Why preserve genetic biodiversity?

Genetic biodiversity allows animals to adapt to different environments, both to changes imposed on animals by mankind in the guise of management or husbandry practices and, in the wider sense, to diseases and climate change (Woelders et al., Reference Woelders, Windig and Hiemstra2012). Selecting only high-producing genotypes may be counter-productive in the long-term if fertility is thereby impaired, as seems to have been the case for dairy cattle (Rodriguez-Martínez et al., Reference Rodriguez-Martinez, Hultgren, Båge, Bergqvist, Svensson, Bergsten, Lidfors, Gunnarsson, Algers, Emanuelson, Berglund, Andersson, Håård, Lindhé, Stålhammar and Gustafsson2008). Conservation of animal genetic resources is essential to provide the genetic biodiversity required to allow animals to adapt to future changes in the environment. As far as farm animals are concerned, this means conserving our indigenous breeds; currently some 20% of livestock breeds are at risk of extinction and data are lacking for a further 36% of breeds. Rather than trying to maintain viable populations of each breed of livestock species (in situ conservation), a more practical method of maintaining genetic diversity is to cryopreserve germplasm (ex situ conservation). Thus by conserving existing genetic diversity, the basis for selecting ‘desirable’ genotypes in the future can be maintained.

With the rapid expansion in global aquaculture, there has been an increased emphasis on the development of reproductive technologies directed towards increased production efficiency in fish farming (Weber & Lee, Reference Weber, Lee, Lamb and DiLorenzo2014). While procedures for sperm cryopreservation have been developed for most major aquaculture species, successful sperm management for many species is currently constrained by the high variability of sperm cryo-resistance, a lack of appropriate biosecurity safeguards, and the absence of cryopreservation technologies sufficiently validated for large-scale aquaculture (Martínez-Páramo et al., Reference Martínez-Páramo, Horváth, Labbé, Zhang, Robles, Herráez, Suquet, Adams, Viveiros, Tiersch and Cabrita2017). In contrast to mammalian vertebrates, fish spermatozoa remain immotile in seminal plasma (Dadras et al., Reference Dadras, Sampels, Golpour, Dzyuba, Cosson and Dzyuba2017), with motility being activated following release into the external environment (sea or freshwater). Following activation, spermatozoa from most fish species remain motile for a short time period, often 1 min or less. To address these challenges, protocols for fish semen processing have focused on developing species-specific extenders to facilitate the collection and storage of inactivated spermatozoa (Tiersch et al., Reference Tiersch, Yang, Jenkins and Dong2007). Recent studies have indicated that temperature affects the lipid composition of motile carp sperm, with greater temperatures being associated with reduced lipid content, increased sperm curvilinear velocity and a decreased duration of the period motility (Dadras et al., Reference Dadras, Sampels, Golpour, Dzyuba, Cosson and Dzyuba2017).

Assisted reproduction technologies

The technologies involved in mammalian ART include a number of methods of bringing together spermatozoa and oocytes, such as artificial insemination (AI), gamete intra-fallopian transfer (GIFT), in vitro fertilization (IVF) and embryo transfer (ET), intra-cytoplasmic sperm injection (ICSI). To facilitate carrying out these techniques, other technologies have been developed such as sperm preservation and cryopreservation, and in vitro maturation (IVM) of oocytes (usually from slaughterhouse material), for subsequent IVF and ET.

By far the most widely used ART for breeding food production animals is AI using fresh or frozen semen. The availability of preserved semen (either cooled or cryopreserved) enables semen to be inseminated into females that are isolated from the male, thus facilitating both genetic improvement and disease control. If frozen, the semen can be used at any time, including after the death of the donor. This feature is particularly important when rare breeds are concerned as the chances of having access to fresh semen exactly when it is needed for insemination are low. However, it is not always possible to collect semen from all individuals, particularly those that have not been trained to ejaculate into an artificial vagina. In some cases, the collection of epididymal spermatozoa after castration or death is feasible (Woelders et al., Reference Woelders, Windig and Hiemstra2012) and can yield high numbers of useable spermatozoa e.g. from rams.

Use of AI has the disadvantage that it focuses mainly on the genetic component of the male; ET should enable genetic progress to be made more rapidly than AI as it enables both the male and female genetic components to be transferred. Worldwide, ET is increasing considerably within the bovine livestock sector, mainly due to an increase in the transfer of in vitro produced (IVP) embryos. Figures from 2012 indicate a current annual transfer of around 500,000 embryos produced by superovulation and 400,000 by in vitro maturation (Galli et al., Reference Galli, Duchi, Colleoni, Lagutina and Lazzari2014). The vast majority of these transfers is occurring in South America, especially Brazil.

