**3. AI techniques in different species**

Insemination techniques used depend on species, type of semen, breeding system, availability of equipment and expertise. Intravaginal (dogs), intracervical (sheep), transcervical intrauterine (cattle, horses, dogs, sheep), transcervical deep horn intrauterine (horses, cattle, pigs), laparoscopic (sheep), surgical intrauterine (dogs) insemination as well as endoscopic semen deposition at the uterotubal junction (horses) is available.

#### **3.1 AI techniques in dogs**

In dogs, fresh, chilled extended and frozen semen can be used. AI using fresh semen is usually performed if the animals will not or cannot copulate naturally. There are certain breeds, like English bulldogs and other brachycephalic breeds that almost always require AI due to their anatomical incompatibility to mate naturally. AI is also used if time constraints are an issue as fresh semen AI in the bitch is much quicker than a natural mating where the coital tie between male and female can last up to 40 minutes or more. AI can be used in bitches with congenital vaginal abnormalities like vaginal strictures or septae which might cause copulation failure. These cases often have ethical implications as well. Owners need to understand that even though AI can be performed easily these bitches might require Cesarian sections as the vaginal abnormalities might also impair natural delivery or that certain vaginal abnormalities are heritable and offspring with the same problem might be produced. AI is also used if the semen quality of the specific male is poor and addition of semen extender or pooling of more than one ejaculate is required for one insemination dose. Fresh semen is deposited into the cranial vagina of the bitch using an insemination pipette that is inserted through the vulva and directed into the vagina. Once the semen has been deposited, the hindquarters of the bitch are usually elevated for a few minutes to facilitate movement of the semen from the vagina through the cervix into the uterus. Vaginal contractions can be elicited for the same reason at the same time by either tickling the vaginal wall or massaging the clitoris. An insemination dose of at least 150 x 106 sperm is recommended and most commonly the whole ejaculate is used which may contain 250-2500 x 106 sperm (Johnston et al., 2001).

Pregnancy rates with the use of fresh semen for insemination is reported to vary between 65-84% on average depending on semen quality, timing of insemination and correct site of semen deposition (Johnston et al., 2001; Linde-Forsberg & Forsberg, 1989).

Artificial Insemination in Veterinary Science 21

to the often very valuable stallion by avoiding live coverings, insemination of mares that do not want to allow a mating or splitting of an ejaculate into several insemination doses if more than one mare is to be inseminated at the same time. Generally accepted insemination doses for fresh semen as supported by the World Breeding Federation of Sport Horses (WBFSH) are 300 x 106 straight forward swimming (progressively motile) spermatozoa (Katila, 2005) which is slightly lower than the originally recommended fresh semen AI dose of 500 x 106 progressively motile spermatozoa (PMS) (Pickett & Voss, 1975). First cycle pregnancy rate for fresh semen AI is approximately 60%. Chilled extended semen is usually used if the mare and the stallion are in distant places. Semen is collected, extended with a suitable semen extender to provide buffers, nutrients, antibiotics etc. in order to prolong lifespan of the spermatozoa and slowly cooled down to 4 degrees Celsius. Usually, special transport containers that form cooling units (e.g. Equitainer®, Hamilton Thorne) are used and the semen of most stallions is viable for at least 48 hours. Most insemination centres try to inseminate mares with chilled extended semen within 24 hours of collection. One billion spermatozoa or 600 x 106 progressively motile spermatozoa (according to WBFSH (Katila, 2005)) form one insemination dose and pregnancy rates of about 60 % (Sieme et al., 2003) can be expected depending on timing of insemination relative to ovulation, AI dose and semen quality after chilling. Some stallions' semen is not suitable for chilling and the semen quality deteriorates very rapidly. It is therefore recommended to perform a trial cooling of equine semen before a shipping is done and to evaluate semen quality parameters over time

The insemination technique for fresh and chilled extended semen is similar in the horse. A transcervical intrauterine insemination is performed where the semen is deposited into the uterine body. A commercially available insemination pipette is inserted manually into the vagina, the external opening of the cervix is located using the index finger and the pipette is guided through the cervical canal into the uterus. The penetration of the cervix is generally very easy as the cervical tissue is smooth muscle that relaxes under estrogen influence. That allows easy access to the equine uterus at the time of insemination. Frozen-thawed semen has first been used to inseminate mares in 1957 (Barker & Gandier, 1957) even though it only gained increasing popularity over the last 15-20 years. Using frozen semen has a lot of benefits: accessibility to semen from stallions in competition or stallions that become ill, injured or overbooked during the breeding season. Using frozen semen eliminates the need to organize stallion availability at the optimum time for breeding of the mare and disease transmission. Chances for injury are decreased as direct mare-stallion contact is avoided. Pregnancy rate per cycle for frozen semen varies between 30-50% on average (Leipold et al., 1998; Metcalf, 2007; Sieme et al., 2003; Vidament, 2005; Vidament et al., 1997). There are, however, some disadvantages regarding frozen semen. The management of mares during estrus is more intense which increases the costs involved for frozen semen inseminations. This is necessary as inseminations with frozen semen should be done as close to ovulation as possible since frozen-thawed spermatozoa have a shortened lifespan. Frozen-thawed spermatozoa survive for about 12 hours in the reproductive tract of mares whereas fresh semen can survive for 48-72 hours. Frozen-thawed equine semen can be inseminated into the uterine body as described for fresh and chilled distended semen if an insemination dose of 250 x 106 progressively motile spermatozoa is available (Katila, 2005). If the insemination dose is lower or a very small volume is to be inseminated (0.25-0.5ml), the semen can be deposited deeply into the uterine horn ipsilateral to the ovary where ovulation will take place or has taken place (Katila, 2005). Other possibilities for low dose (5-25 x 106

to assess longevity.

