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

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

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.,

Artificial Insemination in Veterinary Science 27

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

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

AI has been and still is the most used reproductive technique in animals. A lot of research has been done over the last few decades, constantly improving techniques, methods and applications of AI. Routine AI procedures as well as specialized techniques like low-dose inseminations or use of sexed semen offer a wide variety for application of AI in domestic as

Adams, G. P., Ratto, M. H., Collins, C. W., & Bergfelt, D. R. (2009). Artificial insemination in South American camelids and wild equids. *Theriogenology, 71*(1), 166-175. Amann, R. P., & Pickett, B. W. (1987). Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. *Journal of Equine Veterinary Science, 7*(3),

Andersen, K. (1972). Fertility of frozen dog semen. *Acta Veterinaria Scandinavia, 13*, 128-130. Andersen, K. (1975). Insemination with frozen dog semen based on a new insemination

Barker, C. A. V., & Gandier, J. C. C. (1957). Pregnancy in a mare resulted from frozen epididymal spermatozoa. *Canadian Journal of Comparative Veterinary Medical Science,* 

Blash, S., Melican, D., & Gavin, W. (2000). Cryopreservation of epididymal sperm obtained

at necropsy from goats. *Theriogenology, 54*(6), 899-905.

suggests that bull and boar sperm are well suited for separation in a flow sorter.

low concentrations (5 x 106 sperm cells/ dose).

well as wild and endangered animal species.

technique. *Zuchthygiene, 10*, 1-4.

**6. Conclusion** 

**7. References** 

145-173.

*21*, 47-51.

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 animal species.
