Advancements in microfluidic and electrophoretic techniques for stallion sperm isolation
Ashlee Medica
A * , R. John Aitken A , Aleona Swegen A and Zamira Gibb A
A
Abstract
Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances.
This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproductive success in equine breeding.
Traditional sperm isolation methods, including sperm washing and single-layer centrifugation (SLC), are compared with emerging techniques such as microfluidic-based technologies and electrophoretic separation to assess their ability to improve sperm quality while minimising DNA damage.
While conventional methods are widely used, they present limitations, such as reduced motility, cost, and potential DNA damage. Novel approaches, including the VetMotl™ and Samson™, replicate natural sperm selection to enhance motility while preserving DNA integrity, and the electrophoretic sperm isolation device, Felix™, separates sperm based on surface charge and motility, benefiting cryopreserved samples. These innovations offer promising improvements in ART outcomes, though challenges remain, including high costs and limited sperm yields.
Emerging sperm isolation techniques have the potential to improve ART success, but further research is required to optimise these methods and validate their efficacy in fertility trials.
Advancements in sperm isolation could modernise equine reproductive practices by improving sperm quality and fertility outcomes, though accessibility and practical applications require continued investigation.
Keywords: DNA damage, electrophoresis, ICSI, IVF, microfluidics, single layer centrifugation, stallion fertility, stallion spermatozoa.
Introduction: the challenges of natural and artificial fertilisation in equines
After sperm deposition in the mare’s uterus, only an estimated 0.0007% of spermatozoa successfully navigate to the site of fertilisation (Rigby et al. 2000). This high level of attrition underscores the biological mechanisms aimed at selecting the most viable spermatozoa, ensuring optimal chances for successful fertilisation. However, this natural process may preclude fertility in sub-fertile stallions or following artificial insemination (AI) using spermatozoa that have been damaged during the storage process as it may result in an insufficient number of spermatozoa reaching the site of fertilisation. Additionally, this selection process does not effectively exclude spermatozoa with compromised DNA integrity, allowing damaged spermatozoa to persist in the reproductive tract and potentially contribute to failed or abnormal fertilisation.
Equine assisted reproductive technologies (ARTs), such as AI, intracytoplasmic sperm injection (ICSI), and more recently, in vitro fertilisation (IVF), have been developed to overcome these challenges and have become valuable tools in equine breeding. While ARTs are commonly associated with addressing infertility, they are also increasingly used for logistical reasons in equids. For example, AI can enable breeding between horses or donkeys on opposite sides of the world, thus enhancing access to superior genetic material, increasing the gene pool, and facilitating more strategic breeding choices. Central to the success of these technologies is the ability to effectively purify and prepare spermatozoa for fertilisation. In vitro semen purification techniques are designed to replicate and enhance the natural sperm selection process, focusing on improving sub-fertile ejaculates and maximising fertility (Vaughan and Sakkas 2019). These techniques aim to either remove seminal plasma, which has detrimental effects on sperm survival during storage (Love et al. 2005), or eliminate non-viable and poorly formed spermatozoa, as well as leukocytes, which generate excessive reactive oxygen species (ROS) that damage spermatozoa (Aitken et al. 1995; Whittington and Ford 1999).
A stallion’s reproductive success is often measured by its per-cycle pregnancy rate, calculated as the number of oestrous cycles resulting in pregnancy divided by the total number of oestrous cycles that the mare was bred, either via natural ‘cover’, or via AI (Nath et al. 2010). Failure to adequately remove poor-quality spermatozoa (Love 2011) and other cell types (Maischberger et al. 2008) from an ejaculate can adversely impact this statistic. Over time, various methods have been developed to obtain functional spermatozoa for ARTs, each with varying degrees of success and industry uptake. This review aims to introduce the fundamental techniques of sperm isolation, setting the stage for an exploration of emerging innovations in equine reproductive technologies.
The old workhorses: traditional methods of equine sperm preparation and isolation
Simple dilution method
The simplest and most widely used method for preparing stallion spermatozoa for storage prior to transport and AI is the dilution of semen with an extender immediately after collection. The most common extenders include Kenney’s extender, INRA 96 (IMV Technologies, France), EquiPlus (Minitube, Germany), and the Botu family of stallion semen extenders (Botupharma, Brazil), which generally contain antibiotics to suppress bacterial growth (Kenney et al. 1975; Olivieri et al. 2011). Key components like casein from skim milk bind seminal plasma proteins (Manjunath 2012) and assist with zona pellucida binding (Coutinho da Silva et al. 2012). While energy substrates such as glucose or pyruvate sustain sperm motility (Hernandez-Aviles et al. 2021). However, simply diluting semen does not eliminate the deleterious effect of seminal plasma or poor-quality spermatozoa. Consequently, it is only recommended for highly fertile stallions in which sperm quality is already optimal, and therefore additional selection is unnecessary (Rigby et al. 2001). Therefore, the lack of a selection mechanism renders simple semen dilution an unsuitable method for processing sub-fertile ejaculates or samples requiring optimal sperm longevity during storage.
