embryo transfer manual fao

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embryo transfer manual fao

Nations concerning the legal status of any country, territory, city orOrganization of the United Nations. No part of this publication may be reproduced, stored in aApplications for such permission, with a statement of the purpose and extent of theAgriculture Organization of the United Nations, Via delle Terme di Caracalla, 00100. Rome, Italy. Organization of the United Nations was trulyKeeping thisWe have limited citations to scientific literatureClearly we have borrowedWe acknowledge with gratitude the work of ourThe sole purpose of links to non-FAO sites is to indicate further information available on related topics. University of Florida and his recipient dam (Photo courtesy of UF Health Science Center. Communications) Nations concerning the legal status of any country, territory, city orNo part of this publication may be reproduced, stored in aApplications for such permission, with a statement of the purpose and extent of theAgriculture Organization of the United Nations, Via delle Terme di Caracalla, 00100. Rome, Italy. Most of the techniquesThe sole purpose of links to non-FAO sites is to indicate further information available on related topics. Seng Tan of the Universiti Pertanian. Sitzman, C.G., Wilgenberg, B.,Press. 242 pp. Cambridge University Press. G.E., Jr. 1978. Embryo transfer forG.E., Jr. 1979. Embryo transfer inTheriogenology, Animal Reproduction Laboratory. Bulletin No. 2. Fort Collins. CO, Colorado State University. Press, 43 pp. (also available in Spanish,Stimulation of early embryonic developmentK. 1988. Pregnancies after co-cultureOffice International des Epizooties,Paris. 90 pp. Embryo Movement Symposium, Champaign, IL, International Embryo. Transfer Society. 198 pp. Effect of donor-embryo-recipient interactionsTheriogenology,Street, Champaign, IL 61820. 87 pp. R.P. 1985. Bovine embryo transferL.J. 1986. Synchronization of festrusEmbryo transfer in animals. In. Gwatkin, R.B.L., ed. ManipulationReprod. Artif. Insemin., Dublin, 5:Annu. Meet. Int.


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Embryo Transfer Adams, W.M. 1980. Effects of a progestagenWard, K.A. 1988. Techniques forCode. F-75017 Paris, France, 12 ruePamphlet, Animal Reproduction Laboratory. Colorado State University.Ultrasonic imaging of the ovariesTheriogenology. Seidel, G.E., Jr. 1989. Effects of. GnRH on superovulated cattle. Theriogenology. Genetic engineering in farm animals:Eyestone, W., Northey, D., Gilligan,Moor, R.M. 1971. Production ofGriffin, J.L. 1980. Commercial aspectsJ.D., eds. Fertilization and embryonicInt. Congr. Anim. Reprod. Artif. Insemin.,University. 88 pp. The embryo transfer industry. In. Seidel, S.M., eds. New technologies. FL, Academic Press (also availableAnalysis of application of embryoTheriogenology,EEC Publication EURG.E., Jr. 1987. Transfer of bovineG.E., Jr. 1984. Pregnancy rates withTheriogenology. Quick splitting of bovine embryos.Press. 242 pp. S.M., eds. 1981. New technologiesFL, Academic Press. 268 pp. San Francisco,FL, Academic Press. 566 pp. Vista International. 148 pp. Fort Collins, Colorado State. University. 43 pp. Fort Collins. Colorado State University. 45 pp. New York, Plenum Press.York, Plenum Press. 388 pp. Beltsville Symposia in Agricultural. Research III. Montclair,Street, Champaign, IL 61820. 87 pp. Proceedings of annual conferences.St., Champaign, IL 61820. York, Plenum Press. 371 pp. State University. 337 pp. Fort Collins, Colorado State University.Sales Agents or directly from Distribution and Sales Section, FAO. Via delle Terme di Caracalla, 00100 Rome, Italy. Records frequently permit one to determineIn many countries licences to do embryoEmbryo Transfer Society for registration purposes (Example 1). ManyThe absoluteIt is obvious that usingIn many laboratories, codedThe responsible practitioner signing this certificate is attestingIf frozen embryos are transferred, the Certificate of Embryo Recovery will be completed by theThe practitionerOne application for transfer is required for each change in ownership.


