Introduction
The purpose of artificial insemination in fish is to successfully fertilize the largest number of fertile oocytes with the minimum number of males, bearing in mind the need to maintain a satisfactory gender balance (Billard et al., Reference Billard, Cosson, Perchec and Linhart1995). Therefore, there is a need to establish an inseminating dose (or sperm ratio per oocyte) for each species that can be defined as the amount of sperm needed to fertilize an oocyte (Chereguini et al., Reference Chereguini, De La Banda, Rasines and Fernandez1999). This dose can improve the use of breeders by increasing their reproductive efficiency through artificial reproduction, as it allows rational management of semen, reducing the number of animals and their maintenance cost.
Use of small or excessive sperm concentrations results in lower fertilization rates (Levanduski and Cloud, Reference Levanduski and Cloud1988; Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti, Veríssimo-Silveira and Senhorini2006; Shimoda et al., Reference Shimoda, Andrade, Vidal Júnior, Godinho and Yasui2007). The insemination dose for fish, unlike terrestrial animals, is relatively higher and varies between species (Beirão et al., Reference Beirão, Boulais, Gallego, O’Brien, Peixoto, Robeck and Cabrita2019). In addition, variations in sperm to oocyte ratio may occur based on oocyte characteristics or semen quality (Suquet et al., Reference Suquet, Billard, Cosson, Normant and Fauvel1995), which directly interfere with fertilization potential (Rurangwa et al., Reference Rurangwa, Kime, Ollevier and Nash2004).
Brycon amazonicus, know as matrinxã, is a neotropical fish whose reproductive biology is described, taking into account that many aspects are species specific. Reproduction in B. amazonicus does not always follow a standardized hormone induction protocol, with frequent failures in spawning, fertilization and hatching rates within and between reproductive periods (Romagosa et al., Reference Romagosa, Narahara, Borella and Fenerich-Verani2001; Pardo-Carrasco et al., Reference Pardo-Carrasco, Suarez-Mahecha, Muñoz-Lara, Arias-Castellanos and Hernando2002; Arias, Reference Arias2006; Hainfellner et al., Reference Hainfellner, De Souza, Muñoz, Freitas and Batlouni2012). Females have high relative fecundity (Arias, Reference Arias2006) and the same female can spawn at least twice in the same reproductive cycle when given hormonal therapies (Honczaryk and Inoue, Reference Honczaryk and Inoue2009). However, their large-scale production is limited by their intense cannibalistic behaviour in the early stages of development, with losses of up to 99% (Bernardino et al., Reference Bernardino, Senhorini, Fontes, Bock and Mendonça1993) and a reproductive seasonality that restricts the availability of juveniles to a certain period of the year (Zaniboni-Filho et al., Reference Zaniboni-Filho, Carvalho, Villacorta-Correa and Rezende1988). Therefore, it is important to maximize the use of available gametes to produce a greater number of viable embryos, especially considering that B. amazonicus is an aggressive fish and highly sensitive to the entire process of artificial reproduction, and which justifies avoidance of gamete wastage and unnecessary use of breeders.
There have been studies for this species on its reproductive biology (Zaniboni-Filho et al., Reference Zaniboni-Filho, Carvalho, Villacorta-Correa and Rezende1988; Romagosa et al., Reference Romagosa, Narahara, Borella, Parreira and Fenerich-Verani1999), covering artificial reproduction (Bernardino et al., Reference Bernardino, Senhorini, Fontes, Bock and Mendonça1993; Romagosa et al., Reference Romagosa, Narahara, Borella and Fenerich-Verani2001), cryoprotectants, cryopreservation and conservation of gametes (Cruz-Casallas et al., Reference Cruz-Casallas, Medina-Robles and Velasco-Santamaría2006; Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti, Veríssimo-Silveira and Senhorini2006; Velasco-Santamaría et al., Reference Velasco-Santamaría, Medina-Robles and Cruz-Casallas2006; Bashiyo-Silva et al., Reference Bashiyo-Silva, da Silva Costa, de Castro Ribeiro, Senhorini, Veríssimo-Silveira and Ninhaus-Silveira2015). However, the minimum insemination dose for the species has not yet been reported and unspecified volumes are being used for artificial fertilization, without any concern for better use of gametes. We highlight the importance of this work not only for artificial reproduction of the species, but also for tests in applied biotechnology, as manipulation of gametes, control of fertilization, polyploidy and androgenesis. The objective of this work was to determine the insemination dose for artificial fertilization of B. amazonicus oocytes, to attain a higher reproductive efficiency through the rational use of breeders.
