Artificial Insemination PDF

# CHAPTER 26: Artificial Insemination ## Artificial Insemination R.L. Ax, M.R. DALLY, B.A. DIDION, R.W. Lenz, C.C. LoVE, D.D. Varner, B. HAFEZ, and M.E. BELLIN - Artificial insemination (AI) is the most important single technique devised for the genetic improvement of animals. - A few select mal...

# CHAPTER 26: Artificial Insemination ## Artificial Insemination R.L. Ax, M.R. DALLY, B.A. DIDION, R.W. Lenz, C.C. LoVE, D.D. Varner, B. HAFEZ, and M.E. BELLIN - Artificial insemination (AI) is the most important single technique devised for the genetic improvement of animals. - A few select males produce enough sperm to inseminate thousands of females per year. - In contrast, relatively few progeny per female can be produced per year even by embryo transfer. - The earliest carefully documented use of AI was in 1780 when Spallanzani, an Italian physiologist, obtained beagle puppies. - Other reports appeared in the 19th century, but it was not until 1900 that extensive studies with farm animals began in Russia and shortly thereafter in Japan. - Major advantages of AI are: - Genetic improvement - Control of venereal diseases - Availability of accurate breeding records - Economic service - safety through elimination of dangerous males - AI is facilitated with estrous synchronization programs, and it has been proposed as a means of gender control through separation of spermatozoa containing X and Y chromosomes. ## When properly done, there are few disadvantages to using AI. However, it is necessary to have trained personnel to provide proper technique, and to have appropriate arrangements for corralling females for hormone therapy or detection of heat and insemination, particularly under range conditions. ## Advantages of AI are: - Enables the widespread use of outstanding sires with valuable genetics to any livestock operation. - Facilitates progeny testing under a range of environmental and managerial conditions, thereby further improving accuracy of selection. - Leads to improved performance and potential of the national herd. - Permits crossbreeding to change a production trait. - Accelerates introduction of new genetics. - Enables use of deep-frozen semen after a donor is dead, aiding in the preservation of select lines. - Permits use of semen from incapacitated or oligospermic males. - Reduces risk of spreading sexually transmitted diseases. - Is usually essential after synchronization of estrus in large groups of animals. - May permit males with desirable genetic markers to be used in specific genetic matings. - Provides a useful research tool for investigating many aspects of male and female reproductive physiology. ## The genetic improvement achieved by AI of dairy cattle has resulted from the use of progeny-tested sires - Obtaining semen at the earliest possible age from potential valuable sires being tested is desirable to hasten identification of superior sires. - Ultimately, the genetic impact of a superior sire is limited by the number of sperm produced, which is a direct function of testicular size. ## Carefully selected young males should be sampled as soon as possible after puberty. Performance and progeny test programs are important to make maximal genetic progress with meat-type farm animals. AI facilitates crossbreeding, requiring that only one breed be maintained on the farm. Development of AI in beef cattle has been slower in the United States and some other countries, because of the difficulty of heat detection and insemination under range conditions. - It is also possible to have considerable genetic improvement for highly heritable traits, using performance-tested bulls, without AI ## MANAGEMENT OF MALES/SEMEN COLLECTION When young males are properly fed and managed, semen can be collected successfully at the following approximate ages: - Bulls: 12 months - Rams, goats, and boars: 7 to 8 months - Stallions: 24 months - Of major importance to an AI program is the correct collection of semen. ### Mounts and Teasing Procedures - Live mounts - such as a teaser female, another male, or a castrated male - are the most successful techniques for routine semen collection. - The exception is that teaser animals for stallions are mares rather than males. - Some males, especially boars, can be trained to mount dummies equally well, which avoid disease transmission and provide stability for physical collection. ## Cattle ### Semen Collection - A general routine to follow for mature bulls is a Monday, Wednesday, and Friday schedule. The bull can be collected twice daily for optimal sperm output. - The average ejaculate contains 8 to 16 billion total sperm. A weekly average to strive for is a total of 30 billion sperm cells. - Young bulls can be