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VOLUME 22, NO. 1 WINTER 2007

A Closer LookAMERICAN EMBRYO TRANSFER ASSOCIATION

“THE VANGUARD OF THE EMBRYO TRANSFER INDUSTRY”

President’s Message

What’s Inside . . .

Headquarters Directory .......................................2

AETA Board and Committees .............................3

Abstract from Theriogenology ............................5

Ask John ................................................................7

US Livestock Genetics Export Program

Summary..............................................................10

Article from Journal of Animal Science ............11

AETA Business Office

1111 N. Dunlap Ave., Savoy, IL 61874Phone: 217-398-2217 FAX: 217-398-4119

aeta@assochq.orgwww.aeta.org

AETA PresidentDr. Ron Kling

Think spring!! For much of the United States, winter has hit sudden and hard. Eleven feet of snow in upper New York, heavy snow storms in the West, tornadoes in Florida, and everything in between for many of us. The weather greatly affects our industry and can make the days long. Like the seasons of our life, this season too shall soon end.

The AETA Board of Directors (BOD) met at the FASS headquarters in Savoy, IL, on January 27. Despite cancelled flights, delayed flights, and bad weather, all board members were present at the meeting. Thank you to the entire BOD for your perseverance and sacrifice. The Audit Committee met at FASS on January 25, 2007, to audit the financial records; they found no discrepancies in financial records and verified adherence to all control procedures set up by the BOD. The BOD passed a motion to conduct a professional external audit in 2008. This audit will review all in-house check-and-balance procedures and make recommendations on financial and investment planning.

Michael Phillips, President/CEO of USLGE (US Livestock Genetics Export) attended the board meeting and gave a very informative presentation. The USLGE has worked with the AETA for more than 14 years to provide support and funding for developing international marketing. The USLGE has opened a door for the AETA to market embryos to Russia. In January, members of the Cooperator Committee met with a Russian delegation attending the livestock show in Denver.

The Government Liaison Committee is working with the USDA to develop a health protocol for export of US embryos to Russia. In addition, with funding from USLGE, the Cooperator Committee is continuing to develop a multilingual AETA promotional CD. Michael Phillips is very eager to help the AETA members with the international market. If anyone has ideas to explore new international markets or needs assistance in promotion of US genetics internationally, contact the Cooperator Committee or USLGE.

The AETA has rebounded from the GMO situation and is now financially and managerially stable. We need to continue to fine-tune and improve these areas. However, after talking with various AETA members and the AETA board, it appears we have come to a season of increasing our membership and an awakening to who we are. The Membership Committee over the last 3 years has done an excellent job of building our membership and making an awareness of AETA, especially in universities. The AETA board would like to continue this momentum by researching

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AETA Headquarters DirectoryVicki Paden, AETA Administrative Assistant(aeta@assochq.org) As the AETA Administrative Assistant, Vicki works with the AETA members on day-to-day issues. She updates the AETA membership database, processes

memberships, renewals, meeting registrations, orders, claims, invoices and responds to e-mail. She is also the helpful, friendly voice on the other end of the phone when you call the AETA line.

Christina Tomlinson, AETA Newsletter Coordinator(christinat@assochq.org) Christina handles all coordination for the production of the A Closer Look, including newsletter advertising, article submissions, and announcement postings.

Save These Dates!AETA v 2007 AETA Annual Meeting in conjunction with The Society for Theriogenology Monterey, California August 7–11, 2007

Future Meetings of Interest!AABP v 2007 AABP Annual Meeting Vancouver, BC, Canada September 20–22, 2007IETS v 2008 IETS Annual Meeting Denver, Colorado January 5–9, 2008

Newsletter Advertising 2007

Publication Schedule and DeadlinesThe AETA newsletter is published four times per year and is mailed to all AETA members. Distribution is between 350-400 professionals in the animal embryo transfer industry.

Members – Advertise FREE with us!Members wishing to place an advertisement related to sale of practice, buying and selling of used equipment or employment opportunities may do so free-of-charge (up to 1/8 of a page). The advertising of information (i.e. short courses, seminars, books, etc.) that is clearly to the benefit of the greater good of the AETA membership, and not considered to be of a commercial nature, may also be advertised free-of-charge (up to 1/8 of a page). Standard rates on any advertisements over 1/8 page shall apply. Any advertising request, which does not fit within these guidelines, shall be brought to the Newsletter Committee for approval. The same rationale shall apply to any website advertising.

A Closer Look Advertising Rates for 2007

Business Card $50 per issue1⁄4 Page $75 per issue1⁄2 Page $150 per issueFull Page $200 per issue

Ads are due the 15th prior to each issue month. Online ads are full color and print ads are black and white. Contract terms: All amounts due on invoicing.

AETA accepts electronic or camera-ready ads for publica-tion. Accepted ad formats include: PDF (preferred) or high quality .jpg, .tif, or .eps, Call regarding all other formats.If you would like to advertise in the next issue, please contact AETA at aeta@assochq.org or 217-398-2217.

Issue Winter— February Spring — May Summer — August Fall — November

the method of branding: a process whereby an organization is given a new image and marketing strategy based on the target audience and the needs of the organization. The board is looking at ways to increase educational resources, educational classes on ET and certification, and develop a new promotional brochure. Additional projects the BOD is working on include the following:1. Evaluation and revision of the AETA by-laws,2. Renewal of the CETA/AETA & AETA Joint Conference

Agreement for the next 4 years, and3. Preparation for the 2008 convention to be held in Kansas

City, MO.

Surveys for certified ET businesses have been mailed and are due March 31, 2007. This survey is mandatory to remain certified, and I encourage you to return the survey promptly to save expenses and hassle to the AETA.

The 2007 Joint AETA/SFT (Society for Theriogenology) Meeting in Monterey, CA, is deep into the planning stages by David Duxbury. Speakers have been confirmed, and session topics are scientifically strong, cutting-edge, and practical. One registration fee will allow registrants to attend any of the AETA or SFT scientific sessions. Exhibitor participation should be larger than ever, and huge sponsorships will provide exciting social events. Included in the registration fee is a post-conference symposium for additional CE. The location and timing of this convention also allows for a family vacation. This year also marks the 25th anniversary of the AETA, and special activities are being planned for this convention. Do not miss this unique and great event!