Sperm washing
A step beyond simple dilution involves sperm washing, a process where diluted semen undergoes centrifugation to remove seminal plasma. The pelleted sperm is then resuspended in an extender (Ijaz and Ducharme 1995). While effective at enhancing membrane stability during chilled storage through the removal of seminal plasma (Barrier-Battut et al. 2013), centrifugal sperm washing does not facilitate the selection of high-quality spermatozoa. Furthermore, studies indicate that discarding the supernatant during centrifugation may inadvertently eliminate the most motile spermatozoa (Morrell et al. 2010). Additionally, concerns regarding potential chromatin damage from centrifugation have led to breeders in some countries, such as the UK, France, and Italy, to avoid centrifugation methods completely (Batellier et al. 1998; Rota et al. 2004; Allen 2005). Protocols addressing these risks have incorporated conservative centrifugation forces (400–600g) to minimise damage, but these lower g-forces may fail to pellet the entire sperm population, thereby compromising sperm yield (Macpherson et al. 2002; Ferrer et al. 2012). Despite these limitations, sperm washing remains a practical option in settings where motility and morphology are less critical and seminal plasma removal is the primary goal to improve sperm longevity during storage.
Single-layer centrifugation (SLC)
Introduced in the horse by Morrell et al. (2008), single-layer centrifugation (SLC) represents a significant improvement over traditional sperm washing. By centrifuging semen through a layer of high-density colloidal solution such as EquiPure™ or Androcoll-E™, this method selectively isolates a subpopulation of high-quality spermatozoa based on density (Morrell et al. 2009). Spermatozoa capable of traversing the colloid layer and pelleting at the base of the tube exhibit superior morphology, motility, membrane integrity, and chromatin stability, correlating with enhanced fertility outcomes (Brahem et al. 2011; Jayaraman et al. 2012). Furthermore, SLC removes surface-bound seminal plasma proteins (Kruse et al. 2011), and the majority of microbial contaminants (Morrell et al. 2014; Guimarães et al. 2015; Varela et al. 2018) prolonging sperm viability during chilled storage (Lindahl et al. 2012).
Despite its advantages, SLC is not without drawbacks. Concerns persist regarding the effects of centrifugation forces on sperm integrity, with conflicting evidence on whether motility and chromatin stability are compromised (Cochran et al. 1984; Jasko et al. 1991, 1992). In addition, depending on the quality of the original sample, SLC centrifugation may be unable to isolate sufficient sperm numbers for conventional AI due to a very low proportion of high-quality, ‘dense’ cells in the ejaculate. This leads to the phenomenon of ‘rafting’, whereby the low-density, poor-quality spermatozoa form an impenetrable raft on the top of the colloid, blocking any high-quality sperm from making their way through to the pellet (Morrell et al. 2011). Furthermore, concerns have been raised about the presence of transition metals in some commercial colloid formulations, such as PureSperm (Nidacon, Sweden), which has been associated with exacerbated levels of oxidative DNA damage in human spermatozoa (Aitken et al. 2014). Moreover, the high costs associated with the purchase of colloid media and centrifuges have limited widespread adoption in the equine breeding industry. As a result, the search for cost-effective and scalable alternatives remains a priority.
Migration methods
The swim-up (or swim-down) method, widely used in human ARTs, relies on sperm motility to migrate into a separate medium, yielding a highly motile and morphologically normal sample (Henkel and Schill 2003; Oseguera-López et al. 2019). Although cost-effective and straightforward, these methods are unsuitable for equine AI due to their low yield, recovering only 5–10% of the original sperm population (Casey et al. 1993; Correa and Zavos 1996). However, these methods remain viable options for applications such as ICSI or IVF, where fewer spermatozoa are required. That said, the technique is subject to a high degree of user error, and requires an experienced technician and careful handling to ensure optimal results. Additionally, it poses a risk of seminal plasma contamination, which could compromise sperm quality during in vitro storage if not adequately addressed (Akçay et al. 2006).
