To date, freezing extender containing 23% (v/v) egg yolk, 8% (w/v) lactose monohydrate has been most commonly used extender used to cryopreserve rat sperm [34]. All of these previous studies cooled straws containing rat sperm by holding 2 cm above the liquid nitrogen level (at −150 to −170 °C) for 10–15 min before plunging into LN2 [34], [56], [57] and [58]. However, they did not report exact cooling rates. To date there has been only one fundamental cryobiologic study investigating optimal cooling rate for rat sperm. Hagiwara et al. [19]

evaluated the biophysics (membrane permeability) mTOR inhibitor of rat sperm to better understand the cooling rate response that contributes to cryopreservation GSI-IX cost success. A differential scanning calorimeter studies predicted and experimentally tested optimal cooling rates that ranged from 53 to 70 °C /min for rat sperm. Maximum motility was obtained with cooling rates between 50 and 80 °C/min. This is one of the first studies which aimed at determining optimal cooling rate using a Linkam Cryostage. Optimal cooling rate varies from species to species. It has been shown for mouse sperm that cryo-survival

is depended significantly on the cooling rate, and less strongly associated with the warming rates as long as rapid warming (∼1000 °C/min) is used. In this study we also used the rapid warming (∼1000 °C/min). Cooling rate significantly affected post-thaw motility of SD sperm in TL-HEPES, m-KRB and TES-R extenders and motility of F344 sperm in TL-HEPES, SM, TES-R and TES-S extenders. In these extenders, post-thaw motility decreased

significantly in 10 °C/min cooling rate compared to 100 °C/min cooling rate. The highest motility was obtained oxyclozanide when rat sperm was cooled between 40 and 100 °C/min. This is consistent with the previous report from Hagiwara et al. [19] in that maximum motility was obtained with cooling rates between 50 and 80 °C/min. In this study we did not investigate cooling rate higher than 100 °C/min because of the limitation of Linkam cooling stage. Most commonly used cooling machines in laboratories cannot reach controlled cooling rate of 100 °C/min and above. It is accepted that constant cooling of rat sperm cannot be achieved in LN2 which cools sperm between 100 and 130 °C/min. Stacy et al. [47] has elegantly demonstrated low reproducibility of freezing protocols due mainly to variation in cooling rate in LN2 vapor. For mouse sperm, cooling rate of 114 °C/min resulted in higher motility than cooling rate of 39 °C/min but, cooling rate of 192 °C/min led to the lowest motility [47]. Similarly, Koshimoto and Mazur [27] showed that cooling rate between 27 and 130 °C/min resulted in more motile sperm compared to the lower or higher rates. In this study, freezing and thawing of rat sperm resulted in decrease in motility, plasma membrane integrity, acrosome integrity and MMP.