1. Superovulation of Donor Cows
The cow usually ovulates only one egg during each estrous cycle. Many years ago, it was found that the administration of hormones with FSH-like activity would stimulate extensive development of mature follicles in the ovaries and thus allow for ovulation of a larger number of eggs. The most commonly used hormone preparations for inducing such superovulatory responses are pituitary extracts from swine or sheep containing FSH, and pregnant mare’s serum gonadotropin (PMSG), a hormone with FSH-like activity that is found in the serum of pregnant mares.
Administration of the superovulating hormone should begin 4 to 9 days prior to the expected onset of estrus. When using PMSG, a single injection will suffice. FSH should be given twice daily during the entire period of superovulation. In most cases, prostaglandin F2a (PGF) is used in conjunction with superovulation, so that one can accurately predict the time of the onset of estrus. A typical schedule to superovulate a cow would be to begin injections of FSH 9 to 14 days after the last estrous period and to inject PGF on the fourth day of FSH treatment. The cow would be expected to be in estrus 36 to 60 hours after the injection of PGF.
Superovulation does have some problems, the largest of which is the wide variation in response. Thus, some cow will not superovulate at all. The optimal response, in terms of the number of transferable embryos recovered, appears to be in the order of 10 to 15 ovulations.
Table 1. Bovine Superovulation Protocol
| Day | 0. Estrus |
| Dairy breeds | Dual purpose breed |
| 10
11
12
13
14
15
22 | a.m.* p.m.
a.m. p.m.
a.m. p.m.
a.m.
p.m.
a.m. p.m.
a.m. p.m. | - 2.500 - 3.000 PMSG
- -
- -
Prostaglandin
-
- -
- AI + Anti-PMSG Antiserum | - 7 mg FSH
7 mg FSH 6 mg FSH
6 mg FSH 5 mg FSH
5 mg FSH + Prostaglandin 4 mg FSH
4 mg FSH -
- AI + HCG 3.000
Embryo Collection | - 8 mg FSH
8 mg FSH 7 mg FSH
7 mg FSH 5 mg FSH
5 mg FSH + Prostaglandin 5 mg FSH
5 mg FSH -
- AI + HCG 3.000
|
* Superovulation can be commenced between days 9 and 15
2. Nonsurgical Recovery of Bovine Oocytes
Bovine ovum recovery attempts are made from 6 to 8 days (D 6 – 8) after estrus. Most ova have entered the tip of the uterine horn at this time and still have their shell (zona pellucida) intact (which is believed an advantage when recovering and transferring embryos). Donors are starved and deprived of water for 24 hours prior to ovum recovery, except for lactating dairy cows. Rectal palpation and manipulation (of instruments) are facilitated in a partially starved animal.
Beef cows are usually placed in a squeeze chute and dairy cows in a head bail before preparation. The tail-head is clipped, washed with soap and water, and sterilized using an iodine compound followed by alcohol. A suitable site (between 2 articulating vertebrae in the sterilized area) (intercoccygeal groove 1 – 2) is selected for injecting 5 to 6 ml of a 2% local anaesthetic (Xylocaine HCl, Lidocaine HCl, Procain HCl etc) around the spinal cord (Epidural anaesthetic). The epidural injection prevents the cow from straining on your arm when it is in the rectum palpating the reproductive tract and manipulating instruments. In some older, particularly fat cows, it is difficult to obtain a satisfactory epidural block. If two attempts are unsuccessful, it is recommended to apply 40 mg of an acepromazine intravenously for general relaxation (as tranquilizer). The tail is tied out of the way, and the rectum may be emptied of faeces.
The vulva area is washed with soap and water and sterilized with an iodine compound and alcohol. With the arm in the rectum, ovaries are located, and follicles and corpora lutea are estimated to give an approximate idea of the number of ova released. The vulvar lips are parted, and a plastic pipette is introduced into the vagina and placed in the lumen of the cervix. Under gentle pressure, the pipette is manipulated just through the cervix and then, under vacuum by a 20 cc syringe, slowly pulled backward to aspirate and remove the mucus from the cervix.
