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Ovopel and carp pituitary extract for the reproductive induction of Colossoma macropomum males
Theriogenology 98 (2017) 57e61
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Ovopel and carp pituitary extract for the reproductive induction of Colossoma macropomum males tima Ferreira Martins a, Danilo Pedro Streit Jr. b, Janessa Sampaio de Abreu a, Edenilce de Fa ^a-Filho c, Carlos Antonio Lopes de Oliveira d, Ruy Alberto Caetano Corre Nelson Mauricio Lopera-Barrero e, Jayme Aparecido Povh a, c, * , MT, Brazil Animal Science Graduate Program, Federal University of Mato Grosso, Cuiaba Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil Faculty of Veterinary Medicine and Animal Science, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil d , Maringa , PR, Brazil Department of Animal Science, State University of Maringa e Department of Animal Science, State University of Londrina, Londrina, PR, Brazil a
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2017 Received in revised form 28 April 2017 Accepted 29 April 2017 Available online 30 April 2017
The objective of this study was to evaluate Ovopel and carp pituitary extract (CPE) in the reproductive induction of Colossoma macropomum males. Nine treatments were tested in triplicate, totaling 27 experimental units. C. macropomum breeders were subjected to the following treatments: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg; 2.5 mg CPE/kg (traditional protocol); and a control treatment (no hormone). Breeders under hormone treatment produced a larger (P < 0.05) semen volume (2.4 ± 0.7 to 4.2 ± 0.3 mL) compared with the control (0.9 ± 0.4 mL). Sperm concentration did not differ significantly among treatments (7.2 109 ± 1.7 to 10.8 109 ± 2.6 spermatozoa/mL). Total sperm count was higher (P < 0.05) after treatment with 0.3, 0.4, and 0.6 Ovopel pellet/kg (41.6 ± 9.3 to 42.3 ± 10.5 109 spermatozoa) than the other Ovopel treatments (20.0 ± 2.4 to 26.9 ± 8.2 109 spermatozoa) and control (6.6 ± 1.1 109 spermatozoa), but did not differ significantly from CPE (33.7 ± 3.2 109 spermatozoa). Sperm motility was higher (P < 0.05) in the CPE treated, and 0.2, 0.3, and 0.7 Ovopel pellet/kg (88.3 ± 2.9 to 90.0 ± 5.0) breeders when compared with the other treatments (70.0 ± 10.0 to 78.3 ± 5.8), except for the 0.4 pellet/kg (81.7 ± 2.9) treatment, which did not differ significantly from any of the treatments. The motility period of the spermatozoa did not differ significantly among treatments (93.5 ± 15.7 to 120.0 ± 7.6 s). For the sperm morphological analysis, occurrence of normal spermatozoa was similar across the treatments, with three sperm abnormalities (short tail, bent tail, and detached head) differing (P < 0.05) among the treatments. Ovopel efficiently induced reproduction of C. macropomum breeders, with treatment using 0.3 and 0.4 Ovopel pellet/kg and CPE providing the best semen characteristics. © 2017 Elsevier Inc. All rights reserved.
Keywords: Aquaculture Induced reproduction Rheophilic fish Semen characteristics Tambaqui
1. Introduction In the last few years, tambaqui (Colossoma macropomum Cuvier, 1818) has been one of the most largely produced freshwater fish in South America, especially in Brazil, where it is the second-most produced aquatic species (135,858 tons in 2015) [1]. C. macropomum is a high-performance omnivorous species whose meat is widely accepted by consumers [2].
* Corresponding author. Animal Science Graduate Program, Federal University of , MT, Brazil. Mato Grosso, Cuiaba E-mail address: [email protected] (J.A. Povh). http://dx.doi.org/10.1016/j.theriogenology.2017.04.047 0093-691X/© 2017 Elsevier Inc. All rights reserved.
C. macropomum reaches sexual maturity at three years of age and its reproductive period is usually from September to February [2]. Reproduction in this species can be induced upon hormone application [3]; however, semen quality is dependent on the hormonal inducer used for spermiation [4]. Establishing an adequate reproduction protocol is therefore essential for obtaining highquality spermatozoa and is a pre-requisite for the successful breeding of any aquatic organism [5]. Existing reproduction studies with C. macropomum have evaluated spawning inducers [6], semen cryopreservation [7], determination of the insemination dose [8], and semen characteristics [9e11]. However, an appropriate reproduction protocol has not yet been established for C. macropomum. The human chorionic gonadotropin (HCG) hormone is
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inefficient at inducing C. macropomum spawning [6] and successive use of HCG may provoke an immune reaction in the fish [5,12]. Carp pituitary extract (CPE) is the spawning inducer most widely used for C. macropomum and several other rheophilic fish [13]. However, the use of CPE in aquaculture has some important disadvantages such as (i) lack of standardization for the concentration of gonadotropic hormone, which may generate irregular results in induced reproduction [12]; (ii) the possibility of the fish developing an immune reaction to the gonadotropin or other proteins present in CPE [5]; (iii) the possibility of non-gonadotropic hormones present in CPE causing deleterious or negative effects or change the effect on gonadotropin [14]; and (iv) disease transmission from donor to recipient fish [5,12]. The gonadotropin-releasing hormone (GnRH) is considered the most appropriate spawning inducer for fish reproduction, as it acts at the highest level of the brain-pituitary-gonad axis to stimulate the release of endogenous gonadotropins [5]. Moreover, GnRH does not generate an immune reaction in the fish and does not pose a risk of disease transmission, in addition to being able to stimulate other important pituitary hormones for reproduction [12]. At present, more powerful GnRH analogues (GnRHa) are available [15] that have allowed for improvements in fish sperm quality [16], such as those observed in Oncorhynchus mykiss [17] and Lates calcarifer [18]. The use of GnRHa in association with dopamine inhibitors has also been shown to increase reproduction efficiency, especially in species featuring a strong dopaminergic inhibition of gonadotropin release [5]. However, there remains a dearth of information about the effects of GnRH and its analogues in C. macropomum. The use of gonadotropin-releasing hormones associated with dopamine-receptor blockers (pimozide, domperidone, and metoclopramide) can be a lower-cost alternative with improved efficiency in the reproductive induction of fish [3]. Among these combination compounds is Ovopel, a synthetic product developed by the University of Godollo in Hungary. Ovopel is composed of a mammalian GnRH analogue (D-Ala6, Pro9Net-mGnRH) and dopamine antagonist, metoclopramide, at concentrations of 18e20 mg and 8e10 mg, respectively [19]. Ovopel, has been successfully used as a reproductive inducer in carp, at a dosage of 0.4 and 0.6 Ovopel pellet/kg [19]; Leiarius marmoratus, at a dosage of 0.2, 0.4, and 0.6 pellet Ovopel pellet/kg (the observed characteristics were similar to those obtained with CPE) [3]; and Brycon cephalus at a dosage of 0.7 Ovopel pellet/kg (higher semen volume, sperm motility, and motility period compared to the rates for 0.2 and 1.3 Ovopel pellet/ kg) [20]. The objective of the present study was thus to evaluate the effects of Ovopel and CPE on the reproductive induction of C. macropomum males. 2. Material and methods 2.1. Location and animals Twenty-seven four year old C. macropomum breeders, weighing 6.2 ± 1.5 kg and reared on a fish farm in Nova Mutum, MT, CentralWest Brazil (13 51057.200 S and 5611030.200 W), were used in this study. The experiment took place during the month of December, which was within the species' reproductive period. Prior to the species' reproductive period, the fish were placed in a 500 m2 tank and fed extruded feed (32% crude protein, 4% ether extract, 13% mineral matter, 7% crude fiber, 3.5% calcium, 0.9% phosphorus, and 100 mg/kg vitamin C) twice daily. For reproductive induction, the fish were weighed, identified by a microchip, and allocated to 2000 L tanks (males and females in separate tanks) with continuous water flow (10% renewal per day), at a temperature of 29 C.