The practitionerEach change of ownership must be covered byDanish red) WW - Red Holstein RP - Red Poll RN - Romagnola RO - Rotbunte SA - Salers SG - Santa Gertrudis MS - Shorthorn (milking) SS - ShorthornSire: Disposed. Various suppliersThere is nothing to wash and sterilize; noSome practitioners do not even have a refrigerator,We have organized this chapter by listingWe have listed onlyThese suppliers, however, can give information on distributorshipsInclusion in this list does not signifyUSA. Anpro gas sterilizer and sterilization products. Transfusion bags, Syncromate B. CO 80523 USA. 303-491-5287. Cervical expander. USA. 303-371-5713. Siliconizing agent, culture dishes, biological filters,Sterilization packaging and supplies. Prairie, TX 75051 USA. 214-660-1771; Fax: 214-660-2303. Antibiotics,Englewood, CO 80112 USA or Fisher Scientific International, 50 Fadem. Road, Springfield, NJ 07081 USA. 201-467-6400; Cable: Fishersci, Springfield. NJ; Telex: 475 4246 or 138287; Fax: 201 379 7415. Paraffin oil,Culture media. Estrumate (cloprostenol). Micromanipulator. Valley Road, Tyler, TX 75702 USA. 216-595-2047; Telex: 205997-PETSUR. Fax: 214-592-1525. Antibiotics, artificial insemination equipment,USA. 303-484-0307. Microscopes. Latex tubing, culture dishes, biological filters, pipettes, and many laboratoryAntibiotics, antiseptics, artificial insemination equipment, catheters, dishes,Most people useHigher magnifications areHowever, a 30X to 50X magnification isThus, one needsIn practice,Unless required for some other purpose, such as splitting embryos, stereo-microscopesZoom model SZ-111-100 with transmitted light-base illuminator; American. Optics (now Reichert-Jung) Stereostar 561B or 561C with Starlite illuminator;Nicholas illuminator.


It should be borne in mind that a compoundAlso the process of learning to evaluate embryos is easier withAny of these can be used forThis is easier to use for embryosIn purchasing aNote well, however,Nearly all of theseAll machines require repairs from time toA particularly good approach is a system of shipping a replacement machineThis is especiallyThe second importantTemperatures at the time of seeding and plunging areSoquel, CA 95073 USA Liquid nitrogen vessel Straws lowered into vapour Suite 303 Napa, CA 94558 USA Vessel of liquid nitrogen Slots for straws only Spring Valley, WI 54767 USA Neck of liquid nitrogen tank Small chamber Cost, reliability of service,We have no way of knowing about all possible models of freezing machines. In the majority of embryo transfer programmes,For example, it makes good sense to selectObviously, it is inappropriate to produce animals that will not beThis may beHealthy, cycling cattleYoung cows seem to yield slightlyExtremely fat cows make poor donors, both because they do not respondSick animals usually do not produce many goodSecond, encourage management practicesThird, develop strategies toIn fact, selecting the male is usually moreLikewise, it is necessary to select fertile bulls and fertile semen.


SpermBoth situationsKeeping donors on the farm isAlso, less input for labour andHowever, it is extremely important to have goodPlanning is very complex, since a series ofIn most cases, personnel will visit theHowever, donors can be trucked to the embryoThree to four hours of travel in trucks or trailers does not seem to be aThis includes equipment for handling cattle, such as chutes andPersonnel at the farm must have certain skills and,For best results, palpation skills areOn the average, success rates with donors on the farmNevertheless,In most cases of embryo collection on the farm,Clearly, under these circumstances, facilities for large numbers of donorsConcentrating cattle in this way invitesThe net result is that greatThis includesIf done systematically and conscientiously, herd health programmes tailoredLocal conditions,Proper nutrition is extremelyDonors definitely shouldFrequently, using the farmer'sAnother consideration is management capabilities of the farmer. In.

North America, one simple criterion has been useful to assess managementOne must beHeifers generally have higher fertility than cows,On the other hand, it is more difficult to transferClearly, cows and heifersThe following are essential: Note that severe problems can occur if a herdThis can be prevented by vaccinationThis also provides antibodies inSometimes such considerationsVery large herds without a pronounced breedingEffective herds usually number at least severalThese herds are the mostThe waiting time can be minimizedExcess embryos are frozen when insufficient recipients are available;Despite the advantages of frozen embryos, recipientsMany fail to getBlood or milk can beThe exception is if pregnancy ratesAbout 5 percent ofThere is some evidence that oestrus synchronization with prostaglandinsPhysiological characteristics of the reproductive tract change greatlyOn day 1 after oestrus, forSince considerable behaviouralThus, recipients observed to be in oestrus one-half dayThe method that generally worksDetection cannot be done properly while sitting on the fence, although thisSome cows are more active in mounting other cowsIt may help in the case of such cows to placeDisplay of behaviouralEach animal will be in one ofThe latter two categories should beCows in oestrus stand when mounted by others. Suspicious signsNot every cow showing oneWhen oestrus is detected,Chapter 16, example 4), the information should be transferred to a notebookWhenever dataSuch aids are recommendedIf a teaser animal is used, it should be an androgenized female.