Chilled extended semen is usually used if the male and female are geographically separated. An extender is added to the collected semen before shipment in order to prolong the lifespan of sperm. For the same reason, the extended semen is slowly cooled down to 4 degrees Celsius. The entire ejaculate is usually used for the AI but an insemination dose of at least 150 x 106 sperm is recommended. Average pregnancy rate for chilled extended semen is 50% but can vary between 33-89% (Johnston et al., 2001). Extended semen can be used for up to 4 days after collection but should be inseminated as soon as possible. The insemination technique is the same as described above for fresh semen or transcervical intrauterine insemination can be used. Transcervical AI is performed using special catheters (e.g. Norwegian catheters) or endoscopes. The anatomy and location of the cervix in the bitch is such that it is difficult to penetrate the cervix and perform transcervical intrauterine inseminations due to a vaginal fold that is obscurring direct access to the cervix. Therefore, special equipment is needed for transcervical intrauterine AI. Norwegian catheters have been developed in the 1970s (Andersen, 1972, 1975) and are available in three different sizes to accommodate different sized bitches. For this technique, the cervix must be fixed through abdominal palpation and the catheter is moved slowly to find the cervical canal while the cervix is pulled downward. This method requires a lot of practice and there is a potential danger that inexperienced veterinarians might traumatize the cervix. Used by experienced clinicians this technique offers an inexpensive, fast way of inseminating non anesthetized bitches. Using a rigid endoscope for transcervical AI enables the operator to visualize the cervix, straighten the cervical canal and insert a catheter into the uterus to deposit the semen. The advantage of this technique is that it is not blind and the danger of trauma to the reproductive tract is minimal. Bitches do not usually have to be sedated for the procedure and AI can be repeated a few times if necessary.

For frozen semen, deposition of the semen into the uterine lumen is recommended in order to achieve good pregnancy rates (Thomasse et al., 2001; Thomassen et al., 2006). Even though the first report of successful AI with frozen semen in dogs was a result of intravaginal deposition of the semen (Seager, 1969) it has been shown that success rates with intrauterine inseminations are far superior (Thomassen et al., 2001; Thomassen et al., 2006). Intrauterine insemination can be performed by transcervical intrauterine AI as described above or surgical AI via laparotomy. In cases where transcervical intrauterine AI is unsuccessful or the equipment is not readily available, surgical intrauterine AI is performed. It is usually a quick procedure that requires general anesthesia for about 20 minutes. A small abdominal incision is made, the uterus exteriorized and the semen injected directly into the uterine lumen through the uterine wall. Disadvantages of this procedure are the relatively high costs as well as the anesthetic and surgical risk. Surgical insemination is considered an unethical procedure in some countries (Linde-Forsberg, 1991). Insemination doses are usually composed of 100-150 x 106 motile spermatozoa. Whelping rates for frozen semen AI are reported to be as high as 70-75% if the timing of insemination is accurate, good quality frozen-thawed semen is used and the semen deposition is correct (Thomassen et al., 2001; Thomassen et al., 2006).

#### **3.2 AI techniques in horses**

AI is a widely practiced breeding method in most sport horse breeds worldwide. Fresh, chilled extended and frozen-thawed semen can be used in horses. Common reasons for the use of insemination for fresh semen instead of natural mating are the reduced risk of injury

Chilled extended semen is usually used if the male and female are geographically separated. An extender is added to the collected semen before shipment in order to prolong the lifespan of sperm. For the same reason, the extended semen is slowly cooled down to 4 degrees Celsius. The entire ejaculate is usually used for the AI but an insemination dose of at least 150 x 106 sperm is recommended. Average pregnancy rate for chilled extended semen is 50% but can vary between 33-89% (Johnston et al., 2001). Extended semen can be used for up to 4 days after collection but should be inseminated as soon as possible. The insemination technique is the same as described above for fresh semen or transcervical intrauterine insemination can be used. Transcervical AI is performed using special catheters (e.g. Norwegian catheters) or endoscopes. The anatomy and location of the cervix in the bitch is such that it is difficult to penetrate the cervix and perform transcervical intrauterine inseminations due to a vaginal fold that is obscurring direct access to the cervix. Therefore, special equipment is needed for transcervical intrauterine AI. Norwegian catheters have been developed in the 1970s (Andersen, 1972, 1975) and are available in three different sizes to accommodate different sized bitches. For this technique, the cervix must be fixed through abdominal palpation and the catheter is moved slowly to find the cervical canal while the cervix is pulled downward. This method requires a lot of practice and there is a potential danger that inexperienced veterinarians might traumatize the cervix. Used by experienced clinicians this technique offers an inexpensive, fast way of inseminating non anesthetized bitches. Using a rigid endoscope for transcervical AI enables the operator to visualize the cervix, straighten the cervical canal and insert a catheter into the uterus to deposit the semen. The advantage of this technique is that it is not blind and the danger of trauma to the reproductive tract is minimal. Bitches do not usually have to be sedated for the procedure

