*2.1.4. Kumamoto oyster (Crassostrea sikamea)*

Kumamoto oyster *Crassostrea. sikamea* is a highly valuable mollusc species and cultivated in several countries, so it is important to generate new knowledge for its culture and alternative treatments for its management in the laboratory. Four experiments with *C. sikamea* were performed as described below.

**Trial 1**. An experimental design was applied at CIBNOR for conditioning broodstock with two replicates (25 oysters each) in eight plastic boxes (60 l), in which the following treatments were tested: ViP-ViA (T1) and PhA-SiT (T2) as HOM treatments, and ET (T3) and NT (T4). As treatment vehicle, fully impregnated inert homoeopathic pills were used, one-a-day per oyster.

Oysters were dissected (30 at t<sup>0</sup> and 15 from each replicate at t42) and soft tissues histologically processed by haematoxylin and eosin staining to determine gonadic (GI) and digestive gland (DG) indexes, reproductive stages and theoretical diameter in oocytes (DT) according to Rodriguez-Jaramillo *et al*. [22] and Barber and Blake [24]. Moreover, significant differences were observed (*p* < 0.05) in GI in broodstock oysters with respect to the initial value (39%), and the best results were observed in oysters treated with T2 and T3 (63 and 67%). The highest DI was recorded at the beginning of the experiment (60%) and the lowest (*p* < 0.05) for T2 and T3 (36 and 32%), which suggested a greater energy storage in the gonad for oocyte maturation [24] (**Figure 6**). A high frequency of organisms in post-spawning and gonadic development stage (38 and 52%) was observed at t0 . At t42, the oysters of T2 and T3 showed the highest percentage of full sexual maturity (50%) while the greatest number (35 and 40%) of organisms in gonadic development stage was observed in T3 and T4 (**Figure 6**).

Relative to the final DT (35 μm), which was 28 μm at t<sup>0</sup> , no significant differences were recorded between HOM-treated and control oysters. However, maturity and undifferentiated stages were simultaneously observed in T3 oysters. Since *C. sikamea* matures and partially spawns several times during the same reproductive season, this result suggested a positive effect by

**Figure 6.** Gonadic index (GI) and digestive index (DI) (left) and gonad development (tight) in broodstock oyster *Crassostrea sikamea* conditioned in the laboratory for 42 days. Different letters show significant differences between treatments (*p* < 0.05). Stage 0: Undifferentiated; stage I: Previtellogenesis; stage II: Vitellogenesis; stage III: Posvitellogenic; stage IV: Partial spawning; stage V: Post spawning.

the activation of a new oocyte production cycle associated to HOM T2 (PhA-SiT). In bivalve molluscs, an expenditure of digestive gland reserves is associated to gonad development and maturation of gametes [24], and T2 was associated to a greater number of sexually mature oysters with higher GI and lower ID values. It could be applicable to gonadic conditioning for sexual maturation of broodstock oyster at the hatchery.

nursery confirmed a great potential for aquacultural homoeopathy to strengthen the state-ofthe-art technology in oyster seed production and increase actual productivity and economic

**Figure 5.** Growth rate in shell length (μm day−1) (left) and volumetric biomass of harvested seed (right) of American oyster *Crassostrea virginica* seed, receiving HOM treatments in recirculating upwelling nursery units (40 l) at COTET

Kumamoto oyster *Crassostrea. sikamea* is a highly valuable mollusc species and cultivated in several countries, so it is important to generate new knowledge for its culture and alternative treatments for its management in the laboratory. Four experiments with *C. sikamea* were

**Trial 1**. An experimental design was applied at CIBNOR for conditioning broodstock with two replicates (25 oysters each) in eight plastic boxes (60 l), in which the following treatments were tested: ViP-ViA (T1) and PhA-SiT (T2) as HOM treatments, and ET (T3) and NT (T4). As treatment vehicle, fully impregnated inert homoeopathic pills were used, one-a-day per oyster.

