Características reprodutivas de onças-pintadas (Panthera onca) machos, mantidos em cativeiro

Ronaldo Gonçalves MORATO^1,2; Marcelo Alcindo Barros de Vaz GUIMARÃES^1,3; Fernando FERREIRA⁴; Ieda Terezinha do Nascimento VERRESCHI⁵; Renato Campanarut BARNABE¹

CORRESPONDENCE TO:
Ronaldo Gonçalves Morato
Departamento de Reprodução Animal
Faculdade de Medicina Veterinária e Zootecnia da USP
Cidade Universitária Armando de Salles Oliveira
Av. Orlando Marques de Paiva, 87
05508-000 – São Paulo – SP
e-mail: rgmorato@usp.br

SUMMARY

Ejaculate traits, testicular volume and plasma testosterone levels were determined once every two months for one year in 4 adult male jaguars (Panthera onca) housed at the São Paulo Zoo, SP, Brazil. Semen samples was collected by electroejaculation and analyzed for pH, total volume, motility (0-100%), status (0-5), total spermatozoa count and morphology. Blood samples was obtained by cephalic venipuncture immediately before the onset of electroejaculation and stored at –20ºC until assay of testosterone by RIA. Using a calliper, the length and width of each testis was measured and the values were combined to determine the testicular volume. Semen analyses demonstrated a high percentage of structurally abnormal sperm (mean = 51%) and low rates of motility (50.6%) and status (2.2). No correlation was found between semen traits, plasma testosterone and testicular volume (Spearman’s test). No season variation was detected throughout the year for semen traits, plasma testosterone and testicular volume (p>0.05, Friedman’s test). The results of this study suggest that the captive jaguars in Brazil are not seasonal, and that semen collections can be performed throughout the year without a perturbation in overall ejaculate quality. However, underlying causes of high percentages of structurally abnormal sperm, found in captive jaguars, need to be investigated mainly to improve semen quality.

INTRODUCTION

The jaguar (Panthera onca), the largest cat of the Americas, inhabits different regions from North Mexico to Argentina¹⁹. In Brazil, the jaguar is considered an endangered species by the Brazilian Institute for Environmental and Natural Resources (IBAMA), and numbers in the wild have been reduced by a variety of factors with loss and fragmentation of habitats being as the most prevalent. In captivity, low reproductive success and inbreeding, due to small populations’ size, has been a barrier for the genetic management of this species. Recently Morato; Gasparini¹⁶ observed that 90% of the captive population of Brazilian Zoos is of unknown origin. Considering the importance of captive propagation to the preservation of endangered species, reproduction of wild caught and jaguars of known origin needs to become an integral component of conservation efforts for this species. Assisted reproductive technologies, such as artificial insemination and in vitro fertilization, can be viewed as one potential approach for safeguarding species, based on the highly successful applications to humans and domestic livestock²⁸. However, to apply assisted reproductive strategies in the management of the jaguar, we need to first understand basic reproductive characteristics. Towards this aim, the objectives of this study were: 1) to examine the testicular function (steroidogenic and spermatogenic), and 2) to analyze the influence of season on reproductive activity in males.

MATERIAL AND METHOD

Animals

Four adult (7-10 years) male jaguars were maintained at the São Paulo Zoo, São Paulo, Brazil (23º34’09.5" S and 46º44’19.1" W). All males were not proven breeders. They were housed individually having access to indoor and outdoor enclosures and were exposed to the natural photoperiod throughout the year. A beef-based diet was provided 6 days a week.

Electroejaculation, collection of blood samples, semen evaluation and testicular biometry

Semen from each collection was immediately evaluated for total volume and pH (reagent strips-Merck). Motility (0-100%) and rate of forward progression (status; 0-5) were based on observations of four separated microscopic fields at 100X^9,26. An aliquot was diluted (1:3) in a 10% formaline-saline solution and used to determine the sperm concentration using a hemacytometer (Neubauer). Sperm morphology evaluations were performed after fixing an aliquot in a formaline-saline solution and examining 200 individual sperm cells using phase contrast microscopy (1,000X).

