Do Brine Shrimp Feel Pain When Frozen? The Science Behind These Tiny Creatures

| Method | Effectiveness | Speed ||--------|--------------|-------|| Rapid freeze | High | Very fast || Slow freeze | Low | Slow || With cryoprotectants | Highest | Fast |

Titration of first-step solutions.

Using 95% reagent alcohol (85% ethyl alcohol, 5% isopropyl alcohol, 4% methanol; product no. 9500-1, StatLab Medical Products, McKinney, TX) and tap water, we produced solutions containing 5%, 10%, 20%, 30%, 40%, 50%, 60%, and 70% alcohol; therefore, the total ethanol concentration was 53.7% for the 60% alcohol solution. Although our goal was to maintain a similar salinity to the tank water for all anesthetic solutions to diminish stress, salt precipitated out of solutions containing 30% alcohol or more in tank water; in these cases, the precipitated salt formed a gel-like consistency, which was difficult for the Artemia to swim through. For this reason, we mixed alcohol with tap water; Artemia did not differ between tap water and tank water in preliminary experiments. We pipetted 1.5 mL of solution each into 2 wells of a 24-well plate, and 5 Artemia were then placed in each well. The time to anesthesia and any abnormal behaviors were recorded. Anesthesia was defined as a lack of forward motion and lack of response to stimulation by using a probe. Euthanasia was defined as a lack of thoracopod movement for 10 s of observation.

Eugenol was tested at 1.3 and 2.5 mg/L. For a stock solution, we diluted 99% eugenol (product no. AC119110050, ACROS Organics, Morris, NJ USA) with 95% alcohol, and then with tank water, and stored the resulting solution in an amber bottle at room temperature. The final concentration of alcohol in the 2.5-mg/L dose was 2.4%. We pipetted 1 mL of each solution into each of 2 wells of a 24-well plate, and 5 Artemia were then placed in each well. The time to anesthesia and any abnormal behaviors were recorded.

TMS (product no. NC0135573, Tricaine-S, Western Chemical, Ferndale, WA) was diluted to 10 g/L by using tank water and then buffered to pH 7 to 7.5 with sodium bicarbonate (product no. S233-500, Fisher Scientific, Hampton, NH). The solution was then further diluted with tank water to 1, 2, and 4 g/L; resulting solutions were stored in amber bottles at 4 °C. Each solution was allowed to warm to room temperature prior to exposure. We pipetted 1 mL of each solution into each of 2 wells of a 24-well plate, and 5 Artemia were then placed in each well. The time to anesthesia and any abnormal behavior were recorded.

Artemia were assigned to 4 groups (n = 30 per group) and placed in solutions of 60% alcohol, 4 g/L TMS, 2.5 mg/L eugenol, or tank water (control). In each of six 24-well plates, 20 wells contained either 1 mL of an anesthetic solution or tank water according to a permuted plate randomization (Figure 1). We then used a transfer pipet to add a single Artemia to each well. A maximum of 50 μL of tank water was moved with each shrimp, to minimize dilution of the solution. A treatment-blinded observer used a wooden probe to confirm anesthesia, which was defined as a lack of forward motion and lack of response to the probe. Time to anesthesia was recorded for each animal, with a cut-off time of 60 min. After 5 min of anesthesia, the Artemia were transferred to a euthanasia solution.

Artemia were placed in 1 mL of anesthetic solution in a 24-well plate according to a permuted plate randomization design.

Anesthetized Artemia were divided into subgroups of 10; each subgroup was transferred to a euthanasia solution: 70% alcohol (product no. 7070-1, StatLab), 95% alcohol (product no. 9500-1, StatLab), or 10% NBF (product no. 28600-5, StatLab). Time to euthanasia, defined as a lack of thoracopod movement for 10 s of observation, was recorded.28

Behavior during the first 5 min in the anesthetic solution was scored by a treatment-blinded observer. One point each was given for abnormal posturing, hyperactivity, or seizure-like behavior, for a score of 0 to 3; 0, no abnormal behavior; 1, mild; 2 or greater, severe abnormal behavior.

Anesthesia, euthanasia, and behavior scoring were tested in triplicate on separate days.

Artemia (n = 10) from each anesthetic group were anesthetized in a 24-well plate as described earlier. After 5 min of anesthesia, they were rinsed by placing them in a well of tank water and were immediately transferred into a second well of tank water to assess their ability to recover over a 2-h period. Recovery was considered to be achieved on regaining forward motion. This experiment was repeated in triplicate (total, n = 90).