cally processed by haematoxylin and eosin staining to determine gonadic (GI) and digestive gland (DG) indexes, reproductive stages and theoretical diameter in oocytes (DT) according to Rodriguez-Jaramillo *et al*. [22] and Barber and Blake [24]. Moreover, significant differences were observed (*p* < 0.05) in GI in broodstock oysters with respect to the initial value (39%), and the best results were observed in oysters treated with T2 and T3 (63 and 67%). The highest DI was recorded at the beginning of the experiment (60%) and the lowest (*p* < 0.05) for T2 and T3 (36 and 32%), which suggested a greater energy storage in the gonad for oocyte maturation [24] (**Figure 6**). A high frequency of organisms in post-spawning and gonadic development

percentage of full sexual maturity (50%) while the greatest number (35 and 40%) of organisms

between HOM-treated and control oysters. However, maturity and undifferentiated stages were simultaneously observed in T3 oysters. Since *C. sikamea* matures and partially spawns several times during the same reproductive season, this result suggested a positive effect by

in gonadic development stage was observed in T3 and T4 (**Figure 6**).

and 15 from each replicate at t42) and soft tissues histologi-

. At t42, the oysters of T2 and T3 showed the highest

, no significant differences were recorded

profitability of the commercial production of the species in the hatchery.

*2.1.4. Kumamoto oyster (Crassostrea sikamea)*

performed as described below.

78 Aquaculture - Plants and Invertebrates

hatchery.

Oysters were dissected (30 at t<sup>0</sup>

stage (38 and 52%) was observed at t0

Relative to the final DT (35 μm), which was 28 μm at t<sup>0</sup>

**Trial 2**. Once the broodstock assay finished, a subsequent second bioassay was developed to evaluate larval performance in two different larval cohorts from HOM-treated spawners (T1, T2, T3, T4; three replicates each) and NT spawners (T5, T6, T7, T8; three replicates each). The greatest growth in length was recorded in T5 and T6 (217 and 212 μm) and the lowest in T3 and T4 (197 and 190 μm). A similar growth pattern was observed in height: the greatest in T5–T8 and the lowest in T1–T4. In general, larvae from untreated broodstock grew more. Nevertheless, as a counterpart, the general mean survival of larvae from HOM-treated spawners (T1-T4) was 32.3% higher than those from NT broodstock, and it could be very important to improve hatchery seed production. Those differences in larval survival suggested a favourable effect of HOM treatments in oocyte quality and then a potential effect in the progeny.

**Trial 3**. A 35-day experimental design in triplicate (six treatments and 18 replicates, 120 seed each replicate) was applied at CIBNOR to compare efficiency of antibiotics against homoeopathy in juvenile *C. sikamea*. Juveniles (6.13 ± 0.16 mm) were placed in 2-l plastic containers with filtered (1 μm) and UV-sterilised seawater (23 ± 1°C; 38 psu), continuous aeration and microalgal food. Juvenile oysters received PaV-Pha (T1), PaV-Sit (T2), Pav-MeS (T3) as HOM treatments, ampicillin AMP (T4) as antibiotic treatment, and ethanol ET (T5) and NT (T6) as control treatments. Liquid treatments were applied 100 μl l −1 for homoeopathy and 10 ppm for antibiotic after exchange of seawater every 48 h and before feeding oysters. Evaluations were performed on growth in height (mm), and total wet weight of the shell (mg), biochemical flesh composition (mg g−1) and SOD activity. The biochemical composition was determined by triplicate sampling at the beginning and end of the trial. Samples were stored at −80°C and then lyophilized, rehydrated in 3 ml cold saline solution (35%) and homogenised to obtain crude extracts. Crude extracts were processed at CIBNOR laboratory applying traditional and certified techniques, which are described by López-Carvallo *et al*. [20] and Mazón-Suástegui *et al*. [23]. For SOD analyses, after deep cleaning of shell, soft tissues were dissected and fixed individually in RNAlater®

(Thermo Fisher Scientific, Waltham, MA, USA) at a 1:5 ratio (100 mg tissue: 500 μl RNA-Later®) and then preserve it at −20°C. SOD analyses activity was determined with a commercial kit (SOD Assay Kit #19160, Sigma-Aldrich). Results were expressed as an indirect measure of SOD activity as a per cent of the water soluble tetrazolium salt formazan complex inhibition. The juveniles grew significantly more with HOM T1 (9.27 ± 0.18 mm, 0.073 mm day−1) and T3 (9.36 ± 0.18 mm, 0.076 mm day−1) compared with NT (T6) (8.02 ± 0.23 mm; 0.053 mm day−1), but of all treatments, the ET T5 group was the best (10.17 ± 0.31 mm; 0.105 mm day−1). A significant increase in total wet weight was recorded with HOM T2 (107.33 ± 6.9 mg, 2.0 mg day−1) compared to NT group T6 (76.11 ± 2.8 mg, 1.1 mg day−1) (**Figure 7**). Survival was 100% in all treatments and their replicates. Moreover, the biochemical composition of the juveniles showed significant differences in carbohydrates and lipids but not in proteins. The highest amount of lipids was obtained with HOM T2 (96.32 ± 1.18 mg g−1) and that of carbohydrates in antibiotic T4 (27.48 ± 2.27 mg g−1) compared with the NT (T6) (6.96 ± 1.47 mg g−1). At the end of this trial, SOD activity was higher in HOM T1 (92%) than control T6 (88%) and antibiotic T4 (84%).