Materials and methods
Broodstock maintenance and hormonal induction
We used 4-year-old wild specimens that had been maintained since 6 months of age at the Experimental Farm Aquaculture Station of the Federal University of Amazonas (UFAM) Manaus, Amazonas, Brazil (2°38′56.1′′S, 60°03′14.7′′W). They were placed in earthen ponds (550 m2, ca. 0.1 fish m²) and fed twice daily to their apparent satiation with a commercial fish feed that contained 32% crude protein.
Four breeders were selected in December 2014. Two males that emitted semen under mild abdominal pressure and two females with hyperemic genital papilla, soft, bulging abdomens (Woynarovich and Horváth, Reference Woynarovich and Horváth1983). They had >50% oocytes that presented eccentric germinal vesicles (Arias, Reference Arias2006), which were observed after ovarian biopsy by insertion of a flexible catheter into the genital pore, followed by gentle aspiration and immersion of oocytes in Serra’s fixative solution for a few minutes (Stoeckel, Reference Stoeckel2000). The animals were separated by sex in two circular fibreglass tanks with a capacity of 3000 L each. Water was constantly renewed and aerated, and an average temperature of 27.7 ± 1.0°C and pH 7.9 ± 0.2 was maintained.
Females (1.66 ± 0.16 kg) received 5.5 mg kg–1 carp pituitary extract divided into three doses and males (1.31 ± 0.69 kg) received a single dose of 2.0 mg kg–1 carp pituitary extract (Zaniboni-Filho and Barbosa, Reference Zaniboni-Filho and Barbosa1996), as intramuscular injections in the base of the dorsal fin. The animals had never been previously subjected to hormone therapy.
Gamete collection and evaluation
Each specimen was removed from the tank, and placed in a container containing 50 mg L–1 eugenol solution (Vidal et al., Reference Vidal, Furuya, Graciano, Schamber, da Silva, dos Santos and de Souza2007) to reduce the stress associated with this procedure and to collect the gametes.
Semen were collected 12 h after hormonal injection in ml-graduated tubes (16.5 ± 7.0 ml). Sperm motility was evaluated subjectively (94.16 ± 2.5% of mobile sperm and 33.66 ± 2.9 s of post-activation motility) for each sample in two steps as described by Billard and Cosson (Reference Billard and Cosson1992). The first step consisted of diluting the semen in a 1% NaCl solution (1:40, semen:diluter) and, in the second step, the previously diluted sample was activated with distilled water (1:20, diluted sample:water), on a microscope slide coated with 0.05% bovine serum albumin to prevent sperm from adhering to the slide, under an optical microscope, ×40 magnification objective. Subsequently, a semen pool was constituted. A Neubauer haematimetric chamber was used to determine the pool sperm concentration (1.63 × 1010 sperm ml–1), as recommended by the Brazilian College of Animal Reproduction (Colégio Brasileiro de Reprodução Animal, 2013).
Oocytes were released 5 h after the last hormone dose (ca. 140 accumulated thermal units) into plastic containers.
Insemination doses, artificial fertilization and fertilization rate
The insemination doses were prepared according to the methodology used for Misgurnus anguillicaudatus (Yasui et al., Reference Yasui, Arias-Rodriguez, Fujimoto and Arai2009), with some dosage modifications for B. amazonicus, based on two previous experiments. Sperm were diluted in 1% NaCl solution to attain concentrations of 0.75 × 107, 1.51 × 107, 3.02 × 107, 4.53 × 107 and 6.04 × 107 spermatozoa ml–1. This solution keeps the sperm still and viable until insemination. We pipetted 150 µl of the oocytes (755.2 ± 19.6 oocytes) into 18 glass beakers (50 ml) using a clipped pipette tip. Each oocyte mass was immediately inseminated with 50 µl of the previously diluted sperm solution. After insemination, gametes were activated with 800 µl distilled water, homogenized for 1 min and then hydrated with 20 ml of incubator water (28.9°C). These dilutions resulted in insemination doses of 10,000, 20,000, 40,000, 60,000 and 80,000 spermatozoa oocyte–1 ml–1 (three replicates for each dose were made). A mass of oocytes mixed with only 50 µl of 1% NaCl solution was maintained as a control group.
