*2.1.3. American oyster (Crassostrea virginica)*

The American oyster *Crassostrea virginica* is an important fishery and aquaculture resource in the Atlantic coast in the Caribbean and in the Gulf of Mexico. A commercial oyster laboratory is already operating in Centro Ostrícola Tecnológico de Tabasco (COTET), Mexico, and homoeopathic medicines were assessed to improve the species culture. HOM treatments were assessed in larval culture and settlement and early nursery of on-cultch and cultch-less seed. Also, a transcriptomic focus study was developed in adult broodstock oyster. The results obtained in six experiments conducted with *C. virginica* are detailed below.

**Trial 1**. A study was conducted at CIBNOR to analyse the transcriptomic response to five HOM treatments in broodstock oyster *C. virginica* to understand the response mechanisms that are activated by these treatments, thus helping to characterise its mode of action. Adult oysters (120 g; 10–12 cm) from a homogeneous population were conditioned in 80-l plastic boxes provided with continuous microalgal food at COTET for 45 days. An experimental design (3 replicates/treatment; 25 oyster/replicate) was applied with four HOM treatments: ViP-ViA (T1), PhA-SiT (T2), END (T3), INF (T4) and two controls ET (T5) and NT (T6). As treatment vehicle, fully impregnated inert homoeopathic pills were provided one daily per oyster. Routine techniques of the oyster hatchery COTET were applied for conditioning broodstock oyster, and during the trial period, two samplings (S1 and S2 ) were performed for histological analyses at CIBNOR. As a general rule, at the end of the conditioning assay, all conditioned groups were capable of sexually maturing and spawning in greater or lesser percentage, but specific details were observed that allowed to distinguish effects associated to different treatments and controls. Female oysters treated with HOM T1 attained the highest frequency of gonad in vitellogesis (48%) and postvitellogenesis (30.2%) stages at first sampling (T1-S<sup>1</sup> ) and 16.7% in the second sampling (T1-S2 ). In T2-S2 oysters, the highest frequency (31.6%) showed at resting (undifferentiated) stage and 30.8% post-spawning stage. In ET control T5-S<sup>2</sup> , 38.5% of the female oysters were at resting stage and 30.8% at post-spawning stage. Oysters of NT control group T6-S<sup>2</sup> recorded the highest frequency (37.5%) in postvitellogenic stage, 31.3% in partial spawning stage and 25% in post-spawning stage. In the veterinary HOM T3-S<sup>1</sup> , oysters showed the highest frequency in partial spawning stage (71.4%), and in T4-S1 , the highest frequency (57.1%) was recorded in post-spawning stage. It is important to highlight that even when T1, T2, T3 and T6 promoted maturation and spawning of the male oysters, there were differences between them related to gamete quality since the presence of atretic oocytes was seen in some samples of T5 and T6 control treatments. The veterinary HOM T4 seemed to have promoted damage to gametes because abundant degenerative oocytes were observed. To evaluate the quality of the oocytes matured under different treatments, the Sudan Black histochemical technique was used staining the lipid components of the cells. The triglyceride lipid index (TLI) was calculated as described by Rodríguez-Jaramillo *et al*. [22], and significant differences were found in oocytes from different treatments and sampling times. Lipid content (TLI) was significantly higher (*p* < 0.0001) in oysters receiving HOM T2 in a short conditioning time (S1 ). The rest of the treatments and replicates recorded significantly lower TLI values, including Veterinary HOM T3 and T4. A difference between HOM treatments lies in the fact that some of them seem to trigger a constant production of new generations of oocytes, which may be useful to hatchery purposes because it could be associated to the possibility of several partial spawning events that could derive into several larval batches and more opportunities for seed production.

receptors ER and ERR2- like (*p* < 0.01). Estradiol levels have been reported to have a direct influence on gonadic development and oocyte quality in marine bivalves [21] that allowed

This work has provided a *de novo* transcriptome characterisation for *M. capax* for the first time and together with the evaluation of physiological variables, it constitutes the first research about the beneficial effect of homoeopathy in gonadal maturation of the species, which has a clear technological applicability during broodstock gonadic conditioning for seed spawning and production of the species. It will likely be applicable to other bivalve molluscs with com-

