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Training manual for embryo transfer in cattle

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Chapter 17
Equipment and supplies

EQUIPMENT, SUPPLIES, DRUGS AND REAGENTS

Most embryo transfer practitioners in North America use entirely disposable supplies and purchase sterile saline and complete media. Various suppliers ship these materials with just a telephone call (credit is prearranged). This greatly simplifies operations. There is nothing to wash and sterilize; no medium need be prepared except to add macromolecular and antibiotic solutions with a sterile syringe; there is no need to purify water; there is no danger of spreading disease from farm to farm because everything is disposed of at each farm. Some practitioners do not even have a refrigerator, but depend on each farmer's household refrigerator.

This approach is inappropriate for embryo transfer in many countries because of unreliable access to suppliers, but it should be considered seriously in some situations. We have organized this chapter by listing equipment and supplies needed for basic embryo transfer, and add additional supplies for various functions, such as media preparation and cryopreservation.

Equipment

Supplies

Drugs and reagents

Additional needs if washing and sterilizing capabilities are required for reuse of equipment:

Equipment

Supplies

Drugs and reagents

Additional needs if media are to be prepared at the embryo transfer laboratory:

Equipment

Supplies

Drugs and reagents

Additional needs if embryos are to be cryopreserved:

Equipment

Supplies

Drugs and reagents

Additional needs for micromanipulation:
(Note: Simple bisection of embryos does not require this equipment.)

Equipment

Supplies

Other optional equipment:

Suppliers

The following list of suppliers includes companies which have exhibited at the annual conference of the International Embryo Transfer Society recently or which are listed in Procedures for recovery, bisection, freezing and transfer of bovine embryos (Elsden and Seidel, 1985). We have listed only our local suppliers; it is logistically impossible to list all suppliers worldwide. These suppliers, however, can give information on distributorships for their products in other localities. Inclusion in this list does not signify endorsement nor does exclusion signify lack of endorsement.

American Embryo Systems, 2619 Skyway Drive, Grand Prairie, TX 75051 USA. 214-641-5420. Culture media, serum, antibiotics.

H.W. Andersen Products, P.O. Box 1050, Chapel Hill, NC 27514 USA. Anpro gas sterilizer and sterilization products.

CEVA Laboratories, Inc., 10560 Barkley, Overland Park, KS 66212 USA. Transfusion bags, Syncromate B.

Colorado State University, Embryo Transfer Laboratory, Fort Collins, CO 80523 USA. 303-491-5287. Cervical expander.

Curtin Matheson Scientific, 12950 E. 38th Avenue, Denver, CO 80239 USA. 303-371-5713. Siliconizing agent, culture dishes, biological filters, pipettes, tubing, and many laboratory supplies including plastic ware.

Edwards Agri Supply, P.O. Box 65, Baraboo, WI 53913 USA. 608-356-6641. Artificial insemination equipment; oestrus-detection aids.

Emery Medical Supply. 5601 Gray Street, Arvada, CO 80002 USA. Sterilization packaging and supplies.

EM-TEX Supply Co., Inc., 2741 S. Great Southwest Parkway, Grand Prairie, TX 75051 USA. 214-660-1771; Fax: 214-660-2303. Antibiotics, antiseptics, artificial insemination equipment, catheters, dishes, flushing and freezing media, disposable flush kits, embryo filters, embryo transfer guns and straws, gloves, bovine serum albumin, sera, programmable cryopreservation unit, cervical expanders, drugs for superovulation and oestrus synchronization, connectors, sterilization packaging.

Fisher Scientific Company, 14 Inverness Drive E., Building A, Suite 144, 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 dishes, biological filters, pipettes, tubing, microscopes, and many laboratory supplies including plastic ware.

GIBCO, 3175 Staley Road, Grand Island, NY 14072 USA. Culture media.

Mobay Corp., P.O. Box 390, Shawnee, KS 66201 USA. 913-631-4800. Estrumate (cloprostenol).

IMV, 10, rue Georges Clemenceau, B.P. 76, F-61300 L'Aigle, France. 333-324-0233 or 6870 Shingle Creek Parkway, Suite 100, Minneapolis, MN 55430 USA. 612-560-4986. Artificial insemination equipment, transfer guns, sheaths, straws and polyvinyl chloride powder.

Intermed, Inc., Newfoundland, NH 07435 USA. 201-697-3818. Foley catheters.

Kamar, Inc., P.O. Box 26, Steamboat Springs, CO 80477 USA. 303-879-2591. KaMaR oestrus-detection aids.

M & M Company, 1120 Industrial Ave., Escondido, CA 92025 USA. 619-746-0800; Telex: 607 950. Micromanipulator.