Blood samples (10 ml) were collected by cephalic venipuncture, immediately before the onset of electroejaculation. Samples were centrifuged at ambient temperature (300 g/10 minutes) 1 hour after collection, and the recovered sera stored at –20ºC until hormone analyses by radioimmunoassay.

Clinical evaluations of the testicles were made by digital palpation and then classified as rigid, normal and flaccid. Using a calliper, the length and width of each testis was measured and the values were plotted in a formula (V= LxW²x0.524, where L is the length and W is the width) to determine the testicular volume¹¹. The volumes for the right and left testis were combined to obtain the total testicular volume of each male.

Radioimmunoassay

Plasma testosterone was measured as described by Lox et al.¹², using an antibody produced at the Unidade Edócrina of the Escola Paulista de Medicina of the Universidade Federal de São Paulo^21,25. Tritiated testosterone (1, 2, 6, 7, 16, 17-³H testosterone, approximately 1,000 c.p.m., New England Nuclear, Boston, MA, USA) was added to 0.5 ml of plasma sample for the recovery assay. The tubes were incubated for 30 minutes at room temperature (25ºC) and the testosterone extracted with 3 ml of twice-distilled ether (Merck, USA). After incubation, the tubes were centrifuged and the extracts separated by freezing. The ether fraction was evaporated in a water-bath at 50ºC, and the residue was suspended in 2 ml gelatin buffer. Aliquots (0.1 ml) of this solution were transferred to scintillation tubes for the recovery assay and additional 0.1 ml aliquots were added to 0.1 ml of [1, 2, 6, 7, 16, 17-³H]-testosterone (~7,000 c.p.m.) and 0.1 ml of antibody solution. The preparation was incubated at 4°C overnight and 0.2 ml of dextran-charcoal T-70 solution in gelatin buffer were added and mixed for 5 seconds. The tubes were centrifuged at 800 g for 30 minutes at 4°C, and the supernatant was dispensed into a tube with 10 ml of scintillation liquid, and counted in a gamma counter. The assay sensitivity was 5.0 ng/dl and the net recovery was 90%. All samples were analyzed in duplicate with <10% intra- and inter-assay coefficients of variation.

Statistical analyses

The year was divided into four seasons that were represented for 1 or two months: winter (July), spring (September and November), summer (January) and fall (March and May). For each animal, mean (± SEM) values were calculated for seminal and hormonal characteristics obtained after each ejaculation procedure. The data were then averaged across that season. The parameters for each animal during the seasons of the experiment were analyzed by the Friedman Rank Test using a statistical package (SSPS 6.0 for Windows, SSPS Inc., Chicago, IL). Spearman’s rank correlation coefficient was calculated between seminal traits, testosterone concentrations and testicular volumes⁷.

RESULTS

Table 1 Individual ejaculate traits, testicular volumes and testosterone concentrations (mean ± SEM) of captive male jaguars, São Paulo Zoo, 1995 to 1996.

a range.

Ejaculate traits, testicular volume, and plasma testosterone concentrations were not significantly correlated (p>0.05).

Ejaculate traits, testicular volume and plasma testosterone concentrations were not statistically different for the various seasons (p>0.05) indicating no evidence of seasonal effects on these reproductive parameters (Tab. 2).

]]> Table 2 Seasonal evaluation of ejaculate traits, plasma testosterone and testicular volume (mean ± SEM) in jaguars, São Paulo Zoo, 1995 to 1996.

a range. There is no significant variation of the analyzed parameters throughout the year (p>0.05).

DISCUSSION

Semen collection, by electroejaculation, has been performed in many different cat species⁹. However, repeated collections have not been performed on jaguars. This study presents that repeated collections by electroejaculation in this species did not appear to affect semen quality, gonadal function or general health such as has been reported for tigers³, snow leopards¹¹, Palla’s cats²³ and others South American cats¹⁵.

In general, semen analyses demonstrated a high percentage of structurally abnormal sperm. Although teratozoospermia is a common finding in felids⁸ the percentages of abnormal sperm found in this study is higher than reported by others^9,14.