The American oyster *Crassostrea virginica* is an important fishery and aquaculture resource in the Atlantic coast in the Caribbean and in the Gulf of Mexico. A commercial oyster laboratory is already operating in Centro Ostrícola Tecnológico de Tabasco (COTET), Mexico, and homoeopathic medicines were assessed to improve the species culture. HOM treatments were assessed in larval culture and settlement and early nursery of on-cultch and cultch-less seed. Also, a transcriptomic focus study was developed in adult broodstock oyster. The results

**Trial 1**. A study was conducted at CIBNOR to analyse the transcriptomic response to five HOM treatments in broodstock oyster *C. virginica* to understand the response mechanisms that are activated by these treatments, thus helping to characterise its mode of action. Adult oysters (120 g; 10–12 cm) from a homogeneous population were conditioned in 80-l plastic boxes provided with continuous microalgal food at COTET for 45 days. An experimental design (3 replicates/treatment; 25 oyster/replicate) was applied with four HOM treatments: ViP-ViA (T1), PhA-SiT (T2), END (T3), INF (T4) and two controls ET (T5) and NT (T6). As treatment vehicle, fully impregnated inert homoeopathic pills were provided one daily per oyster. Routine techniques of the oyster hatchery COTET were applied for conditioning broodstock oyster, and during the trial

general rule, at the end of the conditioning assay, all conditioned groups were capable of sexually maturing and spawning in greater or lesser percentage, but specific details were observed that allowed to distinguish effects associated to different treatments and controls. Female oysters treated with HOM T1 attained the highest frequency of gonad in vitellogesis (48%) and postvi-

oysters, the highest frequency (31.6%) showed at resting (undifferentiated) stage and 30.8%

(37.5%) in postvitellogenic stage, 31.3% in partial spawning stage and 25% in post-spawning

It is important to highlight that even when T1, T2, T3 and T6 promoted maturation and spawning of the male oysters, there were differences between them related to gamete quality since the presence of atretic oocytes was seen in some samples of T5 and T6 control treatments. The veterinary HOM T4 seemed to have promoted damage to gametes because abundant degenerative

) were performed for histological analyses at CIBNOR. As a

) and 16.7% in the second sampling (T1-S2

recorded the highest frequency

, 38.5% of the female oysters were at resting stage and

, oysters showed the highest frequency in partial spawning

, the highest frequency (57.1%) was recorded in post-spawning stage.

). In

obtained in six experiments conducted with *C. virginica* are detailed below.

and S2

30.8% at post-spawning stage. Oysters of NT control group T6-S<sup>2</sup>

tellogenesis (30.2%) stages at first sampling (T1-S<sup>1</sup>

post-spawning stage. In ET control T5-S<sup>2</sup>

stage. In the veterinary HOM T3-S<sup>1</sup>

stage (71.4%), and in T4-S1

explaining the high reproductive condition found in the mussels that received T3.

mercial interest.

74 Aquaculture - Plants and Invertebrates

period, two samplings (S1

T2-S2

*2.1.3. American oyster (Crassostrea virginica)*

**Trial 2**. A study was conducted at CIBNOR to analyse the transcriptomic response to five HOM treatments in broodstock oyster *C. virginica* in order to understand the response mechanisms that are activated by these nanomedicines, thus, helping to characterise their mode-of-action. About 25 groups of 25 adult oysters (120 g; 10 ± 12 cm), each from a homogeneous population, were conditioned in 80-l plastic boxes provided with continuous microalgal food at COTET for 45 days. An experimental design with seven different treatments, each one with three replicates, was developed to determine the transcriptomic effect of an *Actinomycetes* strain (1 × 106 CFU ml−1 = RL8) and four homoeopathic drug complexes, alone and in combination: RL8 (T1), ViP-ViA (T2) PhA-SiT (T3), ViP-ViA + RL8 (T4), PhA-SiT+RL8 (T5) and two control groups: ethanol as a positive control (T6) and NT negative control (T7). Five oysters of each homoeopathic and control treatments were initially (t0 ) collected and dissected. The rest of the oysters were collected and dissected at the end of the experiment (t45). Several tissues, including mantle, gills, gonad, muscle and digestive gland (DG), were separately placed on snap-frozen tubes in RNA later® (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and stored at −80°C. The rest of tissue portion were fixed in Davidson solution for histological examination. DG tissues fixed in RNA later® (Thermo Fisher Scientific, Waltham, MA, U.S.A.) from five individuals of each experimental group were subjected to RNA extraction, for the transcriptomic analysis, using Illumina Hiseq 2000 platform (Illumina, San Diego, CA, U.S.A.). The comparison of the transcriptome data with the KEGG database indicated that the treatments influenced associated metabolic pathways in gonadal development and maturation including "Developmental process involved in reproduction", "Meiotic cell cycle process", "Steroid metabolic process", "Response to oestrogen "and" Regulation of reproductive process ". These pathways were assigned to the KEGG categories of "Developmental process involved in reproduction" which was among the most important category, indicating the significance of signal transduction systems and endocrine regulation of gonad development and function in *C. virginica*.

