Article

Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats.

Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
Comparative medicine (Impact Factor: 1.12). 01/2004; 53(6):639-41.
Source: PubMed

ABSTRACT We attempted to cryopreserve spermatozoa from closed colonies (Jcl:SD and Jcl:Wistar), and inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant (Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A], and green fluorescent protein [GFP]) strains of rats. Rat epididymal spermatozoa suspended in cryopreservation solution (23% egg yolk, 8% lactose monohydrate, and 0.7% Equex Stm, pH 7.4, adjusted with 10% Tris [hydroxymethy] aminomethane) were frozen and stored at -196 degrees C. After thawing at 37 degrees C, the spermatozoa were instilled into the tip of each uterine horn of the recipients. A total of five recipient females for each strain were inseminated with cryopreserved spermatozoa, and normal live offspring of all strains (Jcl:SD: 11, Jcl:Wistar: 13, BN/Crj: 9, F344/DuCrj: 28, LEW/Crj: 4, Long-Evans: 6, WKY/NCrj: 8, TRM: 24, WTC.ZI-zi: 27, A: 30 and GFP: 20) were obtained.

0 Bookmarks
 · 
52 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sperm preservation protocols differ among animal species because of different sperm characteristics among species. Rat sperm have extreme sensitivity to suboptimal conditions in centrifugation, pipetting and chilling due to their longer tail, the shape and size of the sperm head, and membrane composition. The aim of this study was to determine optimal conditions for short-term storage of rat sperm by evaluating their motility and membrane and acrosomal integrity in response to various extender solutions, temperatures, and durations. Motility of rat sperm was highest when stored at 22 °C; motility was 28% and 14% at 72 h in TL-HEPES and PBS extenders, respectively. The motility and membrane integrity of rat sperm fell significantly within 24 h at 4 and 37 °C. Although cold storage did not have a detrimental effect on acrosomal integrity of sperm, room temperature storage reduced acrosomal integrity after 24 h. LEY extender caused the highest loss in acrosomal integrity at 48 and 72 h. In conclusion, storage at 4 or 37 ° C reduced the motility and membrane integrity of rat sperm even with short incubation periods. Rat sperm stored in TL-HEPES or PBS remained motile for at least 3 d when held at 22 °C.
    Journal of the American Association for Laboratory Animal Science: JAALAS 01/2013; 52(6):732-7. · 1.15 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Current article explains about the effects of cryopreservation on testicular sperm DNA. Conservation of sperms has several purposes in artificial reproductive technologies (ART), species conservation and clinical medicine. Despite of various advances in cryopreservation methodology, the recovery rate of functional post thawed spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. Sperm cryopreservation and storage facility currently require liquid nitrogen method for long or short term storage. The combinations of sperm storage involved are temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control factors that affect the life-span of spermatozoa. The lack of structural integrity to sperm and damage to DNA of sperm shall have a significant negative impact on oocyte fertilization, embryo development rate, and live-birth rate. Hence the research conducted as on today conclude that cryopreservation of testicular spermatozoa may reduce DNA life expectancy; therefore scientists are yet in a position to rectify this troubleshoot for ultimate shell of life of DNA for prolonged periods without damage. This review describes about the effect of oxidative stress and apoptosis as one of the possible mechanisms involved in sperm cryoinjury.
    International Journal of Pharmacy Research and Technology. 12/2012; 2(4):1-9.
  • [Show abstract] [Hide abstract]
    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.
    Cryobiology 05/2013; · 2.14 Impact Factor