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Methods of cryopreservation of Tambaqui semen, Colossoma macropomum A.S. Varela Junior a,∗ , K.L. Goularte c , J.P. Alves a , F.A. Pereira a , E.F. Silva a , T.F. Cardoso a , R.D. Jardim a , D.P. Streit Jr b , C.D. Corcini c a b c
Compared Animal Reproduction – RAC, Institute of Biological Sciences, Federal University of Rio Grande, Rio Grande, RS, Brazil Aquam, Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil ReproPel, Faculty of Veterinary Medicine, Federal University of Pelotas, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 26 March 2015 Accepted 31 March 2015 Available online xxx Keywords: Freezing Sperm Conservation Methods, Fish
a b s t r a c t This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2 L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2 L, amounting to 12 treatments (four DMSO concentrations × three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P > 0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P < 0.05) as well as for sperm motility and latency time (P < 0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO. © 2015 Published by Elsevier B.V.
1. Introduction Tambaqui – Colossoma macropomum (Cuvier, 1818) is a native migratory species in the Amazon basin and is considered the second largest scale fish of the Amazon and Solimões rivers (Goulding and Carvalho, 1982). The fish inhabit the Madeira River located in the state of Rondonia,
∗ Corresponding author. Tel.: +55 53 32935186; fax: +55 53 32336633. E-mail address: [email protected] (A.S. Varela Junior).
Brazil. In this river, the implementation of a hydroelectric dam (Lopes et al., 2009) may hinder the migration of this species by changing the lotic to lentic system, causing the formation of populations with a high degree of kinship and restricting gene flow, which may result in a decrease of the genetic heterogeneity (Odum, 1971; Braga et al., 2005; Carpio, 2006). In these cases, the preservation of the heterogeneity of the genetic heritage of native species through semen cryopreservation, for example, becomes important to avoid negative impacts on fish populations (Sirol and Britto, 2006).
http://dx.doi.org/10.1016/j.anireprosci.2015.03.017 0378-4320/© 2015 Published by Elsevier B.V.
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C. macropomum is the native Brazilian species commercially produced, with positive results with use of different types of intensive farming (Chellapa et al., 1995; Valenti et al., 2000), and this specie currently participates in a breeding program at the national level (Maria et al., 2011). In commercial production systems, genetic variability is important, because it enables the improvement of production traits (Suquet et al., 2000). Thus, seminal cryopreservation while enhancing the diffusion and improvement of the genetics of fish populations, helps with reduction of personnel costs, and is a great tool for selecting productive characteristics (Suquet et al., 2000), and conservation of genetic variability of the animals in nature by forming a germplasm bank (MartínezPáramo et al., 2009). Information about the most suitable and standardized procedures for handling semen of this species certainly contribute to the technological advancement of its production in captivity (Maria et al., 2011). The use of internal and external cryoprotectants in diluents for freezing semen seeks to protect sperm, and efficiency of technique use depends on the ability to protect cells from damage during freezing, and to inhibit chemical and osmotic toxicity from occurring (Gao et al., 1995) for sperm cells (Squires et al., 2004). Dimethylsufoxide (DMSO) is the most used cryoprotectant in freezing semen of freshwater fish native to Brazil (Viveiros and Godinho, 2009) as an internal cryoprotectant, and its rapid cell penetration is due to its low molecular weight (Lovelock and Bishop, 1959). The development of cryopreservation protocols for Tambaqui sperm and consequently its use depends on the standardization of more appropriate methods of collection and dilution (Maria et al., 2011). The most widely used methods for freezing semen from Brazilian native fish are the Dry Shipper and the Box Conditioner Methods when sperm are located 5 cm from the N2 L (Viveiros and Godinho, 2009). The Vitrification Method has been tested as to improve fish sperm viability. With this cryopreservation technique, a suspension of spermatozoa is plunged directly into N2 L and this cryopreservation technique has been shown to be successful with Oncorhynchus mykiss (Merino et al., 2011, 2012), and Atlantic salmon (Salmo salar) spermatozoa (Figueroa et al., 2015). Therefore, the aim of the present study was to analyze the effect of three different freezing methods using different DMSO concentrations, on structures (mitochondria, plasma membrane and DNA) and functionality (motility, latency time, fertilization rate and hatching rate) of cryopreserved sperm cells of C. macropomum.
2. Materials and methods 2.1. Collection and evaluation of semen The C. macropomum males from Piscigranja Boa Esperanc¸a (11◦ 41 46.95 S and 61◦ 13 47.50 O) located in Pimenta Bueno, in the state of Rondônia, Brazil, were used during the breeding season (December to February) in 2013. With this research, 16 males (5.4 ± 0.4 kg) and 16 females (7.9 ± 0.5 kg) were used.
