Many things can go wrong in an embryo transfer programme. The concept of quality control is to carry out tests on a regular basis to avoid problems or at least to identify them early. Quality control procedures need not be elaborate and, if worked into regular routines, may be effective at a cost of only a small percentage of the budget of a programme.
Frequently we rely on quality control procedures of manufacturers by assuming, for example, that purchased drugs are efficacious and sterile, or that disposable plastic ware is not contaminated with micro-organisms or embryo toxins. Quality control concepts become more critical in an embryo transfer programme as personnel at the embryo transfer unit carry out more steps, such as making culture media from the constituent reagents rather than purchasing complete media, or washing and reusing disposable Foley catheters instead of using new ones each time.
Good records are the foundation of a quality control programme. Recording data as suggested in Chapter 16 is a good start. Being able to correlate events in time by comparing dates is especially useful. For instance, the cause of an unexplained decrease in pregnancy rates may be traced by checking personnel schedules or dates of purchase of lots of reagents or supplies. Records need not be complicated but they need to be complete. It is good to date supplies when the container arrives or when it is opened. This is especially important for drugs and chemicals. Lot numbers should be recorded for products like FSH or paraffin oil that vary from batch to batch with regard to potency or toxicity. For certain procedures, the person doing the work should be recorded, for example, for artificial insemination, oestrus detection, embryo evaluation and embryo transfer.
A number of checks should be scheduled on a regular basis, such as levels of liquid nitrogen in cryogenic storage containers and inventories so that supplies can be ordered as needed. Indicators of proper function of equipment and proper execution of procedures are discussed below in more detail, and a recommended schedule of quality control measures is given in Table 15.
If culture media such as modified Dulbecco's phosphate-buffered saline are made up correctly, they will be suitable for embryos. However, since many steps are involved, it is easy to use an incorrect chemical, make a tenfold error in weighing, use an instrument or container with toxic residues, or fail to effect sterility. If media are made at the embryo transfer unit, they should be checked at the least for pH and osmolality. The latter can be done with an osmometer or, more crudely, by observing shrinking or swelling of red blood cells. If the medium is suspect, the notes recorded when weighing the ingredients should be consulted to see if a weighing error occurred. If the problem is not resolved at this point, the batch of medium should be discarded. It is better to discard an occasional good batch of medium that deviates slightly from acceptable pH and osmolality standards than to have an occasional batch that kills embryos. Note that a somewhat aberrant pH indicates that something is wrong, but pH may not be the factor that kills the embryos. Put another way, adjusting the pH will not solve the problem.
Sterility can be checked by incubating a sample at 37°C overnight and examining it for micro-organisms the next day. Toxicity is more difficult to deal with. Some laboratories incubate two-cell mouse embryos in each new batch of medium to verify that it will support normal development. Paraffin oil, if used, can be checked similarly for sterility and toxicity. If circumstances warrant extra precautions, a sample of each batch of culture medium can be labelled and stored in the freezer until pregnancy rates are available.
Determining water quality is especially difficult. Resistance to electric current is commonly used to measure absence of ions, but this is of little value in detecting organic compounds such as endotoxins. The best course is to pay scrupulous attention to detail in preparing water, cleaning deionizing or distillation systems regularly to prevent micro-organisms from colonizing components, and otherwise following directions. Culture of twocell mouse embryos in medium made with the water in question is probably the best overall test of water quality.
It is convenient to divide equipment and supplies into two groups for purposes of quality control: those that come into contact with embryos and those that do not. Anything that contacts embryos should be non-toxic and sterile. Once a routine has been established, it should not be necessary to check equipment for toxicity and sterility each time. However, it is wise to check various items for sterility every three or four months by rinsing with culture medium followed by incubation of the rinse.
Toxicity of most items contacting embryos, except plastic culture dishes (manufacturers generally exercise stringent control over quality of these items), should be handled in a different way: assume that there is toxic residue and rinse with sterile medium or 0.9 percent saline before use. From time to time toxicity has been demonstrated with syringes, Foley catheters, bacteriological filters, most kinds of rubber tubing and straws. Accordingly, all of these should be rinsed before use. For example, discard the first few ml of medium coming through bacteriological filters, rinse straws before loading embryos, etc. The rubber plungers of certain syringes have been proven to be extremely toxic in the past. Some toxicity is due to sterilization procedures, e.g. with ethylene oxide. If one gets into the habit of always rinsing these items before use, risks of potential toxicity will be markedly lowered. It is of course extremely important not to compromise sterility during rinsing.
Filters for collecting embryos should be examined with a stereomicro-scope before each use, especially if filters are reused. Sometimes they are damaged during the washing and sterilizing procedures, which results in loss of embryos.
Temperature controls on incubators, freezers, warming plates, microscope stages, etc. should be verified on a regular basis. Deviations of only a few degrees can be lethal. For example, incubators set at 38°C that are actually at 40° or 41°C have killed many embryos. Freezing machines set at -6°C for seeding that actually were at -3° or -4°C so that seeding was ineffective have resulted in disastrous pregnancy rates. Refrigerators that should be at 5°C but are actually at 9° or 10°C lead to marked increases in microbial growth. Warming plates and microscope stages can cook embryos. Temperature settings on items of equipment can rarely be trusted, and many laboratory thermometers, especially dial thermometers, are incorrect by several degrees and get worse with age. Always check any thermometer against a second or even a third one of high quality. Further, we suggest that all equipment be checked every three months to verify that the desired temperature is, in fact, the actual temperature.
