title: IVF recovery of mutant mouse lines using sperm cryopreserved with mtg in cryovials.
abstract: BACKGROUND
  Modification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown.
  
  
  OBJECTIVE
  The study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws.
  
  
  METHODS
  This study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds.
  
  
  RESULTS
  Glutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P < 0.05). Sperm cryopreserved in R18S3+MTG had significantly better (P < 0.05) post-thaw progressive motility and IVF rate than when cryopreserved in R18S3 alone, R18S3+Glu (100 mM), or RSGlu87 (15.7% raffinose, 2.6% skim milk, and 87 mM L-glutamine). There was no significant difference in IVF rates among sperm cryopreserved with R18S3+MTG in cryovials or in cryostraws (P > 0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring.
  
  
  CONCLUSIONS
  Mouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.
authors.forename: Ming-Wen
authors.initials: MW
authors.lastname: Li
authors.forename: Jadine M
authors.initials: JM
authors.lastname: Vallelunga
authors.forename: Kristy L
authors.initials: KL
authors.lastname: Kinchen
authors.forename: Karina L
authors.initials: KL
authors.lastname: Rink
authors.forename: Jasmin
authors.initials: J
authors.lastname: Zarrabi
authors.forename: Armen O
authors.initials: AO
authors.lastname: Shamamian
authors.forename: K C Kent
authors.initials: KC
authors.lastname: Lloyd
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D005307:Fertilization in Vitro; D060005:Genotyping Techniques; D005973:Glutamine / Q000494:pharmacology; D005990:Glycerol / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008892:Milk / Q000737:chemistry; D011887:Raffinose / Q000494:pharmacology; D012662:Semen Preservation / Q000295:instrumentation* / Q000379:methods; D013076:Sperm Count; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*
chemical_list: D003451:Cryoprotective Agents; D005973:Glutamine; C009465:thioglycerol; D011887:Raffinose; D005990:Glycerol