PETS, Professional Embryo Transfer Supply, Inc., 27221/2 Garden Valley Road, Tyler, TX 75702 USA. 216-595-2047; Telex: 205997-PETSUR; Fax: 214-592-1525. Antibiotics, artificial insemination equipment, catheters, dishes, flushing and freezing media, disposable flush kits, embryo filters, embryo transfer guns and straws, gloves, bovine serum albumin, sera, polyvinyl chloride powder, sterile water, microscopes, cervical expanders, drugs for superovulation and oestrus synchronization, connectors and sterilization packing.

Reproduction Resources, Inc., P.O. Box 135, Hebron, IL 60034 USA. 815-648-2431. Sani-Shield Protector.

Research Instruments, Ltd., Kernick Road, Penryn, Cornwall, TR10 9DQ, UK. Micromanipulator.

Rocky Mountain Microscope, 440 Link Lane, Fort Collins, CO 80524 USA. 303-484-0307. Microscopes.

Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178 USA. 314-771-5750. Reagents for culture and freezing media.

The Upjohn Company, P.O. Box 108, Kansas City, MO 60901 USA. 616-323-4000. Lutalyse (prostaglandin F2 alpha).

VWR Scientific, P.O. Box 39396, Denver, CO 80239 USA. 303-371-0970. Latex tubing, culture dishes, biological filters, pipettes, and many laboratory supplies including plastic ware.

Veterinary Concepts, 100 McKay Avenue, Spring Valley, WI 54767 USA. Antibiotics, antiseptics, artificial insemination equipment, catheters, dishes, flushing and freezing media, disposable flush kits, embryo filters, embryo transfer guns and straws, gloves, bovine serum albumin, sera, programmable cryopreservation unit, cervical expanders, drugs for superovulation and oestrus synchronization, connectors and sterilization packaging.

United States Biochemical Corporation, P.O. Box 22400, Cleveland, OH 44122 USA. 216-765-5000. Bovine serum albumin.

MICROSCOPES

It is essential to have a stereomicroscope of good quality to search for embryos, and most programmes should have at least two. Most people use a magnification of 8X to 15X to locate embryos. Higher magnifications are unsuitable for this purpose because the field of view is too small, which greatly increases the time required for searching as well as the likelihood that embryos will be overlooked. However, a 30X to 50X magnification is essential for evaluation of embryos once they are located. Thus, one needs a stereomicroscope with at least two magnification settings. In practice, there is usually some zoom or step arrangement to vary magnification from lowest to highest settings.

Stereomicroscopes of good quality are priced in the range of US$1 200–1 500. Sometimes good used instruments can be purchased for much less. Unless required for some other purpose, such as splitting embryos, stereo-microscopes costing US$3 000 and higher are a luxury; they are not any better for routine embryo transfer work than the less expensive ones. Conversely, the microscopes marketed for US$200–300 (price when new) simply are not good enough.

Appropriate stereomicroscopes that we have used include the Olympus Zoom model SZ-111-100 with transmitted light-base illuminator; American Optics (now Reichert-Jung) Stereostar 561B or 561C with Starlite illuminator; and Bausch and Lomb (now Cambridge Instruments) BVB-73 with Nicholas illuminator. Eyepieces of 10–20X magnification are available for most of these. Similar models from other companies are usually satisfactory. Always be sure to obtain a base/stand designed for transillumination of transparent specimens and a good light source (with spare bulbs).

Advice on purchasing a compound microscope for embryo evaluation purposes is similar to that for a stereomicroscope: a sturdy, easy-to-use bright-field microscope without complex accessories is best, generally in the range of US$1 500–2 000. It should be borne in mind that a compound microscope is not absolutely essential, but that a small percentage of embryos cannot be evaluated properly without one, and evaluation of progressive motility of semen requires a microscope with 100–200X magnification. Also the process of learning to evaluate embryos is easier with the improved resolution of a compound microscope.

Many laboratories have compound microscopes for other purposes such as semen evaluation or microbiology studies. Any of these can be used for embryos as well. If a new microscope is to be purchased just for embryos, an inverted type should be considered. This is easier to use for embryos because the objective is below the stage, which reduces the risk of contaminating or spilling embryos. Inverted microscopes, however, are more expensive and generally have slightly poorer resolution. In purchasing a compound microscope, one also should take into account needs such as micromanipulation, for which a fixed stage is required, whether the microscope is inverted or not. Obviously, in some cases it is best to have more than one compound microscope, for example, one with differential interference phase-contrast (Nomarski) optics or phase-contrast optics and one that is less expensive with simple bright-field optics. Note well, however, that embryos can be evaluated perfectly well with a 10X bright-field objective, and for this purpose more sophisticated systems are of little additional value. A 2X or 4X objective is useful for locating embryos prior to examination with the 10X objective.

