Cryopreservation of cooled semen for equine intracytoplasmic sperm injection
Date
2010
Authors
Daigneault, Bradford W., author
Carnevale, Elaine M., advisor
Denniston, David J., committee member
Graham, James K., committee member
Bruemmer, Jason E., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
The development of intracytoplasmic sperm injection (ICSI) for use in the horse has resulted in unique needs for semen. Modifications of ways to handle sperm for ICSI-related procedures were investigated through a series of three experiments. In Experiment 1, semen was cooled for 24 h and then frozen to provide a viable population of sperm for ISCI. The objectives of this study were to compare the efficacy of: (1) two extenders for cooling semen prior to freezing and (2) four extenders for freezing cooled semen. Stallion semen was extended in two commercial cooling extenders (CST and INRA96) and cooled for 24 h before freezing in four extenders. Freezing extenders were a modified- French (FR8), Lactose-EDT A (LAC 10), glycerol (G), and glycerol egg yolk (GEY). Extenders were added directly to cooled semen and diluted to a final concentration of 20 x 106 sperm/mL before freezing. After thawing, sperm were evaluated at 0, 45 and 75 min. Cooling extender did not affect sperm motility after freezing. Total motility and progressive motility was similar (p>0.05) for all freezing extenders at O and 45 min, but sperm frozen in glycerol egg yolk (GEY) yielded the highest total and progressively motile sperm at 75 min. Differences in motility over time were not detected except for sperm frozen in glycerol (G) in which total and progressive motility declined from Oto 75 min. For all extenders, some motile sperm were obtained from semen cooled for 24 h and then frozen. In Experiment 2, five media were evaluated for holding sperm to determine which best maintained the motility of stallion sperm over time. The experiment was designed to determine appropriate media in which to handle sperm for ICSI selection procedures. The media compared were: 1) GIVF, 2) FDCM, 3) TALP, 4) Emcare (EM) and 5) Mare Mojo (MM). The only media designed for holding sperm was T ALP; the remaining media were formulated as holding media for oocytes and embryos. Motility of sperm was analyzed every hour for 5 h. No differences in total or progressive motility were detected among groups until 5 h. At 5 h, sperm held in TALP had higher (p<0.05) total and progressive motility than all other groups. Sperm can be held in any of the five media evaluated and remain motile for ICSI. However, T ALP resulted in the best sperm motility over time. Experiment 3 was performed to evaluate a hyaluronic acid (HA) binding assay for the selection of sperm for ICSI. Sperm binding to hyaluronic acid has been characterized for many species. Sperm that bind to HA have been shown to exhibit more normal chromosomal structures and improve fertilization. The objective of Experiment 3 was to determine if fresh and frozen stallion sperm bind to HA when introduced to premanufactured hyaluronic acid binding kits (HBA, Biocoat Inc., Horsham, PA). Fresh semen pooled from two stallions exhibited approximately 15% of sperm bound to HA. Four frozen samples from Experiment 1 were thawed and pooled. Frozen sperm did not bind to the assays. For fresh and frozen samples, sperm displayed some hyperactivation, uncharacteristic circling, and tails bent at the mid-piece. Sperm were observed to agglutinate in congregations of fifty or more sperm. Hyaluronic acid seemed to have an effect on stallion sperm that has not been described for other species. Further investigation of HA binding for selection of sperm for ICSI is warranted.
Description
Rights Access
Subject
Horses -- Germplasm resources -- Cryopreservation
Stallions -- Spermatozoa