Microfluidics: riding the wave of innovation
Microfluidic-based technologies have emerged as a cutting-edge solution for sperm selection and preparation, offering a more natural approach compared to the traditional aforementioned methods. These technologies aim to replicate the micro-confined geometry of the female reproductive tract, providing a bio-inspired environment that enables sperm to navigate through miniature channels, similar to their journey through the uterus and into the oviduct. This process mirrors the natural mechanisms by which sperm must traverse the mucosal microchannels of the female reproductive tract to reach the egg, a journey that is critical for fertilisation (Tasoglu et al. 2013). Advances in microfluidics have enabled the integration of biochemical gradients similar to those present in the oviductal environment. Early attempts to replicate these gradients in vitro utilised follicular fluid (Eisenbach 1999), as it has been shown to attract sperm via chemotaxis and is strongly linked to oocyte fertilising potential (Ralt et al. 1991). However, its role in vivo is likely limited, as only a small portion of follicular fluid actually enters the oviduct along with the oocyte–cumulus complex (Hansen et al. 1991; Brüssow et al. 1999; Hunter et al. 1999). Moreover, a sustained chemoattractant gradient would be required throughout the oocyte’s fertilisable period (~24 h post-ovulation in humans), whereas follicular fluid is released only momentarily at ovulation. This suggests that sperm chemoattractants are more likely secreted by the cumulus–oocyte complex after ovulation while it resides in the oviduct. Due to the challenges associated with using follicular fluid, research shifted toward employing cultured cumulus cells, which naturally release signalling molecules such as progesterone during ovulation (Oren-Benaroya et al. 2008). These cells provide a more reliable and physiologically relevant model for sperm guidance and selection in microfluidic systems (Xie et al. 2010). Another promising potential advancement is the use of the mammalian sperm chemoattractant allurin. This protein, deposited on the ciliated surfaces of luminal epithelial cells, interacts directly with sperm, forming an allurin-rich jelly coat as spermatozoa travel through the oviduct. This natural chemoattraction process is crucial for successful sperm migration and fertilisation in vivo (Olson et al. 2001; Xiang et al. 2004), aligning it as a strong candidate for microfluidic sperm isolation.
Two standout microfluidic devices in the field of sperm isolation are the VetMotl™ sperm separation device (VetMotl inc, USA) and the Samson™ (Memphasys Ltd., Sydney, Australia). VetMotl™ sperm separation device utilises motility-based sorting within microfluidic channels, isolating spermatozoa based on their natural motility patterns over a period of 30 min, closely mimicking the selection processes that occur within the female reproductive tract. Early applications in low-dose equine AI have shown promise in frozen-thawed spermatozoa (Morris et al. 2024). Similarly, the Samson™ by Memphasys combines microfluidic technology with an innovative filtration process. This device isolates motile spermatozoa through a unique membrane-based filtration system, providing a more efficient and less invasive alternative to traditional sperm preparation methods. The Samson™ has demonstrated promising results in fresh equine spermatozoa, underscoring its potential to isolate a subpopulation of highly motile spermatozoa with significantly lower levels of DNA damage compared to SLC (Medica et al. 2024). However, fertility trials have yet to be conducted to evaluate the device’s impact on pregnancy rates. These trials are essential to confirm whether the improved sperm quality translates into enhanced fertilisation outcomes in vivo and in vitro.
Microfluidic sperm selection technologies offer several advantages, including enhanced precision and a more physiologically relevant approach to sperm isolation. However, challenges remain, particularly with throughput, as microfluidic devices can only isolate a relatively small population of spermatozoa per procedure. Additionally, the costs associated with these technologies are currently higher than traditional sperm isolation methods, which may limit their accessibility and uptake for horse and donkey breeders.
Electrophoretic isolation: sperm on the fast track
There is currently only one commercially available electrophoretic sperm isolation device, the Felix™ (Memphasys Ltd., Sydney, Australia). Unlike microfluidic devices, which focus on sperm behaviour within confined channels, the Felix™, relies on electrical currents to separate spermatozoa based on their membrane surface charge, as well as motility, over a period of only 6 min. While this technology has demonstrated significant success with human spermatozoa (Jayaram and Govindarajan 2023; Kitahara et al. 2024), no published studies exist on its use in other species, including horses. However, preliminary investigations into its application in equine reproduction are reportedly underway (Memphasys Ltd., personal communication).
The Felix™ device isolates spermatozoa using an electrophoretic separation technique, where electrical currents pull high-quality spermatozoa – those with a greater negative surface charge – toward a harvesting chamber (Simon et al. 2016; Orsolini et al. 2022). This negative charge is primarily attributed to the presence of sialic acid residues on the sperm membrane, which are acquired during epididymal maturation (Kallajoki et al. 1986). Sialic acid plays a crucial role in sperm survival by preventing premature immune recognition and clearance within the female reproductive tract, as well as facilitating interactions with the oocyte during fertilisation (Tecle et al. 2019). Higher-quality spermatozoa tend to have more sialic acid residues because they have undergone proper epididymal maturation, which optimises membrane stability, motility, and overall functionality. Studies have shown that the Felix™ can isolate spermatozoa with significantly lower levels of DNA damage and reduced 4-hydroxynonenal (4-HNE) adduction, a marker of oxidative stress, compared to traditional sperm separation methods such as colloid centrifugation in both fresh and frozen-thawed samples (Hungerford et al. 2023; Shapouri et al. 2023; Villeneuve et al. 2023).