collected on Tuesdays and Thursdays. - There are three stimulatory approaches: - The introduction to a different mount animal(s) in the original locale - Change the locale - Change both locale and mount. - If a bull does not mount after 5 min, then it will require more stimulation to provide a satisfactory ejaculate. - The artificial vagina (AV) is made of a cylindrical rubber tube (radiator casing), a thin rubber liner, a thin-walled rubber cone, and a collection vial encased in an insulated jacket. - The space between the wall of the rubber casing and the inner rubber liner is filled with hot water (60°C). A rubber cone is fitted on the end of the rubber casing. To the other end of the cone, a 15 mL graduated test tube is attached. An insulated jacket is used to ensure that the entire AV is kept at approximately 45°C for the time of collection. - A sterile lubricant is applied to the inner liner. - After precollection stimulation, consisting of 3 to 5 false mounts and active restraint, the bull is allowed to mount the teaser animal. - The collector diverts the bull's penis into the AV, which is held alongside the flank of the mount. - Immediately on contact with the warm and lubricated surface, the bull ejaculates into the AV. - Upon ejaculation, the AV is immediately tilted downward toward the test tube. - The semen drains into the collection tube, which is then removed and placed in a water bath maintained at 34°C, before measurement of sperm concentration. - The method of electro-ejaculation is reserved for lame or old bulls, and for bulls that have temporarily lost their desire to serve the AV. - The technique involves introducing either a probe or finger electrodes into the rectum, where the nerves supplying his reproductive organs are electrically stimulated. - Voltage is gradually increased with repeated rhythmic stimulation pulses. - Experience is necessary to cause erection followed by ejaculation. - Ejaculates from this procedure have large volumes. - It is not unusual for a bull to ejaculate 7 to 10 mL of semen, which contains 1.0 to 1.5 billion sperm/mL, with an average progressive motility of 60 to 75%. - A semen packaging machine and a large storage tank for semen in frozen inventory are shown in Figures 26-7 and 26-8, respectively. ### Thawing Semen Thawing 2 or 3 mL straws is accomplished in a 37°C water bath (45 seconds). The inseminator must be able to provide some protection for thawed semen on cold days. Never allow semen to cool down or refreeze. Try to dry the straw and maintain the 37°C temperature while loading the insemination gun in cold weather. Prewarm the gun by vigorous rubbing several times. ### Insemination Technique - The rectovaginal technique of artificial insemination in cattle is one of the most widely used methods and is taught by every AI company. - Excess fecal matter can be removed by a series of gentle raking motions of the inserted hand. - Once excess fecal matter has been expelled, the vulva is wiped clean and dried with an absorbent paper towel to prevent contamination. - If rectal constrictions persist, press down with the palm of the hand and massage back and forth on the floor of the pelvic cavity. - This will also help to locate the cervix, which has been described as feeling like a turkey's neck. - The insemination gun should be held at a 30° angle with the end containing the semen uppermost when entering the reproductive tract. - This is necessary to prevent the inseminating instrument from entering the suburethral diverticulum or urethra, which is located on the floor of the vagina a short way inside the opening of the vulva. - The gun is forward along the roof of the vagina and at the same time the cervix is pushed forward to straighten out any vaginal folds which might otherwise be encountered with the tip of the gun. - The cervical os or opening is usually in the center of the cervix, but one may have to probe very lightly with the gun tip until the opening is found. - To work the cervix onto the gun, keep the first two fingers and the thumb on the gloved hand just behind the tip of the gun to manipulate the cervix. - Having gone through the cervical rings, the gun will slip forward with little resistance. - When this happens, the tip of the gun will be in the uterine body, or perhaps may have slipped even further into the uterine horn. - One will be able to feel the tip since the uterine wall is quite thin. - Next, rotate the gloved hand around the cervix until the hand lies on top. - It will be possible to use the forefinger as an indicator of where the gun tip is located. - Slowly pull the gun back until it is flush with the cervical opening. - Keeping the gun steady with the outside hand, raise the index finger of the