 ± 2.4198.8 ± 35.991.2 ± 2.23.9 ± 1.34.9 ± 0.990.4 ± 0.7
 
3 (Beagle)2.3 ± 0.36.7 ± 0.34.1 ± 1.091.7 ± 3.3310.8 ± 110.792.8 ± 3.22.6 ± 2.14.6 ± 2.889.6 ± 4.2
4 (Beagle)1.8 ± 0.96.7 ± 0.32.5 ± 1.189.6 ± 4.5158.3 ± 81.591.7 ± 3.23.8 ± 2.14.6 ± 2.189.9 ± 4.2
5 (Mongrel)NDNDNDNDNDNDNDNDND
6 (Mongrel)2.0 ± 0.76.8 ± 0.53.2 ± 1.286.3 ± 10.7194.2 ± 94.688.8 ± 4.23.8 ± 2.57.4 ± 3.685.8 ± 5.6
7 (Mongrel)2.0 ± 1.06.6 ± 0.24.0 ± 0.992.9 ± 3.3211.7 ± 131.992.58 ± 3.753.3 ± 1.94.1 ± 2.692.3 ± 3.0
8 (Mongrel)1.0 ± 0.36.8 ± 0.34.1 ± 1.093.6 ± 2.8239.2 ± 62.686.7 ± 7.22.0 ± 0.911.4 ± 7.793.5 ± 2.5
Means ± SD1.8 ± 0.56.7 ± 0.13.6 ± 0.790.8 ± 3.0222.8 ± 57.390.5 ± 2.73.1 ± 0.86.4 ± 3.190.2 ± 2.9

Twelve semen collections were performed at intervals of 7–12 days.

The primary abnormalities are those originating during spermatogenesis, such as micro- and macrocephalic sperms; elongated, pyriform, and double heads; double, swollen, bent, or attached midpieces; and, double and bent tails.

The secondary abnormalities are those that occur during transit from the testes to the epididymis or as a result of the handling of semen, such as detached heads, midpieces, and tails; proximal or distal cytoplasmic droplets; and coiled or bent sperms.

No significant differences were observed (P > 0.05). ND = not determined.

The appearance of the collected semen was whitish or milky for all of the dogs. The ejaculate characteristics of the control and experimental dogs are presented in Table 3. There were no statistically significant differences in the quality of the semen from two healthy dogs and five infected males. Almost all of the sperm quality parameters were in the normal ranges, except that the ejaculate volume of the second fraction was slightly higher and the mass motility was slightly lower in both groups of analyzed dogs. The ranges of the examined samples of the infected group were as follows: semen second-fraction volume between 1.8 and 2.0 mL, pH from 6.6 to 6.8, mass motility between 2.5 and 4.1, percentage of progressively motile sperm between 86.3% and 93.6%, sperm concentration from 158 to 239 × 106 cells/mL, live proportion between 85.8% and 93.5%, and morphologically normal spermatozoa from 86.7% to 92.8%.

In this study, a correlation of r = 1.0 was observed between the scrotal circumference (14.5 ± 2.12 cm) and the sperm concentration (198.75 ± 35.95 × 106 cells/mL) for control healthy dogs. In the chronically infected dogs, the scrotal circumference (15 ± 1.58 cm) was not correlated with the sperm concentration (222.83 ± 57.27 × 106 cells/mL), as determined by regression analysis (r = 0.101).

A comparison of semen parameters was performed among the five dogs that were experimentally infected with different concentrations of metacyclic trypomastigotes. The data analysis showed no significant differences in the macro and microscopic seminal features of these animals. A comparison was made between the ejaculates of healthy and infected Beagle dogs (excluding mongrel dogs). The results showed no significant differences in semen characteristics, with mean values that are very similar to those observed in Table 3.

All the sections of testes showed seminiferous tubules with abundant spermatogonia at different stages of maturation. The epithelium of seminiferous tubules was stratified into 10–14 layers and abundant mitosis indicating active maturation was observed. In the lumen of the seminiferous tubules were observed spermatids, precursors of spermatozoa. The lumen of the epididymal tubes were filled with sperm. No evidence of inflammation related to the T. cruzi infection was detected; neither amastigote nests nor trypomastigotes were found (Figure 1).

Figure 1.
Figure 1.

Micrographs of testicular structures of chronically Trypanosoma cruzi-infected dogs. (A) Seminiferous tubules (H & E × 400), (B) epididymal tubes filled with sperm (H & E × 100). Four hundred fields per dog were analyzed.

