Artificial Insemination and Embryo Transfer in Sheep

CHAPTER 86 Artificial Insemination and Embryo Transfer in Sheep

C.F.B. SHIPLEY, BRIAN C. BUCKRELL, M.J.A. MYLNE, JOHN. POLLARD, J.R. HUNTON

Artificial insemination and embryo transfer in sheep offer many advantages for genetic improvement. They are not new techniques, but several advances in technology have made them somewhat easier for cost-effective implementation. At this time, many of the drugs that are routinely used for synchronization and superovulation are not available to U.S. veterinarians, yet they are commonly used in most foreign countries.

ARTIFICIAL INSEMINATION

Benefits

Artificial insemination (AI) provides many benefits to the producer. They include, but are not limited to, the following:

• Increased genetic gain by allowing increased selection pressure on the ram population to hasten genetic improvement

• Increased offspring from a superior sire

• Availability of a superior sire to small flocks

• Frozen and fresh semen that can be shipped and stored and moved nationally or internationally with fewer health risks and welfare implications than live animals

• Disease control through testing of rams before use and treatment of the semen with antibiotics to lessen the threat of disease

• Semen that can be frozen as insurance against injury or death of a superior sire

• Sire evaluations that can be carried out in many environments to assess the superiority of that sire over many different production types

Selection and Management of the Ram

The ram should have a complete physical examination and be superior in genotype and phenotype. He should be in good body condition (body condition score [BCS] 3–3.5) and be evaluated for locomotion. The reproductive tract should receive special attention and the prepuce examined for lesions and discharge. Testing for any diseases should be done at this time.

The penis should be manually examined for defects or injuries. This can accomplished by setting the ram up on its dock and pushing on the sigmoid flexure while pushing back on the prepuce. There should be no scars, adhesions, or abrasions and the urethral process should be intact.

The scrotal circumference should be measured and compared to normal values for age, breed, and season. Minimum circumferences should be per age and size and carefully adhered to. Sperm production is linked closely to testicle size, as is pubertal age of the offspring.

The testicles should be palpated in a routine fashion so that no abnormalities or defects are missed. The testicles themselves should be firm with no palpable hard or fluctuating lesions that might suggest neoplasm, trauma, abscess, or venous damage. The head, body, and tail of the epididymis should be palpated as well for any lesions with particular attention to the tail of the epididymis and the spermatic cord where varicoceles and sperm granulomas may be found.

The scrotum should be symmetrical and not have any scars, adhesions, or evidence of parasites and it should hang low to help maintain a scrotal temperature 4° to 7° C below body temperature. There should be no signs of scrotal hernia, cryptorchidism, testicular hypoplasia, or other defects. Any such abnormality should exclude the ram from use as an AI sire.

Libido testing should be done if possible. Rams should mount and breed freely and have no feet abnormalities.

All stressful management procedures such as shearing, deworming, vaccinations, vitamin and mineral injections, and hoof trimming should be done 6 to 8 weeks before collection to avoid causing damage to sperm during the spermatogenic cycle.

The rams should be on a rising plane of nutrition and be fed ad lib good quality hay or forage as well as protein and concentrate that is low in Mg²⁺ and Ca²⁺ and may contain 1% ammonium chloride to help prevent urolithiasis. Depending on the area of the country and the quality of forage and concentrate available, supplementation of vitamin E and selenium may be advisable. Low blood copper and zinc have been implicated in reduced libido and fertility, and assessment of these in the diet or blood may be advisable.

Exposure of rams to cycling ewes at this time may also help semen production, especially in rams from more seasonally dependent breeds.

Two weeks before semen collection, rams should be individually housed to help prevent injury, avoid homosexual behavior (especially in ram lambs), and help control any special dietary needs of individual rams. This may also help more submissive rams increase their libido. All contact with ewes should stop at this time, but it is important to maintain ewes within sight, smell, and sound of the rams.