The first good indicatorProgesteroneHowever, with good oestrus detection, oneIn other words,Another point is that neither oestrusThe informationWhen costs of ultrasonographyA serious problemOf course, ultrasound can also be usedWe do not recommend palpation prior to day 45, bothThus, our recommendation is toThe following steps will make it possible for most non-pregnant recipientsFirst, it is imperative not to breedPregnancy diagnosis is then doneWith rectal palpation, this window may extend toNote that a good portion of this window isThis method should not be used, at least not priorThe small percentage ofNutrition is clearly important as well as preventionIt is easy to lose 10It is especially costly to loseOf course, recipients often. However, each step in the process must be done correctly for theThe end result will only be as good as the weakest The second most common problem isUnless large numbers of healthy, thrivingA clean laboratory work area is needed; mobile vans can beObviously, cows must be kept separate from bulls. An unusually common error is that recipients become pregnant from naturalIt is clearly necessaryImplicit in such a facility isFacilities andAlso, the skillsIn fact, we do not recommendFrequently the goal is simply to establishEmbryo transfer should be thought of as aOnly rarely is it the method of choice for reproducingObviously this may change asFor instance, it is misleading to use cost per viableFor a more thorough discussion of uses of. Thus, precise costs mean littleHowever, two generalizationsThe costs of the actual embryo transferA second generalization is that costs per calf areCosts are listed in Table 13 and discussed below (comments are numberedThe cost in feed and lostUnder poor conditions, these losses canIn some countries, there are tax advantages toFor example, an unfavourable (or favourable)Obviously, one must analyse the costs and benefits. When the benefitsHowever, if.

However, factors such as inflation and interest rates complicate evaluationsThis is a marketable product, and other measuresThese steps are summarized in Table 9 along withThe one factor that is nearly impossibleThis causes considerable frustration. One can never be sureWeather cannotCows with histories of infertility generallyOccasionally, results with infertile donors are satisfactory. Usually one hasEither kind of semen works well if handled properly, except that some bullsIf embryos are frozen, the averageTable 11 were collected, so current success rates could be slightly higher atSuccess rates would probably be lower on the farm. Embryo Transfer Unit at Colorado State University. Data include all donorsOften animals with these problems areNote that 47 percent of theIf one superovulates each donor repeatedly, for instance, three timesAbout 90 percent of recipients pregnant. Furthermore, it is much easierRecipients are placed in squeeze chutes thatThe CL is located by rectal palpation and theAbout 60 ml of 2 percent procaine is givenIn everyday practice this seems moreHaving scrubbed, the surgeonThe surgeonThe uterineThe uterine horn itself is very fragile.

A puncture wound is made with aFirst, it is necessary to be able to palpate ovariesPregnancy rates areAlso, recipients should be rejectedEven very experienced palpators make some errors in palpating corporaHeifers present a special challenge because of theThe best training prior to undertakingIdeally, the trainee will have inseminated hundreds of cattle artificially,Some people never master this technique, and others requireThis is not surprising sinceWell-trained inseminators generally requireMost technicians who are successful with non-surgicalOnce the entire sequence of superovulation,The result is that proficiency isEven if this is true, in most circumstances nonsurgicalThis may also obviate the needA plastic 1-cc tuberculin syringe fits snugly over the straw for aspirating andCare must be taken notCassou inseminating gun for French straws (Figure 27). There areMany have been tried in the. Embryo Transfer Laboratory at Colorado State University. None of theseThe top straw is loaded for freezingCassou insemination gun may be advisable. Most instruments designedThis can beThe third situation in which a different instrumentHolstein-Friesian cows. Many of the instruments for non-surgical transferPerhaps the most used of these specialStraws should be shortened byCassou inseminating gun (B), and the sheath (C) Figure 2). This relaxes rectal musculature, making it easier to manipulateThe problem ofA good site to aim at is theSome technicians go a bit furtherThe key is to pass the gun without damaging the endometrium. Therefore,This is definitely trueTwo questions arise: to what extent isSeveral studies with largeThere is a hintPregnancy rates do not decline.

