The effect of sperm and cryoprotectant concentration on the freezing success of sex sorted ram sperm for in vitro fertilization.
The current method used to sex-sort ram sperm resulted in a dilute end product. The obligatory removal of cryopreservation medium during preparation of sperm IVF further reduced sperm number. This study aimed to increase the number of viable, sex-sorted sperm available for IVF by increasing their pre-freeze concentration and assessing the cryodiluent concentration used to accommodate this change. In Experiment 1, semen was collected from Merino rams (n = 3), sex-sorted, and then frozen at concentrations of 20, 40, or 80 x 10(6) sperm/mL in three forms of tris-citrate-glucose cryodiluent containing 5% (L-Cryo), 6% (M-Cryo), and 8% (H-Cryo) (v/v) glycerol. Motility, plasma membrane and acrosome integrity, and mitochondrial activity were assessed at 0, 2, 4, and 6 h post thaw. In Experiment 2, cleavage and blastocyst development rates were compared between non-sorted and sex-sorted sperm frozen at the aforementioned concentrations (in the cryodiluent most effective in Experiment 1). In Experiment 1, total motility between 0 and 6 h was similar for all sperm concentrations when frozen using L-Cryo. Mitochondrial activity was elevated in samples frozen in L-Cryo and M-Cryo at 0 h compared to those preserved in H-Cryo for all concentrations (P < 0.05). In Experiment 2, sex-sorted sperm with a higher pre-freeze concentration yielded a higher sperm concentration after preparation for IVF (8.57 +/- 1.22 sperm/mL), compared to the lowest group (2.96 +/- 0.18 sperm/mL; P < 0.05). There were no significant differences between non-sorted and sex-sorted sperm for rates of embryo cleavage or development. Therefore, sex-sorted sperm was effectively cryopreserved at a higher concentration than conventionally practiced. Although this yielded a higher sperm concentration for IVF, reduced insemination volume, and increased the number of potentially fertile gametes from which to select, fertilisation rate was not significantly improved.
Faculty of Veterinary Science, The University of Sydney, NSW, Australia. kirib@vetsci.usyd.edu.au
K H
KH
Beilby
S P
SP
de Graaf
C G
CG
Grupen
D000177:Acrosome / Q000502:physiology; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents; D047108:Embryonic Development; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D008928:Mitochondria / Q000502:physiology; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012743:Sex Preselection / Q000662:veterinary*; D012756:Sheep; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology*
D003451:Cryoprotective Agents