The effects of ergot alkaloids on the breeding stallion reproductive system.

REASONS FOR PERFORMING STUDY: Ergot alkaloids cause a range of pathological conditions in mares. There is no evaluation of the effects of ergot alkaloids from endophyte-infected tall fescue on the stallion breeding soundness examination spermiogram. OBJECTIVES: The objective of this study was to investigate the effect of ergot alkaloids from endophyte-infected tall fescue on the stallion's reproductive functions. STUDY DESIGN: Crossover toxicology experiment. METHODS: Six stallions were fed either toxic endophyte-infected tall fescue seed or a nontoxic endophyte tall fescue seed (Flecha AR-542, MaxQ). The fescue seed content was compounded at 45% of a grain diet and the stallions were fed the grain diet at 1% of their body weight. The stallions were fed the diet for 70 days, then rested for at least 70 days (no fescue seed) and then fed fescue seed for a second 70 days. At regular intervals blood sampling and a breeding soundness examination were performed. RESULTS: The mean time to maximal systemic toxicity was 8.33 h after starting toxic seed ingestion with a mean toxicity level of 49.98 ng alkaloid/mg creatinine. After cessation of feeding toxic seed, the systemic alkaloid concentration fell to control levels within 48 h. There were no significant changes in sperm motility, sperm concentration, sperm cell morphology, total number of sperm cells, number of breeding doses, testicular volume, baseline and human chorionic gonadotropin stimulated testosterone levels. There were no changes in core body temperature and superficial scrotal temperature. The ejaculate from stallions consuming endophyte-infected tall fescue seed had significantly lower gel-free volume (47.5 ± 4.1 ml) than stallions consuming nontoxic endophyte tall fescue seed (62.8 + 4.3 ml, P<0.01). CONCLUSION: Ergot alkaloids decreased the gel-free volume of stallions consuming high levels of ergot alkaloids but statistically significant effects on the spermiogram of adult breeding stallions were not found.

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

The cellular effects of tall fescue grass-associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cells for 48 h at 5°C was to make the sperm cells less likely to undergo a calcium ionophore-induced acrosome reaction. The toxic ergot alkaloids had no effect on the freezability of sperm cells. However, if yearling colts were fed toxic ergot alkaloids, then the cytological analysis of meiotic chromosome synapsis revealed a significant increase in the proportion of pachytene spermatocytes showing unpaired sex chromosomes compared to control spermatocytes. There was little effect of ergot alkaloids on adult stallions, but there might be a significant effect on yearling colts.

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility.

OBJECTIVE: We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. DESIGN: A randomised 2 x 3 block design was used. PROCEDURE: Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen. RESULTS: There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% (P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF (P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol (P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%. CONCLUSION: In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.

Controlling animal populations using anti-fertility vaccines.

The goal for fertility control of animal populations is the development of a safe, economical and effective contraceptive. One offshoot of the development of this technology is the acquisition of multiple therapeutic strategies for diseases, such as immunotherapy probes for cancer. In the long run, successful population control requires multifactorial strategies. One component of population control is immunocontraception. Development of effective antigens for immunocontraceptive vaccines has been remarkable and has greatly advanced our understanding of the molecular mechanisms of fertilization. The chasm between the discovery of an antigen in the laboratory, to the implementation of an effective field program, is immense. The zona pellucida (ZP) immunocontraceptive that has been most extensively evaluated as a fertility vaccine antigen and the porcine ZP has received particular attention. The long-term goal of population control would be the use of a synthetic vaccine, e.g. the ZP, tailored to a target species. In the future, if populations' levels are to be controlled by fertility vaccines, we should consider that the vaccinated animals could receive other health protective agents at the same time. For example, if a species were immunocontracepted, then they could be simultaneously vaccinated against habitat diseases such as rabies (Plumb et al., Rev Sci Tech, 26, 2007, 229).

Advancements in large animal embryo transfer and related biotechnologies.

Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstration of a new cryopreservation method which involves reduction of the blastocoelic volume and microinjection of cryopreservative. Successful cryopreservation will improve the ability of practitioners to preserve and implant embryos in recipient mares. Recent advances in the use of equine FSH to induce superovulation in mares brings to the forefront the issue of how to best preserve the large number of embryos that are produced. Finally, the use of sexed semen after superovulation will provide the bovine and equine breeding industry the offspring of the desired sex.

Embryo transfer in the dog and cat.

The practicality and feasibility of embryo transfer technology in dogs and cats is quickly becoming a clinical reality. Although progress has been slow, I anticipate that embryo transfer will be a practical and an economical technique in the near future. Most importantly, it is essential that the practical lessons learned with equine and bovine embryo transfer be integrated into the development of canine and feline programs.

Effect of porcine zonae pellucidae immunisation on ovarian follicular development and endocrine function in domestic ewes (Ovis aries).

Domestic ewes (Ovis aries) were immunised with porcine zonae pellucidae (pZP) or pZP conjugated to keyhole limpet haemocyanin (KLH) in adjuvant(s) to examine the feasibility of the species to serve as a model for further development of pZP-based vaccines in ungulates. Two immunisation groups were employed, with a third group receiving only adjuvant (n = 5 per group). Early in the study, oestrous activity was monitored by the use of a vasectomised ram fitted with a marking harness. Eventually, ewes were exposed to an intact ram for breeding. In addition, weekly serum and every-other-day faecal samples were collected to measure pZP antibodies and progesterone metabolite concentrations respectively. At the conclusion of the study, fecundity was established, and ovarian tissue was examined. Ewes immunised against pZP : KLH with adjuvant produced minimal antibody absorbance levels, displayed normal oestrous cycles, became pregnant upon introduction of the intact ram and exhibited normal ovarian histopathology. Ewes immunised against pZP with adjuvant produced high antibody absorbance levels, were acyclic following primary immunisation and were infertile. Examination of the ovarian tissue revealed atrophic changes that included: (1) the absence of growing follicles; (2) significant reduction in the number of primordial follicles; and (3) the presence of abnormal granulosa cell clusters lacking oocytes. Antisera displayed immunoreactivity to the major components of pZP, and immunohistochemical labelling of ovarian tissue showed specificity to the ZP. These data are the first generated in an ungulate species showing deleterious effects of pZP immunisation on folliculogenesis and oestrous cyclicity.

Field applications of immunocontraception in African elephants (Loxodonta africana).

The primary aim of the Makalali elephant immunocontraception programme is to test the efficacy of porcine zona pellucida (PZP) vaccine for practical population control of elephants in small, enclosed reserves, with the goal of stabilizing the current growth rate and reducing it to the 5-10% per annum displayed currently in the Kruger National Park. A secondary aim is to test the hypothesis that PZP treatment does not affect patterns of elephant social behaviour. Eighteen sexually mature cows (age > 12 years) were vaccinated in May 2000 using remote darts. Behavioural observations before, during and after vaccination included noting the activity of individual animals every minute for 15 min. No changes in general behaviour patterns have been noted to date although the animals' spatial use of the reserve was erratic during the period of vaccination, indicating irregular or disturbed patterns associated with vaccination. Normality was resumed on completion of the vaccinations. No aggressive or indifferent behaviour related to nursing, calf proximity or bull-cow interactions have been noted. Ten of the females were in various stages of pregnancy when treated. Subsequently, seven of them gave birth to healthy calves and the other three females are expected to calve shortly. It is too early in the study to draw conclusions about stabilization of growth rates.

Differences in immunocontraceptive responses in white-tailed deer (Odocoileus virginianus) and goats (Capra hircus).