2.2. Hormonal induction For the reproductive induction of C. macropomum, we evaluated seven treatments with different doses of Ovopel (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg) and one treatment with Carp Pituitary Extract (CPE) at the dose of 2.5 mg CPE/kg (traditional protocol). Each Ovopel pellet contained 18e20 mg GnRHa (D-Ala6, Pro9Net-mGnRH) and 8e10 mg metoclopramide [19]. The control group of fish received saline solution (0.9% NaCl) only, without hormone addition. For all treatments, breeders were induced with a single dose of hormone. Each of the eight hormone (seven with Ovopel and one with CPE) and control (without hormone induction) treatments were conducted in triplicate, totaling 27 experimental units. Ovopel was diluted according to the methodology described by Carneiro and Mikos [21], whereas CPE was diluted as described by Woynarovich and Horv ath [22]. After 280 degree-hours, breeders were anesthetized with eugenol (65 mg/L), as recommended by Roubach et al. [23]. Semen was subsequently harvested from all animals by using individual syringes, as per the methodology of Billard et al. [24], and immediately stored in Styrofoam boxes (16.9 C) for analysis. 2.3. Sperm analyses Semen volume was measured directly in a 5-mL graduated syringe and sperm concentration analyzed using a Neubauer counting chamber, as described by Sanches et al. [25]. One microliter of semen was diluted in 500 mL buffered formalin (1:500), mixed, and transferred to the counting chamber via capillary action. After 5e10 min, all sperm cells on 10 grids were counted in a Solaris Bel trinocular light microscope (200). Total sperm count was obtained by multiplying sperm concentration by semen volume for each male. Sperm motility and motility period were evaluated by diluting 1 mL semen in 100 mL distilled water (temperature 29 C and pH 7.0) on a slide, according to the methodology of Varela Junior et al. [7]. Sperm motility was estimated on a scale of 0e100%. The sperm motility period was determined as the interval from activation to the moment the sperm stopped moving. These analyses were performed using a Solaris Bel trinocular light microscope (400). For the sperm morphological analysis, 5 mL of semen was diluted in 500 mL buffered formalin (4.6%), following the method described by Streit Jr. et al. [26]. Two drops of this solution were then transferred to a slide that was tilted at an angle of 45 to allow the drops to cover the slide. Each sample was stained with 10 mL rose bengal and incubated horizontally at 29 C. The slides were examined under a Solaris Bel trinocular light microscope (400). In three Neubauer chamber squares, two hundred spermatozoa were counted (totaling 600 spermatozoa) as described by Herman et al. [27]. The following morphological abnormalities were evaluated: coiled tail, broken tail, short tail, bent tail, detached head, proximal cytoplasmic droplets, distal cytoplasmic droplets, and macrocephalic sperm. 2.4. Statistical analyses The experiment was conducted as a randomized complete design with nine treatments (seven Ovopel doses, one CPE dose, and one control) in triplicate, totaling 27 experimental units. The variables of semen volume, sperm concentration, total sperm count, sperm motility, and motility period were analyzed by the Generalized Mixed Models methodology with Gamma distribution and reverse link function via SAS PROC GENMOD. The sperm morphological analysis involved a Generalized Mixed Models methodology by Poisson distribution with logarithmic link
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function, via SAS PROC GENMOD. The averages were compared by Tukey's test at 5% probability. 3. Results C. macropomum breeders induced with Ovopel and CPE produced increased semen volumes (P < 0.05) relative to the control treatment (no hormone) (Table 1). In contrast, sperm concentration was not significantly different for the hormone treatments when compared with the control, averaging 7.2 109 ± 1.7 to 10.8 109 ± 2.6 spermatozoa/mL. Total sperm count was higher (P < 0.05) in the treatments with 0.3, 0.4, and 0.6 Ovopel pellet/kg compared to the other Ovopel treatments and control, but did not differ significantly from CPE treatment. Sperm motility was higher (P < 0.05) in the breeders treated with CPE and 0.2, 0.3, and 0.7 Ovopel pellet/kg, compared to those subjected to other treatments, except in breeders treated with 0.4 Ovopel pellet/kg, which did not differ significantly from any other treatment (Table 1). Spermatozoa motility period did not differ significantly across treatments, with mean values of 93.5 ± 15.7 to 120.0 ± 7.6 s. The percentage of normal spermatozoa did not differ significantly among the Ovopel, CPE, and control treatments (Table 2). Likewise, the sperm abnormalities of coiled tail and broken tail did not differ significantly between the hormone and control treatments. However, the short tail sperm abnormality was higher (P < 0.05) after the 0.6 Ovopel pellet/kg treatment than that in the control and after