*2.2.1. White shrimp (Litopenaus vannamei)*

The White shrimp *L. vannamei* is a species with rapid growth, high survival and price in the market, which makes this crustacean one of the most important resources at worldwide level.


Data are presented as Mean value ± SD. W = Wet weight.

**Table 2.** *Octopus bimaculoides* juvenile growth and survival during food and HOM treatment assessment.

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 size in a commercial farm (Trial 5).

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

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,

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 compen-

The White shrimp *L. vannamei* is a species with rapid growth, high survival and price in the market, which makes this crustacean one of the most important resources at worldwide level.

**Item. HOM-60°C 60°C HOM-35°C 35 °C No HOM** W Day 1 (g) 0.11 ± 0.05 0.10 ± 0.03 0.10 ± 0.04 0.12 ± 0.04 0.12 ± 0.04 W Day 28 (g) 0.11 ± 0.06 0.11 ± 0.06 0.15 ± 0.06 0.20 ± 0.06 0.20 ± 0.07

Survival (%) 28 46 87 54 93

**Table 2.** *Octopus bimaculoides* juvenile growth and survival during food and HOM treatment assessment.

HOM treatments (**Table 2**).

82 Aquaculture - Plants and Invertebrates

sated by the HOM treatment.

*2.2.1. White shrimp (Litopenaus vannamei)*

Data are presented as Mean value ± SD. W = Wet weight.

**2.2. Crustaceans**

reducing food digestibility and octopus growth [28, 29].

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

**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


Different letters in the same column, indicate significant difference (*p* < 0.05). Length increase (LI), weight increase (WI), daily weight increase (DWI), growth rate (GR), survival (S).

**Table 3.** Growth and survival of *Litopenaeus vannamei* postlarvae treated with homoeopathic treatments during an experimental assay at CIBNOR laboratory.

survival with 64.43 and 56%, respectively, while all those treated with T1 and T5 died. These results suggested that the greatest survival of HOM-treated shrimp could have been related to a stimulation of their immune system, and consequently, to a greater resistance to acute infectious diseases associated with the genus *Vibrio* [32]. Regarding SOD activity, at 70 h after the start of the challenge, the groups treated with T1 and T2 did not show significant differences (34.48 ± 1.87 and 16.32 ± 1.22, respectively) in relation to the control group (41.63 ± 2.59) while T3 and T4 with a SOD activity of (86.43 ± 1.02 and 83.47 ± 5.54, respectively) exhibited significantly higher values than the control group (*p* < 0.05) (41.63 ± 2.59) (**Figure 8**).

between sucrose and non-sucrose fermented colonies; *V. alginolyticus* is sucrose fermenter and shows yellow-coloured colonies while *V. parahaemolyticus* and *V. vulnificus* are non-sucrose fermenters and have green colonies [34]. Although no statistical significant (*p* > 0.05) differences were found in HOM-treated and non-treated PLs, positive effects were apparently attained with HOM treatments (**Figure 9**). Research must continue not only with controlled and multi-replicate experimental designs in the laboratory but also efforts should continue in commercial hatcheries even if obvious difficulties exist because production is at most the first

priority and not necessarily compatible with a strict and traditional scientific research.

weight of the shrimp postlarvae was exponential with a correlation coefficient (r<sup>2</sup>

**Trial 4**. A fourth assessment was made also at Aquacultura Mahr hatchery. An experimental design was applied in six concrete tanks (20 t) with five HOM treatments: BaC-INM, Sit-INM (T2), HeS-INM (T3), PhA-INM (T4), PaV-INM (T5), and two NT control groups (T6 and T7). Liquid homoeopathic dynamisations were provided with pulverised food as vehicle. Growth in

treatment was T5 (PaV-INM). The NT control group had the lowest growth rate and the lowest survival (20.2%), while in the HOM-treated groups, it was 25.6 ± 5.38% (21–34.1%) (**Figure 10**, Left). At the end of the production cycle, gene expression analyses were made at CIBNOR to compare HOM-treated with non-treated postlarvae. The results of gene expression related to the activities of the aminopeptidase (AMP), amylase (AMY), chymotrypsin (CHY) and trypsin (TRY) enzymes showed clear and statistically significant differences (*p* < 0.05; *n* = 30) between the HOM-treated and NT postlarvae (**Figure 10**, Right). These results confirmed a positive impact of the use of homoeopathic medicines in the commercial production of *L. vannamei* postlarvae. **Trial 5**. A fourth experiment consisted of the evaluation of HOM treatments during the mass cultivation of *L. vannamei* from postlarvae to adult size in commercial facilities of the company BCS Camarón, a commercial shrimp farm. The evaluation was performed in six earthen ponds of 10 ha each, initially seeded at a density of 8 PL m−2. An experimental design was applied with six earthen ponds 10 ha, four ponds with HOM treatment and two NT ponds without homoeopathy: PhA-SiT (T1), PaV-ViT (T2), and NT as negative control group (T3). Liquid homoeopathic dynamisations were sprayed in commercially balanced food and applied in the culture ponds, each treatment component on alternate days for

