Open access peer-reviewed chapter
Reproductive efficiency following insemination with spermatozoa after centrifugation through low-density Porcicoll [77].
Abstract
Antimicrobial resistance is a serious global threat requiring a widespread response. Both veterinarians and medical doctors should restrict antibiotic usage to therapeutic use only, after determining the sensitivity of the causal organism. However, the addition of antibiotics to semen extenders for animal artificial insemination represents a hidden, non-therapeutic use of antimicrobial substances. Artificial insemination for livestock breeding is a huge global enterprise with hundreds of million sperm doses prepared annually. However, reporting of antimicrobial resistance in semen is increasing. This review discusses the consequences of bacteria in semen samples, as well as the effect of antimicrobial substances in semen extenders on bacteria in the environment and even on personnel. Alternatives to antibiotics have been reported in the scientific literature and are reviewed here. The most promising of these, removal of the majority of bacteria by colloid centrifugation, is considered in detail, especially results from an artificial insemination study in pigs. In conclusion, colloid centrifugation is a practical method of physically removing bacteria from semen, which does not induce antibiotic resistance. Sperm quality in stored semen samples may be improved at the same time.
Keywords
- antimicrobial resistance
- assisted reproduction technologies
- semen preservation
- environmental bacteria
- food of animal origin
- livestock production
1. Introduction
Increasing use of antimicrobials is driving antimicrobial resistance (AMR), which is amongst this era’s defining global health challenges [1]. By 2030, global antimicrobial use in both humans and food producing animals is projected to increase to 236,757 tons annually [2]. Since the production of milk, meat and eggs requires healthy animals, the ability to treat bacterial infections effectively is of paramount importance to the human population, and therefore the spectre of AMR is an increasing threat in animal husbandry [3]. The latter authors report that a dramatic increase in antimicrobial use in animal production is primarily a consequence of intensive animal production systems introduced to meet growing global food demand [3]. Thus, the situation is complex, involving the interaction between humans, animals and the environment, i.e. One Health [4]. Therefore, an integrated approach to tackling AMR is required, coordinating efforts by the World Health Organisation (WHO), the Food and Agriculture Organisation (FAO) and the World Organisation for Animal Health (OIE) [5].
One area where significant quantities of antibiotics are used is in artificial insemination (AI), which is the method of choice for breeding food-producing animals in most parts of the world. The technique was developed originally to reduce disease transmission since it allows animals to be bred without coming into contact with each other or being transported to different farms for mating [6]. Additional benefits include safety and allowing access to more males, thus permitting more rapid genetic improvement than is possible with natural mating [7]. Success with the technique is dependent on a number of factors, especially a readily available supply of good quality semen. However, regulations stipulate that antibiotics should be added to semen doses for international trade, which contradict current recommendations on prudent use of antimicrobial substances. The latter state that antibiotics should be used only for therapeutic purposes, and only after determining the susceptibility of the causative agent to the relevant antibiotic [8]. Therefore, surprisingly, the subject of widespread use of antibiotics in semen extenders for all animal species has received little attention [9].
The questions surrounding this issue that are relevant to animal breeding are: do we need antibiotics in semen extenders? Is this use of antibiotics in line with recommendations for prudency? Are their alternatives to antibiotics for semen extenders? The purpose of this review is to provide some clarity for these questions.
Advertisement
2. Addition of antibiotics to semen extenders
2.1 Origin of bacteria in semen samples
Almost all semen samples contain some bacteria. The mucosa of the reproductive tract becomes colonised with microbes from the environment and from the animal itself [10]; these bacteria are transferred to semen during ejaculation [11]. The cleanliness of the environment in which the animals are kept plays an important role in the extent of the contamination. Thus, in stables where the bedding was changed every day, there was less microbial contamination of stallion semen than where the bedding was changed less frequently [10].
Post-collection contamination of semen by bacteria from the environment or from personnel can occur during processing; therefore, strict attention to hygiene is required during the whole process. Exposure of sterile semen extender to the air in a semen processing laboratory for a short period followed by culture resulted in the growth of 2000 colony forming units/mL (cfu/mL)
2.2 Effect of bacterial contamination
Bacteria not only compete with spermatozoa for nutrients; they could produce metabolic byproducts and toxins, thus reducing the “shelf-life” of semen doses [9]. Therefore, sperm quality may decline in the presence of bacteria. Indeed, bacteria in semen were shown to be negatively associated with sperm quality and fertility [14]. Bacteria may even cause infection and infertility in the female after insemination, or result in the spread of infection [15].
Since the semen extender used to maintain sperm viability also acts as a nutrient medium for bacteria, the contaminating bacteria are in a favourable environment for multiplication. Although bacterial growth is temperature-dependent, some bacteria continue to grow to some extent during cooling. Most commensal bacteria are mesophiles, with an optimum temperature range between 20 and 40°C. They stop growing at temperatures below 15°C, although some can continue to multiply to some extent at lower temperatures [16]. Therefore, one method for inhibiting bacterial growth in semen is to reduce the temperature. However, spermatozoa are sensitive to cooling below 20°C, with species-specific differences in their susceptibility to “cold shock” [17]. Although protocols have been developed to maintain bull sperm survival during cryopreservation with good results, efforts to achieve a successful outcome for other species have met with more variable success. Thus, although it is possible to freeze semen from at least some boars, pregnancy rate and litter size are lower than with liquid semen, providing little incentive for pig breeders to use frozen semen [11]. Post-thaw sperm motility and fertility is acceptable in some stallion ejaculates, whereas others have very poor post-thaw motility and fertilising capacity. On the other hand, artificial insemination with cooled stallion semen the day after semen collection results in a per cycle pregnancy rate of approximately 65% [18]. Therefore, the majority of artificial inseminations in horses currently use cooled semen rather than cryopreserved semen.
