Archived Protocol

<!doctype html public "-//w3c//dtd html 4.0 transitional//en"> <html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:dt="uuid:C2F41010-65B3-11d1-A29F-00AA00C14882" xmlns:st1="urn:schemas-microsoft-com:office:smarttags" xmlns="http://www.w3.org/TR/REC-html40"> <head> <meta name="Microsoft Theme 2.00" content="topo 011"> <meta http-equiv=Content-Type content="text/html; charset=iso-8859-1"> <meta name=ProgId content=Word.Document> <meta name=Generator content="Microsoft Word 10"> <meta name=Originator content="Microsoft Word 10"> <link rel=File-List href="default_files/filelist.xml"> <link rel=Edit-Time-Data href="default_files/editdata.mso">  <title>Bovine IVF University of Florida</title> <o:SmartTagType namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="place"/> <o:SmartTagType namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="PlaceName"/> <o:SmartTagType namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="PlaceType"/> <o:SmartTagType namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="date"/>  <style>  </style>  <meta name=Author content="Peter J. Hansen">  </head> <body bgcolor=white background="default_files/image001.jpg" lang=EN-US link="#990000" vlink="#006666" style='tab-interval:.5in'>  <div class=Section1> <![if !vml]><img width=180 height=176 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image003.jpg v:shapes="_x0000_s1026"><![endif]><o:p> </o:p> Procedures for In Vitro Production of Bovine Embryos <o:p></o:p> R.M Rivera, J.L. Edwards, A.D. Ealy, V.M. Monterroso, A.C. Majewski, and P.J. Hansen Dept. of Animal Sciences, University of Florida The procedures for in vitro production [IVP; i.e. in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC)] of embryos described here are based on procedures developed in other laboratories at the University of Wisconsin (<a href="http://www.dps.ufl.edu/hansen/ivf/references.htm">Parrish et al., 1986</a>), University of Guelph  (<a href="http://www.dps.ufl.edu/hansen/ivf/references.htm">Xu et al., 1992</a>), and University of Missouri (<a href="http://www.dps.ufl.edu/hansen/ivf/references.htm">Hernandez-Ledezma et al., 1993</a>).  These procedures as used by our group have been published previously (<a href="http://www.dps.ufl.edu/hansen/ivf/references.htm">Edwards et al., 1997; Paula-Lopes et al., 1998</a>).  Keep in mind that the protocols described here are not fixed but rather constantly evolve as new developments take place.  Therefore, practitioners of IVP will be well advised to experiment with the procedures used, especially after reading of improvements made by other laboratories.  This protocol is organized by day of the protocol with d 0 being the day of fertilization. <div class=MsoNormal align=center style='text-align:center'><![if !vml]><img border=0 height=10 width=576 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image004.gif alt=--- v:shapes="_x0000_i1025"><![endif]></div> DAY-BY-DAY PROCEDURE <a href="http://www.dps.ufl.edu/hansen/ivf/day-2.htm">Day -2:  Getting Prepared</a> <a href="http://www.dps.ufl.edu/hansen/ivf/day-1.htm">Day -1:  Collection of Ovaries | Oocyte Collection | Preparation of IVF Medium</a> <a href="http://www.animal.ufl.edu/hansen/ivf/day0.htm">Day 0:   In Vitro Fertilization</a> <a href="http://www.animal.ufl.edu/hansen/ivf/day1-9.htm">Day 1-9:  Culture of Embryos</a> <a href="http://www.animal.ufl.edu/hansen/ivf/shortprotocol.htm">Abbreviated Protocol for the Experienced User</a> <a href="http://www.animal.ufl.edu/hansen/ivf/printing.htm">Instructions for Printing This Manual</a> OTHER INFORMATION<o:p></o:p> <div align=center> <table class=MsoNormalTable border=1 cellpadding=0 width="100%" style='width:100.0%;mso-cellspacing:1.5pt;background:navy;border:outset #FFFF66 1.0pt; mso-border-alt:outset #FFFF66 .75pt;mso-padding-alt:0in 0in 0in 0in' cols=4> <tr style='mso-yfti-irow:0'> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/tips.htm">Helpful Hints</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.dps.ufl.edu/hansen/ivf/stocks.htm">Stock Solutions</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.dps.ufl.edu/hansen/ivf/mediaprep.htm">Media Preparation</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/dishwashing.htm">Dishwashing/Autoclaving</a><o:p></o:p> </td> </tr> <tr style='mso-yfti-irow:1'> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/embryogallery.htm">Embryo Photo