Ron Kling, DVM

Winter 2007 3

AETA Committees 2006-2007AETA Officers and Directors

2006-2007

PresidentRon Kling, DVMNew Vision Transplants456 Springs RoadGrantsville, MD 21536PHONE: (301) 895-5232FAX: (301) 895-5232E-mail: godsmusic7@verizon.net

Vice PresidentDavid B. Duxbury, DVMMidwest Embryo Transfer Service, LLC1299 South Shore DriveAmery, WI 54001PHONE: (715) 268-9900FAX: (715) 268-2691E-mail: dbduxbury@gmail.com

Secretary TreasurerByron Williams, DVMEmQuest ET ServiceBox 504Plymouth, WI 53073-0504PHONE: (920) 892-6878FAX: (920) 893-8083E-mail: emquest@excel.net

Immediate Past PresidentPatrick M. Richards, DVM1215 East 2000 SouthBliss, ID 83314PHONE: (208) 539-3076FAX: (208) 352-1934E-mail: drpat@richards.myrf.net

Directors

Cheryl Nelson, DVMNelson Reproductive Service1735 Pinckard PikeVersailles, KY 40383PHONE: (859) 873-7319E-mail: cnl79@qx.net

Allen Rushmer, VMDNext Generation ET Service3162 Oregon PikeLeola, PA 17540PHONE: (717) 656-6921FAX: (717) 656-6934E-mail: nextgenvet@att.net

Sam Edwards, DVMHarrrogate Genetics Int’l. Inc.Box 1Harrogate, TN 37752(423) 869-3152Fax: (423)869-5546Samedwards62@hotmail.com

Steve Hughes, DVM7732 Garnett StreetLenexa, KS 66214(913) 961-6666Fax: (913) 961-6666Sth2897@yahoo.com

James Spears, Ph. D.Professional Embryo Services5707 Russellville RoadFranklin, KY 42134(270) 586-7430Fax: (270) 586-5697spearsjames@bellsouth.net

AUDIT COMMITTEEDaniel Hornickel, DVM, ChairSunshine Genetics IncW7782 Hwy 12Whitewater, WI 53190PHONE: (262) 473-8905FAX: (262) 473-3660E-mail: Dan@sunshinegenetics.com

Committee MembersEdwin Robertson, DVMRichard Whitaker, DVM

CERTIFICATION COMMITTEEStephen Malin, DVM, ChairMalin Embryo TransferN5404A Highway 151Fond du Lac, WI 54937PHONE: (920) 921-1231FAX: (920) 921-1231E-mail: malin@spiritusa.net

Committee MembersBoyd Henderson, VMDLarry Horstman, DVMJoseph M. Wright, DVMJames K. West, DVM

CONVENTION/PROGRAM COMMITTEEDavid B. Duxbury, DVMMidwest Embryo Transfer Service LLC1299 South Shore DriveAmery, WI 54001PHONE: (715) 268-9900FAX: (715) 268-2691E-mail: dbduxbury@gmail.com

Committee MembersLarry Lanzon, DVM Byron Williams, DVMThomas Rea, DVM

COOPERATOR COMMITTEEScott W. Armbrust, DVM, Co-ChairParadocs Embryo Transfer Inc121 Packerland DriveGreen Bay, WI 54303PHONE: (920) 498-8262FAX: (920) 498-8181E-mail: pdocset@sbcglobal.net

Richard S. Castleberry, DVM, Co-ChairVeterinary Reproductive Services8225 FM 471 SouthCastroville, TX 78009PHONE: (830) 538-3421FAX: (830) 538-3657E-mail: vrsrsc@mail.ev1.net

Committee MembersDarrel DeGrofft, DVMLarry Kennel, DVMGreg Lenz, DVMRobert Leonard, DVMPat Phillips, USLGEJames West, DVMByron Williams, DVM

EXHIBIT COMMITTEEDavid B. Duxbury, DVMMidwest Embryo Transfer Service LLC1299 South Shore DriveAmery, WI 54001PHONE: (715) 268-9900FAX: (715) 268-2691E-mail: dbduxbury@gmail.com

Committee MembersDan Hornickel, DVMMark Steele DVM

GMO RESOLUTION COMMITTEERandall H. Hinshaw, DVM, ChairAshby EmbryosAshby Herd Health Services Inc.2420 Grace Chapel RoadHarrisonburg, VA 22801PHONE: (540) 433-0430FAX: (540) 433-0452E-mail: RHinshaw@RICA.NET

Committee MembersDarrel DeGrofft, DVMStephen Malin, DVMDaniel R. Hornickel, DVM

GOVERNMENT LIAISON COMMITTEERichard O. Whitaker, DVM, ChairNew England Genetic, LLC10 Business Park WayTurner, ME 04282PHONE: (207) 225-2722FAX: (207) 225-3883E-mail: moodoc@megalink.net

Committee MembersDavid Duxbury, DVM

MANUALS, PROMOTIONS AND MEMBERSHIP COMMITTEEThomas A. Borum, DVM, ChairBorum’s Veterinary Embryonics145 East Franklin StreetNatchez, MS 39120PHONE: (601) 442-5522FAX: (601) 442-5523E-mail: aeta@borum.com

Committee MembersRobert Zinnikas, DVMStanley F. Huels, DVM

MEMBERSHIP COMMITTEECheryl Nelson, DVM, Co-chairNelson Reproductive Service1735 Pinckard PikeVersailles, KY 40383PHONE: (859) 873-7319E-mail: cnl79@qx.net

Charles R. Looney, PhD, Co-chairOvaGenix, LPPO Box 3038Bryan, TX 77805PHONE: (979) 731-1043FAX: (979) 731-1086E-mail: charles@ovagenix.com

Committee MembersSam Edwards, DVMStanley Huels, DVMJimmy Webb, DVM

NEWSLETTER COMMITTEEBrad R. Lindsey, PhDRex ConsultingP. O. Box 158Midway, TX 75852PHONE: (979) 450-2599E-mail: brad@consultrex.net

Committee MembersCharles R. Looney, PhDKathy Creighton SmithLarry Kennel, DVM

NOMINATIONS COMMITTEEPatrick M. Richards, DVM1215 East 2000 SouthBliss, ID 83314PHONE: (208) 539-3076FAX: (208) 352-1934E-mail: drpat@richards.myrf.net

Committee MembersPhil Buhman, DVMTom Rea, DMV

PROFESSIONAL REVIEW COMMITTEERon Kling, DVMNew Vision Transplants456 Springs RoadGrantsville, MD 21536PHONE: (301) 895-5232FAX: (301) 895-5232E-mail: godsmusic7@verizon.net

Committee MembersDavid Duxbury, DVMByron Williams, DVMPat Richards, DVM

STATISTICAL INFORMATION COMMITTEEBrad Stroud, DVMStroud Veterinary Embryo Service, Inc6601 Granbury HighwayWeatherford, TX 76087PHONE: (817) 599-7721FAX: (817) 596-5548E-mail: bstroud@biotechproductions.com Committee MembersIrma Robertson Jeanne M. Reyher

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Winter 2007 5

NOTICE TO READERSArticles published in A Closer Look are not necessarily peer-reviewed or refereed. All statements, opinions, and conclusions contained in the articles in A Closer Look are those of the author(s) and are not necessarily those of the American Embryo Transfer Association unless specifically approved by the AETA Board of Directors.