Some in vitro technologies are better suited to particular species than others. The increasing use of bovine IVP embryos is indicative of the success of the technique in this species. However, in horses it has not been possible to develop a reliable method for equine IVF. Thus ICSI is more commonly used than IVF for equids, although the need for specialized equipment and highly trained personnel limit the use of this ART. Conversely, ICSI is not commonly used for bovine oocytes because of technical difficulties, such as inadequate oocyte activation (Malcuit et al., Reference Malcuit, Maserati, Takahashi, Page and Fissore2006) or cytoskeletal damage, e.g. from the large-diameter pipette needed to accommodate the bull spermatozoon (Galli et al., Reference Galli, Vassiliev, Lagutina, Galli and Lazzari2003). In other species, the lack of access to oocytes is a limiting factor in the potential development of an ET program. Protocols for cryopreserving embryos may not be suitable for oocytes, even in the same species; further research is needed to develop better methods of preserving oocytes. Thus it is important not only to choose the technique to fit the species but to develop as many forms of ART as possible, to increase the chances of success in any given species.

Germplasm banking

The term ‘germplasm’ encompasses all reproductive cells that, singly or in combination, lead to the production of offspring. The most commonly encountered form of germplasm banking is sperm cryopreservation, which has been used in livestock breeding for more than 50 years and is considered to be the backbone of modern dairy cattle production. It is also the most readily applicable biotechnology as spermatozoa are the reproductive cells that are most easily obtained and numerous. In contrast, obtaining oocytes is invasive, their numbers are limited and maturation is cycle-dependent in many livestock species. Whereas sperm cryopreservation protocols exist for approximately 300 species, protocols for oocytes and embryos have been developed for fewer than 50 species. Other sources of germplasm include spermatids, spermatogonial stem cells, testicular and ovarian tissue, as well as somatic cells for nuclear transfer.

Embryos and oocytes

Embryos (and, to some extent, oocytes) can be frozen using permeating cryoprotectants and slow freezing protocols in programmable cell freezers, in a similar manner to spermatozoa. Vitrification, particularly of oocytes, is in many cases more successful than conventional slow freezing. Oocytes are particularly sensitive to slow freezing because of the low permeability of the plasma membrane to cryoprotectants but also because of premature cortical granule exocytosis leading to zona hardening, which renders them impenetrable by a spermatozoon for subsequent fertilization (Arav, Reference Arav2014). With vitrification, however, the cells are placed in a solution of high osmolarity but without permeating cryoprotectants, and are then plunged directly into liquid nitrogen. As no ice-formation takes place and the transition to a glass-like state occurs very rapidly, structural damage is minimized (Arav, Reference Arav2014). Table 1 provides a comparison of the two techniques. Despite the success with vitrification of human oocytes, those of cattle are proving more problematic, which may be due to the amount of cytoplasmic lipids and/or the composition of the plasma membranes (Sprícigo et al., Reference Sprícigo, Diógenes, Leme, Guimarães, Muterlle, Silva, Solà-Oriol, Pivato, Silva and Dode2015).

Table 1 Comparison of slow freezing and vitrification for embryos and oocytes

Vitrification has also been attempted with spermatozoa. The problems encountered with cryopreservation-induced sperm damage are well known and include physical damage from ice-crystal formation and dehydration, damage to the sperm mitochondria arising from cold-shock, damage to the plasma and acrosomal membranes (capacitation-like damage) and DNA, as well as toxicity of cryoprotectants (Agha-Rahimi et al., Reference Agha-Rahimi, Khalili, Nabi and Ashourzadeh2014). Theoretically, these problems could be circumvented by vitrification, in which there is no ice-crystal formation and very high rates of cooling are employed that are considered to be too rapid for cold-shock to occur (Isachenko et al., Reference Isachenko, Mallmann, Rahimi, Risopatròn, Schulz, Isachenko, Sànchez and Katkov2012). However, to date, the only species in which sperm vitrification is reported to be successful is the human (Isachenko et al., Reference Isachenko, Mallmann, Rahimi, Risopatròn, Schulz, Isachenko, Sànchez and Katkov2012). Considerable chromatin damage was induced in dog spermatozoa by vitrification, much more so than by conventional freezing (Sánchez et al., Reference Sánchez, Risopatrón, Schulz, Villegas, Isachenko, Kreinberg and Isachenko2011). Even with human spermatozoa there are conflicting reports about whether vitrification offers any advantages over conventional slow freezing (Agha-Rahimi et al., Reference Agha-Rahimi, Khalili, Nabi and Ashourzadeh2014). The size of the sperm head appears to be a crucial factor in determining cryosensitivity during vitrification, with smaller sperm heads being able to withstand the severe osmotic effects better than the larger, paddle-shaped sperm heads of most livestock species. Therefore, further developments are needed before this technique will be able to provide a real alternative to slow freezing of spermatozoa.