With the goal of increasing the volume of semen collected, males were captured, and immediately carp pituitary extract 1 mg/kg was administered in the dorsal region of the animal. The extract was diluted in 0.5 mL of sterile saline (0.9% NaCl). Fish were kept in two handling tanks (no more than four animals in each) with a water column of 70 cm, with steady stream of water flow through the tanks. After 6.5 h at room temperature, the fish were cloth dried, and the urogenital orifice cleaned with a paper towel. Semen was collected in a 15 mL conical tube (one for each animal) through abdominal massage and the volume collected was recorded. Simultaneous extrusion of feces and/or urine was avoided so as to not activate sperm or contaminate samples (Billard et al., 1995). Prior to cryopreservation, sperm motility and latency time of each semen sample was evaluated by using an optical microscopy slide under a cover slip with the same trained technician assessing all samples. Semen was discarded when sperm activation occurred due to contamination with urine, feces or water. When there was no contamination activation, sperm motility was assessed by placing 1 L semen with 99 L distilled water (25 ◦ C) under the coverslip and an optical phase contrast microscope was used for sperm assessments with a magnification of 200× (BX Olympus 41, 400×). Sperm of all males had a motility value of greater than 80% in 10Ys after activation with distilled water. To evaluate the latency time, the time from activation to complete stoppage of progressive sperm movement was measured (in seconds). The sperm concentration was determined using a Neubauer chamber with semen being diluted 1:2000 in 4% formalin. Cells (after settling for 5 min) were counted under an optical phase contrast microscope (Olympus BX41, 400×). 2.2. Cryopreservation of semen The base solution was the diluent Beltsville Thawing Solution (BTS) comprising 37 g of glucose, 6 g of sodium citrate dihydrate, 1.25 g sodium bicarbonate, 1.25 g of ethylene diamine and 0.9 g of potassium chloride in 1YL of distilled water (Pursel and Johnson, 1975). All reagents used in this experiment were obtained from Sigma Chemical Company (St. Louis, MO, USA). Semen was diluted from 16 males and homogenized in BTS at 5%, 10%, 15%, and 20% DMSO with the final concentration being 800 million cells per mL. All treatments (nY= 12) were packaged into 0.25 mL straws and frozen using different freezing methods: Dry Shipper, Box Conditioner – slow freezing and Vitrification Methods. With the Dry Shipper Method, the straws were placed in a cylinder canister with liquid nitrogen vapor (N2 L), dry shipper type (Taylor-Wharton, CP 300 dry shipper model), at −170 ◦ C for 12 h, and were then transferred to a conventional cylinder containing N2 L (MVE, CP-34 model) for storage at −196 ◦ C. On the day preceding sperm processing for the Dry Shipper Method, the cylinder was filled with N2 L, to allow for overnight for stabilization before the sperm were placed in the cylinder. Before starting the process, excess N2 L was drained (Streit Junior et al., 2006). With the Box Conditioner Method, the N2 L was in
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the vapor form and the straws being held at room temperature were placed on a grid of polystyrene at a distance of 5 cm from the N2 L in a styrofoam box designed for this type of semen storage that was 32.5 cm in height that contained 15 cm of N2 L therein. Semen was pre-frozen for 10 min in the N2 L steam and, after this period, the straws were dipped in N2 L. Soon after this processing, the straws were placed in canister and stored in N2 L (−196 ◦ C). With the Vitrification Method, after being filled and sealed, the straws were plunged directly into N2 L (−196 ◦ C) (Isachenko et al., 2004, 2005; Merino et al., 2012).
2.3. Thawing and assessments of semen quality To assess the fertilization rate, 16 females, one for each male was used. Female egg release was induced using carp pituitary extract, applying 5 mg/kg of body weight, diluted in 2 mL of sterile saline (0.9% NaCl) with administration occurring by use of a 5 mL syringe and injection in the dorsal lateral region. After 9 h, the eggs were extruded by abdominal massage and were collected in 1000 mL beaker. Of the eggs, 16 aliquots of 2 g were reserved. To obtain the rate of fertilization, semen samples were thawed in a water bath at 45 ◦ C for 5Ys (Streit Junior et al., 2006). Thawed samples of 12 treatments per male (three freezing methods × four DMSO concentrations), were added in aliquots of eggs for fertilization. For each aliquot of eggs (1800 eggs), 150 L (120 million sperm) were used from the thawed sample of each treatment. Two aliquots in which eggs were fertilized with fresh semen were used for control samples, collected from two other males at the time of preliminary methodology assessments. With fresh semen samples, the samples were evaluated using the same number of sperm cells (120 million) similar to treatments with frozen semen. Aliquots with fresh semen were used to fertilize the first and last aliquot of eggs, the difference in fertilization rate between these two aliquots and fertilization of other eggs from the same female, never exceeded 6%. Rates of fertilization with fresh semen served as control values to ensure the quality of the eggs, and the rate of fertilized eggs in all repetitions were above 70%. In implementing the fertilization tests for both samples of thawed semen, as well as for the rates of fertilization with fresh semen (eggs quality control), sperm cells were placed in contact with the eggs in a 50 mL beaker. After, the material was gently homogenized for about 5Ys, activation of semen was performed by the addition of 2 mL of distilled water at 29 ◦ C by gently homogenizing again (5Ys) with a resting time of 2 min. At the end of this period, 20 mL of distilled water at 29 ◦ C were added to hydrate the fertilized eggs. After these eggs were incubated under gentle steady upward 150 mL/min flow rates of water (29 ◦ C), in incubators (conical) of 2YL, the experimental fertilization rates were evaluated after 8 h of incubation. Evaluations occurred for the total number of dividing eggs compared with the total number of eggs evaluated. Hatching rate was calculated based on the number of embryos hatched compared with the total number of fertilized eggs (1800).
