Hygiene basics for low-bacteria semen production
High quality semen doses must provide fertile sperm cells and avoid the transmittance of pathogens into the female genital tract. During storage at +17°C, extended boar semen is susceptible to bacterial growth. Strict hygienic measures at all stages of semen collection and processing, are therefore fundamental to minimize the bio burden of extended semen. This is also true for antibiotic-free storage of boar semen at +5°C, despite the low storage temperature.
It is scientifically proven, that boar sperm can cope with a certain bacterial load in the diluted semen and that this does not affect the fertility of the semen doses. However, there is threshold for the maximally allowed number of CFU/ml dependent on the bacterial type (Luther et al., 2023). Additionally, it is of outmost importance that none of the bacteria strains is pathogenic for swine.
Besides the prudent use of antimicrobials, the daily general cleanliness, personnel hygiene as well as targeted hygiene and sanitation procedures along the complete semen production chain are the essential fundamentals for a healthy and sustainable boar semen production.
The following hygiene measures are therefore indispensable, when producing boar sperm doses:
Ideal semen collection starts with the separation of the semen collection area from the boar barn to have less airborne particles and bacteria in the semen collection area. The boars should be clean, dry, and free of straw and other particles when used for semen collection.
Hygiene during semen collection includes the use of clean boar dummies with smooth and intact surfaces, clean single-use gloves, germ-free semen collection bags and filters, as well as an appropriate semen collection technique avoiding the contamination of the ejaculate with fluid from the preputial cavity or the first jets from the pre-secretion of the ejaculate.
The use of automated semen collection systems like BoarMatic is advantageous, since the semen is not exposed to (dusty) air during collection. Before and during transport to the laboratory, semen vessels must be kept closed in order to prevent contamination by airborne microorganisms.
Once arrived in the laboratory, any materials coming into contact with the semen extender or extended semen, represent further major potential sources of contamination.
Laboratories should be strictly separated from the collection area including air transmission. Laboratory workflow from semen examination to filling and sealing the semen tubes or bags should be in-line (straight), thereby avoiding cross-traffic between workstations. Only single use disposable wipes should be allowed to prevent cross-contamination. Laboratory desks, floors and walls should be as empty as possible. Any material or equipment is a potential source of contamination and impairs cleaning, especially where corners and niches are formed.
Prepared extenders may become contaminated when stored overnight at room temperature. It is recommended to avoid the usage of extender prepared the day before. If it is necessary, then the leftovers should not be mixed with the freshly prepared extender but used as a separate batch. Semen dyes used in some AI stations to identify different lines of boars, have been identified as a major source of bacterial contamination in extended semen. If stains must be used, a new syringe and needle should be used each day and the cap of the stain bottle must be disinfected several times per day.
Antibiotics are helpful to control bacterial growth in extended semen, however, they cannot compensate for hygienic deficiencies in semen collection or processing. Special care needs to be taken to avoid the development of resistant bacteria in laboratory sinks or drains. Extender or extended semen expose bacteria present in sanitary installations to antibiotics and create a perfect environment for the selection of resistant bacteria. Therefore, a strictly enforced procedure for the disposal of extender or extended semen must be established without using laboratory sinks and drains.
The disposable of semen extender is easier when boar semen is produced antibiotic-free. As no antibiotics are included in the semen extender, left-over extender can easily be discarded in the sewage system. Antibiotic-free boar semen production prevents the contact of any bacteria with antibiotics and therefore prevents the building up of antibiotic resistant bacteria as well. During antibiotic-free semen production and the associated storage of boar semen at +5°C, hygiene protocols are very important to keep the bioburden as low as possible.
A further alternative to prevent discarding extender with antibiotics, is to use the ADA method (accurate dosage of antibiotics). With the ADA method, extender (without antibiotics) and antibiotics are dosed separately and independent from each other during the dilution process. Therefore, left-over extender can easily be discarded, as no now antibiotics are included in the extender. The antibiotics are added in form of an antibiotic concentrate to the boar ejaculate. For further information see the article from Schulze et al. (2017).
