Equine Semen Preservation: Current and Future Trends


department of Animal Pathology, Faculty of Veterinary Medicine, Agroalimentary Institute of Aragon 1A2, University of Zaragoza, Center for Agrifood Research and Technology (CITA), Zaragoza, C/Miguel Servet 133, 50013, Zaragoza, Spain

  • 2 Chief Veterinary Officer of the Veterinary Unit, Zaragoza, Spain
  • *Corresponding author. E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it


Nowadays, stallion semen processing and preservation for artificial insemination (AI) is a widely used tool in modem horse reproduction. During the 20th century, AI had a strong development in most domestic animal species except in equine species, due to economic interests, traditional aspects and mistrust of horse owners. Semen conservation technologies based on temperature decrease to reduce or stop sperm metabolism are necessary to increase the lifespan of sperm and preserving sperm functionality and fertility. Currently. AI is performed mainly with fresh, chilled or frozen semen. Sperm refrigeration at 5°C has several advantages related to easy handling and shipping, low cost because it does not require special equipment and minor legal requirements for import and export. However, cryopreservation is the only viable method for spermatozoa storage during indefinite periods. The main problem of freezing semen is related to the low fertility rates obtained due to wide inter-individual sperm quality variability; therefore only some stallions produce suitable semen for cryopreservation. To solve these disadvantages, new methodologies have been studied during recent years with different results. Vitrification and lyophilization are promising stallion semen preservation techniques that require further study to be applied routinely during long-term periods.


Equine sperm preservation methods have been linked to the development of artificial insemination (AI). The first reports of AI in horses were documented from Arabian texts, in which semen samples were obtained from recently covered mares of rival tribes and then utilized to inseminate their own mares. However, until the end of the 19th century, AI did not obtain promising results in mares due to researches carried out by Ivanow in Russia and Dr. Pearson at Pennsylvania University.

The decrease of equine population after the Second World War and the restrictive regulations regarding the use of AI in several equine breed organizations delayed the progress of this technique in horses. First, studies about stallion semen collection and handling were described by Mckenzie et al. (1939) and Berliner (1942) with suitable sperm quality results. The discovery of glycerol as cryoprotectant agent in 1949 by Polge et al., was the beginning of the development of preservation techniques of biological materials including equine sperm. Next year, Barker and Gandier (1957) obtained the first foal from cryopreserved epidydimal stallion sperm. During the 1970s and 1980s, the utilization of cooled semen on AI programs increased due to the development of a transport container (Equitainer) (Douglas-Hamilton et al., 1984), the use of Kenney extender (Kenney et al., 1975) and the acceptance of reproductive biotechnology by several breeders.

Several advantages show frozen-thawed sperm in comparison to cooled sperm, such as the scheduled use of stallions outside the competition period, easy international transport and the centralized processing of frozen semen by specialized laboratories, which decrease the variability of the seminal quality. Despite the current advances, cryopreserved semen shows some disadvantages, such as low fertility rates, control of the mare cycle, and a high cost along cryopreservation process (Brinsko and Varner, 1992; Samper and Moms, 1998; Loomis and Graham, 2008). Furthermore, it has been estimated that only 30-40% of stallions produce semen that is suitable for cryopreservation, and a large interindividual variation on sperm survival during the freezing and thawing procedures has been also reported (Loomis and Graham, 2008).

Nowadays, AI is the most common biotechnology utilized around the world in the horse industry and it has enabled the development of other technologies, such as sperm sexing, regulation of the estrous cycle, embryo transfer, cloning as well as improvement of semen preservation methods as sperm refrigeration or freezing and, currently, sperm vitrification and lyophilization.


Semen collection is an essential part of preservation protocols and AI programs. The quality of collected semen depends on numerous factors such as libido, season, age, or breed (Samper, 2000). Semen can be collected using different methods; condom, pharmacological induction, manual manipulation or by the use of artificial vagina, the tool most commonly used (Samper, 2000). There are several models of artificial vagina available commercially. The Colorado model is the most widespread at the beginning, but currently the most used is the Missouri model due to the improvements and efficiency.

