Effect of antibacterial agents in semen extender on bacterial growth in extended canine semen held at 5 o C and 20 o C for up to 48 hours Carla Barstow, Margaret V. Root Kustritz College of Veterinary Medicine, University of Minnesota, St. Paul, MN Abstract It has long been believed by some theriogenologists and dog breeders that the antibiotics in commercial semen extenders would control the growth of bacteria introduced with the semen sample. However, manufacturers include those antibiotics to prolong the product shelf life, not for the inhibition of bacterial growth. We hypothesized that the growth of aerobic, anaerobic and Mycoplasma species will be controlled in semen extended with commercial canine extender when held at refrigerator (5 C) or room (20 C) temperatures for up to 48 hours. Semen was collected by manual ejaculation from 14 dogs and the semen split into 11 aliquots. One aliquot each was submitted for aerobic, anaerobic, and mycoplasma cultures. Half of the remaining semen from each dog was extended 1:1 by volume with Fresh Express (Synbiotics, Kansas City, MO) and the other half was extended 1:1 by volume with CaniPro ApX2 Chill 5 (Minitube, Verona, WI). The extended semen was submitted for all three cultures immediately after addition of extender, and at 24 and at 48 hours after extension; half of the semen in each extender was held at 5 C and the rest at 20 C. Bacterial growth was compared between extenders, between storage temperatures, and over time. There was no significant growth in any sample held at 5 0 C. There was bacterial growth in some samples held at room temperature. Percentage progressively motile spermatozoa in a given sample is not a reflection of bacterial growth in the sample. Introduction Biosecurity is a growing social concern. While there is a significant body of research and there are strong regulatory controls for handling, storage, and transport of semen from large animal species, there is little in the veterinary literature specifically about biosecurity concerns for breeding dogs and there are no regulations governing movement of canine semen within the United States. Semen cannot be collected by manual ejaculation without bacterial contamination due to presence of normal bacterial flora on the urethral mucosa. Organisms also can be shed into semen as a component of prostatic or testicular fluid or by hematogenous spread from systemic infection. Not all organisms that may infect the testes may pass into semen and not all organisms shed into semen remain viable in semen. Semen extenders are liquid media that support spermatozoa through changes in temperature and provide nutrients and buffering capacity to offset acid-base changes as the spermatozoa undergo normal metabolism during storage. Antibiotics are added to semen extenders to maintain the shelf-life of the product and are not intended by the manufacturer to control bacterial growth. In stallions, it has been demonstrated that addition of gentamicin, polymyxin B, amikacin, streptomycin, or potassium penicillin were effective at controlling growth of aerobic bacteria in semen held at refrigerator temperature but not in semen held at room temperature. 1,2 Studies specifically looking at Mycoplasma species in bulls and stud dogs have shown ability of extenders containing a mixture of tylosin, gentamycin, lincomycin, and spectinomycin to decrease but not eradicate growth of Mycoplasma over 72 hours in semen held at 5 C. 3,4 In a study evaluating growth of Brucella canis added to extended semen with or without additional antibiotics, it was demonstrated that bacterial growth was not negated by presence of antibiotics. 5 Veterinarians and dog owners should not assume that the antibiotics in semen extender can control bacterial growth, even in samples that are handled properly during shipment. Significant growth of aerobic bacteria from semen is defined as growth of greater than 10 5 CFU/ml. Organisms most commonly cultured from semen from populations of normal dogs and dogs with history of reproductive tract disease were beta-hemolytic Streptococcus sp., Pasteurella multocida, beta-hemolytic E. coli, non-hemolytic E. coli, beta-streptococcus sp., Achromobacter xylosoxidans, Actinomyces pyogenes, Bacillus sp., coagulase-positive Staphylococcus sp., Hemophilus hemoglobinophilus, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, and 231 Clinical Theriogenology Volume 6, Number 3 September 2014