For frozen semen, deposition of the semen into the uterine lumen is recommended in order to achieve good pregnancy rates (Thomasse et al., 2001; Thomassen et al., 2006). Even though the first report of successful AI with frozen semen in dogs was a result of intravaginal deposition of the semen (Seager, 1969) it has been shown that success rates with intrauterine inseminations are far superior (Thomassen et al., 2001; Thomassen et al., 2006). Intrauterine insemination can be performed by transcervical intrauterine AI as described above or surgical AI via laparotomy. In cases where transcervical intrauterine AI is unsuccessful or the equipment is not readily available, surgical intrauterine AI is performed. It is usually a quick procedure that requires general anesthesia for about 20 minutes. A small abdominal incision is made, the uterus exteriorized and the semen injected directly into the uterine lumen through the uterine wall. Disadvantages of this procedure are the relatively high costs as well as the anesthetic and surgical risk. Surgical insemination is considered an unethical procedure in some countries (Linde-Forsberg, 1991). Insemination doses are usually composed of 100-150 x 106 motile spermatozoa. Whelping rates for frozen semen AI are reported to be as high as 70-75% if the timing of insemination is accurate, good quality frozen-thawed semen is used and the semen deposition is correct (Thomassen et al., 2001;

AI is a widely practiced breeding method in most sport horse breeds worldwide. Fresh, chilled extended and frozen-thawed semen can be used in horses. Common reasons for the use of insemination for fresh semen instead of natural mating are the reduced risk of injury

and AI can be repeated a few times if necessary.

Thomassen et al., 2006).

**3.2 AI techniques in horses** 

to the often very valuable stallion by avoiding live coverings, insemination of mares that do not want to allow a mating or splitting of an ejaculate into several insemination doses if more than one mare is to be inseminated at the same time. Generally accepted insemination doses for fresh semen as supported by the World Breeding Federation of Sport Horses (WBFSH) are 300 x 106 straight forward swimming (progressively motile) spermatozoa (Katila, 2005) which is slightly lower than the originally recommended fresh semen AI dose of 500 x 106 progressively motile spermatozoa (PMS) (Pickett & Voss, 1975). First cycle pregnancy rate for fresh semen AI is approximately 60%. Chilled extended semen is usually used if the mare and the stallion are in distant places. Semen is collected, extended with a suitable semen extender to provide buffers, nutrients, antibiotics etc. in order to prolong lifespan of the spermatozoa and slowly cooled down to 4 degrees Celsius. Usually, special transport containers that form cooling units (e.g. Equitainer®, Hamilton Thorne) are used and the semen of most stallions is viable for at least 48 hours. Most insemination centres try to inseminate mares with chilled extended semen within 24 hours of collection. One billion spermatozoa or 600 x 106 progressively motile spermatozoa (according to WBFSH (Katila, 2005)) form one insemination dose and pregnancy rates of about 60 % (Sieme et al., 2003) can be expected depending on timing of insemination relative to ovulation, AI dose and semen quality after chilling. Some stallions' semen is not suitable for chilling and the semen quality deteriorates very rapidly. It is therefore recommended to perform a trial cooling of equine semen before a shipping is done and to evaluate semen quality parameters over time to assess longevity.

The insemination technique for fresh and chilled extended semen is similar in the horse. A transcervical intrauterine insemination is performed where the semen is deposited into the uterine body. A commercially available insemination pipette is inserted manually into the vagina, the external opening of the cervix is located using the index finger and the pipette is guided through the cervical canal into the uterus. The penetration of the cervix is generally very easy as the cervical tissue is smooth muscle that relaxes under estrogen influence. That allows easy access to the equine uterus at the time of insemination. Frozen-thawed semen has first been used to inseminate mares in 1957 (Barker & Gandier, 1957) even though it only gained increasing popularity over the last 15-20 years. Using frozen semen has a lot of benefits: accessibility to semen from stallions in competition or stallions that become ill, injured or overbooked during the breeding season. Using frozen semen eliminates the need to organize stallion availability at the optimum time for breeding of the mare and disease transmission. Chances for injury are decreased as direct mare-stallion contact is avoided. Pregnancy rate per cycle for frozen semen varies between 30-50% on average (Leipold et al., 1998; Metcalf, 2007; Sieme et al., 2003; Vidament, 2005; Vidament et al., 1997). There are, however, some disadvantages regarding frozen semen. The management of mares during estrus is more intense which increases the costs involved for frozen semen inseminations. This is necessary as inseminations with frozen semen should be done as close to ovulation as possible since frozen-thawed spermatozoa have a shortened lifespan. Frozen-thawed spermatozoa survive for about 12 hours in the reproductive tract of mares whereas fresh semen can survive for 48-72 hours. Frozen-thawed equine semen can be inseminated into the uterine body as described for fresh and chilled distended semen if an insemination dose of 250 x 106 progressively motile spermatozoa is available (Katila, 2005). If the insemination dose is lower or a very small volume is to be inseminated (0.25-0.5ml), the semen can be deposited deeply into the uterine horn ipsilateral to the ovary where ovulation will take place or has taken place (Katila, 2005). Other possibilities for low dose (5-25 x 106