gloved hand away from the gun tip. - Be certain to push with the thumb and pull the gun out of the cervix with your fingers. - Slowly deposit the semen, taking at least 5 seconds to push the plunger in. - Depositing semen slowly helps get the maximum distribution of semen. - Following correct insemination procedures will help one achieve better breeding efficiencies. - If the cervical mucus of a cow previously inseminated feels thick or sticky, she may have become pregnant from the previous insemination. - In that case, go halfway through the cervix and deposit semen. - Inspect the tip of the gun for signs of infection. - Make notes for the veterinarian. ## Swine ### Semen Collection - The boar is led to the dummy sow in a collection area with a nonslip surface (i.e., wood-shavings, sawdust, or perforated rubber matting) and the dummy sow should be stabilized by fixing to the floor or wall. - Once the boar has mounted the dummy sow, he will begin thrusting movements. - If possible the preputial fluid retained in the prepuce should be emptied and the prepuce dried with a paper towel. - The penis will emerge and the technician should grasp the end of the penis allowing the boar to lock in it. - Pressure is the stimulus which allows the boar to ejaculate and pressures will vary (slight to hard squeeze) for each boar. - The technician would note the various fractions of the ejaculate taking care to collect the sperm-rich fraction. - Training a new boar to mount a dummy sow is best attempted immediately after another boar has already worked on it. - Boars observing other boars mounting live or dummy sows have enhanced libido or sexual drive, and sexual stimulation of the boar prior to semen collection has been shown to increase the number of sperm in the ejaculate. ### Frequency of Collection - Too frequent collection in the case of AI boars, or overuse of boars for natural mating, is one of the greatest factors causing temporary loss of fertility. - Young boars should not serve more than two sows per week. ### When to Inseminate - Successful AI in swine is influenced by the variation that exists in the onset of heat after weaning, duration of estrus, and the time between estrus onset and ovulation. - Using transcutaneous sonography of weaned sow ovaries, Weitze (11) was able to identify ovulation times relative to estrus onset on 427 sows. - The following recommendations were made with respect to insemination time: - AI sows that show estrus early post-wean: on second and third day of estrus once every 12 h. - AI sows that show estrus at usual time post-wean: at 24 h after detection and once every 12 h later. - AI sows that show estrus late post-wean: as early as possible after detection. ### Insemination Techniques - Several methods are used to impregnate swine: - Natural service - Transcervical with fresh, extended, or frozen semen - Surgical insemination with fresh, extended, or frozen semen. - Farrow rates average 70 to 85% for transcervical insemination with fresh and extended semen; 40 to 70% for transcervical insemination with frozen semen; and 30 to 55% for surgical insemination with fresh, extended, and frozen semen. - For AI the technician inverts the semen dose (plastic bottle/vial/cochette) several times slowly to mix the semen. - The end of the catheter is lubricated beforehand (with K-Y jelly, semen, or extender). - The vulva is wiped clean and the vulva lips are opened to allow the catheter to be inserted in an upwards/forwards motion. - The technician will feed the end of the catheter lock into the cervix. - Attach the semen dose to the catheter and allow semen to flow by gravity into the female. - The normal uterine contractions (1 to 2 per minute) will assist in drawing semen into the uterine horns. - Surgical insemination into the oviduct, or tip of uterine horn, is effected by using a Tomcat Catheter pushed through a hole made in the upper 1.5 cm of the uterine horn. - This procedure is invasive but has merit in situations using semen that has high economic merit. ## Horses ### Collection of Semen - Ejaculated semen is collected from stallions for artificial insemination of mares, or for assessment of semen quality. - To ensure protection of the sperm, semen should be collected using an artificial vagina which has been properly prepared. - Quality of semen collected in a condom is generally inferior to that obtained with an artificial vagina, because spermatozoa can be adversely affected by debris from the penis or toxicants in the condom. ### Artificial Vaginas - Several models of artificial vaginas are available commercially. - Homemade artificial vaginas may also be constructed to meet any specific needs of the user. - It is imperative that all components of an