Overview
ISO 8607:2003 - Artificial insemination of animals - Frozen semen of breeding bulls - Enumeration of living aerobic microorganisms - specifies an internationally harmonized colony-count method for measuring microbiological contamination in frozen bovine semen. The method counts colonies that grow on a solid medium after aerobic incubation at 37 °C, and expresses contamination as colony-forming units (CFU) per millilitre of test sample. It is designed to assess hygienic collection, handling and storage of semen used for artificial insemination (AI).
Key topics and technical requirements
- Scope: Enumeration of living aerobic microorganisms (bacteria, yeasts, moulds) in frozen semen of breeding bulls.
- Sampling & storage: Random selection from a series of doses; store samples in liquid nitrogen; thaw in a 37 °C water bath for 3 minutes.
- Preparation: Initial suspension and decimal dilutions prepared according to ISO 6887-1; higher dilutions recommended because semen extenders often contain antibiotics that inhibit growth.
- Diluent: Peptone–salt solution (enzymatic digest of casein 1.0 g, NaCl 8.5 g per 1 000 ml; pH ~7.0 ± 0.2 after sterilization).
- Agar medium: Specified nutritive medium (meat extract, glucose, yeast extract, peptone, NaCl, Na2HPO4·12H2O, gelatine and agar) with addition of 10% inactivated sterilized bovine or sheep serum when poured.
- Procedure: Pour-plate technique - transfer 1 ml of final dilution (and 1 ml of the next higher dilution) into duplicate Petri dishes, pour ~15 ml agar at ~45 °C, mix, solidify.
- Incubation: Invert plates and incubate at 37 °C for 72 h ± 3 h.
- Controls & interpretation: Include diluent control plates; count colonies and calculate CFU/ml; Annex A gives confidence limits for small counts.
- Equipment: Autoclave, incubator, water baths (37 °C & 45 °C), sterile pipettes, Petri dishes, colony-counting equipment and pH-meter.
Applications
- Quality control in semen collection centers, AI laboratories and cryobanks.
- Routine microbiological monitoring to verify hygienic techniques during collection, processing and storage of frozen bull semen.
- Supporting biosecurity, veterinary reproductive services and contractual quality requirements for semen distribution.
- Investigations into contamination sources and verification of extenders/antibiotic effects on culture-based counts.
Who should use this standard
- Veterinary diagnostic and microbiology laboratories
- Artificial insemination and breeding organizations
- Semen processing facilities and cryopreservation services
- Regulatory bodies and auditors overseeing AI product safety
Related standards
- ISO 6887-1 - Preparation of test samples, initial suspension and decimal dilutions
- ISO 7218 - General rules for microbiological examinations
Keywords: ISO 8607:2003, frozen semen, breeding bulls, enumeration, aerobic microorganisms, CFU/ml, artificial insemination, microbiological contamination, colony-count method.
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