The Use of Antibiotics in Cryopreservation of Ram Sperm

Introduction

The increasing specialization of artificial insemination (AI) programs in sheep production demands that sperm collection and processing centers (SCPC) become adjusted to the requirements of regulatory agencies. The World Organization for Animal Health (OIE, 2009) recommends the inclusion of antibiotics in extenders used in cryopreservation of sperm, in order to control bacterial contamination. In Brazil, these requirements are clearly defined for SCPCs that process sperm from bulls and water buffalos, but no specific regulation is established for ram sperm.

The inclusion of antibiotics in extenders is recommended for the control of several microorganisms that can be present in sperm (Bielansky, 2007; Thibier and Guerin 2000). Although opportunistic contaminants generally do not incur an important health risk for the inseminated females, they can affect sperm quality (Yániz et al., 2010). Antibiotic associations capable of controlling potentially pathogenic agents have been determined for bull sperm, having no negative effects on sperm quality (Lorton and Sulivan, 1988; Shin et al., 1988).For ram sperm, the determination of antibiotic associations mainly focused on cooled sperm (Yániz et al., 2010), and demonstrating different levels of antibiotic resistance during preservation of cooled sperm (Yániz et al., 2010; Azwi, 2012). In cryopreserved sperm, agents such as Brucella ovis and Actinobacilus seminis can be controlled by the inclusion of gentamicin or the association of penicillin and streptomycin in extenders (Moustacas et al., 2010). One might consider that bacterial populations and their levels of resistance can vary, and that the efficiency can be affected by extender components (Azwi, 2012). Additionally, the direct effect of antibiotics on sperm viability should also be considered (Lorton et al., 1988; Yániz et al., 2010; Azwi, 2012).

The objectives of this study were to determine the efficiency of antibiotics in controlling bacterial growth on cooled and thawed ram sperm, and their potential effects on sperm viability after cooling and thawing.

Materials and methods

Sperm Processing and Evaluation

Five 3-4 years old sexually mature Corriedale rams were used as sperm donors, having the follow sperm characteristics: total sperm motility of at least 70%; and score (0-5) of sperm vigor equal or greater than 3. The rams were kept under semi-extensive conditions at the Universidade Federal de Pelotas (UFPel: 31°48’17.91″S; and 52°25’4.36″W), having access to native pasture, mineral salt and water ad libitum during the daytime and being fed concentrated supplements twice a day. At night, the rams were housed in barns covered with wood shaving bedding.

Prior to sperm collection, the preputial ostium was cleaned with sterile saline (0.9% NaCl) and paper towel. Then, a sample of the preputial content was collected with a swab. Sperm collection was performed using an artificial vagina, twice a week for six weeks. All experimental procedures used in this study were approved by the UFPel Ethics and Animal Experimentation Committee (Registration No. 4383/2010).

Sperm samples were submitted to microbiological evaluation prior to dilution with tris-egg yolk extender (Evans & Maxwell, 1987). After dilution, samples with 2 x 109 spermatozoa/mL (determined in a Neubauer chamber) were pooled, so each ram contributed equally to the final sperm concentration. Sperm samples were subsequently diluted in an extender containing: 300 mM Tris-hydroxymethyl-aminomethane; 30 mM fructose; and 94.7 mM citric acid monohydrate (Evans & Maxwell, 1987). After that, sperm samples with concentration of 4 x 108 spermatozoa/mL were split in the following treatments: Control, with no antibiotics; PES, consisting of 100,000 IU/mL penicillin and 100 µg/mL streptomycin (Milli-Q water diluted penicillin g benzathine and streptomycin sulphate, Sigma Chemical Company, St. Louis, MO, USA) (Salamon and Maxwell. 2000); GTLS, consisting of 500 µg/mL gentamicin (Gentrin, Ourofino Agronegócio, São Paulo, SP, Brazil), 100 µg/ml tylosin (Tylan 200, Elanco Animal Health, São Paulo, SP, Brazil), 300 µg/mL lincomycin and 600 µg/mL spectinomycin (LincoSpectin, Pfizer Animal Health, São Paulo, SP, Brazil) (Lorton and Sulivan, 1988); CEF, consisting of 50 µg/mL ceftiofur sodium (Excenel, Pfizer Animal Health, São Paulo, SP, Brazil) (Guilmoto and Regault, 1996) and; ENR, consisting of 1000 µg/mL enrofloxacin (Kinetomax, Bayer Animal Health, Rio de Janeiro, Brazil) (Tomczyk and Minta, 2002). All treatments were also tested at 50, 75, 125 and 150% of the recommended dose (considered as 100%). Thus, considering the control, 21 treatments were tested. Glycerol (Sigma-Aldrich, St. Louis, MO, USA) was added to the extender, at a final concentration of 5%. For each treatment, five 0.25 mL straws containing 50 x 106 spermatozoa were cooled at 5 °C for 2 h and subsequently frozen in vapor of liquid nitrogen (Salamon and Maxwell, 2000). Samples were thawed in water bath at 37o.C during 30 s.