*2.1.5. Geoduck clam (Panopea globosa)*

ET (T6) and NT (T7). Samples were taken at the beginning (t<sup>o</sup>

The geoduck clam *Panopea globosa* is an important marine resource distributed on both coasts of the peninsula of Baja California, Mexico. Its cultivation is still in experimental stage and totally depending on juveniles produced in the laboratory. As with other bivalves, there are limitations in their production due to mortalities of larvae and seeds, associated with the presence of pathogens. This problem has led to the search for new eco-friendly alternatives such as aquaculture homoeopathy, which has a positive effect on nutrition, health and immune response of bivalve molluscs, shrimp and marine fish [5, 23]. Our study evaluated the effect of various HOM treatments on growth, survival and microbiota of the gastrointestinal tract (GIT) of the species. Juvenile (spat) *P. globosa* with an average length of 1.98 ± 0.1 cm were produced in the laboratory and provided by the company Acuacultura Robles, a commercial mollusc hatchery located in La Paz, B.C.S. Mexico. Clams with an average length of 1.98 ± 0.1 cm were produced and provided by the company Acuacultura Robles, acclimatised at CIBNOR and then cultured (21 days) in nursery units previously described for *A. ventricosus*. About 24 upwelling units were used, each one with 52 clams and 13 clams per upweller cylinder; the following HOM treatments and controls were applied: ViP-ViA/a (T1), ViP-ViA/b (T2), AcF-MsS (T3) PhA-SiT (T4), ViT (T5),

randomly selecting eight clams per replica, accounting for a total number of 216. After external deep cleaning, soft tissues were dissected to isolate GIT of each clam to fix individually in RNAlater® (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:5 ratio (100 mg tissue: 500 μl RNA-Later®) and then preserve it at −20°C. The bacterial 16S rDNA was extracted according to Garcia-Bernal *et al*. [3] and amplified using Illumina MiSeq Platform (Illumina, San Diego, CA) in a certified Genomic Services Laboratory (www.langebio.cinvestav.mx; Irapuato, Guanajuato, México). Afterward, a bioinformatic and statistical analysis of the generated database was carried out. Taking as reference the microbial diversity in the GIT of *P. globosa* juveniles, the best results were obtained with HOM T1, a nosode product developed at CIBNOR. T1 favoured dominant abundance of the Proteobacteria phylum and some of its classes as γ-Proteobacteria. In that sense, similarities were observed with the microbiota of other marine species, and that part of the microbiota found in *P. globosa* is associated with stimulation of the immune system. Overall, the results indicated that the HOM treatments modified the abundance of the microbial communities of the species, mainly in the phylotypes related to nutritional processes. On the other hand, significant differences were recorded with respect to growth in weight and length (*p* < 0.0001) between the clams that received HOM treatment and the control groups. The highest growth in weight was recorded in T3, T4 and T5. The difference in growth in length was smaller but equally superior to these HOM treatments. Significant differences (*p* = 0.019) in survival were also observed. The highest value (95%) was recorded in clams with HOM T3 followed by the NT control group T7 (93%) without homoeopathy or ethanol. In contrast, the

lowest survival (76%) was observed in T2 and also in the ET control group T6.

An experimental design in triplicate (10 juveniles/replicate) was developed in 60-l fibreglass units at CIBNOR laboratory to assess growth and survival in juvenile octopus during a 28-day

*2.1.6. Octopus (Octopus bimaculoides)*

) and end of the experiment (t<sup>1</sup>

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030

) by

81

**Trial 4.** Once the previous experiment was completed, a pathogen challenge was made with treated seeds. About 30 juveniles were randomly selected from each previous treatment (in duplicate) and challenged at CIBNOR with a pathogenic strain of *V. alginolyticus* (CAIM57: www.ciad. mx). An initial single dose (1 × 106 CFU ml−1) was given as based on the mean lethal dose (LD50) determined by the Probit method based on the dose–response model described by Finney [19].