INFLUENCE OF THE INTERVAL BETWEEN THAWING TO TRANSFER ON PREGNANCY RATES OF FROZEN/THAWED

DIRECT TRANSFER EMBRYOSG.A. Bo1, L.C. Peres1, D. Pincinato1, M. de la Rey2, and R. Tribulo*1, 1Instituto de Reproduccion Animal Cordoba (IRAC),

J.L. de Cabrera 106, X5000GVD Cordoba, Cordoba, Argentina, 2Embryo Plus, Brits, South Africa.

An experiment was designed to evaluate the effect of the interval between thawing to deposition of the embryo into the uterine horn on pregnancy rates of in-vivo produced frozen/thawed embryos in 1.5 M ethylene glycol (Direct Transfer). Data was collected from 1122 embryo transfers performed in the same farm (Estancia El Mangrullo, Lavalle, Santiago del Estero, Argentina) during the spring and summer of 2004/05 and 2005/06 (6 replicates, ambient temperature between 20 to 40 °C). Recipients used in all replicates were non-lactating, cycling, multiparous Bos taurus x Bos indicus crossbred cows with body condition score between 3 and 4 (1 to 5 scale) that were synchronized using fixed-time embryo transfer protocols. Briefly, the synchronization treatments consisted on the insertion of a Crestar ear implant (Intervet, Brazil) or a progesterone-releasing device (DIB, Syntex SA, Argentina), plus 2 mg of estradiol benzoate (EB; Syntex) intramuscularly (im) on Day 0, and 400 UI of eCG (Folligon 5000, Intervet or Novormon 5000, Syntex) im plus 150 mg D (+) cloprostenol im (Preloban, Intervet or Ciclase Syntex) on Day 5. Progestin devices were removed on Day 8 and all cows received 1 mg of EB im on Day 9. All cows were examined by ultrasonography on Day 16 and those with a luteal area >76 mm2 (by calculating the area of the CL minus the area of the cavity) received, on Day 17, frozen/thawed embryos by non-surgical transfer. All the embryos were Grade 1, and all were frozen in 1.5 M ethylene glycol at the Embryo Plus Laboratory (Brits, South Africa). After being stored in liquid nitrogen, the embryos were plunged directly (no air thawing) in a 30 °C water bath for 30 seconds, and then transferred to the recipient cows by either one of two technicians. Based on the interval between thawing and transfer, the transfers were classified as being in one of three groups: Group 1: < 3 minutes; Group 2: between 3 to 6 minutes; Group 3: between 6 to 16 minutes. The main reason for delayed transfers beyond 6 minutes was the replacement of one recipient for another because of difficulty in threading the cervix (1% of the total transfers) or a recipient falling down into the chute or with very bad disposition and behavior. Pregnancy was determined by ultrasonography 28 to 35 d after fixed-time embryo transfer and data were analyzed by logistic regression. There were no effects of replicate, technician, CL area, recipient body condition score, embryo stage and time from thawing to transfer on pregnancy rates. Pregnancy rates in the three thawing to transfer intervals were: Group 1: 215/385, 55.8%; Group 2: 372/655, 56.8%; Group 3: 42/82, 51.2%; P>0.6. These results may be interpreted to suggest that there is no significant effect of time from thawing to transfer (up to 16 minutes) in Direct Transfer embryos using Bos taurus x Bos indicus recipients transferred at a fixed-time.

The following abstract is reproduced with Elsevier’s permission from Vol. 19 (1) 2007 (Proceedings of the Annual Conference of the IETS, Kyoto, Japan) p. 220 (Abst. #207) [Published by CSIRO Publishing / http://www.publish.csiro.au/journals/rfd ].

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Latest Conference Proceedings available at www.aeta.org

25th Anniversary AETA History BookA history book is being compiled with pictures and past history of the AETA organization. If you have access to any pictures of board members, past presidents, past award winners, and any interesting pictures of the membership along with any information on critical or monumental times in the association’s history, please send them to Ron and Linda Kling, 456 Springs Rd., Grantsville, MD 21536 or e-mail the information to godsmusic7@verizon.net The pictures will be sent back to you. Also, if you have past programs/newsletters with pertinent information, copy those and send them. Any questions, feel free to call 301-895-3964. Your help with this is greatly appreciated!

Winter 2007 7

Ask John . . .

*************************************Questions for “Ask John” may be addressed to:

askjohn@assochq.org

The following question and answer were recently carried on the CETA “Tech Talk” internet site, http://ceta.ca/techtalk/.

QUESTIONS I’m still relatively new to embryo transfer and have a question regarding a couple of recent occurrences. The rst happened today and involves uid disappearing from the uterus. I ushed the rst horn with no problems and switched the catheter to the other side without difculty. The horn initially appeared to ll, and I recovered at least some of this uid.

When attempting to ll the horn for the second time, uid was running in but not palpable within the horn or recoverable. This happened to me once before on a different cow but on the third or fourth time lling the horn, and I ended up still recovering all or most of the embryos. This time I did not. I am as certain as I can be that I did not traumatize or

perforate the uterus, so I am thinking that perhaps the uid is escaping through the utero-tubal junction. Is this possible? Is it common? What do you do?

The second question concerns uterine bleeding. This particular cow had blood present in about the second bolus of uid recovered from the uterus. Subsequent uid put into and recovered from the uterus had blood present in increasing amounts.