Staphylococcus intermedius). 6-8 In one study, 28.4% of samples from dogs presented for breeding soundness examination (n=39), infertility (n=25), or reproductive tract disease (n=30) yielded significant growth of aerobic bacteria. 6 In a longitudinal study of 15 fertile male dogs, 31.2% of samples yielded significant growth of aerobic bacteria. Transfer of bacteria to bitches at the time of copulation was reported in the latter study. 8 Any growth of anaerobic bacteria from semen is considered significant. In one study, there was significant growth of anaerobic bacteria from 13.7% of samples submitted from 95 dogs, with the most common organisms identified being Bacteroides sp., Peptostreptococcus sp., Propionibacterium acne, Clostridium perfringens, Fusobacterium necrophorum, Propionobacterium avidum, and Streptococcus morbillorum. 6 Mycoplasma sp. are part of the normal flora of the distal urethra in dogs. Quantitative culture is difficult so many laboratories report only positive or negative results. In one study, positive growth of Mycoplasma sp. was reported from 57.9% of samples submitted from 95 dogs. 6 In another study, 72% of semen samples were positive for Mycoplasma sp., with higher isolation rate from dogs with a history of infertility or reproductive tract disease. 9 This study is an attempt to mimic what can happen in the real world. With semen being shipped across the country, it is imperative for the receiving veterinarian and brood bitch owner to know that the semen product they are using is safe. Shipments may have been in a hot truck all day with melted ice packs or worse yet, may be left in an uncontrolled environment. The hypothesis evaluated in this study is that growth of aerobic, anaerobic and Mycoplasma species will be controlled in semen extended with commercial canine extender when held at refrigerator (5 C) or room (20 C) temperatures for up to 48 hours. Materials and methods Male dogs were recruited for the study from local breed clubs. Semen was collected from all dogs by the authors. Only dogs that produced at least 6 ml of semen were included in the study. Males were manually collected without the use of a teaser bitch. They were collected using a latex collection device that was washed and dried in between collection of the males, and each dog s ejaculate was collected into a separate sterile centrifuge tube. Immediately after collection, 1mL of semen was placed into anaerobic transport medium and 1mL of semen was placed into the aerobic and mycoplasma/ureaplasma transport container. These were the neat samples. The remaining volume of semen was divided in half and diluted 1:1with semen extender. Half of the semen was extended with Fresh Express and the other half was extended with CaniPro ApX2 Chill 5. One ml of each extended semen sample was then submitted for anaerobic, aerobic and mycoplasma/ureaplasma. These were the Time 0 extended samples. The Fresh Express extended semen was then divided in half. Half of the samples were held at room temperature (20 C) and the other half were stored in the refrigerator (5 C). The same was done for the CaniPro ApX2 Chill 5 samples. After 24 hours semen was submitted for aerobic and mycoplasma/ureaplasma from the Fresh Express semen that was kept at 5 C and 20 C and the CaniPro ApX2 Chill 5 semen that was kept at 5 C and 20 C. After 48 hours semen was submitted for aerobic and mycoplasma/ureaplasma from the Fresh Express semen that was kept at 5 C and 20 C and the CaniPro ApX2 Chill 5 semen that was kept at 5 C and 20 C (Figure). Because the samples would be exposed to air while keeping the samples at their appropriate temperatures, further anaerobic testing was not performed, beyond the neat and Time 0 samples, as those results would have been expected to be negative for growth. All samples were plated at a commercial laboratory using a standard three streak isolation technique and results were provided by microbiologists at that laboratory (Marshfield Laboratories, Marshfield WI). The motility of sperm in these samples was checked two hours after collection after having been held at room temperature and after removal of the aliquots required to complete the study. Further assessment of sperm motility, at 24 and 48 hours, was performed only on the three samples that had greater than 90% motility as neat samples. All refrigerated samples were warmed on the microscope Clinical Theriogenology Volume 6, Number 3 September 2014 232