The ejaculate is a liquid suspension composed of sperm and seminal plasma, which comprised a complex mixture of secretions (fructose, sorbitol, ascorbic acid, lactic acid, citric acid, proteins, enzymes, vitamins, and hormones). Seminal plasma-derived primarily from the epididymis and accessoiy sex glands of the male. It participates in the final speim maturation, modifies spermatic membrane surface, besides acts as a vehicle for the ejaculated sperm and protects the spermatozoa during the female reproductive tract transport (Topfer-Petersen et ah, 2000). In stallion, the seminal plasma is normally separated from the semen during the cryopreservation process, since it has proved to be a harmful medium because decrease the percentage of sperm with progressive motility (Pickett et al., 1975; Jasko et al., 1992). The ejaculate should not be exposed to mechanical damage, light, cold, or heat and the equipment in contact with the speim must be tempered, dry, clean, and free of toxic residues. After ejaculation, the semen will be kept at 30/32°C before the seminal evaluation and subsequent dilution (Brinsko and Varner, 1992).

Another effective way to recover genetic material in horses is to obtain it from the epididymis from castrated or slaughtered animals. This technique allows obtaining sperm from stallions of high genetic value that have suffered sudden death, major injuries, or castration. Different studies have demonstrated the efficiency of epididymal sperm recovery (Tiplady et al., 2002, Monteiro et ah, 2011). In addition, several researchers suggest that sperm can be harvested immediately after orchiectomy or after 24 h of storage at 4°C-5°C without any difference in terms of viability (Bruemmer et ah, 2006; Neild et ah, 2006). Different methods have been used to collect epididymal sperm, including percutaneous epididymal sperm aspiration, the flotation method, the retrograde flush technique, or the standard flush technique of epididymis and ductus deferens (Cary et ah, 2004; Bruemmer, 2006). This teclmique allows recovery of a high number of sperms compared with the collection by artificial vagina (Bruemmer, 2006). The motility of epididymal and ejaculated sperm in stallions has been found comparable and no differences were observed in tenns of morphological defects and sperm viability (Weiss et ah, 2008; Guimaraes et ah, 2012).


The semen quality is assessed immediately after collection. Andrological evaluation verifies the reproductive potential of a stallion in the buying and selling process, before the beginning of the reproductive season and before semen preservation or AI. The general parameters used for the analysis of semen quality include total volume, sperm concentration, motility, and normal morphology. Other tests, such as acrosome and membrane integrity, hypo-osmotic swelling test (HOST), mitochondrial activity, or the thermal resistance test can be used for assessment of semen quality (Love et ah, 2018). However, some animals with apparently normal semen quality show very poor fertility rates; in these cases, a more exhaustive evaluation is necessary.

After the semen quality evaluation, equine ejaculate is usually centrifuged. In some countries, it is a standard practice prior to preparing spenn doses before AI. Centrifugation increases sperm concentration of the ejaculate and also removes the seminal plasma (Parlevliet and Colenbrander, 1999; Hoogewijs et ah, 2010). However, it might generate negative effects on the conservation of equine spenn, decreasing the motility and viability of spenn (Martin et ah, 1979), due to the physical damage on the cells and ROS production (Hoogewijs et ah, 2010).

Contradictory results have been obtained in relation seminal plasma utilization on sperm preservation. Different studies showed that the removal of seminal plasma enhanced the survival spermatozoa (Parlevliet and Colen- brander, 1999; Aurich, 2005); however, the presence of a small amount of stallion seminal plasma (0.6-20%) increased sperm motility, plasma membrane integrity, and fertility (Jasko et al., 1992; Moore et al., 2006; Neuhauser et al., 2015). Beneficial effects may be related to antioxidant properties and the inhibitory effect on the binding of polymorphonuclear neutrophils on the female reproductive tract (Knop et al., 2005).

A special extender based on dense isotonic compounds is utilized during centrifugation. The most common mediums are citrate-ethylene acid (EDTA) (Cochran et al., 1984), Tyrode medium (Ljaz and Ducharme, 1995), and glucose-EDTA (Martin et al., 1979). The extender function is to maintain motility and protect the sperm during the centrifugation process and in a dilution ratio of 1:1 or 1:2. The centrifugation influences on equine sperm by the strength and time of centrifugation, the kind of extender, the presence or absence of the extender before centrifugation, and the concentration of seminal plasma (Pickett et al., 1975; Aurich, 2005). Different protocols have been used as shown in (Table 8.1).

TABLE 8.1 Different Stallion Sperm Centrifugation Protocols.



1000g/5 min

Martin et al., 1979

600g/10 min

Palmer 1984

400g/12 min

Brinsko et al., 2011

400g/15 min

Cochran et al., 1984

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