Although semen may be collected at any time of year, it is important to note that many of the breeds are seasonal and their semen quality and quantity are affected when collected out of the normal breeding season. To this end, it is important to collect those rams during the natural breeding season even if the semen is to be used out of season.

RAM TRAINING FOR SEMEN COLLECTION WITH THE ARTIFICIAL VAGINA

Rams can be trained to mount a teaser ewe and ejaculate into an artificial vagina with a collector kneeling beside him. Many factors affect how long this training will take, but age of ram, breed, season, libido, mating experience and temperament all affect training time. Most mature rams will mate an ewe restrained in a standing position. Ejaculates may be collected daily from healthy mature rams, but sperm concentration and quality should be evaluated on a daily basis and collections adjusted accordingly to achieve optimal use of the ram.

Rams with low libido or inexperience may be taught to mount and collect by following these guidelines:

• Move the rams to the collection area or similar surroundings 2 to 4 weeks before collection and accustom them to hand feeding, gentle handling, and the presence of humans.

• Introduce one or two estrus ewes into the pen and allow the rams to mount normally. Teaser ewes may be made for such use by ovariectomizing them and giving 1 mg of estradiol benzoate once weekly. If two ewes are kept and injected on a Monday/Thursday morning schedule, at least one of them is available throughout the week.

• Let the ram mount a ewe that has been restrained in a head catch with a handler in close proximity.

• After the ram is comfortable with mounting, the handler then starts to gently touch the sheath upon mounting to condition him to handling. Techniques such as changing the ewe, allowing the ram to watch another ram mount, or allowing a ram to choose a ewe may help the slower, more timid ram to mount. Short periods of training are preferable to longer periods and direct contact between rams should be avoided.

• Once regular mounting is achieved, the handler diverts the penis into the artificial vagina (AV) for collection by moving the sheath. Patience and gentle handling go a long way to improving collection and performance of the ram.

The Artificial Vagina

The AV is made up of a liner inside a hard casing that is firmly held in place with rubber bands. These can be purchased commercially or homemade but must be watertight because sperm is killed by any direct contact with water. A stopcock or other device is drilled through the casing to add water and air to control the temperature and the pressure. A glass or plastic collection vessel is attached to one end and a cover or sleeve is applied over it to prevent cold shock to the semen. The open end is lubricated with a nonspermicidal sterile obstetric gel.

Each ram should have his own AV and liner, which should be cleaned with purified water and then rinsed with alcohol and let air-dry between uses. Collectors should wear clean vinyl examination gloves for handling and collecting semen.

Just before use, the AV should be filled half full with water that is 50° to 55° C and the collection container inserted into one end. Correct pressure is attained by blowing air into the AV through the control valve and is easily evaluated by inserting a clean gloved finger into the AV through the open, lubricated end. Exact pressure and temperature will vary between rams, but a final collection temperature (internal) of 40° to 45° C and a pressure that allows easy entry of the penis but still allows for moderate pressure will usually work best.

Semen Collection

The ram will approach the restrained ewe and the collector should be positioned near the ewe, ready to collect with a properly prepared AV. The ram will normally smell, lick, and paw at the ewe, who will respond by urination, vocalization, and tail deviation. Time to mount will vary, but well-teased males tend to produce more semen per collection. When the ram mounts, the collector should deflect the prepuce of the ram to the side, allowing the penis to deviate into the AV. The AV should be applied in such a way that the ram’s ejaculatory thrust is not impeded in an upward manner. The penis itself should not be handled.

Problems that may occur with collection with the AV include failure to ejaculate due to improper AV temperature or pressure or improper collector technique; delayed ejaculation after the AV is removed, usually due to poor technique or to over- or understimulation; and lastly, premature ejaculation due to poor technique, high libido, or overteasing.

Once trained, most rams are easy to handle for collection of semen and the ejaculate is superior to that obtained from electroejaculation because it usually does not contain excessive accessory gland secretions.