A 16 or 18 G (French gauge), sterile, 2-way Foley catheter (depending on the size of the uterus) with a mandarin stiletto of stainless steel inside is gently inserted into the vagina and on into the cervix. It is then manipulated into the selected horn so that the inflatable balloon is situated clearly behind the palpable bifurcation of the two horns. The balloon is gently inflated with a 10 to 15 ml of air, depending on the size of the uterine horn. The balloon should be tight so that medium cannot leak out between the balloon and wall of the uterus. However, if the balloon is too tight, the endometrium will rupture and bleeding occurs, which will adversely affect ovum searching. After the catheter is placed in its final position with the balloon inflated, the mandarin stiletto is removed.
Total of 1.000 ml flushing medium warmed to 37 °C is prepared in a water bath. A Y-junction tubing is connected to the bag with flushing medium and attached to the tube of the flushing catheter. The medium is then flushed slowly into the lumen of the chosen uterine horn (500 ml flushing medium for each horn). Under gentle massage of the uterus wall via rectum the medium is collected through the outlet tubing of the Y-junction and into a filter.
The catheter can be changed to the opposite horn by either removing it completely from the uterus and replacing it, or the catheter can be pulled back into the body of the uterus after deflating the balloon. Then the mandrin is inserted into the lumen of the catheter until it reaches the tip, which can be palpated. After placing the catheter, the process of flushing is repeated with the second horn. The filter filled with flushed medium is taken to the laboratory. The Petri dish is placed for searching the ova under a stereo microscope with a transluminant base at a magnification rate of 10-12 times.
Fig. 1: BoviPro™ flushing medium, Y-junction tubing and Miniflush™ Filter
Location, identification, manipulation and classification of ova and embryos require experience and will only be learned by considerable practice. Ova are easily missed, so each dish should be searched twice by one person and then checked by a second one.
Ova are manipulated with small glass pipettes or capillary tubes, or 0.25 ml straws attached to some suction device. They are retrieved from the medium in about 0.1 ml of fluid and placed in small 5-well dishes with culture medium. When the searching is completed, the ova are classified, based on their morphological appearance at a magnification rate of 40-50 times and washed by being passed through three sterile solutions of holding medium.
The embryos are then stored at room temperature until transfer or freezing. Embryos will continue growing in the dish and, for example, those collected seven days after estrus will change gradually from the morula stage to early blastocysts, which is a good indication of the viability of the embryos and the efficiency of the medium used. In the first 12 hours of culturing at room temperature, very little viability is lost, then, up to 24 hours a little decrease occurs, after 24 hours there is a rapid loss.
After superovulation, the average fertile donor will yield in total 10 ova, fertilized, unfertilized and or degenerated. Usually 5 to 7 embryos are of transferable quality.
The bovine embryo is about 160 μm (0.16 mm) in diameter through day 8 of development. This size is beyond the resolving power of the naked eye. Therefore, a stereo microscope (magnification, 10-50 times) is used to identify embryos in uterine flushing.
Fig 2. Stereo microscope
The morphology of all bovine embryos is similar through day 9 of development, and it is composed of a cellular mass surrounded by an acellular matrix known as the zona pellucida. The area between the embryonic mass and the zona pellucida is known as the perivitelline space. Cells composing the embryo are termed blastomeres. During embryonic development blastomere number increases in a geometric fashion through the 16 cell stage (e.g. 1 – 2 – 4 – 8 – 16 cells). After the 16 cell stage, cell divisions become asynchronous and individual blastomeres become difficult to distinguish. At the 40 to 60 cell stage, the bovine embryo undergoes compaction; that is, the blastomeres loose their spherical shape and begin to adhere to one another. Embryos at this stage of development are termed compacted morula and are most often recovered on day 5 to 7 after standing estrus. During the late morula stage, blastomeres begin to differentiate giving rise to two cell types trophoblast and inner cell mass. These two cell types become readily distinguishable at the blastocyst stage. A blastocyst is a layer of trophoblast cells surrounding a cavity called the blastocoele. The cells of the blastocyst are specialized since the inner cell mass will form the fetus proper and the trophoblast will form the placenta. Blastocysts are found most often 7 to 10 days after estrus.