The transcriptome of T2 and T3 had 998 and 881 genes with a significantly increased level of expression, respectively, compared to the control (*p* < 0.05). In T4 and T5, there were 748 and 789 genes with an increased level of expression, respectively, compared to a separate control (*p* < 0.05). No genes were significantly differentially expressed under T1 compared to a separate T2, T3, T4, T5 and controls (*p* < 0.05). Genes with increased expression following T2 and T3 were associated with biological processes, including metabolic pathways, ribosomal biogenesis, and transport of nitrogen compounds and anions. Following T4 and T5, genes with increased expression were involved in metabolism processes related with response to stress, maintaining protein expression. Genes with increased expression following T1 were associated with protein kinase A (PKA) signalling that regulates stress responses and mitochondrion degradation. The transcriptomic results obtained allowed us to determine that homoeopathic treatments expressed transcripts associated with complex biological processes, such as reproduction, stress response, cell growth and metabolism of the *C. virginica* oyster.

biomass was evaluated. Temperature and salinity were maintained in the range of 27 ± 3°C and 22 ± 7 psu. Liquid HOM treatments were added daily to culture water (0.01%). Based on biometrical data of 30 larvae for each replicate every 48 h, the best results in larval growth were obtained with HOM T1, followed by control (T3) and finally by T2 (**Figure 4**). The highest yield in biomass of mature pedivéliger larva (9.29 g) was obtained with HOM T2, followed by control T3 (7.42 g) and the lowest production (7.04 g) was obtained with T1 (**Figure 4**). Differential results obtained in this study were attributable to better survival of larvae because homoeopathy favours nutrition and assimilation of nutrients, increases stress resistance because of high culture density and strengthens the immune system and resistance to attack by pathogens [5, 23]. A better survival associated to small sized larvae has been observed in other species treated with homoeopathy, such as *Panopea globosa* and *Crassostrea sikamea*

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 77

**Trial 6.** An experimental design was applied at COTET for cultch-less spat culture with two homoeopathic treatments and one control (four replicates each) in recirculating upwelling nursery units (40 l) each one allocating four upwellers of PVC pipe 4″ and airlift devices. As HOM treatments, MaP–FeP (T1) SiT–ViT (T2) and NT control (T3) were assessed. Initial seed density was set on 4750 spat/upweller and *I. galbana* and *C. calcitrans* (1:1) was used as food. The total harvest of seed per treatments and replicates (volume) were measured weekly with a graduated cylinder. Temperature and salinity were maintained in the range of 27 ± 3°C and 24 ± 3 psu. Liquid dynamisation treatments were added daily to culture water (0.02%). Based on initial and weekly biometrical data of 30 spat for each replicate, the best growth results were obtained in the control T3, followed by HOM T2 and finally T1 (**Figure 5**). In contrast, the best results in oyster seed biomass were attained with HOM T1 (268 ml) and T2 (242 ml)

Oyster seed receiving HOM treatments grew less in regard to individual size, but an overall greater volume of marketable juveniles was harvested, which means that survival was greater in HOM treatments. From a commercial hatchery perspective, it is more important to produce live seed biomass even if small sized than dead ones. In real production and profitability terms, the hatchery could produce and sell more seeds if homoeopathic drugs, such as those evaluated in this study were routinely applied. The results in larval settlement and juvenile

**Figure 4.** Growth in size (μm) of veliger larvae (left) and harvest of pediveliger larvae (right) of American oyster

*Crassostrea virginica* treated with HOM in 750-l conical fibreglass tanks in COTET hatchery.

