Influence of addition of different antibiotics in semen diluent on viable bacterial count and spermatozoal viability of Awassi ram semen

The objectives of the present study were to determine the effects of six different antibiotics in controlling the growth of semen contaminating bacteria and if these antibiotics have any adverse effect on Awassi ram spermatozoa. Semen samples from six mature Awassi rams were used in this study. A total number of 120 ejaculates were collected from the rams using an artificial vagina once a week. Semen ejaculates were evaluated for volume, sperm concentration, mass motility, individual motility, percentage live sperm, sperm abnormalities, and viable bacterial count. Semen samples were diluted by sodium citrate-fructose-egg yolk. The diluted semen sample was divided into 7 parts. Six types of antibiotics were added to the semen diluent parts including; penicillin G 1000 IU ml-1 with streptomycin 1 mg ml-1, gentamicin sulphate 250 mg ml-1, tetracycline 0.5 mg ml-1, lincomycin 1 mg ml-1, cefoperazone sodium 1mg ml-1, cefdinir 1 mg ml-1 and the seventh part considered as a control group without antibiotic addition. The diluted semen samples were cooled and preserved at 5 Co for 5 days. Cooled diluted semen samples were examined for individual motility, percent of live sperm, sperm abnormalities, acrosomal defects and bacterial count every 24 h until 5 days. Comparing with the control, all the antibiotics examined were effective in controlling bacterial growth (P


Abstract
The objectives of the present study were to determine the effects of six different antibiotics in controlling the growth of semen contaminating bacteria and if these antibiotics have any adverse effect on Awassi ram spermatozoa.Semen samples from six mature Awassi rams were used in this study.A total number of 120 ejaculates were collected from the rams using an artificial vagina once a week.Semen ejaculates were evaluated for volume, sperm concentration, mass motility, individual motility, percentage live sperm, sperm abnormalities, and viable bacterial count.Semen samples were diluted by sodium citrate-fructose-egg yolk.The diluted semen sample was divided into 7 parts.Six types of antibiotics were added to the semen diluent parts including; - penicillin G 1000 IU ml with streptomycin 1 mg ml , gentamicin sulphate 250 mg ml , tetracycline 0.5 mg ml , - lincomycin 1 mg ml , cefoperazone sodium 1mg ml , cefdinir 1 mg ml and the seventh part considered as a o control group without antibiotic addition.The diluted semen samples were cooled and preserved at 5 C for 5 days.Cooled diluted semen samples were examined for individual motility, percent of live sperm, sperm abnormalities, acrosomal defects and bacterial count every 24 h until 5 days.Comparing with the control, all the o antibiotics examined were effective in controlling bacterial growth (P<0.05)from 24 h to 96 h of preservation at 5 C .Cefdinir and cefoperazone sodium proved to be significantly (P<0.05)effective than other antibiotics in 3 controlling bacterial growth at 96 h of preservation as the bacterial count were 23.3 ± 3.7 x 10 / ml and 25.4 ± 6.2 3 x 10 / ml, respectively.Lincomycin, gentamicin sulphate and tetracycline proved ineffective in controlling 3 3 bacterial growth at 96 h of preservation as the bacterial count were 57.1 ± 20.1 x 10 / ml, 52.5 ± 29.4 x 10 / ml and 3 46.5 ± 8.8 x 10 / ml, respectively.The addition of tetracycline to diluted ram semen significantly reduced (P<0.05)sperm individual motility and percent live sperm and a significant increase (P<0.05)acrosomal defects was observed at 96 h of preservation in comparison to control and other antibiotics.Sperm viability was highly correlated with bacterial count in the control part of diluted semen (r = 0.794; P < 0.01).It could be concluded from the results of the present study that additions of cephalosporins (cefdinir or Cefoperazone sodium) at the A total number of 120 ejaculates were collected in the use of AI, it is important to control from the rams using an artificial vagina once a efficiently the population of micro-organisms in week.For collecting ejaculates, rams were the semen.
penned with ewes in estrus, in the presence of a The most available diluents used for storage handler with an artificial vagina.Ejaculates were of spermatozoa are particularly supportive of evaluated and accepted to include in this study, if microbial growth, it is necessary to include the following criteria were met: volume varying antibiotics to prevent massive proliferation of the 9 between 0.5-2 ml; sperm concentration of 2 × 10 microorganisms present in ejaculated ram semen.sperm/ml; the motile sperms percentage higher The inclusion of antibiotics is necessary whether than 70% and less than 10% abnormal sperm in the spermatozoa are stored short term in liquid total.medium.Antibiotics are added to mostly used Semen analysis : The volume of each ejaculate semen diluents as a prophylactic measures was recorded and sperm concentration was against transmission of pathogenic bacteria as determined using semen diluted with 3% NaCl, well as to reduce the load of non-pathogenic the diluted semen was placed on a hemocytometer organisms that contaminate the semen.In ovine with the sperm counted in five squares of one artificial insemination, benzyl penicillin and chamber.Sperm motility was identified as those streptomycin are the most widely used antibiotics sperm cells that demonstrated progressive (Salamon and Maxwell, 2000).Some problems motility.Sperm motility was scored from zero to with resistance of bacteria in ram semen have 100% by a qualified and experienced investigator.been noted (Ahmad et al., 1999).A new Semen was placed on a heated glass slide, and combination of antibiotics comprised of scoring was performed at microscopic magnicephalosporins and gentamicin has been used fication of 200X.Each sample was evaluated successfully to control certain resistant microtwice.The mean value was used for data analysis.organisms in liquid preserved ram semen (Yániz Assessment of abnormal and normal spermatozoa et al., 2010).The objectives of the present study was performed using an eosin-nigrosin staining were to determine the effects of six different method.For the percent of spermatozoa with antibiotics in controlling growth of semen abnormal acrosomes, fast green stain was used.contaminating bacteria and if these antibiotics

