Husbandry

Holding and producing conditions

How we keep and produce Xenopus tropicalis (February 2019)

System	Tecniplast/MBK recirculating – with UV, particulate, mechanical and carbon filters
Temperature	25.5 oC (range 24-27 oC).
Conductivity	800 uS (range 700-900uS).
pH	7.9 (range7.5-9.0). pH maintained by Sodium Bicrbonate doses.
Water	Portsmouth Mains water. Conductivity maintained by a marine sea-salt.
Salt added	Tropic Marin/Instant Ocean sea salt.
Tank size	4/6/16/27 litres
Stocking density	6/12/23/55 (males/females or same sex tanks)
Flow rate	1.33 litres per minute in a 27 litres tank. Less flow in smaller tanks.
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. Artificial lily pads.

Feeding

Adults	Horizon 23 pellets/Eterna 2.5 –made by Skretting fed 34 times per day, a couple of pellets each.
Froglets	Horizon 23 pellets/Eterna 2.5 –made by Skretting fed 2-3 times per day, fish food+ crushed pellets, a couple of pellets each depending on size.
Tadpoles	Sera-micron algae fed daily, system water flow turned to a slow drip.

All adult frogs are fed 5 days per week. All frogs may be given occasional supplements of lamb’s heart or similar, cut to appropriate size, especially for frogs with weight loss or non-competitors.

Ovulation

Prime females with 10iu hSG a day before boosting injection (or up to 3 days before). 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 followed by Schedule 1 method, for in vitro fertilizations. Testes removed and stored in L15/calf serum or 1 x MBS. 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 testes in L15/calf serum or 1 x MBS, and add to squeezed eggs in a Petri dish using a Finn pipette. Shake dish gently or use pipette tip to ensure good mixing. After 5-10 minutes flood with 0.05 MMR or 0.1 x MBS. All solutions used can be warmed to room temperature, or 25 degrees C, but we have used solutions at 4 degrees straight out of the fridge, and it has worked fine.

How we keep and produce Xenopus laevis (February 2019)

System	Tecniplast/Marine Biotech – 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). ph maintained by Sodium Bicarbonate doses.
Water	Portsmouth Mains water. Conductivity maintained by a salt doser.
Salt added	Tropic Marin/Instant Ocean sea salt.
Tank size	27/23 litres.
Stocking density	7 adult females/18 males per 23 litres. 12 adult females/28 adult males per 27 litres.
Flow rate	1.33 litres per minute in adult’s tanks, less flow in tadpoles tanks.
Lighting	Fluorescent 13 hours light/11 hours dark with night light.
Environmental enrichment	Black plastic guttering and downpipe tubes cut into c.20 cm. lengths.

Feeding

Adults	Horizon 23 trout pellets – fed 2-3 times per day, a few pellets each. All frogs may be given occasional supplements of lambs heart,cut to appropriate size, frozen bloodworm or newborn mouse pup or similar, especially for frogs with weight loss, or non-competitors. Frozen heart is especially well received by frogs, but preparing it is quite time-consuming.
Froglets	Horizon 23 trout pellets – fed 2-3 times per day. A few crushed pellets each + fish food
Tadpoles	Sera-micron algae fed daily, system water flow turned to a slow drip.

All adult frogs are fed 5 days per week.

All frogs may be given occasional supplements of lamb’s 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, usually at c.16:30 the evening before egg are required.

Males are sacrificed using Tricaine/MS222 for in-vitro fertilizations, followed by schedule 1 method.

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 gently to ensure good mixing. After 5-10 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 4C

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:

There must be 3 months or 90 days between ovulations.

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

General appearance: with your experience, you consider it likely to lay well.
Skin texture: smooth and free from abrasions and redness.
Body shape: consistent with history (known slim or plump frog).
Body weight: consistency to be looked at.

Only if re-use 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.