130 days. Three samplings were made: after seeding and 7 days for acclimatisation (t<sup>1</sup>

52 days (t52) and after 130 days post seeding (t130). Growth parameters and biomass production as body weight (BW), total length (TL) and weight gain average (WGA) were determined. As physiological health indicators, the hepatopancreatic coverage index (HCI) was

**Figure 9.** Straight regression showing the increasing trends in the bacterial count of *Vibrio spp*. in culture seawater and macerate of *Litopenaeus vannamei* postlarvae. Petri dish with TCBS culture medium, which was previously inoculated,

showing the presence of sucrose positive (yellow) and sucrose negative (green) colonies of the genus *Vibrio*.

) > 0.95. The best

85

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

); after

Treatment T3 contained PaV used in human medicine as a tranquilliser to reduce stress and improve sleep, in addition to Heel-Mix (Rubiopharma®, Mexico) that contains medicines used for the treatment of enzymatic disorders, infectious diseases and stimulation of the body defences in human beings, whereas T4 is a homoeopathic medicine whose active principle is the venom of the scorpion *Rhopalurus junceus* which is an endemic species of Cuba. These results suggested a potential applicability of the homoeopathic medicines studied as prophylactic treatments to reduce stress and to improve shrimp immune response, which could help reduce the incidence of epizootic diseases and massive mortalities that have been a great problem for the industry due to economic losses associated with *V. parahaemolithycus* in shrimp culture.

**Trial 3**. A third experiment consisted of the evaluation of HOM treatments during the commercial production of *L. vannamei* postlarvae in the facilities of the commercial hatchery Aquacultura Mahr in square concrete tanks with a capacity of 20 t. An experimental design was applied with four tanks with three HOM treatments: MaP-CaP-Fep-Zip-PhA (T1) Hes-Sit-Cap-Pha (T1), INM-PaV-ViT-PhA (T3), and NT as the control group (T4). Liquid homoeopathic dynamisations were added to commercially pulverised food given periodically for a 24-h period (day-night). Shrimp farming has been affected by viral and bacterial diseases principally those associated to a highly virulent strain of *V. parahaemolyticus*; this problem is of primary interest concern [33], so this assay was intended to determine the population dynamics of the bacterial populations mainly *Vibrio* spp. Bacteriological examination of isolated vibrio species depend mainly on using TCBS agar as a selective media to differentiate

**Figure 8.** SOD activity in HOM-treated juvenile shrimp *Litopenaeus vannamei* treated with homoeopathy and then challenged with *Vibrio parahaemolithycus*.

between sucrose and non-sucrose fermented colonies; *V. alginolyticus* is sucrose fermenter and shows yellow-coloured colonies while *V. parahaemolyticus* and *V. vulnificus* are non-sucrose fermenters and have green colonies [34]. Although no statistical significant (*p* > 0.05) differences were found in HOM-treated and non-treated PLs, positive effects were apparently attained with HOM treatments (**Figure 9**). Research must continue not only with controlled and multi-replicate experimental designs in the laboratory but also efforts should continue in commercial hatcheries even if obvious difficulties exist because production is at most the first priority and not necessarily compatible with a strict and traditional scientific research.

survival with 64.43 and 56%, respectively, while all those treated with T1 and T5 died. These results suggested that the greatest survival of HOM-treated shrimp could have been related to a stimulation of their immune system, and consequently, to a greater resistance to acute infectious diseases associated with the genus *Vibrio* [32]. Regarding SOD activity, at 70 h after the start of the challenge, the groups treated with T1 and T2 did not show significant differences (34.48 ± 1.87 and 16.32 ± 1.22, respectively) in relation to the control group (41.63 ± 2.59) while T3 and T4 with a SOD activity of (86.43 ± 1.02 and 83.47 ± 5.54, respectively) exhibited