It should be noted that bacteria can survive to some extent in frozen semen samples, resuming growth after thawing. Therefore, the time between thawing semen and insemination should be kept to a minimum and strict hygiene measures observed at all times.
To avoid these negative effects of bacterial contamination, regulations governing international trade in semen for artificial insemination specify that antibiotics must be added to extended semen, e.g. in Appendix C of the Council of Europe [19]. These regulations state “Where antibiotics or a mixture of antibiotics are added, their bactericidal activity must be at least equivalent to that of the following mixtures in each ml of semen: gentamicin (250 μg), tylosin (50 μg), lincomycin-spectinomycin (150/300 μg); penicillin (500 IU), streptomycin (500 μg), or amikacin (75 μg), divekacin (25 μg)”. Therefore, all semen doses for international trade should contain antibiotics. National regulations governing insemination doses destined for the domestic market may be less rigid; thus, antibiotics might not be added to semen that is to be used for AI on the same premises soon after collection (e.g. 30 minutes). However, it is customary to add antibiotics to all semen doses that are to be cooled and transported to other premises for AI.
2.3 Resistance to antibiotics in semen extenders
Bacteria have been cultured from bull [20, 21] and boar semen [22, 23] despite the presence of antibiotics. Gentamicin, tylosin, spectinomycin and lincomycin did not inhibit the growth of bacteria in bull semen [24] although no growth occurred in semen samples containing ceftiofur/tylosin or ofloxacin. There are anecdotal reports that additional antibiotics are added to boar semen extenders if resistance is suspected, leading to even faster development of antibiotic resistance. Growth of bacteria was reported from stallion semen frozen in extender containing amikacin in a Portugese study [25]. Hernández-Avilés et al. [26] observed that several antibiotics were effective against low levels of
Resistant bacteria have been isolated from the reproductive tract of mares in several studies over the last 20 years (reviewed by Malaluang et al. [28]), although semen extenders may not be the only source of antibiotics in the reproductive tract if the mares had been treated for fertility problems or other diseases. A change in the AMR patterns of vaginal bacteria after exposure to antibiotic in semen extenders was reported in horses [29]. Apart from exposure of the vaginal flora to antibiotics, bacteria in the environment are also exposed since the bulk of the inseminated fluid (semen extender and seminal plasma) is expelled from the reproductive tract via backflow. It is not known how much exposure to antibiotics is needed for AMR to develop: some authors consider that even a small exposure to antibiotics is sufficient to induce AMR [30] whereas others are of the opinion that only therapeutic concentrations are needed. However, the duration and length of exposure are likely to contribute to the resistance-inducing potential of the substance. Since environmental pollution events, such as incorrect disposal of antimicrobials, can result in AMR [31], it is essential that any antibiotic-containing substances are destroyed correctly, i.e. by boiling or incineration [32]. Thus, it is particularly important that unused semen extender and semen doses are not poured down the drain, since they could affect environmental bacteria and thereafter be transferred to humans or animals.
2.4 Potential spread of antimicrobial resistance to personnel
Resistance to antimicrobial substances is passed between bacteria, regardless of where these bacteria are present. Therefore, resistance genes can be transmitted between bacteria in different host species, such as between animals and people, with or without involvement of environmental bacteria [33]. If AMR develops in bacteria in the host animal, these resistant bacteria can be spread in the environment and to the human population. Some of the organisms that WHO is most concerned about in the human population i.e.
Transmission of diseases (and AMR) from animals to humans occurs through a variety of routes, although the food-borne route is probably the most important for enteric organisms, such as Salmonella and
This transfer of AMR is not only from animals to humans: tourists were thought to be the origin of antibiotic-resistant bacteria in the faeces of reptiles on the Galapagos Islands [40]. A recent review on MRSA concluded that continued resistance in farm animals was likely to be due to contact with human carriers [39]. Therefore, it is advisable to try to restrict the development of AMR in livestock and horses, to protect both animal and human populations.
Advertisement
3. Prudent use of antimicrobial substances
As previously mentioned, the addition of antibiotics to semen extenders does not fit with the current recommendations for prudent use of antimicrobial substances, in the light of increasing incidence of AMR [41]. Although some of the bacteria isolated from semen have also been isolated from some cases of endometritis, they are not present in all cases. Therefore, there is no clear evidence of a
Advertisement
4. Alternative to antibiotics in semen extenders
4.1 Reducing the temperature
Low temperature extenders, although recently available for boar semen (e.g. [42, 43]), have not proved popular with the pig breeding industry. Cooling of the semen has to take place over several hours to avoid cold shock, thus interfering with established routines at the semen collection station. It will take time to replace the infrastructure currently in place to support storage at 16–18°C with refrigerators for storage at 4–6°C, and to replace room temperature transport with refrigerated transport. However, even if bacteria do not multiply during refrigeration, some of them will continue to grow and produce toxic substances during the period before refrigeration temperature is reached, as well as after removing the insemination dose from the refrigerator while preparing it for insemination [44]. Thus, the bacteria still have the potential to affect sperm quality and the health of the inseminated sow [15].