stage for 10 seconds before evaluation. All motility assessments were made by one on of the authors (MVRK). Results Semen collection was attempted on 18 male dogs; semen was successfully collected from 14 of those dogs. Breeds represented were Samoyeds (n=6), Malamutes (n=6), and Springer Spaniel and Labrador retriever (n=1 each). Ages ranged from 2 years to 9 years, with a mean age of 4.5 years. Formulations of commercial canine extenders are proprietary. Fresh Express extender from Synbiotics corporation (www.synbiotics.com) contains several antibiotics while CaniPro ApX2 Chill 5 extender from Minitube (www.minitube.com) contains a single antibiotic, gentamycin. These are the two extenders most commonly used by small animal practitioners belonging to the Society for Theriogenology, as determined using an electronic survey. The commercial laboratory reported bacterial growth as scant, light, moderate or numerous. Culture results are shown in Table 1. Overall, 35% (n=5) of the dogs used in the study had significant growth of aerobic bacteria. Thirty-five percent (n=5) of the dogs also had anaerobic growth. Seventyeight percent (n=11) were positive for Mycoplasma growth and 50% (n=7) were positive for Ureaplasma growth at least once during the study. Aerobic growth was considered significant if it was deemed as moderate to numerous. In the aerobic neat samples, 5 dogs had growth that was significant. The most commonly identified bacteria were E. coli, beta hemolytic Streptococcus group G, mixed urogenital/skin flora and Enterobacter aerogenes. Any growth of anaerobic bacteria was considered significant. In the anaerobic neat samples, there was no growth. Samples from five dogs yielded Mycoplasma and samples from five dogs yielded Ureaplasma sp.; samples from some of the dogs yielded both. In the Time 0 samples with Fresh Express none of the dogs had aerobic growth and four dogs had anaerobic growth, with Bacteroides fragilis, Clostridium perfringens and a gram negative bacillus (most likely Hemophilus and Pasteurella), being the most commonly isolated. Samples from four dogs yielded Mycoplasma and samples from two dogs yielded Ureaplasma sp. In the Time zero samples with Chill 5 had one dog with aerobic growth, beta hemolytic Streptococcus group G, and two dogs had anaerobic growth with a gram negative bacillus (most likely Hemophilus and Pasteurella) and Propionibacterium acne. Samples from five dogs yielded Mycoplasma and samples from four dogs yielded Ureaplasma sp. In the 24 hour samples kept under refrigeration in Fresh Express, there was no growth of aerobic or anaerobic bacteria which were cultured for separately from mycoplasma and ureaplasma organisms. Samples from two dogs yielded Mycoplasma and samples from five dogs yielded Ureaplasma sp. In the 24 hour samples kept at room temperature in Fresh Express one dog yielded Enterococcus species. Samples from one dog yielded Mycoplasma and samples from three dogs yielded Ureaplasma sp. In the 24 hour samples kept under refrigeration in Chill 5, there was no growth. Samples from eight dogs yielded Mycoplasma and samples from six dogs yielded Ureaplasma sp. In the 24 hour samples kept at room temperature in Chill 5, there was no growth. Samples from seven dogs grew Mycoplasma and samples from five dogs yielded Ureaplasma sp. In the 48 hour samples kept under refrigeration in Fresh Express, there was no growth of aerobic or anaerobic bacteria which were cultured for separately from mycoplasma and ureaplasma organisms. Samples from six dogs yielded Mycoplasma and samples from six dogs yielded Ureaplasma sp. In the 48 hour samples kept at room temperature in Fresh Express, samples from two dogs had growth, with E. coli and Enterococcus faecalis. Samples from four dogs yielded Mycoplasma and a sample from one dog yielded Ureaplasma sp. In the 48 hour samples kept under refrigeration in Chill 5, there was no growth. Samples from seven dogs yielded Mycoplasma and samples from four dogs yielded Ureaplasma sp. In the 48 hour samples kept at room temperature in Chill 5, the sample from one dog had growth of E. coli. Samples from four dogs yielded Mycoplasma and samples from four dogs yielded Ureaplasma sp. Percentage progressively motile spermatozoa in neat samples ranged from excellent to poor; with half of the dogs having acceptable motility (greater than 70%; Table 2). One dog was azoospermic. 233 Clinical Theriogenology Volume 6, Number 3 September 2014
