CONTROL OF Neospora caninum IN SEMEN OF BULL

The objective of the present study was to develop a diluent of bull semen efficient in the control of Neospora caninum. The antimicrobial test (experiment 1) trimethoprim isolated (TRI) and associated with sulfadiazine (SDT) and sulfamethoxazole (SMT), clarithromycin (CLA), erythromycin (ERI), azithromycin (AZI) and clindamycin (CLI) was performed using cell culture and evaluated for the presence of free tachyzoites and the cytopathic effect in the monolayer. The antimicrobials that proved effective in controlling the tachyzoites in vitro were used in the preparation of the diluent for the semen. Each antimicrobial was evaluated for sperm toxicity (experiment 2) with 8 groups (7 groups received an antibiotic in the diluent except for 1 group control group). In relation to the morphological analysis, the SMT and AZI groups showed more acrosomal and tail defects. The SDT, SMT and AZI groups presented lower values of Average Path Velocity. The Straight-line Velocity was lower in the SMT and AZI groups and the Beat/cross Frequency was also lower in the CLI and AZI groups. The Curvilinear Velocity values of the SDT, SMT and CLA groups were lower. The SMT, ERI and AZI groups showed lower values of Total Motility and Progressive Motility. As for flow cytometry, the SDT, SMT and AZI groups presented higher percentages of acrosome reaction and damaged plasma membrane. The antibiotics can be added to the diluent of bull semen were clindamycin, clarithromycin and trimethoprim.


INTRODUCTION
Knowing the transmission pathways and life cycle of Neospora caninum is essential for the implementation of disease control measures, that include slaughter and replacement of seropositive animals, management techniques, among others (Reichel & Ellis, 2006;Dubey et al., 2007). In N. caninum infected herds, control programs are based on the prevention of vertical transmission, elimination of seropositive animals, and the reduction of horizontal transmission, mainly by controlling the access of definitive hosts considered as sources of contamination, since they eliminate oocysts in the feces (Dubey et al., 2007) on pasture areas. Management measures such as removal of potentially infected tissues from the environment, e.g., aborted fetuses and placenta, which may serve as a source of infection for susceptible hosts, are recommended (Anderson et al., 2000).
The reproductive health of males is a decisive factor for artificial insemination programs. As the DNA of N. caninum was detected in semen of bulls (Ortega-Mora et al., 2003;Caetano-da-Silva et al., 2004;Ferre et al., 2005;Serrano-Martinez et al., 2007a;Doosti et al., 2015), studies evaluated venereal transmission of N. caninum in cattle, classifying it as a horizontal transmission route (Serrano et al., 2006;Serrano-Martinez et al., 2007b). Considering that neosporosis is an important cause of reproductive problems and causes significant economic losses in the agricultural sector, precautionary measures Archives of Veterinary Science, v.26, n.1, p. 80-91, 2021.
are necessary for the use of semen from bulls infected with N. caninum. The objective of this study was to develop an efficient bull's semen diluent to control of N. caninum in the semen.

MATERIAL AND METHODS
Experiment 1: In vitro test of the effective active principles and compounds against N. caninum Tachyzoites of the N. caninum strain NC-1 were inoculated on VERO cells (African Green Monkey kidney cells) in 25 cm 2 Roux flasks and kept in an oven at 37 ° C with 5% CO 2. From two to three times a week, the cell culture monolayer was evaluated using inverted microscope to verify the cytopathic effect, caused by the multiplication of the tachyzoites. The F-10 HAM growth medium (Sigma-Aldrich) with pH adjusted to 7.4 and supplemented with 10% fetal bovine serum, 100 UI/mL potassium penicillin G, 50 µg/mL streptomycin sulfate, and 1.25 µg/mL amphotericin B was used for the exchange of cell culture medium.
The cell culture on culture flasks was scraped and the contents were transferred to sterile Falcon tubes for tachyzoites recovery. The suspension containing tachyzoites, cells and medium was centrifuged at 3000 RPM for 5 minutes, and the supernatant was removed.
The number of recovered tachyzoites was determined by Neubauer® chamber counting. After counting, each culture flask with established VERO cell monolayer was inoculated with 10 4 tachyzoites totaling 8 cell culture flasks. Of these, 7 flasks received their respective antimicrobial which was added to F-10 HAM maintenance medium on the exchange days.
After inoculation of the N. caninum tachyzoites, the flasks were evaluated daily for 7 days using inverted microscope to verify the presence of tachyzoites and the cytopathic effect in the VERO cells monolayer.
Experiment 2: Evaluation of sperm after dilution in diluent containing efficient antimicrobials against N. caninum.
A 96-month-old Nelore bull (Bostaurus indicus) weighing 900 kg and received a diet based on corn silage and ryegrass hay with water and mineral salt ad libitum. The animal was previously evaluated for normal andrological parameters (CBRA, 1998). The ejaculate was collected using an estrus female and an artificial vagina.
With the results obtained in the experiment 1, the antimicrobials that proved efficient in the control of tachyzoites in vitro were used in the preparation of the diluent for the semen. After the collection and evaluation of the semen, the sample was divided into eight experimental groups (same collection), then the diluent Tris-glycerol containing the antimicrobial of each group was added to seven of them, according to Table 1. No antimicrobial was added to the control group. After the semen dilution, the groups were re-evaluated and then placed in 0.25 mL straw with a concentration of 30 million viable spermatozoa each, duly identified and sealed with polyvinyl alcohol. After being filled and closed, the straws were cryopreserved.  obtained: intact plasma and acrosomal membranes (CAT 1); intact plasma membrane and damaged acrosomal membrane (CAT 2); damaged plasma membrane and intact acrosomal membrane (CAT 3); and damaged plasma and acrosomal membranes (CAT 4) (Celeghini et al., 2008). In order to evaluate of spermatozoa with acrosome reaction, CAT 2 and CAT 4 were added together and to evaluate of plasma membrane damage, CAT 1 and CAT 2 were added together.
The results obtained in the experiment were submitted to the Shapiro-Wilk normality test. The variables presented an abnormal distribution, thus the repeated measures RM ANOVA on Ranks was carried out followed by the Dunnett test for comparison of the experimental groups with the control group. The statistical program used was Sigma Plot TM 12.0 (Systat software, San Jose, CA), with significance level of 5%.

RESULTS AND DISCUSSION
All antimicrobials tested (clarithromycin, erythromycin, azithromycin, clindamycin, and trimethoprim alone and associated with sulfadiazine and sulfamethoxazole) were effective in controlling the tachyzoites inoculated in the cell culture flasks. Intracellular tachyzoite groups, cell culture rupture and free tachyzoites were observed only in the control group along the inoculation week. These active principles were also tested by Lindsay et al. (1994) who also observed efficacy against N. caninum. Subsequently, Lindsay et al. (1996) tested the synergism between sulfonamides and DHFR/TS (dihydrofolate reductase/thymidylate synthetase inhibitor). In the present study, the results obtained corroborate the efficiency of the concentrations previously tested.
A crucial point is that the active principles used in semen diluents should not present toxicity to sperm cells. The adverse effect has been associated with some antibiotics (BACK et al., 1975), as they may affect the functionality and integrity of the spermatozoa plasma membrane. To date, there have been no studies testing the interaction of clindamycin, clarithromycin, azithromycin, trimethoprim alone and trimethoprim sulfa with sperm cells.
The results of spermatozoa total motility, progressive motility, acrosomal and tail defects, acrosome reaction and damaged plasma membrane are described in Table 2.
The SMT and AZI groups presented lower values (p <0.05) for TM and PM and more acrosomal and tail defects when compared with the control group. The negative correlation between motility and sperm defects described by Saacke et al. (2000) corroborate these results. The ERI group presented lower values for motility but no more acrosomal and tail defects. However, according to Stallcup & McCartney (1953) et al. (1975), erythromycin proved deleterious to mammalian sperm when added to semen diluents. According to Maia et al. (2009)  in the formulation of the semen diluent medium as they negatively affect sperm motility.
As for the flow cytometry, the SDT, SMT and AZI groups presented significantly higher (p <0.05) percentages of spermatozoa with an acrosome reaction and with damaged plasma membrane compared to the control group. Probably as a consequence of osmolality and pH alterations, and the presence of reactive oxygen species in the dilution medium. In this case, fertilization failure could occur if semen was used in dilution medium containing these antimicrobials.
The acrosome reaction, characterized by the release of the enzymes present in the acrosome, is an important event that allows penetration of the spermatozoa in the zona pellucida and fusion with the oocyte plasma membrane at the time of fertilization (Verstegen et al., 2002). Plasma membrane plays a key role in sperm survival (Parks & Graham, 1992) since it is responsible for the maintenance of sperm cell osmolarity. Thus, it is ideal that the plasma membrane is intact, and the acrosome preserved, as well as its enzymes (Braundmeier & Miller, 2001;Kastelic & Thunsathil, 2008).
In the present study, it was observed that the SMT and AZI groups presented a reduction in sperm motility and higher percentages of spermatozoa with acrosome reaction, corroborating the results obtained by Silva et al. (2006) who observed a negative correlation between acrosome reaction and progressive sperm motility postthawing in goat semen.
The results of sperm kinetics analysis are presented in Table 3  In this study, the antibiotics that showed the best results were clindamycin and trimethoprim alone and, therefore, can be added to the bovine semen diluent. These active principles did not differ statistically when compared to the control group in Archives of Veterinary Science, v.26, n.1, p. 80-91, 2021.
relation to the parameters of sperm viability. However, clarithromycin still proved to be applicable because even reducing the capacity of mitochondria in the production of ATP, the group that received this antimicrobial showed similarity in relation to motility and acrosome defects with the control group, parameters that are important in male fertility.
To date, there are no studies testing the interaction of clindamycin, clarithromycin, trimethoprim alone with animal sperm cells.

CONCLUSION
Thus, under the conditions of this study, it was concluded that the antibiotics effective against N. caninum that were not deleterious to the spermatozoa were clindamycin, clarithromycin, and trimethoprim alone. Therefore, these active principles may be added to the diluent media for cryopreservation of semen from seropositive bulls.