In general, three important factors have been directly identified to be related to semen quality, namely: genetic, nutrition and environmental conditions¹.

Considering the genetic factor, poor semen quality has been associated with inbreeding, has been reported for cheetahs²⁷ and Florida panthers²⁰. Previous studies demonstrated that 90% of captive jaguars in Brazil are from unknown origin¹⁶; therefore, studies need to be conducted to elucidate the influence of reduced heterozygosity on the reproduction of captive jaguars in Brazil. Although the effect of nutrition on reproduction is complex, there is evidence suggesting that the beef-based diets fed to captive jaguars are inherently low in essential vitamins (e.g., A and E)¹⁷. Vitamin A and E deficiency has been associated with compromised spermatogenesis and testicular degeneration, respectively^10,13,22,24. In light of these previous reports, studies need to be performed to evaluate the influence of essential nutrients on reproductive characteristics of captive male jaguars.

It is unlikely that the elevated number of morphologically abnormal spermatozoa in the jaguar was the result of sexual abstinence or degenerative process associated with elimination of aged spermatozoa once we performed two electroejaculations procedures in all jaguars before the beginning of the study. In this case, we observed similar number of defectives forms of spermatozoa.

Testicular volume has been used as an indicator of reproductive capacity and fertility in bulls⁶. Nevertheless, there was no apparent correlation between testicular volume and ejaculate traits in jaguars found in this study. Furthermore, there was no correlation between plasma testosterone concentrations and ejaculate traits, which was similar to that which was reported previously for Siberian tigers³.

The results of this study suggest that the captive jaguars in Brazil are not seasonal, and that semen collections can be performed throughout the year without a perturbation in overall ejaculate quality. However, high percentages of structurally abnormal sperm, found in captive jaguars, need to be investigated further to determine possible underlying causes. The outcome of this study is a first step towards the development of a genome resource bank and assisted reproduction program for the endangered jaguar.

ACKNOWLEDGMENTS

We thank Fundação Parque Zoológico de São Paulo to permit us to use the animals. We also thank Daniel Luzes Fedullo, Sandra Helena Ramiro Correa and Ivone Fonti Bianco for technical support.

RESUMO

Quatro onças pintadas (Panthera onca), machos, adultos, mantidos no Zoológico de São Paulo (SP-Brasil), foram submetidos a avaliação seminal, quantificação hormonal e biometria testicular a cada dois meses pelo período de um ano. As amostras de sêmen foram obtidas por eletroejaculação e analisadas quanto ao pH, volume total, motilidade (0-100%), vigor (0-5), espermatozóides totais e morfologia. Amostras de sangue foram obtidas pela punção da veia cefálica imediatamente antes da eletroejaculação e o plasma foi estocado a –20ºC até a realização do radioimunoensaio para quantificação de testosterona. Comprimento e largura dos testículos direito e esquerdo foram obtidos com auxílio de paquímetro e os valores obtidos foram combinados para a obtenção de volume testicular. As amostras de sêmen mostraram elevado índice de espermatozóides morfologicamente anormais (média = 51%), baixos índice de motilidade (50,6%) e vigor (2,2). Não foi encontrada correlação entre as características seminais, níveis plasmáticos de testosterona e volume testicular (Teste de Correlação de Spearman). Não foi detectado efeito da estação durante o ano para qualidade espermática, testosterona sérica e volume testicular (p>0,05, Teste de Friedman). Os resultados sugerem que as onças pintadas mantidas em cativeiro não são sazonais e que a colheita e avaliação espermática podem ser realizadas em qualquer período do ano sem que haja perda na qualidade espermática. No entanto, as causas de elevado índice de espermatozóides morfologicamente anormais, encontrados neste estudo, devem ser investigadas.

UNITERMOS: Jaguar; Características do sêmen; Testosterona; Sazonalidade.