Citation: The American Society of Tropical Medicine and Hygiene 91, 4; 10.4269/ajtmh.13-0207

Discussion

The pathological effects that are associated with trypanosomatid infections in the genitalia of male domestic, laboratory, and wild animals have been studied and reported.710,12,15,16,1824 However, information is scarce and controversial regarding T. cruzi infections (that affect the reproductive system) and the direct transmission of such infections through coitus.11,13,14 The latest report by Carvalho and others17 showed that T. cruzi can colonize different cells, including myod cells, and suggested the possibility of parasite migration to the seminal fluid in case of a rupture of these cells. Therefore, the CD could potentially be transmitted through sexual intercourse.

In this study, changes did not occur in the testes or prostate glands of the chronically infected animals. However, it has been reported that total scrotal width or scrotal circumference (in addition to testicular volume) is highly correlated with testicular size. Some authors have reported that these are useful predictors for daily spermatozoa production and, consequently, for fertility in bulls, rams, dogs, and other species including humans.3537 In this study, it was shown that the sperm counts of healthy animals but not of chronic chagasic dogs are dependent on the scrotal perimeter. This finding suggests that the scrotal circumference could be useful for predicting the production and ejaculation of sperm only in healthy males. These data are similar to those obtained by Olar and others35 who found a high correlation (r = 0.75) between testicular volume and sperm ejaculated daily in dogs. However, the data are discordant with those of England38 and Cortez and others,39 whose results showed low correlations between the parameters.

The mass motility parameter showed means of 3.5 and 3.6 in healthy and chronically infected chagasic dogs, respectively. Both values were slightly lower than those reported by others (who obtained averages between 4.0 and 4.6).40,41

There are no reports available that mention if the pH is affected by the trypanosomatid infection. The pH values reported for the semen of healthy dogs vary from 6.3 to 6.7.39 In this study, the pH values of the semen in healthy and chronically infected dogs were within the reported range, 6.5 and 6.7, respectively. The progressive motility, sperm concentration, abnormalities of spermatozoids, and viability exhibited means (in both healthy control and chronically infected dogs) that are in accordance with the reference values.30,39,40

These results indicate that chronic infection by T. cruzi had no effect on the reproductive parameters of male dogs. This result differs from the findings reported by other authors who carried out experiments in Leishmania chagasi-infected dogs,20 T. congolense-infected New Zealand White rabbits,21 and T. brucei-infected West African dwarf bucks,18 or red fronted gazelles22 during the acute stage of infection.

These differences are attributable to the variety of the animal species that were analyzed and are observed because those authors conducted their evaluations during the acute stage of infection: 5 weeks,21 7 weeks,22 and 10 weeks18 post infection, when both hyperthermia and inflammation are present. It is well known that elevated body temperature adversely affects spermatogenesis, suggesting the presence of testicular degeneration that involves thrombosis of the testicular vessels leading to ischemic necrosis. Thermal degeneration is experienced especially at the peak of hyperthermia and anoxia.18 These findings suggest that the reproductive vitality is reestablished once the acute stage has passed or when appropriate drugs are administered, such as diminazene aceturate9,21,22 or combined dexamethasone and gonadotropin-releasing hormone (GnRH).12

It is likely that the infective parasitic form used, the doses used, and the route of administration performed also influence the pathological effects of CD on the reproductive system. The doses of blood trypomastigotes used in other studies18,21,22 vary from 4.8 × 105 to 10 × 106, and differ from our study by more than an order of magnitude. The routes of inoculation were intraperitoneal,18,22 intravenous,22 and a natural infection.20 In this study, the metacyclic trypomastigotes (the natural infective form) were inoculated by intraperitoneal means (which simulate the natural route of infection by the transmission vector) at doses of 5 × 104 to 1 × 106.

The abnormalities in the sperm morphology during the acute phase of the disease, the shedding of T. cruzi through the semen of acute infected dogs, the parasitic colonization in the reproductive organs, and the effects on the reproductive system of the different strains, inocula and/or routes of administration all require further elucidation. In conclusion, the T. cruzi Ninoa (MHOM/MX/1994/Ninoa) strain did not cause significant alterations in dog semen quality during the chronic phase of the CD at doses of 5 × 104 to 1 × 106 metacyclic trypomastigotes per animal (intraperitoneally inoculated).

ACKNOWLEDGMENTS

We thank Benito Chávez Rentería for providing technical assistance with specimen processing for the histopathological studies.

  • 1.

    OPS/OMS, 2005. Plan Estratégico Nacional de Chagas 2003–2007. Honduras: Enfermedad de Chagas: Modelo de la Armonización de la Cooperación Interagencial, 535.

    • Search Google Scholar
    • Export Citation
  • 2.