To obtain greater clarity in the results, in addition to the groups treated with homoeopathy, antibiotic and ethanol, two new groups were included, which did not receive any previous treatment; one of which was infected with the pathogen (CTRL **+**) and another one that was not infected with the pathogen (CTRL -). Survival (%) of juveniles was evaluated at 0, 24, 48, 72 and 120 h after infection and the SOD activity (using a commercial kit; SOD Assay Kit #19160, Sigma-Aldrich) of before infection and at 2, 24, 48 and 72 post-infection was determined. After being infected with the pathogen, all the juveniles survived and no significant differences were observed between treatments and controls with respect to SOD activity. Up to date, that result has no coherent explanation yet; unless the species is highly resistant, the pathogenic strain has not got sufficient virulence or a wrong (lower) dose was applied because 1 × 107 CFU ml−1 was applied to juvenile scallop *A. ventricosus* by Mazón-Suástegui [23].

**Figure 7.** Growth in size (mm day−1) (left) and total wet weight (mg day−1) (right) in juveniles of the oyster Kumamoto *Crasssostrea sikamea* treated with homoeopathic medicines.

## *2.1.5. Geoduck clam (Panopea globosa)*

(Thermo Fisher Scientific, Waltham, MA, USA) at a 1:5 ratio (100 mg tissue: 500 μl RNA-Later®) and then preserve it at −20°C. SOD analyses activity was determined with a commercial kit (SOD Assay Kit #19160, Sigma-Aldrich). Results were expressed as an indirect measure of SOD activity as a per cent of the water soluble tetrazolium salt formazan complex inhibition. The juveniles grew significantly more with HOM T1 (9.27 ± 0.18 mm, 0.073 mm day−1) and T3 (9.36 ± 0.18 mm, 0.076 mm day−1) compared with NT (T6) (8.02 ± 0.23 mm; 0.053 mm day−1), but of all treatments, the ET T5 group was the best (10.17 ± 0.31 mm; 0.105 mm day−1). A significant increase in total wet weight was recorded with HOM T2 (107.33 ± 6.9 mg, 2.0 mg day−1) compared to NT group T6 (76.11 ± 2.8 mg, 1.1 mg day−1) (**Figure 7**). Survival was 100% in all treatments and their replicates. Moreover, the biochemical composition of the juveniles showed significant differences in carbohydrates and lipids but not in proteins. The highest amount of lipids was obtained with HOM T2 (96.32 ± 1.18 mg g−1) and that of carbohydrates in antibiotic T4 (27.48 ± 2.27 mg g−1) compared with the NT (T6) (6.96 ± 1.47 mg g−1). At the end of this trial, SOD

activity was higher in HOM T1 (92%) than control T6 (88%) and antibiotic T4 (84%).

Mazón-Suástegui [23].

80 Aquaculture - Plants and Invertebrates

*Crasssostrea sikamea* treated with homoeopathic medicines.

**Trial 4.** Once the previous experiment was completed, a pathogen challenge was made with treated seeds. About 30 juveniles were randomly selected from each previous treatment (in duplicate) and challenged at CIBNOR with a pathogenic strain of *V. alginolyticus* (CAIM57: www.ciad. mx). An initial single dose (1 × 106 CFU ml−1) was given as based on the mean lethal dose (LD50) determined by the Probit method based on the dose–response model described by Finney [19]. To obtain greater clarity in the results, in addition to the groups treated with homoeopathy, antibiotic and ethanol, two new groups were included, which did not receive any previous treatment; one of which was infected with the pathogen (CTRL **+**) and another one that was not infected with the pathogen (CTRL -). Survival (%) of juveniles was evaluated at 0, 24, 48, 72 and 120 h after infection and the SOD activity (using a commercial kit; SOD Assay Kit #19160, Sigma-Aldrich) of before infection and at 2, 24, 48 and 72 post-infection was determined. After being infected with the pathogen, all the juveniles survived and no significant differences were observed between treatments and controls with respect to SOD activity. Up to date, that result has no coherent explanation yet; unless the species is highly resistant, the pathogenic strain has not got sufficient virulence or a wrong (lower) dose was applied because 1 × 107 CFU ml−1 was applied to juvenile scallop *A. ventricosus* by

**Figure 7.** Growth in size (mm day−1) (left) and total wet weight (mg day−1) (right) in juveniles of the oyster Kumamoto