treatment with 0.3, 0.4, and 0.7 Ovopel pellet/kg. The bent tail sperm abnormality occurred with a higher frequency (P < 0.05) after 0.1 Ovopel pellet/kg treatment than that observed in the control and after other treatments, except for treatment with 0.4 Ovopel pellet/kg. The detached head sperm abnormality was more frequent (P < 0.05) after the 0.7 Ovopel pellet/kg treatment than that in the control and after treatment with 0.2, 0.4, and 0.5 Ovopel pellet/kg. Of the 27 breeders evaluated, only three displayed proximal cytoplasmic droplets (following treatment with CPE and 0.2 and 0.3 Ovopel pellet/kg), distal cytoplasmic droplets (Control and following treatment with 0.2 and 0.6 Ovopel pellet/kg), and macrocephalic sperm (following treatment with CPE and 0.2 and 0.3 Ovopel pellet/kg), all with an incidence of 0.2%. 4. Discussion Semen volume in C. macropomum breeders increased in response to all Ovopel and CPE doses, with values similar to the 4.5 mL obtained by Varela Junior et al. [7] in the same species after induction with 1.0 mg CPE/kg. Larger C. macropomum semen volumes (10.2 and 12.6 mL) were obtained in two separate studies by Maria et al. [9,10] following induction with two CPE doses (0.25 and
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2.5 mg/kg, respectively; at an 8-h interval). Differences in the semen volumes observed in these studies may be due to the climate where the experiments were conducted, considering they influence fish reproduction [4], and because of the reproduction protocol adopted since semen volumes can be increased with higher dosage and number of CPE doses. In the case of Ovopel, however, higher doses did not increase semen volume. Other factors can also interfere with the semen volume; e.g., age, weight, and maturity stage of the fish, feeding (quantity and quality), sampling period, and water physical and chemical properties. Sperm concentrations were similar between hormone treated breeders and control breeders, indicating that hormonal induction did not reduce spermatozoa concentrations, despite an increase in semen volumes relative to the control. Sperm concentrations calculated in the present study were similar to those previously observed in C. macropomum by Maria et al. in two separate studies [9,10] and Varela Junior et al. [7], which were 9.1 109, 7.9 109, and 8.7 109 spermatozoa/mL, respectively. Although semen volume and sperm concentration did not differ between hormone treatments, the total sperm count indicated that the breeders treated with 0.3, 0.4, and 0.6 Ovopel pellet/kg produced more spermatozoa in relation to the other treatments, except CPE. Application of these hormone treatments to induce reproduction may therefore increase reproductive efficiency. This is important, given that the adequate insemination dose for C. macropomum is 102,000 sperm per oocyte [8]. Sperm motility was higher when treated with 0.2, 0.3, and 0.7 Ovopel pellet/kg and CPE, indicating improved sperm quality following these treatments. Sperm motility is directly related to fertilization capacity [28]; for this reason, it is desirable that this trait be as high as possible. In separate studies, Maria et al. [9,10] and Varela Junior et al. [7] found similar values to those obtained in the present study for the best C. macropomum hormone treatments: 96.0%, 94.5%, and 95.7%, respectively. The lowest sperm motility was found in the control breeders and in breeders treated with 0.1, 0.5, and 0.6 Ovopel pellet/kg; however, adequate sperm motility values (for different proportions of sperm and oocytes) have not yet been established for good fertilization rates in C. macropomum More in-depth analyses of sperm motility using computer-assisted sperm analysis, as proposed by Gallego et al. [11], may be useful for establishing semen qualitative parameters. The spermatozoa motility period was similar across all treatments, and correlated closely with the 122.6 s found by Varela Junior et al. [7] in C. macropomum. The fertilization rate can be affected when the motility period of the spermatozoa or the insemination dose (number of spermatozoa/oocytes) are not taken into account in reproduction [8]. The percentage of normal spermatozoa was similar between the hormone treated and control breeders. These values correlated well with the 84.2% [9] and 85.0%
Table 1 Semen characteristics of Colossoma macropomum induced with Ovopel and Carp Pituitary Extract (CPE). Treatment Ovopel (pellet/kg) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CPE (mg/kg) 2.5 Control
Sperm concentration (109/mL)
Total sperm count (109)
0.7a 0.7a 0.5a 0.3a 0.9a 0.3a 1.0a
9.7 ± 0.5 8.2 ± 1.6 10.7 ± 2.7 9.8 ± 3.1 7.2 ± 1.7 10.8 ± 2.6 7.5 ± 1.2
23.7 26.3 42.3 41.6 20.0 41.6 26.9
3.3 ± 0.6a 0.9 ± 0.4b
10.4 ± 2.4 7.9 ± 2.0
33.7 ± 3.2ab 6.6 ± 1.1c
Semen volume (mL) 2.4 3.1 3.9 4.2 3.2 3.8 3.5
± ± ± ± ± ± ±
± ± ± ± ± ± ±
5.8b 8.5b 10.5a 12.6a 2.4b 9.3a 8.2b
Values (mean ± SD) superscripted by different letters within the same column are significantly different (P < 0.05).