    World Health Organization, 2012. Global polio eradication initiative: 7th meeting of the Independent Monitoring Board. Wkly Epidemiol Rec 87: 509526.

    • Search Google Scholar
    • Export Citation
  • 3.

    Sosa-Jurado F, Zumaquero-Rios JL, Reyes PA, Cruz-Garcia A, Guzman-Bracho C, Monteón VM, 2004. Factores bióticos y abióticos que determinan la seroprevalencia de anticuerpos contra Trypanosom acruzi en el municipio de Palmar de Bravo, Puebla, México. Salud Publica Mex 46: 3948.

    • Search Google Scholar
    • Export Citation
  • 4.

    Estrada-Franco JG, Bhatia V, Diaz-Albiter H, Ochoa-Garcia L, Barbabosa A, Vazquez-Chagoyan JC, Martinez-Perez MA, Guzman-Bracho C, Garg N, 2006. Human Trypanosoma cruzi infection and seropositivity in dogs, Mexico. Emerg Infect Dis 12: 624630.

    • Search Google Scholar
    • Export Citation
  • 5.

    Jiménez-Coello M, Ortega-Pacheco A, Guzmán-Marin E, Guiris-Andrade DM, Martínez-Figueroa L, Acosta-Viana KY, 2010. Stray dogs as reservoirs of the zoonotic agents Leptospira interrogans, Trypanosoma cruzi and Aspergillus spp. in an urban area of Chiapas in southern México. Vector Borne Zoonotic Dis 10: 135141.

    • Search Google Scholar
    • Export Citation
  • 6.

    Balán LU, Yerbes IM, Piña MA, Balmes J, Pascual A, Hernández O, López R, Monteón V, 2011. Higher seroprevalence of Trypanosoma cruzi infection in dogs than in humans in an urban area of Campeche, México. Vector Borne Zoonotic Dis 11: 483484.

    • Search Google Scholar
    • Export Citation
  • 7.

    Ikede BO, 1979. Genital lesions in experimental chronic Trypanosoma brucei infection in rams. Res Vet Sci 26: 145151.

  • 8.

    Anosa VO, Isoun TT, 1980. Further observations on the testicular pathology in Trypanosoma vivax infection of sheep and goats. Res Vet Sci 28: 151160.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ikede BO, Akpavie SO, 1982. Delay in resolution of trypanosome-induced genital lesions in male rabbits infected with Trypanosoma brucei and treated with diminazene aceturate. Res Vet Sci 32: 374376.

    • Search Google Scholar
    • Export Citation
  • 10.

    Anosa VO, Kaneko JJ, 1984. Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus). Light and electron microscopic study of testicular lesions. Rev Vet Pathol 21: 238246.

    • Search Google Scholar
    • Export Citation
  • 11.

    Lamano-Carvalho TL, Ribeiro RD, Lopes RA, 1991. The male reproductive organs in experimental Chagas' disease. I. Morphometric study of the vas deferens in the acute phase of the disease. Exp Pathol 41: 203214.

    • Search Google Scholar
    • Export Citation
  • 12.

    Boly H, Humblot P, Tillet Y, Thibier M, 1994. Effects of Trypanosoma congolense infection on the pituitary gland of Baoulé bulls: immunohistochemistry of LH- and FSH-secreting cells and response of plasma LH and testosterone to combined dexamethasone and GnRH treatment. J Reprod Fertil 100: 157162.

    • Search Google Scholar
    • Export Citation
  • 13.

    Lenzi HL, Castelo-Branco MT, Pelajo-Machado M, Oliveira DN, Gattass CR, 1998. Trypanosoma cruzi: compromise of reproductive system in acute murine infection. Acta Trop 71: 117129.

    • Search Google Scholar
    • Export Citation
  • 14.

    Herrera L, Urdaneta-Morales S, 2001. Experimental transmission of Trypanosoma cruzi through the genitalia of albino mice. Mem Inst Oswaldo Cruz 96: 713717.

    • Search Google Scholar
    • Export Citation
  • 15.

    Diniz SA, Melo MS, Borges AM, Bueno R, Reis BP, Tafuri WL, Nascimento EF, Santos RL, 2005. Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs. Vet Pathol 42: 650658.

    • Search Google Scholar
    • Export Citation
  • 16.

    Adamu S, Fatihu MY, Useh NM, Mamman M, Sekoni VO, Esievo KA, 2007. Sequential testicular and epididymal damage in Zebu bulls experimentally infected with Trypanosoma vivax. Vet Parasitol 143: 2934.

    • Search Google Scholar
    • Export Citation
  • 17.