Treatment T3 contained PaV used in human medicine as a tranquilliser to reduce stress and improve sleep, in addition to Heel-Mix (Rubiopharma®, Mexico) that contains medicines used for the treatment of enzymatic disorders, infectious diseases and stimulation of the body defences in human beings, whereas T4 is a homoeopathic medicine whose active principle is the venom of the scorpion *Rhopalurus junceus* which is an endemic species of Cuba. These results suggested a potential applicability of the homoeopathic medicines studied as prophylactic treatments to reduce stress and to improve shrimp immune response, which could help reduce the incidence of epizootic diseases and massive mortalities that have been a great problem for the industry due to economic losses associated with *V. parahaemolithycus* in shrimp culture.

**Trial 3**. A third experiment consisted of the evaluation of HOM treatments during the commercial production of *L. vannamei* postlarvae in the facilities of the commercial hatchery Aquacultura Mahr in square concrete tanks with a capacity of 20 t. An experimental design was applied with four tanks with three HOM treatments: MaP-CaP-Fep-Zip-PhA (T1) Hes-Sit-Cap-Pha (T1), INM-PaV-ViT-PhA (T3), and NT as the control group (T4). Liquid homoeopathic dynamisations were added to commercially pulverised food given periodically for a 24-h period (day-night). Shrimp farming has been affected by viral and bacterial diseases principally those associated to a highly virulent strain of *V. parahaemolyticus*; this problem is of primary interest concern [33], so this assay was intended to determine the population dynamics of the bacterial populations mainly *Vibrio* spp. Bacteriological examination of isolated vibrio species depend mainly on using TCBS agar as a selective media to differentiate

**Figure 8.** SOD activity in HOM-treated juvenile shrimp *Litopenaeus vannamei* treated with homoeopathy and then

challenged with *Vibrio parahaemolithycus*.

84 Aquaculture - Plants and Invertebrates

significantly higher values than the control group (*p* < 0.05) (41.63 ± 2.59) (**Figure 8**).

**Trial 4**. A fourth assessment was made also at Aquacultura Mahr hatchery. An experimental design was applied in six concrete tanks (20 t) with five HOM treatments: BaC-INM, Sit-INM (T2), HeS-INM (T3), PhA-INM (T4), PaV-INM (T5), and two NT control groups (T6 and T7). Liquid homoeopathic dynamisations were provided with pulverised food as vehicle. Growth in weight of the shrimp postlarvae was exponential with a correlation coefficient (r<sup>2</sup> ) > 0.95. The best treatment was T5 (PaV-INM). The NT control group had the lowest growth rate and the lowest survival (20.2%), while in the HOM-treated groups, it was 25.6 ± 5.38% (21–34.1%) (**Figure 10**, Left). At the end of the production cycle, gene expression analyses were made at CIBNOR to compare HOM-treated with non-treated postlarvae. The results of gene expression related to the activities of the aminopeptidase (AMP), amylase (AMY), chymotrypsin (CHY) and trypsin (TRY) enzymes showed clear and statistically significant differences (*p* < 0.05; *n* = 30) between the HOM-treated and NT postlarvae (**Figure 10**, Right). These results confirmed a positive impact of the use of homoeopathic medicines in the commercial production of *L. vannamei* postlarvae.

**Trial 5**. A fourth experiment consisted of the evaluation of HOM treatments during the mass cultivation of *L. vannamei* from postlarvae to adult size in commercial facilities of the company BCS Camarón, a commercial shrimp farm. The evaluation was performed in six earthen ponds of 10 ha each, initially seeded at a density of 8 PL m−2. An experimental design was applied with six earthen ponds 10 ha, four ponds with HOM treatment and two NT ponds without homoeopathy: PhA-SiT (T1), PaV-ViT (T2), and NT as negative control group (T3). Liquid homoeopathic dynamisations were sprayed in commercially balanced food and applied in the culture ponds, each treatment component on alternate days for 130 days. Three samplings were made: after seeding and 7 days for acclimatisation (t<sup>1</sup> ); after 52 days (t52) and after 130 days post seeding (t130). Growth parameters and biomass production as body weight (BW), total length (TL) and weight gain average (WGA) were determined. As physiological health indicators, the hepatopancreatic coverage index (HCI) was

**Figure 9.** Straight regression showing the increasing trends in the bacterial count of *Vibrio spp*. in culture seawater and macerate of *Litopenaeus vannamei* postlarvae. Petri dish with TCBS culture medium, which was previously inoculated, showing the presence of sucrose positive (yellow) and sucrose negative (green) colonies of the genus *Vibrio*.

