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Cryopreservation of rooster sperm using methyl cellulose
J J Phillips1, R K Bramwell, J K Graham
1Department of Physiology, Colorado State University, Fort Collins 80523, USA.
Abstract:
Experiments were designed to determine when, during the cryopreservation process, sperm lose fertilizing capacity and whether the cryoprotectant, methyl cellulose (MC), could be used in combination with glycerol to cryopreserve sperm and remain in the inseminate without reducing fertility. Semen diluted in Minnesota Avian extender (MNA) and inseminated immediately had greater fertility (75%) than semen processed for cryopreservation (12 to 60%). The largest decreases in fertility were due to addition of glycerol to sperm and to cryopreservation. In another experiment, fertility of inseminates containing 0, 1, and 2% glycerol were 82, 29, and 21%, respectively, for eggs collected 2 to 5 d after insemination. When 0.5% MC was added to the same three treatments, fertility rates were 88, 63, and 69%, respectively. Semen cryopreserved in MNA containing 9% glycerol; MC + 3% glycerol; MC + 4% glycerol; MC + 9% glycerol; or 9% glycerol with the cryoprotectant removed post-thaw by dilution and subsequent centrifugation exhibited 59, 30, 35, 60, and 69% viable cells, respectively; and 65, 38, 46, 69, and 65% motile sperm, respectively. Sperm cryopreserved with MC and either 4 or 9% glycerol exhibited similar numbers of sperm binding to chicken perivitelline layers in vitro as did fresh sperm, whereas sperm frozen with MC and 3% glycerol bound oocytes with only 31% efficiency (P < 0.05). The extent to which cryopreserved sperm penetrated the perivitelline layer in vitro was independent of glycerol concentration, but was four times more efficient than that of fresh sperm (P < 0.05). The fertility rates of fresh semen, semen frozen in 9% glycerol with the cryoprotectant removed after thawing, and semen frozen in MC with either 3 or 4% glycerol were 87.4, 27.6, 0.8, and 0.5%, respectively (P < 0.05). The MC reduces the contraceptive effects of glycerol when inseminated with fresh sperm, but does not maintain fertilizing capacity in frozen-thawed sperm when used in combination with 3 or 4% glycerol.
Insights
Methyl cellulose (MC) reduces glycerol
Area of Science:
- Animal Science
- Reproductive Biology
- Cryobiology
Background:
- Cryopreservation of avian sperm is crucial for genetic resource preservation.
- Glycerol is a common cryoprotective agent but can reduce sperm fertility.
- Methyl cellulose (MC) is investigated as a potential adjunct to glycerol.
Purpose of the Study:
- To determine when sperm lose fertilizing capacity during cryopreservation.
- To evaluate the efficacy of methyl cellulose (MC) in combination with glycerol for avian sperm cryopreservation.
- To assess if MC can be included in inseminates without compromising fertility.
Main Methods:
- Semen was cryopreserved using various concentrations of glycerol and MC.
- Fertility was assessed by insemination and egg collection.
- Sperm viability, motility, and in vitro binding to perivitelline layers were evaluated.
Main Results:
- Cryopreservation significantly reduced sperm fertility compared to fresh semen.
- MC mitigated the negative effects of glycerol on fresh sperm fertility.
- Sperm cryopreserved with MC and 4% or 9% glycerol showed good viability and motility, but fertility outcomes varied.
- Cryopreserved sperm penetration of the perivitelline layer was more efficient than fresh sperm.
Conclusions:
- Methyl cellulose (MC) can reduce the contraceptive effects of glycerol in fresh avian sperm.
- MC does not fully maintain fertilizing capacity in cryopreserved sperm when used with 3-4% glycerol.
- Further optimization of cryopreservation protocols involving MC and glycerol is needed for avian sperm.