Welfare of invertebrates: a pilot study on a new land snail stunning technique

The almost complete absence of regulations to protect invertebrates is a common condition in legal systems, including the European one, especially when it comes to invertebrates intended for human consumption. Thus, in the vast majority of cases, edible invertebrates do not receive even the most basic protection at slaughter. Despite recent research indicating that invertebrates are capable of feeling pain and stress, the humane step of stunning is not used on them. This is also the case for land snails, which are gastropod invertebrates whose consumption has now reached significant levels, already involving tonnes and that is expected to increase significantly as edible snail farming becomes more popular as a relatively low-cost, easy-to-perform, and sustainable alternative animal husbandry, thereby making land snails an increasingly economically important species. This paper presents and investigates a proposed stunning method based on the immersion of mollusks in CO2-supplemented and refrigerated water that could be used in the snail meat production chain to reduce the slaughter suffering of millions of these invertebrates. To this end, body condition descriptors (hemolymph parameters) in snails were determined before and after CO2 treatment in cold water, while generating useful data for defining a preliminary set of reference intervals for basal values.


Cornu aspersum's biological and anatomical characteristics
Cornu aspersum (former Helix aspersa) is a common snail naturally found throughout the Italian peninsula, particularly in the Mediterranean basin (Fig. 1).It is a terrestrial gastropod mollusk of the Helicidae family.Its shell is cone-shaped, with 3-4 coils developed around a central axis (columella) and can hold the animal's cephalo-podal mass.In fact, the snail has a ventral muscular foot that allows it to move.The internal organs are contained within the body in a single cavity, which is protected by a mantle that lines the inside of the shell.
Snails are pulmonated animals.Their nervous system is ganglionic, with ganglia of nerve cells interconnected by bundles of nerve fibers.Although they lack a true brain, snails have been observed to have associative thinking skills, implying that this learning ability, while primitive, allows them to experience pleasure and pain (as sentient beings) 14,15 .The evolution of terrestrial gastropods has revealed that they evolved from aquatic forms 16 .The importance of water in terrestrial gastropod survival has been extensively studied [17][18][19] .Even today, land snails contain a large amount of water and maintain a constant, complete aqueous body cover due to the production of a hygroscopic slime.This condition allows them to withstand more water immersion before drowning than other pulmonated organisms 20 .There are also terrestrial snails' species that can survive underwater 21 .

Terrestrial gastropods' respiratory system
Snails breathe through a valved opening called a pneumostome that connects to a rudimentary lung made up of an evolved lung sac in the mantle cavity.The pneumostome opening regulates both internal CO 2 pressure and the need to inhale oxygen-rich fresh air.It has been demonstrated that oxygen is introduced into the respiratory surfaces of lung gastropods via water.In fact, an aqueous layer coats both the walls of the lung cavity and the outer surface of the body, allowing skin respiration 22 .Furthermore, the snail lung can adapt to aquatic respiration, albeit with less advantageous gas exchanges than air respiration.The exchange of gases within the organism is based on the phenomenon of diffusion, which is influenced by the difference in partial pressures of each of them between the environment outside and inside the organism itself and, as previously stated, occurs primarily through the pneumostome, the opening of which is stimulated by the lowering of oxygen pressure inside the mollusk as well as by hypercapnia, with a response mediated by CO 2 -sensitive cells present in the central nervous system of the snail 23 .Some research suggests that hypoxia with hypercapnia, as well as subsequent acidification of intracellular and tissue pH, may govern a reversible transition phase between the active and quiescent condition in lung mollusks 24,25 .These findings lend support to the hypothesis that hypercapnia slows metabolism in such animals, allowing them to enter a dormant state.It is a reversible state of quiescence that, in snails, is comparable to stunning.In fact, according to Regulation (EC) No. 1099/2009 on the protection of animals at the time of killing, 'stunning' means any intentionally induced process which causes loss of consciousness and sensibility without pain, including any process resulting in instantaneous death (Art.2, letter f).Contrary to what generally happens in vertebrate species when using the stunning methods listed in Regulation (EC) No. 1099/2009, snails are able to recover after the treatment described 26 .

The legislation governing the slaughter of invertebrates
Existing general requirements for their killing and related operations require that animals be spared avoidable pain, distress, or suffering (Regulation No. 1099/2009/EC, Art. 3).To that end, stunning before killing has been made mandatory.This practice, however, does not apply to the slaughter of invertebrates, which are exempt from the application of Regulation No. 1099/2009/EC, establishing criteria for the protection of animals used for food production that are limited to vertebrate animals.
In the absence of current regulations requiring prior stunning, land snails produced by heliciculture and intended for gastronomic consumption are slaughtered directly with the traditional boiling of live and conscious mollusks.

Summary statistics
In Table 1, summary statistics are shown for each variable given the selected treatment after removing extreme observations (outliers).In Table 1S of the Supplementary Material, summary statistics are also calculated but using raw data, that is without removing extreme observations (outliers).Box plots based on the quantiles shown in Table 1 are also included in the Supplementary Material.

Inferring intervals of commonly observable values for pretreatment variables
The procedure to estimate endpoints of intervals describing common observable values is summarized below for the pH variable.
Using the LOO criterion, the skew-normal distribution was selected for variable pH (pretreatment).In Fig. 2, 200 draws from the posterior predictive distribution have been performed and the correspondent estimates of the pdf are shown in light grey, where the thick black line is the estimate based on the collected sample.
Using 5000 draws from the posterior predictive distribution, the first and last percentile were calculated, together with realized min and max values.Table 2 below contains the inferred endpoints of intervals to recommend inspection of extreme points.The complete data can be found in Table 2S (Supplementary material).