Artificial Insemination in Veterinary Science 23

sizes) similar to or better than those resulting from natural matings (Roca et al., 2006). Apart from good conception rates, there are other reasons for the dramatic increase in AI utilization for pig breeding systems. On one hand there was the change of breeding and farrowing units that became larger and more specialized, and that the application of AI mating technologies became more feasible and cost effective on the other hand (Singleton, 2001). At the same time, the pork industry initiated payment programs based on actual carcass value instead of live weight basis. Genetic evaluation programs were used to implement genetic improvement programs through the use of AI to produce higher quality pork carcasses. For most commercial breeders it was the prospect of genetic improvement that was the major incentive to engage in AI (Brassley, 2007). Semen processing centres (boar studs) or on-farm collection facilities provide boar semen used for AI programs. More than 99% of AIs are performed with semen extended in a liquid state that can be stored at 15-20°C for up to 3 days (cooled semen)(Roca et al., 2006). The number of viable sperm per dose ranges between 2.5- 4 billion motile spermatozoa. Dose volumes range from 80-100ml for fresh liquid semen. The insemination procedure, called intra-cervical insemination (intra-CAI), involves the deposition of spermatozoa into the posterior part of the cervix using a catheter that engages with the folds of the cervix, stimulating the corkscrew tie of the boar's penis (Roca et al., 2006). This easy and quick procedure has been developed in the mid 1950s, standardized in the 1970s and is still used worldwide today (Roca et al., 2006). The remaining 1% of AIs utilizes frozen-thawed semen at doses of 5-6 x 109 spermatozoa. Even though large sperm numbers are used, fertility is substantially lower than that obtained with cooled semen. Due to the lowered reproductive performance, frozen-thawed boar semen is mostly limited to specialized breeding programs, research and for export puposes (Singleton, 2001; Wongtawan et al. , 2006). There are two insemination procedures available that allow insemination with low numbers of spermatozoa: post-cervical insemination (post-CAI) and deep uterine insemination (DUI). Both techniques facilitate deposition of semen into the uterus. Using post-CAI, semen is placed into the uterine body while DUI facilitates placement of semen in the proximal 1/3 of one uterine horn (Martinez et al., 2005). Similar fertility results can be expected for DUI using 600 x 106 spermatozoa and 1-1.5 x 109 spermatozoa for post-CAI as compared to intra-CAI using 3000 x 109 sperm per dose (Roca et al., 2006). Or in other words, a threefold reduction of fresh sperm numbers using post-cervical AI, and for DUI a 20-fold reduction for fresh and sixfold reduction for frozen semen can achieve acceptable pregnancy rates (J. M. Vazquez et al., 2008). Other possibilities for inseminations with low sperm numbers are surgical intrauterine inseminations where high pregnancy rates (89%) can be obtained with as few as 10 x 106 cooled stored spermatozoa that are placed on the uterotubal junction (Krueger & Rath, 2000; Krueger et al. , 1999). A further reduction in sperm numbers to 5 x 106 can be achieved if spermatozoa are placed close to the uterotubal junction by laparoscopy (Fantinati et al., 2005). A new procedure where semen is placed into the oviduct via laparoscopy also displays an opportunity for the use of diluted and sex sorted spermatozoa (Vazquez et al., 2008). Other insemination techniques like intraperitoneal insemination, where semen was deposited directly into the abdominal cavity, have been used to investigate insemination possibilities other than intra-CAI (Hunter, 1978). This technique, however, has never come to use in the AI industry due to ineffective sperm transport to the oviducts, reduced fertility and difficulty of the procedure when compared to intra-CAI. It has also been shown that

progressively motile sperm) or very low volume (0.02-0.2 ml) inseminations (Katila, 2005) are endoscopic deposition of semen at the utero-tubal junction right in the tip of the uterine horn ipsilateral to the ovary where ovulation will take place (Lindsey et al. , 2001; Lindsey et al. , 2002; L. H. A. Morris, 2004), and surgical deposition of spermatozoa directly into the oviduct (McCue et al. , 2000).

#### **3.3 AI techniques in dairy cattle**

In Britain, AI in dairy cattle began to be available in 1942, and by 1950 20% of dairy cattle were being inseminated. By 1960, more than 2 million cows were inseminated yearly, which was about 80% of the maximum level that AI would reach (Brassley, 2007). The established procedure for AI in cattle since the 1960s is transcervical deposition of semen into the uterine body. This technique replaced the original vaginal or shallow cervical insemination performed in the 1940s as the intrauterine method proved to be more efficient and resulted in higher fertility (Lopez-Gatius, 2000). Transcervical intrauterine AI involves the technique of cervical fixation per rectum to facilitate easier penetration of the cervical rings with a stainless steel Cassou device (AI pistolette) (Noakes et al. , 2001). Other techniques like unicornual or bicornual insemination where semen is deposited into one or both uterine horns, and intraperitoneal insemination have been investigated (Lopez-Gatius, 2000) but could not replace the transcervical intrauterine AI with semen deposition into the uterine body. For commercial AI in cattle, frozen-thawed semen is routinely used and a generally accepted insemination dose contains 10-20 x 106 spermatozoa. Deep horn AI close to the uterotubal junction has been investigated and facilitates AI with a conventional number of spermatozoa reduced x 100 or if very small volumes of semen (0.1-0.25 ml) are to be used (Hunter, 2003). Potential advantages of deep horn AI include: raising fertility of genetically valuable bulls whose non-return rates to estrus are sub-optimal, reducing the number of sperm per AI dose, facilitating the use of limited numbers of sex selected sperm cells available from flow cytometry, and breeding from valuable but oligospermic (too few sperm in ejaculate) bulls (Hunter, 2003).

