Susan Novak, PhD

Stallions are selected mainly for their genetic potential, not their reproductive potential.

Horse reproduction has seen much great advancement in recent years, including the successful cloning of the first horse. However, the traits contributing to overall fertility are poorly understood in the horse and also in most species. For example, it has been recently shown that over 200 different genes may affect male fertility.

Susan Novak

Dr. Susan Novak completed her B.Sc. (Animal Science) in 1995, and Ph.D. (Reproductive Physiology) in 2000, both at the University of Alberta. She finished a post-doctoral fellowship at Université Laval in Québec where she studied in vitro reproductive technologies.  For the past two years, she has joined Alberta Agriculture, Food and Rural Development as an Equine Research Scientist, aiming to improve reproductive technologies in horses. She also teaches Equine Physiology and Nutrition at the University of Alberta.


The use of artificial insemination (AI) in horses is becoming more popular, yet the semen quality needs to be improved before the technology is widely accepted and used. Conception rates from cooled transported semen are lower than for natural breeding, and conception rates from cryopreserved semen are very low. The improvement of the quality of semen, either cooled or frozen, will improve conception rates and reproductive performance.

What if we could select a stallion for breeding, whether for natural breeding or AI based on their potential fertility?

We currently assess the fertility of the stallion through microscopic evaluation to scrutinize sperm motility, morphology and concentration. These criteria do help evaluate the suitability of the use of semen for insemination, but they are not very reliable indicators of fertility. A stallion with poor motility may be a very fertile stallion when used for natural mating, give poor results with artificial insemination (AI), and improve when the semen is frozen for AI. This is because there are many factors involved in the production of semen and sperm that can influence reproductive performance. But what if we could find a marker of fertility?

Determination of a reliable in vitro semen characteristic that is associated with stallion fertility is very useful.

• Identification of protein markers of fertility in stallion semen (identification stallions suitable for cryopreservation or transported semen technologies).

• Establishment of good criteria for evaluation of semen quality in vitro and how this relates to in vivo fertility.

• The development of a screening test for stallion fertility that may also be applied to young stallions to determine breeding potential.

• Improvement of protocols for processing of fresh and cryopreserved semen for AI using fertility markers as indicators of success.

• The fertility markers, or proteins and other additives may be added to semen extenders to improve the shelf life of transported equine semen from 2 days to 4 or more days

What do we know about semen production in the stallion and how these components impact fertility?

Stallion semen is composed of two main components, the sperm and the seminal plasma. The production of sperm takes place in the epididymus, and the creation of mature spermatozoa takes 60 days. The seminal plasma is produced by the associated seminal vesicles (i.e. prostate), and functions to provide nutrients, buffers and other factors aid the sperm before and during fertilization. The sperm, once deposited into the uterus of the mare migrate to the opening of the oviduct (the site of fertilization) and stay there until ovulation occurs. Final sperm maturation occurs in the oviduct of the female, and an important component of fertilization is the ability of the sperm to capacitate, or become hyperactive, at the appropriate moment right before fertilization. The seminal plasma also aids in coating the sperm with proteins to protect it from capacitating too early.

The proteins present on the sperm surface will enable it to attach to the lining of the oviduct to await ovulation, allow for appropriate capacitation and also for fertilization of the egg. Similarly, the proteins in the seminal plasma are important for coating the sperm surface to allow the events around fertilization occur, and to provide a good, nourishing environment for the sperm.

The proteins on the sperm surface and in the seminal plasma are good potential fertility markers because of these important functions in fertilization, see summary on Table 1. We are thus screening the proteins in the sperm and seminal plasma to determine their relationship with fertility.

