Fulminant and fatal encephalitis caused by Acanthamoeba in a kidney transplant recipient: case report and literature review.

Acanthamoeba is the most common cause of granulomatous amebic encephalitis, a typically fatal condition that is classically described as indolent and slowly progressive. We report a case of Acanthamoeba encephalitis in a kidney transplant recipient that progressed to death within 3 days of symptom onset and was diagnosed at autopsy. We also review clinical characteristics, treatments, and outcomes of all published cases of Acanthamoeba encephalitis in solid organ transplant (SOT) recipients. Ten cases were identified, and the infection was fatal in 9 of these cases. In 6 patients, Acanthamoeba presented in a fulminant manner and death occurred within 2 weeks after the onset of neurologic symptoms. These acute presentations are likely related to immunodeficiencies associated with solid organ transplantation that result in an inability to control Acanthamoeba proliferation. Skin lesions may predate neurologic involvement and provide an opportunity for early diagnosis and treatment. Acanthamoeba is an under-recognized cause of encephalitis in SOT recipients and often presents in a fulminant manner in this population. Increased awareness of this disease and its clinical manifestations is essential to attain an early diagnosis and provide the best chance of cure.

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival.

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1mg CLC/120 × 10(6)sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.

Response of boar sperm to the treatment with cholesterol-loaded cyclodextrins added prior to cryopreservation.

Cryopreserved boar sperm is not used extensively for artificial insemination, owing to the poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged as the cells are cooled from body temperature to 5°C (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could help them survive cryopreservation, similar to sperm from other species that are cold shock sensitive. The aim of this study was to determine the optimal cholesterol-loaded cyclodextrin (CLC) concentration to use for boar sperm cryopreservation, and the influence of CLCs on the cryosurvival of sperm from boars classified as good or poor freezers. Treating boar sperm with 1 mg of CLC/120 × 10(6) sperm slightly improved (p < 0.05) the percentage of viable sperm after freezing-thawing. On the other hand, sperm, from both good and poor freezers, responded similarly to CLC treatment. Nevertheless, additional studies will be needed to study the effect of this treatment on other parameters of sperm quality.

Use of cholesterol in sperm cryopreservation: present moment and perspectives to future.

CONTENTS: Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm.

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5mg CLC (positive control); 0.75 mg or 1.5mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 degrees C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 degrees C over a 2h. Upon reaching 5 degrees C, the samples were diluted 1:1 with T diluent containing 10% EY+16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686-699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments (P<0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing (P>0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control (P<0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.

Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols.

One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen production, it is likely that more than one method for cryopreserving sperm will be necessary. This manuscript reviews some of the processes involved in cryopreservation, how individual sperm physiology affects the ability to survive freezing and thawing, and how cryopreservation protocols can be customized to maximize sperm cryosurvival on an individual stallion basis.

In vitro evaluation of sperm quality.

This paper highlights selected laboratory analyses that are currently used to evaluate sperm, and describes why results from these assays do not consistently correlate with sperm fertility. Reasons for the disconnect between the two are due in part to the definition and reliability of the fertility data collected, to the complexity of the spermatozoon itself, to imprecision of some measurements, and to uncontrollable factors not associated to either the laboratory analysis or the sperm sample. Each sperm must possess a number of different attributes to fertilize an oocyte, and individual laboratory assays measure only one or a few of these attributes. Current and past data, correlating laboratory assay data with sperm fertility are presented in an effort to determine which types of assays are important to conduct and when to conduct them. Even though laboratory assay results do not allow accurate evaluation of the fertilizing potential of a semen sample, these assays are important to enable culling of poor quality samples.

Molecular effects of nicarbazin on avian reproduction.

Nicarbazin (NCZ) is an anticoccidial drug routinely used in the poultry industry that can negatively affect reproduction by reducing egg production, egg weight, and egg hatchability. The molecular mechanisms by which NCZ affects reproduction are unknown. Lipoprotein lipase, vitellogenin, transglutaminase, and calcium are all involved in egg formation and embryogenesis. Therefore, in vitro assays were used to evaluate 4 potential mechanisms of action of NCZ on egg formation and embryogenesis. First, a lipoprotein lipase assay was conducted to determine if NCZ increases lipoprotein lipase activity. Second, vitellogenin phosphorylation was evaluated to determine if NCZ acts as a vitellogenin phosphatase. Third, transglutaminase activity was measured to determine if NCZ inhibits transglutaminase activity. Finally, bull sperm was used as a model to determine if specific channel-mediated calcium uptake can be blocked by NCZ. Nicarbazin increased the activity of lipoprotein lipase in vitro at 3.9 and 7.8 microg of NCZ/mL. Nicarbazin increased intracellular calcium levels in bull sperm, suggesting it also acts as a calcium ionophore. The portion of the NCZ molecule responsible for the increase in intracellular calcium is 2-hydroxy-4,6-dimethylpyrimidine. Nicarbazin affected vitellogenin phosphorylation but only at a concentration many times higher than expected plasma values. Nicarbazin also inhibited transglutaminase activity in vitro. Whereas the 4,4'-dinitrocarbanilide portion of the NCZ molecule inhibited transglutaminase activity, the 2-hydroxy-4,6-dimethylpyrimidine portion increased transglutaminase activity. All of these assays were conducted in vitro; therefore these results should be viewed as preliminary findings to aid in directing further research on the effect of NCZ on reproduction in vivo. Because NCZ increases lipoprotein lipase activity and acts as a calcium ionophore, future experiments should investigate these effects in particular.

Effect of method of delivering nicarbazin to mallards on plasma 4,4'-dinitrocarbanilide levels and reproduction.

Nicarbazin (NCZ), a coccidiostat used in the poultry industry, has been developed as a contraceptive for resident Canada geese. We tested the efficacy of NCZ as a contraceptive using mallards (Anas platyrhynchos) as a model for Canada geese. Nicarbazin-treated corn was fed ad libitum for 14 d at 0, 750, 1,000, or 1,500 ppm. Plasma and egg levels of 4,4'-dinitrocarbanilide (DNC), the active anticoccidial component of NCZ, differed among treatment groups in a dose-response relationship, but plasma levels did not differ between sexes. Nicarbazin caused a decrease in egg weight, but there was no effect of NCZ on the numbers of eggs laid per female per day. Nicarbazin did not significantly impact bird health. An additional trial tested the effect of the method of NCZ delivery on plasma DNC levels. Mallards were given NCZ daily for 12 d either by gavage with a corn oil suspension, gavage with a water suspension, peroral administration of a capsule, or feeding 500 mg of NCZ/kg of pelleted feed ad libitum. The method of delivery significantly affected plasma DNC levels, with the highest levels in the corn oil suspension group and the lowest levels in the pelleted feed group. This is likely due to decreased availability of NCZ in a pellet compared with gavage with a suspension or capsule. Mallards receiving 34.2 mg of NCZ/kg of BW when fed cracked corn coated with NCZ daily for 14 d had higher plasma DNC levels than those obtained by liquid gavage, capsule, or pelleted NCZ feed. For maximum effect in the field, NCZ should be coated onto corn. A higher concentration of NCZ is needed in pelleted feed to obtain comparable plasma DNC levels to allow for the decreased absorption of DNC.

Evaluation of nicarbazin as a potential waterfowl contraceptive using mallards as a model.

Contraception may provide a useful nonlethal management tool to reduce wild bird populations. We tested the efficacy of nicarbazin (NCZ) as a contraceptive for waterfowl and assessed health effects of NCZ, using domestic mallards (Anas platyrhynchos) as a model for Canada geese (Branta canadensis). Mallards were given gelatin capsules containing 0, 8.5, 17.0, or 33.75 mg of NCZ/kg of BW perorally once daily for 14 d. Fecal 4,4'-dinitrocarbanilide (DNC) and fluorescein were evaluated as potential markers of plasma and egg DNC levels. Plasma, egg, and fecal DNC levels differed among treatment groups in a dose response relationship. There were no significant effects on the numbers of eggs laid per female per day, proportion of fertile eggs, proportion of eggs hatching, or egg yolk mottling. Hatchability was 0.55 +/- 0.1 in the control group compared with 0.26 +/- 0.1 in the 33.75 mg/kg of BW group. Degeneration of the vitelline membrane was evident at all treatment levels; severity was dose-related and greater in the outer vitelline membrane than the inner vitelline membrane. No significant health effects were observed for birds treated with NCZ. The heterophil:lymphocyte ratio was elevated during the treatment and posttreatment periods in all groups, indicating birds were experiencing stress due to handling. Fecal DNC levels did not correlate well with plasma DNC levels, likely due to NCZ being administered as a bolus dose rather than being fed ad libitum. Fluorescein correlated well with plasma DNC levels during the treatment period and can therefore be used successfully as a noninvasive marker to determine the approximate amount of NCZ a bird is consuming. As a contraceptive, NCZ likely would have minimal adverse health effects on the target animal, although field studies with the species of interest need to be conducted. Further research using higher NCZ levels needs to be conducted to determine whether NCZ can inhibit reproduction in waterfowl.

Removing seminal plasma improves bovine sperm sex-sorting.

Bull ejaculates with sperm concentrations of less than 1 billion sperm sort poorly for sex chromosomes, but whether this is because of the sperm concentration or the concomitant seminal plasma content has not been elucidated. Experiments were conducted to determine why ejaculates with lower sperm concentrations sort poorly and develop a protocol to increase sorting efficiency. In Experiment I, spermatozoa at 160 or 240 × 106 sperm/mL were stained at 49, 65 or 81 µm Hoechst 33342 with 0 or 10% seminal plasma and then sex-sorted. In Experiment II, seminal plasma was adjusted to create samples with sperm concentrations of 0.7, 1.4 and 2.1 × 109 sperm/mL, prior to sex-sorting. In Experiment III, spermatozoa were diluted to 0.7, 1.4 and 2.1 × 109 sperm/mL using TALP containing 0 or 10% seminal plasma prior to sex-sorting and cryopreservation. In Experiment I, the optimal staining combination was 160 × 106 sperm/mL stained with 65 µm Hoechst 33342 and no seminal plasma. In Experiment II, the percentages of membrane-impaired sperm were lower for sample concentrations of 2.1 × 109 sperm/mL (15%) than for samples at 1.4 × 109 (17%) or 0.7 × 109 sperm/mL (18%; p < 0.01). The X sort rate was slower for samples stored at 0.7 × 109 sperm/mL (3.45 × 103 sperm/sec) than for samples stored at 1.4 × 109 and 2.1 × 109 sperm/mL (3.85 and 3.94 × 103 sperm/sec, respectively; p < 0.05). In Experiment III, samples containing 0% seminal plasma had higher percentages of live-oriented cells (54 vs. 50%; p < 0.05), fewer dead sperm (19 vs. 22%; p < 0.01) and higher post-thaw motility (41 vs. 35%; p < 0.05) than samples containing 10% seminal plasma. Ejaculates with high sperm concentrations result in superior sorting because these samples have less seminal plasma during staining than ejaculates with lower initial sperm concentrations as all samples are diluted to 160 × 106 sperm/mL for staining. Therefore, sorting efficiency appears to be affected by seminal plasma concentration, not by the original sperm concentration.

Fertility evaluation of frozen/thawed semen.

In vitro semen analyses have been used for more than half a century to estimate the fertilizing potential of a semen sample. Unfortunately, none of the assays developed provide results that consistently correlate well with fertility. The reasons for this lack of consistency, due in part to the complexity of the spermatozoon itself, the collection of fertility data, and factors beyond control of the semen analyses themselves, are discussed. Different spermatozoal attributes that are necessary for a spermatozoon to fertilize an oocyte are presented and assays used to evaluate each attribute described. Although laboratory assay results do not correlate well with semen fertility, the importance of conducting laboratory assays on every semen sample used for artificial insemination or to attempt to determine causes for infertility, is discussed.

Effect of seminal plasma on the cryopreservation of equine spermatozoa.

Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80% seminal plasma for 15 min, prior to being diluted to a final concentration of 50 x 10(6)sperm/mL in a Lactose-EDTA freezing diluent and cryopreserved. Sperm motility was analyzed at 10 and 90 min after thawing, while sperm viability and acrosomal integrity were analyzed 20 min after thawing. Seminal plasma did not affect sperm motility, viability or acrosomal integrity (P>0.05). Experiment 2 tested the main affects of seminal plasma level (5 or 20%), incubation temperature (5 or 20 degrees C) and incubation time (2, 4 or 6 h) prior to cryopreservation. In this experiment, spermatozoa were incubated with 5 or 20% seminal plasma for up to 6h at either 5 or 20 degrees C prior to cryopreservation in a skim milk, egg yolk freezing extender. Samples cooled immediately to 5 degrees C, prior to freezing had higher percentages of progressively motile spermatozoa than treatments incubated at 20 degrees C (31 versus 25%, respectively; P<0.05), when analyzed 10 min after thawing. At 90 min post-thaw, total motility was higher for samples incubated at 5 degrees C (42%) compared to 20 degrees C (35%; P<0.05). In addition, samples containing 5% seminal plasma had higher percentages of total and progressively motile spermatozoa (45 and 15%) than samples exposed to 20% seminal plasma (33 and 9%; P<0.05). In conclusion, although the short-term exposure of sperm to seminal plasma had no significant effect on the motility of cryopreserved equine spermatozoa, prolonged exposure to seminal plasma, prior to cryopreservation, was deleterious.

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa.

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.

Cholestanol-loaded-cyclodextrin improves the quality of stallion spermatozoa after cryopreservation.

This study was to compare the effect of adding cholesterol or cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of eight stallions were diluted to 120 million cells in a S-MEDIUM diluent. The diluted sperm were sub-divided into three treatments: no additive (control); 0.75mg of cyclodextrin pre-loaded with cholesterol (CLC)/120 million sperm (positive control); 1.5mg CLC/120 million sperm; 0.75mg of cyclodextrin pre-loaded with cholestanol (CnLC)/120 million sperm; and 1.5mg CnLC/120 million sperm. To set the experiments, the treated sperm were incubated for 15min at 22°C to allow for the incorporation of cholesterol or cholestanol. In each experiment, treated sperm incubated for 15min at 22°C to allow for incorporation of cholesterol or cholestanol. The samples were then diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5°C over a 2h period. Loaded into 0.25ml polyvinylchloride straws, frozen in liquid nitrogen vapor for 10min, and then plunged into liquid nitrogen until further use. Higher percentages of motile sperm and viable cells were achieved after thawing for stallion sperm treated with CLC and CnLC compared to control (P<0.05). Addition of CnLC also resulted in more number sperm binding to chicken egg perivitelline membrane (CEPM) after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CnLC and CLC improved the percentage of post-thaw motility of equine sperm and CnLC provided greater binding efficiency.

Cryopreservation of mammalian sperm: what we ask them to survive.

Techniques for freezing bull sperm developed over the past 40 years have not yielded protocols for preserving sperm from other species. Recent advances in our understanding of cell membrane structure function and metabolism now permit alternative modes of investigation. These data will allow development of unique studies which should have a higher probability of yielding successful protocols for sperm from other species. In this review the authors will: (1) provide a general overview of cryopreservation; (2) review emerging concepts of membrane structure and the relationship of membrane composition to water and cryoprotectant movement; (3) emphasize how these parameters affect cell volume and surface areas; (4) focus attention on the concept that cryoprotectants will alter membrane structure and function in addition to their well-recognized effects on bulk solvent; and (5) emphasize the effect of the processing protocol on metabolic balance. These concepts are reintroduced in the context of the established and successful protocol for freezing bull sperm to illustrate the molecular responses that may be necessary to survive a freeze-thaw cycle.

Blepharoplasty in patients with an anophthalmic orbit.

Blepharoplasty in the patient with an anophthalmic orbit is discussed. The different principles involved in dealing with lids on the normal side and the anophthalmic side are outlined.

Fistulas of the penile urethra.

Urethral fistula, the most common complication of urethroplasty, is discussed. There has been no classification for these disorders, and the plethora of reconstruction procedures available often leads to confusion. The classification presented here for acquired fistulas attempts to give direction in the selection of appropriate management in individual cases. Early, acute fistulas are managed conservatively. In the case of mature single fistulas, local tissue may be used for surgical repair. These are subdivided, depending on the size of the opening. Chronic, multiple, large lesions draining the urethra require tissue from a distance for repair. In severe surgical cripples, a total new urethral reconstruction will be required. A new technique to expose the urethra, allowing correction of urethral fistulas with stricture and diverticulum, is described.

Assessment of sperm quality: a flow cytometric approach.

For many years, scientists have sought to develop laboratory assays that accurately predict the fertilizing capacity of a semen sample. This goal, however, has proven elusive and will most likely be very difficult to achieve, due to the complex nature of the problem. Part of the problem results from the many attributes that a spermatozoon must possess to fertilize an egg, and how laboratory assays can evaluate all of these attributes simultaneously. The percentage of motile sperm in a sample is most commonly used to evaluate semen quality. This assay, however, is not highly correlated with the fertilizing capacity of semen samples. One reason motion assays do not correlate well with fertility is that we are evaluating only one of many attributes that a sperm must possess to fertilize an oocyte. One of the problems of measuring multiple sperm attributes is the time and cost required. Using flow cytometric assays, multiple sperm attributes, including cell viability, acrosomal integrity, and mitochondrial function, can be measured simultaneously in sperm cells. In addition, the ability of sperm to undergo capacitation and the acrosome reaction, as well as the chromosomal integrity of sperm can be measured using flow cytometry. Flow cytometry permits us to evaluate 50,000 sperm in less then 1 min and at reasonable cost. Although flow cytometry is a powerful tool for evaluating many sperm attributes, it cannot evaluate all of the attributes a sperm cell requires to fertilize an oocyte. Therefore, laboratory assays are also being developed to evaluate the ability of sperm: (1) to bind to the oocyte, by evaluating the ability of sperm to bind to the perivitelline membrane of the hen egg in vitro; (2) to undergo an acrosome reaction in vitro, after treatment with membrane destabilizing compounds; and (3) to penetrate oocytes in vitro. When data from multiple sperm assays are used, higher correlations with the fertilizing potential of a semen sample is achieved. For example, in a study conducted utilizing five stallions, the percentage of motile sperm in semen samples correlated poorly with fertility (r(2)=0.22), however, when data for sperm motility, viability and penetration rates into zona-free hamster oocytes were utilized together, these data explained 72% of the differences in the fertility of the stallions (r=0.849; [Theriogenology 46 (1996) 559]). Armed with a battery of tests, which evaluate many different sperm attributes, researchers should be able to more accurately estimate the fertilizing potential of semen samples.

A new pellet technique for cryopreserving ram and bull spermatozoa using the cold surface of cattle fat.

A technique for freezing ram and bull spermatozoa in pellet form, using the cold surface of cattle fat was compared to other freezing procedures. Three freezing methods were compared to cryopreserve ram spermatozoa: 0.25 ml straws, pellets frozen on the cold surface of paraffin wax and pellets frozen on the cold surface of cattle fat. In addition, two cryoprotectants, glycerol or sucrose, in an egg yolk-Tris diluent were compared. Ram spermatozoa frozen as pellets on cattle fat exhibited higher percentages of motile cells after thawing (54%) than spermatozoa frozen in straws (49%) or as pellets on paraffin wax (42%, S.E.M. = 1; P < 0.05). However, the percentages of acrosome intact cells were similar for spermatozoa frozen as pellets (49%) and spermatozoa frozen in straws (48%; P > 0.05), but higher than for spermatozoa frozen as pellets on paraffin wax (39%, S.E.M. = 1; P > 0.05). Ram spermatozoa exhibited higher percentages of motile cells after thawing when the cryoprotectant was sucrose (51%) compared to glycerol (46%; P < 0.05). Similarly, acrosomal integrity was greater with sucrose (49%) than with glycerol (42%; P < 0.05). Bull spermatozoa exhibited higher percentages of motile cells after thawing, when cells were frozen in straws (47%) than in the pellet form, regardless of the surface on which the pellets were frozen (31-37%, S.E.M. = 3; P < 0.05). However, bull spermatozoa exhibited higher percentages of motile cells when frozen as pellets on the surface of cattle fat (66%) or dry ice (61%), than when frozen on paraffin wax (53%, S.E.M. = 4; P < 0.05). In conclusion, although bull spermatozoa survive cryopreservation more effectively in straws, ram spermatozoa can be cryopreserved as pellets on the cold surface of cattle fat using sucrose as the cryoprotectant. This technique is simple, requires little equipment, is less expensive than using straws and may prove useful for cryopreserving ram and possibly bull spermatozoa in developing countries.