Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro.
Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.
Cesari
A
A
Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, UNMdP. CC:1245 (7600) Mar del Plata, Buenos Aires, Argentina. acesari@mdp.edu.ar
Kaiser
G G
GG
Mucci
N
N
Mutto
A
A
Vincenti
A
A
Fornés
M W
MW
Alberio
R H
RH
D000177:Acrosome / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D002417:Cattle / Q000502:physiology*; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D002499:Centrifugation, Density Gradient / Q000662:veterinary; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D008297:Male; D046529:Microscopy, Electron, Transmission / Q000379:methods / Q000662:veterinary; D008856:Microscopy, Fluorescence; D009865:Oocytes; D011205:Povidone / Q000494:pharmacology; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary; D012744:Sex Ratio; D012822:Silicon Dioxide / Q000494:pharmacology; D013076:Sperm Count / Q000662:veterinary; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure*
C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone