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.

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.

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols.

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10(6) sperm) and bull (2 mg CLC/120×10(6) sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.

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.

Seminal plasma effects on sex-sorting bovine sperm.

The objective was to determine which characteristics of bovine ejaculates affected efficacy of sex sorting bovine sperm by flow cytometry. The effects of first versus second ejaculates, seminal plasma content, addition of BSA, and seminal plasma from different bulls during staining were all studied, as was the effect of 8-hour storage with and without seminal plasma. Semen collected by artificial vagina was centrifuged at 1000 ×g for 15 minutes to separate sperm from seminal plasma; seminal plasma was clarified by 10 minutes of additional centrifugation at 2000 ×g. Sperm were rediluted to 160 × 10(6) sperm per mL with: Tyrode's medium plus albumin, lactate, and pyruvate (TALP) containing 0%, 5%, 10%, or 20% homologous seminal plasma, TALP containing 10% heterologous seminal plasma, or TALP containing 0.3% (control), 0.6%, or 1.2% BSA. After incubation with Hoechst 33342 for 45 minutes, an equal volume of TALP containing red food dye was added, and sperm were analyzed by flow cytometry/cell sorting to determine percent of live-oriented sperm, X sort rate, percent of membrane-impaired sperm, and split (degree of separation between X- and Y-bearing sperm populations). The percent of live-oriented sperm was higher for sperm incubated with 0% seminal plasma (64%) than for sperm incubated with 5%, 10%, or 20% seminal plasma (60%, 59%, and 58%, respectively; P < 0.05). The X sort rate was higher for sperm incubated with 0% seminal plasma than sperm with 20% seminal plasma (4.26 vs. 3.61 × 10(3) sperm per second). When seminal plasma was exchanged between bull ejaculates, only one bull had seminal plasma that was detrimental to sperm, resulting in 31% membrane-impaired sperm compared with a range of 16% to 19% for seminal plasmas from other bulls (P < 0.05). The addition of BSA did not affect sort efficiency at the concentrations studied. Sperm from six bulls stored for 8 hours without seminal plasma had more membrane-impaired sperm (which were discarded) during sorting (28%) than with seminal plasma (19%; P < 0.01), but higher postthaw motility postsorting (63%) than with seminal plasma (52%; P < 0.05). In conclusion, the presence of seminal plasma during staining and sorting decreased sort rates and percent of live-oriented sperm, and storing sperm without seminal plasma increased postthaw motility.

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.

Cholesterol-loaded-cyclodextrins and fertility potential of stallions spermatozoa.

Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acrosome react, and bind to the zona pellucida in vitro, and to fertilize oocytes in vivo. When CLC-treated cryopreserved stallion spermatozoa were treated with various agents to induce capacitation and the acrosome reaction (AR), dilauroylphosphatidylcholine (PC-12) and lysophosphatidylcholine (LPC) induced the AR in control cells (62% and 55%, respectively) but not in CLC-treated cells (17% and 14%, respectively, P<0.05). However, the calcium ionophore A23187 induced the AR in both control- and CLC-treated cells equally well (39%, P>0.05). Control- and CLC-treated stallion spermatozoa bound to ZP of cattle oocytes equally well (0.44+/-0.16 vs. 0.25+/-0.09, respectively; P>0.05). In addition, the fertility rates of mares inseminated with control- and CLC-treated sperm were similar (P>0.05).

Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival.

This study compared the effect of adding other cholesterol conjugates, which should incorporate into and increase sperm membrane fluidity at low temperatures thereby increasing cryosurvival. Ejaculates from each of four bulls were diluted to 120 million cells/ml in a Tris diluent and used in two experiments. Each experiment contained four treatments: No additive (control); 1.5mg CLC/120 million sperm (positive control); and 1.5mg cyclodextrin pre-loaded with cholestanol or desmosterol/120 million sperm. In the first experiment, fresh sperm were treated with cyclodextrins that were pre-loaded with cholesteryl conjugates and incubated for 15 min at 22 degrees C to allow incorporation of the conjugates. The percentages of motile sperm after exposure to solutions ranging from 0 to 1200 mOsm were used to evaluate the osmotic tolerance of the cells. The ability of treated sperm to bind to the bovine zona pellucida (ZP) and chicken egg perivitelline membrane (CEPM) was evaluated to determine if altering the sperm membrane affected the ability of sperm to bind to oocytes. In the final experiment, the cryosurvival rates of control and treated sperm were determined. Control and treated sperm were cryopreserved in a Tris diluent containing 10% egg yolk (EY) and 8% glycerol (final concentrations). Samples were thawed to determine the motility and ability of sperm to bind to the ZP and to CEPM using a CASA and epifluorescence microscope, respectively. Fresh sperm treated with CLC resulted in more binding to the ZP compared to all other treatments (P<0.05). No differences were observed between ZP and CEPM binding (P>0.05). The percentages of motile sperm were greater for fresh samples treated with cholesterol, cholestanol or desmosterol loaded cyclodextrin than control cells (P<0.05) when the sperm were exposed to anisosmotic conditions, and then returned to isosmolality. After cryopreservation the percentages of motile sperm and number of sperm binding to each CEPM were similar for sperm treated with CLC and cholestanol compared to sperm treated with desmosterol (P>0.05). All treatments provided greater motility and binding efficiency than control sperm (P<0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival.

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.

The hypoosmotic swelling test in fresh rabbit spermatozoa.

The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the membrane integrity of spermatozoa of various domestic animals including cattle, horses, and swine. However, the best approach for using this technique in rabbit semen has not been tested. The present study aimed to establish the best hypoosmotic solution (HS) for testing membrane integrity in fresh rabbit semen. Sucrose solutions with the following osmolarities were used: 50, 60, 75, 100, 125 and 150mOsm/L. Semen samples (n=30) were collected from five mature White New Zealand rabbits (six collections per rabbit) at 72h intervals. After macroscopic evaluation, 10microL of semen was immediately added to 2mL of each solution and incubated for 1h at 37 degrees C. Sequentially, 20microL of semen diluted in HS were evaluated with oil immersion using a phase-contrast microscope. A total of 200 spermatozoa were counted in at least five different fields, and sperm tails were classified as non-coiled, coiled, and strongly coiled. The respective percentages of spermatozoa with coiled tails (coiled plus strongly coiled) in the six solutions listed above were 54.8, 65.2, 54.3, 53.9, 38.9 and 29.4%. Percentage of strongly coiled spermatozoa was: 40.2, 51.0, 43.2, 41.5, 32.7 and 26.9 for the six solutions, respectively. According to total and strong coiling 60mOsm/L was superior to others treatments (P<0.05). Results suggest that the 60mOsm/L solution would be most desirable for use in HOST in fresh rabbit spermatozoa.

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.

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.

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.

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.

Cholesterol-loaded cyclodextrins added to fresh bull ejaculates improve sperm cryosurvival.

Damage occurring to spermatozoa during cryopreservation results in a loss of motile cells and cells that are functionally normal, compared with fresh sperm samples. Treating bull sperm with cholesterol-loaded cyclodextrins (CLC) before cryopreservation results in increased sperm cryosurvival. However, in previous studies, CLC were always added to sperm samples that had been highly diluted. The aim of this study was to develop a procedure for adding CLC to whole bull ejaculates that would optimize sperm cryosurvival. Adding 2 or 4 mg of CLC/120 x 10(6) sperm to sperm samples ranging in concentration from 120 to 2,000 x 10(6) sperm/mL resulted in greater (17 to 28 percentage points; P < 05) numbers of live cells compared with control samples (no CLC treatment), regardless of the sperm concentration, except for samples at 120 x 10(6) sperm/mL treated with 4 mg of CLC. Incubating sperm with CLC at 23 or 37 degrees C before cryopreservation resulted in similar sperm cryosurvival. The cooling rate used to cryopreserve CLC-treated cells did not affect sperm cryosurvival. Finally, adding CLC to undiluted ejaculates (2 mg of CLC/120 x 10(6) sperm) resulted in greater percentages of live sperm compared with the control samples (62 vs. 45%; P < 0.05), although the percentages of motile sperm were similar for both CLC-treated and control samples (58%). In conclusion, bull sperm cryo-survival can be improved if spermatozoa are treated with CLC before freezing. In addition, CLC can be added to fresh ejaculates at either 23 or 37 degrees C. This technique is simple, practical, and can be easily integrated into current cryopreservation protocols.

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.