Electroejaculation for Semen Collection

Electroejaculation of the ram is a relatively simple and easy procedure with the correct equipment. In rams that are going to be used extensively in an AI program, it cannot be recommended because of the repeated stresses on the animal, but for minimal collections or animals that are too debilitated to mount a teaser, it is a viable alternative for the practitioner. It is also useful when examining a large number of rams in a day, for collection of vasectomized rams before use or for rams that refuse to serve an AV. It has also been observed to increase libido in some low-libido rams.

Most common equipment includes either a Bailey or a Ruakura ram probe, although other probes by other manufacturers will also work. These types of probes have a simple on/off switch that delivers stimulation of 10 to 15 volts of 30 to 50 sine or square waves. Operators should thoroughly familiarize themselves with whatever equipment they are using and use it appropriately for the situation.

Rams may be collected with or without sedation depending on their disposition, skill of the operator and assistants, as well as the frequency and objectives of the collection. Xylazine (15–20 mg IM) or acepromazine (10 mg IM) have been very effective in the authors’ experience. The ram should be restrained in lateral recumbency and the penis exteriorized (may be easiest to accomplish while the ram is set up on its dock and then lowered to lateral recumbency). The penis is grasped just below the glans with a sterile gauze sponge and the glans and urethral process directed into the collection container. Care must be taken not to cold shock the semen, so either a cover or water bath around the collection vessel is appropriate. The penis and prepuce may be cleaned and clipped at this time to decrease chances of contamination of the semen. Phosphate buffered saline (PBS) or saline may be used so if contamination with them occurs, it will not affect semen. The rectum is then cleared of any feces by a gloved hand and lubrication applied liberally to the probe and anus. Gentle massage of the accessory glands with either the fingers or probe will help to stimulate the ram and may ease collection. Generally this is done for 10 to 15 seconds and then stimulation applied with the probe in a rhythmic on/off sequence of 3 to 5 seconds on and 5 to 15 seconds off with gentle downward pressure toward the pelvic floor. Gentle movement of the probe during the on/off sequence may help the collection and help to find the proper depth of the probe for maximum stimulation. Individual rams vary in their response, but generally, ejaculation occurs after 3 to 5 stimulations and if collecting for freezing and maximum sperm numbers are desired, stimulation may continue for several more stimulation sequences until ejaculation is not apparent or sufficient semen is obtained. If no ejaculation occurs after several stimulations, the probe should be removed and cleaned of feces and the battery checked on the electroejaculator. Having several collection containers available may help to fractionate the sample so only the sperm-rich semen is collected with minimal accessory gland contamination or, in some cases, urine. Encouraging the ram to urinate before collection is also helpful to eliminate or reduce the chance of contamination with urine. Any accessory fluid that is collected should be discarded, or in some cases, the semen may be centrifuged so that the accessory fluid can be discarded. Once collected, the semen is handled and examined as in AV collection.

HANDLING, EXAMINATION, ASSESSMENT, AND EVALUATION OF SEMEN

Semen must be handled carefully to avoid heat shock; cold shock; contamination with water, disinfectants, sunlight, and air; and any other process or product that may decrease viability. Sperm will die if temperatures exceed 45° C and any increase above 37° C will increase metabolic rate and thus decrease sperm life. Cold shock may be avoided if all equipment that semen comes into contact with is kept at 30° to 37° C. All containers that come into contact with the semen should be either plastic or glass and cleaned with laboratory type cleaners, or in the authors’ opinion, single-use containers are preferred, if practical.

Semen should be examined as soon as possible after collection and transferred to a sterile collection tube held in a 30° C water bath. The criteria used for a breeding soundness examination of rams should be followed in the examination and evaluation of the semen. Color should be milky off-white. Pink usually indicates contamination with blood, and a gray or brown color may be indicative of a reproductive tract infection. Urine contamination is usually yellow with a characteristic odor and the semen will appear dilute. Any off-quality semen should be discarded and not used for AI.

