**5. Use of sexed semen for AI**

Another aspect of artificial insemination in animals is the use of sex sorted spermatozoa. Separation of the X and Y bearing sperm is desirable in animals as one sex has significantly more value than the other in certain species. For dairy cattle for example, cows as the "milk producers" are the main source of income for the industry while bull calves are of less value as lactation is limited to females. For other food producing animals a higher percentage of male offspring might be beneficial as they grow faster and produce more meat. In many large, long-lived species like elephants, rhinoceroses, dolphins that are kept in captivity, sex selection could ease and avoid housing problems with males of these species (Durrant, 2009).

It has first been established for human spermatids in 1979 that there is a difference in DNA content between the mammalian X-chromosome-bearing spermatozoa and the Ychromosome (Otto et al., 1979). Since then, DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species (Garner, 2006; Garner et al., 1983; Lu et al., 2010; Pinkel et al., 1982), and offspring have been produced from sexed sperm of at least seven species, including rabbits (Johnson et al., 1989), humans (Levinson et al., 1995), cattle (Cran et al., 1993), horses (Buchanan et al., 2000), sheep (Catt et al., 1996), dogs (Meyers et al., 2008), cats (Pope et al., 2008), elk (Schenk & DeGrofft, 2003), buffalo (Presicce et al., 2005) and dolphins (O'Brien & Robeck, 2006). The first offspring born with flow cytometrically sex sorted spermatozoa was in rabbits after surgical AI into the oviduct (Johnson et al., 1989).

The Beltsville Sperm Sexing technology (Garner, 2006) uses the difference in DNA content of the X- and Y-chromosome to sort the sex-determining gametes. The procedure is called fluorescence-activated cell separation (FACS) where ejaculated spermatozoa are treated with a DNA stain (called a flourochrome) and due to the fact that X-chromosomes contain more DNA, the stain take-up will be higher for the X-chromosome bearing spermatozoa than the Y-chromosome bearing spermatozoa. This difference in stain absorption is used in a flow cytometer chamber where the fluorescent stain in the spermatozoa is excited by a laser. Each live sperm produces an emission with an intensity that is directly related to the quantity of DNA within the sperm head. The X bearing spermatozoa emit more intense light than the Y bearing spermatozoa. A high speed computer is used to analyze the relative fluorescence of the X- and Y-sperm populations as they flow through a cytometer chamber. Spermatozoa are then assigned either a negative or positive charge depending on the DNA content while passing by charged plates. An electromagnetic field separates the X- and Ychromosome bearing spermatozoa (Senger, 2003). This separation technology has a 85-95% success rate (Garner, 2006).

2006), Spanish Ibex (Santiago-Moreno et al., 2006), African buffalo (Herold et al., 2006), North American buffalo (Lessard at al., 2009) and monkeys (Goff et al., 2009; Ng et al., 2002). Pregnancies and offspring after AI with epididymal spermatozoa have been produced amongst others in horses (Barker & Gandier, 1957; Heise et al., 2010; Morris et al., 2002; Papa et al., 2008), dogs (Hori, Hagiuda, Kawakami, & Tsutsui, 2005) and Spanish Ibex (Santiago-Moreno et al., 2006). Application of AI for epididymal spermatozoa holds tremendous potential for future use of valuable genetics not only in domestic but also especially in wild

Another aspect of artificial insemination in animals is the use of sex sorted spermatozoa. Separation of the X and Y bearing sperm is desirable in animals as one sex has significantly more value than the other in certain species. For dairy cattle for example, cows as the "milk producers" are the main source of income for the industry while bull calves are of less value as lactation is limited to females. For other food producing animals a higher percentage of male offspring might be beneficial as they grow faster and produce more meat. In many large, long-lived species like elephants, rhinoceroses, dolphins that are kept in captivity, sex selection could ease and avoid housing problems with males of

