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Effects of different doses of eugenol on plasma cortisol levels and the quality of fresh and frozen-thawed sperm in South American catfish (Rhamdia quelen)
Theriogenology 125 (2019) 135e139
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Effects of different doses of eugenol on plasma cortisol levels and the quality of fresh and frozen-thawed sperm in South American catfish (Rhamdia quelen) ^ mulo B. Rodrigues a, Maira N. Corso a, Lis S. Marques a, *, Luis F.G. Gracia a, Ro b, c a Leonardo J.G. Barcellos , Danilo P. Streit Jr. a b c
~o em Zootecnia, Universidade Federal do Rio Grande do Sul, 91540-000, Porto Alegre, RS, Brazil s-Graduaça Programa de Po ~o em Farmacologia, Universidade Federal de Santa Maria, 97105-900, Santa Maria, Rio Grande do Sul, Brazil s-Graduaça Programa de Po ~o em Bioexperimentaça ~o, Universidade de Passo Fundo, 99052-900, Passo Fundo, Rio Grande do Sul, Brazil s-Graduaça Programa de Po
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2018 Received in revised form 19 September 2018 Accepted 30 October 2018 Available online 31 October 2018
The production of captive fish is only possible through artificial reproduction, but manipulation is a known stressor stimulus. Thus, the objective of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during reproductive management of Rhamdia quelen. Seventy-five mature male R. quelen were randomly distributed among the five treatments, and blood samples were collected at the time of semen collection to measure plasma cortisol. The following parameters were evaluated in the fresh semen samples: motility, motility duration, concentration and fertilization rate. The following parameters were evaluated in the frozen semen samples: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The animals anesthetized with eugenol at concentrations of 40 and 50 mg/L had lower levels of plasma cortisol (88.4 and 83.3 ng/mL, respectively) than the control (147.1 ng/mL). For fresh semen, the control treatment presented the highest rate and time of motility but differed (P < 0.05) only from the animals treated with 60 mg/L eugenol. For the cryopreserved semen the highest rates and motility time were observed in the control treatment and in the animals anesthetized with 40 mg/L eugenol, differing (P < 0.05) from anesthetized animals with 50 and 60 mg/L. Mitochondrial functionality was higher in fish anesthetized with 30 mg/L eugenol differing only for animals anesthetized with 60 mg/L. There was no difference between treatments for sperm concentration and fertilization rate of fresh semen. There were no differences (P > 0.05) between treatments in the parameters of membrane integrity, DNA integrity and% of normal spermatozoa after thawing of the cryopreserved semen samples. The use of 30, 40 and 50 mg/L eugenol maintained the seminal quality of the fresh semen, and the quality of the thawed semen was maintained with 30 and 40 mg/L eugenol. These results show that stress reduction can be reconciled with reproductive management without compromising reproductive performance. © 2018 Elsevier Inc. All rights reserved.
Keywords: Cortisol levels Fish anesthesia Reproductive management Seminal quality Fish reproduction
1. Introduction In most captive fish species, artificial reproduction is optimized by hormonal induction, which enables the final maturation and release of gametes, thus improving management. In addition, combining hormonal therapy with semen freezing allows the number of broodstock to be reduced and is effective in breeding programs [1]. However, reproduction is a physiological process that
* Corresponding author. E-mail addresses: [email protected], [email protected] (L.S. Marques). https://doi.org/10.1016/j.theriogenology.2018.10.033 0093-691X/© 2018 Elsevier Inc. All rights reserved.
is particularly sensitive to the effects of stress, and fish management during hormonal therapy is a stressor [2,3]. Stress resistance requires great energy expenditure, and these costs can be considered to compete with those necessary to perform other reproductive functions, such as the production of high-quality gametes [4]. Knowing that gamete quality is one of the main factors limiting reproductive success [3], our hypothesis is that using anesthetics during the reproductive management of male fishes alleviates stress, thus improving reproductive performance. When selecting an anesthetic for a specific purpose, the user should consider the following factors: convenience of use, efficacy,
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physiological disturbances, and cost as well as fish, human and environmental safety [5]. Eugenol [2-methoxy-4- (2-propenyl) phenol] is among the most commonly used anesthetics for fish [6], and it is widely available on the market, affordable, and considered safe for the handler and the environment [7]. Eugenol is derived from the stem, leaves and shoots of the Eugenia aromatica tree [8]. Eugenol have limited water solubility; thus, it must be dissolved in ethanol (1:9) before dilution with water [6]. Stress induces cortisol secretion by the adrenal glands, and this hormone causes various secondary stress responses, including decrease in the immunologic capacity. In a previous study, the eugenol-based anaesthetics (clove oil and Aqui-S™) showed stressreducing capabilities for Atlantic salmon [7]. According to the authors, it is likely that eugenol blocks the transmission of sensory information to the hypothalamus. Studies have shown the effects of broodstock stress on subsequent gamete quality. Authors reported that rainbow trout males confined alone in small tanks had cortisol levels four times higher than the controls, and sperm motility less than 10% when compared to control groups [3]. In addition, stress hormones may interfere with plasma osmolarity and, consequently, affect sperm quality [9]. Therefore, the aim of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during the reproductive management of R. quelen on male cortisol levels and sperm quality parameters from fresh and frozen semen samples. 