artificial vagina which come in contact with semen are nontoxic to sperm. - For disinfectant, rubber liners can be submerged in 70% isopropyl or ethyl alcohol for 20 minutes. - Rubber liners of some artificial vaginas can be toxic to spermatozoa. - If possible, sterile, nontoxic disposable equipment should be used to avoid contamination of semen with toxic chemicals, and to minimize transmission of venereal diseases. - A filter should be installed in the artificial vagina prior to collection of semen. - This filter allows the sperm-rich portion of an ejaculate to pass into the semen receptacle, but retains the gel fraction. - In stallions with suspected inflammatory conditions (seminal vesiculitis), or plugged ampullae or urospermin, the filter should be carefully inspected, since evidence of these conditions can be trapped in the filter. - The result is a higher usable sperm harvest because far fewer spermatozoa become entrapped in the gel during collection. - The internal temperature of the artificial vagina normally should not exceed 45 to 48°C at the time of semen collection, because irreversible damage to spermatozoa can result from exposure (even short-term) to temperatures above this level. - If the glans penis is beyond the water jacket of the artificial vagina at the time of ejaculation (as typically occurs when using the Missouri-model artificial vagina), the temperature of the water jacket can be adjusted to 50 to 60°C without any temperature-related injury incurred by ejaculated spermatozoa. - Only lubricants which are nonspermicidal should be used in the interior of the artificial vagina. ### Other Considerations - Prior to collecting semen, the stallion's penis (particularly the distal portion) should be cleansed with water, then thoroughly dried. - Semen is generally collected after allowing the stallion to mount a breeding dummy, or a receptive and properly-restrained mare. - Stallions can also be trained to ejaculate while standing. - Ejaculated semen is promptly transported to the laboratory (maintaining 35 to 37°C) for evaluation and further processing. - Upon arrival the semen should be promptly placed in an incubator adjusted to 37 to 38°C, then mixed with an appropriate extender. ### Semen Extenders - Semen collected for artificial insemination should always be placed in a semen extender, which may be purchased commercially, or prepared in the laboratory. - If semen is to be used for insemination soon after collection, it should be mixed with semen extender at a 1:1 to 1:3 ratio (semen to extender). - More extensive dilution is recommended if semen is to be stored for a prolonged period of time prior to insemination. ### AI Technique - Artificial insemination is permitted by the vast majority of United States breed associations. - Only sterile, nontoxic, disposable equipment should be used for the procedure. - All inseminations should be performed using a minimum-contamination technique. - With the mare adequately restrained, and the tail wrapped and elevated, the area between the base of the tail and ventral commissure of the vulva is thoroughly scrubbed, rinsed, and dried. - Semen contained within a syringe is deposited into the anterior uterine body through a 22-inch sterile insemination pipette. - A sterile or clean plastic shoulder-length sleeve should be worn when passing the pipette through the cervix to the uterine body where the semen is to be deposited. - Mares are generally inseminated with 250 × 10º to 500 × 106 progressively motile spermatozoa, contained in a semen extender. - The volume of the inseminate typically ranges from 5 to 20 mL. - For maximum pregnancy rates, it is best to inseminate mares within 12 to 24 h prior to ovulation, although pregnancies have been achieved at a similar rate when mares are inseminated within 12 h following ovulation. - Using semen from highly fertile stallions, mares may sometimes be inseminated 48 to 72 h prior to ovulation with no depression in pregnancy rate. - Containers for transporting stallion semen and straws for packaging stallion semen are illustrated in Figures 26-17 and 26-18. ## Sheep ### Semen Collection - There are two methods by which semen may be collected from the ram: electro-ejaculation (EE), or using an artificial vagina (AV). - The EE procedure is performed by inserting a bipolar electrical probe into the ram's rectum. - Low voltage electrical stimulation is given for 2 to 4 seconds at 10-second to 20-second intervals until an ejaculation occurs. - EE collections are highly variable in both sperm concentrations and volume. - Contamination with urine can be a problem when semen is collected by EE. - EE is stressful on the ram, so it should only be used in extreme cases. - The AV is the