The percentage progressively motile spermatozoa in the three dogs that were followed for the duration of the study declined during the first 24 hours. Change in percentage progressive motility was variable thereafter, with individual dog difference in the different extenders and at differing temperatures (Table 3). At the 48 hour mark, only 3 of the 12 samples would be deemed to have adequate motility ( 50%) in either the refrigerated or room temperature samples (Table 3). Discussion This study had several limitations. A larger sample size with greater breed diversity would have better reflected the population of dogs from which semen is collected and shipped in the United States. Adding a neat sample as an un-extended control to determine if the bacterial population changed over the 48 hour time period and the differences noted between refrigeration and room temperature also would have added value to the study. The authors chose not to add this sample because it does not mirror what happens in veterinary practice. A final limitation was lack of assessment of sperm motility in all samples immediately after collection and evaluation of samples from all dogs throughout the study. This would have better permitted the investigators to assess correlation between decreased motility and increased bacterial counts. Thirty-five percent of dogs had significant growth of bacteria in their semen; this is in accord with information from the literature. 1 Bacterial growth was controlled in samples that were held at refrigerator temperature, but not in all samples that were held at room temperature. One dog had growth of bacteria that were not originally present; this may be because the antibiotic in the extender killed the bacteria in the neat sample and allowed organisms that were present in very small amounts to proliferate. The same can be seen in the Time 0 extended samples; anaerobic bacteria that were not seen in the neat sample were able to proliferate without the competition of other bacteria. This may potentially be a concern, as more pathogenic bacteria can survive in the face of antibiotics. It also makes one concerned for the possibility of increasing bacterial resistance. There was no discernible pattern to growth of Mycoplasma and Ureaplasma sp. in this study. Extenders were not used in accordance with manufacturers protocols, which recommend extension of the sperm-rich fraction only, at a dilution of 1:3 to 1:5. Again, the authors chose to mimic veterinary practice. Results may have been different if the samples had been extended as the manufacturer recommends, as the resulting extended sample would automatically have a lower concentration of bacteria due to the effect of dilution. The authors note that reported percentage progressively motile spermatozoa in neat samples probably was not a true reflection of the dogs semen quality, as samples were handled multiple times and were held at room temperature for two hours before evaluation. One dog was azoospermic. This could have been due to the fact that he was nervous for the collection or would have responded better if a teaser bitch had been present; this was not pursued as it was outside the purview of the study. The percentage of progressive sperm motility that was determined throughout the study showed that the sperm responded differently to the two extenders and that motility in most cases was adequate at the 24 hour mark. Because the entire ejaculate, not just the sperm-rich fraction, was used and manufacturers protocols were not followed could explain why motility was less than that which may be seen in practice. The sperm did show some agglutination and it is not known what effect it would have on fertility. Conclusion Practicing veterinarians should use caution when using semen samples that may not have been handled appropriately or that arrive warm. If the sample arrives within the appropriate time and is still chilled, it appears that clinicians need not worry about excessive bacterial growth in those samples. Sperm motility was not affected by the presence of bacteria or Mycoplasma and Ureaplasma sp. therefore cannot be used to determine whether or not there are bacteria present in the sample. Clinical Theriogenology Volume 6, Number 3 September 2014 234
