Effect of feeding fescue seed containing ergot alkaloid toxins on stallion spermatogenesis and sperm cells

 Effect of feeding fescue seed containing ergot alkaloid toxins on stallion spermatogenesis and sperm cells

Richard Fayrer-Hosken1, Donna Kelley2 and Bob Kelley2,

SoRhoVet LLC, Winterville GA 30683 and Friesian Night Farms, 433 Thousand Oaks Drive, Danielsville

(With sincere thanks to Sylvia Bedford-Guaus).

Summary

Fescue is a highly successful pasture grass and is pest and cold resistant. In the southeastern United States tall fescue is one of the most important pasture grasses for horses and is an extremely important grass as it survives through the winter. Most of the wild type grasses have a commensal fungal endophyte, Neotyphodium coenophialum, and the fungal presence protects the growing fescue. The fungal endophyte secretes ergot alkaloids and the ergot alkaloids confer protection of the fescue grass from insect infestation (Fayrer-Hosken et al. 2008). The physiological effects of tall fescue grass and its associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were the objective of our studies. Previously studies have investigated the stallion spermiogram. In the subsequent research we examined the effect of ergot alkaloids on chilling or freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 hours at 5C, conferred more stability in sperm cells in extender. The ergot alkaloids had no effect on the ability to freeze sperm cells. Most concerning from the research is that it showed the sperm production of yearling colts fed ergot alkaloids was impaired. The cellular effects showed problems with the X and Y chromosome formation. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts.
Introduction

Tall fescue in the southeastern United States is one of the most important pasture grasses, and it is an extremely successful grass that is protected by a commensal fungal endophyte Neotyphodium coenophialum. The endophyte secretes ergot alkaloids and these bestow a variable degree of protection to the grass from insect infestation (Fayrer-Hosken et al. 2008, Fayrer-Hosken et al. 2011, Fayrer-Hosken 2011). Generally, fescue (toxic or non-toxic) is grazed by multiple species without any problems. Ruminants and horses that graze toxic fescue grass do have clinical problems. Practically, the diseases caused by fescue associated ergot alkaloids have significant financial impact on the cattle and horse industries in the states with affected grasses (Hoveland 1993).

Studies in the mare have demonstrated (Blodgett 2001, Copetti et al. 2002, Cross et al. 1995, Evans 2002, Fiserova et al. 1997, Lehner et al. 2008, Marek and Roth 1980, Porter and Thompson 1992, Ryan et al. 2001, Youngblood et al. 2004) that consuming toxic endophyte-infected grasses during the last 3 months of gestation leads to a high incidence of foal loss and other associated medical problems. In the mare, the effects of the ergot alkaloids include decreased conception or increased early embryonic mortality in days 15 to 30 of gestation, abortions, prolonged gestation (Putnam et al. 1991), decreased relaxin (Ryan et al. 2001), dystochia, thickened (Loch et al. 1987) (edema) or retained placental membranes, agalactia (Copetti et al. 2002), and increased rates of newborn mortality. Treatment of the multiple clinical manifestations involves removal from the toxin source 60-90 days prior to foaling. Symptoms of agalactia in the mare can be alleviated by domperidone administration (Redmond et al. 1994), which is a dopamine receptor antagonist.
The profound effects of ergot alkaloids in mares prompted the research to investigate the impact of ergot alkaloids on stallion reproduction and spermiogram (Fayrer-Hosken et al. 2011, Fayrer-Hosken 2011). This was a two stage research project with the initial stage examining the general physiological effects of fescue in stallions (Fayrer-Hosken et al. 2011, Fayrer-Hosken 2011). The initial study (Fayrer-Hosken et al. 2011, Fayrer-Hosken 2011) examined the general physiological parameters and showed there were no statistical changes in sperm motility, sperm number (concentration), sperm cell morphology, number of breeding doses produced, testicular size and volume, degree of bactospermia, diurnal testosterone levels, and inducible steroidogenic capability of the Leydig cells. Also there were no changes in core body temperature and superficial scrotal temperature. The only statistically altered parameter in the stallions consuming toxic endophyte-infected tall fescue seed was that they had significantly lower gel-free volumes than stallions consuming non-toxic endophyte tall fescue seed without affecting the numbers of ejaculated sperm cells.
Spermatogenesis in the stallion (Freidman et al. 1991, Thompson et al. 1979, Einarsson et al. 2009, Roser 2008, Roser 2001, Johnson et al. 1997, Rodriguez-Martinez 1992, Hurtgen 1992, Johnson et al. 1991, Amann et al. 1979, Swierstra et al. 1975, Gebauer et al. 1974) requires 62 days for the testicular phase and then 13 days for the epididymal phase. This provides a spermatogenic cycle for sperm production of 75 days. The stallion sperm cell is sensitive to cooling and freezing, similar to other mammalian sperm cells (Pommer et al. 2003, Thomas et al. 2006) in that they exhibit cryocapacitation during chilling or freezing.
Cryocapacitation (Einarsson et al. 2009, Cormier and Bailey 2003, Amann et al. 1979, Abad et al. 2007b, Abad et al. 2007a) induces a series of complex biochemical changes in the plasma and outer acrosomal membranes of the cryopreserved sperm cell. The range of cryocapacitational states predisposes the sperm cells to a premature inducible acrosome reaction. We reasoned that ingested ergot alklaoids could induce biochemical or structural defects in sperm cells during spermatogenesis and/or epididymal maturation. If the sperm cells were affected, then one postulate is that cryocapacitated sperm cells would be more likely to undergo an induced acrosome reaction. Clinically, chilled and frozen sperm cells would be more fragile and less fertile. Toxic ergot alkaloids can induce motility changes in bull sperm cells through alpha-adrenergic receptors (Wang et al. 2009). Ergot alkaloids have also been shown to induce abnormal proliferation in bovine vascular smooth muscle cells resulting in diminished blood flow to organs. Altered blood flow could potentially disrupt testis thermoregulatory mechanisms (Strickland et al. 1996) and this might affect critical stages of spermatogenesis such as the meiotic prophase I. The hypothesis for this research was that if stallions ingest toxic levels of ergot alkaloids the sperm cells might be more fragile. In stallions fed ergot alkaloids their sperm cells would be more sensitive to an induced acrosomal reaction induction than control stallions. In colts we theorized that there probably would be effects on the peri-pubertal spermatogenesis, and these might be chromosomal defects.