#### **3.4 AI techniques in pigs**

Even though AI in pigs has been used since the 1930s, wider commercial application only started in the 1980s, with a dramatic increase in the use of AI in pork production in the last 20 years. International comparison shows that in 1990, 80% of pigs in former Eastern Germany were bred by AI, compared to only 23% in West Germany, while the figures for Norway were 71% and for the Netherlands 51%. France and the UK were about the same with 10% and the USA was only 7% (Brassley, 2007). By the end of the 1990s, close to 50% of the worldwide gilts and sows were inseminated (Roca et al., 2006). Nowadays, AI is used routinely in pig companies. In some European countries, like Belgium, Italy, the Netherlands, Norway and Spain, more than 80% of females are bred by AI and in North America and Brazil the percentage has reached 75% (Roca et al., 2006). One reason for the slow take-off for AI in pigs were initial conception rates which were as low as 18.1% in 1955 rising to 62% in 1961 while natural conception rates were expected to be close to 90% (Brassley, 2007). Conception rates could be increased to about 80% by the mid 1970s. Consistent 80-90% fertility rates with the use of liquid extended semen are common on many farms by now with an overall fertility success (measured as farrowing rates and litter

progressively motile sperm) or very low volume (0.02-0.2 ml) inseminations (Katila, 2005) are endoscopic deposition of semen at the utero-tubal junction right in the tip of the uterine horn ipsilateral to the ovary where ovulation will take place (Lindsey et al. , 2001; Lindsey et al. , 2002; L. H. A. Morris, 2004), and surgical deposition of spermatozoa directly into the

In Britain, AI in dairy cattle began to be available in 1942, and by 1950 20% of dairy cattle were being inseminated. By 1960, more than 2 million cows were inseminated yearly, which was about 80% of the maximum level that AI would reach (Brassley, 2007). The established procedure for AI in cattle since the 1960s is transcervical deposition of semen into the uterine body. This technique replaced the original vaginal or shallow cervical insemination performed in the 1940s as the intrauterine method proved to be more efficient and resulted in higher fertility (Lopez-Gatius, 2000). Transcervical intrauterine AI involves the technique of cervical fixation per rectum to facilitate easier penetration of the cervical rings with a stainless steel Cassou device (AI pistolette) (Noakes et al. , 2001). Other techniques like unicornual or bicornual insemination where semen is deposited into one or both uterine horns, and intraperitoneal insemination have been investigated (Lopez-Gatius, 2000) but could not replace the transcervical intrauterine AI with semen deposition into the uterine body. For commercial AI in cattle, frozen-thawed semen is routinely used and a generally accepted insemination dose contains 10-20 x 106 spermatozoa. Deep horn AI close to the uterotubal junction has been investigated and facilitates AI with a conventional number of spermatozoa reduced x 100 or if very small volumes of semen (0.1-0.25 ml) are to be used (Hunter, 2003). Potential advantages of deep horn AI include: raising fertility of genetically valuable bulls whose non-return rates to estrus are sub-optimal, reducing the number of sperm per AI dose, facilitating the use of limited numbers of sex selected sperm cells available from flow cytometry, and breeding from valuable but oligospermic (too few sperm

Even though AI in pigs has been used since the 1930s, wider commercial application only started in the 1980s, with a dramatic increase in the use of AI in pork production in the last 20 years. International comparison shows that in 1990, 80% of pigs in former Eastern Germany were bred by AI, compared to only 23% in West Germany, while the figures for Norway were 71% and for the Netherlands 51%. France and the UK were about the same with 10% and the USA was only 7% (Brassley, 2007). By the end of the 1990s, close to 50% of the worldwide gilts and sows were inseminated (Roca et al., 2006). Nowadays, AI is used routinely in pig companies. In some European countries, like Belgium, Italy, the Netherlands, Norway and Spain, more than 80% of females are bred by AI and in North America and Brazil the percentage has reached 75% (Roca et al., 2006). One reason for the slow take-off for AI in pigs were initial conception rates which were as low as 18.1% in 1955 rising to 62% in 1961 while natural conception rates were expected to be close to 90% (Brassley, 2007). Conception rates could be increased to about 80% by the mid 1970s. Consistent 80-90% fertility rates with the use of liquid extended semen are common on many farms by now with an overall fertility success (measured as farrowing rates and litter

oviduct (McCue et al. , 2000).

**3.3 AI techniques in dairy cattle** 

in ejaculate) bulls (Hunter, 2003).