REFERENCES

1- BARTH, A.D.; OKO, R.J. Abnormal morphology of bovine spermatozoa. Ames : Iowa State University Press, 1989. p.19-53: Normal bovine spermatogenesis and sperm maturation.        [ Links ]

2- BRONSON, F.H.; HEIDEMAN. Seasonal regulation of reproduction in mammals. In: KNOBIL, E.; NEILL, J.D. The physiology of reproduction, 2.ed., New York : Raven Press Ltd., 1994. p.541-83.        [ Links ]

3- BYERS, A.P.; HUNTER, A.G.; SEAL, U.S.; GRAHAM, E.F.; TILSON, R.L. Effect of season on seminal traits and plasma hormone concentrations in captive male Siberian tiger (Panthera tigris). Journal of Reproduction and Fertility, v.90, n.1, p.119-25, 1990.        [ Links ]

4- CRAWSHAW JR., P.G.; QUIGLEY, H.B. A ecologia do jaguar ou onça-pintada (Panthera onca) no Pantanal Mato-grossense. Relatório final para o Instituto Brasileiro de Desenvolvimento Florestal-IBDF, Brasília : IBDF, 1984.        [ Links ]

5- EWER, R.F. The carnivores. New York : Cornell University Press, 1973. p.494.        [ Links ]

6- HAHN, J.; FOOTE, R.H.; SEIDEL, G.E. Testicular growth and related sperm output in dairy bulls. Journal of Animal Science, v.29, n.1, p.41-7, 1969.        [ Links ]

7- HOLLANDER, M.; WOLFE, D.A. Non Parametrics Statistical Methods. New York : John Wiley, 1973. p.528.        [ Links ]

8- HOWARD, J.G.; BUSH, M.; HALL, L.L.; WILDT, D.E. Morphological abnormalities in spermatozoa of 28 species of non-domestic felids. In: INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, 10., Urbana-Champaign, 1984. Proceedings. p.57.        [ Links ]

9- HOWARD, J.G. Semen collection and analysis in carnivores. In: FOWLER, M.E., Zoo and wildlife medicine. Philadelphia : W.B. Saunders, 1993. p.390-8.        [ Links ]

10- HUANG, H.F.S.; DYRENFURTH, I.; HEMBREE, W.C. Endocrine changes associated with germ cell loss during vitamin A-induced recovery of spermatogenesis. Endocrinology, v.112, n.4, p.1163-71, 1983.        [ Links ]

11- JOHNSTON, L.A.; ARMSTRONG, D.L.; BROWN, J.L. Seasonal effects on seminal and endocrine traits in the captive snow leopard (Panthera uncia). Journal of Reproduction and Fertility, v.102, n.1, p.229-36. 1994.        [ Links ]

12- LOX, C.D.; CHRSTIAN, C.D.; HEINE, M.W. A simple radioimmunoassay for testosterone. American Journal of Obstetrics and Gynecology, v.118, n.1, p.114-8, 1974.        [ Links ]

13- MATHUR, U.; DATTA, S.L.; MATHUR, B.B.L. The effect of aminoprotein-induced folic acid deficiency on spermatogenesis. Fertility and Sterility, v.28, n.12, p.1356-61, 1977.        [ Links ]

14- MIES-FILHO, A.; TELECHEA, N.L.; BOHRER, J.L.; WALLAWER, W.P. Produção espermática de Panthera onca. Arquivos da Faculdade de Medicina Veterinária da Universidade Federal do Rio Grande do Sul, v.2, n.1, p.55-65, 1974.        [ Links ]

15- MORAIS, R.N.; MOREIRA, N.; MORAES, W.; MUCCIOLO, R.G.; LACERDA, O.; GOMES, M.L.F.; SWANSON, W.F.; GRAHAM, L.H.; BROWN, J.L. Testicular and ovarian function in South American small felids assessed by fecal steroids. In: CONGRESS OF AMERICAN ASSOCIATION OF ZOO VETERINARIANS, Guadalajara. 1996. Proceedings. p.561-5.        [ Links ]

16- MORATO, R.G.; GASPARINI, R.L. Levantamento preliminar sobre a situação da onça-pintada (Panthera onca) em cativeiro. In: CONGRESSO DA SOCIEDADE DE ZOOLÓGICOS DO BRASIL, 13., Rio de Janeiro. Anais. 1994. p.56.        [ Links ]

17- NATIONAL RESEARCH COUNCIL. Nutrient requeriments of cats. Washington : National Academy of Press, 1986. p.78.        [ Links ]

18- NOWAK; PARADISO (1983) apud OLIVEIRA (1994), p.76.