Primordial germ cells (PGCs)

The conservation of both maternal and paternal genetic information is essential for conservation initiatives (germplasm banking). As successful cryopreservation of both oocytes and embryos in teleost fish remains prohibitively difficult, attention has switched to preserving alternative forms of germplasm, notably PGCs, which are the precursors of both oogonia and spermatogonia. PGCs are formed during the early stages of embryonic development and, in fish, these cells undergo active proliferation following migration to the genital ridges. Recent studies have now demonstrated that functional spermatozoa and eggs can be obtained following the transplantation of either PGCs or functionally similar spermatogonial stem cells in a number of fish species (Okutsu et al., Reference Okutsu, Suzuki, Takeuchi and Yoshizaki2006; Kobayashi et al., Reference Kobayashi, Takeuchi, Takeuchi and Yoshizaki2007; Morita et al., Reference Morita, Kumakura, Morishima, Mitsuboshi, Ishida, Hara, Kudo, Miwa, Ihara, Higuchi, Takeuchi and Yoshizaki2012). For example, thawed PGCs, isolated from adult rainbow trout (Oncorhynchus mykiss), when transplanted into the peritoneal cavity of newly hatched embryos differentiated into mature spermatozoa and eggs in the recipient gonads. Furthermore, the donor-derived spermatozoa and eggs obtained from the recipient fish were able to produce normal offspring.

Interest in PGC transplantation techniques increased further after it was demonstrated that PGCs from one species could be successfully transplanted into the embryo of another, in which they proliferate, yielding viable gametes of the donor species. This procedure, termed surrogate reproduction, was first demonstrated with trout PGCs successfully transplanted into triploid masu salmon (Oncorhynchus masou) embryos (Okutsu et al., Reference Okutsu, Shikina, Kanno, Takeuchi and Yoshizaki2007). The principle of this reproductive technology is relatively simple. Progenitor cells of eggs and sperm, either PGCs or spermatogonia, are isolated from the target species and transplanted into a closely related recipient species. Upon maturation, donor-derived eggs and sperm are then produced in the recipient (Fig. 1). Surrogate broodstocking has major implications to both aquaculture production and conservation, especially as studies now show that PGCs can be transplanted across species, genus, and even family barriers (Saito et al., Reference Saito, Goto-Kazeto, Fujimoto, Kawakami, Arai and Yamaha2010, Yoshizaki et al., Reference Yoshizaki, Okutsu, Morita, Terasawa, Yazawa and Takeuchi2012). There is now considerable interest in applying surrogate broodstock technologies in the aquaculture of species that are difficult or costly to breed in captivity, such as the sturgeons (family Acipenseridae) and the bluefin tuna (Thunnus thynnus). For example, transplantation of germ cells from the valuable bluefin tuna into the earlier maturing and easier to maintain chub mackerel (Scomber japonicus) is being viewed as a possible answer to the aquaculture production of this difficult species (Yoshizaki et al., Reference Yoshizaki, Okutsu, Morita, Terasawa, Yazawa and Takeuchi2012). It is envisaged that both conservation and efficient utilization of genetic resources (aquaculture) can now be more efficiently achieved in fish through surrogate production combined with the cryopreservation of PGCs.

Figure 1 Principle of surrogate reproduction in fish. Conservation of a threatened species can be achieved by transplanting frozen PGCs or spermatogonia into recipient embryos of a closely related species (adapted from Yoshizaki et al., Reference Yoshizaki, Okutsu, Morita, Terasawa, Yazawa and Takeuchi2012).