Dilution of semen and addition of antibiotics :
have any adverse effect on Awassi ram spermatozoa.
Semen samples were diluted by sodium citrate-

Materials and methods
fructose-egg yolk (sodium citrate 2.9 g, fructose 1 g, double distilled water 80 ml and egg yolk 20 Animals and semen collection: Semen samples ml).Semen quality was re-evaluated to ensure from six mature Awassi rams (2-3 years of age) that the dilution has not affected the semen used in this study, and maintained using conventional quality.The diluted semen sample was divided feeding, housing and lighting conditions.The into 7 parts.Six types of antibiotics were added to bacteria present in each Petri dish was calculated semen diluent parts including penicillin G 1000 by multiplying the number of colonies with the -1 -1 dilution rate at which the colonies developed.IU ml with streptomycin 1 mg ml , gentamicin -1 -1 sulphate 250 mg ml , tetracycline 0.5 mg ml , Statistical analysis : Data were expressed as -1 lincomycin 1 mg ml , cefoperazone sodium 1mg means (±S.E.) and statistical analyses were -1 -1 ml , cefdinir 1 mg ml and the seventh part performed with the software (Sigma Stat, Jandel considered as a control group without antibiotic scientific software V2.0, Richmond, CA, 2004).addition.The diluted semen samples were cooled The differences between means of the same o and preserved at 5 C for 5 days.Cooled diluted parameter were tested by the analysis of variance semen samples were examined for individual (ANOVA) and least significance differences motility, percent of live sperm, sperm abnormalities, (LSD).For the determination of correlation acrosomal defects and bacterial count every 24 h coefficient between bacterial contamination and until 5 days.sperm viability changes of two variables, the Pearson product moment correlation analysis Bacteriological count : For the determination of was used.viable bacterial count, the medium was prepared

by dissolving 23 g of dehydrated nutrient agar in 1 L of deionized doubled distilled water and
Results of the present study showed a heating it to boiling point.The medium was decreased viable bacterial count of all samples autoclaved at 121°C under 15 lb/inch pressure for o including control part stored at 5 C for 72 h of 30 minutes.By cooling down to 50-55°C 10% preservation.Comparing with the control, all the fresh de-fibrinated sheep blood was added and antibiotics examined were effective in controlling subsequently poured in sterilized Petri dishes.In bacterial growth (P<0.05)from 24 h to 96 h of order to check the sterility of media, Petri dishes o preservation at 5 C. Cefdinir and cefoperazone were kept in incubator at 37°C for 24 hours.The sodium proved to be significantly (P<0.05)effective viable bacterial count in all diluted semen than other antibiotics in controlling bacterial samples was determined by using spread plate growth at 96 h of preservation as the bacterial method (Harry and Paul, 1981).As a procedure, 3 3 cont were 23.3 ± 3.7 x 10 / mland 25.4 ± 6.2 x 10 / 0.1 ml of diluted semen was added into a test tube ml, respectively, as shown in Table -1.having 0.9 ml of phosphate buffer solution Lincomycin, gentamicin sulphate, and stepwise 1:10, 1:1000, and 1:10.000dilutions tetracycline proved ineffective in controlling were obtained.By using double set of Petri dishes bacterial growth at 96 h of preservation as the for each dilution, 0.5 ml from each diluted 3 bacterial cont were 57.1 ± 20.1 x 10 , 52.5 ± 29.4 samples was spread on nutrient agar plates.The x 10 and 46.5 ± 8.8 x 10 , respectively.The Petri dishes were then incubated at 37°C for a difference in bacterial count between penicillin period of 24 hours and a colony counter counted with streptomycin and cephalosporins (cefdinir the number of colonies that arose.The number of