4.2 Unconventional antimicrobial substances
Other methods to inhibit bacteria in semen are based on the addition of unconventional antimicrobial substances, such as plant extracts [45, 46] or antimicrobial peptides [47]. However, addition of these novel antimicrobial agents does not preclude the emergence of bacterial resistance, as bacteria adopt new survival mechanisms to evade their effect.
4.2.1 Plant extracts
The effects of extracts from 45 plant species added to semen extenders are reported in the scientific literature, as reviewed by Ros-Santaella et al. [46]. Most of the beneficial effects were due to antioxidant activity but a few extracts also had antibacterial activity. Thus, rosmarinic acid was reported to have an antibacterial effect in boar semen [45, 46, 48, 49] but, according to another report, lacked such an effect in bull semen [50]. Whether these contrasting results reflect the species of animal, type of bacteria present or the source of the rosmarinic acid, or could be attributable to the development of resistance, is not known. Moringa had an antibacterial effect in bull and ram semen [51, 52]. Furthermore, it removed all bacterial contamination from banana shrimp spermatophores [53]; ginger had a similar effect. Tea tree oil exerted an antibacterial effect in boar semen [45, 54]. Omaji was reported to be effective against Gram positive bacteria in bull semen [55], although the minimum inhibitory concentration values shown were for cultured bacterial strains rather than on bacteria in semen.
Antimicrobial peptides are substances produced by the immune system of some mammals and are active against a range of microorganisms [56]. They have a cationic charge, exerting a selective action on negatively charged lipids in bacterial membranes [57]. A cationic peptide derived from human semenogelin was found to have antimicrobial activity [58], although the mechanism of action was not described. Another peptide, GL13K, was found to be active against
4.2.2 Nanoparticles
Nanoparticles were reported to have antimicrobial activity against certain bacteria [63]. The addition of iron oxide (Fe3O4) nanoparticles during boar semen processing was reported to produce a slight antibiotic effect with no adverse effects on sperm characteristics [64]. In contrast, although iron oxide nanoparticles were not toxic to ram spermatozoa, they did not have the desired antibacterial effect [65]. A combination of silver and iron oxide nanoparticles produced a greater antibacterial effect than iron oxide but showed higher spermatotoxicity. Therefore, more research is needed before these nanoparticles can be a contender to inhibit the presence of bacteria in semen. Interestingly, selenium nanoparticles [66] and zinc nanoparticles [67] were thought to improve membrane integrity of bull spermatozoa; although no microbiological analysis of the samples was reported, it is conceivable that the improved membrane integrity observed could have been due to an antimicrobial effect. A reduction in microbial activity would theoretically result in a reduction in the production of reactive oxygen species and hence a decrease in sperm membrane damage [9].
Other nanoparticles that have been investigated for their potential antibacterial effect in semen extenders include chitosan, a glycosaminoglycans that interacts with bacterial cell membranes causing lysis, together with ethylene diaminetetraacetic acid (EDTA), which increases the permeability of the cell wall of gram negative bacteria, and bestatin [68]. The combination was considered to have a bacteriostatic effect without causing a decrease in sperm quality.
4.3 Colloid centrifugation
Apart from potentially inducing AMR, one of the problems with antibiotics is that the killed bacteria remain in the sperm suspension after death. Therefore, intracellular substances and reactive oxygen species, or endotoxins from the lipopolysaccharides of the outer membrane of the cell wall of Gram-negative bacteria, are released into the extender and can have a negative effect on sperm quality. Intuitively, it might be better to remove the bacteria from the sperm sample rather than inhibiting their growth or killing them and leaving them in situ. One such method for separating spermatozoa from bacteria is colloid centrifugation [12, 69, 70]. The method used in these latter studies was a modified density gradient in which only one layer of colloid was used, hence its name “Single Layer Centrifugation” (SLC). This simplified method enables large volumes of semen to be processed easily [71]; even voluminous ejaculates can be processed, provided that the centrifuge rotor can accommodate large tubes. A detailed description of the methodology for 50 mL tubes can be found in Morrell and Nunes [72] and for 500 mL tubes in Morrell et al. [71].
4.3.1 Single layer centrifugation
Initially, SLC was shown to be effective in separating aliquots of boar sperm samples from bacteria in 12 ml tubes [69]. A scaled-up version of this technique was used with stallion semen samples that had been inoculated with various bacterial suspensions [12]. In this case, the usual SLC was modified by inclusion of an inner tube - a 5 mL plastic semen straw or similar tubing - inserted through a hole in the cap before loading the semen on the colloid, to facilitate retrieval of the sperm pellet after centrifugation. The sperm sample, inoculated with known amounts of
In the study by Al-Kass et al. [70, 74], split ejaculates were used, with antibiotic added to half of each ejaculate. This protocol enabled comparisons to be made of control samples with and without antibiotics, as well as SLC samples with and without antibiotics. Although there were clear differences in sperm quality between the SLC and corresponding control samples, in favour of SLC, there were few obvious differences between the samples with and without antibiotics within a treatment. In other words, the presence of antibiotics had little effect on sperm quality. The only exception was for the DNA fragmentation index, where the control samples with antibiotics showed more chromatin damage than control samples without antibiotics. Whether this increased damage was due to the effects of the antibiotics
The experiments by Al-Kass et al. [70, 74] show several important points. The first is that it is possible to remove most of the bacteria from stallion semen relatively easily using SLC. Second, any bacteria remaining in the sperm samples did not have an adverse effect on sperm quality during cooled storage for 96 h. Third, the addition of antibiotics did not enhance sperm quality during storage and, in the case of sperm DNA fragmentation, actually increased DNA damage in the control samples by 96 h. Thus,
Experiments with boar semen have progressed further. The economics of pig production are such that the cost of the high-density colloid used for sperm selection would be prohibitive for the industry. Thus, instead of using a high-density colloid to select robust spermatozoa from the rest of the ejaculate, a low-density colloid was tested to determine if the majority of the spermatozoa could be separated from the seminal plasma and its bacterial load [76]. Since the price of the colloid formulations is determined by the cost of the silane-coated silica base, using a formulation with a lower content of this material will be cheaper to manufacture than formulations with a higher density. Preparing the semen using the low-density colloid formulation for boar spermatozoa, Porcicoll, it was possible to remove most of the bacteria from boar semen, and sperm quality did not decline during subsequent storage of the processed samples [76]. In a small AI trial, pregnancy rates and litter sizes were not adversely affected by the low-density SLC-preparation of the sperm samples, and there were fewer mummified piglets in the litters derived from SLC sperm samples than in the controls (Table 1; [77]). At current prices (i.e. not adjusting for economies of scale) the cost of processing the whole ejaculate by low-density colloid would add approximately $2 to the price of each insemination dose. Economies of scale in the manufacture of the colloid could further reduce its cost. These results are very encouraging and a larger breeding trial in pigs is planned; the possibility of using this method with stallion semen should be investigated.