Acknowledgements The authors wish to thank Synbiotics and Minitube for donating the extender that was used in the study. References 1. Vaillancourt D, Guay P, Higgins R: The effectiveness of gentamicin or polymyxin B for the control of bacterial growth in equine semen stored at 20 C or 5 C for up to forty-eight hours. Can J Vet Res 1993;57:277-280. 2. Varner DD, Scanlan CM, Thompson JA, et al: Bacteriology of preserved stallion semen and antibiotics in semen extenders. Theriogenology 1998;50:559-573. 3. Visser IJR, TerLaak EA, Jansen HB: Failure of antibiotics gentamycin, tylosin, lincomycin and spectinomycin to eliminate Mycoplasma bovis in artificially infected frozen bovine semen. Theriogenology 1999;51:689-697. 4. Becher A, Spergser J, Aurich C, et al: Cold storage of canine semen: the in vitro effect of different concentrations of a combination of antibiotics on bacterial growth [abstract]. Proc Int Symp Canine Feline Reprod; 2012. 5. Makloski C, Lamm C, Love B: Bacterial growth and semen viability in canine semen extenders inoculated with Brucella canis [abstract]. Clin Therio 2012;4:423. 6. Root Kustritz MV, Johnston SD, Olson PN, et al: CJ. Relationship between inflammatory cytology of canine seminal fluid and significant aerobic bacterial, anaerobic bacterial or Mycoplasma cultures of canine seminal fluid: 95 cases (1987-2000). Theriogenology 2005;64:1333-1339. 7. Furneaux RW: Recurrent infertility and venereal transmission of infection in the dog. Aust Vet J 1968;44:101-102. 8. Bjurstrom L, Linde-Forsberg C: Long-term study of aerobic bacteria of the genital tract in stud dogs. Am J Vet Res 1992;53;670-673. 9. Doig PA, Ruhnke HL, Bosu WT: The genital Mycoplasma and Ureaplasma flora of healthy and diseased dogs. Can J Comp Med 1981;45:233-238. Figure: Schematic view of study Dog Collected Neat Sample: Anaerobic, Aerobic and Mycoplasma Time 0 Fresh Express: Anaerobic, Aerobic and Mycoplasma Time 0 Chill 5: Anaerobic, Aerobic and Mycoplasma Time 24 Fresh Express: Aerobic and Mycoplasma @ 5 0 Time 24 Fresh Express: Aerobic and Mycoplasma @ 20 0 Time 24 Chill 5: Aerobic and Mycoplasma @ 5 0 Time 24 Chill 5: Aerobic and Mycoplasma @ 20 0 Time 48 Fresh Express: Aerobic and Mycoplasma @ 5 0 Time 48 Fresh Express: Aerobic and Mycoplasma @ 20 0 Time 48 Chill 5: Aerobic and Mycoplasma @ 5 0 Time 48 Chill 5: Aerobic and Mycoplasma @ 20 0 235 Clinical Theriogenology Volume 6, Number 3 September 2014
Table 1: Culture results of the dog s semen neat, in both extenders at Time 0, 24 hour and 48 hours at 5 and 20 C. Animal Neat FE Time Chill 5 FE @ 5 FE @ 20 C5 @ 5 C5 @ 20 FE @ 5 FE @ 20 C5 @ 5 C5 @ 20 Number Time 0 0 Time 0 Time 24 Time 24 Time 24 Time 24 Time 48 Time 48 Time 48 Time 48 101 S1, L4 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 105 S1 15 S3 Neg Neg Neg Neg Neg Neg Neg Neg 106 S1, L2, M4 107 N2, M3, S1 S5, L6 S6 Neg Neg 15 15 Neg Neg Neg Neg S2, S3 L2, M3 S2, S3 S2 L2, L3 S2, L3 S2 N2 S2, L3 N2 108 S1, L3 S3, S6 S3 Neg Neg S3 Neg Neg Neg Neg Neg 112 L1, Neg Neg Neg 15, Neg Neg 113 L1, N2, M3, 15, 114 L1, M2, M3, 15 S1, L2, L7 S1, L2, L7 S1, L2, L3, 15, S1, L2, L3, 15 S1, L2 S1, L2 L2, L3, 15, S1, L2, 15, L2, L3, 15, S2, 15, L2, 15 S1, L2, L3, 15 L2, 15 L1, 15 S1, L2, 15 L2, 15 L2, 15 S1, L2, L3, 15 S1, L2, 15 115 L1 L8, S9 L1, 15 S1 S8, M10 S8 L1, 15 S1, 15 N11, 15 S1, 15 S8, S12 L2, 15 1 L1, 15 S3, L6, S12, 15 117 M1, 15, S1, L8 15, S1, L3, 15 S3, S12, 15, S1, L8, S1, S3, 15, S1, 15, 15, S1, L3, 15, 15, S8, S1, L8, 15, L1, L1, 15, L1, 15 L1, 15, L1, 118 L1, S1, S1, S1, S8, S1, 15, S1, S8, S13, S1 S1, 119 L1 Neg Neg Neg Neg Neg 15 15 Neg 15 Neg 120 L1, 15, 15 S14, 15 Neg 15, 15 Neg 15, 15 Key: S=Scant; L=Light; M=Moderate; N=Numerous; 1=Mixed Urogenital/Skin Flora; 2=E. coli; 3=Beta-hemolytic Streptococcus group G; 4=Enterobacter aerogenes; 5=Bacteroides fragilis; 6=gram negative bacillus (Hemophilus or Pasteurella); 7=Hemophilus hemoglobinophilus; 8=Staphylococcus warnei; 9=Clostridium perfringens; 10=Enterococcus sp.; 11=Enterococcus faecalis; 12=Bacillus sp.; 13=Coryneform bacilli; 14=Propionibacterium acnes; 15=Mycoplasma; =Ureaplasma Clinical Theriogenology Volume 6, Number 3 September 2014 236
Table 2: Percentage progressively motile spermatozoa in neat samples Animal Identification Motility results 101 50% 105 20% 106 Azoospermic 107 10% 108 >90% 112 >90% 113 70% 114 70% 115 60% 1 >90% 117 60% 118 20% 119 80% 120 70% Table 3: Percentage progressively motile spermatozoa from three dogs by extender, temperature, and time in storage DOG DOG 120 DOG 112 DOG 108 NEAT >90% >90% >90% FE C5 FE C5 FE C5 TIME 0 70 80 90 95 90 95 24 HRS 5 C 30 50 50* 35* 20 50 24 HRS 20-25 C 70 40* 10* 70* 55* 15* 48 HRS 5 C 25* 40* 0* 50* 0 0 48 HRS 20-25 C 50* 50* 5 10 10 0 *denotes sperm agglutination 237 Clinical Theriogenology Volume 6, Number 3 September 2014
Clinical Theriogenology Volume 6, Number 3 September 2014 238