2. Materials and methods 2.1. Experimental design Seventy-five sexually mature male R. quelen (500 ± 10 g) were randomly assigned to five treatments, four concentrations of eugenol (30, 40, 50 and 60 mg/L) and a control (0 mg/L eugenol), and each male was considered an experimental unit (15 replicates per treatment). The anesthetic baths and blood collection to measure plasma cortisol were performed before semen collection. The semen obtained from each fish was divided into three parts that were subjected to the following procedures: analysis of fresh semen, fertilization and freezing. The following sperm quality parameters were evaluated in the fresh semen samples: motility, motility duration, morphology, concentration and fertilization rate. In the frozen/thawed semen samples, the following sperm quality parameters were evaluated: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The Ethics Committee of our Institution approved all the protocols reported in this study. 2.2. Anesthetic baths The fish were placed in containers containing 10 L of water (6 mg O2/L water) and different concentrations of eugenol (30, 40, 50 and 60 mg/L) that had been previously dissolved in ethanol (1:9), a control group where animals were not anesthetized. The animals were kept in the anesthetic bath until they manifested the fourth stage of anesthesia, according to Woody et al. [10]. Five animals were anesthetized in each bath, and the baths were repeated three times at different days. 2.3. Hormonal induction and semen collection A single dose of 2.5 mg/kg of carp pituitary extract was administered intraperitoneally under the right pectoral fin of the fish. The temperature was maintained at 22.0 ± 1.0 C and monitored
frequently. The male gametes were collected after 240 hours or accumulated thermal units (ATU), and the reproducers were contained and dried with a cloth and a paper towel. Before gamete collection, the fish were submitted to the anesthetic baths (0, 30, 40, 50 and 60 mg/L), and semen collection was initiated after the fish reached the fourth stage of anesthesia by massaging the ventral region of the animal in the cerebrum-caudal direction [11], avoiding contamination with water or urine. 2.4. Plasma cortisol To analyze plasma cortisol, 1e3 mL of blood from each fish was collected by puncturing the caudal vessel after fish reached the fourth stage of anesthesia. Then blood was centrifuged (3000g/ 10 min). Plasma was collected with a Hamilton syringe, transferred to 1.5-mL microtubes and stored at 25 C until analysis. Cortisol was measured in duplicate samples of non-extracted plasma with the commercially available (125I) DPC cortisol RIA test (Coat-ACount®; DPC Los Angeles, CA). The results were validated based on the standard curve of the kit. The intra-assay coefficient of variation was 6% with a sensitivity of 50 pg/mL. 2.5. Motility, motility duration, concentration and sperm morphology The semen sample was diluted with water to a 1:10 ratio (semen: distilled water), for the activation of sperm motility. The motility rate was evaluated by optical microscopy (40X) and quantified as the percentage of motile spermatozoa, from 0 to 100%, in the optical field. The motility duration was determined as the time from the moment of sperm activation in distilled water until the sperm ceased moving in the optical field [12]. The sperm concentration was determined in a Neubauer chamber after diluting the semen in formaldehyde-buffered saline (1:2000) [13]. Sperm morphology (% of normal spermatozoa) was evaluated in fresh and thawed semen by diluting the semen in formaldehydebuffered saline solution (1:2000) followed by staining with Rose Bengal. One hundred sperm cells from each sample were evaluated under an optical microscope (100X). Three replicates of the samples were used for each test. 2.6. Fertilization The same hormonal induction protocol described above was applied to four sexually mature female R. quelen (500 ± 10 g). A pool of oocytes was formed from the total oocytes extruded from the females, from which 5.0-g samples (4274.45 ± 344.68 oocytes) were collected. Each oocyte sample was mixed with 1 mL of semen from each male (approximately 90,000 sperm: oocyte), and the spermatozoa were activated by immersion distilled water for 60 s. Then, the probable fertilized eggs were distributed in conical experimental incubators made of PVC with a useful volume of 2.5 L. The water from the incubators was kept warm at 22.0 ± 1.0 C, and after 6 h of incubation at 75e90% epiboly stage, the fertilization rate (% fertilized eggs out of 100 eggs) was determined for each experimental unit. 2.7. Freezing The semen was diluted to a 1:3 ratio (semen: diluent) in 90% Beltsville Thawing Solution (BTS) with 10% methanol. The samples were packed in 250-mL tubes and stored for 12 h in liquid nitrogen vapor through a dry shipper. Afterward, the tubes were transferred to a liquid nitrogen cylinder (196 C) and stored for 30 days.