| No. of sows | No. Farrowing | Total born/litter | Live born/litter | Born dead/litter | Mummified/litter |
|---|---|---|---|---|---|
| Control (17) | 13 | 14.5 ± 5.1 | 13.6 ± 5.1 | 0.9 ± 1.0 | 1.1 ± 1.8* |
| SLC (12) | 9 | 16.0 ± 3.4 | 15.3 ± 2.9 | 0.8 ± 0.9 | 0.7 ± 0.71* |
Table 1.
p < 0.022.
4.3.2 Advantages and disadvantages of single layer centrifugation
At this point, it is worth considering both a risk–benefit analysis of the use of antibiotics in semen extenders (Table 2; [44]) and a cost–benefit analysis of SLC.
| Risk | Benefit | |
|---|---|---|
| Bacteria compete with sperm for nutrients, produce toxic byproducts and LPS that cause decreased sperm quality; can cause disease in inseminated females. | No antibiotic toxicity to spermatozoa; no risk of spreading antibiotic resistance. | |
| Can be toxic to spermatozoa; sperm quality may be affected by dead bacteria; bacteria in semen may become resistant; contamination of the environment with antibiotics from inseminated mares; can contribute to the spread of antibiotic resistance in animals and personnel. | Kill most contaminating bacteria before they have a chance to grow; no competition for nutrients in semen extender; bacteria are not transferred to the female via AI unless resistant. |
Table 2.
Risk–benefit analysis of antimicrobial substances in semen extenders (modified from [44]).
A cost–benefit analysis of performing SLC will include the advantages and disadvantages of not using antibiotics, as summarised in Table 2, but will also include the cost of purchasing a centrifuge (unless one is already available), extra time spent by personnel in processing semen, and the cost of the colloid. In many cases, the stud will already possess a centrifuge, for example, if semen is being frozen on the premises or removal of some seminal plasma by sperm washing is done routinely in the preparation of cooled stallion semen samples [78]. The centrifugation time is longer for SLC than for sperm washing (20 minutes versus 10 minutes), but personnel are able to perform other duties while the centrifugation is operating. At present, the cost of Equicoll would add approximately $10–$15 to the cost of the semen dose for equine AI, which is insignificant compared to the price of most stallion semen doses. However, if it were possible to use the low-density colloid instead of the usual colloid formulation for stallion semen, as discussed previously for pig semen, the cost of the colloid would be approximately halved. Perhaps the question should be re-phrased: can we afford
It should be noted that the efficiency of the technique will be affected by increasing bacterial load. Therefore, SLC should not be a substitute for attention to hygiene in animal husbandry, or in semen collection and processing protocols. Strict attention to hygiene should occur at all times when dealing with breeding animals and semen handling [79].
Advertisement
5. Conclusion
Bacteria appear in ejaculates during semen collection and processing. Some types of bacteria have a detrimental effect on sperm quality during storage and some may cause disease in inseminated females. The addition of antibiotics to semen extenders reduces the bacterial load in semen doses for artificial insemination but can facilitate the development of antimicrobial resistance. Furthermore, this application is non-therapeutic and does not fit with current guidance on the prudent use of these substances. Bacterial growth is reduced or prevented by cooling semen below 15°C but resumes when the temperature rises again in preparation for artificial insemination. Alternatives to antibiotics, such as plant-based extracts and nanoparticles, are available but may not be effective in all situations or may be spermatotoxic, which is counter-productive. It remains to be seen whether bacteria can develop resistance to such plant-based extracts. Colloid centrifugation of semen separates spermatozoa from most bacteria, thus avoiding the possibilities of AMR development. Since the process is purely physical, bacteria cannot mutate as an avoidance mechanism. The technique can be used to prepare whole stallion and boar ejaculates at the semen collection center and is cost-effective compared to the price of AMR. It could be used in conjunction with reducing the temperature of the semen. However, strict attention to hygiene should still occur at all stages of semen collection and processing, and in the routine husbandry of breeding males.
Advertisement
Acknowledgments
Funded by the Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, FORMAS Stockholm (Research council for sustainable development; project number 2017-00957), and Carl Tryggers Foundation (project number CTS 21: 1163), both awarded to JMM. The funders were not involved in the writing of this article or in the decision to publish.
