**7.1 Collection of mussels**

The study was conducted at ICAR-CIFA, Kausalyaganga farm, Bhubaneswar, Odisha, India (Lat. 20° 11′ 06′′-20° 11′ 45′′N; Long.85° 50′52′′-85° 51′35′′E). Mussels are hand-picked and harvested by competent employees from culture pond, ideally early in the morning. The mussels are harvested and stored in water for short distance travels to avoid stress during transportation. While collecting mussels, the mussel size requirement is the most significant consideration. It has been proven that pearl mussels with a shell length of 8–12 cm and a weight of 35 g or more can produce pearls.

#### **7.2 Pre-operative conditioning**

Pre-operative conditioning allows appropriate relaxation of the adductor muscles and keeps the animal's metabolic rate low to get the best possible result throughout the surgical implantation process. Prior to surgery, pearl mussels are subjected to pre-operative conditioning for 24 to 48 hours after being collected from cultured ponds. The mussel was stocked in 200 litre FRP tanks at 60 mussels per tank during acclimation. The experimental tank was filled with filtered pond water. Green water should be offered to them as a source of nutrients. During the study, the following physico-chemical water quality parameters were measured: dissolved oxygen 5.6 to 6.5 mg l−1; pH 7.5 to 7.6; nitrites 0.020 to 0.030 mg l−1; ammonia 0.05 to 0.08 mg l−1 and temperature 27 to 29°C. Further, 5% mortality of the mussel was also observed during this period.

#### **7.3 Preparation and surgical implantation of nuclei**

It is the most crucial stage in the pearl-growing process. The cultivated pearl design that will be used as the end result will define the type of nucleus to be developed. Some of the nuclei that can be made are designer, half-round, and round nuclei. The nucleus can be prepared using one of two methods. The first step involves mixing acrylic powder with its solvent in a 1:1 ratio to make slurry that is utilised to prepare all of the nucleus forms. The second method, on the other hand, comprises a time-consuming technique that involves bleaching, powdering, and sieving dead mussel shells to get a finely crushed shell powder. The shell powder is then mixed with araldite glue to make dough, which is then cast on moulds to make designer and half-round nuclei, or rolled on the palm to make circular nuclei. Prior to either the acrylic powder-solvent slurry or the shell powder-araldite dough to the moulds, make sure they are lubricated to make it easier to remove the nuclei from the moulds.

The ultimate pearl to be generated determines the method of implantation to be used. There are three different kinds of implantation procedures. The list is as follows:

#### *7.3.1 Mantle cavity implantation*

This is the easiest way of the three, requiring the least amount of skill and expertise. The nucleus is implanted into the cavity between the outer mantle layer and the mussel shell's inner surface in this procedure. The outer mantle layer serves as the source of nacre secretion; hence mantle graft is not employed in this procedure.

#### *7.3.2 Mantle tissue implantation*

The nucleus, together with the mantle graft, is implanted into pockets formed in the recipient mussel's mantle tissue on the posterior side, in both the left and right lobes (**Figure 5**).

#### *7.3.3 Gonadal implantation*

A small incision is made in the recipient mussel's gonad and the nucleus together with a single mantle graft or sandwiched between two pieces of graft are then put into the incision. Along with the round nucleus, a live graft of 2 to 3 mm is introduced from the pallial mantle ribbon. Sometimes, infections can occur in the mussel after the graft and nucleus have been implanted; these infections can be treated with broad-spectrum antibiotics, which also assist to restore the immunological status of the operated mussel.

The success rate of pearl generation in the mantle cavity and mantle tissue implantation methods is 60–70 percent, but it is 25–30 percent in gonadal implantations.

#### **7.4 Post-operative care**

The implanted mussels' recuperation necessitates post-operative care, which is a key phase in the production of freshwater pearls. In this step, 7–10 days post the implantation the mussels are monitored closely. The shell valves are given enough care to guarantee that they can open and close freely for respiration. The use of a broad-spectrum antibiotic in the water in post-operative care units at a rate of 1–2 ppm is favourable to the survival and wound healing of the implanted mussels. Mussels in post-operative care are given green algae grown in the lab. During postoperative care, there is a risk of rejection of the implanted nucleus and graft, which can be reduced by lowering stress in the post-operative care tanks by maintaining water level, feeding, and aeration. It is vital to keep in mind that proper post-operative care reduces the likelihood of abnormal pearls by preventing nucleus extrusion soon after implantation.