preferred method for collecting semen. - Rams must be trained to use the AV, but this is not difficult and most rams can be trained within a week. - It is best to collect semen during the peak of the breeding season. - The AV consists of a hose 20 to 25 cm in length and 5 to 7 cm in diameter, with a rubber liner. - It is critical to have the interlining lubricated, with a temperature of the AV between 42 and 46°C. - As the ram mounts the ewe, his penis is gently guided inside the AV. - The amount of inter-AV pressure will vary among rams. - It is also important to have the glass collecting tube warm (37°C) to avoid cold shock. -After the ram ejaculates, the glass tube containing the semen is removed and placed in a waterbath at 30°C until the semen reaches that temperature. ### Semen Diluents and Diluents - Motility and concentration govern the ratio which semen is diluted. Semen which score 5 in both motion and concentration may be diluted as high as a 4:1 ratio. - Most ejaculates are diluted at a 2:1 ratio. - Semen scoring 2 should not be diluted and should only be used in the fresh, undiluted state. - Natural or synthetic diluent can be used. - Cow milk is the most commonly used natural diluent. - When whole, skim, or powdered milk is used, it must be heated to 92 to 95°C in a water bath for 8 to 10 min. - If ultra-heat-treated milk is used, no additional heating is required. - Table 26-5 lists a commonly used synthetic diluent. - Diluent and semen should be at the same temperature (30°C) when dilution is performed; adding the diluent to the semen will reduce shock to the sperm. - Never add the semen to the diluent. - The mixture should be gently mixed, with evaluation of semen after dilution, to confirm sperm viability. ### Buck - Buck semen is less concentrated than ram semen, therefore the dilution ratio is lower. - When egg yolk is used in a diluent, amount added is significantly less than in a ram's diluent. - Buck semen's seminal plasma is removed by centrifuging the semen. - This method also requires the sperm concentration to be determined in the centrifuged sample before dilution. ### Estrus Synchronization for AI - AI can be successful only if it is performed during the last half of the estrous period. - Synchronizing the ewe's estrus with that of other ewes permits the Al technician to inseminate a group in one session and facilitates insemination during the period of optimal conception. - Synchronization can be accomplished naturally or pharmacologically. - The natural method involves the use of a teaser ram or testosterone-treated wether checking for estrus twice daily at 12-hour intervals. - Ewes found to be in estrus are artificially inseminated 12 and 24 h after detection. - If checked only once a day, insemination would be performed 13 to 17 h after detection. - This method is labor intensive but does not incur any drug costs. - There are two commonly used pharmacologic procedures to synchronize ewes: progesterone and prostaglandin therapy. - Progesterones can be administered by several means. - They can be fed daily, implanted, injected intramuscularly, or inserted into the ewe in the form of a vaginal pessary or sponge. - The pessary method is the most widely used and convenient one. - A pessary impregnated with progesterone is inserted into the ewe's vagina and remains in place for 12 to 14 days. - Any naturally occurring corpus luteum on the ovaries will regress during this period, leaving the exogenous progesterone as the only progesterone source. - At pessary removal, the ewes are given a intramuscular injection of pregnant mare serum gonadotropin (PMSG). - PMSG causes a closer synchronization and increases ovulation rate. - PMSG doses of 400 to 500 IU or 600 to 750 IU are commonly recommended during the natural breeding season and nonbreeding season, respectively, except in small-bodied breeds when PMSG doses of as little as 250 IU are recommended. - As PMSG dosages increase, ovulation rates increase; however, at levels above 800 IU the conception rate may decrease. - Another intravaginal device, the controlled internal drug release (CIDR) dispenser, is used to synchronize ewes. - The CIDR contains 9 to 12% progesterone in a silicone elastomer. - No difference in conception rates is observed between the two intravaginal therapy methods. - Prostaglandin (PG) therapy depends on the presence of an active corpus luteum and therefore can be used only during the breeding season. - Prostaglandin causes the corpus luteum to regress and thereby inhibits progesterone production. - It is effective only between days 5 and 14 of the estrous cycle. - Therefore, two injections of PG are required 10 to 14 days apart to be effective in synchronizing the entire breeding