Advertisement
Conflict of interest
The author, Prof. Jane M. Morrell, is the inventor and one of the patent holders of the colloids mentioned in this article.
References
- 1.
Prestinaci F, Pezzotti P, Pantosi A. Antimicrobial resistance: A global multifaceted phenomenon. Pathogens and Global Health. 2015; 109 :309-138 - 2.
Schar D, Klein EY, Laxminarayan R, Gilbert M, van Boeckel TP. Global trends in antimicrobial use in aquaculture. Scientific Reports. 2020; 10 :21878. DOI: 0.1038/s41598-020-78849-3 - 3.
Tiseo K, Huber L, Gilbert M, Robinson TP, van Boeckel TP. Global trends in antimicrobial use in food animals from 2017 to 2030. Antibiotics. 2020; 9 :918-931. DOI: 10.3390/antibiotics9120918 - 4.
Eriksen J, Björkman I, Röing M, Essack SY, Stålsby LC. Exploring the one health perspective in Sweden’s policies for containing antibiotic resistance. Antibiotics. 2021; 10 :526-538. DOI: 10.3390/antibiotics10050526 - 5.
Aslam B, Khurshid M, Arshad MI, Muzammil S, Rasool M, Yasmeen N, et al. Antibiotic resistance: One health one world outlook. Frontiers in Cellular and Infection Microbiology. 2021; 11 :771510 (20 pages). DOI: 10.3389/fcimb.2021.771510 - 6.
Givens MD. Review: Risks of disease transmission through semen in cattle. Animal. 2018; 12 (s1):s165-s171. DOI: 10.1017/S1751731118000708 - 7.
Morrell JM. Artificial insemination: Current and future trends. In: Manafi M, editor. Artificial Insemination in Farm Animals [Internet]. London: IntechOpen; 2011. Available from: https://www.intechopen.com/chapters/16096 . DOI: 10.5772/17943 - 8.
Teale CJ, Moulin G. Prudent use guidelines: A review of existing veterinary guidelines. Revue Scientifique et Technique. 2012; 31 :343-354. DOI: 10.20506/rst.31.1.2119 - 9.
Morrell JM, Wallgren M. Alternatives to antibiotics in semen extenders: A review. Pathogens. 2014; 3 :934-946. DOI: 10.3390/pathogens3040934 - 10.
Picket BW, Voss JL, Jones RL. Control of bacteria in stallions and their semen. Journal of Equine Veterinary Science. 1999; 19 :424-441. DOI: 10.1016/S0737-0806(99)80254-8 - 11.
Nitsche-Melkus E, Bortfeldt R, Jung M, Schulze M. Impact of hygiene on bacterial contamination in extended boar semen: An eight-year retrospective study of 28 European AI centers. Theriogenology. 2020; 146 :133-139.DOI: 10.1016/j.theriogenology.2019.11.031 - 12.
Morrell JM, Klein C, Lundeheim N, Erol E, Troedsson MHT. Removal of bacteria from stallion semen by colloid centrifugation. Animal Reproduction Science. 2014; 145 :47-53. DOI: 10.1016/j.anireprosci.2014.01.005 - 13.
Viudes-de-Castro MP, Mocé E, Lavara R, Marco-Jiménez F, Vicente JS. Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate. Theriogenology. 2014; 81 :1223-1228. DOI: 10.1016/j.theriogenology.2014.02.003 - 14.
Bennett DG. Therapy of endometritis in mares. Journal of the American Veterinary Medical Association. 1986; 188 :1390-1392 - 15.
Kuster CE, Althouse GC. The impact of bacteriospermia on boar sperm storage and reproductive performance. Theriogenology. 2016; 85 :21-26. DOI: 10.1016/j.theriogenology.2015.09.049 - 16.
Nedwell DB. Effect of low temperature on microbial growth: Lowered affinity for substrates limits growth at low temperature. FEMS Microbial Ecology. 1999; 30 :101-111. DOI: 10.1111/j.1574-6941.1999.tb00639.x - 17.
Pickett BW, Komarek RJ. Effect of cold shock and freezing on loss of lipid from spermatozoa. Journal of Dairy Science. 1967; 59 :753-757 - 18.
Aurich J, Aurich C. Developments in European horse breeding and consequences for veterinarians in equine reproduction. Reproduction in Domestic Animals. 2006; 41 :275-279. DOI: 10.1111/j.1439-0531.2006.00719 - 19.
Council of Europe. Directive 92/65/EEC of 13 July 1992, laying down animal health requirements governing trade in and imports into the community of animals, semen, ova and embryos not subject to animal health requirements laid down in specific community rules referred to in annex a to directive 90/425/EEC. Official Journal of the European Communities. 1992; 268 :54-72. Updated 2017 - 20.
Zampieri D, Santos VG, Braga PA, Ferreira CR, Ballottin D, Tasic L, et al. Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Theriogenology. 2013; 80 :337-345. DOI: 10.1016/j.theriogenology.2013.04.020 - 21.
Kilburn C, Rooks DJ, McCarthy AJ, Murray RD. Antimicrobial resistance in some gram-negative bacteria isolated from the bovine ejaculate. Reproduction in Domestic Animals. 2013; 48 :525-528. DOI: 10.1111/rda.12127 - 22.
Bresciani C, Cabassi CS, Morini G, Taddei S, Bettini R, Bigliardi E, et al. Boar semen bacterial contamination in Italy and antibiotic efficacy in a modified extender. Italian Journal of Animal Science. 2014; 13 :3082. DOI: 10.4081/ijas.2014.3082 - 23.