FREEZING MACHINES

More than 20 models of freezing machines are currently being manufactured by approximately 12 companies in eight countries. Nearly all of these machines work satisfactorily. All machines require repairs from time to time, so arrangements for service are important, particularly in remote areas. A particularly good approach is a system of shipping a replacement machine on loan while the malfunctioning machine is being repaired. An obvious generalization is that more can go wrong with complex machines than with simple ones; however, this does not always apply because some of the more complex machines are particularly well made.

The reason for purchasing more complex freezing machines is that they are easier to use; most have automatic functions so that little or nothing need be done except to add the straws or ampoules to the freezing chamber at the beginning of the process and remove them prior to plunging into liquid nitrogen. A somewhat incongruous situation is that companies and organizations in developing countries tend to purchase complex and expensive freezing machines. Success rates are not usually improved with more complex machines; they just save (and replace) labour. This is especially ironic since capital is short and labour is in excess in many countries.

There are two important criteria for evaluating performance of freezing machines. The first is whether the machine cools embryos at the assigned rate. The smoothness of the cooling curve is frequently overemphasized. Fluctuations in temperature of 0.5-1°C from a perfect, straight-line cooling curve are not of much consequence as long as the average cooling rate is correct. The latter capability is essential, however. The second important criterion is whether the temperature being recorded in the freezing chamber is, in fact, correct. Temperatures at the time of seeding and plunging are critical, and drifts in thermometer readings of 2-3°C can lead to catastrophic results. In fact, it is a good idea independently to check temperatures in freezing chambers on a regular basis, perhaps every few months, as a quality control measure.

TABLE 14
Information on some commercially available freezing machines

Brand nameAddress of companySource of coolantDescription of chamber
Bio-CoolFTS Systems, Inc.
P.O. Box 158
Stone Ridge, NY 12484
USA; 914-687-7664
Mechanical refrigeratorAlcohol bath
Cryoembryo-PSPHoxan
Hoxan Bldg.
2 Nishi 1-chome
Kito 3-jo
Chuo-ku Sapporo 060
Japan
Vessel of liquid nitrogenSlots for straws only
Cryo GeneticCryo-Genetic Technology
400 Hoover Rd.
Soquel, CA 95073 USA
Liquid nitrogen vesselStraws lowered into vapour
Cryo-MedCryo-MedN
49659 Leona Dr.
Mt. Clemens, MI 48045
USA; 323-371-5713
Liquid nitrogen tankLarge chamber
CTELabortechnik
Postfach 1107
D-3406 Bovenden—Göttingen
Fed. Rep. Germany;
(0551) 82835
Liquid nitrogen tankOpen vessel
Freeze ControlFreeze Control USA
3377 Solano Ave.
Suite 303
Napa, CA 94558
USA
Vessel of liquid nitrogenSlots for straws only
Glacier TechnologyGlacier Technology
404 Europe St.
Baton Rouge, LA 70802
USA
Peltier effect/electricitySmall chamber
Mini CoolCFPO
B.P. 15
F-38360 Sassenage
France
Liquid nitrogen tankLarge chamber
McDonaldVeterinary Concepts
303 South McKay Ave.
Spring Valley, WI
54767 USA
Neck of liquid nitrogen tankSmall chamber
PlanerPlaner Products, Ltd.
Windmill Road
Sunbury-on-Thames
Middlesex TW16 7HD
UK
Liquid nitrogen tank15 cm diameter × 20 cm high cylinder
RPEPeter Elsden & Assoc.
P.O. Box 9677
Fort Collins, CO 80525
USA; 303-223-6665
Neck of liquid nitrogen tankSlots for straws only

One other criterion for selecting a freezing machine is ease of use. Things to consider are weight, if it must be moved from place to place, ease of access to the cooling chamber, simplicity of programming, systems for holding straws, vials or ampoules, depending on which is to be used, and ease of diagnosing problems and fixing them. Cost, reliability of service, reputation of the manufacturer and dealer, and similar factors also need consideration. A brief description of freezing machines is given in Table 14. We have no way of knowing about all possible models of freezing machines available and have provided information on those companies that have contacted us in recent years.

One final point is that embryos can be cooled perfectly adequately with dry ice and alcohol in DeWar flasks (see Maurer, 1978, for examples). This is more labour intensive and requires conscientious personnel, but if done correctly results will be as good as with freezing machines.


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