group. - AI conception rates are lower when PG therapy is used than with the progesterone pessary method. - PG can be used in combination with short periods of progesterone therapy and PMSG. - This method requires a 7-day treatment period with a vaginal pessary, and on the sixth day a single injection of PG is administered. - The PMSG is given at pessary removal. ### Ram Effect - Teaser rams can be used to synchronize ewes naturally, because pheromones produced by the ram stimulate the advancement of the breeding season by 2 to 3 weeks and synchronize the ewes. - Pheromones are specialized hormones released by the ram and smelled by the ewe. - They are found in the wool and wax that accumulate around the ram's eyes. - Visual or physical contact with the ram is not necessary to stimulate the ewe. -Wool from a Dorset ram was rubbed on the muzzle of a group of ewes 3 times a day for 2 days. - This treatment resulted in half of the ewes being stimulated. - Different breeds of rams may differ in their ability to stimulate ewes. - When Dorset rams were used as teasers, 67% of the ewes were bred during the first two weeks of their breeding season, compared to only 34% with Romney rams. - No significant difference was found between Dorset and Suffolk teaser rams in their ability to stimulate onset of estrus during May. - Within 10 min of the introduction of the ram or pheromones, ewes that are stimulated will start secreting gonadotrophins. - The hormones cause the ewe to ovulate within three days. - Ewes must be completely isolated from rams for approximately two months before introduction of the teaser rams in order to have a stimulation effect from the ram pheromones. - It is also suggested that rams be kept at least a half mile away from the ewes to prevent the ewes from picking up the scent of the rams' pheromones during this period. - A ratio of one teaser ram per 100 ewes is normally required to assure a stimulating effect. - Vasectomized, epididectomized, or intact rams can be used to stimulate ewes. - However, if an intact ram is allowed to be in direct contact with the ewes, any ewe that has a behavioral estrus during the teasing period might be bred. - Since direct contact with the ram is not required to stimulate the ewe, it is advisable when using intact rams to place them in an adjacent field. - Long periods of teasing are not required; studies have shown that allowing ewes to be exposed to rams for 2 days is as effective as teasing for 17 days. - Wethers that have been treated with testosterone propionate are almost as effective at stimulating ewes as rams. - Buck goats have also been found to have a stimulating effect on ewes. ### Insemination Methods - There are four methods of artificially inseminating the ewe: vaginal (VAI), cervical (CAI), transcervical (TAI), and laparoscopic (LAI) or intrauterine. - Each method has its advantages and disadvantages. - The first sheep was successfully artificially inseminated in 1936. - Today sheep AI is widely used throughout the world. - Insemination procedures have been previously published. - Various insemination methods are summarized in Table 26-6 and Figures 26-19 to 26-22. ## Time of Insemination - When progesterone pessary and PMSG are used in combination, and VAI or CAI is performed, a single insemination should be conducted 54 to 56 h after the pessary is removed. - If the ewes are to be inseminated twice, the first insemination should take place 48 h after pessary removal and the second 12 h later. - Double insemination normally results in a 5 to 10% increase in conception rate and increases prolificacy as well. - When ewe lambs are inseminated it is recommended that insemination take place 50 h after pessary removal, since ewe lambs come into estrus earlier than mature ewes. - When the TAI is performed, insemination should take place between 48 and 56 h following pessary removal. - If the CIDR without PMSG is used to synchronize estrus, insemination is performed 48 to 57 h after removal. - If the short term progesterone therapy combined with prostaglandin and PMSG is used, insemination is performed 48 to 60 h following pessary removal. - When LAI is performed and the progesterone and PMSG therapy is used, insemination should be conducted 56 to 62 h following pessary removal. - If the ewes are superovulated, given higher levels of PMSG or FSH, insemination should take place earlier. - When fresh semen is used the insemination is performed 36 to 48 h after withdrawing the pessary. - Superovulation insemination should be conducted 44 to 48 h after pessary withdrawal when frozen-thawed semen is used. - LAI is performed 48 to 52 h after CIDR is withdrawn. ## References 1. 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