The geoduck clam *Panopea globosa* is an important marine resource distributed on both coasts of the peninsula of Baja California, Mexico. Its cultivation is still in experimental stage and totally depending on juveniles produced in the laboratory. As with other bivalves, there are limitations in their production due to mortalities of larvae and seeds, associated with the presence of pathogens. This problem has led to the search for new eco-friendly alternatives such as aquaculture homoeopathy, which has a positive effect on nutrition, health and immune response of bivalve molluscs, shrimp and marine fish [5, 23]. Our study evaluated the effect of various HOM treatments on growth, survival and microbiota of the gastrointestinal tract (GIT) of the species. Juvenile (spat) *P. globosa* with an average length of 1.98 ± 0.1 cm were produced in the laboratory and provided by the company Acuacultura Robles, a commercial mollusc hatchery located in La Paz, B.C.S. Mexico. Clams with an average length of 1.98 ± 0.1 cm were produced and provided by the company Acuacultura Robles, acclimatised at CIBNOR and then cultured (21 days) in nursery units previously described for *A. ventricosus*. About 24 upwelling units were used, each one with 52 clams and 13 clams per upweller cylinder; the following HOM treatments and controls were applied: ViP-ViA/a (T1), ViP-ViA/b (T2), AcF-MsS (T3) PhA-SiT (T4), ViT (T5), ET (T6) and NT (T7). Samples were taken at the beginning (t<sup>o</sup> ) and end of the experiment (t<sup>1</sup> ) by randomly selecting eight clams per replica, accounting for a total number of 216. After external deep cleaning, soft tissues were dissected to isolate GIT of each clam to fix individually in RNAlater® (Thermo Fisher Scientific, Waltham, MA, USA) at a 1:5 ratio (100 mg tissue: 500 μl RNA-Later®) and then preserve it at −20°C. The bacterial 16S rDNA was extracted according to Garcia-Bernal *et al*. [3] and amplified using Illumina MiSeq Platform (Illumina, San Diego, CA) in a certified Genomic Services Laboratory (www.langebio.cinvestav.mx; Irapuato, Guanajuato, México). Afterward, a bioinformatic and statistical analysis of the generated database was carried out. Taking as reference the microbial diversity in the GIT of *P. globosa* juveniles, the best results were obtained with HOM T1, a nosode product developed at CIBNOR. T1 favoured dominant abundance of the Proteobacteria phylum and some of its classes as γ-Proteobacteria. In that sense, similarities were observed with the microbiota of other marine species, and that part of the microbiota found in *P. globosa* is associated with stimulation of the immune system. Overall, the results indicated that the HOM treatments modified the abundance of the microbial communities of the species, mainly in the phylotypes related to nutritional processes. On the other hand, significant differences were recorded with respect to growth in weight and length (*p* < 0.0001) between the clams that received HOM treatment and the control groups. The highest growth in weight was recorded in T3, T4 and T5. The difference in growth in length was smaller but equally superior to these HOM treatments. Significant differences (*p* = 0.019) in survival were also observed. The highest value (95%) was recorded in clams with HOM T3 followed by the NT control group T7 (93%) without homoeopathy or ethanol. In contrast, the lowest survival (76%) was observed in T2 and also in the ET control group T6.

### *2.1.6. Octopus (Octopus bimaculoides)*

An experimental design in triplicate (10 juveniles/replicate) was developed in 60-l fibreglass units at CIBNOR laboratory to assess growth and survival in juvenile octopus during a 28-day period. This assay was intended to study and compare the effects of fresh crab *Callinectes belicosus* and squid *Dosidicus gigas* meat, as raw or thermically processed food (35°C and 60°C). Also, a HOM treatment was added to culture water, as a digestive system enhancer to *Octopus bimaculoides*. Two processed food treatments (35°C and 60°C), two HOM treatments (HOM-35 and HOM-60), and a positive control treatment (unprocessed raw food) were assessed. As HOM treatment, PhA-SiT was added directly to culture water alternating each medicine every day from Monday to Saturday. Food was provided *ad libitum* once a day. Wet weight (day 0 and 17) and survival percentage (day 17) were recorded for all 15 groups (**Table 2**). As expected for a positive control with a traditionally used raw food, the highest survival was attained in juvenile octopus fed on raw meat (97%) but also with HOM-35 (93%) and HOM-60 (86%) the lowest survival was seen in octopuses fed processed food 60°C without giving them HOM treatments (**Table 2**).