Thirteen deer (Odocoileus virginianus) and three goats (Capra hircus) were given an initial immunization of 300 microg porcine zona pellucida (PZP) combined with 50 mg synthetic trehalose dicorynomycolate ml(-1) (STDCM) in drakeol i.m. in the semitendinosus and semimembranosus muscles. This immunization was followed by two booster injections of 300 microg PZP, 2 weeks apart. The vaccinations were made from the same purified batch of PZP and lot of adjuvant. All the doses were made at the same time and injected on the same day. The immune response was quantified by measuring serum anti-PZP IgG antibody concentration by ELISA. The results showed that the mean serum IgG concentration of the deer increased from 0.000 +/- 0.003 to 0.083 +/- 0.023 absorbance units, whereas in the goats the mean IgG concentration increase was from 0.044 +/- 0.019 to 1.245 +/- 0.774 absorbance units. Both the goats and deer showed a significant increase in IgG concentration (P < 0.05) after the final booster injection compared with preimmune serum. The final goat IgG concentration was significantly (P < 0.05) higher than that of the deer. If PZP and Freund's adjuvant are administered to deer, immunocontraception is achieved, but the IgG concentration in the deer in this study did not appear to be compatible with immunocontraception.

Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants.

A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.

Effects of an extract of Serenoa repens on dogs with hyperplasia of the prostate gland.

OBJECTIVE: To determine effects of an extract of Serenoa repens on dogs with prostatic hyperplasia. ANIMALS: 20 mature male dogs with benign prostatic hyperplasia. PROCEDURE: Dogs were assigned to 3 comparable groups on the basis of prostatic volume per kg of body weight and degree of prostatic hyperplasia determined histologically. Dogs in 2 groups were treated for 91 days (8 received 500 mg, PO, q 8 h [1,500 mg/d], and 6 received 100 mg, PO, q 8 h [300 mg/d]). The control group of 6 dogs did not receive medication. Effects of treatment on prostatic volume, prostatic weight, prostatic histologic characteristics, radiographic and ultrasonographic assessment of prostatic size, results of CBC, serum biochemical analyses, and urinalysis, serum testosterone concentration, and semen characteristics were determined. At the termination of the study, all dogs were euthanatized, and necropsies were performed. Investigators conducting tests and interpreting results were not aware of treatment group of each dog. RESULTS: Treatment did not affect prostatic weight, prostatic volume, or prostatic histologic scores, libido, semen characteristics, radiographs of the caudal portion of the abdomen, prostatic ultrasonographs, or serum testosterone concentrations. Results of CBC, serum biochemical analyses or urinalysis, and body weights did not change during treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with an extract of S repens for 91 days did not significantly affect the prostate gland of dogs. Adverse effects were not evident. Although products containing extracts of S repens are widely advertised for men with prostatic hyperplasia, beneficial or harmful effects of this plant extract were not found in dogs with prostatic hyperplasia.

Immunocontrol in dogs.

Population control in dogs and cats is an important goal for many groups. Control measures over the years has included surgery, hormonal therapy and more recently immunological control. The current presentation discusses dog population control with an emphasis on immunologic control. Specifically, vaccination with purified zona pellucida (ZP) glycoproteins leads initially to immunocontraception and then to the profound and irreversible changes of immunosterilization. The preliminary studies are extremely encouraging on developing a vaccine for lasting canine population control.

Evaluation of somatic and reproductive immunotoxic effects of the porcine zona pellucida vaccination.

Immunological, immunocytochemical and fertility analyses were performed to determine the potential toxic side effects of porcine zona pellucida (pZP) vaccinations on target animals, including horses and dogs. The study was designed to determine the effect of antibodies, raised against highly purified pZP, on somatic tissues. Immunocytochemical studies performed with fixed tissues showed that rabbit anti-pZP antiserum did not crossreact with brain, heart, lung, kidney, liver, bladder, stomach, small intestine, large intestine, muscle, skin, spleen, pancreas, or lymph node of either the dog or horse. To determine the effect or oral intake on nontarget animals, female rabbits were fed a contraceptive vaccine containing pZP glycoproteins and the synthetic trehalose dicorynomycolate in drakeol (S-TDCM) adjuvant. Enzyme-linked immunoadsorbent assay (LISA) analyses showed that rabbits fed with the adjuvanted pZP proteins did not develop circulating anti-pZP IgG antibodies that crossreacted with pZP. Furthermore, fertility studies performed on rabbits fed with adjuvanted pZP revealed no significant differences in the number of embryos or stage of the embryos produced between the treated and control animals. Results of these studies suggest that the pZP vaccine delivered to dogs or horses in field studies have no recognizable somatic tissue effects. Moreover, there were no side effects on nontarget animals should they eat the vaccine. This substantiates field trials results about the safety of the pZP immunocontraceptive vaccine.