Sperm motility (%)
Motility period (s)
10.0b 2.9a 5.0a 2.9ab 5.8b 10.4b 5.0a
117.7 ± 14.2 93.5 ± 15.7 120.0 ± 7.6 110.3 ± 44.7 98.3 ± 33.3 117.7 ± 2.5 102.7 ± 46.1
88.3 ± 2.9a 78.3 ± 10.4b
109.7 ± 46.3 109.0 ± 20.5
70.0 88.3 90.0 81.7 78.3 78.3 90.0
± ± ± ± ± ± ±
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Table 2 Sperm morphological parameters of Colossoma macropomum induced with Ovopel and Carp Pituitary Extract (CPE). Treatment
Normal spermatozoa (%)
Sperm abnormality (%) Coiled tail
Ovopel (pellet/kg) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CPE (mg/kg) 2.5 Control
87.5 85.8 87.5 91.0 91.0 88.0 86.8
± ± ± ± ± ± ±
8.4 9.0 8.4 4.6 2.5 2.3 1.0
88.2 ± 4.9 89.3 ± 0.6
5.0 7.0 5.8 4.4 4.8 4.5 6.2
± ± ± ± ± ± ±
1.0 2.0 1.1 2.8 1.2 1.7 2.2
5.2 ± 1.1 4.4 ± 0.8
Broken tail 1.0 1.6 2.1 1.4 1.2 1.7 2.1
± ± ± ± ± ± ±
0.3 0.7 0.9 0.5 0.2 0.6 0.8
1.5 ± 0.4 3.0 ± 0.6
Short tail 1.1 2.0 0.7 0.4 1.0 2.2 0.5
± ± ± ± ± ± ±
0.4ab 0.7ab 0.6b 0.2b 0.4ab 1.9a 0.4b
0.9 ± 0.2ab 0.5 ± 0.4b
Bent tail 4.0 2.0 2.0 2.4 1.2 1.4 1.5
± ± ± ± ± ± ±
3.0a 0.7b 0.9b 0.7ab 0.8b 0.9b 0.7b
1.9 ± 2.1b 1.7 ± 0.6b
Detached head 1.4 1.0 1.5 0.4 0.8 2.0 2.9
± ± ± ± ± ± ±
1.5abc 1.2bc 0.7abc 0.2c 0.6bc 1.7ab 1.1a
1.9 ± 0.9ab 0.9 ± 0.5bc
Values (mean ± SD) superscripted by different letters within the same column are significantly different (P < 0.05).
[10] obtained for C. macropomum by Maria et al. in two separate studies. Hormone treatment did not change the coiled tail and broken tail abnormalities relative to the control. However, short tail, bent tail, and detached head were more frequent at the highest (short tail and detached head: 0.6 and 0.7 pellet/kg, respectively) and lowest (bent tail: 0.1 pellet/kg) dosages of Ovopel, suggesting a possible effect on sperm abnormalities. The occurrence of proximal cytoplasmic droplets, distal cytoplasmic droplets, and macrocephalic sperm was extremely low (0.2%), and only found in three of the 27 breeders assessed. These sperm morphological traits should be further studied and analyzed with respect to their effects on the fertilization rate. Similar sperm morphological results were observed in two separate studies of C. macropomum by Maria et al. [9,10], who also found degenerated head, degenerated midpiece, and microcephalic sperm, in addition to the sperm abnormalities observed in the present study. Moreover, the main sperm abnormality (bent tail: 7.81 and 6.77%) observed by them differed from that observed in the present study. These variations are possibly more closely related to factors other than the spawning inducer. According to Herman et al. [27], the origin of sperm abnormalities may be related to the age of the breeders, their diet, climatic conditions, inbreeding, and disease. GnRHa (without any dopamine inhibitor) is a spawning inducer that has shown good results in some fish species, e.g. Oncorhynchus mykiss, at a rate of 4e16 mg/kg [17], and Lates calcarifer, at a rate of 20 mg/kg [18]. However, Alavi et al. [16] observed that only 25e50% of male Acipenser ruthenus spermiated after the use of GnRHa (25 and 75 mg/kg), whereas 100% of males induced with CPE (4 mg/kg) or Ovopel (3 pellets/kg) spermiated and were found to have increased semen volumes, indicating possible dopamine inhibition that was not manifested in the Ovopel treatment due to the presence of metoclopramide (dopamine inhibitor). Although those authors worked with a higher dose of Ovopel for induced spawning of A. ruthenus (temperate-climate species), tropical fish species have displayed better results with lower doses of Ovopel [3,20]. Information is scarce regarding the efficiency of GnRHa (with or without dopamine inhibitor) on the spermiation of C. macropomum, as well as other tropical fish species. Studies with breeders of other fish species have indicated that Ovopel was efficient in the breeding induction of Indian carps at a dosage of 0.4e0.6 pellet/kg [19], Leiarius marmoratus at a dosage of 0.2e0.6 pellet/kg (similar semen characteristics observed between treatments with Ovopel and CPE) [3], and Brycon cephalus at a dosage of 0.7 pellet/kg (higher volume, motility, and motility period in relation to treatment with 0.3 and 1.3 pellet/kg) [20]. In conclusion, Ovopel was efficient in the induced reproduction of
C. macropomum breeders. Treatment with 0.3 and 0.4 Ovopel pellet/kg and with CPE was found to yield the best semen characteristics. Acknowledgements This study was sponsored by the Foundation for Research Support of the State of Mato Grosso (FAPEMAT). The authors thank the staff of Piscicultura Buriti (Nova Mutum, MT, Brazil) for their invaluable contributions to this study. References [1] IBGE, Brazilian Institute of Geography and Statistics. Aquaculture production (Portuguese). 2017. http://www.sidra.ibge.gov.br/bda/tabela/listabl.asp? c¼3940&z¼t&o¼21 (Accessed 09 January 2017). [2] Lopera-Barrero NM, Ribeiro RP, Povh JA, Mendez LDV, Poveda-Parra AR. Production of aquatic organisms - an overview in Brazil and in the world (Portuguese). Guaíba: Agrolivros; 2011. [3] Araújo JEXS, Streit Jr DP, Ribeiro JSA, Martins EFF, Souza FN, Oliveira CAL, et al. Ovopel and carp pituitary extract as spawning inducers in males of the Amazon catfish Leiarius marmoratus (Gill, 1970). Braz Arch Biol Technol 2014;57:882e6. [4] Mylonas CC, Fostier A, Zanuy F. Broodstock management and hormonal manipulations of fish reproduction. Gen Comp Endocrinol 2010;165:516e34. [5] Mylonas CC, Duncan NJ, Asturiano JF. Hormonal manipulations for the enhancement of sperm production in cultured fish and evaluation of sperm quality. Aquaculture 2016. http://dx.doi.org/10.1016/j.aquaculture.2016.04.021. [6] Chellappa S, Cacho MSRF, Humtingford FA, Beveridge MCM. Observations on induced breeding of the Amazonian fish tambaqui, Colossoma macropomum (Cuvier) using CPE and HCG treatments. Aquac Res 1996;27:91e4. [7] Varela Junior AS, Corcini CD, Gheller SMM, Jardim RD, Lucia Jr T, Streit Jr DP, et al. Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum. Theriogenology 2012;78:244e51. [8] Leite LV, Melo MAP, Oliveira FCE, Pinheiro JPS, Campello CC, Nunes JF, et al. Determination of insemination dose and embryonic development in the artificial fertilization of tambaqui (Colossoma macropomum) (Portuguese). Arq Bras Med Vet Zootec 2013;65:421e9. [9] Maria AN, Azevedo HC, Santos JP, Silva CA, Carneiro PCF. Semen characterization and sperm structure of the Amazon tambaqui Colossoma macropomum. J Appl Ichthyol 2010;26:779e83. [10] Maria AN, Azevedo HC, Santos JP, Carneiro PCF. Hormonal induction and semen characteristics of tambaqui Colossoma macropomum. Zygote 2012;20: 39e43. [11] Gallego V, Cavalcante SS, Fujimoto RY, Carneiro PCF, Azevedo HC, Maria AN. Fish sperm subpopulations: changes after cryopreservation process and relationship with fertilization success in tambaqui (Colossoma macropomum). Theriogenology 2017;87:16e24. [12] Evans DH, Claiborne JB. The physiology of fishes. 3th ed. Boca Raton: Taylor & Francis; 2006. [13] Zaniboni-Filho E, Weingartner M. Induced breeding in migratory fishes (Portuguese). Rev Bras Reprod Anim 2007;30:367e73. [14] Hoar WS, Randall DJ, Donaldson EM. Fish physiology. New York: Academic Press; 1983. [15] Harvey B, Carolsfeld J. Induced breeding in tropical fish culture. Ottawa: IDRC; 1993. [16] Alavi SMH, Hatef A, Mylonas CC, Gela D, Papadaki M, Rodina M, et al. Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants. Fish Physiol Biochem
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Ovopel and carp pituitary extract for the reproductive induction of Colossoma macropomum males tima Ferreira Martins a, Danilo Pedro Streit Jr. b, Janessa Sampaio de Abreu a, Edenilce de Fa ^a-Filho c, Carlos Antonio Lopes de Oliveira d, Ruy Alberto Caetano Corre Nelson Mauricio Lopera-Barrero e, Jayme Aparecido Povh a, c, * , MT, Brazil Animal Science Graduate Program, Federal University of Mato Grosso, Cuiaba Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil Faculty of Veterinary Medicine and Animal Science, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil d , Maringa , PR, Brazil Department of Animal Science, State University of Maringa e Department of Animal Science, State University of Londrina, Londrina, PR, Brazil a
b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2017 Received in revised form 28 April 2017 Accepted 29 April 2017 Available online 30 April 2017
The objective of this study was to evaluate Ovopel and carp pituitary extract (CPE) in the reproductive induction of Colossoma macropomum males. Nine treatments were tested in triplicate, totaling 27 experimental units. C. macropomum breeders were subjected to the following treatments: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg; 2.5 mg CPE/kg (traditional protocol); and a control treatment (no hormone). Breeders under hormone treatment produced a larger (P < 0.05) semen volume (2.4 ± 0.7 to 4.2 ± 0.3 mL) compared with the control (0.9 ± 0.4 mL). Sperm concentration did not differ significantly among treatments (7.2 109 ± 1.7 to 10.8 109 ± 2.6 spermatozoa/mL). Total sperm count was higher (P < 0.05) after treatment with 0.3, 0.4, and 0.6 Ovopel pellet/kg (41.6 ± 9.3 to 42.3 ± 10.5 109 spermatozoa) than the other Ovopel treatments (20.0 ± 2.4 to 26.9 ± 8.2 109 spermatozoa) and control (6.6 ± 1.1 109 spermatozoa), but did not differ significantly from CPE (33.7 ± 3.2 109 spermatozoa). Sperm motility was higher (P < 0.05) in the CPE treated, and 0.2, 0.3, and 0.7 Ovopel pellet/kg (88.3 ± 2.9 to 90.0 ± 5.0) breeders when compared with the other treatments (70.0 ± 10.0 to 78.3 ± 5.8), except for the 0.4 pellet/kg (81.7 ± 2.9) treatment, which did not differ significantly from any of the treatments. The motility period of the spermatozoa did not differ significantly among treatments (93.5 ± 15.7 to 120.0 ± 7.6 s). For the sperm morphological analysis, occurrence of normal spermatozoa was similar across the treatments, with three sperm abnormalities (short tail, bent tail, and detached head) differing (P < 0.05) among the treatments. Ovopel efficiently induced reproduction of C. macropomum breeders, with treatment using 0.3 and 0.4 Ovopel pellet/kg and CPE providing the best semen characteristics. © 2017 Elsevier Inc. All rights reserved.
Keywords: Aquaculture Induced reproduction Rheophilic fish Semen characteristics Tambaqui
1. Introduction In the last few years, tambaqui (Colossoma macropomum Cuvier, 1818) has been one of the most largely produced freshwater fish in South America, especially in Brazil, where it is the second-most produced aquatic species (135,858 tons in 2015) [1]. C. macropomum is a high-performance omnivorous species whose meat is widely accepted by consumers [2].
* Corresponding author. Animal Science Graduate Program, Federal University of , MT, Brazil. Mato Grosso, Cuiaba E-mail address: [email protected] (J.A. Povh). http://dx.doi.org/10.1016/j.theriogenology.2017.04.047 0093-691X/© 2017 Elsevier Inc. All rights reserved.
