Capture Protocol, Sampling and Storage

Wild grey and harbour seals are regularly captured for sample collection for a variety of projects at SMRU. All plasma samples from adult individuals were taken using the same methodology, while the protocol for juveniles, weaners and pups was slightly different (outlined below). The protocol for treatment and storage of samples after collection was the same across all species and age groups.

2.4.1. Adult animals – Grey Seals

Adult grey seals sampled for this study were all breeding females on either North Rona or the Isle of May colonies. The majority were ‘study females’ with a history of pupping on the colony and a record of prior captures and data. Mothers and their dependant pups were caught twice in one season, once 2-4 days after birth and then approximately 10 days later in late lactation. Mothers were approached and

administered 1ml/kg of Zoletil® (Virbac) intramuscularly using blow darts, and were then left for ten minutes to allow the drug to take effect. Mothers were then restrained in a net for sampling as the drug would not render them fully unconscious, and their pups were caught and restrained manually without drugs. Plasma was drawn into heparin vacutainers (unless otherwise stated in the methods of individual experiments) from the extradural vein and samples were immediately put on ice until they could be spun and frozen at the end of the day. Milk samples were obtained by injecting 1ml of oxytocin (Oxytocin-S, Intervet UK Ltd) intravenously to stimulate milk let down. Samples were then collected by vacuum pump and put on ice immediately until they could be frozen. Obtaining milk samples without oxytocin injection was extremely difficult. Only two samples out of 250 were obtained without the use of the drug. These were used to allow detection of any interference of the injection with the natural levels in the milk. Mothers were monitored through out the capture period for signs of respiratory distress, and if this occurred the individual was intubated and given Dopram® (Phzer) to resuscitate them. Mothers coming out of the drugged state early were given additional intravenous Zoletil® if required. Pups were replaced with mothers as soon in the capture procedure as possible to minimise the risk of the pair becoming separated after the capture. All mothers were left to recover naturally with

motility and awareness rapidly as Zoletil® induces short duration anaesthesia lasting only approximately 20 minutes.

2.4.2. Adult animals – Other Species

The protocol for taking a plasma sample in other species in this study was the same as outlined above, with differences in the methods used to capture and induce

anaesthesia. Harbour seals caught on haul out locations were approached rapidly in boats and captured when they attempted to go to sea using a pop-up net deployed just offshore, which was placed there earlier in the week. The net was then pulled onto land and individual seals restrained in hoop nets until they could be drugged using an intravenous injection of Zoletil® into the extradural vein. Individual Weddell seals were approached on ice floe haul outs, darted and caught before they could re-enter the water.

2.4.3. Juveniles, Weaners and Pups

The protocol for taking plasma samples from non-adult individuals differed from that for adults in the methods used to capture them, the use of drugs to chemically restrain individuals and the site from which the plasma sample was drawn. For non-adult individuals, the sampling procedure used was determined by their age. Animals over eight months of age were physically restrained and then chemically immobilised using an intravenous injection of Zoletil® for plasma sampling. Individuals younger than this were categorized as either ‘weaners’ (pups from the current breeding season that had not been seen with a mother for two consecutive days, generally over 20 days of age) or ‘pups’ (still dependant on their mothers and suckling, generally under 20 days old). Both of these groups received no drugs during sampling (unless otherwise specified in the methods of individual experiments) and were physically restrained for sampling. Most plasma samples were drawn from the extradural vein but for some individual’s samples were taken from the plantar network of veins on the ventral surface of the hind flippers.

2.4.4. Plasma Collection, Processing and Storage

All plasma samples were kept on ice or chilled using ice packs until they could be brought back to a laboratory environment for processing and storage. Oxytocin has a

Chapter 2: General Methodology

that samples are kept chilled until they can be frozen (C.S. Carter 2010 pers. comm). The enzyme-linked immunosorbent assay (ELISA) protocol for the kit (Assay Designs Inc, Ann Arbor, MI, USA) used in this study recommends the use of

Ethylenediaminetetraacetic acid (EDTA) vacutainers, the addition of Aprotonin to the plasma after collection and storage at -70°C to ensure preservation of all oxytocin in the samples. This study did not use Aprotonin due to difficulties in obtaining the now discontinued substance, but used heparin vacutainers and froze samples at -20ºC (C.S. Carter 2010 pers. comm). Please see Chapter 3 for the experiments investigating and justifying these deviations from the recommended protocol.

Samples taken at the SMRU captive facility were put on ice immediately and then spun, aliquoted and frozen within half an hour of collection. Samples taken in the field were placed immediately in a cool box with multiple ice packs to keep them chilled, and were kept this way until the team returned to the laboratory at the end of the day. They were then spun immediately, aliquoted and frozen until they could be transported back to the main laboratory at SMRU for analysis.