M.N. Corso et al. / Theriogenology 125 (2019) 135e139
The tubes were thawed in a water bath at 45 C for 8 s, and two tubes from each treatment were thawed and diluted in 400 mL of BTS (1:3) at 22 C. Motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality were analyzed. An epifluorescence microscope with 40X magnification was used to count and evaluate 200 spermatozoa in each sample. 2.8. Membrane integrity To evaluate membrane integrity, a 10-mL semen sample was diluted in 40 mL of isotonic saline solution containing 1.7 mM formaldehyde, 20 mM carboxyfluorescein diacetate (CFDA) and 7.3 mM propidium iodide (PI). When green fluorescence occurred, the membranes of the spermatozoa were considered intact because they did not allow the carboxyfluorescein diacetate to emerge from the cytoplasm. The membranes of the spermatozoa showing red or red and green fluorescence on the head were not considered intact [14]. The percentage of sperm with intact membranes was determined as the proportion of those who emitted green fluorescence relative to the total number of spermatozoa (green, red or red and green fluorescence emitters). 2.9. DNA integrity DNA integrity was assessed after diluting 45 mL of semen in 50 mL of TNE (0.01 M Tris-HCl, 0.15 M NaCl, 0.001 M EDTA, pH 7.2). After 30 s, 200 mL of 1X Triton solution were added, and 50 mL of acridine orange (2 mg/mL in deionized water) was added after an additional 30 s. After 5 min, 200 spermatozoa were counted. Those with green fluorescence were considered to have intact DNA, while those with red or orange fluorescence were considered to have non-intact DNA [15]. The DNA integrity rate was determined as the proportion of spermatozoa emitting green fluorescence relative to the total number of spermatozoa (green, red or orange fluorescence).
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(P < 0.05, Fig. 1). In the thawed semen, the motility rate was lower under the 50 mg/L eugenol treatment (36.7%) than under 40 mg/L treatment (64%) and the control (63.3%), and the rate under the 60 mg/L eugenol treatment (17.7%) also differed from that under the 30 mg/L eugenol treatment (52%), Fig. 1. The motility duration of the sperm in fresh semen under the 60 mg/L eugenol treatment was inferior (P < 0.05) to that of the control; there was no significant difference among the other treatments. In thawed semen, motility duration was lower in the treatments with 50 and 60 mg/L eugenol when compared to the control and 40 mg/L treatment. The motility duration under 60 mg/L eugenol treatment also differed from that with 30 mg/L eugenol (Fig. 1). In the evaluation of fresh semen, there were no differences in fertilization rate and sperm concentration between treatments. The mean values observed between the treatments for the fertilization rate was 59.44 ± 3.85% and for sperm concentration it was 22.25 ± 0.91 106 spermatozoa/mL. In the thawed semen samples, mitochondrial functionality only differed (P < 0.05) between the highest (60 mg/L) and lowest (30 mg/L) concentrations of eugenol used as anesthetic treatments (Fig. 2). No differences were observed in membrane and DNA integrity between treatments. In terms of sperm morphology, there were also no differences in the percentage of normal spermatozoa among treatments. The mean values observed among the treatments for membrane integrity, DNA integrity and percentage of normal spermatozoa were 71.02 ± 1.25%, 98.70 ± 0.63% and 54.74 ± 2.46%, respectively. The plasma cortisol concentration was significantly lower in treatments using 40 (88.4 ng/mL) and 50 mg/L (83.3 ng/mL) eugenol compared to the control (147.1 ng/mL and 0 mg/L eugenol) (Fig. 3).
2.10. Mitochondrial functionality Mitochondrial functionality was evaluated after incubating 10 mL of semen with 40 mL of Rhodamine 123 (13 mM) solution at 20 C for 10 min. Spermatozoa with positive rhodamine staining (green fluorescence) were considered to have functional mitochondria; spermatozoa with nonfunctional mitochondria did not show fluorescence, that is, negative rhodamine staining [16]. The proportion of spermatozoa emitting green fluorescence relative to the total spermatozoa (green or no fluorescence) represented the mitochondrial functionality rate. 2.11. Statistical analysis Samples obtained from the treatments (control, 30, 40, 50 and 60 mg/L eugenol) were considered independent variables, and the qualitative and quantitative semen parameters and plasma cortisol were dependent variables. The normality of the data was tested by the Shapiro-Wilk test. Data that were not normally distributed following data transformation were subjected to non-parametric analysis of variance, and the treatments were compared by the Kruskal-Wallis test. For the plasma cortisol analysis, the data were log transformed, and the Tukey test was used to compare the means. 3. Results The sperm motility rate was significantly lower in the fresh and thawed semen samples subjected to the 60 mg/L eugenol treatment
Fig. 1. Motility rate (A) and motility time (B) of fresh and thawed R. quelen semen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different upper case letters in the columns indicate statistically significant differences (P < 0.05) between treatments for the fresh semen samples. a, b, c Different lowercase letters in the columns indicate significant differences (P < 0.05) between treatments for the frozen semen samples. Mean ± SEM.
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Fig. 2. Mitochondrial functionality of thawed R. quelen semen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different letters in the columns indicate significant differences (P < 0.05) between treatments. Mean ± SEM.