C. macropomum reaches sexual maturity at three years of age and its reproductive period is usually from September to February [2]. Reproduction in this species can be induced upon hormone application [3]; however, semen quality is dependent on the hormonal inducer used for spermiation [4]. Establishing an adequate reproduction protocol is therefore essential for obtaining highquality spermatozoa and is a pre-requisite for the successful breeding of any aquatic organism [5]. Existing reproduction studies with C. macropomum have evaluated spawning inducers [6], semen cryopreservation [7], determination of the insemination dose [8], and semen characteristics [9e11]. However, an appropriate reproduction protocol has not yet been established for C. macropomum. The human chorionic gonadotropin (HCG) hormone is
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inefficient at inducing C. macropomum spawning [6] and successive use of HCG may provoke an immune reaction in the fish [5,12]. Carp pituitary extract (CPE) is the spawning inducer most widely used for C. macropomum and several other rheophilic fish [13]. However, the use of CPE in aquaculture has some important disadvantages such as (i) lack of standardization for the concentration of gonadotropic hormone, which may generate irregular results in induced reproduction [12]; (ii) the possibility of the fish developing an immune reaction to the gonadotropin or other proteins present in CPE [5]; (iii) the possibility of non-gonadotropic hormones present in CPE causing deleterious or negative effects or change the effect on gonadotropin [14]; and (iv) disease transmission from donor to recipient fish [5,12]. The gonadotropin-releasing hormone (GnRH) is considered the most appropriate spawning inducer for fish reproduction, as it acts at the highest level of the brain-pituitary-gonad axis to stimulate the release of endogenous gonadotropins [5]. Moreover, GnRH does not generate an immune reaction in the fish and does not pose a risk of disease transmission, in addition to being able to stimulate other important pituitary hormones for reproduction [12]. At present, more powerful GnRH analogues (GnRHa) are available [15] that have allowed for improvements in fish sperm quality [16], such as those observed in Oncorhynchus mykiss [17] and Lates calcarifer [18]. The use of GnRHa in association with dopamine inhibitors has also been shown to increase reproduction efficiency, especially in species featuring a strong dopaminergic inhibition of gonadotropin release [5]. However, there remains a dearth of information about the effects of GnRH and its analogues in C. macropomum. The use of gonadotropin-releasing hormones associated with dopamine-receptor blockers (pimozide, domperidone, and metoclopramide) can be a lower-cost alternative with improved efficiency in the reproductive induction of fish [3]. Among these combination compounds is Ovopel, a synthetic product developed by the University of Godollo in Hungary. Ovopel is composed of a mammalian GnRH analogue (D-Ala6, Pro9Net-mGnRH) and dopamine antagonist, metoclopramide, at concentrations of 18e20 mg and 8e10 mg, respectively [19]. Ovopel, has been successfully used as a reproductive inducer in carp, at a dosage of 0.4 and 0.6 Ovopel pellet/kg [19]; Leiarius marmoratus, at a dosage of 0.2, 0.4, and 0.6 pellet Ovopel pellet/kg (the observed characteristics were similar to those obtained with CPE) [3]; and Brycon cephalus at a dosage of 0.7 Ovopel pellet/kg (higher semen volume, sperm motility, and motility period compared to the rates for 0.2 and 1.3 Ovopel pellet/ kg) [20]. The objective of the present study was thus to evaluate the effects of Ovopel and CPE on the reproductive induction of C. macropomum males. 2. Material and methods 2.1. Location and animals Twenty-seven four year old C. macropomum breeders, weighing 6.2 ± 1.5 kg and reared on a fish farm in Nova Mutum, MT, CentralWest Brazil (13 51057.200 S and 5611030.200 W), were used in this study. The experiment took place during the month of December, which was within the species' reproductive period. Prior to the species' reproductive period, the fish were placed in a 500 m2 tank and fed extruded feed (32% crude protein, 4% ether extract, 13% mineral matter, 7% crude fiber, 3.5% calcium, 0.9% phosphorus, and 100 mg/kg vitamin C) twice daily. For reproductive induction, the fish were weighed, identified by a microchip, and allocated to 2000 L tanks (males and females in separate tanks) with continuous water flow (10% renewal per day), at a temperature of 29 C.
2.2. Hormonal induction For the reproductive induction of C. macropomum, we evaluated seven treatments with different doses of Ovopel (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg) and one treatment with Carp Pituitary Extract (CPE) at the dose of 2.5 mg CPE/kg (traditional protocol). Each Ovopel pellet contained 18e20 mg GnRHa (D-Ala6, Pro9Net-mGnRH) and 8e10 mg metoclopramide [19]. The control group of fish received saline solution (0.9% NaCl) only, without hormone addition. For all treatments, breeders were induced with a single dose of hormone. Each of the eight hormone (seven with Ovopel and one with CPE) and control (without hormone induction) treatments were conducted in triplicate, totaling 27 experimental units. Ovopel was diluted according to the methodology described by Carneiro and Mikos [21], whereas CPE was diluted as described by Woynarovich and Horv ath [22]. After 280 degree-hours, breeders were anesthetized with eugenol (65 mg/L), as recommended by Roubach et al. [23]. Semen was subsequently harvested from all animals by using individual syringes, as per the methodology of Billard et al. [24], and immediately stored in Styrofoam boxes (16.9 C) for analysis. 2.3. Sperm analyses Semen volume was measured directly in a 5-mL graduated syringe and sperm concentration analyzed using a Neubauer counting chamber, as described by Sanches et al. [25]. One microliter of semen was diluted in 500 mL buffered formalin (1:500), mixed, and transferred to the counting chamber via capillary action. After 5e10 min, all sperm cells on 10 grids were counted in a Solaris Bel trinocular light microscope (200). Total sperm count was obtained by multiplying sperm concentration by semen volume for each male. Sperm motility and motility period were evaluated by diluting 1 mL semen in 100 mL distilled water (temperature 29 C and pH 7.0) on a slide, according to the methodology of Varela Junior et al. [7]. Sperm motility was estimated on a scale of 0e100%. The sperm motility period was determined as the interval from activation to the moment the sperm stopped moving. These analyses were performed using a Solaris Bel trinocular light microscope (400). For the sperm morphological analysis, 5 mL of semen was diluted in 500 mL buffered formalin (4.6%), following the method described by Streit Jr. et al. [26]. Two drops of this solution were then transferred to a slide that was tilted at an angle of 45 to allow the drops to cover the slide. Each sample was stained with 10 mL rose bengal and incubated horizontally at 29 C. The slides were examined under a Solaris Bel trinocular light microscope (400). In three Neubauer chamber squares, two hundred spermatozoa were counted (totaling 600 spermatozoa) as described by Herman et al. [27]. The following morphological abnormalities were evaluated: coiled tail, broken tail, short tail, bent tail, detached head, proximal cytoplasmic droplets, distal cytoplasmic droplets, and macrocephalic sperm. 2.4. Statistical analyses The experiment was conducted as a randomized complete design with nine treatments (seven Ovopel doses, one CPE dose, and one control) in triplicate, totaling 27 experimental units. The variables of semen volume, sperm concentration, total sperm count, sperm motility, and motility period were analyzed by the Generalized Mixed Models methodology with Gamma distribution and reverse link function via SAS PROC GENMOD. The sperm morphological analysis involved a Generalized Mixed Models methodology by Poisson distribution with logarithmic link