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To perform the seminal analysis, samples were transported to the Laboratory of Animal Reproduction, Faculty of Veterinary Medicine of the Federal University of Pelotas, REPROPEL (31◦ 48 8.62 S and 52◦ 24 44.97 ). At this stage of the study, two straws of each treatment from each male were thawed (45 ◦ C/5Ys), and each sample was resuspended in 400 L BTS (1:3, v/v) at a temperature of 22 ◦ C in a 1.5 mL conical tube to minimize the toxicity of cryoprotectant after thawing. The seminal analyses conducted after thawing were sperm: motility, latency time, mitochondrial function, DNA integrity, membrane integrity and cell viability. Mitochondrial functionality was evaluated by fluorescent staining Rhodamine 123, placing 10 L of thawed sample and 40 L of the working solution of rhodamine 123 (13 M), and incubation occurring at 20 ◦ C for 10 min (He and Woods, 2004). For the integrity of DNA, 45 L thawed semen was diluted in 50 L TNE (0.01 M Tris–HCl, 0.15 M NaCl, 0.001 M EDTA, pH 7.2), and after 30 s 200 L 1× Triton solution was added, then 50 L of Acridine Orange (2 mg per mL in deionized H2 O) was added, waiting 5 min to perform the assessment, not exceeding 1 min exposure of the blade fitted to fluorescence during evaluation (Bencharif et al., 2010). The membrane integrity of spermatozoa was assessed using the fluorescent probes carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) by adding 40 L of working solution to 10 L of thawed sample and incubating at 20 ◦ C for 10 min (Harrison and Vickers, 1990). The evaluations of the mitochondria functionality, DNA integrity and membrane integrity were performed using an epifluorescence microscope (Olympus BX 51, America, Inc., São Paulo, Brazil) with the samples being held in 1 L of solution with sperm on slides under a cover slip. There were evaluations of 200Ycells per sample. Rates were expressed as the percentage of intact/functional cells divided by the total number of evaluated cells. Cell viability was performed using histochemical eosin nigrosin dyes (Maria et al., 2012). There was 10 L of diluted semen in 1 mL of staining solution (1.6 g of eosin Y and 6 g of nigrosin in BTS) that were homogenized with the smear being applied after 2 min. Sperms were observed after drying the slides in brightfield (Olympus BX41) with an oil immersion objective (100×). There were 200Ycells evaluated per sample. 2.4. Statistical analysis For analysis of normality, the Shapiro–Wilk test was performed. Then analysis of variance was performed with subsequent comparison between averages through the Tukey test. Concentrations of DMSO and different freezing methods were considered independent variables, and the variables of sperm motility, latency time, mitochondria functionality, DNA integrity, membrane integrity, cell viability, fertilization rate as well as egg hatching rates were considered dependent variables. The model of analysis of variance considered the combinations between the concentrations of DMSO and freezing method, totalling 12 treatments. All data were expressed as an average ± standard deviation (SD). All analyses were performed using Statistix 9.0 (2008) software.