Problems may be encountered with blastocysts collected after day 8 because the zona pellucida is lost between day 8 and 10. When zona-free blastocycts are collected, a greater risk of damage due to handling is encountered. Additionally, zona-free blastocysts are “sticky” and may adhere to tubing and glassware.
Identification of embryos in uterine flushings is based on several of the above mentioned morphological features. First, the zona pellucida is used as a landmark. The zona, being a translucent structure present on all embryos through day 9, clearly distinguishes embryos from other cellular debris in flushing. Second, the color of the embryo (a dark amber) facilitates identification because it is usually darker than other uterine debris. Third, the embryo is spherical and tends to roll along the bottom surface of the searching dish. Finally, knowing the stage of the embryo to be collected, from the day estrus, speeds identification of embryos in the collection medium.
To date there is no single parameter of embryo morphology which correlates with pregnancy rate, except that unfertilized ova will not result in calves. Therefore, several characteristics or recovered embryos are assessed before transfer. Recovered embryos are classified from excellent to poor based on their gross morphological appearance.
- Excellent embryos have no visible imperfections.
- Good embryos show a single small imperfection such as an extruded blastomere, asymmetrical shape, or slightly retarded development compared to other embryos in the same flush.
- Fair embryos possess more than one of the above imperfections and are usually 1 to 2 days retarded in development.
- Poor embryos show signs of cellular degeneration, little or no cellular organization and are 2 to 3 days retarded in development.
- Several degenerated embryos are more than 3 days retarded in development.
- Unfertilized ova do not show any sign of development.
After embryos have been classified as to quality, individual imperfections are recorded on an embryo description sheet. The age of the embryo, from estrus to collection is recorded, as it is an estimate of the number of cells present in the embryo. Compactness of the blastomeres is estimated; loosely arranged blastomeres may indicate poor development or degeneration. Very dark or light blastomeres may indicate that degenerative processes have occurred. The cellular mass of the embryo should be spherical and symmetrical. Large vesicles in blastomeres are indicative of some degree of degeneration. Blastomeres may be extruded during any stage of development. A small number of extruded blastomeres is usually not detrimental to further embryonic development, but a large number (over 5) of extruded blastomeres indicate that the embryo may be undergoing more degenerative than development processes. The proportion of the perivitelline space occupied by the embryo is also recorded. Generally, the percentage of perivitelline space occupied by the embryo is least at the compact morula stage. Finally, the stage of the embryo is recorded, for example morula, blastocyst, etc.
Normally developed embryos:
Early morula Compacting morula Beginning blastocyst
Beginning blastocyst Blastocyst
Fig 3. Bovine normal embryos
Partially degenerated embryos:
16-cell-stadium (retarded) Degenerated blastomere Shrunken morula
Morula with degenerated blastomeres
Useless embryos:
1-cell-stadium Empty zona (broken) Irregular divisions (unfertilised)
Fig 4. Abnormal bovine embryosBlastomeres falling apart
Both the physical and chemical environments of the recovered embryos must be maintained close to those of the uterine environment to insure maximum pregnancy rates after transfer. Embryos are fragile and will not tolerate small changes in either environment.
Embryos are often maintained at room temperature for several hours without adverse effects. Caution should be employed since repeated sudden changes in temperature may have adverse effects on embryo viability.
As embryos are identified in the flushing medium, they are immediately transferred to a small petri dish containing fresh sterile (0.20 μm filtered) culture medium (= holding medium). Embryos are tentatively classified as good or bad and recorded so that a quick estimate of embryo number can be made. When all embryos are recovered, they are serially washed through small dishes of sterile culture medium and finally placed into a fresh well containing sterile culture holding medium. Embryos are then for transfer or cryopreservation.
Fig 5. 5-well culture dish
Recipients should be healthy, mature heifers or young cows in good condition. At least two normal cycles should have been established before use to determine whether they are in natural heat on the same day as the donor.