**3.4 AI techniques in pigs** 

sizes) similar to or better than those resulting from natural matings (Roca et al., 2006). Apart from good conception rates, there are other reasons for the dramatic increase in AI utilization for pig breeding systems. On one hand there was the change of breeding and farrowing units that became larger and more specialized, and that the application of AI mating technologies became more feasible and cost effective on the other hand (Singleton, 2001). At the same time, the pork industry initiated payment programs based on actual carcass value instead of live weight basis. Genetic evaluation programs were used to implement genetic improvement programs through the use of AI to produce higher quality pork carcasses. For most commercial breeders it was the prospect of genetic improvement that was the major incentive to engage in AI (Brassley, 2007). Semen processing centres (boar studs) or on-farm collection facilities provide boar semen used for AI programs. More than 99% of AIs are performed with semen extended in a liquid state that can be stored at 15-20°C for up to 3 days (cooled semen)(Roca et al., 2006). The number of viable sperm per dose ranges between 2.5- 4 billion motile spermatozoa. Dose volumes range from 80-100ml for fresh liquid semen. The insemination procedure, called intra-cervical insemination (intra-CAI), involves the deposition of spermatozoa into the posterior part of the cervix using a catheter that engages with the folds of the cervix, stimulating the corkscrew tie of the boar's penis (Roca et al., 2006). This easy and quick procedure has been developed in the mid 1950s, standardized in the 1970s and is still used worldwide today (Roca et al., 2006). The remaining 1% of AIs utilizes frozen-thawed semen at doses of 5-6 x 109 spermatozoa. Even though large sperm numbers are used, fertility is substantially lower than that obtained with cooled semen. Due to the lowered reproductive performance, frozen-thawed boar semen is mostly limited to specialized breeding programs, research and for export puposes (Singleton, 2001; Wongtawan et al. , 2006). There are two insemination procedures available that allow insemination with low numbers of spermatozoa: post-cervical insemination (post-CAI) and deep uterine insemination (DUI). Both techniques facilitate deposition of semen into the uterus. Using post-CAI, semen is placed into the uterine body while DUI facilitates placement of semen in the proximal 1/3 of one uterine horn (Martinez et al., 2005). Similar fertility results can be expected for DUI using 600 x 106 spermatozoa and 1-1.5 x 109 spermatozoa for post-CAI as compared to intra-CAI using 3000 x 109 sperm per dose (Roca et al., 2006). Or in other words, a threefold reduction of fresh sperm numbers using post-cervical AI, and for DUI a 20-fold reduction for fresh and sixfold reduction for frozen semen can achieve acceptable pregnancy rates (J. M. Vazquez et al., 2008). Other possibilities for inseminations with low sperm numbers are surgical intrauterine inseminations where high pregnancy rates (89%) can be obtained with as few as 10 x 106 cooled stored spermatozoa that are placed on the uterotubal junction (Krueger & Rath, 2000; Krueger et al. , 1999). A further reduction in sperm numbers to 5 x 106 can be achieved if spermatozoa are placed close to the uterotubal junction by laparoscopy (Fantinati et al., 2005). A new procedure where semen is placed into the oviduct via laparoscopy also displays an opportunity for the use of diluted and sex sorted spermatozoa (Vazquez et al., 2008). Other insemination techniques like intraperitoneal insemination, where semen was deposited directly into the abdominal cavity, have been used to investigate insemination possibilities other than intra-CAI (Hunter, 1978). This technique, however, has never come to use in the AI industry due to ineffective sperm transport to the oviducts, reduced fertility and difficulty of the procedure when compared to intra-CAI. It has also been shown that

Artificial Insemination in Veterinary Science 25

pelvic rim (Morrell, 1995). Semen is deposited intravaginally. Due to the fact that rabbits have two separate uterine horns and cervices, intracervical AI is not performed as it would be required to release semen into both cervices. Other peculiarities regarding AI in rabbits are that females should be kept separate from males for about 19 days before breeding to exclude pseudopregnancy, that timing of AI is based on general behaviour and vulva colour of the doe, and that rabbits are induced ovulators that always require induction of ovulation when AI is used. Three methods for induction of ovulation are available: 1) mating with a vasectomized buck, 2) administration of human chorionic gonadotropin (hCG) or 3) administration of gonadotropin releasing hormone (GnRH)

AI in non-human primates is based on captive colony management and propagation of endangered or valuable founder animals. Its application, (although limited due to high costs, substantial technical requirements and limited available captive populations), has found use in Great Apes as well as Old World and New World Macaques (Wolf, 2009). Initially, intravaginal AI was performed while intrauterine insemination is the technique of

Application of AI in South American camelids has been challenging due to the inconsistent success in collecting semen from males. Llamas as well as alpacas copulate for extended periods of time (10-60 min), display a recumbent mating posture, deposit semen into the uterus, and the semen is very viscous (Adams et al., 2009). Camelids are not as easily trained to mount an AV as rams, bulls or stallions. It is necessary to maintain a stable AV temperature during prolonged copulation. Other semen collection attempts using condoms or intravaginal sacs, vaginal sponges, electro-ejaculation, post-coital vaginal aspiration, and fistulation of the penile urethra were associated with recovery of poor semen samples and contamination of semen samples with blood (Adams et al., 2009). For AI, semen is deposited into the uterus transcervically or via laparoscopy. Reported pregnancy rates after AI vary