Possible mechanisms of mammalian immunocontraception.

Ecological and conservation programs in ecosystems around the world have experienced varied success in population management. One of the greatest problems is that human expansion has led to the shrinking of wildlife habitat and, as a result, the overpopulation of many different species has occurred. The pressures exerted by the increased number of animals has caused environmental damage. The humane and practical control of these populations has solicited the scientific community to arrive at a safe, effective, and cost-efficient means of population control. Immunocontraception using zona pellucida antigens, specifically porcine zona pellucida (pZP), has become one of the most promising population control tools in the world today, with notable successes in horses and elephants. A conundrum has risen where pZP, a single vaccine, successfully induces an immunocontraceptive effect in multiple species of mammals. This review describes the most current data pertaining to the mammalian zona pellucida and immunocontraception, and from these studies, we suggest several potential mechanisms of immunocontraception.

Contraceptive potential of the porcine zona pellucida vaccine in the African elephant (Loxodonta africana).

Immunocontraception has been successful in controlling free-roaming equids; however, what is the potential for the immunocontraceptive control of the African elephant (Loxodonta africana)? The porcine zona pellucida (pZP) glycoproteins share antigenic domains with the African elephant zona pellucida (elZP) glycoproteins, and anti-zona pellucida serum antibodies have been successfully stimulated. To determine the cross-reactivity of the pZP and elZP, immunocytochemistry was evaluated by light and electron microscopy. Specifically, the binding of polyclonal antibodies against total heat-solubilized-porcine zona pellucida to fixed elephant ovary sections was evaluated. The elZP of primary, secondary and tertiary follicles was recognized by the rabbit-anti-pZP serum, but there was no apparent recognition of the primordial follicles. The ability of anti-pZP antibodies to recognize the elZP demonstrates that there is molecular homology between the pZP and elZP glycoproteins. This homology makes the African elephant a candidate for pZP immunocontraception. Three captive elephants were vaccinated with 400 micrograms pZP with a synthetic trehalose dicorynomycolate (S-TDCM) adjuvant. The elephants received 2 boosters of 600 micrograms pZP at 4 wk and 10 m.o. after the primary vaccination. The vaccinated female elephants developed significant (P < 0.05) titers to pZP over prevaccination levels. These levels persisted for 12 to 14 m.o. after the third vaccination. This preliminary evidence shows that the female elephant can develop significant serum antibody levels to pZP. These levels of antibodies are comparable to those required in horses for successful immunocontraception. Thus, porcine zona pellucida immunocontraception might be used to control elephant populations.

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility.

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.

Urinary and biliary excretion of ergot alkaloids from steers that grazed endophyte-infected tall fescue.