The fertilized oocytes were transferred to 18 cylindrical incubators (10 × 12 cm), with screened bottoms, properly identified and packed in a 3000 L fibreglass water tank with constant water and aeration renewal, at a temperature of 28.9 ± 1.0°C and a pH of 7.8 ± 0.1. Fertilization rates were calculated relative to the total amount of eggs ca. 6 h after artificial insemination, under a stereomicroscope, when the eggs were in the blastopore closure phase (Nakaghi et al., Reference Nakaghi, Neumann, Faustino, Mendes and de Braga2014). In this phase of development, eggs that were whitish or broken were considered unfertilized or dead, and eggs that were translucent with perfect embryos inside the chorion were considered fertilized.
Statistical analysis
The regression equation that fitted the data for the variation in fertilization rates was estimated as a function of spermatozoa count per oocyte by applying the segmented regression model ‘Linear Response Plateau’ in the R statistical program. The appropriate ratio of spermatozoa to oocyte was calculated using this equation. Data were first tested for normality (Shapiro–Wilk test). Data are presented as the mean ± standard error. A P-value < 0.05 was considered to be significant.
Results
The estimated regression equation was Ⓨ = 14.3415 + 0.0007836X (P < 0.05), where Ⓨ = fertilization rate and X = spermatozoa per oocyte (P < 0.05; R2 = 0.827). The fertilization rate increased linearly until it reached the maximum rate of 63.34%, remaining constant and starting to plateau at a ratio of 62,524 spermatozoa oocyte–1 ml–1 (Fig. 1). This starting point of the plateau represents the minimum ratio required to achieve maximum fertility. Although the insemination dose increased after this point, the fertilization rate remained constant on a plateau of 63.34%.
Values for oocyte fertilization rates of Brycon amazonicus as a function of spermatozoa oocyte–1 ml–1.
Discussion
Knowledge of the appropriate inseminating dose is important for the development of semen and/or oocyte cryopreservation programmes, intended either for the conservation of genetic biodiversity of the species or even for breeding programmes in farms, in addition to the possibility of gamete optimization in aquaculture stations (Denniston et al., Reference Denniston, Michelet, Godke, Tiersch and Mazik2000).
The quality of gametes observed in this study corroborates the results of other authors (Cruz-Casallas et al., Reference Cruz-Casallas, Medina-Robles and Velasco-Santamaría2006, Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti, Veríssimo-Silveira and Senhorini2006) and fertilization rates were similar to those reported by other authors (Romagosa et al., Reference Romagosa, Narahara, Borella and Fenerich-Verani2001; Arias Reference Arias2006). Therefore, the animals used were fit and the gamete quality parameters can be considered satisfactory for the artificial reproduction process.
According to Rurangwa et al. (Reference Rurangwa, Kime, Ollevier and Nash2004), fertilization rates are related to sperm motility and may be influenced by the inseminating dose used, but it is poorly reported for fish species and presents great variation between species, which can probably be related to the characteristics of gametes, such as oocyte diameter, speed of spermatozoa, uptime and the distance travelled by the spermatozoa to reach the micropyle of the oocyte (Suquet et al., Reference Suquet, Billard, Cosson, Normant and Fauvel1995; Lahnsteiner Reference Lahnsteiner2000; Gage et al., Reference Gage, Macfarlane, Yeates, Ward, Searle and Parker2004; Bombardelli et al., Reference Bombardelli, Mörschbächer, Campagnolo, Sanches and Syperreck2006).
Suquet et al. (Reference Suquet, Billard, Cosson, Normant and Fauvel1995) found that the insemination dose for Scophthalmus maximus was 6000 spermatozoa oocyte–1 with 87.3% fertilization. These authors concluded that this species needed a small amount of semen because of the size of its oocytes (0.91–1.2 mm) and also the semen had a long activation time. B. amazonicus oocytes are small (1.11–1.30 mm in diameter; Nakaghi et al., Reference Nakaghi, Neumann, Faustino, Mendes and de Braga2014), as reported for another species of the same genus, B. insignis (1.3 mm; Shimoda et al., Reference Shimoda, Andrade, Vidal Júnior, Godinho and Yasui2007). However, the insemination dose for fertilization of B. insignis oocytes (314,481 spermatozoa oocyte–1) was much higher than that found in our study, which suggested that other factors may influence interspecifically, such as contact time between gametes and the fertilization protocol used (Rurangwa et al., Reference Rurangwa, Kime, Ollevier and Nash2004).
According to Cruz-Casallas and Velasco-Santamaría (Reference Cruz-Casallas and Velasco-Santamaría2004) a proportion of 50,000 fresh sperm or 75,000 thawed sperm per oocyte is sufficient to obtain maximum fertilization percentages in artificial insemination of Brycon siebenthalae. In this study, the fertilization results showed effect pertinent to the treatments, presenting positive linear behaviour up to the proportion of 62,524 spermatozoa oocyte–1. The fertilization rates reached a plateau at this ratio, and had no effect at the higher doses, remaining constant thereafter.