I switched lters and ushed the other side, and the exact same thing happened. The only thing I can think of is that I may have overinated the cuff on the rst horn. This cow had a large uterus and I put in a little more air than normal, about 20 cc. I was careful not to put as much in the second horn. I felt that the catheter placement was reasonably smooth on this cow, and the initial uid appeared to be clear. Is this problem generally caused by poor technique or is it likely to happen occasionally, even to experienced practitioners?

RESPONSE received from Dr. John Hasler:It is not impossible to run uid from the uterus up through the utero-tubal junction and into the oviduct. Were it impossible, we would not have been successful in ushing hundreds of donors via a midline surgical approach at Colorado State University during the early 1970s. The pressure necessary to produce the backow is moderate, but greater than what is normally produced during nonsurgical embryo recoveries. I don’t think there is a problem with backow during nonsurgical recoveries. The failure to get the backow described by Jack Reeb may have been because it was a tract removed from a dead cow.

I agree with Reuben Mapletoft that often when a signicant amount of recovery uid is lost, it ends up in the broad ligament. I have seen a liter of uid lost and the cow temporarily ending up in an obviously uncomfortable state and acting colicky. We trained a number of practitioners over the years at Em Tran, Inc., and nearly everyone experienced a few cases of ruptured endometrium during the learning phase, especially with horn ushes. I think uterine rupture is a less common problem when the catheter balloon is inated in the internal os of the cervix for a body ush, as some practitioners prefer. Of course, that approach has its own particular challenges and problems. Last, I think there is the occasional old cow with a particularly friable uterus, and uid may be lost by even the most experienced practitioners.

John Hasler

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The AETA Statistics Committee is requesting your ET data for the 2006 calendar year. As you know, it’s a requirement for Certified ETBs to annually report their embryo collection and transfer data to the Statistics Committee, but we’re strongly encouraging non-certified ETBs to do the same. Those seeking opportunities to provide goods and services to our industry depend on us to provide data, such as rising or falling trends in certain areas, so they can make intelligent business decisions.

Watch for a hard copy of the survey to arrive in the mail! This year, in addition to a hard copy paper questionnaire, the committee has produced an Excel spreadsheet (AETA ETB Stats Spreadsheet) for those who don’t have computer software to automatically compute the requested data. The spreadsheet is an adjunct, not a replacement, to the hard copy questionnaire.

The Stats Committee is requesting that each ETB return either the hard copy version or the Excel spreadsheet version of the questionnaire to the AETA office by March 31, 2007. It can be sent by mail, fax, or e-mail in the case of the spreadsheet.

Thanks in advance for your cooperation and quick response!

Respectfully,AETA Stats Committee

Remember March 31, 2007

Winter 2007 9

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FY 2007 MAP Branded Livestock ProgramAdministered by US Livestock Genetics Export, Inc. Program Summary413 N. Broadway, Suite C, Salem, IL 62881

The US Livestock Genetics Export (USLGE) has received funds that will be available to private livestock breeders, companies, or cooperatives interested in promoting livestock, semen, or embryo sales in international markets through December 31, 2007. These funds are available through the Market Access Program (MAP) of the Foreign Agricultural Services (FAS) of the US Department of Agriculture.

The USLGE sponsors and administers the branded program with the goal of helping the US livestock industry increase the international demand for US livestock genetics.

MAP funding is used to supplement but not supplant private funds that would be used for promotion activities.

The MAP branded program provides for partial reimbursement (up to 50%) of approved activities such as international advertising, the development, translation and distribution of promotional materials, and participation in foreign trade shows and exhibitions. Funds cannot be used for travel or personnel reimbursement. An administrative fee is charged to participate in the program.

The total amount of funds available to USLGE for brand promotions is set by FAS. The allocation of these funds will be made to eligible participants on a fair and equitable basis as set by FAS and consistent with the goals and objectives of the MAP program as outlined by Congress. Funding criteria is based, in part, upon available funding, anticipated economic impact, and the completeness of the application.

Interested parties should request a FY07 MAP Branded Application and Program Guidelines booklet from US Livestock Genetics Export, Inc., 413 N. Broadway, Suite C, Salem, IL 62881. Phone: 618/548-9154, fax 618/548-9709, e-mail: uslge@uslge.org

Applications will be considered throughout the year pending the availability of funding.

Winter 2007 11

Reprinted by permission of Journal of Animal Science (vol 85:138-142). doi:10.2527/jas.2006-258© 2007 American Society of Animal Science

INTRODUCTION

Natural disease resistance refers to the inherent capacity of an animal to resist disease when exposed to pathogens, without prior exposure or immunization

(Adams and Templeton, 1998; Caron et al., 2004). Previous studies conducted in our laboratory resulted in the identification of a Black Angus herd sire which was confirmed to be genetically resistant to in vitro and in vivo challenge virulent with Brucella abortus (Qureshi et al., 1996; Adams et al., 1999). Brucellosis is an important zoonotic disease of mammals caused by Brucella spp. and is characterized by its ability to cause abortions, birth of weak or nonviable offspring, and infertility in males and females. The disease-resistant sire was utilized to initiate breeding studies, determine if natural resistance to bovine brucellosis was heritable, and to identify and characterize the genes involved. Bovine Solute Carrier 11A1 (SLC11A1) also known as natural resistance associated macrophage protein gene 1 (NRAMP 1), which had been previously identified as Lsh/Ity/Bcg gene in mice and humans (Mock et al., 1990, Vidal et al., 1993, Cellier et al., 1994), is conserved on Bos taurus autosome, BTA 2 (Beever et al., 1994; Feng et al., 1996, Barthel et al., 2001) and was identified as the major candidate gene for controlling natural resistance and susceptibility to bovine brucellosis in cattle (Harmon et al., 1985; Harmon et al., 1989; Qureshi et al., 1996). Unfortunately, our foundation sire died in 1996. In addition, all frozen semen derived from this bull was

Rescuing valuable genomes by animal cloning: A case for naturaldisease resistance in cattle1,2

M. E. Westhusin,3 T. Shin, J. W. Templeton, R. C. Burghardt, and L. G. Adams4

Texas A&M University, College of Veterinary Medicine & Biomedical Sciences, College Station, TX