19- OLIVEIRA, T.G. Cats: ecological and conservation. São Luís : Edusma, 1994. p.244.        [ Links ]

20- ROELKE, M.E.; MARTENSON, J.S.; O’BRIEN, S.J. The consequences of demographic reduction and genetic depletion in the endangered Florida panther. Current Biology, v.3, n.6, p.340-50, 1993.        [ Links ]

21- RUSSO, E.K.; VIEIRA, J.G.H.; NOGUTI, K.O.; VERRESCHI, I.T.N. Purificação de antisoros para radioimunoensaio de testosterona. In: CONGRESSO BRASILEIRO DE ENDOCRINOLOGIA E METABOLOGIA, 12., Salvador, 1976. p.188.        [ Links ]

22- SCOTT, P.P.; SCOTT, M.G. Vitamin A and reproduction in the cat. Journal of Reproduction and Fertility, v.8, n.2, p.270-1, 1964.        [ Links ]

23- SWANSON, W.F.; BROWN, J.L.; WILDT, D.E. Influence of seasonality on reproductive traits of the male pallas’cat (Felis manul) and implications for captive management. Journal of Zoo and Wildlife Medicine, v.27, n.2, p.234-40, 1996.        [ Links ]

24- THRELFALL, W.R.; LEPINE, A.J.; BURR, J.R. The influence of diet on sperm quality and quantity. In: CAREY, D.P.; NORTON, S.A.; BOLSER, S.M. Recent advances in canine and feline nutritional research, 1996. p.99-114.        [ Links ]

25- VIEIRA, J.G.H.; RUSSO, E.M.K.; VERRESCHI, I.T.N.; MACIEL, R.M.B.; LIMA, M.C. Caracterização de antisoro antitestosterona para uso em radioimunoensaio. In: CONGRESSO BRASILEIRO DE ENDOCRINOLOGIA E METABOLOGIA, 12., Salvador, 1976. p.186.        [ Links ]

26- WILDT, D.E.; BUSH, M.; HOWARD, J.G.; O´BRIEN, S.J.; MELTZER, D.; VAN DYK, A.; EBEDES, H.; BRAND, D.J. Unique seminal quality in the South African cheetah and a comparative evaluation in the domestic cat. Biology of Reproduction, v.29, n.4, p.1019-25. 1983.        [ Links ]

27- WILDT, D.E.; BROWN, J.L.; BUSH, M.; BARONE, M.A.; COOPER, K.A.; GRISHAM, J.; HOWARD, J.G. Reproductive status of cheetahs (Acinonyx jubatus) in North American zoos: the benefits of physiological surveys for strategic planning. Zoo Biology, v.12, n.1, p.45-80. 1993a.        [ Links ]

28- WILDT, D.E.; SEAL, U.S.; RALL, W.F. Genetic resource banks and reproductive technology for wildlife conservation. In: CLOUD, J.G.; THORGAARD, G.H. Genetic conservation of salmonid fishes. New York : Plenum Press, 1993b. p.159-73.        [ Links ]

Received: 09/11/1998
Accepted: 23/04/1999

^* This research was supported by CNPq (Conselho Nacional de Pesquisa) and CAPES (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).
¹ Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da USP - SP
² Pró-Carnívoros /CENAP/IBAMA. São Paulo - SP
³ Associação Fundação Parque Zoológico de São Paulo - SP
⁴ Departamento de Medicina Veterinária Preventiva e Saúde Animal da Faculdade de Medicina Veterinária e Zootecnia da USP - SP
⁵ Unidade Endócrina da Escola Paulista de Medicina da UNIFESP, São Paulo - SP