4. Discussion There is a great divergence in the literature about the ideal concentration of eugenol for anesthetic induction and recovery in fish. A previous study suggested that the concentration should be between 25 and 60 mg/L [6], and in R. voulezi, the best concentration for anesthetic induction and recovery was reported to be 50 mg/L [17]. In contrast, some authors state that sedation in R. quelen is achieved with a dose of 20 mg/L eugenol, but the concentrations for anesthetization should not be less than 40 mg/L [18]. However, Bolner and Baldisserotto [19] used 32 mg/L to anesthetize juveniles of this species. In the present study, the fourth stage of anesthesia, a parameter that indicates anesthetic action, was reached in male R. quelen with all concentrations of eugenol tested (30, 40, 50 and 60 mg/L). Management during artificial reproduction causes stress in fish, which release corticosteroids, such as cortisol, as a primary response [20]. The baseline cortisol concentration in R. quelen males is 15.86 ng/mL, but after acute stress, the concentration
Fig. 3. Plasma cortisol level of male R. quelen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different letters in the columns indicate statistically significant differences (P < 0.05) between treatments. Mean ± SEM.
increases to 158.12 ng/mL [9]. In the present study, male R. quelen that were not anesthetized with eugenol during reproductive management reached a peak plasma cortisol concentration of 147.1 ng/mL, and the lowest levels were recorded in the 40 mg/L (88.4 ng/mL) and 50 mg/L (83.3 ng/mL) eugenol treatments, demonstrating that the use of anesthetic during handling reduced reproductive stress. In contrast, plasma cortisol levels increased to 108.2 ng/mL in the treatment containing the highest concentration of eugenol (60 mg/L), and there was no significant difference between the control (0 mg/L eugenol) and 30 mg/L eugenol treatments. The control of the stress response depends on the type and concentration of anesthetic used because the anesthetic itself can be a stressor to the animal [21], which likely explains the increased stress in male R. quelen subjected to 60 mg/L eugenol. Moreover, the lowest sperm motility rate and duration as well as reduction of mitochondrial functionality were recorded in fish treated with 60 mg/L eugenol. It is known that increased plasma cortisol levels resulting from stress have deleterious effects on the reproductive performance of fish [20]. Depending on the duration and intensity of the application, a stressor may decrease sperm motility, the fertilization rate, and the quality of gametes and offspring [3,22,23]. In a study with rainbow trout (Oncorhynchus mykiss), a decrease in sperm concentration was reported after repeated acute stress events [24]. In contrast, there was no change in the sperm concentration of rainbow trout subjected to chronic stress [2]. Our results also showed no differences in sperm concentration or the fertilization rate of R. quelen treated with different concentrations of eugenol and subjected to artificial reproduction, which suggests that these reproductive parameters are not directly affected by increased plasma cortisol levels. Therefore, it is possible to reduce reproductive plasma cortisol while maintaining good reproductive performance, so reducing stress with eugenol can reconcile animal welfare with animal production. In the present study, the different concentrations of eugenol (0, 30, 40, 50 and 60 mg/L) did not affect sperm motility in male catfish, but the sperm motility duration in fresh semen under the treatment with 60 mg/L eugenol was lower than that of the control. According to Wagner et al. [21], the percentage of motile spermatozoa is not affected by the type or concentration of anesthetic, but motility duration is inversely proportional to the concentration of eugenol administered. This corroborates our results for frozen semen that showed decreasing motility duration with increasing eugenol concentrations. Moreover, the percentage of motile cells in frozen semen was lower under the two highest eugenol concentrations (50 and 60 mg/L) compared to the control and the 40 mg/L concentration of eugenol. Because motility is the main indicator of sperm quality in fish [25], eugenol concentrations of 50 and 60 mg/ L are not recommended in the reproductive management of R. quelen if the semen will be frozen. In addition to cortisol, anesthetics may also interfere with sperm motility. A study carried out with rainbow trout males observed that direct contact between the tricaine methanesulfonate anesthetic (MS-222) and spermatozoa decreased the duration of sperm motility [21]. In the frozen semen samples, mitochondrial functionality was affected by the 60 mg/L eugenol concentration. It is known that the levels of ATP in fish spermatozoa are only sufficient to maintain motility from a few seconds to a maximum of a few minutes [26]. Thus, the reduction in the number of functional mitochondria can reduce the amount of intracellular ATP available for cellular consumption, resulting in inadequate functioning of the ionic pumps [27]. Therefore, the lower motility observed in the 60 mg/L eugenol treatment is a consequence of the low levels of ATP resulting from the stress generated by the high dose of anesthetic.
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5. Conclusion It is possible to reconcile the well-being of R. quelen breeders with good reproductive indexes because plasma cortisol levels were reduced in the treatments with 40 and 50 mg/L eugenol while the same reproductive performance as the control group was maintained. The seminal quality was compromised by the administration of 60 mg/L eugenol in fresh semen and 50 or 60 mg/L in frozen semen. Therefore, anesthesia with eugenol 40 mg/L is recommended because, despite offering the same reproductive performance as non-anesthetized men, anesthesia has reduced animal stress and, consequently, can avoid possible disorders caused to the immune system of animals by stress in the reproductive management. Preserve the integrity of animal health in order to maintain reproductive performance in future semen collections, in addition to reducing the risk of disease and mortality. Author's contributions Conception and design of the study: MNC, DPS. Acquisition of data: MNC, LFGG. Analysis and interpretation of data: MNC, LSM, DPS. Drafting the article or revising it critically for important intellectual content: MNC, LSM, DPS, LJGB. All authors read and approved the final version to be submitted.