Given the current limitations in the ability to freeze fish oocytes and embryos there is increasing interest in cryobanking PGCs and spermatogonia of threatened fish species. Thus PGCs have been successfully transplanted from the critically endangered Chinese sturgeon to a closely related but not endangered species, the Dabrey's sturgeon (Ye et al., Reference Ye, Li, Yue, Du, Yang, Yoshino, Hayashida, Takeuchi and Wei2017). While there is still much work to do to develop this technique, these results show that transplantation of PGCs, and their survival in the recipient, is possible.

While public concern has focused primarily on the conservation of terrestrial species, especially iconic mammalian species, aquatic species including teleost fishes are considered more vulnerable to anthropogenic impacts. Indeed, according to the IUCN Red List of Threatened Species (International Union for Conservation of Nature, 2016), 58 ray-finned fish species (Actinopterygii), as well as one lamprey species, have become extinct in recent years. As such, further advances in the cryopreservation of fish germ cells and reproductive technologies such as surrogate broodstocking will be a major benefit not only to aquaculture, but also for the conservation of threatened fish species (Comizzoli & Holt, Reference Comizzoli, Holt, Holt, Brown and Comizzoli2014). The principle of surrogate reproduction in fish is depicted in Fig. 1.

Pitfalls with germplasm cryopreservation

Although there are many indications for the use of germplasm banking in livestock production, major technological limitations still exist in its application to all types of germplasm and to all species. Cryopreservation tends to decrease the viability and fertilizing capability of germplasm, and this is more so where pieces of tissue are frozen rather than gametes. In addition, there may be epigenetic changes brought about by the cryoprotective agents and techniques used (Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014) which cannot be studied effectively until enough offspring are produced from these procedures to provide a representative population. A complicating factor in such studies with rare breeds or endangered species is the lack of suitable breeding animals to provide gametes, or to test the effect of proposed changes in handling procedures on fertility (Woelders et al., Reference Woelders, Windig and Hiemstra2012).

Species-specific protocols may be needed as existing ones cannot be extrapolated to all other species. Tissue-specific protocols may be needed: for example, cryopreserving epididymal spermatozoa may require a different protocol to ejaculated samples. Cryopreserving embryos allows the full genetic complement of both sire and dam to be preserved whereas sperm freezing preserves only the male genetic component, but the transfer of the material to recipient animals may be technically more demanding for embryos than for spermatozoa, depending on the species. In many sheep, for example, it is not possible to pass a catheter through the cervix; while semen can be deposited vaginally, deposition of embryos requires a surgical or laparoscopic approach, which is more technically demanding than vaginal deposition of semen. In addition, awareness of biosecurity has increased considerably over the last few decades; it is now known that not only must the donor animals be free of disease but also that material of animal origin in the cryopreservation medium should be avoided or, when this is impossible, the material should be from specific pathogen-free sources. Furthermore, liquid nitrogen itself may act as a vector for transmitting pathogens (Bielanski & Vajta, Reference Bielanski and Vajta2009). Relying on antibiotics to control bacterial contamination is not a sustainable course of action and may result in many of the stocks of frozen germplasm being unusable in the future if legislation on antimicrobials changes. Use of cryopreserved tissues (as opposed to gametes or embryos) necessitates surgical transplantation of the tissue to the recipient, which is not a trivial procedure.

Cloning

The cloning of domestic livestock by somatic cell nuclear transfer (SCNT) has been possible for several years but the procedure remains technically demanding and expensive. In this procedure, a donor cell is injected into, and fused with, a mature, good quality, enucleated oocyte, followed by activation and culture of the product. However, there are high rates of pregnancy loss and also abnormal placental development and pathologies during the neonatal period, which is a major limiting factor in the adoption of this technique. As biobanking of somatic cells is increasing around the world, material from rare livestock breeds could be available for SCNT. While the technology is still in its infancy for conservation purposes with low success rates in wild species (Holt, Reference Holt2008), it is possible that it could provide a means of rescuing rare livestock breeds because of the availability of oocytes and recipient females for the cloned embryos among production animals. Several wild species of cattle and sheep have already been cloned (Ryder & Benirschke, Reference Ryder and Benirschke1997; Critser et al., Reference Critser, Riley, Prather, Holt, Pickard, Rodger and Wildt2003; Holt et al., Reference Holt, Pickard and Prather2004). However, it should be noted that many attempts are needed in order to obtain some success; only 1–5% of porcine SCNT embryos develop into piglets (Grupen, Reference Grupen2014). Production of offspring unfortunately does not mean that a healthy adult animal, capable of reproducing, will result (Holt, Reference Holt2008). Furthermore, the production of a few cloned individuals is unlikely to conserve much genetic diversity; an extensive biobank of material from existing individuals would be needed to ensure that as much as possible of the current gene pool is retained. Such biobanking for all livestock species is likely to require considerable commitment in time and resources.