ABSTRACT: Tissue banking and animal cloning represent a powerful tool for conserving and regenerating valuable animal genomes. Here we report an example involving cattle and the rescue of a genome affording natural disease resistance. During the course of a 2-decade study involving the phenotypic and genotypic analysis for the functional and genetic basis of natural disease resistance against bovine brucellosis, a foundation sire was identified and confirmed to be genetically resistant to Brucella abortus. This unique animal was utilized extensively in numerous animal breeding studies to further characterize the genetic basis for natural disease resistance. The bull died in 1996 of natural causes, and no semen was available for AI, resulting in the loss of this valuable genome. Fibroblast cell lines had been established in 1985, cryopreserved, and stored in liquid nitrogen for future genetic analysis. Therefore, we decided to utilize these cells for somatic cell nuclear transfer to attempt the production of a cloned bull and salvage this valuable genotype. Embryos were produced by somatic cell nuclear transfer and transferred to 20 recipient cows, 10 of which became pregnant as determined by ultrasound at d 40 of gestation. One calf survived to term. At present, the cloned bull is 4.5 yr old and appears completely normal as determined by physical examination and blood chemistry. Furthermore, in vitro assays performed to date indicate this bull is naturally resistant to B. abortus, Mycobacterium bovis, and Salmonella typhimurium, as was the original genetic donor.

Key words: animal cloning, genetic conservation, natural disease resistance

© 2007 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2007. 85:138–142doi:10.2527/jas.2006-258

1Mycobacterium bovis BCG Montreal Strain 9003 was kindly provided by Danuta Radzioch, McGill University, Montreal.

2We gratefully appreciate the assistance of Doris Hunter with establishment of the original ear punch fibroblast cell lines; Dana Dean for electron microscopy and immunocytochemistry; Robert Schnabel, Jim Derr, and the Core Technologies Lab, Texas A&M University, with microsatellite analysis; Bhanu Chowdhary for karyotyping; Charles Looney for embryo transfer; and Juan Romano, Wesley Bisset, Amy Plummer, Peter Rakestraw, and Thomas Kasari with surgeries and neonatal care of the cloned calf in the Large Animal Clinic, Texas A&M University.

3Corresponding author: mwesthusin@cvm.tamu.edu4L. G. Adams’ laboratory was supported by the Texas

Advanced Technology Program grant No. 999902-045, USDA, Cooperative State Research Education and Extension Service grants no. 90-37241-5583 and 93-37204-9491, Texas Agricultural Experiment Station Project TEXO H-6194 and TEXO H-8409.

Received April 24, 2006.Accepted August 8, 2006.

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accidentally destroyed due to improper maintenance in a liquid nitrogen (LN2) storage tank. As a result, the ability to conserve and propagate the genome of this unique animal and produce additional offspring was lost. Given our previous success with cloning cattle by somatic cell nuclear transfer (Hill et al., 2000), we decided to employ this technique to rescue the genotype of this bull using cryopreserved fibroblasts.

MATERIALS AND METHODS

All procedures involving animals were approved by the Texas A&M University Institutional Animal Use and Care Committee operated under the guidelines and certification of the Association for Assessment and Accreditation of Laboratory Animal Care, USDA, and Texas Animal Health Commission.

Production of Cloned Bull Adult fibroblast cell lines were established in 1985 from an ear punch obtained from the disease-resistant bull (referred to as Bull 86). These were expanded in culture using medium composed of Dulbecco Modified Eagle medium (DMEM; Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco), then frozen and stored in LN2. In 2000, (approximately 15 yr later) the cell lines were thawed and plated into 4-well dishes (Nunc Inc., Naperville, IL) containing DMEM/F-12 medium (Gibco) supplemented with 10% FCS + 1% penicillin/streptomycin (10,000 U/mL of penicillin G, 10,000 µg/mL of streptomycin, Gibco) and maintained for 3 to 5 d. For recombination with the enucleated oocytes, the cultured donor cells were treated with trypsin-EDTA solution (Sigma, St. Louis, MO) for less than 1 min with gentle pipetting. After the addition of 3 mL of HEPES-buffered Tissue Culture Medium (TCM 199, Gibco) supplemented with 10% FCS, the donor cells were washed by centrifugation (3 min, 200 × g), then resuspended in the medium. Bovine cumulus cell oocyte complexes were collected from abattoir-derived ovaries and matured for 20 h in TCM 199 supplemented with 10% FCS and 1% penicillin/streptomycin and, on a per milliliter basis, 0.1 units of FSH (Sioux Biochem, Sioux City, IA), 0.1 units of LH (Sioux Biochem), 1 µg of estradiol (Sigma), 28 µg of pyruvate (Sigma), and 0.05 µg of epidermal growth factor (EGF, Sigma). After in vitro maturation, expanded cumulus-oocyte complexes were denuded by vortexing for 3 min in 0.1% hyaluronidase (Sigma) in Tyrodes Lactate, HEPES buffered medium (TL-HEPES, Gibco), then washed and placed in TCM 199 with 10% FCS. Oocytes were enucleated at 21 h postmaturation. Before enucleation, they were placed for 10 min in HEPES-buffered TCM 199 supplemented with 10% FCS and containing (per mL) 5.0 µg of cytochalasin B (Sigma) and 5 µg of Hoechst 33342 (Sigma). All oocytes were carefully selected for the presence of the first polar body and a

