Breeding of African Cichlids Facility: Aquatic Animal Research Laboratory (AARL) Rooms 1-2
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Powder SOP BR-02-02 Page 1 of 4 Effective Date: 06-05-2018 Title: Breeding of African cichlids Facility: Aquatic Animal Research Laboratory (AARL) Rooms 1-2 Author(s): Date: 06-05-2018 Kara E. Powder Principle Investigator AARL Facility Date: __________ Manager: John A. Smink Attending Date: __________ Veterinarian: Dr. John Parrish 1.0 OBJECTIVE This SOP describes husbandry, embryo collection, and embryo housing for Lake Malawi cichlids. 2.0 HEALTH AND SAFETY All personnel will be enrolled in the Clemson University Medical Surveillance Program. Attire appropriate to the task must be worn at all times. 3.0 PERSONNEL/TRAINING/RESPONSIBILITIES All personnel must have completed the required Animal Care and Use Committee on-line training. Thereafter, any Clemson employee familiar with the techniques and trained in this and referenced SOPs may perform this procedure. Prior to being assigned full responsibility for performing this procedure, personnel must have demonstrated proficiency in the use of the technique in a closely supervised environment. Documentation of training should be kept for each person performing the procedure. 4.0 REQUIRED AND RECOMMENDED MATERIALS 4.1 Required Materials net, collection dish (roughly 4L), transfer pipette, petri dishes, permanent markers for labeling of dishes, 250 mL flask, 3L tanks, 70% ethanol. 5.0 GUIDELINES The following procedures were developed to meet or exceed the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. 6.0 PROCEDURE 6.1 Tank environment, cichlid mating, and identification of holding females Powder SOP BR-02-02 Page 2 of 4 Effective Date: 06-05-2018 Cichlid breeding groups consist of a single male with several females (generally 15-20 animals with a male:female ratio of 1:4-6). Inclusion of physical structures ("substrate") such as empty ceramic flower pots, PVC pipes, and concrete blocks mimics the fishes' natural, rocky environment while promoting breeding, enabling establishment of territories, and minimizing inter-fish aggression. Cichlid mating is highly ritualized behavior including various approaches culminating in a mating “dance.” Therefore, we allow adults to mate naturally in their standard housing. Please don’t disturb the fish (e.g. moving substrate or catching another fish in the tank with a net) while they are demonstrating mating behavior. Our species of Lake Malawi cichlids mouth brood, which means the female holds fertilized eggs in their mouth. In the wild, the mother will starve for 3-4 weeks, until the young are ready to feed on their own. Holding females are readily observable through a distended buccal cavity. Record holding females on the Cichlid Brood Log. 6.2 Collection of embryos We will collect embryos from mouth brooding females 2-5 days after they are first observed to be holding. 1. Remove the brooding female with a net and transfer her to a 4 L (or appropriately sized) container of system water. 2. Gently but firmly hold the brooding female under water, GENTLY opening her mouth with your finger or a transfer pipette. The eggs will fall to the bottom of the tank. 3. Before returning the female to the tank, verify you have gotten all of the embryos by looking in her mouth. 4. Pipette embryos into a marked petri dish. 5. Update the Cichlid Brood Log. 6.3 Care of embryos As needed, use microscope to observe embryos in the petri dish. As described in SOP TR-01-01, to bring fish to the main Powder lab in 060C, petri dishes containing system water are carried in a secondary container. Any dead or non-developing embryos will be removed from the petri dish immediately using transfer pipettes. Dead tissue is disposed of as in Powder SOP EU-03-01. Determine the brood date of birth and age using the cichlid aging charts and guides in the lab, for example Fujimura and Okada (2007) and de Jong et al. (2009). Mark date on labels. Embryos are maintained in a circulating flask until they develop into freely feeding larvae with a fully absorbed yolk. This water flow promotes circulation of embryos and thus inhibit fungal growth. Using a transfer pipette, move embryos to a 250 mL flask within a 3L tank that has a baffle on the back. Mark the tank using tape and permanent marker with the species name, generation, and birth date. Ensure water flow is just enough to keep eggs moving gently, but not enough that lifts eggs from the bottom of the flask. Check daily for any mortality. As necessary, remove any dead or non-developing embryos. Powder SOP BR-02-02 Page 3 of 4 Effective Date: 06-05-2018 Fish that have yolk should not be fed (it promotes fungal growth). Once fish have fully absorbed yolk and are free-feeding (roughly 16-21 days post fertilization), gently dump fish from the flask into the surrounding 3L tank. At this point, they will be fed standard flake mix. 6.4 Sanitation Collection dishes will be manually scrubbed and put on the rack to dry after each use. Dishes will be disinfected periodically using a bleach solution (1/4 cup or 60mL sodium hypochlorite in 10 gallons of water). Dishes should be rinse VERY well after bleaching and allowed to dry completely before use. Sanitation containers should be labeled with date solution was made and an expiration date of 5 days after “Made” date. Petri dishes will be manually scrubbed and sprayed with 70% ethanol prior to reuse. 6.5 Specialized Care of Fish A. Mortality or cloudy water Embryos and larvae are fragile and may experience high mortality rates without any obvious cause. However, if possible, identify the source of mortality as quickly as possible. An immediate change of water is indicated if water becomes cloudy. B. Euthanasia Larvae are sacrificed as described in Powder SOP EU-03-01, EU-04-01, and EU-05-01. 7.0 QUALITY CONTROL CHECKS AND ACCEPTANCE CRITERIA All procedures are subject to review by the Attending Veterinarian and the Institutional Animal Care and Use Committee. 8.0 LITERATURE CITED de Jong IM, Witte F, Richardson MK. Developmental stages until hatching of the Lake Victoria cichlid Haplochromis piceatus (Teleostei: Cichlidae). J Morphol. 2009 May;270(5):519-35. Fryer, G. and T.D. Iles, 1972, The Cichlid Fishes of the Great Lakes of Africa: Their Biology and Evolution. THR Publications Fujimura K and Okada N. Development of the embryo, larva and early juvenile of Nile tilapia Oreochromis niloticus (Pisces: Cichlidae). Dev Growth Differ. 2007 May;49(4):301-24. Konings, A., 2007, Malawi Cichlids: in their natural habitat. 4th edition. Cichlid Press. 9.0 APPENDICES Cichlid Brood Log Powder SOP BR-02-02 Page 4 of 4 Effective Date: 06-05-2018 CICHLID BROOD LOG Date Species Tank Noticed Collect Collect Comments by Date by Initials Initials .