Anaesthetic effect of sodium bicarbonate (NaHCO₃, baking soda) and its effect on Hematological parameters of three spotted tilapia (Oreochromis andersonii, Castelnau 1861) broodstock

ABSTRACT

This study aimed to determine the optimal concentration of sodium bicarbonate (NaHCO₃) and its effect on the Hematological parameters of three-spotted tilapia (Oreochromis andersonii) broodstock. Using a Completely Randomized Design, 150 broodstock (weight: 180 ± 10 g) were immersed in nine NaHCO₃ concentrations (0–50 g/L) in triplicate. Induction and recovery times were recorded, and Red Blood Cells (RBC), White Blood Cells (WBC), Packed Cell Volume (PCV), and Hemoglobin (Hb) were measured. Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), and Mean Corpuscular Hemoglobin Concentration (MCHC) were calculated. Induction time decreased and recovery time increased with higher NaHCO₃ concentrations, without any mortality. A 10 g/L concentration only significantly affected RBC and PCV. At 45 and 50 g/L there were significant changes to MCH, while significant increase was observed at 10 and 15 g/L in Hb and WBC, respectively. The optimal concentrations established for routine handling in hatcheries were 30 and 35 g/L.

KEYWORDS:

• Anaesthesia effect
• Oreochromis andersonii
• Induction and recovery time
• Haematological parameters
• Sodium bicarbonate (baking soda)

Introduction

Three Spotted Tilapia (Oreochromis andersonii) holds significant importance within Zambia’s aquaculture sector and is farmed in most provinces excluding the northern regions (Hasimuna et al. 2021; Maulu et al. 2019; Mphande et al. 2023; Siavwapa et al. 2022). Recognizing its pivotal role, the Department of Fisheries has adopted this fish as a leading species for aquaculture development and is currently undergoing a genetic improvement program through selective breeding to enhance its growth performance (Siavwapa et al. 2022), thus potentially escalating the demand for broodstock within and beyond Zambia (Siavwapa et al. 2022). However, ensuring sustainable production of high-quality fingerlings hinges greatly on the mitigation of stress during routine operations by fish farmers and hatchery proprietors (Mphande et al. 2023; Siavwapa et al. 2022), given that stress significantly predisposes fish to disease outbreaks (Maulu et al. 2021). In contemporary aquaculture and hatchery management, stress is minimized by using anaesthetics during activities like artificial reproduction, weighing, injecting, marking, blood sampling, tagging, experimental surgery, and veterinary procedures (Gabriel, Erasmus, and Namwoonde 2020; Githukia, Kembenya, and Opiyo 2016; Hasimuna et al. 2021; Mphande and Chama 2015; Opiyo, Ogello, and Charo-Karisa 2013; Siavwapa et al. 2022; Simfukwe, Limuwa, and Njaya 2022). Anaesthetics induce reversible, controlled drug-induced sedation of the central nervous system (CNS), effectively preventing the perception or recollection of noxious stimuli (Donohue, Hobson, and Stephens 2013). Essential attributes for anaesthetic agents in aquaculture include effectiveness, safety, affordability, and accessibility (Hasimuna et al. 2020; Mphande et al. 2023; Velisek et al. 2007). In the aquaculture industry, the commonly used anaesthetics are Tricaine methanesulfonate (MS-222) (Burka et al. 1997), benzocaine (Bolasina, de Azevedo, and Petry 2017) quinaldine sulfate (Massee et al. 1995; Rust, Hardy, and Stickney 1993), clove oil and powder (Weber et al. 2009), eugenol, 2-phenoxyethanol, metomidate (King et al. 2005; Palić et al. 2006), and carbon dioxide (CO₂) (Fish 1943; Hasimuna et al. 2020; Mbewe et al. 2024; Rairat et al. 2021; Siavwapa et al. 2022). This is even more pivotal in broodstock as it is a major factor that compromises egg and sperm quality, hatchability, fry survival rate, and reproductive performance (Mphande et al. 2023; Siavwapa et al. 2022). However, there is a call to use ecologically friendly anaesthetics which do not comprise the welling of the fish, are cheaper, readily available, and safe for the user and the consumer (Gabriel, Erasmus, and Namwoonde 2020; Hasimuna et al. 2020; Mphande et al. 2023; Siavwapa et al. 2022). In the pursuit of ecologically friendly and cost-effective anaesthetics, research endeavors in the aquaculture sector persist in exploring alternatives to conventional agents, especially in resource-limited settings (Gabriel, Erasmus, and Namwoonde 2020; Hasimuna et al. 2021). Sodium bicarbonate (NaHCO₃), commonly known as baking soda, has emerged as a promising anaesthetic due to its nontoxic nature to fish, humans, and the environment. It can be administered directly through air-stone diffusion or indirectly by adding NaHCO₃ to water, releasing CO₂ (Fish 1943). Numerous studies have investigated NaHCO₃ as an anaesthetic for various fish species, including Oreochromis niloticus, Clarias gariepinus, Oreochromis mossambicus, Oreochromis andersonii, Oreochromis macrochir, and Cyprinus carpio, affirming its effectiveness and recommending dosage adjustments based on species, size, and sex (Akinrotimi, Gabriel, and Deekae 2014; Gabriel, Erasmus, and Namwoonde 2020; Gajutos and Gajutos 2023; Hasimuna et al. 2020; Opiyo, Ogello, and Charo-Karisa 2013; Siavwapa et al. 2022).

