Modification of the short osmotic resistance test for evaluating the functional integrity of boar spermatozoa

Teste de resistência osmótica modificado (sort modificado) na avaliação da integridade funcional do espermatozóide de suíno

Varrão; espermatozóide; resistência osmótica

Boar; spermatozoa; osmotic resistance

COMMUNICATION

[Comunicação]

Modification of the short osmotic resistance test for evaluating the functional integrity of boar spermatozoa

[Teste de resistência osmótica modificado (sort modificado) na avaliação da integridade funcional do espermatozóide de suíno]

J.K.F. Nascimento¹, E.F. Nascimento¹, M.I.V. Melo²

¹Escola de Veterinária da Universidade Federal de Minas Gerais

Caixa Postal 567

30123-970 - Belo Horizonte, MG

2PUC MG – Belo Horizonte

Recebido para publicação, após modificação, em 16 de outubro de 2000.

Evaluation of functional integrity of the spermatozoal plasma membrane, particularly the acrosome, is one of the most efficient ways to recognize the fertilization capacity of males in many species. Among the methods commonly used for this purpose are the hypoosmotic and osmotic resistance evaluation tests.

It is also well known that spermatic quality varies among boars of the same breed and even among ejaculates within animals. Under certain circunstances, the morphological examination of spermatozoa is not enough to characterize the spermatic longevity and the real reproductive potential of males. In these cases, the evaluation of the acrosome membrane functionality can be more precise and may predict the classification of males as better semen donors, particularly when large periods of storage are involved.

The traditional osmotic resistance test (ORT), described by Schilling et al. (1984), proposes the incubation of semen samples in a 150mOsmol solution for two hours, which is a considerable long period of time. Pérez-Llano et al. (1998) carried out a study with the objective of reducing this incubation period and were able to decrease it to five minutes using 75mOsmol hypoosmotic and 300mOsmol isosmotic solutions incubated at 37ºC. This latter approach was refered to as short osmotic resistence test (SORT).

Regarding the SORT test presents no difficulties in counting cells in the desired dilution of fresh semen; however, cell count is difficult in diluted and cooled semen under phase-contrast microscopy at 1,000´ magnification. Therefore, in the present paper, the objective was to establish a possible correlation between the original SORT test, in which samples are incubated in 300 and 75mOsmol solutions, and the modified SORT, where samples from sperm morphology were used instead of those incubated in a 300mOsmol solution, aiming to achieve the proportion of normal acrosomes (NAR).

Fifty samples of semen were diluted for both the SORT test (Perez -Llano et al., 1998) and for the modified SORT. The SORT test was carried out as described by Pérez-Llano et al. (1999), except for the hypoosmotic solution used, sodium citrate/fructose, which is the most appropriate solution for swine semen.

After the preparation of 300mOsmol and 75mOsmol SORT solutions, using sodium citrate/fructose, the test preparation involved the following steps: 1- addition of 100µl of semen (fresh or cooled) to 1,500µl of a 300mOsmol solution, incubation at 37ºC for five minutes and addition of 500µl of a formalin-saline solution (at 37ºC); 2- addition of 100µl of semen to 1,500µl of a 75mOsmol solution, incubation at 37ºC for five minutes, and addition of 500µl of a formalin-saline solution (at 37ºC); 3- morphological evaluation of spermatozoa under phase-contrast microscopy; 4- results are applied to the SORT formula:

SORT (5 min)=1/2[%NAR in 300mOsmol solution (5 min) + % NAR in 75mOsmol solution (5 min)]

The modified SORT was carried out according to the above items, except for the step of incubation in 300mOsmol sodium citrate/fructose which was omitted. Thus, modified SORT (5 min)=1/2[%NAR in samples from sperm morphology + % NAR in 75mOsmol solution (5 min)], being NAR = % of normal acrosomes

In order to investigate the possibility of replacing the 300mOsmol incubation by the evaluation of sperm morphology, the t test was applied (software Excel 7.0 Microsoft). This test showed no significant differences between the percentage of normal acrosomes in samples which had been incubated in isosmotic solution and those from sperm morphology. A significant correlation of 0.98 (Excel, 7.0) was observed between the results obtained from the SORT and from the modified SORT. This facilitates the test, since sperm morphology is easier to evaluate and there is no need to incubate samples twice. The modified SORT had the advantage of eliminating one incubation solution and reducing the lecture time.

The results demonstrate that the modified SORT can be used to evaluate the osmotic resistance of boar spermatozoa.

Figures 1 and 2 show some examples of spermatozoa found after incubation in a 75mOsmol sodium citrate/fructose solution. Figure 1 depicts a spermatozoon with intact acrosome membranes, even after the osmotic stress induced by the test, while in Figure 2, the spermatozoon is seen with coiled tail and swollen acrosome.

Keywords: Boar, spermatozoa, osmotic resistance

RESUMO

Neste trabalho descreve-se a técnica do teste de resistência osmótica modificado (SORT modificado) e sua eficiência na avaliação da integridade da membrana do acrossoma do espermatozóide do varrão. Modificaram-se as soluções usadas nos testes de resistência osmótica atuais, eliminando-se também a incubação em solução isosmótica (300 mOsml). Esta foi substituída pela percentagem de NAR das amostras de morfologia espermática.

Palavras-chave: Varrão, espermatozóide, resistência osmótica

• PEREZ-LLANO, B., SANCHEZ-SANCHEZ, J.L., LORENZO-GONZALEZ, P. et al. Study of relationship between host and fertility in boar semen. In: International Conference on Boar Semen Preservation, 4, 1999, Beltsville. Proceedings.. Beltsville: IPVS, 1999, p.14.
• PEREZ-LLANO, B., SANCHEZ-SANCHEZ, J.L., LORENZO-GONZALEZ, P. et al. A short version of the osmotic resistance test for boar semen. In: Congress of the International Pigs Veterinary Society, 15, 1998, Birmingham. Proceedings.. Birmingham: IPVS, 1998, p.61, a.
• SCHILLING, E., VENGUST, M., SMIDT, D. ORT - A new test to predict the freezebility and storage of boar spermatozoa. In: Congress of the International Pigs Veterinary Society, 8, 1984, Ghent. Proceedings.. Ghent: IPVS, 1984, p. 296.

Publication Dates

• Publication in this collection
  07 June 2002
• Date of issue
  June 2001

History

• Received
  16 Oct 2000