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Table 1 Rates of fertilization and hatching (average ± standard deviation) obtained with Tambaqui (Colossoma macropomum) fresh and thawed semen after different cryopreservation techniques. Cryopreservation technique
Concentration DMSO (%)
Fertilization rate
5 10 15 20 5 10 15 20 5 10 15 20
84.9 55.5 51.9 41.4 32.8 43.0 37.8 21.6 5.4 7.0 2.9 0.0 0.0
Fresh semen Box conditioner
Dry shipper
Vitrification
± ± ± ± ± ± ± ± ± ± ± ± ±
6.5a 13.0b 13.0bc 12.6de 8.4e 11.1cd 10.8de 16.9f 2.9g 6.3g 3.3g 0.0g 0.0g
Hatching rate 75.2 44.1 40.6 31.2 21.4 29.1 27.5 11.1 2.1 0.4 0.6 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
11.6a 16.3b 14.9bc 10.9cd 13.6de 12.5d 13.2d 12.8ef 2.6fg 0.7g 0.9fg 0.0g 0.0g
The variable of plasma membrane integrity when the Box Conditioner Method was used with 5% DMSO was superior as compared with values for this variable when other treatments were used, independent of DMSO concentrations (P < 0.05). The DNA integrity with use of the Box Conditioner Method at all DSMO concentrations, and with use of the Vitrification and Dry Shipper Methods with 5% and 10% DMSO was similar (P > 0.05) and greater than with use of the Vitrification Method when 20% DMSO was used (P < 0.05). With the use of increased concentrations of DMSO, the DNA integrity was reduced. The mitochondria functionality was similar when the Box Conditioner Method was used with 5%, 10% and 15% DMSO and Dry Shipper Method was used with 10% and 15% of cryoprotectant (P > 0.05). Use of the Vitrification Method with 5% and 10% DMSO had the smallest mitochondrial function (P < 0.05) compared with the other groups (Table 3).
DMSO, dimethyl sulfoxide; a–g: averages ± SEM with different superscripts in the same column are different (P < 0.001).
4. Discussion 3. Results The average volume of fresh semen collected (n = 16), motility and latency time were, respectively, 4.5 ± 0.3 mL, 95.7 ± 2.0% and 122.6 ± 5.0Ys. Fertilization and hatching rates using the Box Conditioner Method with BTS added and using 5% and 10% DMSO were similar (P > 0.05). With use of a concentration of 5% DMSO, these rates were greater than for the other treatments tested (P < 0.05). With use of the Vitrification Method, rates were less for fertilization and hatching (Table 1). Sperm motility, viability and latency time were influenced by the freezing method and concentration of DMSO used (Table 2). Sperm motility and the latency time after cryopreservation when the Box Conditioner Method was used at the 5% DMSO concentration were greater than when other concentrations of cryoprotectants were used with the same methods of freezing as well as when all concentrations of DMSO were used in samples cryopreserved using the Dry Shipper and Vitrification Methods (P < 0.05). The viability rate, however, was greatest when the Dry Shipper Method was used with 20% DMSO and this rate was similar to that when the Box Conditioner Method was used with 15% DMSO (P > 0.05).
The present study is the first to simultaneously evaluate the Vitrification, Box Conditioner and Dry Shipper Methods for freezing of semen of fish. The Dry Shipper Method is the most widely used method in semen storage for species of migratory Brazilian fish (Viveiros and Godinho, 2009). Maria et al. (2012) found that the Dry Shipper Method was superior to the automated system of semen freezing (TK 4000® Machine, Uberaba, MG, Brazil) in maintaining the mobility and membrane integrity of frozen sperm of Tambaqui. In the present study, it was demonstrated by assessing different variables that the Box Conditioner Method of freezing in nitrogen vapor at a 5 cm spacing from the N2 L when the quality of sperm cells were compared to those of sperm cells where freezing occurred with the Dry Shipper and Vitrification Methods. Evaluation of alternative methods for fish sperm freezing is important because of the inherent shortfalls of each method, such as the cost of using a bottled dry-shipper, the great expense of N2 L with use of the Box Conditioner Method. It is also important to ascertain the most efficient method for preservation of the quality of the C. macropomum sperm cells. The use of the Vitrification Method for semen cryopreservation of C. macropomum was not satisfactory based on
Table 2 Motility and sperm viability and latency time (average ± standard deviation) after use of different techniques of cryopreservation of Tambaqui (Colossoma macropomum) semen with different DMSO concentrations. Cryopreservation technique
Box conditioner
Dry shipper
Vitrification
DMSO concentration (%)
Motility (%)
5 10 15 20 5 10 15 20 5 10 15 20
51.2 35.0 26.1 3.1 19.4 15.0 1.3 0.6 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ±
17.3a 21.4b 9.0c 5.3d 8.2c 4.6c 3.5d 1.2d 0.0d 0.0d 0.0d 0.0d
Latency time (s) 67.4 37.1 28.0 4.1 25.4 22.7 2.4 1.4 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ±
13.6a 16.9b 5.9c 4.0d 8.9c 7.6c 3.7d 3.9d 0.0d 0.0d 0.0d 0.0d
Sperm viability 56.9 57.0 59.0 54.3 48.5 52.4 57.7 70.9 52.3 49.9 41.6 43.7
± ± ± ± ± ± ± ± ± ± ± ±
16.7bc 15.5bc 20.2ab 23.6bcd 9.7bcd 4.9bcd 10.8ab 12.5a 8.0bcd 6.5bcd 7.7d 16.3cd
DMSO, dimethyl sulfoxide; a–c: average ± SEM with different superscripts in the same column are different (P < 0.001).