Gòdia YR-C, Zingaretti L, Darwich L, López S, et al. A pilot RNA-seq study in 40 pietrain ejaculates to characterize the porcine sperm microbiome. Theriogenology. 2020; 157 :525-533. DOI: 10.1016/j.theriogenology.2020.08.00 - 24.
Gloria A, Contri A, Wegher L, Vignola G, Dellamaria D, Carluccio A. The effects of antibiotic additions to extenders on fresh and frozen-thawed bull semen. Animal Reproduction Science. 2014; 150 :15-23. DOI: 10.1016/j.anireprosci.2014.08.012 - 25.
Guimaraes T, Lopes G, Pinto M, Silva E, Miranda C, Correia MJ, et al. Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics. Theriogenology. 2015; 83 :186-191. DOI: 10.1016/j.theriogenology.2014.09.003 - 26.
Hernández-Avilés C, Serafini R, Love CC, Teague SR, LaCaze KA, Lawhon SD, et al. The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen. Theriogenology. 2018; 122 :23-29. DOI: 10.1016/j.theriogenology.2018.08.022 - 27.
Goularte KL, Voloski FLS, Redú JFM, Ferreira CER, Vieira AD, Duval EH, et al. Antibiotic resistance in microorganisms isolated in a bull semen stud. Reproduction in Domestic Animals. 2020; 55 :318-324. DOI: 10.1111/rda.13621 - 28.
Malaluang P, Wilén E, Lindahl J, Hansson I, Morrell JM. Antimicrobial resistance in equine reproduction. Animals. 2021; 11 :3035-3047. DOI: 10.3390/ani11113035 - 29.
Malaluang P, Wilén E, Hansson I, Lindahl J, Morrell JM. Antibiotic resistance in vaginal flora of inseminated mares. Reproduction in Domestic Animals. 2021; 57, S2 :YSC7 - 30.
Johansson A, Greko C, Engström BE, Karlsson M. Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes. Veterinary Microbiology. 2004; 99 :251-257. DOI: 10.1016/j.vetmic.2004.01.009 - 31.
Martinez JL, Rojo F. Metabolic regulation of antibiotic resistance. FEMS Microbiology Reviews. 2011; 35 :768-789. DOI: 10.1111/j.1574-6976.2011.00282 - 32.
Anwar M, Iqbal Q , Saleem F. Improper disposal of unused antibiotics: An often overlooked driver of antimicrobial resistance. Expert Review of Antibiotic Therapy. 2020; 18 :697-699. DOI: 10.1080/14787210.2020.1754797 - 33.
Young C, Kharmacharya D, Bista M, Sharma A, Goldstein T, Anthony SJ, et al. Sharing of antimicrobial resistance genes among animals, humans, and the environment in Nepal: A one health case study. International Journal of Infectious Diseases. 2019; 79 :20 - 34.
Lyashchenko KP, Greenwald R, Esfandiari J, Lecu A, Waters WR, Posthaus H, et al. Pulmonary disease due to mycobacterium tuberculosis in a horse: Zoonotic concerns and limitations of antemortem testing. Veterinary Medicine International. 2012:642145. 6 pages - 35.
EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). The European Union one health 2020 Zoonoses report. EFSA Journal. 2021; 19 (12):6971, 324. DOI: 10.2903/j.efsa.2021.6971 - 36.
WHO Estimates of the Global Burden of Foodborne Diseases: Foodborne Disease Burden Epidemiology Reference Group 2007-2015. World Health Organization. ISBN: 978 92 4 156516 5 - 37.
Kuhn KG, Falkenhorst G, Emborg HD, Ceper T, Torpdahl M, Krogfelt KA, et al. Epidemiological and serological investigation of a waterborne campylobacter jejuni outbreak in a Danish town. Epidemiology and Infection. 2017; 2017 (145):701-709. DOI: 10.1017/S0950268816002788 - 38.
Hyllestad S, Iversen A, MacDonald E, Amato E, Borge BÅS, Bøe A, et al. Large waterborne campylobacter outbreak: Use of multiple approaches to investigate contamination of the drinking water supply system, Norway, June 2019. Euro Surveillance. 2020; 25 :2000011. DOI: 10.2807/1560-7917.ES.2020.25.35.2000011. Erratum in: Euro Surveill. 2020;25(43) - 39.
Crespo-Piazuelo D, Lawlor PG. Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) prevalence in humans in close contact with animals and measures to reduce on-farm colonisation. Irish Veterinary Journal. 2021;74 :21. DOI: 10.1186/s13620-021-00200-7 - 40.
Wheeler E, Hong PY, Bedon LC, Mackie RI. Carriage of antibiotic-resistant enteric bacteria varies among sites in Galapagos reptiles. Journal of Wildlife Diseases. 2012; 48 :56-67. DOI: 10.7589/0090-3558-48.1.56 - 41.
Ungemach FR, Müller-Bahrdt D, Abraham G. Guidelines for prudent use of antimicrobials and their implications on antibiotic usage in veterinary medicine. International Journal of Medical Microbiology. 2006; 296 (S2):33-38. DOI: 10.1016/j.ijmm.2006.01.059 - 42.