With embryos of lower qualityFurthermore,In some circumstances, natural synchrony isUnder some circumstances, one of these, Syncromate B (norgestomet)Despite anecdotal reports of problems in the field, each of aThis also synchronizes all donors andMost of Table 8 is self-explanatory, but we callOn day 27, potential recipients are given prostaglandinActually, most. On day 37, afterHowever, it does illustrate the principles involved. If procedures are carried outThe following protocol has workedEmbryos should be frozen within three to fourAll of the above steps are done atThe end is then sealed with heat (for example,Paraffin oilA second benefit of paraffin oil is to prevent embryos from entering the airThis results in death of the embryo duringBe sure that they remain seeded. Seeding is accomplished by touching the side of the embryo container withAutomatic seeding occursThe equipment to cool embryos canThe only advantage of complex equipment is savingThis is done by cutting the heat-sealed end ofTheoretically,Instead of 0.4 percent BSA, 10 percent serum canBoth procedures lead to similar results, but theIf recipients are available, transferA few will turn into calves. Some people use glass containers instead of plastic straws. These thaw moreHowever, these cannot yet be. The conceptQuality control procedures need not beBeing able to correlate eventsFor instance, the cause ofRecords need not be complicated but they need to be complete. It is goodLot numbers should beFor certain procedures, the person doingIndicators of proper function of equipmentTable 15.

However, since manyThe latter can be done withNote that a somewhat aberrant pHToxicity is more difficult toIf circumstancesResistance to electricThe best courseCulture of twocellHowever, it is wise toAccordingly,For example, discard the first fewThe rubber plungers of certain syringes have beenSome toxicity is due to sterilizationIf one gets into the habit of alwaysSometimes they areDeviations of only a fewWarming plates and microscope stages can cook embryos. TemperatureAlways check any thermometer against aFurther, we suggest that allHealth statusExcellent quality control information on nutritionThis may notThis is especially a problem whenTherefore, it is recommended that a dropThis should be doneIn most cases, it is not necessaryThe main error with artificialQuality control for this is best accomplishedIf this can be arranged easily, itIf the majority ofErrors in palpation of corpora lutea areEfficacy of isolating embryos from theThe reproductive tract of a cow that received a shamThe site of deposition of the embryo is moreAttitude problems may be due to health, personalIn any case, workers should beDifferences among persons can be determined by studying such records asWhen marked, consistent differencesThis has beenIf retraining isIf the volume of embryo transfer at. Embryos of various stages are illustrated in. Figures 14 to 25. During this timeOnly at the morula stage does theHoard, Dairyman, 10. March 1988, p. 246) During compaction,Compaction is an excellent sign that the embryo is developing normally;Thus blastocystConversely, lack of blastocoele formation by seven to eight days afterIf the zonaWhen the blastocoele becomesThis is the expanded blastocyst stage. AfterHatched blastocystsObviously, there is noBoth errors are common when people are firstOne must also learn how to manipulate and examine embryos.

ExperienceIdeally hundreds of embryos should be studied under the guidance ofPhotographs, drawings or slides ofHowever, they can only substituteExperienced personnel can evaluate more thanHowever, a small percentage of embryos require aFor learning purposes, a compound microscope isThese limitations make it easy to spillNomarski optics. (B) Follicular oocyte afterNomarski optics. (C) Normal appearing 1-cell ovum recovered five daysBright-field optics. (D) Normal,Bright-field optics. (FiguresAlthough it isDeviations from normal includeMost of these abnormalities areNote that pregnancy rates with bisected embryosNomarski optics. (B) Same ovum as inNomarski optics. (D) Unfertilized oocyte recovered six days after oestrus. Note blisters of clearNomarski optics. (B). Unfertilized ovum with two fragments of cytoplasm. Note large vesicles within cytoplasm. Bright-field optics. (C) Fragmented ovum, likely unfertilized recovered five days after oestrus. Bright-field optics. (D) Disintegrated ovum, probably unfertilized. Bright-field optics Note clear cytoplasmNomarski optics. (D) A 2-cell embryo recovered five days after oestrus. Note clear cytoplasm. Nomarski optics As mentioned earlier, presenceDay-8 embryos should have a largeA distinct, inner cell massAs with day-6 embryos, various imperfections are notBright-field optics. (B) SameUncompacted morula recovered three days after oestrus; dark cytoplasm. (C) Severely retardedAll are bright-field optics. About one-third of these have beenThe single most difficult task for people learning toBright-field optics. (B). Compacted morula recovered seven days after oestrus with several excluded cells; goodNomarski optics. (C) Compacted morula recovered seven and a halfBright-fieldHowever, the small, compactedBright-field optics Bright-field optics.Note the thinned zona pellucida. Bright-field optics. (D). Hatching blastocyst typically found nine days after oestrus.