homogeneous cytoplasm. Enucleation was performed using a beveled 18- to 20-µm o.d. glass pipette mounted on Narishige micromanipulators (Medical Systems Corp., Great Neck, NY) and a Zeiss Microscope (Axiovert 135, Zeiss, Germany). Only oocytes in which the removal of the polar body and the metaphase chromosomes was confirmed by observation under UV light were used. After trypsin/EDTA treatment of cultured donor cell lines, fibroblasts of a median (18- to 20- m) size and a morphologically round, smooth shape were combined with enucleated oocytes. The oocyte-fibroblast couplets were then placed into fusion medium consisting of 275 mM mannitol (Sigma), 0.1 mM CaCl2 (Sigma), and 0.1 mM MgSO4 (Sigma). After equilibration, the couplets were transferred into a 1-mm fusion chamber containing fusion medium, and fused with two 25-µsec, 2.3-kV/cm, direct current pulses delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA). Couplets were then moved to TCM 199 supplemented with 10% FCS and containing 5.0 µg of cytochalasin B (Sigma)/mL and cultured for 1 h before being transferred to cytochalasin B-free medium for an additional 1 h. Fusion was then evaluated, and successfully fused embryos were selected and subjected to an activation treatment. Embryo activation was performed by a 4-min incuba-tion in 5 µM ionomycin (Calbiochem, San Diego, CA) followed by 4 min in TL-HEPES with 30 mg of BSA (Sigma)/mL. Embryos were then washed twice in TL-HEPES supplemented with 3 mg/mL of BSA. Successfully fused and activated embryos were then placed in a culture well (Nunc) containing 500 µL of TCM 199 supplemented with 10% FCS, 10 µg/mL of CHX (Sigma), and 5 µg/mL of cytochalasin B (Sigma) for 5 h. After activation, cloned embryos were cultured in G1.2/G2.2 media (Colorado Center for Reproductive Medicine, Englewood, CO) for 7 d as previously described (Gardner et al., 1994). Embryo development was assessed 7 d after cell fusion. In some cases, embryos that had developed to the blastocyst stage were nonsurgically transferred into synchronized recipient cows. Pregnancy status of cows receiving embryos was assessed by tran-srectal ultrasonography (Aloka 500, 5-MHz transducer, Aloka Co., Tokyo, Japan) at 40 d after nuclear transfer and rechecked on a routine basis. To confirm that the bull calf was genetically identical to the original cell donor, genotyping was conducted as described previously (Schnabel et al., 2000). In brief, genomic DNA was isolated from white blood cells using the Super Quick-Gene DNA isolation kit (Analytical Genetic Testing Center Inc., Denver, CO). Polymerase chain reaction was utilized, and the products were separated on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and sized relative to the internal size standard, Mapmarker low (Bioventures, Murfreesboro, TN). Fluorescent signals from the dye-labeled microsatel-lites were detected using GeneScan 3.1 software (Applied

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Biosystems). Alleles were assigned relative to the allelic ladder. Twelve microsatellite markers were utilized for the analysis.

In Vitro Phenotyping for Disease Resistance The original purebred Angus bull (Bull 86) and his clone (Bull 862) were sequentially studied in 1992 and 2003, respectively. An in vitro, bacterial-killing (expressed as percentage reduction in intracellular survival) assay was employed to phenotype and compare Bull 86, Bull 862, and control animals for disease resistance (Campbell et al., 1994). Bull 86 was characterized as resistant to conjunctival challenge with 107 cfu of live B. abortus S2308 and the ability of his peripheral blood monocyte-derived macrophages to control intracellular proliferation of B. abortus (Campbell et al., 1994; Qureshi et al., 1996; Barthel et al., 2001). To perform the assay, venous blood was collected by aseptic venipuncture into 7.5-mL acid-citrate-dextrose (ACD, Sigma). The blood was diluted with an equal volume of PBS, pH 7.39, containing 13 mM sodium citrate (PBS-C, Gibco). Mononuclear cells were collected by separation of the diluted blood on Percoll (Pharmacia LKB, Uppsala, Sweden) cushions (specific gravity 1.077) by centrifugation at 1,000 × g for 30 min in polypropylene centrifuge tubes. The interface cells, consisting of monocytes, lymphocytes, platelets, and rarely, basophils, were transferred to a clean polypropylene centrifuge tube using disposable polypropylene transfer pipettes and washed 3 times by low speed centrifugation (200 × g) in cold PBS-C to remove platelets and any contaminating Percoll particles. After washing, the cells were suspended at 5 × 106 cells/mL in Roswell Park Memorial Institute (RPMI-1640) medium (Gibco) supplemented with 4 mM L-glutamine (Gibco), 1 mM nonessential amino acids (Hazelton Research Products Inc., Lenexa, KS), 1 mM sodium pyruvate, 7.5% sodium bicarbonate, and 4% fresh autologous serum. The cells were transferred in 5-mL aliquots to 50-mL Teflon Erlenmeyer flasks (Nalge Company, Rochester, NY) and incubated for 2 h at 38°C in a humidified atmosphere of air with 5% CO2 to allow adherence of monocytes. Nonadherent cells were removed by agitation of the flasks, followed by transfer of the supernatant to a clean 50-mL centrifuge tube. The nonadherent cells were enumerated and used for culture of antigen-responsive lymphocytes for additional studies. Five milliliters of supplemented RPMI-1640 with 12.5% fresh autologous serum were added to each flask, and the flasks were incubated at 38°C in 5% CO2 in air for an additional 48 h, at which time any further nonadherent cells were removed and discarded. The cells were cultured for 10 d, with medium changes at 5 to 7 d to allow maturation to macrophages before use in the assays. All experiments were performed on cells in culture for 10 to 28 d. The bacterial strains used in this assay were B. abortus ATCC Strain 2308, Mycobacterium bovis BCG Montreal

Strain 9003, and Salmonella dublin ATCC Strain 5631. B. abortus and S. dublin were grown on tryptose soy agar (TSA) media plates, whereas M. bovis BCG was grown on Middlebrook 7H10 media plates (BBL, Becton Dickinson Microbiological Systems, Cockeysville, MD). Bacterial stocks were stored at –70°C as a suspension of 107 cfu/mL in RPMI-1640 supplemented with 15% FCS containing amino acids, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma). Incubation conditions were 37°C with 5% CO2 in air under humidified conditions. Monocyte-derived macrophages were harvested by chilling the culture flasks on ice for 10 to 15 min, followed by agitation and pipetting to dislodge adherent cells. The harvested cells were enumerated and resuspended at a cell density of 1 million/mL in supplemented RPMI-1640 with 12.5% fresh autologous serum. Cell suspensions (10 µL, 10,000 cells/mL) were transferred to triplicate wells of tissue culture, 60-well, HL-A plates (Nunc) at 750 × g for 5 min and incubated at 38°C in 5% CO2 in air for 12 to 16 h prior to assay. To prevent desiccation of the well contents, 50 to 100 µL of sterile, distilled water were pooled in each corner of the culture plate. After the 12- to 16-h incubation, the well contents were aspirated, and 5 µL of a suspension of B. abortus S2308 (10 million cells/mL) was added to each well. The plates were centrifuged at 750 × g for 5 min and incubated at 38°C in a humidified atmosphere supplemented with 5% CO2. After a 30-min incubation to allow bacteria-cell interaction, 10 µL of a 37.5 µg/mL solution of gentamycin sulfate (Gibco) were added to each well to a final concentra-tion of 25 µg/mL, with further incubation for 1 h. As a control for the killing of extracellular bacteria, 0.5 mL of bacterial suspension was added to 1 mL of antibiotic solution and incubated concomitantly with the culture plates. This kept bacteria and antibiotic in the same relative concentrations as in the test wells and provided a large enough sample to facilitate handling. An additional control for adequacy of bacterial washing included a tube containing 0.5 mL of bacterial suspension and 1.0 mL of RPMI-1640. After the antibiotic treatment, all wells were washed 4 times with fresh, unsupplemented medium, followed by lysis of the macrophages by addition of 10 µL of 0.5% Tween-20 (Sigma) in sterile, distilled water. Well contents were serially diluted in sterile, distilled water, and 100 µL of the dilutions was plated on TSA for enumeration of colony-forming units. The contents of the antibiotic treatment control and bacterial wash control tubes were washed 3 times with distilled water, resuspended to 2 mL of total volume, and 100 µL was plated on TSA. This corresponded in concentration to the first dilution tube after well harvest. At 12 h, the wells of the duplicate culture plate were similarly harvested. The percent survival of bacteria was determined for triplicate samples over the 12-h incubation period. Because the populations of cattle were specifically chosen from or included members of pedigreed families based on the response to a challenge