Epigenetics

Epigenetic changes can be defined as stable alterations of DNA-associated molecules that may cause gene expression to be altered. This alteration is brought about by changes in methylation that affect gene expression (Jovanovic et al., Reference Jovanovic, Ronneberg, Tost and Kistensen2010; Munro et al., Reference Munro, Farquhar, Mitchell and Ponampalam2010). It is now known that factors such as nutrition (under- or over-nutrition) can affect methylation, as can various environmental toxins (Schagdarsurengin & Steger, Reference Schagdarsurengin and Steger2016). It is not known, however, if sperm handling procedures such as preservation or cryopreservation, may have an epigenetic effect. Certainly the more handling procedures to which germplasm is subjected, the more chances there are that an epigenetic effect will occur. Speculation exists of a link between various ART and imprinting disorders, occurring via altered DNA-methylation patterns and histone coding (Urrego et al., Reference Urrego, Rodriguez-Osorio and Niemann2014).

Epigenetic effects can influence reproductive efficiency through alterations in the viability of embryos, foetuses and the neonate, and also through control of endometrial gene expression which may alter implantation. They may also influence health, especially the occurrence of cancer through regulation of proto oncogenes and suppression of tumour suppressor genes, and phenotypic performance for a variety of functions or traits. Some epigenetic modifications can even be transmitted to subsequent generations (Schagdarsurengin & Steger, Reference Schagdarsurengin and Steger2016).

Recent advances in sperm handling for livestock species

The spermatozoa of some species, e.g. stallions, survive cryopreservation better if most of the seminal plasma is removed before the cryoprotectant is added (Loomis & Graham, Reference Loomis and Graham2008). Further improvements can be seen if living spermatozoa are separated from dead and dying cells prior to freezing (Hoogewijs et al., Reference Hoogewijs, Morrell, Van Soom, Govaere, Johannisson, Piepers, De Schauwer, de Kruif and De Vliegher2011). Various methods have been advocated for selecting viable spermatozoa and removing them from seminal plasma (Morrell & Rodriguez-Martínez, Reference Morrell and Rodriguez-Martinez2016) but, to date, only colloid centrifugation of stallion spermatozoa seems to be feasible for field use. These methods select spermatozoa based on certain functional attributes, such as motility or intact plasma or acrosomal membranes and are, therefore, believed to mimic the selection occurring in the female reproductive tract (Suarez, Reference Suarez2007). Some of these techniques will now be described briefly.

In migration techniques, e.g. ‘swim-up’, motile spermatozoa swim away from the bulk of the sample to an area containing few spermatozoa. Passage through glass wool or Sephadex gel physically impedes spermatozoa with reacted acrosomes, damaged membranes or abnormal morphology. Colloid centrifugation selects motile spermatozoa with intact membranes and also those with mature chromatin from those with immature or damaged chromatin. Magnetic activated cell sorting (MACS) has been used in combination with a marker, such as annexin V, to remove apoptotic spermatozoa from the sample. Annexin V has a high affinity for phosphatidylserine, which is thought to be externalized on the sperm plasma membrane in the early stages of apoptosis. When conjugated to magnetic microspheres, annexin V can bind to, and retain, apoptotic spermatozoa within an affinity column. This selection technique has been reported for processing of human spermatozoa for fertility treatments (reviewed by Gil et al., Reference Gil, Sar-Shalom, Sivira, Carreras and Checa2013) but has not been widely adopted for processing of animal spermatozoa, apart from occasional research reports, e.g. for rabbit (Vasicek et al., 2013), bull (Faezah et al., 2014) and Senegalese sole spermatozoa (Valcarce et al., Reference Valcarce, Herráez, Chereguini, Rodríguez and Robles2016). The method may not be amenable for processing large volumes of semen.

One selection technique that is showing promise in the preparation of spermatozoa for ART is colloid centrifugation. This technique has been available for preparing spermatozoa, mainly human spermatozoa for fertility treatments or bovine spermatozoa for IVF, for at least 20 years but the technique has only been adopted more generally for sperm preparation with the development of single layer centrifugation (SLC) in the last few years (Morrell & Wallgren Reference Morrell and Wallgren2011). The chronology of these techniques is shown in Table 2. The reasons for this late adoption were mainly that the method first described – density gradient centrifugation – was considered to be too impractical for use when preparing large volumes of semen or for ejaculates with a high sperm concentration. The development of a technique using only a single layer of colloid has revolutionised sperm preparation as it is much easier to use and allows the technique to be scaled up to process large volumes of semen (Morrell et al., Reference Morrell and Rodriguez-Martinez2009). This technique was named single layer centrifugation by its developer, one of the authors of the present paper (Morrell & Rodriguez-Martínez, 2011).