Table 1. Fertility markers in semen
Proteins in seminal plasma may be involved with: Proteins in the sperm or on sperm surface may be involved with:
Sperm acrosome reaction Plasma membrane integrity
Sperm capacitation Sperm capacitation (gain ability to fertilize)

Adhesion to sperm membrane Binding to oviduct
Effects on the uterus Binding to egg surface for fertilization
Longetivity of the sperm

Are there some fertility markers already known?
Current research efforts are focused on identifying reliable markers of fertility, specifically the proteins found in semen. In various species it has been shown that some proteins in seminal plasma are associated with fertility (cow: Killian et al. 1993, pig: Flowers et al., 2001). In horse seminal plasma, one protein is positively associated with fertility (72 kDa, pI 5.6, osteopontin) and three that are negatively associated with fertility (Brandon et al., 1999). More recently, IGF-I concentrations in seminal plasma have also shown a relationship to stallion fertility (Macpherson et al., 2002). The sperm membrane has also received attention and three proteins (SNARE proteins, caveolin-1 and NSF) have recently been identified in stallion sperm that are thought to be implicated in fertility (Gamboa and Santos, 2005)

How do we go about looking for these markers?
With the recent efforts of sequencing the human, mouse, bovine, swine genomes and now the horse genome, we are now able to examine the genes and proteins that are expressed in different tissues in the horse and associate them with performance characteristics. The use of new technologies, such as genomics and proteomics, allows us to scan the genome or proteome for differences in expression.

In our research, we used proteomics technology to map the proteins in sperm and in seminal plasma of stallions to evaluate differences between them. In a completed study, cryopreserved semen was obtained from Dr. Claire Card, and was used to determine if there were differences in protein expression between stallions that were good freezers versus those that were not. Figure 1 is a picture of a 2D acrylamide gel of stallion sperm, which is the basic tool used in proteomics to visualize the proteins present. Figure 2 shows a differential map of proteins expressed in stallions that were good freezers (cryopreservation) and those that froze poorly.

These results were very promising as we found many proteins that were present in good quality semen (good freezers) compared to those stallions that did not freeze well. These proteins will be subjected to mass spectrometry analysis to identify them in the future.

Another study was conducted this summer using fresh semen obtained from Sandy Ridge Stallion Station (Bassano, Alberta). Sperm and seminal plasma were analysed on 2D protein gels like those shown in Figures 2 and 3, but this time the proteins present will be related to the fertility or reproductive performance of the stallions. At time of writing, we are in the process of collecting the information from the breeding records from these stallions. The preliminary analysis has already targeted 60 proteins that are different across stallions. The results of these studies will be presented at the conference.

Conclusion
The identification of semen markers associated with fertility will be useful in determining which stallions are potentially fertile, even at a young age. In addition, these fertility markers will become reliable criteria for use to improve semen quality and distinguish between stallions that can be used for AI, whether for fresh or frozen semen. The identification of proteins that are related to fertility will result in improvement in sperm processing methods, and / or extender characteristics, which will increase semen quality and longevity after storage.

References
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA. 1999. Two–dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility. Theriogenology 52:863-873.
Flowers WL. 2001 Relationship between seminal plasma proteins and boar fertility. North Caroline State University. College of Animal Science Annual Swine Report.

Gamboa S, Ramalho-Santos J. 2005. SNARE proteins and caveolin-1 in stallion spermatozoa: possible implications for fertility. Theriogenology 64:275-291.

Killian GJ, Chapman DA, Rogowski LA. Fertility-Associate Proteins in Holstein Bull Seminal Plasma. Biology of Reproduction 49:1202-1207.

Macpherson ML, Simmen RC, Simmen FA, Hernandez J, Sheerin BR, Varner DD Loomis P, Cadario ME, Miller CD, Brinsko SP, Rigby S, Blanchard TL. 2002 Insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -5 in equine seminal plasma: association with sperm characteristics and fertility. Biol Reprod 67:648-654.

Acknowledgements
This research study was generously funded with help from Alberta Agriculture, Horse Racing Alberta, Horse Industry Association of Alberta and University of Alberta. In-kind contributions of semen were kindly made by Dr. Claire Card (University of Saskatchewan) and Sandy Ridge Stallion Station, Bassano, Alberta.

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