During the original experimental period, eugenol consistently induced anesthesia, as shown in the Results section. Approximately 4 mo later, we attempted to repeat anesthetic induction of Artemia and were unable to obtain similar results with 2.5 mg/L eugenol. Troubleshooting was performed, including purchasing a new bottle of eugenol. We began a series of titrations of eugenol to determine what concentration would produce results similar to our previous experiment. Four titrations of eugenol were performed over a 7 mo period to account for possible seasonal variation. Eugenol was prepared as described earlier: 99% eugenol was first diluted with 95% alcohol and then further diluted with tank water. Each solution was plated in duplicate by pipetting 1 mL into 2 wells of a 24-well plate, and 5 Artemia were then placed in each well. Concentrations tested included 0.125, 1.3, 2.5, 13, 25, 75, and 130 mg/L; the total alcohol concentration in the 130-mg/L dose was 12.4%. As a control, 2 wells of 60% alcohol each containing 5 Artemia were tested also. The time to anesthesia was recorded.

Kaplan–Meier survival and cumulative morbidity curves were drawn for each treatment group, and differences were analyzed by using log-rank statistics for anesthesia and euthanasia, respectively. Proportional odds logistic regression was used to determine cumulative odds ratios of abnormal behavior between groups. The Kruskal–Wallis ANOVA was used to compare behavior scores between groups. Logistic regression analysis was used to compare the significance of anesthetic recovery rates among the 3 anesthetic groups. One-way ANOVA was used to determine significance of replication anesthetic times of alcohol. A P value of less than or equal to 0.05 was considered significant. Statistical analyses were performed by using Stata version 14 (StataCorp, College Station, TX) or Prism 7.03 (GraphPad Software, La Jolla, CA).

One Artemia in the eugenol group and 3 in the TMS group were removed from the study results, due to recording errors.

Amanda K Darbyshire1Division of Comparative MedicineFind articles by

Received 2018 Apr 6; Revised 2018 May 11; Accepted 2018 May 31; Issue date 2019 Jan. © American Association for Laboratory Animal Science

Invertebrates are often overlooked as laboratory animals, yet they are commonly used in toxicology, developmental, cellular and molecular biology, and radiation studies with euthanasia as an endpoint. Little is known regarding appropriate euthanasia methods for invertebrate species, particularly for Artemia. Here, we evaluated the AVMA-recommended 2-step method of euthanasia in brine shrimp (Artemia franciscana). Artemia were exposed first to anesthetic solutions of 60% alcohol, 2.5 mg/L eugenol, or 4 g/L tricaine methanesulfonate (TMS) and then were transferred to euthanasia solutions of 70% alcohol, 95% alcohol, or 10% neutral buffered formalin. We examined time to anesthesia, behavioral response to anesthesia, anesthesia recovery, and time to euthanasia. Our results show that 2.5 mg/L eugenol and 4 g/L TMS inconsistently achieved anesthesia. Although 60% alcohol produced anesthesia, the time to anesthesia varied among replicate groups, and exposure resulted in an increase in abnormal behavior. We therefore do not recommend any of the tested anesthetic solutions for use in Artemia. Although all 3 euthanasia solutions were effective, more research is needed to provide recommendations regarding euthanasia for this species.

Abbreviations: NBF, neutral buffered formalin; TMS, tricaine methanesulfonate

Brine shrimp (Artemia spp.) are branchiopod crustaceans found along coastlines and in salt lakes. They are used in research for toxicology, developmental, cellular and molecular biology, and radiation studies and as food for aquatic laboratory species, including zebrafish.2 At our institution, a pilot experiment showed that 95% alcohol was an effective euthanasia agent but produced abnormal behavior in Artemia, yet 5% alcohol, recommended by the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition (AVMA Guidelines) as a first-step agent for euthanasia of aquatic invertebrates, was ineffective at producing anesthesia.8 Information regarding euthanasia techniques for this species is sparse currently. As our culture becomes more sensitive to the possibility that invertebrates may experience pain and distress, guidelines for humane treatment and euthanasia should be established.

The AVMA Guidelines provide euthanasia recommendations for both terrestrial and aquatic invertebrates.8 Recommendations comprise a 2-step process involving first anesthesia or presumptive death, followed by an adjunct method to either chemically or physically destroy the brain or major ganglia. The use of an adjunct method alone is described as not acceptable. Recommended first-step solutions include eugenol, 1% to 5% ethanol, and magnesium salts, and second-step, adjunct methods include 70% ethanol, formalin, freezing, boiling, or pithing. It is unacceptable to remove invertebrates from water to desiccate, to leave them in unaerated water to become hypoxic, or to use caustic substances or traumatic techniques.8

For testing, we selected 3 first-step (that is, anesthetic) solutions—alcohol, eugenol, and tricaine methanesulfonate (TMS)—in light of their practicality. No data regarding the dosage of these agents for Artemia have been published, and only sparse, widely disparate data are available regarding their dosage in other invertebrate species.