For Brycon insignis (Shimoda et al., Reference Shimoda, Andrade, Vidal Júnior, Godinho and Yasui2007), the fertilization rate increased to 87.8% and remained on this plateau at a ratio of 314,481 spermatozoa oocyte–1. Other authors have also found this plateau from a given ratio of spermatozoa to oocyte on the fertilization rate of some species, such as Astyanax altiparanae (75.4%, 2390 spermatozoa oocyte–1, Pereira-Santos et al., Reference Pereira-Santos, Shimoda, de Andrade, Silva, Fujimoto, Senhorini, Yasui and Nakaghi2017), Colossoma macropomum (84%, 102,486 spermatozoa oocyte–1; Leite et al., Reference Leite, Melo, Oliveira, Pinheiro, Campello, Nunes and Salmito-Vanderley2013), Misgurnus anguillicaudatus (62.84%, 684.42 spermatozoa oocyte–1; Yasui et al., Reference Yasui, Arias-Rodriguez, Fujimoto and Arai2009) Salminus brasiliensis (57.1%, 30,722 spermatozoa oocyte–1; Sanches et al., Reference Sanches, Bombardelli, Baggio and Souza2009) Rhamdia quelen (86.68%, 89,472 spermatozoa oocyte–1; Bombardelli et al., Reference Bombardelli, Mörschbächer, Campagnolo, Sanches and Syperreck2006).
Determination of insemination dose is important for evaluation of semen cryopreservation procedures and better economical use of large-scale artificial reproduction procedures, as lower insemination doses may trigger lower fertilization rates, and excess sperm reflects the inefficient use breeders. Therefore, fertilization tests, if performed with excessive amounts of semen, may not provide suitable conditions to identify the best treatment to be used, as eventual differences between methodologies may be attenuated by increase in the ratio of sperm to oocyte (Shimoda et al., Reference Shimoda, Andrade, Vidal Júnior, Godinho and Yasui2007). In addition, sperm economy will multiply the reproductive potential of higher performing or genetic breeding stock and, as a result, will benefit semen banks for species conservation and will also enable higher profitability for breeding companies in aquaculture.
Considering that the sperm concentration of B. amazonicus is ca. 1.4 × 109 ml–1 (Cruz-Casallas et al., Reference Cruz-Casallas, Medina-Robles and Velasco-Santamaría2006, Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti, Veríssimo-Silveira and Senhorini2006) and the number of oocytes per gram is estimated at 1500 cells and, based on the results found, it can be recommended that 7 ml of semen per 100 g of oocytes would be sufficient for good fertilization rates in this species. However, the use of a higher proportion of sperm per oocyte should be considered, if the semen has been cryopreserved, and to evaluate sperm concentration and quality under favourable incubation conditions. The sperm to oocyte ratio should only be considered as a reference value and not as an absolute or unalterable value for fertilization trials (Beirão et al., Reference Beirão, Boulais, Gallego, O’Brien, Peixoto, Robeck and Cabrita2019).
The insemination dose for fertilization of B. amazonicus oocytes will be useful for optimizing the use of breeding stock in the artificial propagation process, even more considering that this species is an extremely aggressive fish during reproductive management. This study will be the basis for conducting future studies of induced reproduction and gamete manipulation of fish.
In conclusion, the highest oocyte fertilization rates for B. amazonicus may be obtained with the use of a proportion of 62,524 spermatozoa oocyte–1 ml–1.
Acknowledgements
The authors thank George Shigueki Yasui (University of São Paulo – USP) for his scientific and methodological contributions. We are also grateful to Eduardo Antônio Sanches and Rafael Vilhena Reis Neto (São Paulo State University – UNESP) for contribution in data analysis.
Data availability statement
Research data are not shared.
Financial support
This study was funded by Fundação de Amparo à Pesquisa no Amazonas – FAPEAM (project grant no. 1271/2012) and Financiadora de Estudos e Projetos – FINEP/Projeto Desenvolvimento da Aquicultura e Recursos Pesqueiros da Amazônia – DARPA (protocol no. 01.09.0472.00).
Conflict of interest
The authors report no conflict of interest
Ethical approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Ethics Committee on Animal Use ‘CEUA’ of Federal University of Amazonas, protocol no. 019/2014). The Brazilian College of Animal Experimentation (COBEA) guidelines for the care and use of laboratory animals were followed.