Lipids are critical to capacitation and the cholesterol:phospholipid ratio changes over time in the female reproductive tract. The molar amounts of cholesterol and phospholipids (Brinsko et al. 2007) do not differ between sperm cells of fertile and subfertile stallions. But the cholesterol and phospholipids ratio was higher in sperm of subfertile stallions compared to the fertile stallions (Brinsko et al. 2007), emphasizing the importance of the cholesterol:phospholipid ratio in sperm functionality. For stallions with the subfertile sperm, a possible explanation (Brinsko et al. 2007) is that the altered cholesterol:phospholipid ratio resulted in a decreased rate of cholesterol depletion from the sperm cells. The decreased rate of cholesterol efflux could reduce time of capacitation. Ergot alkaloids might affect sperm cell lipids and thus perturb capacitation. Such membrane problems might manifest as changes in fertility.

Discussion

The impetus for the study was to examine the effect of fescue alkaloids in stallions and possible financial implications. Fescue is an extremely ubiquitous and successful pasture grass in the southeastern USA. From our initial examination (Fayrer-Hosken et al. 2011, Fayrer-Hosken 2011) of six stallions, a double blind crossover study, stallions consuming endophyte-infected fescue seed had higher (P < 0.01) urinary alkaloid (lysergic acid derivatives) levels than stallions consuming the non-toxic endophyte tall fescue MaxQ seed. The mean time to maximal systemic toxicity was 8.33 hours after starting toxic seed ingestion and at that time a mean toxicity level of 49.98 ng alkaloid/mg creatinine was reached. After cessation of feeding toxic seed the systemic levels fell to control (baseline) levels within 48 hours. For the reproductive parameters measured in the stallions, there were no statistical changes in sperm motility, sperm number (concentration), sperm cell morphology, number of breeding doses produced, testicular size and volume, degree of bactospermia, diurnal testosterone levels, and inducible steroidogenic capability of the Leydig cells. Also there were no changes in core body temperature and superficial scrotal temperature. The only statistically affected parameter in stallions consuming toxic endophyte-infected tall fescue seed was that they had significantly lower gel-free volumes than stallions consuming non-toxic endophyte-infected tall fescue seed. The lack of definable effects supported the findings of a preliminary reproductive study that ergot alkaloids had minimal effects of stallion reproduction. There are no comparative fertility data or conception rates reports to demonstrate that ergot alkaloids have a positive or negative impact on shipped semen. It is possible to argue that the decreased propensity of toxic ergot alkaloid exposed sperm to undergo a prematurely induced acrosome reaction might actually enhance fertility of chilled stallion semen.
Failure to partially synapse the XY chromosome bivalent was detected in about 32% of equine pachytene spermatocytes following ergot alkaloid consumption suggesting a potential effect on meiotic progression in a sub-population of meiocytes in this model. The presence of mature sperm in cytological preparations together with physiological fertility parameters investigated indicates that, although ergot alkaloid exposure might impair spermatogenesis in a proportion of cells, stallion reproduction is minimally affected. The mechanisms involved in this process require further investigation.

Horse owners should keep their current fescue pastures, as there is little or no impact on the lactating mare and foal or grazing stallions or fillies. Current established fescue pastures provide quality nutrition for these horses and the cost of change the grass type in the pastures would not be sound financial wisdom. Further studies are required to determine the functional implications of the chromosomal effects observed in a subpopulation of meiocytes of colts, but the ergot alkaloids have little to no effect on stallions. The lack of definable effects supported the findings of a preliminary reproductive study that ergot alkaloids have minimal effects of stallion reproduction.
 

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