Variable pH: the hypothesis of null treatment effect
In Fig. 3, the quantile-quantile plot of post-pre treatment differences are shown to appreciate possible departures from normality.
After removing an observation (top right), the above mentioned three models (Normal, Student t, Skew Normal) were fitted and the posterior distribution of µ D,1 (the mean value of the difference due to treatment) for the best model was estimated by MCMC simulation: the Student-t model was selected by the LOO criterion and in particular no sampled value was greater than zero, i.e. the estimated probability is P µ D,1 > 0 < 0.002 .Tail probabilities for µ D,i are shown in Table 3.

Stun induction
In terms of stun induction, all snails immersed in CO 2 -enriched water were shown to achieve an acceptable level of unconsciousness, resulting in the stunning outcome described above.

Discussion
In this pilot study, we proposed and investigated an original stunning method based on water supplemented with CO2 that could be used safely and humanely in the snail meat production chain.A Bayesian statistical analysis revealed substantial changes in many hemolymph components, like pH, Na, and Cl, in snails after immersion in gaseous water for a time sufficient to get them stunned.This finding is consistent with the literature-supported hypothesis that hypercapnia allows these animals to enter a dormant state.Furthermore, plausible physiological ranges, outside of which observed values should be investigated for the potential presence of large measurement errors and/or atypical responses of snails to treatment, were identified through analysis of the measured basal values of hemolymph parameters.
From a physiological perspective, the exposure of snails to cold and gaseous water facilitates the induction of torpor in snails, as well as a physiological response, as evidenced by changes in hemolymph components, confirming that CO 2 has an effect on mollusks' metabolism, depressing it and driving them to unconsciousness in a relatively short period of time.A remarkable feature of this procedure is its reversibility: even when applied several times to snails, they are able to recover without side effects.For that reason, too, this method of stunning can be considered humane for animals.Another major advantage is that it is possible to perform group stunning, which is a significant asset in favor of the future adoption of this method in industrial practice.

Animals and methods
The research was carried out on a sample of 75 Cornu aspersum (weight: 10-12 g) alive and vital, purchased from a local retail market where the vendor was directly a local snail farmer.The animals were placed in clean plastic containers (Fig. 4), exposed to a natural light-dark cycle, and kept under environmental temperature and humidity conditions similar to those present in the source area, as the farm of origin was located not far from the research facility.No nutrition was provided to maintain the same conditions in which the snails are kept ready to be prepared as food.Each snail was identified by drawing an indelible ID number on each shell (Figs. 5 and 6).
In the first phase of the study, snails were subjected to hemolymph sampling (Figs. 7, 8 and 9), using the method described by J.E. Cooper 29 .Hemolymph sampling was carried out through the sinus region with a sterile butterfly needle (G23 X ¾) and 300mm length tubing.The method does not require the sacrifice of the mollusk and does not determine death or long-term negative impacts on the health of the animal, so as to allow the periodic extraction of hemolymph on the same source animal, if needed.The volume of hemolymph collected from each snail ranged between 0.3 and 0.6 ml.The freshly drawn hemolymph was immediately transferred into a sterile insulin syringe (Fig. 10) and analyzed, using the IDEXX VetStat® Electrolyte Blood Gas Analyzer for veterinary use, which measured the following sixteen parameters: acidity (pH), carbon dioxide partial pressure (pCO 2 ), oxygen partial pressure (pO 2 ), sodium concentration (Na + ), potassium concentration (K + ), chlorine concentration (Cl − ), ionized calcium concentration (Ca ++ ), carbon dioxide concentration (TCO 2 ), (ionized calcium normalized to PH 7.4) nCa, transcutaneous acidity (pH(TC)), transcutaneous carbon dioxide partial pressure (pCO 2 (TC)), transcutaneous oxygen partial pressure (pO 2 (TC)), standard bicarbonate concentration (SBC), bicarbonate ion concentration (HCO 3 − ), Alkalosis (A), osmolarity (Osm).These values were statistically processed in order to determine the plausible physiological range of each variable.
In the second phase of the study, a quota of the specimens was tested for stunning by immersion in mineral drinking water enriched with a high concentration (10 g/l) of food-grade CO 2 (E290) and maintained at 6-8 °C.www.nature.com/scientificreports/This step of the procedure was aimed at assessing their sensitivity to CO 2 -supplemented and refrigerated water and to observe their reaction to the point of verifying the induction of their stunning.The temperature of the water was chosen to match the temperature at which the snails are typically kept in order to preserve them before industrial slaughter.The low temperature also helps to maintain the level of dissolved CO 2 in the water, as CO 2 solubility is temperature dependent and is better maintained in cold water.Furthermore, it has been found that temperatures below 10°C induce and maintain torpor in snails, as is the case during their winter dormancy 30 .The surface of the water was covered with a plastic contact film to limit the dispersion of CO 2 into the air during the test.The snails were immersed completely in water and observed while the immersion time was recorded.The expected outcome of stunning was identified as follows: an animal that was completely out of its shell and relaxed, with no reactivity to even the stimulus of the eye tentacles.The time required to stun each snail was

Figure 2 .
Figure 2. Kernel density estimation of the marginal probability density function of variable pH (dark thick black) is compared with 200 estimates of the same density function based on samples from the predictive posterior distribution (light grey).

Figure 4 .
Figure 4. Snails placed in a clean, plastic container.

Figure 10 .
Figure 10.Transfer of hemolymph into a sterile insulin syringe.

Table 3 .
Final models selected for each variable that describe body state.