Development of a protocol for multiple staining with fluorochromes to assess the functional status of boar spermatozoa.
The aim of this study was to design a simple and reliable method for the simultaneous evaluation of the nucleus, the acrosome, and the mitochondrial sheath of boar spermatozoa. Sperm samples coming from healthy and sexually mature Pietrain boars were incubated with two nuclear fluorochromes--bis-benzamide specific for viable cells, and propidium iodide specific for nonviable cells--the fluorochrome Mitotracker Green FM specific for functional mitochondria, and the lectin Trypsin inhibitor from Soybean (SBTI) conjugated with the fluorochrome Alexa Fluor 488 specific for proacrosin. The results obtained from assessing the functional status of the spermatozoa using fluorochromes were compared with the conventional sperm parameters of sperm vitality using the eosin exclusion test (EE test), and sperm motility and morphology using the computer-assisted semen analyzer SCA 2002Producció. Applying the multiple staining test, it was found that the frequency of viable spermatozoa with intact acrosome and intact mitochondria was not different from the frequency of viable spermatozoa obtained with the EE test, and also correlated positively with the frequency of motile spermatozoa and the frequency of mature spermatozoa. Therefore, this technique is useful to characterize the status of boar spermatozoa by assessing the nuclear, acrosomal, and mitochondrial integrity. Moreover, it provides reliable diagnostic information about the fertility potential of boars.
Bussalleu
Eva
E
Biotechnology of Porcine Reproduction, Department of Biology-INTEA, University of Girona,Campus de Montilivi s/n, 17071 Girona, Spain. eva.bussalleu@udg.es
Pinart
Elisabet
E
Yeste
Marc
M
Briz
Mailo
M
Sancho
Silvia
S
Garcia-Gil
Nuria
N
Badia
Elena
E
Bassols
Judit
J
Pruneda
Anna
A
Casas
Isabel
I
Bonet
Sergi
S
D000177:Acrosome / Q000648:ultrastructure; D000818:Animals; D005456:Fluorescent Dyes / Q000378:metabolism*; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000648:ultrastructure; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure; D013194:Staining and Labeling / Q000379:methods*; D034421:Sus scrofa
D005456:Fluorescent Dyes