The potential benefits of the Felix™ device in equine ARTs such as ICSI and IVF are particularly noteworthy. If the Felix™ device can demonstrate efficacy in samples with compromised semen parameters – such as low motility, poor morphology, or elevated DNA fragmentation – while maintaining or enhancing sperm quality during prolonged liquid storage, it has the potential to revolutionise equine ART outcomes, particularly when using cryopreserved spermatozoa. However, one limitation that must be addressed is sperm yield, as the relatively low recovery rates associated with electrophoretic separation may impact its suitability for certain equine AI applications, where larger sperm numbers are traditionally required.
Conclusions: the future of stallion sperm isolation
The landscape of equine reproductive technologies has evolved significantly in recent years, with various techniques and innovations aimed at improving sperm selection and preparation. The relatively low conception rates in natural equine breeding, influenced by factors such as the prolonged transport of spermatozoa through the female reproductive tract, limited fertile lifespan of both gametes, and the requirement for multiple matings per cycle, combined with challenges posed by sub-fertile stallions or specific breeding goals, has highlighted the importance of ARTs such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF). Central to the success of these technologies is the effective isolation of high-quality spermatozoa, which directly impacts fertilisation outcomes and overall reproductive success. Traditional methods, like centrifugal sperm washing and SLC, have long been employed in equine AI regimens, but they come with limitations, such as potential DNA damage and high costs. These shortcomings have driven the search for more cost efficient, and physiologically relevant sperm selection technologies.
Microfluidic devices have emerged as a promising solution, offering a more natural, bio-inspired approach to sperm isolation. By mimicking the journey of spermatozoa through the female reproductive tract, microfluidic devices such as the VetMotl™ sperm separation device and the Samson™ provide enhanced sperm sorting based on motility and morphology. Additionally, the Samson™ has demonstrated significant potential in isolating highly motile spermatozoa with reduced DNA damage compared to traditional methods, though further fertility trials are necessary to evaluate its true impact on pregnancy rates.
Alternatively, electrophoretic sperm isolation such as that offered by the Felix™ device, represents a novel, fast, and highly promising approach to sperm separation that may be of use to horse and donkey breeders in the future. Unlike microfluidic devices, the Felix™ utilises electrophoresis to separate sperm based on their charge and motility. While currently marketed primarily for human applications, this technology has shown significant success in isolating spermatozoa with reduced oxidative damage and DNA fragmentation, which is critical in ARTs. The ability of the Felix™ to work with both fresh and cryopreserved spermatozoa offers a potential solution to the challenges of sperm preservation in equines, especially in the context of cryopreservation, where DNA integrity may be compromised following thawing (Linfor and Meyers 2002).
Despite the impressive advancements in sperm isolation technologies, challenges remain. The current high costs associated with both microfluidic and electrophoretic devices, along with the low sperm yield, present barriers to widespread adoption, particularly in equine AI, where large volumes of semen are traditionally processed, and relatively high sperm numbers are required for conventional trans-cervical AI (typically 200–500 million progressively motile spermatozoa per dose). However, the emergence of low-dose AI, which requires significantly fewer spermatozoa (as low as 3 million when inseminated at the tip of the uterine horn or uterotubal junction), may help mitigate these limitations. While originally performed hysteroscopically, many veterinarians now use deep-horn catheters guided by palpation, making the procedure more accessible. That said, low-dose AI remains a relatively new technique (Morris et al. 2000; Morris and Allen 2002) and its widespread adoption is limited by the availability of trained practitioners and specialised equipment. As proficiency in this method increases, the sperm yield constraints of newer isolation techniques may become less of a barrier. Furthermore, while these technologies have shown great promise in improving sperm quality and longevity during in vitro storage, further research is needed to evaluate their practical applications in equine ARTs and their ability to meet the industry’s demands.
In conclusion, while significant strides have been made in the development of advanced sperm isolation technologies, including microfluidic and electrophoretic devices, further research, validation, and cost-reduction efforts are needed to make these technologies more accessible and applicable in the equine industry. The continued exploration of these innovative approaches holds the potential to revolutionise equine breeding, offering more effective and efficient solutions for sperm selection and preparation, ultimately improving reproductive success and ARTs in the industry.
Data availability
This study is a literature review and does not involve the generation or analysis of new data. All data used in this review were obtained from publicly available sources, which are cited appropriately within the manuscript.
Conflicts of interest
Memphasys Reproductive Biotechnology is currently a formal collaborator of the University of Newcastle as an industry partner on an ARC Linkage Project. R. John Aitken is Scientific Director at Memphasys Reproductive Biotechnology.
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