However, it’s imperative to not solely rely on behavioral assessments but also evaluate the impact of anaesthetics on Hematological parameters when determining appropriate dosages (Aydın and Barbas 2020; Mirghaed et al. 2018; Yousefi et al. 2018). This is crucial as certain anaesthetic agents, such as essential oils, have been observed to elicit undesirable effects, such as increased cortisolemia, in fish species (Cárdenas et al. 2016; Mazandarani, Hoseini, and Dehghani Ghomshani 2017). Hematological and biochemical analyses of fish blood serve as vital indicators of physiological status and welfare, facilitating the detection of environmental stressors and toxicants (Kanu, Okoboshi, and Otitoloju 2023). Consequently, they play a fundamental role in both ecotoxicological and pharmacotoxicological studies by elucidating the structural and functional implications of exposure to contaminants (Fazio et al. 2019) and serve as early warning indicators of environmental threats (Brosset et al. 2021)

Despite the need for a comprehensive understanding of both behavioral and physiological response of fish species to anaesthetic substances when determining the ideal concentration to use for various routine operations in aquaculture and hatcheries in particular, to the best of our knowledge there are no studies that have been done to use behavioral and physiological response of O. andersonii broodstock to NaHCO₃ and it the effect on Hematological parameters.

For this reason, this study aimed to determine the ideal (appropriate) concentration level (s) of sodium bicarbonate (NaHCO₃) and its effect on the Hematological parameters of three spotted tilapia (Oreochromis andersonii) broodstock.

Materials and methods

Experimental site

This study was carried out 20 kilometers North-East of Lusaka at Kalimba Farm (Figure 1) which is adjacent to Ngwerere River.

Figure 1. Map showing the location of Kalimba farm.

Ethical consideration

Ethical approval for this was granted by the Tropical Diseases Research Centre (TDRC) Ethics Research Committee (ERC) number 00003729 at Ndola Teaching Hospital subsequent to the comprehensive review of protocols governing fish experimentation. Furthermore, all experimental fish were handled according to the Canadian Council’s guide on the care and use of experimental animals (Olfert, Cross, and McWilliam 1993).

Source of sodium bicarbonate

Sodium bicarbonate (NaHCO₃) commonly referred to as baking soda or Chapa Mandashi locally, was sourced from a local super-market in Matero township in Lusaka, Zambia.