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infection with live B. abortus, the data were not normally distributed. Data were analyzed for significance by the nonparametric Mann-Whitney U-test.

RESULTS

Of 493 1-cell (fused) cloned embryos, 223 (45%) developed to the blastocyst stage after in vitro culture. Of these, 39 expanded or hatching blastocysts were selected and transferred to 20 synchronized recipient cows (all but 1 recipient received 2 embryos at the time of transfer). Ten recipients (50%) were diagnosed pregnant at 40 d by ultrasonography. Nine of 10 pregnancies spontaneously aborted (4 at d 44 to 45, 4 at d 68 to 92, and 1 at d 214 of gestation, respectively). One survived to term resulting in a live bull calf at d 283 of gestation. The bull calf, which was delivered by Caesarian section after induction with i.v. glucocorticoid dexamethasone (Azium, Schering-Plough Inc., Kenilworth, NJ) treatment (20 mL of 2 mg/mL) in order to facilitate maturation of the fetal respiratory system, appeared normal and healthy at birth. The genotype of the cloned bull matched the original donor DNA at all 12 markers, indicating it was a clone of the original cell donor. The cloned bull (Bull 862) is now 4.5 yr old. A picture of Bull 862 at 2 yr of age is provided in Figure 1. The results of the in vitro killing assays performed in 2003 demonstrated that macrophages cultured from Bull 862 (cloned bull) reduced bacterial survival of B. abortus S2308 (59.6%), M. bovis BCG (64.0%), and S. dublin (73.3%). These results were similar when compared with macrophages cultured from Bull 86 for reduction of bacterial survival of B. abortus S2308 (61.0%), M. bovis BCG (66.3%), and S. dublin (76.6%) performed in 1993 using the identical procedures, instrumentation, and technical personnel (Table 1). Thus the in vitro microbial killing profiles of the macrophages of Bull 86 and Bull 862 were virtually identical. Clearly, Bull 86 and Bull

862 had significantly greater pathogen killing capacities as compared with previously published reports for the pathogen survival capacities of susceptible cattle respectively for B. abortus S2308 (120 to 180%), M. bovis BCG (125 to 225%), and S. dublin (150 to 425%) (Harmon et al., 1985;; Campbell et al., 1994; Qureshi et al., 1996).

DISCUSSION

In this study, we demonstrated that long-term cryo-preserved somatic cells (15 yr) can be successfully reprogrammed and result in the live birth of a cloned offspring through nuclear transfer technology. The cell donor was a deceased purebred black Angus bull registered with the American Black Angus Association and previously recognized as one that had inherited the SLC11A1 gene, which imparts resistance against bovine brucellosis (Feng et al., 1996). Due to advances with animal cloning technology, we attempted to clone this bull by using frozen-thawed cells that had been collected from ear skin in 1985. As far as we know, this is the first cloned bull produced from a frozen-thawed tissue sample after long-term storage for 15 yr in LN2. The cells used for cloning in this study were frozen more than 10 yr

Figure 1. Cloned Bull 862 at 2 yr of age.

Table 1. Comparison of the original (Bull 86) with the cloned (Bull 862) bull for in vitro, peripheral blood-derived, macro-phage killing of Brucella abortus, Mycobacterium bovis, and Salmonella dublin1,2

B. abortus M. bovis S. dublinItem survival,% survival, % survival, %

Replicates Clone 862

95 50 70 52 76 98 32 66 52 Mean 59.6 64.0 73.3 SD 32.3 13.1 23.2

Original 86

Replicates 55 74 70 67 59 92 61 66 68 Mean 61.0 66.3 76.7 SD 6.0 7.5 13.3862 vs. 86 P = 0.7 P = 1 P = 1

1Intracellular survival of each bacterium in peripheral blood monocyte-derived macrophages from Bull 86 and Bull 862 was analyzed at time 0 h and 12 h postinfection in duplicate culture plates. The percent survival of each bacterium was determined in triplicate samples (replicates), and the data were analyzed for significance by the Mann-Whitney U-test.

2Percent reduction of bacterial survival data for Bull 86 was generated in 1992 before his death, whereas the percent reduction in bacterial survival data for Bull 862 were necessar-ily determined in 2003 using the identical procedures, instru-mentation, and technical personnel.

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before the first report of successful cloning from adult cells (Wilmut et al., 1997) and at a time when cloning animals using somatic cells was thought to be biologically impossible. The relevance of this lies in the fact that more than a dozen mammalian species have now been cloned by somatic cell nuclear transfer. Although difficult to predict, there could easily be thousands of potentially valuable animal genomes stored in the form of somatic cells in LN2 over the last several decades that could be thawed and utilized to recover valuable genotypes. Previous studies have demonstrated that cloned offspring do not always exhibit phenotypes identical to the original cell (nucleus) donor (Shin et al., 2002; Archer et al., 2003). We and others have shown this phenomenon likely due to inadequate reprogramming of the donor nucleus after nuclear transplantation, resulting in abnormal epigenetic regulation of gene expression (De Sousa et al., 1999; Humpherys et al., 2002). Therefore, we were not certain whether Bull 862 would exhibit a disease-resistant phenotype. In vitro bacterial killing assays were performed and demonstrated that (similar to the original Bull 86), the cloned bull (Bull 862) is resistant to brucellosis, indicating that the genes affording disease resistance were not affected by the nuclear transfer procedure and are functioning properly. Had Bull 862 not exhibited a disease-resistant phenotype as a result of inadequate nuclear reprogramming, it is unlikely that this phenotype would be passed on to his offspring. Previous studies in mice have clearly demonstrated that although cloned animals may exhibit abnormal gene expression patterns, this problem is corrected when these animals are used for breeding, and the offspring of clones exhibit normal gene expression (Tamashiro et al., 2002). To date, we have not produced any offspring using semen collected from Bull 862, but based on the in vitro data, we predict all his offspring will exhibit the genotype and phenotype of the original Bull 86. Furthermore, we reproduced a desired genetically identical animal that can allow us further study of the genetic background related to disease resistance. This study provides direct evidence for the feasibility of long-term conservation by cryopreservation and rescue of genetic materials when combined with animal cloning technologies.