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function, via SAS PROC GENMOD. The averages were compared by Tukey's test at 5% probability. 3. Results C. macropomum breeders induced with Ovopel and CPE produced increased semen volumes (P < 0.05) relative to the control treatment (no hormone) (Table 1). In contrast, sperm concentration was not significantly different for the hormone treatments when compared with the control, averaging 7.2 109 ± 1.7 to 10.8 109 ± 2.6 spermatozoa/mL. Total sperm count was higher (P < 0.05) in the treatments with 0.3, 0.4, and 0.6 Ovopel pellet/kg compared to the other Ovopel treatments and control, but did not differ significantly from CPE treatment. Sperm motility was higher (P < 0.05) in the breeders treated with CPE and 0.2, 0.3, and 0.7 Ovopel pellet/kg, compared to those subjected to other treatments, except in breeders treated with 0.4 Ovopel pellet/kg, which did not differ significantly from any other treatment (Table 1). Spermatozoa motility period did not differ significantly across treatments, with mean values of 93.5 ± 15.7 to 120.0 ± 7.6 s. The percentage of normal spermatozoa did not differ significantly among the Ovopel, CPE, and control treatments (Table 2). Likewise, the sperm abnormalities of coiled tail and broken tail did not differ significantly between the hormone and control treatments. However, the short tail sperm abnormality was higher (P < 0.05) after the 0.6 Ovopel pellet/kg treatment than that in the control and after treatment with 0.3, 0.4, and 0.7 Ovopel pellet/kg. The bent tail sperm abnormality occurred with a higher frequency (P < 0.05) after 0.1 Ovopel pellet/kg treatment than that observed in the control and after other treatments, except for treatment with 0.4 Ovopel pellet/kg. The detached head sperm abnormality was more frequent (P < 0.05) after the 0.7 Ovopel pellet/kg treatment than that in the control and after treatment with 0.2, 0.4, and 0.5 Ovopel pellet/kg. Of the 27 breeders evaluated, only three displayed proximal cytoplasmic droplets (following treatment with CPE and 0.2 and 0.3 Ovopel pellet/kg), distal cytoplasmic droplets (Control and following treatment with 0.2 and 0.6 Ovopel pellet/kg), and macrocephalic sperm (following treatment with CPE and 0.2 and 0.3 Ovopel pellet/kg), all with an incidence of 0.2%. 4. Discussion Semen volume in C. macropomum breeders increased in response to all Ovopel and CPE doses, with values similar to the 4.5 mL obtained by Varela Junior et al. [7] in the same species after induction with 1.0 mg CPE/kg. Larger C. macropomum semen volumes (10.2 and 12.6 mL) were obtained in two separate studies by Maria et al. [9,10] following induction with two CPE doses (0.25 and
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2.5 mg/kg, respectively; at an 8-h interval). Differences in the semen volumes observed in these studies may be due to the climate where the experiments were conducted, considering they influence fish reproduction [4], and because of the reproduction protocol adopted since semen volumes can be increased with higher dosage and number of CPE doses. In the case of Ovopel, however, higher doses did not increase semen volume. Other factors can also interfere with the semen volume; e.g., age, weight, and maturity stage of the fish, feeding (quantity and quality), sampling period, and water physical and chemical properties. Sperm concentrations were similar between hormone treated breeders and control breeders, indicating that hormonal induction did not reduce spermatozoa concentrations, despite an increase in semen volumes relative to the control. Sperm concentrations calculated in the present study were similar to those previously observed in C. macropomum by Maria et al. in two separate studies [9,10] and Varela Junior et al. [7], which were 9.1 109, 7.9 109, and 8.7 109 spermatozoa/mL, respectively. Although semen volume and sperm concentration did not differ between hormone treatments, the total sperm count indicated that the breeders treated with 0.3, 0.4, and 0.6 Ovopel pellet/kg produced more spermatozoa in relation to the other treatments, except CPE. Application of these hormone treatments to induce reproduction may therefore increase reproductive efficiency. This is important, given that the adequate insemination dose for C. macropomum is 102,000 sperm per oocyte [8]. Sperm motility was higher when treated with 0.2, 0.3, and 0.7 Ovopel pellet/kg and CPE, indicating improved sperm quality following these treatments. Sperm motility is directly related to fertilization capacity [28]; for this reason, it is desirable that this trait be as high as possible. In separate studies, Maria et al. [9,10] and Varela Junior et al. [7] found similar values to those obtained in the present study for the best C. macropomum hormone treatments: 96.0%, 94.5%, and 95.7%, respectively. The lowest sperm motility was found in the control breeders and in breeders treated with 0.1, 0.5, and 0.6 Ovopel pellet/kg; however, adequate sperm motility values (for different proportions of sperm and oocytes) have not yet been established for good fertilization rates in C. macropomum More in-depth analyses of sperm motility using computer-assisted sperm analysis, as proposed by Gallego et al. [11], may be useful for establishing semen qualitative parameters. The spermatozoa motility period was similar across all treatments, and correlated closely with the 122.6 s found by Varela Junior et al. [7] in C. macropomum. The fertilization rate can be affected when the motility period of the spermatozoa or the insemination dose (number of spermatozoa/oocytes) are not taken into account in reproduction [8]. The percentage of normal spermatozoa was similar between the hormone treated and control breeders. These values correlated well with the 84.2% [9] and 85.0%
Table 1 Semen characteristics of Colossoma macropomum induced with Ovopel and Carp Pituitary Extract (CPE). Treatment Ovopel (pellet/kg) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CPE (mg/kg) 2.5 Control
Sperm concentration (109/mL)
Total sperm count (109)
0.7a 0.7a 0.5a 0.3a 0.9a 0.3a 1.0a
9.7 ± 0.5 8.2 ± 1.6 10.7 ± 2.7 9.8 ± 3.1 7.2 ± 1.7 10.8 ± 2.6 7.5 ± 1.2
23.7 26.3 42.3 41.6 20.0 41.6 26.9
3.3 ± 0.6a 0.9 ± 0.4b
10.4 ± 2.4 7.9 ± 2.0
33.7 ± 3.2ab 6.6 ± 1.1c
Semen volume (mL) 2.4 3.1 3.9 4.2 3.2 3.8 3.5
± ± ± ± ± ± ±
± ± ± ± ± ± ±
5.8b 8.5b 10.5a 12.6a 2.4b 9.3a 8.2b
Values (mean ± SD) superscripted by different letters within the same column are significantly different (P < 0.05).