**Figure 10.** Growth in weight (left) and gene expression (right) in *Litopenaeus vannamei* postlarvae or adult treated and with homoeopathic medicines in a commercial shrimp hatchery (Acuacultura Mahr). Aminopeptidase (AMP), amylase (AMY), chymotrypsin (CHY), trypsin (TRY).

evaluated as a morpho-histological index variable [HCI (%) = hepatopancreatic coverage area/cephalothorax coverage area × 100]. From an initial time characterised by no significant differences in BW and TL after 130 days of culture both HOM (T1 and T2) produced the best growth results in BW, TL and WGA compared to NT ponds (**Table 4**).

nanoparticle content [35]. The presence of nanoparticles has been demonstrated in highly homoeopathic ultra-diluted medicines [8]. On the other hand, the same homoeopathic medicines used in shrimp ponds, (PaV, PhA, SIT and ViT) outperformed antibiotics in juvenile scallop *A. ventricosus* [23] These results enhanced the evidences that aquacultural homoeopathy has applicability in shrimp commercial culture to improve the productivity of the shrimp industry.

**Figure 11.** Hepatopancreatic coverage index (HCI %) of adult shrimp in a commercial shrimp farm (earth ponds 10 h)

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 87

One of the most promising and novel fields that strengthen the immune system in marine organisms is the use of immunostimulants that are natural compounds modulating the immune system and increasing resistance of the host against disease mainly those caused by bacteria [18]. Regarding immunostimulants, homoeopathy has been proposed as a novel alternative in aquaculture practices to improve health and strengthen the organism's immune response [5, 23]. The production of marine mollusc, shrimp and fish around the world still challenged every year by the propagation and emergence of new diseases, mainly those related to viruses and bacteria such as *Vibrio spp.* and *Aeromonas spp.*, which are treated with conventional methods as antibiotics [2, 14]. The findings in our research suggested that homoeopathic medicines have a great potential to increase health and performance in marine

Findings in the scallop *A. ventricosus* support the fact that homoeopathic medicines do not act directly over the disease-cause *per-se*, killing the bacteria or removing the stressing agent but enhancing the capacity of the HOM-treated organisms to resist the infection or overpass stressful conditions. Some results in molluscs have shown a greater effect of homoeopathic medicines on survival than on growth of larvae and seeds. However, it is necessary to consider that greater survival implies maintaining a higher density in larval culture and that not only larvae of larger size are able to settle and become marketable seeds with a good performance in the field. From a commercial point of view, it is more important to attain bigger biomass of successfully setting larvae even if small sized because independently of their

mollusc including bivalves and octopus, shrimp and marine fish.

**3. Conclusion**

treated with homoeopathic medicines.

The shrimp treated with PaV-VIT after 52 days of treatment (T1) achieved the highest HCI (32.32 ± 0.61%) and differed from PhA − SIT and un-treated control ponds. (28.34 ± 0.87%; 26.58 ± 0.64%, respectively). At the end of the experiment (130 days), both homoeopathic treatments showed better HCI; in relation to the negative control group (PhA − SIT *=* 33.87 ± 1.02%; PaV − VIT = 33.31 ± 0.77% control T3 = 26.54 ± 0.56%) as shown in **Figure 11**.

The positive effect of the HOM treatments evaluated in growth and morpho-histological index of *L. vannamei* could be attributable to smaller micelle and higher activity in water with


Body weight (BW), Total length (TL), weight gain average (WGA). Values within the same row with different letters represent significant differences (*p* < 0.05).

**Table 4.** Growth of *Litopenaeus vanamei* treated with homoeopathy while cultured in semi-intensive system in a commercial shrimp farm (BCS Camarón Farm; six earthen ponds, 10 ha).

**Figure 11.** Hepatopancreatic coverage index (HCI %) of adult shrimp in a commercial shrimp farm (earth ponds 10 h) treated with homoeopathic medicines.

nanoparticle content [35]. The presence of nanoparticles has been demonstrated in highly homoeopathic ultra-diluted medicines [8]. On the other hand, the same homoeopathic medicines used in shrimp ponds, (PaV, PhA, SIT and ViT) outperformed antibiotics in juvenile scallop *A. ventricosus* [23] These results enhanced the evidences that aquacultural homoeopathy has applicability in shrimp commercial culture to improve the productivity of the shrimp industry.