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[12]
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[18]
Acknowledgement
[19]
The study was supported by CAPES Foundation e Brazilian Ministry of Education.
[20]
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Effects of different doses of eugenol on plasma cortisol levels and the quality of fresh and frozen-thawed sperm in South American catfish (Rhamdia quelen) ^ mulo B. Rodrigues a, Maira N. Corso a, Lis S. Marques a, *, Luis F.G. Gracia a, Ro b, c a Leonardo J.G. Barcellos , Danilo P. Streit Jr. a b c
~o em Zootecnia, Universidade Federal do Rio Grande do Sul, 91540-000, Porto Alegre, RS, Brazil s-Graduaça Programa de Po ~o em Farmacologia, Universidade Federal de Santa Maria, 97105-900, Santa Maria, Rio Grande do Sul, Brazil s-Graduaça Programa de Po ~o em Bioexperimentaça ~o, Universidade de Passo Fundo, 99052-900, Passo Fundo, Rio Grande do Sul, Brazil s-Graduaça Programa de Po
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2018 Received in revised form 19 September 2018 Accepted 30 October 2018 Available online 31 October 2018
The production of captive fish is only possible through artificial reproduction, but manipulation is a known stressor stimulus. Thus, the objective of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during reproductive management of Rhamdia quelen. Seventy-five mature male R. quelen were randomly distributed among the five treatments, and blood samples were collected at the time of semen collection to measure plasma cortisol. The following parameters were evaluated in the fresh semen samples: motility, motility duration, concentration and fertilization rate. The following parameters were evaluated in the frozen semen samples: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The animals anesthetized with eugenol at concentrations of 40 and 50 mg/L had lower levels of plasma cortisol (88.4 and 83.3 ng/mL, respectively) than the control (147.1 ng/mL). For fresh semen, the control treatment presented the highest rate and time of motility but differed (P < 0.05) only from the animals treated with 60 mg/L eugenol. For the cryopreserved semen the highest rates and motility time were observed in the control treatment and in the animals anesthetized with 40 mg/L eugenol, differing (P < 0.05) from anesthetized animals with 50 and 60 mg/L. Mitochondrial functionality was higher in fish anesthetized with 30 mg/L eugenol differing only for animals anesthetized with 60 mg/L. There was no difference between treatments for sperm concentration and fertilization rate of fresh semen. There were no differences (P > 0.05) between treatments in the parameters of membrane integrity, DNA integrity and% of normal spermatozoa after thawing of the cryopreserved semen samples. The use of 30, 40 and 50 mg/L eugenol maintained the seminal quality of the fresh semen, and the quality of the thawed semen was maintained with 30 and 40 mg/L eugenol. These results show that stress reduction can be reconciled with reproductive management without compromising reproductive performance. © 2018 Elsevier Inc. All rights reserved.
Keywords: Cortisol levels Fish anesthesia Reproductive management Seminal quality Fish reproduction
1. Introduction In most captive fish species, artificial reproduction is optimized by hormonal induction, which enables the final maturation and release of gametes, thus improving management. In addition, combining hormonal therapy with semen freezing allows the number of broodstock to be reduced and is effective in breeding programs [1]. However, reproduction is a physiological process that
* Corresponding author. E-mail addresses: [email protected], [email protected] (L.S. Marques). https://doi.org/10.1016/j.theriogenology.2018.10.033 0093-691X/© 2018 Elsevier Inc. All rights reserved.
is particularly sensitive to the effects of stress, and fish management during hormonal therapy is a stressor [2,3]. Stress resistance requires great energy expenditure, and these costs can be considered to compete with those necessary to perform other reproductive functions, such as the production of high-quality gametes [4]. Knowing that gamete quality is one of the main factors limiting reproductive success [3], our hypothesis is that using anesthetics during the reproductive management of male fishes alleviates stress, thus improving reproductive performance. When selecting an anesthetic for a specific purpose, the user should consider the following factors: convenience of use, efficacy,
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physiological disturbances, and cost as well as fish, human and environmental safety [5]. Eugenol [2-methoxy-4- (2-propenyl) phenol] is among the most commonly used anesthetics for fish [6], and it is widely available on the market, affordable, and considered safe for the handler and the environment [7]. Eugenol is derived from the stem, leaves and shoots of the Eugenia aromatica tree [8]. Eugenol have limited water solubility; thus, it must be dissolved in ethanol (1:9) before dilution with water [6]. Stress induces cortisol secretion by the adrenal glands, and this hormone causes various secondary stress responses, including decrease in the immunologic capacity. In a previous study, the eugenol-based anaesthetics (clove oil and Aqui-S™) showed stressreducing capabilities for Atlantic salmon [7]. According to the authors, it is likely that eugenol blocks the transmission of sensory information to the hypothalamus. Studies have shown the effects of broodstock stress on subsequent gamete quality. Authors reported that rainbow trout males confined alone in small tanks had cortisol levels four times higher than the controls, and sperm motility less than 10% when compared to control groups [3]. In addition, stress hormones may interfere with plasma osmolarity and, consequently, affect sperm quality [9]. Therefore, the aim of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during the reproductive management of R. quelen on male cortisol levels and sperm quality parameters from fresh and frozen semen samples. 