(Mazón-Suástegui *et al*., unpublished).

and the lowest production (236 ml) with control T3 (**Figure 5**).

**Trial 3**. An experimental design was applied at COTET for oyster larval culture assessment including HOM treatments: SiT (T1), ViT (T2), FeP (T3), MaP (T4) and NT (T5) as a control treatment. The experimental larviculture was performed in 20-l plastic buckets three replicates per treatment with filtered seawater, gentle and continuous aeration and the microalga *I. galbana* and *C. calcitrans* (1:1) as food. Three-day old veliger larvae from a mass production fibreglass tank (25 t) were placed in buckets at initial density of 2 larvae ml−1. Liquid HOM treatments were added daily to culture water (0.01%). The larviculture test finalised when the first pedivéliger larva with a retractable foot was detected but without reaching the final stage of eyed and fully developed pediveliger. Analyses were based on initial and three sequential biometrical data from 30 larvae for each treatment replicate. The HOM larval groups showed better development, survival and setting efficiency in comparison with untreated groups. As seen in **Figure 3**, *C. virginica* larvae that received T1 (162 ± 3.08 μm), T2 (162 ± 2.84 μm) and T4 (162 ± 3.13 μm) reached the largest sizes but T5 (145 ± 2.97 μm) reached the lowest sizes, while those larvae receiving T2 reached the highest setting efficiency (83 ± 7.98%) and the highest seed survival (36.5 ± 4.9%).

**Trial 4**. To assess larval settlement, mature eyed pediveliger larvae were obtained by selective screening of the same bulk culture tank; then, they were placed in 20-l buckets at a seeding density of 0.25 larvae ml−1. The same HOM treatments: SiT (T1), ViT (T2), FeP (T3), MaP (T4) and NT (T5) were applied at COTET during the on-cultch setting process. Seawater change, aeration and microalgae were provided as food, and 50 clean oyster shells/buckets were placed as a natural settlement (cultch) substrate. Liquid homoeopathic treatments were added daily to the culture water (0.02% v/v). The results achieved are shown in **Figure 3**.

**Trial 5.** A pilot experimental design in triplicate was applied at COTET for larval culture: MaP–FeP (T1), SiT–ViT (T2) and NT (T3) as control. Four-day old larvae were placed in nineconical fibreglass tanks (750 l) at initial density of 5.33 larvae ml−1 and the microalgae *I. galbana and C. calcitrans* (1:1) was provided as food. Larval growth and harvest of pediveliger larval

**Figure 3.** Larval growth (left), larval setting efficiency (Centre) and seed survival in 20-l plastic buckets (right) of American oyster *Crassostrea virginica*, treated with homoeopathic drugs in COTET oyster hatchery.

biomass was evaluated. Temperature and salinity were maintained in the range of 27 ± 3°C and 22 ± 7 psu. Liquid HOM treatments were added daily to culture water (0.01%). Based on biometrical data of 30 larvae for each replicate every 48 h, the best results in larval growth were obtained with HOM T1, followed by control (T3) and finally by T2 (**Figure 4**). The highest yield in biomass of mature pedivéliger larva (9.29 g) was obtained with HOM T2, followed by control T3 (7.42 g) and the lowest production (7.04 g) was obtained with T1 (**Figure 4**). Differential results obtained in this study were attributable to better survival of larvae because homoeopathy favours nutrition and assimilation of nutrients, increases stress resistance because of high culture density and strengthens the immune system and resistance to attack by pathogens [5, 23]. A better survival associated to small sized larvae has been observed in other species treated with homoeopathy, such as *Panopea globosa* and *Crassostrea sikamea* (Mazón-Suástegui *et al*., unpublished).

