ISO 8607:2003

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.