Ergot alkaloids cause fescue toxicosis when livestock graze endophyte-infected (E+) tall fescue. Little is known about the bioavailability of the ergot alkaloid classes (lysergic acid, lysergic acid amides, or ergopeptine alkaloids) in livestock, and this hampers development of pharmacological strategies to ameliorate the toxicosis. One method used to determine bioavailability of ergot alkaloids is to examine urinary and biliary excretion patterns. Thus, our objectives were to compare ergot alkaloid excretion via urinary or biliary systems and to determine the rate of appearance or clearance of these alkaloids in cattle that were grazing E+ or endophyte-free (E-) tall fescue. In autumn 1996, bile and urine samples were collected from eight steers (203 kg), each grazing E+ and E- tall fescue, and total alkaloid excretion was quantified using competitive ELISA. Approximately 96% of the ergot alkaloids were excreted in urine. The same steers were used to examine the rate of appearance in, or clearance from, urine when switched from E+ to E-, or from E- to E+, pastures in comparison with steers that were continuously grazing E+ or E- tall fescue at 0, 2, 5, and 7 d. Steers were returned to their original pastures after 7 d, and urine was collected at 2, 5, and 7 d. Urinary alkaloid concentrations in steers switched from E- to E+ pastures were similar (P = .55) to those in steers that continuously grazed E+ tall fescue after 2 d. Steers switched from E+ to E- pastures had urinary alkaloid concentrations similar (P = .91) to those in steers that continuously grazed E- pastures after 2 d. In 1997, two trials were conducted in which steers (191 kg) were switched or remained on E+ or E- pastures, and urine was collected at 0, 12, 24, 48, and 96 h to estimate rate of alkaloid appearance or clearance. Steers switched from E- to E+ 1) had about 33% as much urinary alkaloids as steers grazing E+ pasture after 12 h, 2) were not different after 24 h (P = .76), 3) had twice those of the E+ steers at 48 h (P < .05), and 4) were not different after 96 h. Steers switched from E+ to E- tall fescue had approximately 33% less (P < .05) urinary alkaloids than those grazing E+ at 12 h, 67% less (P < .05) at 24 and 48 h, and were not different (P = .86) from steers continuously grazing E- pastures after 96 h. Urinary alkaloid excretion patterns were similar to ergot alkaloid solubility patterns from in vitro digestion of E+ tall fescue. We suggest that alkaloids, liberated from the forage by ruminal microorganisms, were rapidly absorbed as lysergic acid amides and biotransformed ergopeptine alkaloids.

The importance of smoking education and preventative health strategies for people with intellectual disability.

Tobacco smoking is a major health problem within our community. To date, little attention has been focused on determining the prevalence of smoking in people with disabilities or in developing appropriate strategies to assist them to stop smoking. Funding from the Victorian Health Promotion Foundation enabled the Developmental Disability Unit at Monash University, Box Hill, Victoria, Australia, to undertake a project investigating smoking in a population of people with intellectual disability. In the first part of the project, the smoking rate among a geographically defined group of people with mild intellectual disability was investigated. A smoking prevalence of 36% was found within the sample group, as compared to a prevalence of 26% within the general Victorian adult population. An association was found between the motivation to quite and the recall of advice to stop smoking. The second part of the project comprised the development and piloting of a smoking education course for people with intellectual disability. The pilot sample was small, but the results were encouraging. Fifty-five per cent of the group either quit smoking or cut down their intake significantly. Seventy-three per cent expressed a desire to stop smoking at the completion of the course, and all participants expressed an increased concern and knowledge about the effects of smoking on their health after completing the course.

Extraction and quantification of acrosin, beta-N-acetylglucosaminidase, and arylsulfatase-A from equine ejaculated spermatozoa.

Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in an acrosin-acrosin inhibitor complex; this complex was dissociated after incubating the extract in 2 mM HCl. Significant (P < 0.001) increases in acrosin activity were found after-acid extraction from 0.076 U/mg after Method B to 0.327 U/mg after Method A. Arylsulfatase A activity was found to have a higher mean activity (P < 0.03) after Method A (0.012 U/mg) as opposed to Method B (0.007 U/mg). Similarly, beta-N-acetylglucosaminidase was found to have a higher mean specific activity (P < 0.001) after Method A (0.037 U/mg) as compared to Method B (0.008 U/mg). This is the first report of the quantification of these enzymes from equine spermatozoa which can ultimately be used as an index of acrosomal damage in cryopreserved semen, and provide additional insight into biochemical alterations between normal vs. abnormal semen.