stress, maintaining protein expression. Genes with increased expression following T1 were associated with protein kinase A (PKA) signalling that regulates stress responses and mitochondrion degradation. The transcriptomic results obtained allowed us to determine that homoeopathic treatments expressed transcripts associated with complex biological processes, such as reproduction, stress response, cell growth and metabolism of the *C. virginica* oyster. **Trial 3**. An experimental design was applied at COTET for oyster larval culture assessment including HOM treatments: SiT (T1), ViT (T2), FeP (T3), MaP (T4) and NT (T5) as a control treatment. The experimental larviculture was performed in 20-l plastic buckets three replicates per treatment with filtered seawater, gentle and continuous aeration and the microalga *I. galbana* and *C. calcitrans* (1:1) as food. Three-day old veliger larvae from a mass production fibreglass tank (25 t) were placed in buckets at initial density of 2 larvae ml−1. Liquid HOM treatments were added daily to culture water (0.01%). The larviculture test finalised when the first pedivéliger larva with a retractable foot was detected but without reaching the final stage of eyed and fully developed pediveliger. Analyses were based on initial and three sequential biometrical data from 30 larvae for each treatment replicate. The HOM larval groups showed better development, survival and setting efficiency in comparison with untreated groups. As seen in **Figure 3**, *C. virginica* larvae that received T1 (162 ± 3.08 μm), T2 (162 ± 2.84 μm) and T4 (162 ± 3.13 μm) reached the largest sizes but T5 (145 ± 2.97 μm) reached the lowest sizes, while those larvae receiving T2 reached

76 Aquaculture - Plants and Invertebrates

the highest setting efficiency (83 ± 7.98%) and the highest seed survival (36.5 ± 4.9%).

daily to the culture water (0.02% v/v). The results achieved are shown in **Figure 3**.

**Trial 4**. To assess larval settlement, mature eyed pediveliger larvae were obtained by selective screening of the same bulk culture tank; then, they were placed in 20-l buckets at a seeding density of 0.25 larvae ml−1. The same HOM treatments: SiT (T1), ViT (T2), FeP (T3), MaP (T4) and NT (T5) were applied at COTET during the on-cultch setting process. Seawater change, aeration and microalgae were provided as food, and 50 clean oyster shells/buckets were placed as a natural settlement (cultch) substrate. Liquid homoeopathic treatments were added

**Trial 5.** A pilot experimental design in triplicate was applied at COTET for larval culture: MaP–FeP (T1), SiT–ViT (T2) and NT (T3) as control. Four-day old larvae were placed in nineconical fibreglass tanks (750 l) at initial density of 5.33 larvae ml−1 and the microalgae *I. galbana and C. calcitrans* (1:1) was provided as food. Larval growth and harvest of pediveliger larval

**Figure 3.** Larval growth (left), larval setting efficiency (Centre) and seed survival in 20-l plastic buckets (right) of

American oyster *Crassostrea virginica*, treated with homoeopathic drugs in COTET oyster hatchery.

**Trial 6.** An experimental design was applied at COTET for cultch-less spat culture with two homoeopathic treatments and one control (four replicates each) in recirculating upwelling nursery units (40 l) each one allocating four upwellers of PVC pipe 4″ and airlift devices. As HOM treatments, MaP–FeP (T1) SiT–ViT (T2) and NT control (T3) were assessed. Initial seed density was set on 4750 spat/upweller and *I. galbana* and *C. calcitrans* (1:1) was used as food. The total harvest of seed per treatments and replicates (volume) were measured weekly with a graduated cylinder. Temperature and salinity were maintained in the range of 27 ± 3°C and 24 ± 3 psu. Liquid dynamisation treatments were added daily to culture water (0.02%). Based on initial and weekly biometrical data of 30 spat for each replicate, the best growth results were obtained in the control T3, followed by HOM T2 and finally T1 (**Figure 5**). In contrast, the best results in oyster seed biomass were attained with HOM T1 (268 ml) and T2 (242 ml) and the lowest production (236 ml) with control T3 (**Figure 5**).

Oyster seed receiving HOM treatments grew less in regard to individual size, but an overall greater volume of marketable juveniles was harvested, which means that survival was greater in HOM treatments. From a commercial hatchery perspective, it is more important to produce live seed biomass even if small sized than dead ones. In real production and profitability terms, the hatchery could produce and sell more seeds if homoeopathic drugs, such as those evaluated in this study were routinely applied. The results in larval settlement and juvenile

**Figure 4.** Growth in size (μm) of veliger larvae (left) and harvest of pediveliger larvae (right) of American oyster *Crassostrea virginica* treated with HOM in 750-l conical fibreglass tanks in COTET hatchery.

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

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

**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;

**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 treat-

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®

−1 for homoeopathy and 10 ppm for antibiotic

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 79

sexual maturation of broodstock oyster at the hatchery.

stage IV: Partial spawning; stage V: Post spawning.

ments. Liquid treatments were applied 100 μl l