**Figure 5.** *Implantation of live graft pieces into the mantle of mussel.*

### **7.5 Culture in ponds**

To prevent post-operative mussel mortality and nucleus bead rejection, freshwater mussel implantation is done all year in India, except during the extreme hot months (May to June). Pearl culture operations can be carried out in traditional carp culture ponds (2.5 m deep) with a clay-soil basis and somewhat alkaline waters. Ponds free from aquatic macrophytes and algal blooms, such as *Microcystis* and *Euglena*, are perfect for pearl culture. Bamboo poles are used as rafts in the ponds to hang the pearl mussels that have been implanted at a stocking density of 50,000/ha. The mussels are hanged in 1.5 mesh sized nylon bags of 30 cm x 13 cm @ 2 mussels per bag.

Pond management is crucial during the culture stage, especially in terms of natural food production and water quality control via liming or fertilisation, as it affects the quality and quantity of pearl production. The addition of green water (*Chlorella sp., Chlorococcum sp., and Scenedesmus sp*.) to the pearl culture ponds at regular intervals as direct mussel feed has been revealed to be the most effective technique for maintaining the pearl yielding mussel standing crop. The "open culture method" can be used to cultivate green algae in ferro-cement tanks (200 litres) positioned along pond dykes. Cow dung (10,000 kg/ha/yr), urea (100 kg/ha/yr), and single super phosphate (100 kg/ha/yr) are used to fertilise the water in the tanks in equal monthly instalments. When the fertilisers degrade in 10 to 15 days and green water appears, the improved water is placed into the pearl culture ponds. According to reports, freshwater mussels feed on algae belonging to the Chlorophyceae (green algae), Bacillariophyceae (diatoms), and Cyanophyceae (blue green algae) families. Diatoms, green algae (*Chlorella, Chlorococcum, Scenedesmus*, etc.) and blue-green algae (*Spirulina*) are the most usually favoured algal species by the freshwater mussel *L. marginalis*. Mussels may ingest a wide variety of particulate organic materials since they are mucoid filter feeders. A routine health check-up of the cultured mussels should be done at biweekly intervals because there is still a high risk of mortality of operated mussels due to internal incision, limited food availability, and parasitic infection. As a result, mussels in net bags should be removed, inspected, and cleaned before being returned. Because the mussels remain sedentary and static inside the enclosures, algal development can occur as a result of heavy nutrient loading, which should be avoided at all costs. The pond's physico-chemical parameters and water level are monitored during the culture period. Temperatures between 25 and 30 degrees Celsius are optimal.

#### **7.6 Harvest of pearls**

Depending on the implantation method utilised, the size and quantity of nuclei implanted, the health of the mussels, and the pond environment, pond culture of operated mussels can take anywhere from a year to 18 months. Harvesting occurs at the end of the culture period, where the mussels are checked and processed individually in order to obtain the ultimate product, pearls. A biological system produces pearls through a natural process. Because pearls are created through a natural process, they have a wide range of look and quality. After harvest, the pearls are subjected to value addition through surface cleaning, bleaching and dyeing, or both cleaning and bleaching, in order to maintain uniformity in colouring and quality, which may improve their marketability.

## **8. Pearl quality, factors affecting and quality enhancement**

The value of a pearl, like any other valuable gem, is decided by its quality, which is decided by several qualities of the pearl such as shape, size, colour, lustre, and

surface complexion [69, 77]. Given that a pearl is the result of a complex biological phenomenon, diversity in pearls is unavoidable and is influenced by both the genetic makeup of the individual and the impact of many environmental factors. It can be deduced that genetics, environment, and genotype-by-environment interactions influence the overall quality of pearls [78, 79]. Pearls are classified into three varieties based on their appearance to the naked eye: nacreous pearls, prismatic pearls, and organic pearls, with nacreous pearls being the most valuable [80]. Although controlling and regulating the quality of cultured pearls at the genetic level is a time-consuming task that necessitates extensive research, changes in culture methods and environmental conditions can significantly improve pearl quality. Pearl quality is also affected by other factors like the host and donor oysters [70], or by exogenous physico-chemical and biological stressors [81]. Various factors that can be regulated to enhance the pearl quality are listed below.
