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Semen extenders
CHAPTER 1.2
Many extenders for equine semen have been developed and most of them include egg yolk,
milk, milk by-products or chemicals to regulate osmolarity and pH (Pickett and Amann, 1987). The
most popular extenders worldwide are similar in composition to the original recipe published by
Kenney et al. in 1975. These extenders are inexpensive, easy to prepare and can be stored in frozen
form (Aurich, 2008). In the early days of modern AI, milk was used as a semen extender after heating
it. Heating raw milk was necessary to inactivate lactenin, so that sperm could survive in it for a
prolonged time (Thacker and Almquist 1951, 1953; Flipse et al., 1954). Ever since, people have been
refining the diluters and developing media to diminish detrimental components (Aurich, 2008).
Battelier et al. (1997) investigated the ability of different purified milk fractions to preserve equine
sperm and found very differing protection levels, varying from harmful to sperm to supporting a
better survival when compared to commercial heated milk. Native phosphocaseinate (NPPC) and β-
lactoglobulin were both protective for sperm storage, without synergetic effect between them, NPPC
afforded higher protection to spermatozoa. When NPPC was added to Hank’s salts solution
supplemented with Hepes, glucose and lactose (HGLL), it was equally capable of maintaining
spermatozoal motility compared to INRA82, a classical skimmed milk diluter which was commonly
used in Europe (the composition of INRA82 is presented in table 3c). However, higher fertility results
were obtained using this NPPC-HGLL extender in comparison to INRA82, (Batellier et al., 1997). The
discovery of this protective effect of NPPC, which is a direct effect since there is no evidence of its
binding to sperm membranes (Battelier et al., 2000), led to the development of INRA96, a chemically
defined diluter containing Hank’s salts, Hepes, glucose, lactose and NPPC. Another chemically
defined extender, EquiPro, contains a combination of defined caseinates and whey proteins. When
compared to a classical skim milk-glucose extender (Kenney extender), EquiPro was capable of
preserving the in vitro assessed quality of equine semen in a comparable (24h) or even better (≥48h)
way than the skim milk extender (Pagl et al., 2006). Finally, a defined extender containing soybean
lecithin has been developed in order to prevent possible disease transmission by animal proteins
(AndroMed ® , Minitüb) and can also be used for cooled storage of equine semen (Aurich, 2005; Aurich
et al., 2007).
Cooling of equine semen
Generally, it is believed that equine semen is best preserved when chilled at 4 -5 °C (Varner
et al., 1988; Aurich, 2008) in absence of air (Douglas-Hamilton et al., 1984), and in combination with
a slow cooling rate (Province et al., 1985; Varner et al., 1988). Storage at ambient temperature is only
advised if insemination can occur within 12h following collection (Varner et al., 1988). Diluted semen
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