Volume will range from 0.5 to 1.5 ml and may vary based on frequency and type of collection. Repeated collections over several days will decrease volume. Motility will normally be wave-like on gross examination under low power (10–50×) on a prewarmed slide without a coverslip. The motion is scored as in Table 86-1 and any sample scoring 2 or below is discarded.

Table 86-1 Scoring System for Wave Motion Under Low-Power Microscopy, 10× to 50×

Score	Class	Description
5	Very good	Dense, very rapidly moving waves; individual sperm cannot be observed; >90% of the sperm are active
4	Good	Vigorous movement, however waves and eddies are not as rapid as those in score 5; 70 to 90% of the sperm are active
3	Fair	Only small, slowly moving waves; individual sperm may be observed; 40 to 65% of sperm are active
2	Poor	No wave motion forming, but some movement of sperm visible; 20 to 40% of sperm are active
1	Very poor	Less than 10% of sperm active; possible to observe slight “flickering” of sperm with poor motility
0	Dead	No movement apparent

Concentration should be calculated accurately because a reliable number of sperm per insemination is critical to pregnancy rates. An average ejaculate will contain 3.5 to 6.0 billion sperm per milliliter. A hemocytometer, spectrophotometer, computer-assisted analysis, or other accurate method of counting sperm is best, but under some field conditions, concentration of the sperm may be estimated by consistency of the sample using Table 86-2. Thick, creamy samples contain more sperm than those of a more dilute, watery consistency.

Table 86-2 Concentration of Ram Semen Assessed for Consistency

Other tests that may be useful include a morphologic and acrosome integrity test using eosin-nigrosin stain or hypo-osmotic swelling tests. Individual sperm motility and progressive forward movement by dilution of the sample with extender, sodium citrate, or phosphate buffered saline. Longevity of sperm using various extenders and examination at set intervals (usually 1–2 hours) over time may be used to evaluate both the sperm and which extender gives greatest sperm life. Temperature and holding conditions must be carefully monitored to be sure that the sperm is not affected by outside factors and the evaluation is valid.

DILUTION AND INSEMINATION DOSE USING FRESH SEMEN

During natural mating, a mature fertile ram normally deposits approximately 3 billion sperm. Of these, only 100 million to 150 million penetrate the cervix and get into the uterus. Doses of semen containing 100 million sperm have achieved acceptable conception rates when deposited into the cervix. If undiluted, this small volume is very hard to handle and measure accurately. Dilution not only solves this problem, it provides the sperm with a suitable environment for preservation during the insemination and handling process.

Various extenders are available for fresh dilution, but the two most commonly used are heat-treated cow milk or Dulbecco’s phosphate buffered saline (with or without 2% equine serum albumin or 10% fetal calf serum). Ultra-high temperature milk may be purchased or made by heating whole milk to 92° to 95° C for 8 to 10 minutes. Water must not splash into the milk, which must not be allowed to boil. The extender must be either cooled or warmed to 30° C and then added slowly to the extender as soon as possible after collection and used as soon as practical (within 1 hour).

Freezing Semen

There are many protocols for freezing ram semen. One-step and two-step methods have been described, but because of its ease, the one-step method is the most commonly used. Semen is diluted to the final prefreezing dilution rate at 30° C with the cryoprotectant. Straws are then loaded and cooled slowly (using a water bath) by placing them in a refrigerator and cooling to 5° C over 1.5 to 2 hours. One formula that can be used is tris(hydroxymethyl) aminomethane (24.2 g) plus citric acid (13.6 g) plus fructose (10.0 g) plus glycerol (64 ml) plus egg yolk (200 ml) in sufficient quantity to produce 1 L at a pH of 6.8. Commercial extenders have been used with success by the authors and for small numbers and ease of use are very satisfactory.