Automated semen processing allows a hygienic production of semen doses. However, daily care of the machine and especially the tubing is mandatory. Repeated use, cleaning, sterilization, and even simple aging affect surface properties of silicone tubing which may ultimately evolve in the formation of biofilms as a source of persistent contamination. Water and extender tubing are particularly susceptible to the formation of biofilms. Once created, biofilms are tenacious and hard to remove. It is therefore recommended as preventive measure that all tubing coming into contact with semen must be cleaned and sanitized daily and changed regularly at intervals of 2 to 4 months. All tubing used for extender must be changed every 4 to 8 months.
All surfaces within the laboratory must be easy to reach and easy to clean. Special benches with rounded edges and partially integrated equipment are available. The surface of the bench material is totally sealed and can effectively be disinfected.
The boar stud personnel must be instructed and involved in the realization of a hygiene targeted semen quality control plan to create a high consciousness in hygiene.
The instructions for cleaning and disinfection must be clear and should cover the following points:
In conclusion, the application of adequate hygienic guidelines with regards to the design and maintenance of the semen collection area and the laboratory as well as preventive action by the AI stud personnel are fundamental. A profound hygienic consciousness of AI staff must be created and trained regularly.
Adapted from Prof. Karl-Fritz Weitze and Prof. Dagmar Waberski, Unit of Reproductive Medicine, University of Veterinary Medicine Hanover
References
Luther, A.-M., Beckermann, C., Nguyen, T. Q., Verspohl, J., & Waberski, D. (2023). Growth Dynamic and Threshold Values for Spermicidal Effects of Multidrug-Resistant Bacteria in Extended Boar Semen. Microorganisms, 11(3), 788. https://doi.org/10.3390/microorganisms11030788
Schulze, M., Grobbel, M., Riesenbeck, A., Brüning, S., Schaefer, J., Jung, M., & Grossfeld, R. (2017). Dose rates of antimicrobial substances in boar semen preservation—time to establish new protocols. Reproduction in Domestic Animals, 52(3), 397–402. https://doi.org/10.1111/rda.12921
It is scientifically proven, that boar sperm can cope with a certain bacterial load in the diluted semen and that this does not affect the fertility of the semen doses. However, there is threshold for the maximally allowed number of CFU/ml dependent on the bacterial type (Luther et al., 2023). Additionally, it is of outmost importance that none of the bacteria strains is pathogenic for swine.
Besides the prudent use of antimicrobials, the daily general cleanliness, personnel hygiene as well as targeted hygiene and sanitation procedures along the complete semen production chain are the essential fundamentals for a healthy and sustainable boar semen production.
The following hygiene measures are therefore indispensable, when producing boar sperm doses:
- Adequate facility design, including boar barn and laboratory
- Establishment of sanitation guidelines
- Training of personnel in individual and general hygiene
- Definition of critical control points
- Regular monitoring of the bio burden in these critical control points
- Enforcement and validation of corrective measures
- Adequate and prudent use of antibiotics
Ideal semen collection starts with the separation of the semen collection area from the boar barn to have less airborne particles and bacteria in the semen collection area. The boars should be clean, dry, and free of straw and other particles when used for semen collection.
Hygiene during semen collection includes the use of clean boar dummies with smooth and intact surfaces, clean single-use gloves, germ-free semen collection bags and filters, as well as an appropriate semen collection technique avoiding the contamination of the ejaculate with fluid from the preputial cavity or the first jets from the pre-secretion of the ejaculate.
The use of automated semen collection systems like BoarMatic is advantageous, since the semen is not exposed to (dusty) air during collection. Before and during transport to the laboratory, semen vessels must be kept closed in order to prevent contamination by airborne microorganisms.
Once arrived in the laboratory, any materials coming into contact with the semen extender or extended semen, represent further major potential sources of contamination.
Laboratories should be strictly separated from the collection area including air transmission. Laboratory workflow from semen examination to filling and sealing the semen tubes or bags should be in-line (straight), thereby avoiding cross-traffic between workstations. Only single use disposable wipes should be allowed to prevent cross-contamination. Laboratory desks, floors and walls should be as empty as possible. Any material or equipment is a potential source of contamination and impairs cleaning, especially where corners and niches are formed.
