UTILIZAÇÃO DE GLICEROL E ETILENOGLICOL COMO CRIOPROTETORES NA CONGELAÇÃO DO SÊMEN CAPRINO
Ciência Animal Brasileira
Dez amostras de sêmen de dois reprodutores caprinos, da raça Parda-alpina, colhidas em vagina artificial, foram submetidas a quatro tratamentos para avaliação da eficiência do etilenoglicol e do glicerol, associados ou não ao EDTA, na criopreservação da célula espermática caprina. O diluente usado era à base de Tris-gema de ovo contendo 7% de glicerol (glicerol E glicerol+EDTA) ou 7% de etilenoglicol (etilenoglicol e etilenoglicol + EDTA), sendo que nos grupos glicerol+EDTA e etilenoglicol+EDTA foi associado ao diluente 0,1% de EDTA. As amostras foram mantidas por 60 minutos em geladeira a 40C, onde então era efetuada a congelação em nitrogênio (-1960C). A descongelação foi realizada em banho-maria a 370C por 30segundos. As médias obtidas para motilidade (%) a descongelação, para os grupos glicerol, glicerol+EDTA, etilenoglicol, etilenoglicol+EDTA, foram, respectivamente, 51%; 61%; 10% e 12%. Os grupos que utilizaram o glicerol como crioprotetor obtiveram melhores taxas de motilidade pós-descongelação, principalmente quando foi associado ao diluidor o EDTA (grupo glicerol+EDTA). Porém, esse resultado foi comprometido pelos maiores índices de alterações patológicas nos grupos que continham o glicerol.
PALAVRAS-CHAVE: Caprinos, criopreservação, sêmen, glicerol, etilenoglicol, EDTA
Available from: Eunice Oba
Available from: Alexandre Silva
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ABSTRACT: Background: The development of semen cryopreservation protocols for collared peccaries (Pecari tajacu) would contribute to its preservation and multiplication. Nowadays, studies on this subject report the use of Tris and powdered coconut water (ACP (R)-116c) as extenders. However, such studies are limited to the post-thawing immediate evaluation of samples. The knowledge on the sperm survival after thawing would be important in order to schedule the moment and the vial for artificial insemination. Therefore, the aim of the present research was to verify the effect of Tris and ACP extenders on the sperm survival of frozen-thawed semen, centrifuged or not, derived from collared peccaries. Materials, Methods & Results: Semen from 12 adult males collared peccaries were obtained by electroejaculation. Ejaculates were immediately evaluated for sperm motility, vigor, viability, functional membrane integrity and morphology. Samples were diluted in Tris or ACP, both plus 10% egg yolk and 3% glycerol. Then, they were packaged into 0.25 mL straws, and stored in liquid nitrogen. After one week, samples were thawed at 38 degrees C/1 min and divided into two aliquots: one immediately diluted in the same extender, and the other submitted to centrifugation and then re-diluted. A thermorresistance test was conducted including assessments at 0, 15, 30, 45, and 60 min as described for fresh semen. After analyzing the data immediately after thawing, we verify that coconut water provided a better preservation of sperm motility (41.3 +/- 6.9%) and vigor (2.9 +/- 0.2) than Tris, 33.1 +/- 5.9% motile sperm with vigor 2.5 +/- 0.2 (P < 0.05). During thermorresistance test, however, semen parameters in re-diluted samples rapidly decline after 15 min incubation, in the use of both extenders, independently of the centrifugation (P < 0.05). After 60 min, values for sperm motility were lower than 10% for all the samples, corresponding to the values found for sperm viability. In this moment, no statistical differences were verified between extenders in the samples centrifuged or not (P > 0.05). Discussion: As the first study at evaluating the effect of different extenders on collared peccary sperm survival, it was observed that its frozen-thawed semen samples centrifuged or not, present a very low post-thawing longevity in the use of Tris or ACP extenders. These are, nowadays, the unique extenders reported for collared peccary semen cryopreservation and both seems to be efficient for this proposal. However, an immediate use of the thawed samples is recommended, especially for intrauterine artificial insemination. A reduced longevity after thawing is also observed for swine sperm, the domestic animal more closely related to the peccaries. It is known that swine sperm present great amounts of unsaturated lipids on its plasma membrane, and due to this characteristic, it is highly sensible to freezing-thawing procedures. Due to similarities among swine and peccaries, such characteristic could possibly be extrapolated to the peccaries, but further studies regarding its sperm membrane composition should be conducted. In conclusion, collared peccaries sperm presents a short post-thawing survival, both in the use of Tris or ACP extenders, and its immediate use is recommended. Furthermore, no benefits are derived from the centrifugation process that seems to be unnecessary for frozen-thawed semen samples.
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