Single layer centrifugation through puresperm® 80 improves quality of cryopreserved dog spermatozoa

LUCRECIA ALCARAZ; MANUEL HIDALGO; MARIA JOSE GALVES; DANIEL ACHA VALLS; ISABEL ORTIZ; SEBASTIÁN DEMYDA PEYRÁS; CARLOS GONZALES; JOSE MANUEL PORTERO; FABIOLA QUESADA; LUISA RAMIREZ; JESUS DORADO

Hannover

Congreso; 39th Conference of the International Embryo Society; 2013

International Embryo Transfer Society

Density gradient centrifugation with PureSperm
(PureSperm
40þPureSperm
80; Nidacon International, Mo¨lndal, Sweden) has been satisfactorily used to enhance the quality of dog semen samples; however, no studies have been performed on the effect of single layer centrifugation (SLC) with PureSperm
on frozen–thawed dog semen. The aim of this study was to investigate if SLC with PureSperm
80 can improve the postthaw semen quality of dog. Semen from 5 dogs was collected by digital manipulation. Two ejaculates from each dog were centrifuged with Tris-based extender, supernatant was removed, and sperm pellet was suspended to a final concentration of 300–400106 spermmL 1 with CaniPROTM Freeze Aplus 20%egg yolk at 228C. Extended semenwas cooled to 58Cwithin an hour and then diluted to a final concentration of 150–200106 spermmL 1 in CaniPROTM Freeze B plus 20% egg yolk at 58C, loaded in 0.5-mL plastic straws and frozen horizontally in ranks placed 4 cm above the surface of liquid nitrogen vapors for 10 min, after which they were directly placed in liquid nitrogen. After 24 to 48 h of storage, straws were thawed in a water bath at 378C for 30 s. After thawing, semen samples were divided in 2 aliquots: one of them was used as control and the other one was processed by SLC PureSperm
80. Assessment of sperm motility (assessed by computerized-assisted semen analysis), morphology (Diff-Quick staining), and viability [triple fluorescent stain of propidium iodine/isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin/Rhodamine 123] were evaluated in control and treated semen samples. Data were studied by ANOVA. Results are expressed as meanSEM. Significant (P,0.001) differences were found between SLC-treated and control semen for sperm motility (percentage of total motile spermatozoa: 93.650.05 v. 83.790.13; percentage of progressive motile spermatozoa: 79.386.66 v. 54.6116.11), morphology (86.450.01 v. 83.510.01), and viability (percentage of viable sperm with an intact acrosome: 58.320.04 v. 36.500.17; percentage of viable sperm with an acrosome reaction: 2.810.01 v. 9.740.21). Based on our results, we can conclude that SLC with PureSperm
80 is an alternative and successful method for improving the quality of frozen–thawed dog spermatozoa, selecting good-quality spermatozoa (motile, morphologically normal, viable, and acrosome intact spermatozoa) from the rest of the semen sample.