Bright-field optics Occasionally theyAn excellentBright-field optics. (C) Degenerate,Those classified incorrectly should be placed in theIn cases in whichFor those frozenEven two-or three-cell embryos may in fact beTable 6 (from Elsden et al., 1978) provides aClearly, this classification systemOf course, it is far fromAs a rule of thumb, only good andResults of freezing fair quality embryos. Carelessness in even the smallest of details, however, greatly diminishesPrior to collection, an embryo is exposed onlyThus, even when theViruses are rarely found at infective levels in uterine fluid. Furthermore,Pathogens can be removed entirelyOf great importanceIn additionThe presence of pathogens on or in the embryo cannot be diagnosed byIn most cases, it does not because the verySociety, 1987). Minimum standards for the sanitary handling of embryosSociety. This manual is updated regularly to include recommendationsInternational Zoosanitary Code. Currently recommended handling proceduresChapters 6 and 15). Care must be taken to avoid contamination of equipmentFigure 32). No more than 10 embryos should be placedThe container should be agitated gently. TheIt is important to transfer as little fluid as possibleContainers should be covered to avoidContainers and pipettesThe trypsin enzyme used to prepare the washesA ready-to-use solution is available commercially. After embryos have been washed, they should beFor purposes of disease control, only embryos that have an intact zonaAlthough current diagnostic proceduresThe fluid from the last four washesEvery detail is important. Nevertheless, most are based on the principles discussed in this. To date, no chemically defined mediumHowever, several mediaAn embryo's range of tolerance for these properties is narrow.

InadequateSaline will not support embryonic development,Moreover, saline can be steam-sterilized (at leastA serious problem with saline,BSA and serumCommerciallyThe CaCl 2 and MgSO 4 can also beIf these areDissolve mixture One in 2Add these 2 litres to the 8 litres stirring constantly. Other methods ofSterilize medium by passageMedium-199 (TCM-199), Ham's F-10 medium, and Brinster's Mouse Ova. Culture Medium-3 (BMOC-3). All of these are commercially available. TCM-199 with Hank's salts does not depend on CO 2 for buffering, but. TCM-199 with Earle's salts as well as Ham's F-10 and BMOC-3 must beHowever, macromolecules derived from serum are also a possible vectorBSA and bovine serum are currently recommendedPolyvinyl alcohol and polyvinylThese macromolecules do not functionThe powder should be poured very gently on the surface of the medium andThe medium should then be. Purer types of. BSA are also acceptable. BSA can also be purchased as an aqueous solution,The sediment is discardedThis step is repeated once again. The finalIn order to inactivate the proteinSerum can be frozen and stored for up to eight months provided that theFor quality control, one aliquot from eachSociety, 1987). Heat-inactivated, sterilized serum is added to mediumStandard PasteurGlass, in 15-cm lengths, is heated inAfter pipettes have beenPipettes are made in batches ofIn practice, we discardAlternatives to making pipettes are to use tomcat.

Because of low reproductive rates and longCattle may be valuable for many reasons, including scarcity, proven geneticIt is possible toThis is becauseThis waiting can be minimized with good management, and is often justifiedAn exception to decreasedSuch twinningThere is even a bias in scientific reports becauseThus one must beSuccess rates can be especially poorAlthough success rates are low, it is possible toFor example, cows with cystic ovaries might be mostAnimals have the advantage of beingThe disadvantages are that costs,Moreover, if cows are imported, the genetic influenceThe relativePenicillin or oxytetracycline may be infused for three to four days but, if not done carefully, can do more harm than good. Normal embryo transfer procedures can be followed after the next oestrus. In cases of subclinical or recurrent endometritis, repeated superovulation and embryo transfer may result in the “rescue” of viable embryos from the toxic environment to develop in the healthy uterus of the recipient. With normally cycling, parous cows one should try at least three times to recover a single ovum to diagnose if there is ovulation, and if the ovum has been fertilized. It may be helpful to use raw semen. If a morphologically normal embryo at the expected stage of development is recovered, consider progesterone therapy during gestation. If the cystic ovarian condition appears to be hereditary, no propagation should be attempted. A combination of superovulation with surgical recovery or laparoscopy helps to diagnose the cause of the infertility and can result in recovery of viable embryos for transfer. Some conditions can be corrected surgically (e.g., flushing plugs of debris from the oviduct), although relieving adhesions is rarely of lasting benefit. In the future, it may be efficacious to recover oocytes from the ovaries of such cows laparoscopically for fertilization in vitro. Code, 1986).