Waberski D, Luther A-M, Grünther B, Jäkel H, Henning H, Vogel C, et al. Sperm function in vitro and fertility after antibiotic-free, hypothermic storage of liquid preserved boar semen. Scientific Reports. 2019; 9 :14748. DOI: 10.1038/s41598-019-51319-1 - 43.
de Menezes TA, APG M, da Silva Oliveira G, Bernardi ML, Wentz I, da Ulguim RR, et al. Antibiotic-free extended boar semen preserved under low temperature maintains acceptable in-vitro sperm quality and reduces bacterial load. Theriogenology. 2020; 149 :131-138. DOI: 10.1016/j.theriogenology.2020.03.003 - 44.
Morrell JM. Antimicrobials in boar semen extenders – A risk/benefit analysis. Journal of Antimicrobial Agents. 2016; 2 :1-2. DOI: 10.4172/2472-1212.1000107 - 45.
Elmi A, Prosperi A, Zannoni A, Bertocchi M, Scorpio DG, Forni M, et al. Antimicrobial capabilities of non-spermicidal concentrations of tea tree (Melaleuca alternifolia) and rosemary (Rosmarinus officinalis) essential oils on the liquid phase of refrigerated swine seminal doses. Research in Veterinary Science. 2019; 127 :76-81. DOI: 10.1016/j.rvsc.2019.10.014 - 46.
Ros-Santaella JL, Kadlec M, Pintus E. Pharmacological activity of Honeybush (Cyclopia intermedia) in boar spermatozoa during semen storage and under oxidative stress. Animals Open Access Journal MDPI. 2020; 10 :772-796. DOI: 10.3390/ani10030463 - 47.
Schulze M, Junkes C, Müller P, Speck S, Rüdiger K, Dathe M, et al. Effects of cationic antimicrobial peptides on liquid-preserved boar spermatozoa. PLoS One. 2014; 9 :e100490. DOI: 10.1371/journal.pone.0100490 - 48.
Malo C, Gil L, Gonzalez N, Martínez F, Cano R, de Blas I, et al. Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: Comparison between cysteine and rosemary (Rosmarinus officinalis). Cryobiology. 2010; 61 :142-147. DOI: 10.1016/j.cryobiol.2010.06.009 - 49.
Elmi A, Ventrella D, Barone F, Filippini G, Benvenuti S, Pisi A, et al. Thymbra capitata (L.) cav. And Rosmarinus officinalis (L.) essential oils: In vitro effects and toxicity on swine spermatozoa. Molecules. 2017; 22 :2162-2176. DOI: 10.3390/molecules24061071 - 50.
Hagos L, Cojkic A, Hansson I, Johannisson A, Ntallaris T, Morrell JM. Effect of rosmarinic acid on bull sperm quality and bacteria in semen. Animal Reproduction Science. 2020; 220 :19 - 51.
Sokunbi OA, Ajani OS, Lawanson AA, Amao EA. Antibiotic potential of Moringa leaf (Moringa oleifera lam.) crude extract in bull semen extender European. Journal of Medicinal Plants. 2015; 9 :1-8. DOI: 10.9734/EJMP/2015/18546 - 52.
Carrera-Chávez JM, Jiménez-Aguilar EE, Acosta-Pérez TP, Núñez-Gastélum JA, Quezada-Casasola A, Escárcega-Ávila AM, et al. Effect of Moringa oleifera seed extract on antioxidant activity and sperm characteristics in cryopreserved ram semen. Journal of Applied Animal Research. 2020; 48 :114-120. DOI: 10.1080/09712119.2020.1741374 - 53.
Nimrat S, Noppakun S, Sripuak K, Boonthai T, Vuthiphandchai V. Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of medicinal plant extracts: Development of a programmable controlled-rate method and a practical method. Aquaculture. 2020; 515 :734537. DOI: 10.1016/j.aquaculture.2019.734537 - 54.
Elmi A, Ventrella D, Barone F, Carnevali G, Filippini G, Pisi A, et al. In vitro effects of tea tree oil (Melaleuca alternifolia essential oil) and its principal component terpinen-4-ol on swine spermatozoa. Molecules. 2019; 24 :1071. DOI: 10.3390/molecules24061071 - 55.
Tvrdá E, Michalko J, Árvay J, Vukovic NL, Ivanišová E, Duracka M, et al. Characterization of the Omija (Schisandra chinensis) extract and its effects on the bovine sperm vitality and oxidative profile during in vitro storage. Evidence-Based Complementary and Alternative Medicine. 2020; 2020 :7123780. DOI: 10.1155/2020/7123780 - 56.
Hall-Stoodley L, Stoodley P, Kathju S, Hoiby N, Moser C, Costerton WJ, et al. Towards diagnostic guidelines for biofilm-associated infections. FEMS Immunology and Medical Microbiology. 2012; 65 :127-145. DOI: 10.1111/j.1574-695X.2012.00968 - 57.
Coelho ML, Duarte AFS, Bastos MCF. Bacterial labionin-containing peptides and sactibiotics: Unusual types of antimicrobial peptides with potential use in clinical settings (a review). Current Topics in Medicinal Chemistry. 2017; 17 :1-22. DOI: 10.2174/1568026616666160930144809 - 58.
Bourgeon F, Evrard B, Brillard-Bourdet M, Colleu D, Jegou B, Pineau C. Involvement of semenogelin-derived peptides in the antibacterial activity of human seminal plasma. Biology of Reproduction. 2004; 70 :768-774. DOI: 10.1095/biolreprod.103.022533 - 59.
Hirt H, Gorr S-U. Antimicrobial peptide GL13K is effective in reducing biofilms of Pseudomonas aeruginosa . Antimicrobial Agents and Chemotherapy. 2013;57 :4903-4910. DOI: 10.1128/AAC.00311-13 - 60.