Recipients are placed in chutes or in head bails. A small epidural anaesthesia of 5ml 2% lidocaine is infused around the spinal cord. It takes about 5 minutes for the drug to take full effect.
Sterile 0,25ml straws and transfer sheaths are prepared. The embryo is sucked up into the straw by attaching a suction device to the end containing the plug. About 0,1ml of holding medium is placed behind the front bubble which helps to wash the embryo into the uterus.
The loaded straw is placed in the transfer instrument and covered by a sheath. With the hand in the rectum, the corpus lutem on the ovary is located. The vulval lips of the recipient are parted by an assistant, and the transfer instrument is placed in the vagina without touching the vulval lips or the first part of the vagina. Since most embryos are transferred 6, 7 or 8 days after heat, the cervix is tightly closed, especially in heifers. Therefore, it is usually much more difficult to pass the instrument then than during estrus. Once through the cervix, the device is directed to the selected horn. Remember, the true bifurcation is only about 1cm from the cervix, so as the rod leaves the cervix, it must be immediately inclined toward the required horn. The horn is raised and straightened ahead of the tip of the gun before it is gently pushed up the horn. Once resistance is met, the embryo is expelled and the rod removed. An experienced person averages about 50 seconds to implant the embryo. The problems encountered using this method are:
1) Placing the transfer instrument through the closed cervix in heifers
2) Depositing the embryo relatively high up in the uterine horn without causing extensive damage to the delicate lining of the uterus (endometrium)
3) Considerable natural manual dexterity and experience are required
In fact, pregnancy rates depend upon many events, such as quality of embryos and recipients, the skill of embryo transfer technicians, and the preparation and management of the recipients.
Prostaglandin F2a (PG) is efficiently used for embryo transfer programs in both donors and recipients.
Following the recording of regular estrous cycles, donors give a much more consistent response to the gonadotropins. Usually PG is given 3 or 4 days after the first injection of gonadotropin. Heat or estrus then occurs, in the superovulated cow only 48 hours later. Even after 4 superovulation treatments, the response to the PG remains consistent, with the donor averaging 50 hours from injection to heat. The manufacturer’s recommended dose is 25 mg intramuscularly, however, 35 mg may be used on heavy donors (more than 500 kgs body weight). The dose of PGF can be used as an intrauterine infusion with only 5 mg of PGF (1/5 dose of intramuscular dose).
Another use for PG treatment on donors is to terminate an unwanted pregnancy. Following superovulation and recovery, an embryo is occasionally not flushed out and the pregnancy continues. These early pregnancies are efficiently removed by injecting 25 mg of PG. In 72 hours, the pregnant donor comes into heat and aborts the foetus.
Frequently, and especially for on-the-farm embryo transfer, it is an advantage to superovulate 3 to 5 donors at one time. PG can be used successfully between days 6 and 17 of the donor’s estrous cycle. The standard 25 mg dose is sufficient.
Recipients must be in heat either one day before, the same day, or the day after the donor’s heat. With PG, a relatively small group of recipients can be synchronized into heat the same day as the donor.
There are two regimens which can be used:
1) Palpate recipients for corpora lutea (CL) and inject 25 mg of PG into those with a CL. Heat occurs 48 to 96 hours later, peaking at 72 hours.
2) Inject all recipients with PG regardless of the presence or absence of a CL. Repeat the 25 mg injection of PG 11 days later. Estrous behaviour will again peak between 2 and 3 days. Those recipients not responding on the first occasion because they were in the first 5 days of the estrous cycle, respond the second time as they are then in the mid-to-late period of the cycle, while those recipients that did respond are in the early to mid-cycle period at the time of the second PG injection.
Since the average donor yields 7 transferable embryos, ten recipients per donor is a reasonable number to prepare, to have eight on the average suitably synchronized with the donor. The recipients must be injected with PG on the same day as the donor for the final synchronization.
1) On day 7 after estrus donor cows are flushed non-surgically for embryo recovery.
2) Embryos are washed at least three times in holding medium.
