Subfertility in Stallions Associated with Spermatozoal Acrosome Dysfunction
Dickson D Varner, Steven P Brinsko, Terry L Blanchard,
Charles C Love, Rebecca Heck, Margo L Macpherson, and Larry Johnson
Departments of Large Animal Medicine & Surgery and Veterinary Anatomy and Public Health
College of Veterinary Medicine Texas A&M University, College Station, Texas USA and 
Department of Large Animal Clinical Sciences, College of Veterinary Medicine
University of Florida, Gainesville, Florida USA

Introduction

Some stallions pass a routine breeding soundness examination, yet they are unable to impregnate mares, or do so very inefficiently.  Among human patients with acceptable levels of spermatozoal numbers, motility and morphology, 15-20% produce ejaculates in which the spermatozoa fail to acrosome react within the normal range.^1,3  The acrosome plays a pivotal role in establishing the fertilizing potential of spermatozoa.⁵ This  membrane-bound organelle covers the anterior portion of the spermatozoal nucleus.  A portion of the acrosomal membrane is adjacent to the overlying plasma membrane.   Modification, fusion and vesiculation of these membranes is term the acrosome reaction.  This reaction releases enzymes from the acrosomal matrix which facilitate spermatozoal penetration of the investments of the oocyte.  Because a properly functioning acrosome is considered essential to fertilization, this study was conducted to test the hypothesis that acrosome dysfunction occurs in a subset of subfertile stallions, similar to that described in men.

Materials and Methods

Five stallions (4 Thoroughbreds and 1 Quarter Horse) with a history of subfertility or infertility (pregnancy rate less than 20% per cycle) were included in the project. Semen quality and testicular size for each of these stallions were considered satisfactory, based on the findings of a routine a breeding soundness examination.  Five stallions with satisfactory semen quality and known good fertility were used as control animals.

Gel-free semen was mixed with warmed (37^oC) milk-glucose-sucrose extender at a ratio of 1 part semen to 2 parts extender.  Extended semen was incubated at 37^oC for two hours.  Aliquots of extended semen from each stallion were exposed to a potent stimulant of the acrosome reaction, a divalent ionophore termed A23187 (final concentration of 10µM).  Aliquots of extended semen which contained no A23187 were used as negative control samples. Following the two-hour incubation period, samples were fixed in 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer.  The fixed semen was then centrifuged at 800 x g for 10 minutes, and the sperm pellets were processed for transmission electron microscopy.   Sagittal views of spermatozoal heads were evaluated for presence or absence of the acrosome reaction, based on vesiculation of the outer acrosomal and plasma membranes.   Data were analyzed by a two-way analysis-of-variance procedure to evaluate the effects of stallion status (fertile or subfertile) and A23187 on the incidence of

 acrosome-reacted spermatozoa.

Results

The effects of stallion status and the calcium ionophore, A23187, on the incidence of spermatozoal acrosome reactions is presented in Table 1.  In samples containing no A23187, the mean percentage of acrosome-reacted spermatozoa tended to differ  (p = 0.1) between fertile stallions (10.4 ± 11.6) and subfertile stallions (0.8 ± 1.8).  Upon exposure of extended semen to A23187, the mean percentage of acrosome-reacted spermatozoa was markedly different (p=0.001) between fertile stallions (84.0 ± 4.2) and subfertile stallions (5.6^d ± 7.7).

Table 1.  The effect of the calcium ionophore, A23187, on induction of spermatozoal acrosome reactions in subsets of fertile and subfertile stallions following incubation for 2 hours at 37^oC in a milk-based extender.

Discussion

The acrosome reaction in stallion spermatozoa has been characterized,⁴ but the relationship of acrosomal dysfunction to fertility in stallions is poorly understood.  Meyers and coworkers reported that physiological acrosome reactions in stallions is mediated by a plasma membrane progesterone receptor and that response to progesterone differs between fertile and subfertile stallions.² The present study indicates that some stallions with unexplained subfertility, based on a routine breeding soundness examination, may have a defect in acrosomal structure and function.  The prevalence of this condition in breeding stallions is unknown, but may parallel similar findings described for the human population.

This study revealed that transmission electron microscopy may reveal a difference in acrosomal responsiveness of spermatozoa from the fertile versus subfertile stallions.  However, the difference between the two groups was magnified considerably when the spermatozoa were exposed to the calcium ionophore, A23187.

In summary, use of more sophisticated techniques may be required to identify stallions with acrosomal dysfunction than those employed in routine breeding soundness examinations.  The underlying cause of this dysfunction may be associated with alterations in structural membrane composition or cellular messenger systems. Studies are currently being directed at determining the underlying cause of this defect, as well as development of therapeutic strategies.

References

1.        Calvo L, Dennison-Lagos L, Banks SM, Sherins RJ 1994a. Characterization and frequency of acrosome reaction among normal and infertile men. Human Reproduction 9: 1875-1879.

2.        Meyers SA, Overstreet JW, Liu IK, Drobnis EZ 1995. Capacitation in vitro of stallion spermatozoa: comparison of progesterone-induced acrosome reactions in fertile and subfertile males. Journal of Andrology 16: 47-54.

3.        Moghissi KS, EE Wallach 1983.  Unexplained infertility. Fertility and Sterility 39: 5-21.

4.        Varner DD, Ward C, Storey BT, Kenney RM 1987. Induction and characterization of acrosome reaction in equine spermatozoa. American Journal of Veterinary Research 48: 1383-1389.

5.        Yanigimachi R  1994. Mammalian fertilitzation. In Knobil E, Neill JD (eds), The Physiology of Reproduction, 2nd edition. New York: Raven Press, 189-317.

Acknowledgments