LITERATURE CITEDAdams, L. G., R. Barthel, J. A. Gutiérrez, and J. W. Templeton. 1999.

Bovine natural disease resistance macrophage protein 1 (NRAMP1) gene. Arch. Tierz. 42:42–55.

Adams, L. G., and J. W. Templeton. 1998. Genetic resistance to bacterial diseases of animals. Rev. Sci. Tech. 17:200–219.

Archer, G. S., S. Dindot, T. H. Friend, S. Walker, G. Zaunbrecher, B. Lawhorn, and J. Piedrahita. 2003. Hierarchical phenotypic and epigenetic variation in cloned swine. Biol. Reprod. 69:430–436.

Barthel, R., J. Feng, J. A. Piedrahita, D. N. McMurray, J. W. Temple-ton, and L. G. Adams. 2001. Stable transfection of the bovine NRAMP1 gene into murine RAW264.7 cells: Effect on Brucella abortus survival. Infect. Immun. 69:3110–3119.

Beever, J. E., Y. Da, M. Ron, and H. A. Lewin. 1994. A genetic map of nine loci on bovine chromosome 2. Mamm. Genome 5:542–545.

Campbell, G. A., L. G. Adams, and B. A. Sowa. 1994. Mechanisms of binding of Brucella abortus to mononuclear phagocytes from cows naturally resistant or susceptible to brucellosis. Vet. Immunol. Immunopathol. 41:295–306.

Caron, J., D. Malo, C. Schutta, J. W. Templeton, and L. G. Adams. 2004. Genetic susceptibility to infectious diseases linked to Nramp1 gene in farm animals. Pages 16–28 in The Nramp Family. M. Cellier and P. Gros, ed. Landes Biosciences – Kluwer Academic/Plenum Publishers, Georgetown, TX.

Cellier, M., N. Groulx, E. Schurr, T. Kwan, F. Sanchez, G. Govoni, S. Vidal, J. Liu, and E. Skamene. 1994. Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal map-ping, genomic organization, and tissue-specific expression. J. Exp. Med. 180:1741–1752.

De Sousa, P. A., Q. Winger, J. R. Hill, K. Jones, A. J. Watson, and M. E. Westhusin. 1999. Reprogramming of fibroblast nuclei after transfer into bovine oocytes. Cloning 1:63–69.

Feng, J., Y. Li, M. Hashad, E. Schurr, P. Gros, L. G. Adams, and J. W. Templeton. 1996. Bovine natural resistance associated macro-phage protein 1 (NRAMP1) gene. Genome Res. 6:956–964.

Gardner, D. K., M. Lane, A. Spitzer, and P. A. Batt. 1994. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: Amino acids, vitamins, and culturing embryos in groups stimulate development. Biol. Reprod. 50:390–400.

Harmon, B. G., L. G. Adams, J. W. Templeton, and R. Smith, III. 1989. Macrophage function in mammary glands of Brucella abortus-infected cows and cows that resisted infection after inoculation of Brucella abortus. Am. J. Vet. Res. 50:459–465.

Harmon, B. G., J. W. Templeton, R. P. Crawford, F. C. Heck, J. D. Wil-liams, and L. G. Adams. 1985. Macrophage function and immune response of Brucella abortus naturally resistant and susceptible cattle. Pages 345–354 in Genetic Control of Host Resistance to Infection and Malignancy. E. Skamene, ed. Alan R. Lis Inc., New York, NY.

Hill, J. R., C. R. Long, C. R. Looney, J. A. Thompson, and M. E. Westhusin. 2000. Development rates of male bovine nuclear trans-fer embryos derived from adult and fetal cells. Biol. Reprod. 62:1135–1140.

Humpherys, D., K. Eggan, H. Akutsu, A. Friedman, K. Hochedlinger, R. Yanagimachi, E. S. Lander, T. R. Golub, and R. Jaenisch. 2002. Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proc. Natl. Acad. Sci. USA 99:12889–12894.

Mock, B., M. Krall, J. Blackwell, A. O’Brien, E. Schurr, P. Gros, E. Skamene, and M. Potter. 1990. A genetic map of mouse chromo-some 1 near the lsh-ity-bcg disease resistance locus. Genomics 7:57–64.

Qureshi, T., J. W. Templeton, and L. G. Adams. 1996. Intracellular survival of Brucella abortus, Mycobacterium bovis bcg, Salmonella dublin, and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus. Vet. Immunol. Immuno-pathol. 50:55–65.

Schnabel, R. D., T. J. Ward, and J. N. Derr. 2000. Validation of 15 microsatellites for parentage testing in North American bison, Bison bison and domestic cattle. Anim. Genet. 31:360–366.

Shin, T., J. Pryor, L. Liu, J. Rugila, L. Howe, S. Buck, K. Murphy, L. Lyons, and M. E. Westhusin. 2002. A cat cloned by nuclear transplantation. Nature 415:859.

Tamashiro, K. L., T. Wakayama, H. Akutsu, Y. Yamazaki, J. L. Lachey, M. D. Wortman, R. J. Seeley, D. A. D’Alessio, S. C. Woods, R. Yanagimachi, and R. R. Sakai. 2002. Cloned mice have an obese phenotype not transmitted to their offspring. Nat. Med. 8:262–267.

Vidal, S. M., D. Malo, K. Vogan, E. Skamene, and P. Gros. 1993. Natural resistance to infection with intracellular parasites: Isola-tion of a candidate for bcg. Cell 73:469–485.

Wilmut, I., A. E. Schnieke, J. McWhir, A. J. Kind, and K. H. Campbell. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813.

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