3) 2 cryoprotectants are available for freezing embryos:
1. Ethylen glycol, allowing direct transfer after freezing and thawing, is today
mostly used (BoviPro™ Ethylene Glycol with sucrose, Ref. No. 19982/1302).
2. Glycerol requires stepwise exposure to the embryo before freezing and
stepwise removal after thawing (BoviPro™ Glycerol with sucrose, Ref. No.
19982/1303, BoviPro™ Embryo Rehydration Kit, Ref. No. 19982/1404).
Cryoprotectants function by lowering the amount of intracellular ice at any
given temperature and may stabilize cellular membranes, thus reducing
damage during freezing and thawing.
4) Embryos are suspended in the freezing medium. Straws are loaded with the embryos. In the case of Ethylene Glycol freezing, straws are loaded as described in the picture:
5) The straws are then placed into a programmable freezer, precooled at -7 °C, working either by vaporizing liquid nitrogen or by a mechanically refrigerated alcohol bath.
6) After three minutes of equilibration in the -7 °C chamber the straws are “seeded” by local cooling of the straw wall with a forceps which has been precooled in liquid nitrogen. This process causes ice to form in the solution and enables the required slow and uniform cooling rate to be achieved. Ice formation starts the process of cellular dehydration, thus reducing the amount of intracellular ice.
7) The embryos in the straws are then cooled slowly at 0,3 °C/min to -35 °C.
8) The straws are then plunged into liquid nitrogen.
The whole freezing process takes about 2 ½ hours and it is speculated that the embryos, once frozen, will have a storage life similar to that of frozen spermatozoa.
Basically, 2 types of freezing machines are available for slow freezing of embryos: alcohol bath freezers and liquid nitrogen freezers are both suitable.
Fig 6. Bovine embryo freezing machines
According to the two different types of cryoprotectants the embryos are thawed as follows:
a) Ethylen glycol: No special thawing procedure is required.
b) Glycerol:
- The straw is removed from the nitrogen container, placed into a water bath and gently agitated until all ice is melted.
- The straw is then emptied into a prepared small Petri dish, containing a mixed 10% glycerol and sucrosis in holding medium and kept for 5 minutes.
- Then the embryo is transferred into another small Petri dish, with 5% glycerol and sucrosis solution in holding medium.
- Finally the embryos are placed in normal holding medium for 10 minutes before evaluation.
- The thawed embryos are transferred.
Organisation and Preparation of an Embryo Transfer (E.T.) Programme
The success and the cost involved in embryo transfer depend upon the conditions under which the E.T. is carried out.
1) Only animals with undisturbed general well being and healthy and fit are suitable donors.
2) Animals with hoof problems, mastitis, uterus infections, metabolic disturbances or any other disease are not suitable as donors.
3) Donors with calving problems, premature births, abortions, retention of placenta or any other disturbances must be treated before preparation for embryo transfer.
4) Problem animals (repeat breeders) must be announced as such and treated according to their pre-history because sterile cows do not produce viable embryos.
5) The superovulation should be started after the cow has shown at least 2 visible heats (oestrus) after calving. Cows with a history of veterinary treatment, or after disease should show at least one visible heat before
programming for superovulation.
6) To recognise functional disturbances early, the first heat and the subsequent heat should be confirmed with the Milk Progesterone test, i.e. one sample (after milking) on the day of heat and a second sample one week after heat are taken for testing. Further samples are taken and tested during the superovulation program.
Fig 7. : eProCheck® for estrus checking
7) Embryo Transfer becomes problematic in herds where cows are stabled on steel grids and fixed with chains. This changes the spontaneous behaviour of the cow into restrained behaviour. Under these conditions not all animals adapt (behave) in the same way and therefore cows which undergo treatment for Embryo Transfer should be moved to a place where there is minimum stress.
8) The occurrence of an error or disorder whilst preparing the program should be announced as quickly as possible to the responsible person so that the error or the disorder can be compensated.
Very common and accidental errors or disorders are:
- Cow suddenly gets mastitis, goes lame or goes off feed (digestive disorders)
- Incorrect substance injected
- Error in injection schedule
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