The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Abstract

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10–12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4°C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P<0.05). Sperm motility increased as cooling rate was increased for both strains (P<0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C /min and 100 °C /min cooling rate improved post-thaw motility of rat sperm.

Influence of extenders and cooling rates on epididymal sperm of Lewis rat strain

“…The optimal cooling rate was reported to between 76 and 140°C/min in bull (51), 30°C /min in boar (12), 27°C-130 °C /min in mouse (25) and 10°C/min for human (16). An appropriate cooling rate for rat sperm has been reported to be 40 and 100°C/min in F344 and SD rats (46). Similarly one study (14) found optimum cooling rate between 53 and 70°C/min in SD rats.…”

“…The difficulty of freezing rat sperm is probably due to extreme sensitivity of membrane integrity and mitochondrial membrane to freezing process (46). Also an acceptable motility and fertility rate of rat sperm have not been reached (17,18,31,46,52,54), compared with mouse sperm (29,43).…”

“…Mouse sperm is successfully cryopreserved with a basic freezing medium consisting of 18% raffinose and 3% skim milk (29). However skim milk has been demonstrated to be inefficient in rat strains (46). Another important chemicals for rat sperm cryopreservation is sodium dodecyl sulphate (SDS)-based products, which play important role to improve the effectiveness of EY during sperm freezing in rat (31,46,53) mouse (37), cat (4), dog (36), and pig (8).…”

“…However, raffinose is an effective cryoprotectant for mouse and rat (24, 34,52). Also sucrose has a cryoprotective capability similar to raffinose in rat (46).…”

“…However skim milk has been demonstrated to be inefficient in rat strains (46). Another important chemicals for rat sperm cryopreservation is sodium dodecyl sulphate (SDS)-based products, which play important role to improve the effectiveness of EY during sperm freezing in rat (31,46,53) mouse (37), cat (4), dog (36), and pig (8). Glycerol is the most common cryoprotectant used to freeze sperm from different species (38,39).…”