2. Materials and methods 2.1. Experimental design Seventy-five sexually mature male R. quelen (500 ± 10 g) were randomly assigned to five treatments, four concentrations of eugenol (30, 40, 50 and 60 mg/L) and a control (0 mg/L eugenol), and each male was considered an experimental unit (15 replicates per treatment). The anesthetic baths and blood collection to measure plasma cortisol were performed before semen collection. The semen obtained from each fish was divided into three parts that were subjected to the following procedures: analysis of fresh semen, fertilization and freezing. The following sperm quality parameters were evaluated in the fresh semen samples: motility, motility duration, morphology, concentration and fertilization rate. In the frozen/thawed semen samples, the following sperm quality parameters were evaluated: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The Ethics Committee of our Institution approved all the protocols reported in this study. 2.2. Anesthetic baths The fish were placed in containers containing 10 L of water (6 mg O2/L water) and different concentrations of eugenol (30, 40, 50 and 60 mg/L) that had been previously dissolved in ethanol (1:9), a control group where animals were not anesthetized. The animals were kept in the anesthetic bath until they manifested the fourth stage of anesthesia, according to Woody et al. [10]. Five animals were anesthetized in each bath, and the baths were repeated three times at different days. 2.3. Hormonal induction and semen collection A single dose of 2.5 mg/kg of carp pituitary extract was administered intraperitoneally under the right pectoral fin of the fish. The temperature was maintained at 22.0 ± 1.0 C and monitored
frequently. The male gametes were collected after 240 hours or accumulated thermal units (ATU), and the reproducers were contained and dried with a cloth and a paper towel. Before gamete collection, the fish were submitted to the anesthetic baths (0, 30, 40, 50 and 60 mg/L), and semen collection was initiated after the fish reached the fourth stage of anesthesia by massaging the ventral region of the animal in the cerebrum-caudal direction [11], avoiding contamination with water or urine. 2.4. Plasma cortisol To analyze plasma cortisol, 1e3 mL of blood from each fish was collected by puncturing the caudal vessel after fish reached the fourth stage of anesthesia. Then blood was centrifuged (3000g/ 10 min). Plasma was collected with a Hamilton syringe, transferred to 1.5-mL microtubes and stored at 25 C until analysis. Cortisol was measured in duplicate samples of non-extracted plasma with the commercially available (125I) DPC cortisol RIA test (Coat-ACount®; DPC Los Angeles, CA). The results were validated based on the standard curve of the kit. The intra-assay coefficient of variation was 6% with a sensitivity of 50 pg/mL. 2.5. Motility, motility duration, concentration and sperm morphology The semen sample was diluted with water to a 1:10 ratio (semen: distilled water), for the activation of sperm motility. The motility rate was evaluated by optical microscopy (40X) and quantified as the percentage of motile spermatozoa, from 0 to 100%, in the optical field. The motility duration was determined as the time from the moment of sperm activation in distilled water until the sperm ceased moving in the optical field [12]. The sperm concentration was determined in a Neubauer chamber after diluting the semen in formaldehyde-buffered saline (1:2000) [13]. Sperm morphology (% of normal spermatozoa) was evaluated in fresh and thawed semen by diluting the semen in formaldehydebuffered saline solution (1:2000) followed by staining with Rose Bengal. One hundred sperm cells from each sample were evaluated under an optical microscope (100X). Three replicates of the samples were used for each test. 2.6. Fertilization The same hormonal induction protocol described above was applied to four sexually mature female R. quelen (500 ± 10 g). A pool of oocytes was formed from the total oocytes extruded from the females, from which 5.0-g samples (4274.45 ± 344.68 oocytes) were collected. Each oocyte sample was mixed with 1 mL of semen from each male (approximately 90,000 sperm: oocyte), and the spermatozoa were activated by immersion distilled water for 60 s. Then, the probable fertilized eggs were distributed in conical experimental incubators made of PVC with a useful volume of 2.5 L. The water from the incubators was kept warm at 22.0 ± 1.0 C, and after 6 h of incubation at 75e90% epiboly stage, the fertilization rate (% fertilized eggs out of 100 eggs) was determined for each experimental unit. 2.7. Freezing The semen was diluted to a 1:3 ratio (semen: diluent) in 90% Beltsville Thawing Solution (BTS) with 10% methanol. The samples were packed in 250-mL tubes and stored for 12 h in liquid nitrogen vapor through a dry shipper. Afterward, the tubes were transferred to a liquid nitrogen cylinder (196 C) and stored for 30 days.
