Functional integrity of Colossoma macropomum (Cuvier, 1816) sperm cryopreserved with enriched extender solutions

Abstract

Cryoprotectant solutions are used to protect the sperm from alterations caused by the low temperature in the cryopreservation process. We evaluated the quality of Colossoma macropomum semen after freezing, using dimethyl sulfoxide (DMSO) as a cryoprotectant, combined with two extender solutions (T1 -Solution 1: Glucose 90.0 g/L, Sodium Citrate 6.0 g/L, EDTA 1.5 g/L, Sodium Bicarbonate 1.5 g/L, Potassium Chloride 0.8 g/L, Gentamycin Sulphate 0.2 g/L, and T2 -Solution 2: Glucose 90.0 g/L, ACP ® -104 10.0 g/L). Motility rate and motility time did not differ between T1 and T2 and were lower than fresh semen. The number of normal sperm was significantly different in treatments T1 (15.1%) and T2 (21.9%), and both showed a reduction in the percentage of normal sperm compared to fresh semen (57.4%). The values found for the rates of fertilization and hatching, mitochondrial functionality and sperm DNA, did not differ between the treatments (T1 and T2). Regarding membrane integrity, there was a higher percentage of spermatozoa with intact membranes in T1 (53.4%) than T2 (43.7%). The extender solutions, combined with 10% DMSO, maintained the sperm DNA intact in almost all the C. macropomum sperm cells, however there was a loss in their functionality.As soluções crioprotetoras são utilizadas para proteger os espermatozoides das alterações causadas por baixas temperaturas durante o processo de criopreservação. Avaliamos a qualidade do sêmen de Colossoma macropomum após o congelamento, utilizando dimetilsulfóxido (DMSO) como crioprotetor, combinado com duas soluções diluidoras (T1 -Solução 1: Glicose 90,0 g/L, Citrato de Sódio 6,0 g/L, EDTA 1,5 g/L, Bicarbonato de Sódio 1,5 g/L, Cloreto de Potássio 0,8 g/L, Sulfato de Gentamicina 0,2 g/L, e T2 -Solução 2: Glicose 90,0 g/L, ACP®-104 10,0 g/L). A taxa de motilidade (%) e o tempo de motilidade (s) não diferiram entre T1 e T2, porém foram mais baixos do que no sêmen fresco. O número de espermatozoides normais foi significativamente diferente nos tratamentos T1 (15,1%) e T2 (21,9%), e ambos mostraram uma redução na porcentagem de espermatozoides normais, comparado ao sêmen fresco (57,4%). Os valores encontrados para as taxas de fertilização e eclosão, funcionalidade mitocondrial e DNA do esperma, não diferiram entre os tratamentos (T1 e T2). Para a integridade da membrana, houve uma porcentagem mais elevada de espermatozóides com a membrana intacta em T1 (53,4%) do que T2 (43,7%). As soluções diluentes combinadas com DMSO a 10% preservaram o DNA espermático intacto em quase todas as células do sêmen de C. macropomum, mas houve perda na funcionalidade dos mesmos.

Crioconservación seminal de Colossoma macropomum como estrategia de producción y conservación en la Orinoquia Colombiana

“…Por otra parte, Varela et al, (2012) concluyen que la adición de DMSO a una concentración de 10% se asoció con una mejor calidad del semen postdescongelación de Tambaquí, cuando comparado con concentraciones de 5, 15 y 20%. No obstante, García et al, (2015) demuestran que el uso se soluciones extensoras combinadas con un 10% de DMSO, mantuvieron intacto el ADN del espermatozoide en casi todas las células espermáticas de C. macropomum, aun cuando hubo pérdida importante en su funcionalidad. Aunque en éste estudio no fue evaluada la integridad del ADN ni los efectos del tiempo de crioconservación sobre la calidad seminal postdescongelación y desarrollo larval, Calizaya y Yam (2013) reportan efectos negativos importantes sobre la progenie cuando comparado con semen fresco, variable que debe ser estudiada con fines de transferencia de la biotecnología a productores.…”

Semen Cryopreservation in Brazilian Freshwater Fish: Advances and Main Challenges

“…Currently, the BTS™ (Beltsville Thawing Solution-Minitub), which is formulated for swine semen is the basic solution that has been used in the composition of extenders [16][17][18][19]. Similarly, ACP-104™ (ACP Biotechnology-UECE), which was originally formulated for goats semen was also tested in some fish species [15,17,20,21]. However, seminal viability results obtained in the literature are very variable, indicating that there is a need to seek efforts to formulate specific extenders, based on biochemical composition of seminal plasma from each fish species.…”

“…However, for P. lineatus the thawing temperature may vary from 30 to 60°C for 8 or 16 s, respectively, without major changes in motility rate when using 0.5 ml straws [18]. For C. macropomum semen, a thawing temperature of 45°C for 5-8 s [20,24] when using 0.25 ml straw and for 0.5 ml straw 37°C/30 s or 60°C/8 s was used [13,21]. However, at larger scale using 1.6 or 4.5 ml straws these rates can be of 60°C for 90 s [25].…”

Cryopreservation of sperm in annual fish Austrolebias minuano (Cyprinodontiformes; Rivulidae)

“…Dimethyl sulfoxide (DMSO), glycerol and methyl glycol (MG) are common intracellular cryoprotectants used for cryopreservation of fish spermatozoa (Viveiros et al, ). Indeed, alcohol‐based cryoprotectants are widely used for cryopreservation of sperm from freshwater fish: Piracanjuba ( Brycon orbignyanus ; Perry et al, ), Tambaqui ( Colossoma macropomum ; Garcia et al, ; Varela Junior et al, ), Curimba ( Prochilodus lineatus ; Miliorini et al, ; Viveiros et al, ), Pacu ( Piaractus mesopotamicus ; Pires et al, ).…”