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New methods for cryopreserving rooster spermatozoa

Date

2013

Authors

Tarvis, Kimberly Margaret, author
Graham, James K., advisor
Purdy, Phillip H., committee member
Veeramachaneni, D. N. Rao, committee member
Pabilonia, Kristy L., committee member

Journal Title

Journal ISSN

Volume Title

Abstract

Rooster spermatozoa sustain permanent membrane damage during cooling, thawing and dilution due to inappropriate changes during the phase transition and osmotic intolerance. Glycerol is a common cryoprotectant used to protect cells during freezing, but its relatively large molecular weight and relatively low membrane permeability (compared to water) creates relatively long aniosmotic conditions when glycerol is added or removed. Glycerol-exposed rooster spermatozoa exhibit poor fertility, therefore, sperm must undergo an extensive dilution and sperm reconcentration process to remove glycerol and mitigate its negative effects on sperm. Alternative cryoprotectants with lower molecular weight than glycerol cross the membrane more quickly and induce less osmotic damage. Cryoprotectants that do not need to be removed from spermatozoa prior to insemination may eliminate the need for processing the sperm after thawing. The first experiment determined the percentages of motile and membrane intact cells of frozen/thawed spermatozoa cryopreserved in different cryoprotectants. Samples exposed to glycerol exhibited higher percentages of total motile and membrane-intact sperm (54% and 58%, respectively) compared with sperm treated with methylacetamide (48% & 53%, respectively), dimethylacetamide (41% & 45%, respectively), ethylene glycol (38% and 39%, respectively), or methylformamide (28% and 37%, respectively; P < 0.05). Exposure of sperm to a cryoprotectant for a prolonged period of time may have negative effects on the sperm. Rooster sperm, when thawed for insemination, may be kept undiluted and exposed to cryoprotectant for an extended period of time before used. The second experiment evaluated if exposing sperm to methylacetamide for a prolonged period of time affected sperm motility. Exposure of sperm to methylacetamide did not decrease the percentage of total motile sperm over a 1 hour period (> 50%, P > 0.05). When sperm are cooled from 5&deg;C to &ndash;196&deg;C, membrane damage occurs due to membrane changes during the phase transition. This damage, for sperm from many species, can be mitigated by adding cholesterol or unsaturated phospholipids to the membrane. In the next set of experiments, cholesterol-loaded cyclodextrins (CLC) did not change rooster sperm osmotic tolerance limits or improve cryosurvival rates, even though cellular cholesterol levels were increased 2&ndash;fold. Similarly, treating sperm with lipid-loaded cyclodextrins (LLC) failed to improve sperm cryosurvival rates. Diluent composition, cryoprotectant, sperm concentration and freezing procedure affect rooster spermatozoa cryosurvival, with each diluent having its own freezing protocol. Two different diluents, a glutamate-based and a trehalose-based diluent were used to cryopreserve rooster spermatozoa, since sperm in the latter diluent has been reported to have increased fertility when left undiluted with an methylacetamide. The last set of experiments evaluated the percentages of total motile cells of frozen/thawed when glycerol or methylacetamide were used to cryopreserve rooster spermatozoa in trehalose-based or glutamate-based diluents packaged in 0.5&ndash;mL or 0.25&ndash;mL straws. Samples frozen in 0.5&ndash;mL straws exhibited higher percentages of motile cells (> 45%) than sperm frozen in 0.25&ndash;mL straws (< 45%; P < 0.05). Within the samples frozen in 0.5&ndash;mL straws, sperm cryopreserved in trehalose-based diluent exhibited higher percentages of motile cells (> 60%) than sperm frozen in glutamate-based diluent with glycerol as the cryoprotectant (46%; P < 0.05). The last experiment evaluated the percentages of motile and membrane-intact cells of frozen/thawed glycerol-exposed or methylacetamide-exposed rooster spermatozoa in trehalose-based or glutamate-based diluents. These sperm were frozen in 0.5&ndash;mL or 0.25&ndash;mL straws at two different sperm concentrations, 500 million cells/mL or 1 billion cells/mL. Sperm cryopreserved in trehalose-based medium and packaged in 0.5&ndash;mL straws can be frozen at 1 billion cells/mL or 500 million cells/mL, indicating that sperm can be effectively frozen at sufficiently high concentrations to permit freezing, thawing and inseminating sperm without having to concentrate the sperm prior to insemination. If methylacetamide does not have to be removed from the sperm prior to insemination, this technique could be easily incorporated by breeders and researchers to use frozen sperm. Rooster spermatozoa are unique cells that can tolerate a variety of environments when cooled, but after cryopreservation, many different changes occur to the sperm membrane that can cause infertility. Methylacetamide-exposed sperm elicit smaller osmotic changes during cooling, thawing and dilution, has been shown to increase sperm cryosurvival.

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Subject

cryobiology
cryoprotectant
cyclodextrin
extender
rooster
sperm

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