Luther A-M, Nguyen TQ , Verspohl J, Waberski D. Antimicrobially active semen extenders allow the reduction of antibiotic use in pig insemination. Antibiotics. 2021; 10 :1319. DOI: doi.org/10.3390/antibiotics10111319 - 61.
Hensel B, Jakop U, Scheinpflug K, Mühldorfer K, Schröter F, Schäfer J, et al. Low temperature preservation of porcine semen: Influence of short antimicrobial lipopeptides on sperm quality and bacterial load. Scientific Reports. 2020; 10 :13225. DOI: 10.1038/s41598-020-70180-1 - 62.
Vickram AS, Kuldeep D, Archana K, Parameswari R, Ramesh Pathy M, Iqbal HMN, et al. Antimicrobial peptides in semen extenders: A valuable replacement option for antibiotics in cryopreservation—A prospective review. J. Exp. biol. Agricultural Sciences. 2017; 5 :578-588. DOI: 10.18006/2017.5(4).578.588 - 63.
Guzman M, Dille J, Godet S. Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria. Nanomedicine. 2012; 8 :37-45. DOI: 10.1016/j.nano.2011.05.007 - 64.
Tsakmakidis IA, Samaras T, Anastasiadou S, Basioura A, Ntemka A, Michos I, et al. Iron oxide nanoparticles as an alternative to antibiotics additive on extended boar semen. Nanomaterials. 2020; 10 :1568-1583. DOI: 10.3390/nano10081568 - 65.
Tsakmakidis IA, Samaras T, Anastasiadou S, Basioura A, Ntemka A, Michos I, et al. Toxic and microbiological effects of iron oxide and silver nanoparticles as additives on extended ram semen. Animals. 2021; 11 :1011-1026. DOI: 10.3390/ani11041011 - 66.
Khalil WA, El-Harairy MA, Zeidan AEB, Hassan MAE. Impact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation. Theriogenology. 2019; 126 :121-127. DOI: 10.1016/j.theriogenology.2018.12.017 - 67.
Jahanbin R, Yazdanshenas P, Rahimi M, Hajarizadeh A, Tvrda E, Ataei Nazari S, et al. In vivo and I vitro evaluation of bull semen processed with zinc (Zn) nanoparticles. Biological Trace Element Research. 2021; 199 :126-135. DOI: 10.1007/s12011-020-02153-4 - 68.
Viudes-de-Castro MP, Marco-Jimenez F, Vicente JS, Marin C. Antibacterial activity of some molecules added to rabbit semen extender as alternative to antibiotics. Animals. 2021; 11 :1178. DOI: 10.3390/ani11041178 - 69.
Morrell JM, Wallgren M. Removal of bacteria from boar ejaculates by single layer centrifugation can reduce the use of antibiotics in semen extenders. Animal Reproduction Science. 2011; 123 :64-69. DOI: 10.1016/j.anireprosci.2010.11.005 - 70.
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Morrell JM. Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen. Reproduction in Domestic Animals. 2018; 54 :342-349. DOI: 10.1111/rda.13366 - 71.
Morrell JM, van Wienen M, Wallgren M. Single layer centrifugation can be scaled-up further to process up to 150mL semen. ISRN Veterinary Science. 2012; 2011 :6 pages. DOI: 10.5402/2011/183412 - 72.
Morrell JM, Nunes MM. Practical guide to single layer centrifugation of stallion semen. Equine Veterinary Education. 2018; 30 :392-398. DOI: 10.1111/eve.12658 - 73.
Varela E, Plaza E, Muñoz de Propios P, Ortiz-Rodríguez JM, Álvarez M, Anel-López L, et al. How does the microbial load affect the quality of equine cool-stored semen? Theriogenology. 2018; 2018 (114):212-220. DOI: 10.1016/j.theriogenology.2018.03.028 - 74.
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Morrell JM. Effect of presence or absence of antibiotics and use of modified single layer centrifugation on bacteria in pony stallion semen. Reproduction in Domestic Animals. 2019; 54 :342-349. DOI: 10.1111/rda.13366 - 75.
Morrell JM, Johannisson A, Dalin A-M, Hammar L, Sandebert T, Rodriguez-Martinez H. Sperm morphology and chromatin integrity in Swedish warmblood stallions and their relationship to pregnancy rates. Acta Veterinaria Scandinavica. 2008; 50 :2-8. DOI: 10.1186/1751-0147-50-2 - 76.
Morrell JM, Nunez-Gonzalez A, Crespo-Felez I, Martinez-Martinez S, Martinez Alborcia MJ, Fernandez-Alegre E, et al. Removal of bacteria from boar semen using a low-density colloid. Theriogenology. 2019; 126 :272-278. DOI: 10.1016/j.theriogenology.2018.12.028 - 77.
Morrell JM, Martinez-Alborcia M-J, Martinez-Pastor F, Bolarin A. Centrifugation of boar spermatozoa through low density Porcicoll to separate them from bacteria does not affect fertility after insemination. JIOMICS. 2021; 11 :12 I - 78.
Love CC, Thompson JA, Lowry VK, Varner DD. Effect of storage time and temperature on stallion sperm DNA and fertility. Theriogenology. 2002; 57 :1135e42. DOI: 10.1016/s0093-691x(01)00689-6 - 79.
World Organisation for Animal Health (OIE). 2019. Terrestrial Animal Health Code available at: https://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_general_hygiene_semen.pdf [Accessed: July 13, 2021]