Nonetheless, the production of this important resource has been hindered by recurrent epizootic outbreaks and sudden mortalities caused by pathogen microorganisms. To face the challenge, several chemical and antibiotic products have usually been applied whose prophylactic application was initially an effective strategy. However, they have caused the development of resistant bacteria making it necessary to reduce their application. These problems have led the shrimp industry to explore and develop new and more strategies, as effective as or better than antibiotics, eco-friendly and with long-term sustainability. Previous studies have indicated that homoeopathic medicines stimulated the immune system and caused specific organic responses [31, 32]. Therefore, we evaluated the effect of homoeopathic medicines in growth and survival of *L vannamei* postlarvae under controlled laboratory conditions (Trial 1); the survival and antioxidant response through superoxide dismutase (SOD) activity in juveniles when challenged (1 x 10<sup>6</sup> CFU ml−1) with a pathogenic strain of *V. parahaemolyticus* (CAIM-170) at CIBNOR, in a biosecurity laboratory (Trial 2); during the production of postlarvae in a commercial hatchery, focusing on dynamics of the bacterial populations (Trial 3); zootechnical (growth) results and gene expression (Trial 4); and growing-out to marketable

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 83

**Trial 1.** To evaluate growth and survival of postlarvae, an experimental design was applied at CIBNOR with three HOM treatments: ViP-ViA (T1), PhA-SiT (T2), ViP-ViA + PhA-SiT (T3) and ET (T4) as control. HOM treatments were applied for 30 days, spraying liquid dynamisations in commercial pelleted food, administered *ad-libitum*. In general, the best results were

**Trial 2.** To assess survival and SOD activity in juveniles, an experimental design with four HOM treatments: INM (T1), PaV (T2), INM-PaV (T3), ViT (T4) and NT (T5) was applied at CIBNOR. HOM treatments were applied to juveniles 7 days prior to challenge and 5 days during challenge. Liquid dynamisations were sprayed in commercial pelleted food, which was supplied *ad-libitum*, 7 days prior to and during challenge. At 70 h after the start of the challenge, SOD was determined in shrimp tissue. At the end of the challenge (120 h postinfection), the shrimp treated with T2, T3 and T4, exhibited significantly higher average survival (*p* < 0.05) than the control group T5. Juveniles treated with T3 and T4 showed the highest

**T1 T2 T3 T4**

Different letters in the same column, indicate significant difference (*p* < 0.05). Length increase (LI), weight increase (WI),

**Table 3.** Growth and survival of *Litopenaeus vannamei* postlarvae treated with homoeopathic treatments during an

LI (cm) **4.64 ± 0.94 b** 4.61 ± 0.94 a 3.92 ± 0.93 a 4.59 ± 0.70 a WI (g) 2.82 ± 0.60 a **2.92 ± 0.65 b 3.15 ± 0.74 b** 2.62 ± 0.53 a DWI (g day−1) 0.018 ± 0.007 ab 0.02 ± 0.009 ab **0.052 ± 0.018 c** 0.016 ± 0.007 a GR 0.094 ± 0.020 a 0.097 ± 0.021 b **0.105 ± 0.024 c** 0.087 ± 0.017 a

S (%) 99.30 97.33 **99.33** 98.00

obtained in T3 (T1 + T2), showing a clear synergy between T1 and T2 (**Table 3**).

size in a commercial farm (Trial 5).

**Parameter Treatments**

daily weight increase (DWI), growth rate (GR), survival (S).

experimental assay at CIBNOR laboratory.

Knowledge concerning octopus culture is recent and scarce even when completing the life cycle successfully in captivity which is possible with some species [25]. One of the main obstacles to achieve production level is the lack of an industrialised food to be physiologically and economically viable since to date the only efficient food is fresh flesh or live preys [26]. In meal production, raw material goes through aggressive thermal processes that induce protein denaturalisation, carbonylation, hydrophobicity and aggregation [27]. Octopus digestive enzymes are sensitive to these effects; therefore, they cannot hydrolyse their substrates, reducing food digestibility and octopus growth [28, 29].

Heat treatment to raw crab and squid meat to obtain meal ingredients to formulate a balanced diet is not traditionally preferred because this process denaturalises proteins, reduces digestibility and assimilation [28], and lipids can oxidise [30]. Experimental results suggested an enhancement of enzymatic function in *O. bimaculoides* promoted by HOM (PhA-SiT) treatment dissolved into culture water when food was not processed. HOM treatment seemed to have increased the digestive capability in juvenile octopus and the assimilability of processed food (35°C), but the loss of quality in crab and squid meals in 60°C food could not be compensated by the HOM treatment.