Sperm motility (%)
Motility period (s)
10.0b 2.9a 5.0a 2.9ab 5.8b 10.4b 5.0a
117.7 ± 14.2 93.5 ± 15.7 120.0 ± 7.6 110.3 ± 44.7 98.3 ± 33.3 117.7 ± 2.5 102.7 ± 46.1
88.3 ± 2.9a 78.3 ± 10.4b
109.7 ± 46.3 109.0 ± 20.5
70.0 88.3 90.0 81.7 78.3 78.3 90.0
± ± ± ± ± ± ±
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Table 2 Sperm morphological parameters of Colossoma macropomum induced with Ovopel and Carp Pituitary Extract (CPE). Treatment
Normal spermatozoa (%)
Sperm abnormality (%) Coiled tail
Ovopel (pellet/kg) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CPE (mg/kg) 2.5 Control
87.5 85.8 87.5 91.0 91.0 88.0 86.8
± ± ± ± ± ± ±
8.4 9.0 8.4 4.6 2.5 2.3 1.0
88.2 ± 4.9 89.3 ± 0.6
5.0 7.0 5.8 4.4 4.8 4.5 6.2
± ± ± ± ± ± ±
1.0 2.0 1.1 2.8 1.2 1.7 2.2
5.2 ± 1.1 4.4 ± 0.8
Broken tail 1.0 1.6 2.1 1.4 1.2 1.7 2.1
± ± ± ± ± ± ±
0.3 0.7 0.9 0.5 0.2 0.6 0.8
1.5 ± 0.4 3.0 ± 0.6
Short tail 1.1 2.0 0.7 0.4 1.0 2.2 0.5
± ± ± ± ± ± ±
0.4ab 0.7ab 0.6b 0.2b 0.4ab 1.9a 0.4b
0.9 ± 0.2ab 0.5 ± 0.4b
Bent tail 4.0 2.0 2.0 2.4 1.2 1.4 1.5
± ± ± ± ± ± ±
3.0a 0.7b 0.9b 0.7ab 0.8b 0.9b 0.7b
1.9 ± 2.1b 1.7 ± 0.6b
Detached head 1.4 1.0 1.5 0.4 0.8 2.0 2.9
± ± ± ± ± ± ±
1.5abc 1.2bc 0.7abc 0.2c 0.6bc 1.7ab 1.1a
1.9 ± 0.9ab 0.9 ± 0.5bc
Values (mean ± SD) superscripted by different letters within the same column are significantly different (P < 0.05).
[10] obtained for C. macropomum by Maria et al. in two separate studies. Hormone treatment did not change the coiled tail and broken tail abnormalities relative to the control. However, short tail, bent tail, and detached head were more frequent at the highest (short tail and detached head: 0.6 and 0.7 pellet/kg, respectively) and lowest (bent tail: 0.1 pellet/kg) dosages of Ovopel, suggesting a possible effect on sperm abnormalities. The occurrence of proximal cytoplasmic droplets, distal cytoplasmic droplets, and macrocephalic sperm was extremely low (0.2%), and only found in three of the 27 breeders assessed. These sperm morphological traits should be further studied and analyzed with respect to their effects on the fertilization rate. Similar sperm morphological results were observed in two separate studies of C. macropomum by Maria et al. [9,10], who also found degenerated head, degenerated midpiece, and microcephalic sperm, in addition to the sperm abnormalities observed in the present study. Moreover, the main sperm abnormality (bent tail: 7.81 and 6.77%) observed by them differed from that observed in the present study. These variations are possibly more closely related to factors other than the spawning inducer. According to Herman et al. [27], the origin of sperm abnormalities may be related to the age of the breeders, their diet, climatic conditions, inbreeding, and disease. GnRHa (without any dopamine inhibitor) is a spawning inducer that has shown good results in some fish species, e.g. Oncorhynchus mykiss, at a rate of 4e16 mg/kg [17], and Lates calcarifer, at a rate of 20 mg/kg [18]. However, Alavi et al. [16] observed that only 25e50% of male Acipenser ruthenus spermiated after the use of GnRHa (25 and 75 mg/kg), whereas 100% of males induced with CPE (4 mg/kg) or Ovopel (3 pellets/kg) spermiated and were found to have increased semen volumes, indicating possible dopamine inhibition that was not manifested in the Ovopel treatment due to the presence of metoclopramide (dopamine inhibitor). Although those authors worked with a higher dose of Ovopel for induced spawning of A. ruthenus (temperate-climate species), tropical fish species have displayed better results with lower doses of Ovopel [3,20]. Information is scarce regarding the efficiency of GnRHa (with or without dopamine inhibitor) on the spermiation of C. macropomum, as well as other tropical fish species. Studies with breeders of other fish species have indicated that Ovopel was efficient in the breeding induction of Indian carps at a dosage of 0.4e0.6 pellet/kg [19], Leiarius marmoratus at a dosage of 0.2e0.6 pellet/kg (similar semen characteristics observed between treatments with Ovopel and CPE) [3], and Brycon cephalus at a dosage of 0.7 pellet/kg (higher volume, motility, and motility period in relation to treatment with 0.3 and 1.3 pellet/kg) [20]. In conclusion, Ovopel was efficient in the induced reproduction of
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