Streptomycin
Means for each parameter in the same column, with different superscript differ significantly (P < 0.05).
Influence of addition of antibiotics in semen diluent on viable bacterial count and spermatozoal viability of Awassi ram and cefoperazone sodium) used in this study was simultaneously resistant to penicillin and streptomycin, the most common preservative antibiotic not significant.The addition of tetracycline to diluted ram semen significantly reduced (P<0.05)combination used in ovine semen diluents.sperm individual motility and percent live sperm Results of this study indicated that dilution of ram and a significant increase (P<0.05)acrosomal semen in sodium citrate-fructose-egg yolk defects was observed at 96 h of preservation in diluent with antibiotics has resulted in significant comparison to control and other antibiotics.(P<0.05)decrease in the viable bacterial count Higher sperm individual motility were observed compared to semen diluted in the same diluent in semen diluted with the addition of lincomycin without antibiotic (control).Decreased viable and cefdinir (63.6 ± 1.8 % and 62.9 ± 2.7 %, bacterial count of all samples including the respectively) which differ significantly (P<0.05)control stored at 5 C for 72 h indicate that storage from tetracycline and gentamicin sulphate (37.5 ± of semen at 5°C affected the viable bacterial 2.9 % and 47.9 ± 2.7 %, respectively).count.Sperm motility, percent live sperm, sperm These results are in accordance with the abnormalities and sperm acrosomal defect were findings of Qureshi et al. (1993) in diluted bull not significantly affected in diluted semen with semen.Tetracycline addition in our study led to cefoperazone sodium, cefdinir, lincomycin, penicillin lower individual motility and higher acrosomal with streptomycin and control at 96 h, but the defects in comparison to the addition of other preservation had lowered semen quality (as antibiotics in the same diluent used in the present measured by sperm motility, percent live sperm study.Similarly, Shin et al. (1988) and Alaviand abnormal morphology) more effective than Shoushtari et al. (2007) demonstrated that the use antibiotics.Sperm viability was highly correlated of tetracycline in the buffalo semen diluent had a with bacterial count in the control part of diluted harmful effect on spermatozoa.Antibiotics with semen (r = 0.794; P < 0.01).Other ram semen higher antimicrobial activities in the present diluted parts containing antibiotics showed no study were cefdinir and cefoperazone sodium.correlation coefficient between semen viability Their inclusion in the composition of ram semen and bacterial contamination.
diluent have proven an effective inhibition of bacterial growth and no apparent adverse effects Discussion on spermatozoa with higher sperm individual Quality of the ejaculate and the diluted motility.These results are in agreement with the semen portion are fundamental for successful findings of Yániz et al. (2010) who achieved artificial insemination.Semen is qualified as good antimicrobial activity with addition of good when the there is a minimum contamination ceftiofur (third generation of cephalosporins). of bacteria (plus meeting the standards of motility Reports of the use of cephalosporins in the and morphology).When the seminal dose is extension and cooled storage of ram semen are -1 contaminated, semen viability decreases within a scarce.Although lincomycin (1 mg ml ) showed short period of time sperm and death occurs.As a poor ability to inactivate bacterial growth in ram result, the risk of pathologies in the female reprosemen.However, higher rate of sperm individual ductive tract increases, resulting in impairment of motility was observed in the addition of lincomycin fertility.There are many efforts in order to to ram semen diluent.Other concentrations of substitute penicillin with streptomycin in egg this antibiotic should be investigated.yolk-based diluents by other antibiotics, to High correlation between sperm viability minimize the bacterial contamination to dilute and bacterial contamination in the control group sperm during a long-term storage (Maxwell and found in this study is in agreement with Moretti et al. (2009).Microorganisms can affect the semen Salamon, 1993; Salamon and Maxwell, 2000).
et al viability directly, causing the agglutination of Yániz .(2010) demonstrated that 13% of isolated bacteria from ram-diluted semen were motile sperm, reducing the ability of acrosome Kaur et al. 1986), and inducing animals were kept in open front barrens, were fed acrosome reaction (El-Mulla et al. 1996).Microbes individually with concentrated mixture of 1 kg can also have an indirect effect by producing per ram per day, and were given water ad libitum.toxins (Morrell 2006; Fraczek et al., 2007).Thus,