Prepared extenders may become contaminated when stored overnight at room temperature. It is recommended to avoid the usage of extender prepared the day before. If it is necessary, then the leftovers should not be mixed with the freshly prepared extender but used as a separate batch. Semen dyes used in some AI stations to identify different lines of boars, have been identified as a major source of bacterial contamination in extended semen. If stains must be used, a new syringe and needle should be used each day and the cap of the stain bottle must be disinfected several times per day.
Antibiotics are helpful to control bacterial growth in extended semen, however, they cannot compensate for hygienic deficiencies in semen collection or processing. Special care needs to be taken to avoid the development of resistant bacteria in laboratory sinks or drains. Extender or extended semen expose bacteria present in sanitary installations to antibiotics and create a perfect environment for the selection of resistant bacteria. Therefore, a strictly enforced procedure for the disposal of extender or extended semen must be established without using laboratory sinks and drains.
The disposable of semen extender is easier when boar semen is produced antibiotic-free. As no antibiotics are included in the semen extender, left-over extender can easily be discarded in the sewage system. Antibiotic-free boar semen production prevents the contact of any bacteria with antibiotics and therefore prevents the building up of antibiotic resistant bacteria as well. During antibiotic-free semen production and the associated storage of boar semen at +5°C, hygiene protocols are very important to keep the bioburden as low as possible.
A further alternative to prevent discarding extender with antibiotics, is to use the ADA method (accurate dosage of antibiotics). With the ADA method, extender (without antibiotics) and antibiotics are dosed separately and independent from each other during the dilution process. Therefore, left-over extender can easily be discarded, as no now antibiotics are included in the extender. The antibiotics are added in form of an antibiotic concentrate to the boar ejaculate. For further information see the article from Schulze et al. (2017).
Automated semen processing allows a hygienic production of semen doses. However, daily care of the machine and especially the tubing is mandatory. Repeated use, cleaning, sterilization, and even simple aging affect surface properties of silicone tubing which may ultimately evolve in the formation of biofilms as a source of persistent contamination. Water and extender tubing are particularly susceptible to the formation of biofilms. Once created, biofilms are tenacious and hard to remove. It is therefore recommended as preventive measure that all tubing coming into contact with semen must be cleaned and sanitized daily and changed regularly at intervals of 2 to 4 months. All tubing used for extender must be changed every 4 to 8 months.
All surfaces within the laboratory must be easy to reach and easy to clean. Special benches with rounded edges and partially integrated equipment are available. The surface of the bench material is totally sealed and can effectively be disinfected.
The boar stud personnel must be instructed and involved in the realization of a hygiene targeted semen quality control plan to create a high consciousness in hygiene.
The instructions for cleaning and disinfection must be clear and should cover the following points:
- What is to be cleaned and disinfected (structure, equipment, utensils coming into contact with water, extender or semen)
- What chemicals and equipment are to be used
- The method of cleaning or disinfection
- What protective clothing should be worn and what precautions are to be taken
- When it is to be cleaned/disinfected (after use, daily, weekly, monthly)
- Who will carry out cleaning/disinfection
- Monitoring arrangements
In conclusion, the application of adequate hygienic guidelines with regards to the design and maintenance of the semen collection area and the laboratory as well as preventive action by the AI stud personnel are fundamental. A profound hygienic consciousness of AI staff must be created and trained regularly.
Adapted from Prof. Karl-Fritz Weitze and Prof. Dagmar Waberski, Unit of Reproductive Medicine, University of Veterinary Medicine Hanover
References
Luther, A.-M., Beckermann, C., Nguyen, T. Q., Verspohl, J., & Waberski, D. (2023). Growth Dynamic and Threshold Values for Spermicidal Effects of Multidrug-Resistant Bacteria in Extended Boar Semen. Microorganisms, 11(3), 788. https://doi.org/10.3390/microorganisms11030788
Schulze, M., Grobbel, M., Riesenbeck, A., Brüning, S., Schaefer, J., Jung, M., & Grossfeld, R. (2017). Dose rates of antimicrobial substances in boar semen preservation—time to establish new protocols. Reproduction in Domestic Animals, 52(3), 397–402. https://doi.org/10.1111/rda.12921