Experimental fish and conditioning

One hundred and fifty (150) broodstocks (weight: 180 ± 10 g) were obtained from an outdoor earthen pond of an approximate size of 20 m × 30 m using a seine net. The fish were placed in four black plastic troughs of about 2 m × 2 m × 6 m each having about 50 fish for conditioning in one-third (1/3) of borehole water which was aerated the whole night. The fish were starved for 24 h before the experiment to ensure gut evacuation (Hasimuna et al. 2020; Siavwapa et al. 2022). The period was picked to mimic the real routine handling of fish in hatcheries at fish farms. All the fish were in good health based on external examination (Fazio et al. 2017).

Induction and recovery time

After the conditioning, fish underwent exposure to nine different concentration levels of NaHCO₃ (0 [Control], 10, 15, 20, 25, 30, 35, 40, 45, and 50 g/L) along with a control through bath immersion in 20 L transparent plastic containers, with each concentration tested in triplicate using a Completely Randomized Design (CRD) to ensure impartiality. Fifteen (15) fish were immersed at each concentration levels, which were selected based on prior research on similar species, such as Cyprinus carpio (Altun, Bilgin, and Danabaş 2009) and Oreochromis macrochir (Hasimuna et al. 2020). The duration of induction and recovery stages was recorded using a stopwatch following the stages of anaesthesia and description as shown in Table 1. Once fully anesthetized, fish were transferred to tanks containing fresh water for recovery, with recovery time also recorded (Hasimuna et al. 2021; Sorensen et al. 2023).

Table 1. Behavioral stages of anaesthesia and recovery.

Stage of Anaesthesia	Description
I	Loss of equilibrium
II	Loss of gross body movement but with continued opercula movement
III	As in Stage II with cessation of opercula movement
Stage of Recovery	Description
I	Body immobilized but opercula movement just starting
II	Regular opercula movements and gross body movements beginning
III	Equilibrium regained and pre-anaesthetic appearance

Adapted from Siavwapa et al. (2022).

Blood sampling and analysis

Once the fish had reached stage three of induction, three fish per concentration (1 per replicate) were obtained for blood sampling. About 2 mL of blood was sampled within a minute via puncturing the caudal vein using a 20 G 1½ syringe and collected in microtubes containing Ethylene Diamine Tetraacetic Acid (EDTA) as the anticoagulant agent (Fazio et al. 2012). All 30 blood samples were transported on dry ice to the University Teaching Hospital (UTH) for Hematological analysis.

Hematological analysis

The protocols outlined by Fazio et al. (2013) and Shah and Altindağ (2005) were employed to analyze Red Blood Cells (RBC), White Blood Cells (WBC), Packed Cell Volume (PCV), and Hemoglobin (Hb). RBC counts were manually determined using a Neubauer hemocytometer, while PCV and Hb levels were assessed through microhematocrit centrifugation and cyanmethemoglobin, respectively. In brief, each 20 μl of whole blood was accurately diluted with 0.98 ml of Dacie’s fluid, containing 40% of formaldehyde, 3.13 g trisodium citrate, and 0.1 g brilliant cresyl blue dissolved in 100 ml of distilled water. The prepared mixture was then stirred to dissolve the blood cells then this was followed by drawing the solution using a disposable plastic pipette and a few drops discarded and only a single touch drop placed between the counting chamber of Neubauer hemocytometer and a cover slip. After which, RBCs were counted under a microscope in five secondary squares at a magnification of 640X. As of the WBC count, 20 μl of blood was diluted with Turk’s diluting fluid, which is a solution of 1% glacial acetic acid and 0.3% Gentian violet dissolved in distilled water. After which, four large (1 square millimeter) corner squares of the hemocytometer were then counted under a microscope at 640X magnification to obtain the WBC. In addition, PCVs were determined through microhematocrit centrifugation. A microcapillary tube was filled with blood mixture then plugged with clay and then subjected to centrifugation at 19,000 g 5 min. There after, the length of column containing packed red cells and packed red cells plus the supernatant were measured, and the hematocrit was calculated as the ratio of packed red cells to packed red cells plus supernatant, expressed as a percentage. Likewise, Hb levels were measured using a test kit utilized the cyanmethemoglobin method.