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Table 3 Integrity of plasma membrane. DNA integrity and mitochondrial functionality (average ± standard deviation) after use of different techniques of cryopreservation of Tambaqui (Colossoma macropomum) semen with different concentrations of DMSO. Cryopreservation technique
Box conditioner
Dry Shipper
Vitrification
DMSO concentration (%) 5 10 15 20 5 10 15 20 5 10 15 20
Integrity of plasma membrane 65.9 56.5 26.1 10.4 47.9 45.4 30.0 26.9 10.9 9.9 5.7 4.1
± ± ± ± ± ± ± ± ± ± ± ±
a
21.7 28.2ab 9.0c 18.0d 17.3b 4.2b 13.0c 13.4c 10.8d 9.8d 5.3d 4.6d
DNA integrity 98.0 99.0 97.1 96.4 98.4 98.7 95.5 94.7 97.5 96.6 95.2 91.7
± ± ± ± ± ± ± ± ± ± ± ±
ab
13.1 8.9a 17.4abcd 18.9abcd 12.4a 15.8a 5.5bcd 16.2d 18.5abc 8.0abcd 13.3cd 8.8e
Mitochondrial functionality 90.7 76.6 82.9 59.7 73.1 86.7 75.1 72.0 26.0 40.4 44.4 48.0
± ± ± ± ± ± ± ± ± ± ± ±
7.6a 8.6ab 10.6ab 21.2cd 13.0bc 5.9ab 9.2abc 21.6bc 21.0f 21.0f 22.8de 18.8de
DMSO, dimethylsulfoxide; a–g: averages ± SEM with different superscripts in the same column differ (P < 0.001).
the results from the present study. The values for variables evaluated after the freezing and thawing processes were generally less than when the other methods were used, indicating the Vitrification Method is not an acceptable method for sperm storage with this species. If the Vitrification Method is used, the use of cryoprotectants at concentrations other than those used in the present study should be considered. Semen cryopreservation with use of the Vitrification Method, ultra-fast freezing, is an unconventional process for the C. macropomum cell type, being mainly used for freezing of embryos and tissues where it is important that the formation of intra- and extra-cellular ice crystals is prevented (Vajta and Kuwayama, 2006). Results using the Vitrification Method for rabbit sperm storage were not promising with poor or no survival of rabbit sperm (Rosato and Iaffaldano, 2013) and use of this method had a negative impact on sperm variables in humans (Khalili et al., 2014). The detrimental impacts when using this method occurs due to high-speed freezing, the lack of appropriate dehydrating and thus a lethal formation of ice crystals (Yang and Tiersch, 2009). Fertilization and hatching rates were greater with the use of fresh semen (P < 0.05) in the present study, which was expected, because the sperm cells did not suffer the stress of freezing and thawing. Among the freezing techniques, the use of the Box Conditioner Method with 5% DMSO achieved the most desirable fertilization and hatching rates (P < 0.05), similar to with the use of the Box Conditioner Method with 10% cryoprotectant (P > 0.05). These are, therefore, effective techniques for obtaining desirable fertilization rates after freezing sperm cells from C. macropomum. With semen freezing of Lopardus pardalis, the sperm quality did not differ (P > 0.05) when the Dry Shipper and Box Conditioner Methods were used with these methods being superior to freezing semen with dry ice (Stoops et al., 2007). The equipment used with the Dry Shipper Method for semen freezing is practical and safe for the cryopreservation process, and the equipment is convenient and safe for transporting with leakage problems being minimized. This equipment has already been used for cryopreservation of semen of some species of fish (Viveiros and Godinho, 2009), and has been used for the freezing of genetic material of
the C. macropomum (Varela Junior et al., 2012; Maria et al., 2012). However, the equipment needed for the Dry Shipper Method has a significant acquisition cost, so alternatives that allow the cryopreservation of semen with similar or superior results for sperm quality being achieved to that obtained with Dry Shipper Method will allow for a wider dissemination of the biotechnique with increase in the use of frozen semen in Brazilian fish farming. Sperm motility and latency time after cryopreservation using the Box Conditioner Method at the 5% DMSO concentration were superior compared to values for these variables when other techniques and concentrations of cryoprotectants were used (P < 0.05). Even though values for both variables were different when the Box Conditioner Method was used at the 5% and 10% DMSO concentrations, the values for these variables when both concentrations were used were superior to these values when than other groups were evaluated and compared with the values when the Box Conditioner Method was used (P < 0.05). The results obtained with the use of Box Conditioner Method at a distance of 5 cm from the N2 L in the present study are promising, indicating the possibility of its use in breeding systems, enabling for a reduction of cost at the time of freezing for the preservation of genetic material. When the Box Conditioner Method was used, sperm membrane integrity with use of 5% and 10% DMSO was similar (P > 0.05). However, results with use of the lesser DMSO concentration were superior to when other techniques with different DMSO concentrations were used (P < 0.05). Results with use of Dry Shipper Method for sperm storage in the present study with 5% and 10% of cryoprotectant were similar to the results with the Box Conditioner Method with 10% DMSO (P > 0.05), and greater than the results for the other groups (P < 0.05), except for when the Box Conditioner method was used with 5% DMSO. The use of the Vitrification Method resulted in injuries in the sperm plasma membrane, yielding a maximum average of 10.9 ± 10.8 intact cells when the smallest concentration of DMSO was used, similar to the value when 20% cryoprotectant was used with the Box Conditioner Method indicating there may be a concentration–toxicity relationship with cryoprotectant with practically all groups in the present study.
