Abstract

COMMUNICATION COMUNICAÇÃO

Evaluation of fish sperm concentration using two counting chambers

Estimativa da concentração espermática em peixes utilizando-se duas câmaras de contagem

P.F. Taitson^I; H.P. GodinhoI, II, ^* * Autor para correspondência: E-mail: hgodinho@pucminas.br

^ILaboratório de Criobiologia, Instituto de Reprodução Humana

^{I, II}Programa de Pós-Graduação em Zoologia de Vertebrados Pontifícia Universidade Católica de Minas Gerais Avenida Dom José Gaspar, 500 30535-610 - Belo Horizonte, MG

Keywords: fish, sperm counting, Makler chamber, Neubauer chamber

RESUMO

A concentração espermática do sêmen do peixe anostomídeo Leporinus elongatus (Valenciennes, 1849) foi estimada nas câmaras de Makler e de Neubauer, tendo-se obtido, respectivamente, 10,9 ± 2,8 ´ 10⁹ e 11,9 ± 3,7 ´ 10⁹ espermatozóides/ml de sêmen sem que ocorresse diferença significativa entre essas médias (P > 0,05). A estimativa da concentração espermática do sêmen na câmara de Makler apresentou vantagens em relação à da câmara de Neubauer. A contagem na câmara de Makler pôde ser feita sem a necessidade de se esperar pela deposição dos espermatozóides no seu fundo. A baixa diluição do sêmen (1:1.000) necessária para se estimar a concentração espermática pode ser considerada também como vantagem pois altas diluições (como na câmara de Neubauer) são fontes consideráveis de erro. Dessa forma, a câmara de Makler pode ser utilizada na determinação da concentração espermática do sêmen de peixes e, possivelmente, de outros vertebrados, incluindo a de animais domésticos.

Palavras-chave: peixe, concentração espermática, Câmera de Makler, Câmara de Neubauer

Sperm concentration is one of the main parameters in evaluating fish sperm quality (Billard et al., 1996). The Neubauer chamber is the most commonly used sperm counting tool for both human (World..., 1999) and fish (Billard et al., 1996). The sperm. Makler chamber (Makler, 1978) is largely used in human andrology (Borges Júnior, 2000) and has recently been introduced in fish sperm research (Ravinder et al., 1997). In this communication, the application of Makler chamber for the determination of fish sperm concentration is evaluated.

Milt from nine individuals (0.45± 0.12g of body weight; 43.9± 7.6in total length) of the neotropical freshwater anostomid fish Leporinus elongatus (Valenciennes, 1849) was used. The fish were captured in the São Francisco River basin, South-eastern Brazil. They were held in captivity in a private fish hatchery for over one year, in 100m² earthen ponds, at a stocking rate of 1 kg of fish/5-10m² of pond and fed on commercial feed, 1-3% of body weight/day. The experiment was conducted in mid-January, 2000. The fish were gently wrapped in cloth towels to reduce stress. The region of the genital papilla was dried and cleaned with a paper towel and care was taken to avoid contamination with urine, faeces and water. Milt (1.33± 0.3ml) was collected after gentle abdominal pressure into a dry graduated polyethylene tube. Then, 0.2-0.4ml milt aliquots from each fish spermwere diluted 1:1,000 in NaCl 200 mM solution (previously established as a non-activating sperm motility solution). Ten microliters of this mixture were placed the Makler chamber to estimate its sperm concentration. Similar countingsCounts were also performed in the Neubauer chamber after diluting the milt 1:10,000 in the NaCl 200mM solution. In both cases, five counts were performed for each fish in the light microscope at 400´.

Sperm concentrations were 10.9± 2.8´ 10⁹ and 11.9± 3.7´ 10⁹ spermatozoa/ml of milt, respectively for the Makler and Neubauer chamber. The difference between the two means was not significantly different (Student t test; P>0.05). Estimates of sperm concentration in fish usually vertebrates present some technical difficulties. Counting errors may be produced in the Neubauer (Dacie, Lewis, 1984) as well in the Makler chamber. These errors may be related to the sample, operator or to the microscopic field (Dacie, Lewis, 1984). Regarding the Neubauer chamber, these errors may be related to the sample, operator and to the microscopic field (Dacie & Lewis, 1984).The Makler chamber is very thin and all spermatozoa within it can be seen immediately and in a single focal plane. No spermatozoon sticking to the chamber glass was observed in this work. Counting can be performed in this chamber with no need to wait for the spermatozoa to be precipitated on the bottom of the chamber, a process which takes about a few minutes when using the Neubauer chamber (Makler, 1978). The low sperm dilution rate in the Makler chamber (1:1,000) may be considered an additional advantage since high dilutions are an important source of error (Makler, 1980). Possibly, The use of the Makler chamber revealed easiness and reliability. Thus,W the former Makler chamber may constitute a practical, alternative tool in the ddetermining ation of sperm emen concentration in fish and possibly in other vertebrates, including farm animals. This is probably the first work to compare data on fish sperm concentration obtained by Makler and Neubauer chamber.

Recebido para publicação em 3 de abril de 2002

Recebido para publicação, após modificações, em 18 de setembro de 2002

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