Calculation of erythrocyte indices

All the three erythrocyte indices, namely Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Hemoglobin (MCH), and Mean Corpuscular Volume (MCV) were calculated indirectly using the following formulas as prescribed by Fazio et al. (2013).

1. MCHC = Hb x 100/PCV

2. MCH = Hb/RBC

3. MCV = PCV x 10/RBC

Water parameters

Four water quality parameters (temperature, dissolved oxygen, ammonia, and pH) were determined before administering the test chemical using a water test kit (Sunpu Test ZL 2010 2 0,681,385. X – Beijing Sunpu Biochem. Tech. CO., LTD). The values obtained were 23.8 ± 0.5°C, 5.7 ± 0.4 mg/L, ˂ 0.1 mg/L and 7.8 ± 0.2 (Unitless) for temperature, dissolved oxygen, ammonia, and pH, respectively.

Data analysis

Shapiro-Wilk and Levene’s tests were performed to check for normality and homogeneity assumptions of analysis of Variance (ANOVA), respectively. One-way ANOVA was used to analyze the differences in the means since the data met the two assumptions. To establish where differences in the means existed, Turkey’s honestly significant difference test was performed. The differences were considered significant at p < .05 and all tests were executed in Statistical Package for Social Scientists (SPSS) version 22. Additionally, polynomial regression was used to plot the concentration as independent variable against induction and recovery times as dependent variables, including their optimum times.

Results

NaHCO₃ effect on induction time and recovery time

NaHCO₃ exhibited concentration-dependent effects on both induction and recovery times. Induction time, ranging from 0.11 to 9.55 minutes (Table 2), decreased with increasing NaHCO₃ concentration. Similarly, recovery time, spanning from 2.45 to 18.06 min (Table 2), increased with higher NaHCO₃ concentration. In addition, polynomial regression analysis identified optimal concentrations for NaHCO₃, suggesting 30 g/L for induction time less than 3 min and 35 g/L for recovery time less than 9 min (Figure 2).

Figure 2. Polynomial regression of concentration, induction and recovery time and their optimum.

Table 2. O. andersonii broodstock exposed to various concentrations of NaHCO₃ shows effects on induction and recovery time.

Concentration (g/L)	Induction time (Minutes)	Recovery time (Minute)
0	0	0
10	9.55 ± 0.04^i	2.45 ± 0.01^a
15	7.55 ± 0.02^h	3.46 ± 0.03^b
20	6.54 ± 0.01^g	5.03 ± 0.04^c
25	5.03 ± 0.02^f	6.03 ± 0.02^d
30	3.55 ± 0.03^e	7.04 ± 0.01^e
35	1.65 ± 0.01^d	8.46 ± 0.03^f
40	1.08 ± 0.03^c	9.03 ± 0.01^g
45	0.54 ± 0.01^b	12.03 ± 0.02^h
50	0.11 ± 0.02^a	18.06 ± 0.04^i

Different letters within a column indicate significant differences among the concentrations (p < .05) of NaHCO₃. Data are expressed as the means ± SE (n = 10).

NaHCO₃ influences the haematological parameters of O. andersonii broodstock

NaHCO₃ influenced the measured Hematological parameters of O. andersonii broodstock, demonstrating a statistically significant effect (p < .05), as shown in Table 3. Red Blood Cell (RBC) and Packed Cell Volume (PCV) exhibited significant effects at all concentrations, while Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin (MCH) showed significant effects only at higher concentrations. Additionally, no significant difference was attained at all concentration levels in Mean Corpuscular Hemoglobin Concentration (MCHC), while Hemoglobin (Hb) and White Blood Cell (WBC) counts showed significant difference at concentrations exceeding 10 and 15 g/L, respectively.

Table 3. NaHCO₃ influences the Hematological parameters of O. andersonii broodstock.