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The DNA integrity was apparently unaffected by freezing method or by DMSO, with over 90% of cells with intact DNA in all groups. It has been previously reported that alterations in the DNA integrity occurred that were cryopreservation-induced, whereas others have reported no influence of sperm freezing conditions on this variable (Duru et al., 2001; Waterhouse et al., 2010; Zribi et al., 2010; Isachenko et al., 2004). In the present study, there was only a detrimental impact on DNA integrity when the Vitrification Method was used with 20% DMSO (P < 0.05) but even under these conditions there were 90% of cells with intact DNA. The inconsistent results between the present and some previous research may result from different freezing processes, methods for preparation of semen before cryopreservation and in the DNA integrity tests utilized in the studies (Zribi et al., 2010). Semen cryopreservation processes can lead to two types of damage to mitochondria, affecting sperm motility: direct damage to the genetic material or its membrane, and indirect damage caused by the fragmentation of nuclear DNA (Kurland and Andersson, 2000). The sperm mitochondria functionality was less when the Vitrification Method was used in the present study with the sperm motility after cryopreservation being zero, reinforcing the importance of the integrity of this organelle for sperm movement. A possible explanation for the damage to mitochondria in the present study was the direct damage to the sperm cell membrane because the nuclear DNA was not significantly affected. Thus, the results of the present study provide evidence that under the experimental conditions imposed, the freezing with the Box Conditioner Method using 5% or 10% DMSO was effective and superior to other freezing techniques, demonstrating that the use of a thermal ice chest for cryopreservation of C. macropomum semen is a fiscally inexpensive and viable alternative in global fish farming enterprises with this species. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was sponsored by FAPERGS (ARD 2012 process n◦ 141212-5). The authors thank the staff of Piscicultura Boa Esperanc¸a (Pimenta Bueno, RO, Brazil) for their invaluable contributions to this study and to CAPES for the financial support to the second author. C.D. Corcini is a research fellow from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) process n◦ 306356/2014-7. References Bencharif, D., Amirat, L., Pascal, O., Anton, M., Schmitt, E., Desherces, S., Delhomme, G., Langlois, M.-L., Barrière, P., Larrat, M., Tainturier, D., 2010. The advantages of combining low-density lipoproteins with glutamine for cryopreservation of canine semen. Reprod. Domest. Anim. 45, 189–200. Billard, R., Cosson, J., Crim, L.W., 1995. Broodstock management and seed quality – general considerations. In: Bromage, N., Roberts, R.J. (Eds.),
Broodstock Management and Egg Larval Quality. Blackwell Science, Oxford, pp. 1–24. Braga, B., Hespanhol, I., Conejo, J.G., 2005. Introduc¸ão à Engenharia Ambiental, 2nd ed. Pearson Prentice Hall, São Paulo, pp. 3222. Carpio, M.J., 2006. Analisis do losstudios de impacto ambiental del complejo hidroelléctrico Del Rio Madeira: hidrologia y sedimentos. Ministerio de Agricultura y Ganaderia, La Paz, Bolívia, pp. 28. Chellapa, S., Chellapa, N.T., Barbosa, W.T., Huntigord, F.A., Beveridge, M.C.M., 1995. Growth and production of the Amazonian tambaqui in fixed cages under different feeding regimes. Aquac. Int. 3, 11–21. Duru, N.K., Morshedi, M.S., Schuffner, A., Oehninger, S., 2001. Cryopreservation thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalisation and not DNA fragmentation. J. Androl. 22, 646–651. Figueroa, E., Merino, O., Risopatrón, J., Isachenko, V., Sánchez, R., Effer, B., Isachenko, E., Farias, J.G., Valdebenito, I., 2015. Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification. Theriogenology 83, 238–245. Gao, D.Y., Liu, J., Liu, C., Mcgann, L.E., Watson, P.F., Kleinhans, F.W., Mazur, P., Critser, E.S., Critser, J.K., 1995. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum. Reprod. 10, 1109–1122. Goulding, M.A., Carvalho, M.L., 1982. Life history and management of the tambaqui (Colossoma macropomum, Characidae): an important Amazonian foodfish. Rev. Bras. Zool. 1, 107–138. Harrison, R.A.P., Vickers, S.E., 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88, 343–352. He, S., Woods, L.C., 2004. Efects of dimethyl sulfoxide and glycine on cryopreservation induced damage of plasma membrane and mitochondria to striped bass (Morone saxatilis) sperm. Cryobiology 48, 254–262. Isachenko, V., Isachenko, E., Katkov, I.I., Montag, M., Dessole, S., Nawroth, F., Ven, H.V.D., 2004. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol. Reprod. 71, 1167–1173. Isachenko, V., Isachenko, E., Montag, M., Zaeva, V., Krivokharchenko, A., Nawroth, F., Dessole, S., Katkov, I., Van der Ven, H., 2005. Clean technique for cryoprotectant – free vitrification of human spermatozoa. Reprod. Biomed. Online 10, 350–354. Khalili, M.A., Adib, M., Halvaei, I., Nabi, A., 2014. Vitrification of neat semen alters sperm parameters and DNA integrity. Urol. J. 11, 1465–1473. Kurland, C.G., Andersson, S.G.E., 2000. Origin and evolution of the mitochondrial proteome. Microbiol. Mol. Biol. Rev. 64, 786–820. Lopes, T.S., Streit Junior, D.P., Ribeiro, R.P., Povh, J.A., Lopera-Barrero, N.M., Vargas, L., Pinto Filho, C., Queiroz, J.R., 2009. Genetic variability of tambaqui (Teleostei: Characidae) from different regions of Brazil. Braz. J. Vet. Anim. Sci. 61, 728–735. Lovelock, J.E., Bishop, M.W., 1959. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183, 1394–1395. Maria, A.N., Azevedo, H.C., Carneiro, P.C.F., 2011. Protocolo para Criopreservac¸ão do Sêmen de Tambaqui (Colossoma macropomum). Comun. Téc. 112, 8 (ISSN 1678-1937). Maria, A.N., Azevedo, H.C., Santos, J.P., Carneiro, P.C., 2012. Hormonal induction and semen characteristics of tambaqui Colossoma macropomum. Zygote 20, 39–43. Martínez-Páramo, S., Pérez-Cerezales, S., Gómez-Romano, F., Blanco, G., Sánchez, J.A., Herráez, M.P., 2009. Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential. Theriogenology 71, 594–604. Merino, O., Risopatrón, J., Sánchez, R., Isachenko, E., Figueroa, E., Valdebenito, I., Isachenko, V., 2011. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim. Reprod. Sci. 124, 125–131. Merino, O., Sánchez, R., Risopatrón, J., Isachenko, E., Katkov, I.I., Figueroa, E., Valdebenito, I., Mallmann, P., Isachenko, V., 2012. Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report. Andrologia 44, 390–395. Odum, E.P., 1971. Fundamentals of Ecology, 3rd ed. W.B. Saunders Company, Philadelphia, pp. 927. Pursel, V.G., Johnson, L.A., 1975. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99–102. Rosato, M.P., Iaffaldano, N., 2013. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology 79, 508–516.
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Valenti, W.C., Poli, C.R., Pereira, J.A., Borghetti, J.R., 2000. Aqüicultura no Brasil. CNPQ: MCT, Brasilia, pp. 399. Varela Junior, A.S., Corcini, C.D., Gheller, S.M.M., Jardim, R.D., Lucia Jr., T., Streit Jr., D.P., Figueiredo, M.R.C., 2012. Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum. Theriogenology 78, 244–251. Viveiros, A.T.M., Godinho, H.P., 2009. Sperm quality and cryopreservation of Brazilian freshwater fish species: a review. Fish Physiol. Biochem. 35, 137–150. Waterhouse, K.E., Gjeldnes, A., Tverdal, A., DE Angelis, P.M., Farstad, W., Håård, M., Kommisrud, E., 2010. Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen. Anim. Reprod. Sci. 117, 34–42. Yang, H., Tiersch, T.R., 2009. Current status of sperm cryopreservation in biomedical research fish models: zebrafish, medaka, and xiphophorus. Comp. Biochem. Physiol. C 149, 224–232. Zribi, N., Chakroun, N.F., El Euch, H., Gargouri, J., Bahloul, A., Keskes, L.A., 2010. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil. Steril. 93, 159–166.