Gallery</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/suppliers.htm">Suppliers</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/references.htm">References</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/otherembryoprot.htm">Other Embryo Protocols</a><o:p></o:p> </td> </tr> <tr style='mso-yfti-irow:2'> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/Protocols/list.htm">Other Protocols</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/embryosites.htm">Other Embryo Sites</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/acknowledgements.htm">Acknowledgements</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu/hansen/ivf/pjhnotes.htm">Notes for PJH Lab</a><o:p></o:p> </td> </tr> <tr style='mso-yfti-irow:3;mso-yfti-lastrow:yes'> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <o:p> </o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <o:p> </o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.dps.ufl.edu/hansen">PJ Hansen Home Page</a><o:p></o:p> </td> <td style='border:inset #FFFF66 1.0pt;mso-border-alt:inset #FFFF66 .75pt; padding:.75pt .75pt .75pt .75pt'> <a href="http://www.animal.ufl.edu">UF Dept. of Animal Sciences</a><o:p></o:p> </td> </tr> </table> </div> <a href=http://www.dps.ufl.edu/hansen/ivf/new.htm><img border=0 width=32 height=24 id="_x0000_i1026" src=http://www.animal.ufl.edu/hansen/IVF/new04.gif align=ABSCENTER></a>Click on the icon to see recent changes in the IVP Protocol Do you want a hard copy of our <a href="embryogallery.htm">embryo poster</a>? Drop us a line by email with your shipping address and we will send you at no charge an 8.5" x 11" color poster illustrating all of the developmental endpoints observed during IVP of bovine embryos.  If you would like a more elaborate poster, we will be happy to make special arrangements to do so. Contact the Authors: <a href="mailto:rivera@dps.ufl.edu"><img border=0 width=25 height=17 id="_x0000_i1027" src=http://www.animal.ufl.edu/hansen/IVF/envelop7.gif align=ABSCENTER></a>  Rivera <a href="mailto:hansen@animal.ufl.edu"><img border=0 width=25 height=17 id="_x0000_i1028" src=http://www.animal.ufl.edu/hansen/IVF/envelop7.gif align=ABSCENTER></a>  Hansen.  We want your feedback!! Let us know whether this protocol works for you. <div class=MsoNormal align=center style='text-align:center'><![if !vml]><img border=0 height=10 width=576 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image004.gif alt=--- v:shapes="_x0000_i1029"><![endif]></div> The image shown on this page is of a day 6 bovine embryo subjected to staining using the TUNEL reagent and propidium iodide and analyzed using a Zeiss Axioplan microscope using DIC. The nucleus staining yellow is positive for apoptosis. Image by Paolete Soto (© Soto and Hansen, 2002).<o:p></o:p> This page is a contribution to the International Dairy Heat Stress Consortium and was funded in part by a grant from the USDA Initiative for Future Agricultural and Food Systems (USDA CSREES# 2001-52101-11318). <o:p></o:p> All original text and images in this site © Rivera, Edwards, Ealy, Monterroso, Majewski and Hansen, 2000-2002  this site was created <st1:date Month="4" Day="10" Year="2000">4-10-00</st1:date>. This page was last updated <st1:date Year="2002" Day="9" Month="7">7-9-02</st1:date><o:p></o:p> Links to commercial sites do not constitute endorsement by the authors or the <st1:place><st1:PlaceType>University</st1:PlaceType> of <st1:PlaceName>Florida</st1:PlaceName></st1:place> <![if !vml]><a href="http://www.animal.ufl.edu/"><img border=0 width=122 height=117 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image006.gif v:shapes="_x0000_s1031"></a><![endif]><![if !vml]><a href="http://www.ufl.edu/"><img border=0 width=210 height=68 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image007.gif v:shapes="_x0000_s1029"></a><![endif]><![if !vml]><a href="http://www.animal.ufl.edu/dairy"><img border=0 width=254 height=52 src=http://www.animal.ufl.edu/hansen/IVF/default_files/image009.jpg v:shapes="_x0000_s1030"></a><![endif]> <o:p> </o:p>   <o:p> </o:p> <o:p> </o:p>  <applet codebase="http://vc.webcounter.com/" code="vc7.class" width="88" height="31"> <param name=a value=44141><a href=http://www.webcounter.com/> <img src=http://vc.webcounter.com/0/t?a=44141&s=&g= width=88 height=31></a> </applet> <o:p></o:p>  since <st1:date Year="2000" Day="25" Month="9">9-25-00</st1:date><o:p></o:p> </div> </body> </html>