    Carvalho LO, Abreu-Silva AL, Hardoim D de J, Tedesco RC, Mendes VG, da Costa SC, Calabrese KS, 2009. Trypanosoma cruzi and myoid cells from seminiferous tubules: interaction and relation with fibrous components of extracellular matrix in experimental Chagas' disease. Int J Exp Pathol 90: 5257.

    • Search Google Scholar
    • Export Citation
  • 18.

    Raheem AK, Fayemi EO, Leigh OO, Ameen SA, 2009. Selected fertility parameters of West African dwarf bucks experimentally infected with Trypanosoma congolense. Folia Vet 53: 6871.

    • Search Google Scholar
    • Export Citation
  • 19.

    Silva FL, Oliveira RG, Silva TM, Xavier MN, Nascimento EF, Santos RL, 2009. Venereal transmission of canine visceral leishmaniasis. Vet Parasitol 160: 5559.

    • Search Google Scholar
    • Export Citation
  • 20.

    Assis VP, Ribeiro VM, Rachid MA, Castro AC, Valle GR, 2010. Dogs with Leishmania chagasi infection have semen abnormalities that partially revert during 150 days of Allopurinol and Amphotericin B therapy. Anim Reprod Sci 117: 183186.

    • Search Google Scholar
    • Export Citation
  • 21.

    Leigh OO, Fayemi OE, 2010. Ejaculate characteristics of rabbits infected with Trypanosoma congolense and changes caused offer treatment with Diminazene aceturate (Diminaveto). Rev Int J Morphol 28: 471475.

    • Search Google Scholar
    • Export Citation
  • 22.

    Mbaya AW, Nwosu CO, Kumshe HA, 2011. Genital lesions in male red fronted gazelles (Gazella rufifrons) experimentally infected with Trypanosoma brucei and effect of melarsamine hydrochloride (Cymelarsan) and diminazene aceturate (Berenil) in its treatment. Theriogenology 76: 721728.

    • Search Google Scholar
    • Export Citation
  • 23.

    Manna L, Paciello O, Morte RD, Gravino AE, 2012. Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report. Parasites & Vectors 5: 216.

    • Search Google Scholar
    • Export Citation
  • 24.

    Kegler K, Habierski A, Hahn K, Amarilla SP, Seehusen F, Baumgärtner, 2013. Vaginal canine transmissible venereal tumor associated with intra-tumoral Leishmania spp. amastigotes in an asymptomatic female dog. J Comp Path 149: 156161.

    • Search Google Scholar
    • Export Citation
  • 25.

    Monteón VM, Furuzawa-Caballeda J, Alejandre-Aguilar R, Aranda-Fraustro A, Rosales Encina JL, Reyes PA, 1996. American trypanosomosis: in situ and generalized features of parasitism and inflammation kinetics in a murine model. Exp Parasitol 83: 267274.

    • Search Google Scholar
    • Export Citation
  • 26.

    Espinoza B, Solorzano-Domínguez N, Vizcaino-Castillo A, Martínez I, Elias-López AL, Rodríguez-Martínez JA, 2011. Gastrointestinal infection with Mexican TcI Trypanosoma cruzi strains: different degrees of colonization and diverse immune responses. Int J Biol Sci 7: 13571370.

    • Search Google Scholar
    • Export Citation
  • 27.

    WHO Technical Report Series, 2002. Control of Chagas Disease. Second report of the WHO expert committee. Geneva: World Health Organization, 1109.

    • Search Google Scholar
    • Export Citation
  • 28.

    Rodríguez-Morales O, Ballinas-Verdugo MA, Alejandre-Aguilar R, Reyes-López PA, Arce-Fonseca M, 2011. Trypanosoma cruzi connatal transmission in dogs with Chagas disease. Experimental case report. Vector Borne Zoonotic Dis 11: 13651370.

    • Search Google Scholar
    • Export Citation
  • 29.

    Norma Oficial Mexicana NOM-0062-ZOO-1999, 1999. Especificaciones Técnicas para el Cuidado y Uso de Animales de Laboratorio. Mexico: Diario Oficial de la Federación, 158.

    • Search Google Scholar
    • Export Citation
  • 30.

    Linde-Forsberg C, 1991. Achieving canine pregnancy using frozen or chilled extended semen. Vet Clin North Am Small Anim Pract 21: 467485.

    • Search Google Scholar
    • Export Citation
  • 31.

    Dostal LA, Juneau P, Rothwell CE, 2001. Repeated analysis of semen parameters in Beagle dogs during a 2-year study with the HMG-CoA reductase inhibitor, atorvastatin. Toxicol Sci 61: 128134.