Another interesting field for application of AI is the use of epididymal spermatozoa. Harvesting of epididymal sperm enables storage and usage of valuable genetic material of males after death or shortly before death if unexpected accidents or health problems occur. The epididymis is part of the male reproductive tract, is connected to the testis and forms a site for sperm maturation and storage. The epididymis can be dissected free from the testis after castration and epididymal spermatozoa can be harvested by flushing or slice-and-dice techniques (Bruemmer, 2006). Aspiration of spermatozoa from the epididymis has also been performed. Interestingly, the first reported pregnancy in a mare after AI was achieved with

Epididymal spermatozoa have been harvested from a variety of species like cats (Filliers et al., 2008; Hermansson & AxnÈr, 2007), dogs (Garcia-Macias et al., 2006; Hewitt et al., 2001; Nothling et al., 2007; Ponglowhapan et al., 2006), rats (Yamashiro et al., 2007), horses (Braun et al., 1994; Bruemmer, 2006; Heise et al., 2011; Johnson & Coutinho da Silva, 2008; Melo et al., 2008), cattle (Goovaerts et al., 2006; Martins, Rumpf, Pereira, & Dode, 2007), pigs (Ikeda et al., 2002), sheep (Garcia-Macias et al., 2006), goats (Blash, Melican, & Gavin, 2000), red deer (Fernandez-Santos et al., 2006; Garcia-Macias et al., 2006; MartÌnez-Pastor et al.,

frozen-thawed epididymal stallion spermatozoa in 1957 (Barker & Gandier, 1957).

analogues (Morrell, 1995).

widely between 2-68% (Adams et al., 2009).

**4. Use of epididymal spermatozoa for AI** 

choice today.

specific boar stimuli (such as olfactory and tactile stimuli) at or around the time of AI positively influence reproductive performance through an effect on reproductive processes like sperm transport and ovulation (Soede, 1993).

### **3.5 AI in sheep (Youngquist & Threlfall, 2007)**

Fresh, chilled and frozen semen is utilized for AI in sheep. Methods used are vaginal, cervical, laparoscopic intrauterine and transcervical intrauterine insemination. Vaginal insemination can be used for fresh and chilled semen and is also referred to as the "shot in the dark", or SID, method as the semen is blindly deposited into the cranial vagina. Pregnancy rates performing vaginal AI for fresh and chilled semen are acceptable while they are not for frozen semen. The technique is very fast but the use of semen is inefficient as AI doses have to contain large numbers of sperm. For cervical insemination, the hindquarters are elevated while the ewe is restrained "over the rail". The cervix is visualized using a speculum and a light source. Semen is deposited into the cervix with an angled tip insemination gun.

In contrast to horses and cattle, a successful method for transcervical intrauterine insemination in sheep has not been well established due to the specific anatomy of the ovine cervix (Halbert et al. , 1990). The cervical canal is approximately 7 cm long with a series of 6-8 rearward facing, offset rings that make transcervical insemination difficult to impossible. The Guelph system for transcervical AI (GST-AI) has been developed which requires special positioning of the ewe, cervical retraction and stabilization, and the use of specially designed instruments. Trained, experienced inseminators may penetrate the cervix in as much as 75-85% of ewes. Cervical injury, abscesses, infections and poor pregnancy rates are associated with this technique. Surgical AIs (i.e. via midventral laparotomy) are effective, but they are costly, time consuming, require technical proficiency, limit the number of times ewes can be used and require anesthesia (Evans & Maxwell, 1987). Laparoscopic intrauterine AI is used for frozen semen, and requires laparoscopic equipment as well as technical expertise. Animals are usually sedated and restrained in a laparoscopic cradle in dorsal recumbency. The laparoscope is used to identify the uterus and a loaded insemination pipette directed to the uterus to facilitate intrauterine deposition of the semen. Three hundred or more ewes can be inseminated per day using this technique if an experienced team works in a well-equipped and organized operation. Insemination doses for fresh as well as frozen-thawed semen are 400 x 106, 200 x 106, 20 x 106, 100 x 106 progressively motile spermatozoa for vaginal, cervical, laparoscopic and transcervical AI, respectively. Expected lambing rates for fresh semen are 20-60%, 40-80%, 70-100%, 40-80%, and 5-20%, 25-60%, 40-80%, 30-70% for frozen semen for vaginal, cervical, laparoscopic and transcervical AI, respectively.