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The tubes were thawed in a water bath at 45 C for 8 s, and two tubes from each treatment were thawed and diluted in 400 mL of BTS (1:3) at 22 C. Motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality were analyzed. An epifluorescence microscope with 40X magnification was used to count and evaluate 200 spermatozoa in each sample. 2.8. Membrane integrity To evaluate membrane integrity, a 10-mL semen sample was diluted in 40 mL of isotonic saline solution containing 1.7 mM formaldehyde, 20 mM carboxyfluorescein diacetate (CFDA) and 7.3 mM propidium iodide (PI). When green fluorescence occurred, the membranes of the spermatozoa were considered intact because they did not allow the carboxyfluorescein diacetate to emerge from the cytoplasm. The membranes of the spermatozoa showing red or red and green fluorescence on the head were not considered intact [14]. The percentage of sperm with intact membranes was determined as the proportion of those who emitted green fluorescence relative to the total number of spermatozoa (green, red or red and green fluorescence emitters). 2.9. DNA integrity DNA integrity was assessed after diluting 45 mL of semen in 50 mL of TNE (0.01 M Tris-HCl, 0.15 M NaCl, 0.001 M EDTA, pH 7.2). After 30 s, 200 mL of 1X Triton solution were added, and 50 mL of acridine orange (2 mg/mL in deionized water) was added after an additional 30 s. After 5 min, 200 spermatozoa were counted. Those with green fluorescence were considered to have intact DNA, while those with red or orange fluorescence were considered to have non-intact DNA [15]. The DNA integrity rate was determined as the proportion of spermatozoa emitting green fluorescence relative to the total number of spermatozoa (green, red or orange fluorescence).
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(P < 0.05, Fig. 1). In the thawed semen, the motility rate was lower under the 50 mg/L eugenol treatment (36.7%) than under 40 mg/L treatment (64%) and the control (63.3%), and the rate under the 60 mg/L eugenol treatment (17.7%) also differed from that under the 30 mg/L eugenol treatment (52%), Fig. 1. The motility duration of the sperm in fresh semen under the 60 mg/L eugenol treatment was inferior (P < 0.05) to that of the control; there was no significant difference among the other treatments. In thawed semen, motility duration was lower in the treatments with 50 and 60 mg/L eugenol when compared to the control and 40 mg/L treatment. The motility duration under 60 mg/L eugenol treatment also differed from that with 30 mg/L eugenol (Fig. 1). In the evaluation of fresh semen, there were no differences in fertilization rate and sperm concentration between treatments. The mean values observed between the treatments for the fertilization rate was 59.44 ± 3.85% and for sperm concentration it was 22.25 ± 0.91 106 spermatozoa/mL. In the thawed semen samples, mitochondrial functionality only differed (P < 0.05) between the highest (60 mg/L) and lowest (30 mg/L) concentrations of eugenol used as anesthetic treatments (Fig. 2). No differences were observed in membrane and DNA integrity between treatments. In terms of sperm morphology, there were also no differences in the percentage of normal spermatozoa among treatments. The mean values observed among the treatments for membrane integrity, DNA integrity and percentage of normal spermatozoa were 71.02 ± 1.25%, 98.70 ± 0.63% and 54.74 ± 2.46%, respectively. The plasma cortisol concentration was significantly lower in treatments using 40 (88.4 ng/mL) and 50 mg/L (83.3 ng/mL) eugenol compared to the control (147.1 ng/mL and 0 mg/L eugenol) (Fig. 3).
2.10. Mitochondrial functionality Mitochondrial functionality was evaluated after incubating 10 mL of semen with 40 mL of Rhodamine 123 (13 mM) solution at 20 C for 10 min. Spermatozoa with positive rhodamine staining (green fluorescence) were considered to have functional mitochondria; spermatozoa with nonfunctional mitochondria did not show fluorescence, that is, negative rhodamine staining [16]. The proportion of spermatozoa emitting green fluorescence relative to the total spermatozoa (green or no fluorescence) represented the mitochondrial functionality rate. 2.11. Statistical analysis Samples obtained from the treatments (control, 30, 40, 50 and 60 mg/L eugenol) were considered independent variables, and the qualitative and quantitative semen parameters and plasma cortisol were dependent variables. The normality of the data was tested by the Shapiro-Wilk test. Data that were not normally distributed following data transformation were subjected to non-parametric analysis of variance, and the treatments were compared by the Kruskal-Wallis test. For the plasma cortisol analysis, the data were log transformed, and the Tukey test was used to compare the means. 3. Results The sperm motility rate was significantly lower in the fresh and thawed semen samples subjected to the 60 mg/L eugenol treatment
Fig. 1. Motility rate (A) and motility time (B) of fresh and thawed R. quelen semen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different upper case letters in the columns indicate statistically significant differences (P < 0.05) between treatments for the fresh semen samples. a, b, c Different lowercase letters in the columns indicate significant differences (P < 0.05) between treatments for the frozen semen samples. Mean ± SEM.
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Fig. 2. Mitochondrial functionality of thawed R. quelen semen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different letters in the columns indicate significant differences (P < 0.05) between treatments. Mean ± SEM.