    • Search Google Scholar
    • Export Citation
  • 32.

    Gutiérrez Y, Hernández J, Fernández F, Cruz H, 2004. Congelación de espermatozoides obtenidos de colas de epidídimos de bovinos. Rev Salud Anim 26: 192196.

    • Search Google Scholar
    • Export Citation
  • 33.

    Rodríguez-Morales O, Pérez-Leyva MM, Ballinas-Verdugo MA, Carrillo-Sánchez SC, Rosales-Encina JL, Alejandre-Aguilar R, Reyes PA, Arce-Fonseca M, 2012. Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model. Vet Res 43: 79.

    • Search Google Scholar
    • Export Citation
  • 34.

    Rodríguez-Morales O, Carrillo-Sánchez SC, García-Mendoza H, Aranda-Fraustro A, Ballinas-Verdugo MA, Alejandre-Aguilar R, Rosales-Encina JL, Vallejo M, Arce-Fonseca M, 2013. Effect of the plasmid-DNA vaccination on macroscopic and microscopic damage caused by the experimental chronic Trypanosoma cruzi infection in the canine model. Biomed Res Int 2013: 826570.

    • Search Google Scholar
    • Export Citation
  • 35.

    Olar TT, Amann RP, Pickett BW, 1983. Relationships among testicular size, daily production and output of spermatozoa, and extragonadal spermatozoa reserves of the dog. Biol Reprod 29: 11141120.

    • Search Google Scholar
    • Export Citation
  • 36.

    Souza FF, Leme DP, Uechi E, Trinca LA, Lopes MD, 2004. Evaluation testicular fine needle aspiration cytology and serum testosterone levels in dogs. Braz J Vet Res Anim Sci 41: 98105.

    • Search Google Scholar
    • Export Citation
  • 37.

    Reyes LA, Segovia FJ, Cerpa RE, 2011. Volumen testicular: el tamaño sí importa. (Testicular volumen: size does matter). Rev Colomb Radiol 22: 33483351.

    • Search Google Scholar
    • Export Citation
  • 38.

    England GC, 1991. Relationship between ultrasonographic appearance, testicular size, spermatozoal output and testicular lesions in the dog. J Small Anim Pract 32: 306311.

    • Search Google Scholar
    • Export Citation
  • 39.

    Cortez AA, Aquino-Cortez A, Silva AR, Cardoso Rc S, Silva LD, 2002. Relação entre perímetro escrotal e concentração espermática emcães, clinicamentenormais, da raça Pastor Alemão. Arq Bras Med Vet Zootec 54: 549550.

    • Search Google Scholar
    • Export Citation
  • 40.

    Corti L, 2003. Evaluación de la capacidad fecundante del semen congelado del perro (Canis familiaris), en ova recuperadas de perras de celo inducido. Bachelor Thesis for Doctor in Veterinary Medicine. Facultad de Ciencias Veterinarias de Chile, 171.

    • Search Google Scholar
    • Export Citation
  • 41.

    Kawakami E, Hori T, Tsutsui T, 1998. Changes in semen quality and in vitro sperm capacitation during various frequencies of semen collection in dogs with both asthenozoospermia and teratozoospermia. J Vet Med Sci 60: 607614.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Minerva Arce-Fonseca, Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez,” Juan Badiano No. 1, Col. Sección XVI, Tlalpan, Mexico City, 14080, Mexico. E-mail: mini_arce@yahoo.com.mx

Financial support: This work was supported by a grant from the Instituto de Ciencia y Tecnología del Distrito Federal (ICyTDF), Mexico (Grant 236/2010).

Authors' addresses: Olivia Rodríguez-Morales, Elvia Pedro-Martínez, and Minerva Arce-Fonseca, Department of Molecular Biology, Instituto Nacional de Cardiología “Ignacio Chávez,” Mexico City, Mexico, E-mails: rm.olivia@gmail.com, mvz.elvia@hotmail.com, and mini_arce@yahoo.com.mx. José Ernesto Hernández-Pichardo, Department of Agricultural and Animal Production, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma Metropolitana Unidad Xochimilco, Mexico City, Mexico, E-mail: ehernan@correo.xoc.uam.mx. Ricardo Alejandre-Aguilar, Department of Parasitology, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico, E-mail: rialejandre@yahoo.com.mx. Alberto Aranda-Fraustro and Verónica Graullera-Rivera, Department of Pathological Anatomy, Instituto Nacional de Cardiología “Ignacio Chávez,” Mexico City, Mexico, E-mails: arandafraustro@yahoo.com.mx and grauvero@yahoo.com.mx.

Save