Freezing semen in straws is similar to freezing bull semen, but the extender, dilution rates, cooling rates, and other factors will vary from laboratory to laboratory. Ram semen is more difficult to freeze than bull semen and some rams (5–10%) will not freeze successfully with current techniques and extenders that are available. Normally ram semen is diluted to at least 1:8 with the chosen extender with final dilution depending on the type of insemination (laparoscopic AI versus cervical AI versus transcervical AI). Doses of semen for the various techniques can be found in Table 86-3.

Table 86-3 Typical Dose Rates and Inseminate Volumes Required for Different AI Techniques

Semen can be loaded into straws or cooled before loading into straws, then cooled slowly to 4° to 5° C over a period of 1.5 to 2 hours. Depending on the laboratory or extender, semen is then either frozen over nitrogen vapor (4 cm for 0.25-ml straws or 6 cm for 0.5-ml straws) on a chilled (5° C) rack or held for several more hours to allow equilibration and then frozen as described previously. After 8 to 10 minutes in the vapor, the semen is then plunged into the liquid nitrogen and from there loaded into canes and transferred to a tank for storage using standard techniques. When using a programmable freezer, chilled filled straws are loaded into the freezer and cooled at the following rate: 4° C per minute until semen reaches −12° C, then 40° C per minute from −12° to −40° C, then finally 50° C per minute from −40° to −140° C. After this point is reached, straws are loaded into canes and stored as described previously.

Semen can also be frozen in pellets by using a block of dry ice into which small depressions have been punched using a metal rod or die for multiple depressions. Semen frozen in pellets is usually diluted to only 1 : 3 to 1 : 4, depending on sperm evaluation. Then 0.1 to 0.15 ml of semen is pipetted into each depression and after 2 to 3 minutes the pellets are transferred to plastic goblets, labeled, and put in liquid nitrogen. For thawing, pellets are put into an extender prewarmed to 40° C, or two to three pellets are placed in a sterile dry test tube or Whirl Pak bag and put in a water bath at 40° C and shaken or stirred vigorously till the pellets melt. The semen is then transferred to a 30° C water bath, where it may be held for up to 18 hours, till insemination. This technique does not require expensive or sophisticated equipment, but labeling and identity of the semen are harder to maintain.

Frozen ram semen can be expected to achieve conception rates of 65% when using laparoscopic intrauterine AI.

EWE SELECTION AND MANAGEMENT

The success of the AI program depends as much on the successful management of the ewes as it does on the other techniques mentioned previously. Ewes must be in good health and condition and be free of any diseases. They should be neither fat nor thin; have sound feet, legs, udder, and teeth; and be free of heritable faults. Lambs should be weaned at least 6 to 8 weeks before insemination, and stressful procedures such as shearing, vaccination, and deworming should also be avoided during this time as well as for 4 weeks after insemination. Best conception rates and lambing percentage will be obtained when ewes are flushed (increasing calories and protein) 2 to 3 weeks before breeding. Identification of the ewes and accurate records are very important, especially when multiple sires are being used. Best conception rates are obtained when breeding during the natural breeding season.

SYNCHRONIZATION AND DETECTION OF ESTRUS

To achieve optimal pregnancy rates, it is imperative to time insemination to estrus and ovulation. Accurate detection of estrus in the ewe for insemination using a marking harness on a teaser ram can be used to detect heat with the expectation of 6% of ewes to be in heat each day over a 17-day estrous cycle.

Synchronization programs for AI, especially for out-of-season breeding, may make utilization of time, personnel, and equipment more efficient. This will reduce the time spent in a program and allow for selection of the optimal number of ewes to be inseminated each day. The following is a typical program:

Day 0: progesterone started (CIDR [controlled intravaginal drug release] or sponge or Synchromate implant)

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Sep 3, 2016 | Posted by admin in SUGERY, ORTHOPEDICS & ANESTHESIA | Comments Off

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