Parameter	Concentrations (g/L)
	0	10	15	20	25	30	35	40	45	50
RBC (10^6/µl)	0.36 ± 0.01^a	0.54 ± 0.01^b	0.64 ± 0.01^c	0.77 ± 0.01^d	0.84 ± 0.01^d	0.836 ± 0.01^d	1.00 ± 0.02^e	1.54 ± 0.01^g	1.66 ± 0.01^h	1.39 ± 0.03^f
PCV (%)	14.57 ± 0.09^e	14.11 ± 0.10^d	13.96 ± 0.01^d	13.27 ± 0.09^c	13.85 ± 0.02^d	13.21 ± 0.01^c	13.05 ± 0.03^bc	12.87 ± 0.01^b	12.04 ± 0.02^a	11.94 ± 0.01^a
Hb (g/dl)	0.40 ± 0.06^a	0.80 ± 0.06^a	1.27 ± 0.09^b	2.20 ± 0.06^c	2.50 ± 0.06^cd	2.9033 ± 0.06^de	3.04 ± 0.01^e	3.05 ± 0.03^e	3.30 ± 0.06^e	2.87 ± 0.22^de
WBC (10^3/µl)	54.25 ± 0.58^a	63.08 ± 1.32^a	70.78 ± 0.48^a	88.57 ± 0.53^b	94.02 ± 0.88^bc	110.71 ± 4.02^c	253.89 ± 5.24^d	314.26 ± 5.89^e	560.21 ± 6.30^f	999.32 ± 0.19^g
MCV (fl)	149.16 ± 0.12^a	150.60 ± 0.21^ab	149.05 ± 0.30^a	150.80 ± 0.68^ab	147.06 ± 0.30^a	148.13 ± 0.91^a	150.88 ± 0.73^ab	147.53 ± 1.55^a	150.19 ± 1.65^ab	154.3 3± 0.53^b
MCH (pg)	38.69 ± 0.51^a	38.63 ± 0.76^a	38.70 ± 0.55^a	39.40 ± 0.06^a	38.87 ± 0.12^a	38.83 ± 0.43^a	39.60 ± 0.32^a	39.36 ± 0.62^a	42.34 ± 0.31^b	42.71 ± 0.03^b
MCHC (g/l)	24.63 ± 0.07^a	24.07 ± 0.17^a	23.37 ± 0.09^a	22.97 ± 1.38^a	23.37 ± 0.32^a	24.47 ± 0.38^a	24.05 ± 0.02^a	23.93 ± 0.26^a	24.96 ± 0.01^a	25.04 ± 0.02^a

Different letters within a row indicate significant differences among the concentrations (P < 0.05) of NaHCO₃. Data are expressed as the means ± SE (n = 10)