Table 2 Chronology of colloid centrifugation as a sperm preparation technique

Centrifugation of sperm samples through a colloid allows robust spermatozoa i.e. motile spermatozoa with normal morphology, intact plasma membranes and intact chromatin, to be selected from the rest of the ejaculate. The colloid works by presenting a physical barrier to the passage of immotile spermatozoa or those with abnormal morphology and damaged membranes; thus such spermatozoa are prevented from pelleting during centrifugation. Spermatozoa are also separated from seminal plasma and any non-sperm cells in the semen, such as epithelial cells from the lining of the reproductive tract or bacteria contaminating the ejaculate during semen collection. Removal of dead or dying spermatozoa and cellular debris aids survival of living spermatozoa and may also improve sperm cryosurvival in some species (Hoogewijs et al., Reference Hoogewijs, Morrell, Van Soom, Govaere, Johannisson, Piepers, De Schauwer, de Kruif and De Vliegher2011; Martínez-Alborcia et al., Reference Martinez-Alborcia, Morrell, Barranco, Maside, Gil, Parrilla, Vazquez, Martinez and Roca2013). Various ready-to-use species-specific colloid formulations have been produced; these formulations are adjusted according to the semen characteristics of different species and therefore it is important to use the correct formulation for the species in question.

Apart from improving sperm survival there are other reasons for using colloid centrifugation to prepare sperm samples: if the spermatozoa are to be used for IVF, they should first be separated from seminal plasma which contains decapacitating factors, and from cryoprotectants (Morrell & Rodriguez-Martínez, Reference Morrell and Rodriguez-Martinez2009). Traditionally this separation has been achieved with a technique known as ‘swim-up’, wherein motile spermatozoa swim away from the mixture of seminal plasma and cryoprotectant into fresh medium. However, this method does not select spermatozoa with normal morphology or with intact chromatin, and usually only 10–20% of the spermatozoa are recovered (Hallap et al., Reference Hallap, Håård, Jaakma, Larsson and Rodriguez-Martínez2004). Colloid centrifugation with Percoll was suggested as a means of selecting the best spermatozoa for IVF as well as separating them from the seminal plasma but lost favour when it was reported that some batches of Percoll were toxic to spermatozoa (Avery & Greve, Reference Avery and Greve1995). A further disadvantage of the original Percoll protocol was that it did not take into account species differences in semen characteristics (physical properties such as osmolarity and pH, as well as chemical composition) that affect the yield of spermatozoa recovered after the centrifugation (Morrell et al., Reference Morrell, van Wienen and Wallgren2011). The SLC technique with our colloid has also been used to separate sturgeon spermatozoa from cryomedium (Y. Horokhovatskyi & B. Dzyuba, unpublished data).

Colloid centrifugation selects sperm with intact chromatin, which can be important in species such as horses and some exotic animals. Spermatozoa with damaged chromatin are still capable of moving and of fertilizing oocytes, which means that they effectively compete with normal spermatozoa. At some stage after fertilization, however, development is halted and the pregnancy fails. In AI trials, SLC-selected stallion sperm samples produced more pregnancies than their corresponding control samples (Morrell et al., Reference Morrell, Richter, Martinsson, Stuhtmann, Hoogewijs, Roels and Dalin2014), which may be due, at least in part, to the selection of spermatozoa with intact chromatin. In addition, colloid centrifugation separates spermatozoa from pathogens in the ejaculate, which may enable a reduction in the use of antibiotics (Morrell & Wallgren, Reference Morrell and Wallgren2014). The inclusion of antibiotics in semen extenders represents a non-therapeutic use of antimicrobials, which is not consistent with current policies to reduce antibiotic usage. It has become apparent that using any antibiotics can lead to the development of resistance in bacterial species other than the target species, which is readily transferred to bacterial populations in other animal species, including potentially humans. As such, the application of procedures such as SLC will result in increased biosecurity, and importantly will facilitate the reduction in antibiotic use in trading and use of animal semen.