Parameters of sperm quality were evaluated after 2 h of cooling at 5 °C and also after thawing, keeping the samples incubated in a water bath at 37 °C for 10 min. Sperm motility was determined with an optical microscope equipped with heating stage at 37 °C (Olympus BX 51, Sapporo, Japan). Sperm membrane and acrosome integrity were both determined with an epifluorescence microscope (Eclipse 80i, Nikon Instruments Inc., Melville, NY, USA). Carboxifluorescein diacetate and propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) were used to evaluate membrane integrity (Harrison and Vickers, 1990), whereas propidium iodide and lectin from Arachis hypogaea conjugate (Sigma-Aldrich, St. Louis, MO, USA) were used to evaluate acrosome integrity (Kawamoto et al., 1999).

Microbiological Evaluation and Antibiogram

Microbiological evaluation was done to determine the bacteria present in samples from the preputial ostium, in the extender with no antibiotics and in fresh, cooled and thawed sperm. The samples (swabs) were streaked onto agar with 5% sheep blood and incubated at 37 °C in aerobiosis for 48 h. After incubation, the colonies were visualized and a Gram stain performed (Maza et al., 2004). The different types of colonies were isolated and incubated for 24 h in 10 mL tubes with Brain Heart Infusion (BHI) medium (Acumedia, São Paulo, SP, Brazil). Then, the culture were spread in the surface of the culture media with a Drigalski loop in the Petri plates containing the following media Brain Heart Infusion Agar (BHI, Acumedia®), Chapman (Acumedia®) and MacConkey (Acumedia®), in duplicate, respectively. The plates were incubated at 37 ºC for 24 h. Then bacteria growth (colony forming units, CFU/mL) counting was carried out. To evaluate the total bacteria numbers we consider the growth that was record in BHI, for Staphylococcithe growth in Chapman, and for Enterobacteriaceae the growth in MacConkey media.  The cultures were submitted to carbohydrate battery and Gram stain, respectively (Carter, 1988).

The number of CFU/mL was quantified in sperm samples collected at the end of the cooling period and after freezing-thawing. A 100 µL aliquot of diluted sperm was streaked onto BHI agar with 5% sheep blood and incubated at 37 °C for 48 h, as described above.

An antibiogram was performed using autoclave-sterilized paper filter disks impregnated with 2µL of the tested antibiotics (CLSI, 2009). The antibiotic concentrations in the disks were: 10µg penicillin and 40µg streptomycin; 88µg gentamicin; 40µg tylosin; 100µg lincomycin and 200µg spectinomycin; 100µg ceftiofur sodium; and 200µg enrofloxacin. The disks were distributed in Mueller-Hinton agar plates (Acumedia) previously streaked with the isolated colonies. Plates were kept in an incubator at 37 °C for 24 h and the inhibition halos were measured (CLSI, 2009).

Statistical Analysis

The Shapiro-Wilk test indicated that no responses of interest followed normal distributions. Therefore, sperm motility and integrity of sperm membrane and acrosome were transformed to the arcsine scale, and the number of CFU/mL was transformed to the logarithmic scale. All parameters were compared across treatments by analysis of variance, with comparisons of means by Tukey’s test. The results referring to parameters of sperm quality were reported in their original scale for the sake of interpretation. All statistical analyses were conducted with (Statistix®, 2009).

Results

For fresh sperm, motility was 80.3 ± 1.1%, membrane integrity was 56.8 ± 6.5% and acrosome integrity was 76.6 ± 23.9%. After cooling at 5 ºC for 2 h, motility was 70.9 ± 3.0 %, membrane integrity was 69.5 ± 1.7% and acrosome integrity was 82.5 ± 2.3 %. Membrane and acrosome integrity did not differ across treatments (P > 0.05) for cooled sperm. However, even though motility of cooled sperm was generally similar among treatments, it was lower for 150% GTLS and for ENR at both 125% and 150% (P0.05), with concentrations range between 50-125%. However, at 150%, all three showed a reduced CFU/mL compared with the others concentrations and control (Table 1). No difference in CFU/mL was observed for CEF (P>0.05), among the tested concentrations.

After thawing, sperm motility was 33.4 ± 2.3%, membrane integrity was 18.2 ± 1.1% and acrosome integrity was 73.5 ± 2.3 %. Sperm motility for PES, GTLS and CEF was similar (P>0.05) to that of the control group (Table 1). However, with ENR at doses greater than 0.5µg/mL, sperm motility was reduced as compared to the control group (P<0.05). After thawing, the integrity of sperm membrane and acrosome, and the number of CFU/mL did not differ among treatments (P<0.05).

Genera such as, Staphylococcus sp., Klebsiella sp., Corynebacterium sp. and Bacillus sp. were identified in samples from fresh sperm and preputial ostium. Bacterial growth was not significant (less than 30 CFU/mL) in samples of extenders without antibiotics. In the antibiogram, Staphylococcus sp. and Klebsiella sp. presented resistance to tylosin and to the penicillin and streptomycin association.