Appropriate herd health programmes are critical and deserve particular focus when cattle are constantly moving in and out of a facility. Health status can be determined and controlled as described in Chapter 3. Each animal should be observed daily for indications of disease or injury when oestrus detection is carried out. Excellent quality control information on nutrition programmes can be obtained by weighing cattle periodically. This may not be necessary in all embryo transfer programmes, but weights every three or four months will indicate if animals are, in fact, gaining weight. This does not provide information on all aspects of nutrition, but it is the single most important indicator of a good nutrition programme.
One other item worth checking is possible presence of abortifacient or teratogenic weeds in corrals and pastures. This is especially a problem when animals are moved to areas of new plant growth at certain times of the year.
The reproductive status of donors and recipients can be checked by palpation of the ovaries and by studying the intervals between oestrus, when appropriate. Periodic measurement of concentrations of progesterone in milk or blood may be helpful when conditions of management or climate are less than ideal. For example, if heat stress has reduced the efficacy of oestrus detection, screening donors before beginning superovulatory treatment and recipients before transfer on the basis of progesterone may improve success rates.
While most bull studs exercise good control over the quality of semen processing, control over conditions of shipping and storage of semen is often in the hands of individuals who have no knowledge of how to handle semen and no vested interest in its quality. Therefore, it is recommended that a drop of semen from one straw from each lot of semen be examined microscopically for the percentage of progressively motile sperm. This should be done carefully and the result should be recorded. In most cases, it is not necessary to examine sperm for intact acrosomes or morphological abnormalities.
Correct procedures for thawing semen are straightforward, but it is amazing how frequently this is done incorrectly. The main error with artificial insemination is semen placement. Quality control for this is best accomplished by inseminating animals about to be slaughtered with a thick dye, and then examining the reproductive tracts. If this can be arranged easily, it is well worth the effort to check the insemination technique.
The most important overall quality control information comes from studying records, particularly returns to oestrus for the earliest indication of pregnancy rates. These records should be scrutinized monthly. A good quality control of oestrus detection procedures is to take blood or milk samples to determine if progesterone levels are, in fact, low at presumed oestrus. An even simpler procedure is to study intervals between oestrus of cows that are not resynchronized or used as recipients. If the majority of intervals are not 17–24 days, there is a problem.
Ultrasonography is a marvellous tool for quality control of skills in palpation per rectum, and may replace palpation when equipment becomes less expensive. Ultrasonography can be used for pregnancy diagnosis and definitively locating corpora lutea. Errors in palpation of corpora lutea are frequent, and the incidence of such errors can be determined.
Reflushing a random subset of donors periodically will establish whether most embryos are being recovered. Efficacy of isolating embryos from the collection fluid can be evaluated by accumulating the contents of every container throughout the day in a 2-litre cylinder after presumably all embryos have been found and siphoning off the top and re-examining the remaining fluid as described in Chapter 5. An embarrassing number are missed, even by conscientious personnel, but this loss is costly and should be kept to a minimum.
Suggested schedule of quality control measures
|Item or procedure||Quality control measure||Frequency|
|Culture medium, if made at embryo transfer centre||Measure pH Check sterility by culture overnight||Each batch|
Every 3 months
|Serum, if processed at embryo transfer centre||Check sterility by culture overnight Heat-inactivation by culturing mouse embryos or sparing use until pregnancy data available||Every batch|
|Sterility of equipment and supplies||Rinse with sterile medium and culture overnight||Every 3–6 months|
|Temperature controls||Independent thermometer||Every 3–6 months|
|Embryo filters||Stereomicroscopic examination||Every use|
|Cycling of new cattle||Palpate sample for CLs||Every group|
|Nutrition of cattle||Weigh cattle||Every 3–4 months|
|Semen||Determine percentage progressive motility||Each code of each bull|
|Site of semen deposition||Placement of dye in reproductive tract of slaughter cattle||Annually|
|Efficacy of oestrus detection||Study inter-oestrus intervals, measure blood or milk progesterone||Every 3–6 months|
|Efficacy of non-surgical embryo recovery||Reflush donors||5 cows every 6 months|
|Efficacy of isolating embryos||Collect and examine medium from 5 cows||Every 6 months|
|Accuracy of CL palpation||Ultrasonographic evaluation||Check until proficient|
|Non-return to oestrus after ET||Study records||Monthly|
|Pregnancy rate||Study records||Monthly|
|Accuracy of pregnancy diagnosis||Ultrasonographic confirmation||Check until proficient|
|Technician differences in percentage fertilized embryo recovery pregnancy rates||Study records||Every 6 months|
With some instruments, transfer of the embryo can be verified by rinsing the transfer device and examining the fluid under the microscope for presence of the embryo. The reproductive tract of a cow that received a sham transfer just before slaughter can be examined to see if undue trauma of the endometrium has been caused. The site of deposition of the embryo is more critical than that of semen, and this can be checked in a similar way by transfer of 0.25 cc of dye.
Performance of technicians may vary because of innate ability, attitude, training and other factors. Attitude problems may be due to health, personal problems, tension in the laboratory, etc. In any case, workers should be sufficiently professional that they will agree to sign their name to their work. Differences among persons can be determined by studying such records as pregnancy rates after non-surgical transfer. When marked, consistent differences occur among technicians, retraining is indicated. This has been highly successful with artificial insemination technicians. If retraining is unsuccessful or inappropriate, reassignment of responsibilities is often a good option.
Not all of the procedures in Table 15 are carried out at every embryo transfer facility, so some measures of quality control are irrelevant. In many cases, the frequency of quality control checks should be increased when people are learning techniques; likewise, some steps may not be necessary at all with very experienced personnel. If the volume of embryo transfer at a given facility is very low, checking at the suggested frequencies may be misleading because of inadequate sample size; checking less frequently would give a more representative summary.