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.
Beilby
K H
KH
Faculty of Veterinary Science, The University of Sydney, NSW, Australia. kirib@vetsci.usyd.edu.au
de Graaf
S P
SP
Grupen
C G
CG
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