European Xenopus Resource Centre

Holding and producing conditions

How we keep and produce Xenopus tropicalis (at 12/05/09)

System	Tecniplast - recirculating – with UV, particulate, mechanical and carbon filters
Temperature	25.5 ^oC (range 24-27 ^oC )
Conductivity	550 uS (range 500-700uS)
pH	7.9 (range7.5-9.0)
Water	Portsmouth Mains water. Conductivity maintained by a salt doser although mains water is usually within parameters).
Salt added	Tropic Marin /Instant Ocean sea salt
Tank size	27 litres
Stocking Density	25-35 adult females per 27 litres (same sex tanks) 25-40 adult males per 27 litres
Flow rate	1.33 litres per minute
Lighting	fluorescent 13 hours light/11 hours dark with night light
Environmental Enrichment	Dark coloured plastic guttering and downpipe tubes cut into 20 cm lengths.
Feeding
Adults	Omega 23 pellets-fed 2-3 times per day, a couple of pellets each
Froglets	Omega 23 pellets-fed 2-3 times per day, a couple of pellets each+ fish food.
Tadpoles	A filtered suspension of Rabma (Rabbit pellets) and Sanatogen (high protein powder) in system water. Fed daily, system water flow turned off.All frogs may be given occasional supplements of lambs heart or similar, cut to appropriate size, especially for frogs with weight loss or non-competitors.
Ovulation
Females are primed with Chorulon 10 iu HcG in water, the day before the eggs are required.A boosting dose of 100 iu is given as early as possible on the day the eggs are required. Egg laying starts normally 3-5 hours after the boosting dose and may last for several hours. ’Squeezes’ are restricted to 45 minute intervals. Males are sacrificed using Tricaine/MS222 for in-vitro fertilizations. Testes are removed and stored in L15/calf serum at room temperature. These should be used as quickly as possible as there is a dramatic loss in fertility.If the testes are stored for any length of time. Mash 1 testis in L15/calf serum and add to squeezed eggs in a Petri dish using a pipette. Shake dish to ensure good mixing. After 2-3 minutes flood with 0.05 x MMR. All solutions used are warmed to room temperature, 25^oC. Some labs use solutions at 4 degrees.

How we keep and produce Xenopus laevis (At 12/05/09)

System	Tecniplast - recirculating – with UV, particulate, mechanical and carbon filters
Temperature	18.5 ^oC (range 17-19 ^oC )
Conductivity	1550 uS (range 1500-1600uS)
pH	7.9 (range7.5-9.0)
Water	Portsmouth Mains water. Conductivity maintained by a salt doser
Salt added	Tropic Marin /Instant Ocean sea salt
Tank size	27 litres
Stocking Density	5-10 adult females per 27 litres (same sex tanks) 10-15 adult males per 27 litres
Flow rate	1.33 litres per minute
Lighting	Fluorescent 13 hours light/11 hours dark with night light
Environmental Enrichment	Dark coloured plastic guttering and downpipe tubes cut into 20 cm lengths.
Feeding
Adults	Omega 23 pellets-fed 2-3 times per day, a couple of pellets each
Froglets	Omega 23 pellets-fed 2-3 times per day, a couple of pellets each+ fish food
Tadpoles	A filtered suspension of Rabma (Rabbit pellets) and Sanatogen (high protein powder) in system water. Fed daily, system water flow turned off.All frogs may be given occasional supplements of lambs heart or similar, cut to appropriate size, especially for frogs with weight loss or non-competitors.
Ovulation
Females injected with Chorulon 450-600 iu HcG in water, usually at c.16:30 the evening before eggs are required. Males are sacrificed using Tricaine/MS222 for in-vitro fertilizations. Testes removed and stored in 1.0 MBS in fridge. These can be kept for over a week with no recorded loss in fertility. Mash about one third of 1 testis in .0.5ml of 0.1 MBS and add to squeezed eggs in a Petri dish using a dropper pipette. Shake dish to ensure good mixing. After 10 minutes flood eggs with 0.1 MBS. Egg laying starts normally at about 08:00 and may last for several hours. ’Squeezes’ are restricted to 45 minute intervals. All solutions used come straight from the fridge at 4 oC. A target fertilisation rate of >95% is the normal result.

How animals are assessed as suitable for re-use:

Xenopus are individually identified by appropriate non-invasive methods such as digital photography and/or written descriptions of dorsal skin patterns or other distinguishing features. Plain frogs that are indistinguishable from each other are kept separate by placing them uniquely amongst small groups of others that are distinguished by pattern or colour.

This ability to track individuals is at the heart of our database driven reuse protocol, aimed at making judgements on Xenopus reuse more informed as it is based on the frog’s individual history and performance. The current protocol (although this may be subject to minor amendment) is:

Xenopus reuse protocol

Traffic light system

Absolute

There must be 3 months or 90 days between ovulations.

Judgemental - The following criteria must be considered when selecting frogs for reuse:

1. General appearance: With your experience. You consider it likely to lay well.

2. Skin texture: Smooth and free from abrasions and redness.

3. Body shape: Consistent with history (known slim or plump frog?)

4. Body weight: Consistency to be looked at.

Mandatory - only if reuse is carried out

Body weight must be taken, compared with history and recorded on database.

Person declaring fit to reuse must also record their details on database.