It has first been established for human spermatids in 1979 that there is a difference in DNA content between the mammalian X-chromosome-bearing spermatozoa and the Ychromosome (Otto et al., 1979). Since then, DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species (Garner, 2006; Garner et al., 1983; Lu et al., 2010; Pinkel et al., 1982), and offspring have been produced from sexed sperm of at least seven species, including rabbits (Johnson et al., 1989), humans (Levinson et al., 1995), cattle (Cran et al., 1993), horses (Buchanan et al., 2000), sheep (Catt et al., 1996), dogs (Meyers et al., 2008), cats (Pope et al., 2008), elk (Schenk & DeGrofft, 2003), buffalo (Presicce et al., 2005) and dolphins (O'Brien & Robeck, 2006). The first offspring born with flow cytometrically sex sorted

spermatozoa was in rabbits after surgical AI into the oviduct (Johnson et al., 1989).

The Beltsville Sperm Sexing technology (Garner, 2006) uses the difference in DNA content of the X- and Y-chromosome to sort the sex-determining gametes. The procedure is called fluorescence-activated cell separation (FACS) where ejaculated spermatozoa are treated with a DNA stain (called a flourochrome) and due to the fact that X-chromosomes contain more DNA, the stain take-up will be higher for the X-chromosome bearing spermatozoa than the Y-chromosome bearing spermatozoa. This difference in stain absorption is used in a flow cytometer chamber where the fluorescent stain in the spermatozoa is excited by a laser. Each live sperm produces an emission with an intensity that is directly related to the quantity of DNA within the sperm head. The X bearing spermatozoa emit more intense light than the Y bearing spermatozoa. A high speed computer is used to analyze the relative fluorescence of the X- and Y-sperm populations as they flow through a cytometer chamber. Spermatozoa are then assigned either a negative or positive charge depending on the DNA content while passing by charged plates. An electromagnetic field separates the X- and Ychromosome bearing spermatozoa (Senger, 2003). This separation technology has a 85-95%

animal species.

**5. Use of sexed semen for AI** 

these species (Durrant, 2009).

success rate (Garner, 2006).

Not all mammalian sperm are equally suitable for sex sorting of spermatozoa. Apart from the DNA content difference of the X- and Y-bearing spermatozoa, the head shape of the spermatozoa plays a role as well. Flattened, oval shaped sperm heads (e.g. bull, boar, ram spermatozoa) are more readily oriented in a sperm sorter using hydrodynamics than those gametes with more round or angular head shapes (rodent spermatozoa) (Garner, 2006). The area of the flat profile of the sperm head can be multiplied times the difference in DNA content of the X-and Y-chromosome bearing sperm to give the sorting index. This index suggests that bull and boar sperm are well suited for separation in a flow sorter.

Initially, the use of sex sorted spermatozoa was limited due to the slow separation process where only a few hundred thousand sperm per hour could be sorted. Newer sperm sorter systems are able to sort 20,000 sperm/s resulting in up to 6000 or more sperm/s each of Xand Y-sperm at 90% accuracy (Garner & Seidel Jr, 2008). Due to low sperm numbers acquired with sex sorting, initial efforts to predetermine the sex required surgical insemination. Later, with improvement of the equipment, quantities were sufficient for in vitro fertilization (IVF). Today, sexed sperm are commercially available for cattle where the standard insemination doses of 2 x 106 sexed sperm achieve 70-80% of the pregnancy rates achieved with non-sorted sperm in doses of 10-20 x 106 (Bodmer et al., 2005; Garner, 2006). In pigs, low dose (70 x 106 spermatozoa) AI with flow cytometrically sorted sperm deep into the uterine horn resulted in pregnancy rates of 35-45.6% (Vazquez et al., 2003). In horses, hysteroscopic insemination into the uterine horn (Lindsey et al., 2002; Morris & Allen, 2002) and ultrasound guided deep uterine AI were performed using sex sorted spermatozoa in low concentrations (5 x 106 sperm cells/ dose).