Alcohol may be used as an anesthetic in invertebrates.10,16,24 The mechanism of action is not fully known but is likely multifactorial.19 In mollusks, alcohol inhibits neuronal sodium and calcium channels.21 In crustaceans, there is evidence of neuromuscular junction depression of the excitatory postsynaptic potentials.3,19 Alcohol is cost-effective and easily available. The AVMA Guidelines recommend using 1% to 5% ethanol as the first step in a 2-step euthanasia process and suggest that concentrations above 70% should be used only as a second-step solution.8 Ethanol at a concentration of 10% has been used as an anesthetic in giant tiger shrimp (Penaeus monodon).23

Eugenol is commonly used as an anesthetic in both fish and crustaceans.5 This organic phenol is the main constituent of clove oil and has a low potential for toxicity and side effects. Its mechanism of action in fish is thought to involve the antagonism of vanilloid receptor 1; it also has a demonstrated affinity for GABAA and NMDA glutamate receptors.17 Eugenol is cost-effective and readily available. The AVMA Guidelines recommend using 0.125 mL/L (125 mg/L) for euthanasia or anesthetic induction; lower concentrations should be used for anesthesia alone.8 For anesthesia, a dose range of 0.03 to 1 mL/L (30 to 1000 mg/L) has been recommended for crustaceans.24 A safe dose of eugenol for the sedation of postlarvae of white Indian shrimp (Fenneropenaeus indicus) was 1.3 mg/L; other doses tested were 2.5 and 3.7 mg/L.5 In Norway lobsters (Nephrops norvegicus), a eugenol concentration of 900 μL/L (900mg/L) has been determined to be effective.12

TMS (also known as MS222) is an FDA-approved agent for the anesthesia of fish, amphibians, and other aquatic, cold-blooded animals. It is a sulfonated isomer of benzocaine. The mechanism of action is unconfirmed but is thought to be similar to that of benzocaine, which blocks action potential conductance through voltage-gated sodium channels.27 Although TMS has been suggested to be ineffective in crustaceans,9,13,24 successful anesthesia was shown in ostracods.29 The minimum effective anesthetic dose in the ostracod Eucypris virens was 500 mg/L, with induction times ranging from 20 s to 2.5 min depending on concentration.29 The effective dose for cherry shrimp, Neocardinina denticulate, was determined to be 2500 mg/L in a 20-min bath.20 A well-known reference work recommends a dose of 100 mg/L for the anesthesia of aquatic invertebrates.17 Although more costly than the other agents, a small amount of TMS could be used to euthanize large numbers of Artemia.

Adjunct agents recommended by the AVMA for the second step of the euthanasia process include 70% alcohol, 95% alcohol, and 10% neutral buffered formalin (NBF).8 These agents are all common preservatives used in labs, are cost-effective, and are expected to be compatible with post-euthanasia histology. Because researchers at our institution need to analyze euthanized Artemia histologically, we did not assess other AVMA-recommended methods, such as boiling, freezing, and pithing, in this study.8

The purpose of this study was to determine a method for efficiently euthanizing Artemia by using a 2-step approach. After titrating first-step solutions for anesthetic efficacy, we hypothesized, given our previous experience with 95% alcohol, that 60% alcohol would likely cause abnormal behavior and consequently be deemed unsuitable as a first-step agent. We also hypothesized that TMS and eugenol would provide adequate anesthesia and that eugenol would provide more consistent anesthesia than TMS. Finally, after Artemia were anesthetized by using a first-step solution, we anticipated that 70% alcohol would be inadequate for euthanasia, whereas 95% alcohol and 10% NBF would be effective.

Adult A. franciscana (The Aquatic Critter, Nashville, TN) were maintained in 7.5 L artificial seawater (made by using tap water and Instant Ocean [catalog no. SS15-10, Spectrum Brands, Blacksburg, VA]) at 25 °C, salinity of 1.030 g/dL (40 parts per thousand), and pH 8.0. The Artemia were fed spirulina (Whole Foods, Nashville, TN) once daily, and API Stress Coat (85A, Mars Fish Care North America, Chalfont, PA) was added to the water to remove chlorine and chloramines. LED lighting was provided on a 12:12-h light:dark cycle, and moderate aeration was provided at all times. Although our institution does not require an IACUC-approved protocol for invertebrate use, the current research was performed in accordance with the animal use policies and procedures of Vanderbilt University Medical Center. Concentrations of the anesthetic solutions were determined according to the results of the titration trials (described later).

Why I Freeze my shrimp Alive