#### **3.6 AI in other species**

Since the 1920s, AI has been a reproductive tool in commercial rabbitries that permits more controlled management and better planning (e.g. in batch parturition and weaning) than natural mating (Morrell, 1995). Conception rates after AI can be equivalent to or better than that achieved with natural breeding (Morrell, 1995). Natural matings in healthy individuals can result in conception rates of 85%. A single ejaculate can be split into 20-50 insemination doses and used for AI (Lavara et al. , 2005). For the AI procedure, the female is placed into a restraining box, the tail is lifted and an insemination pipette with a bend approximately 8cm from the end is inserted into the vagina at an angle of 45º in order progress beyond the

specific boar stimuli (such as olfactory and tactile stimuli) at or around the time of AI positively influence reproductive performance through an effect on reproductive processes

Fresh, chilled and frozen semen is utilized for AI in sheep. Methods used are vaginal, cervical, laparoscopic intrauterine and transcervical intrauterine insemination. Vaginal insemination can be used for fresh and chilled semen and is also referred to as the "shot in the dark", or SID, method as the semen is blindly deposited into the cranial vagina. Pregnancy rates performing vaginal AI for fresh and chilled semen are acceptable while they are not for frozen semen. The technique is very fast but the use of semen is inefficient as AI doses have to contain large numbers of sperm. For cervical insemination, the hindquarters are elevated while the ewe is restrained "over the rail". The cervix is visualized using a speculum and a light source. Semen is deposited into the cervix with an angled tip

In contrast to horses and cattle, a successful method for transcervical intrauterine insemination in sheep has not been well established due to the specific anatomy of the ovine cervix (Halbert et al. , 1990). The cervical canal is approximately 7 cm long with a series of 6-8 rearward facing, offset rings that make transcervical insemination difficult to impossible. The Guelph system for transcervical AI (GST-AI) has been developed which requires special positioning of the ewe, cervical retraction and stabilization, and the use of specially designed instruments. Trained, experienced inseminators may penetrate the cervix in as much as 75-85% of ewes. Cervical injury, abscesses, infections and poor pregnancy rates are associated with this technique. Surgical AIs (i.e. via midventral laparotomy) are effective, but they are costly, time consuming, require technical proficiency, limit the number of times ewes can be used and require anesthesia (Evans & Maxwell, 1987). Laparoscopic intrauterine AI is used for frozen semen, and requires laparoscopic equipment as well as technical expertise. Animals are usually sedated and restrained in a laparoscopic cradle in dorsal recumbency. The laparoscope is used to identify the uterus and a loaded insemination pipette directed to the uterus to facilitate intrauterine deposition of the semen. Three hundred or more ewes can be inseminated per day using this technique if an experienced team works in a well-equipped and organized operation. Insemination doses for fresh as well as frozen-thawed semen are 400 x 106, 200 x 106, 20 x 106, 100 x 106 progressively motile spermatozoa for vaginal, cervical, laparoscopic and transcervical AI, respectively. Expected lambing rates for fresh semen are 20-60%, 40-80%, 70-100%, 40-80%, and 5-20%, 25-60%, 40-80%, 30-70% for frozen semen for

Since the 1920s, AI has been a reproductive tool in commercial rabbitries that permits more controlled management and better planning (e.g. in batch parturition and weaning) than natural mating (Morrell, 1995). Conception rates after AI can be equivalent to or better than that achieved with natural breeding (Morrell, 1995). Natural matings in healthy individuals can result in conception rates of 85%. A single ejaculate can be split into 20-50 insemination doses and used for AI (Lavara et al. , 2005). For the AI procedure, the female is placed into a restraining box, the tail is lifted and an insemination pipette with a bend approximately 8cm from the end is inserted into the vagina at an angle of 45º in order progress beyond the

like sperm transport and ovulation (Soede, 1993).

**3.5 AI in sheep (Youngquist & Threlfall, 2007)** 

vaginal, cervical, laparoscopic and transcervical AI, respectively.

insemination gun.

**3.6 AI in other species** 

pelvic rim (Morrell, 1995). Semen is deposited intravaginally. Due to the fact that rabbits have two separate uterine horns and cervices, intracervical AI is not performed as it would be required to release semen into both cervices. Other peculiarities regarding AI in rabbits are that females should be kept separate from males for about 19 days before breeding to exclude pseudopregnancy, that timing of AI is based on general behaviour and vulva colour of the doe, and that rabbits are induced ovulators that always require induction of ovulation when AI is used. Three methods for induction of ovulation are available: 1) mating with a vasectomized buck, 2) administration of human chorionic gonadotropin (hCG) or 3) administration of gonadotropin releasing hormone (GnRH) analogues (Morrell, 1995).

AI in non-human primates is based on captive colony management and propagation of endangered or valuable founder animals. Its application, (although limited due to high costs, substantial technical requirements and limited available captive populations), has found use in Great Apes as well as Old World and New World Macaques (Wolf, 2009). Initially, intravaginal AI was performed while intrauterine insemination is the technique of choice today.

Application of AI in South American camelids has been challenging due to the inconsistent success in collecting semen from males. Llamas as well as alpacas copulate for extended periods of time (10-60 min), display a recumbent mating posture, deposit semen into the uterus, and the semen is very viscous (Adams et al., 2009). Camelids are not as easily trained to mount an AV as rams, bulls or stallions. It is necessary to maintain a stable AV temperature during prolonged copulation. Other semen collection attempts using condoms or intravaginal sacs, vaginal sponges, electro-ejaculation, post-coital vaginal aspiration, and fistulation of the penile urethra were associated with recovery of poor semen samples and contamination of semen samples with blood (Adams et al., 2009). For AI, semen is deposited into the uterus transcervically or via laparoscopy. Reported pregnancy rates after AI vary widely between 2-68% (Adams et al., 2009).