Discussion

The present study delved into the potential of NaHCO₃ as an anaesthetic agent for Oreochromis andersonii broodstock, indicating its efficacy, suitability, effect on Hematological parameters and as an alternative to conventional anaesthetic substances employed in aquaculture operations. In terms of induction time, an inverse relationship was observed between NaHCO₃ concentration levels and the time required for induction. This phenomenon can be attributed to the increased release of CO₂ from NaHCO₃, leading to a reduction in dissolved oxygen levels in the water, which in turn may induce anesthesia. The increase in CO₂ from NaHCO₃ could have been the cause of a concussion O. andersonii broodstock. This aligns with previous studies (Fish 1943; Mbewe et al. 2024; Siavwapa et al. 2022) suggesting CO₂-induced anesthesia in fish species. Conversely, a direct correlation was noted between NaHCO₃ concentration and recovery time, likely due to elevated CO₂ levels in the fish blood, necessitating extended recovery periods for CO₂ elimination. This finding resonates with previous research on Oreochromis species (Avillanosa and Caipang 2019; Gabriel, Erasmus, and Namwoonde 2020; Hasimuna et al. 2020; Siavwapa et al. 2022), indicating prolonged recovery times associated with higher NaHCO₃ concentration levels. Importantly, no mortality was observed across varying NaHCO₃ concentration levels in O. andersonii broodstock, consistent with prior studies (Hasimuna et al. 2020; Opiyo, Ogello, and Charo-Karisa 2013; Siavwapa et al. 2022). This lack of mortality could be attributed to minimal alterations in erythrocyte indices at lower NaHCO₃ concentration levels, suggesting the preservation of blood morphology and characteristics, crucial for fish health and survival. The study established optimal NaHCO₃ concentration levels of 30 and 35 g/L for effective anesthesia induction and recovery within 3 and 9 minutes, respectively. These concentrations partly agree with the commendation made by Siavwapa et al. (2022) who stated that 30 g/L could be used for male O. andersonii broodstock and disagreed with the recommendation of 25 g/L for female species of the O. andersonii and further aligns with recommendations for routine hatchery operations (Hasimuna et al. 2021). This disagreement is ascribed to differences in fish sizes or age investigated (Effati and Bahrekazemi 2017). Additionally, our recommendation disagrees with the concentration of 50 g/L for C. gariepinus juveniles reported by Githukia, Kembenya, and Opiyo (2016). The differences in the concentrations commended for use can be attributed to differences in the species. It’s not surprising that C. gariepinus, despite being juveniles, required higher concentrations than broodstock of O. andersonii because C. gariepinus is a very hard species and can tolerate low levels of dissolved oxygen than Oreochromis species like O. andersonii investigated in the present study. These concentration levels demonstrated safety and effectiveness, without significant impact on Hematological parameters or mortality, thereby presenting a viable option for aquaculture handling activities. Regarding Hematological responses, an increase in both RBC and WBC counts was observed with rising NaHCO₃ concentration levels, consistent with findings in other fish species exposed to various anaesthetic agents (Gressler et al. 2014; Jia et al. 2022; Shaluei et al. 2012; Yousefi et al. 2022; Abd El-Hack et al. 2022). This physiological response, characterized by heightened immune activity and oxygen transport, likely aids in fish recovery from anesthesia-induced stress. Furthermore, PCV exhibited a reduction while Hb levels increased with increasing NaHCO₃ concentration levels, in agreement with previous studies on different fish species and anaesthetic agents (Bagheri and Imanpour 2011; de Oliveira et al. 2019; Olufayo and Adeyanju 2012; Zahran, Risha, and Rizk 2021). These Hematological alterations reflect adaptive responses to anesthesia-induced stress, emphasizing the resilience of O. andersonii broodstock to NaHCO₃ exposure. Erythrocyte indices (MCV, MCHC and MCH) remained relatively stable at lower NaHCO₃ concentration levels, with significant differences observed only at higher levels. This aligns with findings in other fish species exposed to anaesthetic agents (Adeshina, Adewale, and Yusuf 2016; Akinrotimi et al. 2014), suggesting a low-cost response to anesthesia-induced stress. This stability in erythrocyte indices further supports the suitability of NaHCO₃ as an anaesthetic substance for O. andersonii broodstock.

Conclusion

The anaesthetic effect of sodium bicarbonate and its effect on Hematological parameters of Oreochromis andersonii broodstock has been investigated. Furthermore, the study has demonstrated the efficacy of NaHCO₃ as an effective anaesthetic for Oreochromis andersonii broodstock, with concentration levels of 30 and 35 g/L identified as suitable for anesthesia induction. Notably, no mortality was observed across varying NaHCO₃ concentration levels, underscoring its safety for fish. Furthermore, NaHCO₃ exhibited minimal impact on Hematological parameters and erythrocyte indices, particularly at lower concentration levels.

Based on these findings, NaHCO₃ is recommended for anaesthetizing O. andersonii broodstock, and this has the potential to enhance stress management and welfare of broodstock fish in Zambian hatcheries. To provide comprehensive insights into the broader impact of NaHCO₃ anesthesia on O. andersonii broodstock physiology and health, further research is necessary that will investigate the effects of NaHCO₃ on the biochemical parameters and determine its acute toxicity levels. This will help to formulate appropriate, timely, and practical stress management in hatchery across the country.

Acknowledgments

The authors wish to thank all individuals that assisted in the execution of this study in any form.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data used to support the findings of this study are available and can be requested from the corresponding author.

Additional information

Funding

The work was supported by the DAAD as part of the In-Country/In-Region scholarship program Sub-Saharan Africa [Grant number 91973270].