4. Discussion There is a great divergence in the literature about the ideal concentration of eugenol for anesthetic induction and recovery in fish. A previous study suggested that the concentration should be between 25 and 60 mg/L [6], and in R. voulezi, the best concentration for anesthetic induction and recovery was reported to be 50 mg/L [17]. In contrast, some authors state that sedation in R. quelen is achieved with a dose of 20 mg/L eugenol, but the concentrations for anesthetization should not be less than 40 mg/L [18]. However, Bolner and Baldisserotto [19] used 32 mg/L to anesthetize juveniles of this species. In the present study, the fourth stage of anesthesia, a parameter that indicates anesthetic action, was reached in male R. quelen with all concentrations of eugenol tested (30, 40, 50 and 60 mg/L). Management during artificial reproduction causes stress in fish, which release corticosteroids, such as cortisol, as a primary response [20]. The baseline cortisol concentration in R. quelen males is 15.86 ng/mL, but after acute stress, the concentration
Fig. 3. Plasma cortisol level of male R. quelen subjected to five concentrations of eugenol (0, 30, 40, 50 and 60 mg/L; n ¼ 15). A, B Different letters in the columns indicate statistically significant differences (P < 0.05) between treatments. Mean ± SEM.
increases to 158.12 ng/mL [9]. In the present study, male R. quelen that were not anesthetized with eugenol during reproductive management reached a peak plasma cortisol concentration of 147.1 ng/mL, and the lowest levels were recorded in the 40 mg/L (88.4 ng/mL) and 50 mg/L (83.3 ng/mL) eugenol treatments, demonstrating that the use of anesthetic during handling reduced reproductive stress. In contrast, plasma cortisol levels increased to 108.2 ng/mL in the treatment containing the highest concentration of eugenol (60 mg/L), and there was no significant difference between the control (0 mg/L eugenol) and 30 mg/L eugenol treatments. The control of the stress response depends on the type and concentration of anesthetic used because the anesthetic itself can be a stressor to the animal [21], which likely explains the increased stress in male R. quelen subjected to 60 mg/L eugenol. Moreover, the lowest sperm motility rate and duration as well as reduction of mitochondrial functionality were recorded in fish treated with 60 mg/L eugenol. It is known that increased plasma cortisol levels resulting from stress have deleterious effects on the reproductive performance of fish [20]. Depending on the duration and intensity of the application, a stressor may decrease sperm motility, the fertilization rate, and the quality of gametes and offspring [3,22,23]. In a study with rainbow trout (Oncorhynchus mykiss), a decrease in sperm concentration was reported after repeated acute stress events [24]. In contrast, there was no change in the sperm concentration of rainbow trout subjected to chronic stress [2]. Our results also showed no differences in sperm concentration or the fertilization rate of R. quelen treated with different concentrations of eugenol and subjected to artificial reproduction, which suggests that these reproductive parameters are not directly affected by increased plasma cortisol levels. Therefore, it is possible to reduce reproductive plasma cortisol while maintaining good reproductive performance, so reducing stress with eugenol can reconcile animal welfare with animal production. In the present study, the different concentrations of eugenol (0, 30, 40, 50 and 60 mg/L) did not affect sperm motility in male catfish, but the sperm motility duration in fresh semen under the treatment with 60 mg/L eugenol was lower than that of the control. According to Wagner et al. [21], the percentage of motile spermatozoa is not affected by the type or concentration of anesthetic, but motility duration is inversely proportional to the concentration of eugenol administered. This corroborates our results for frozen semen that showed decreasing motility duration with increasing eugenol concentrations. Moreover, the percentage of motile cells in frozen semen was lower under the two highest eugenol concentrations (50 and 60 mg/L) compared to the control and the 40 mg/L concentration of eugenol. Because motility is the main indicator of sperm quality in fish [25], eugenol concentrations of 50 and 60 mg/ L are not recommended in the reproductive management of R. quelen if the semen will be frozen. In addition to cortisol, anesthetics may also interfere with sperm motility. A study carried out with rainbow trout males observed that direct contact between the tricaine methanesulfonate anesthetic (MS-222) and spermatozoa decreased the duration of sperm motility [21]. In the frozen semen samples, mitochondrial functionality was affected by the 60 mg/L eugenol concentration. It is known that the levels of ATP in fish spermatozoa are only sufficient to maintain motility from a few seconds to a maximum of a few minutes [26]. Thus, the reduction in the number of functional mitochondria can reduce the amount of intracellular ATP available for cellular consumption, resulting in inadequate functioning of the ionic pumps [27]. Therefore, the lower motility observed in the 60 mg/L eugenol treatment is a consequence of the low levels of ATP resulting from the stress generated by the high dose of anesthetic.
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5. Conclusion It is possible to reconcile the well-being of R. quelen breeders with good reproductive indexes because plasma cortisol levels were reduced in the treatments with 40 and 50 mg/L eugenol while the same reproductive performance as the control group was maintained. The seminal quality was compromised by the administration of 60 mg/L eugenol in fresh semen and 50 or 60 mg/L in frozen semen. Therefore, anesthesia with eugenol 40 mg/L is recommended because, despite offering the same reproductive performance as non-anesthetized men, anesthesia has reduced animal stress and, consequently, can avoid possible disorders caused to the immune system of animals by stress in the reproductive management. Preserve the integrity of animal health in order to maintain reproductive performance in future semen collections, in addition to reducing the risk of disease and mortality. Author's contributions Conception and design of the study: MNC, DPS. Acquisition of data: MNC, LFGG. Analysis and interpretation of data: MNC, LSM, DPS. Drafting the article or revising it critically for important intellectual content: MNC, LSM, DPS, LJGB. All authors read and approved the final version to be submitted.
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Acknowledgement
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The study was supported by CAPES Foundation e Brazilian Ministry of Education.
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