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Methods of cryopreservation of Tambaqui semen, Colossoma macropomum A.S. Varela Junior a,∗ , K.L. Goularte c , J.P. Alves a , F.A. Pereira a , E.F. Silva a , T.F. Cardoso a , R.D. Jardim a , D.P. Streit Jr b , C.D. Corcini c a b c
Compared Animal Reproduction – RAC, Institute of Biological Sciences, Federal University of Rio Grande, Rio Grande, RS, Brazil Aquam, Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil ReproPel, Faculty of Veterinary Medicine, Federal University of Pelotas, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 26 March 2015 Accepted 31 March 2015 Available online xxx Keywords: Freezing Sperm Conservation Methods, Fish
a b s t r a c t This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2 L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2 L, amounting to 12 treatments (four DMSO concentrations × three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P > 0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P < 0.05) as well as for sperm motility and latency time (P < 0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO. © 2015 Published by Elsevier B.V.
1. Introduction Tambaqui – Colossoma macropomum (Cuvier, 1818) is a native migratory species in the Amazon basin and is considered the second largest scale fish of the Amazon and Solimões rivers (Goulding and Carvalho, 1982). The fish inhabit the Madeira River located in the state of Rondonia,
∗ Corresponding author. Tel.: +55 53 32935186; fax: +55 53 32336633. E-mail address: [email protected] (A.S. Varela Junior).
Brazil. In this river, the implementation of a hydroelectric dam (Lopes et al., 2009) may hinder the migration of this species by changing the lotic to lentic system, causing the formation of populations with a high degree of kinship and restricting gene flow, which may result in a decrease of the genetic heterogeneity (Odum, 1971; Braga et al., 2005; Carpio, 2006). In these cases, the preservation of the heterogeneity of the genetic heritage of native species through semen cryopreservation, for example, becomes important to avoid negative impacts on fish populations (Sirol and Britto, 2006).
http://dx.doi.org/10.1016/j.anireprosci.2015.03.017 0378-4320/© 2015 Published by Elsevier B.V.
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C. macropomum is the native Brazilian species commercially produced, with positive results with use of different types of intensive farming (Chellapa et al., 1995; Valenti et al., 2000), and this specie currently participates in a breeding program at the national level (Maria et al., 2011). In commercial production systems, genetic variability is important, because it enables the improvement of production traits (Suquet et al., 2000). Thus, seminal cryopreservation while enhancing the diffusion and improvement of the genetics of fish populations, helps with reduction of personnel costs, and is a great tool for selecting productive characteristics (Suquet et al., 2000), and conservation of genetic variability of the animals in nature by forming a germplasm bank (MartínezPáramo et al., 2009). Information about the most suitable and standardized procedures for handling semen of this species certainly contribute to the technological advancement of its production in captivity (Maria et al., 2011). The use of internal and external cryoprotectants in diluents for freezing semen seeks to protect sperm, and efficiency of technique use depends on the ability to protect cells from damage during freezing, and to inhibit chemical and osmotic toxicity from occurring (Gao et al., 1995) for sperm cells (Squires et al., 2004). Dimethylsufoxide (DMSO) is the most used cryoprotectant in freezing semen of freshwater fish native to Brazil (Viveiros and Godinho, 2009) as an internal cryoprotectant, and its rapid cell penetration is due to its low molecular weight (Lovelock and Bishop, 1959). The development of cryopreservation protocols for Tambaqui sperm and consequently its use depends on the standardization of more appropriate methods of collection and dilution (Maria et al., 2011). The most widely used methods for freezing semen from Brazilian native fish are the Dry Shipper and the Box Conditioner Methods when sperm are located 5 cm from the N2 L (Viveiros and Godinho, 2009). The Vitrification Method has been tested as to improve fish sperm viability. With this cryopreservation technique, a suspension of spermatozoa is plunged directly into N2 L and this cryopreservation technique has been shown to be successful with Oncorhynchus mykiss (Merino et al., 2011, 2012), and Atlantic salmon (Salmo salar) spermatozoa (Figueroa et al., 2015). Therefore, the aim of the present study was to analyze the effect of three different freezing methods using different DMSO concentrations, on structures (mitochondria, plasma membrane and DNA) and functionality (motility, latency time, fertilization rate and hatching rate) of cryopreserved sperm cells of C. macropomum.
2. Materials and methods 2.1. Collection and evaluation of semen The C. macropomum males from Piscigranja Boa Esperanc¸a (11◦ 41 46.95 S and 61◦ 13 47.50 O) located in Pimenta Bueno, in the state of Rondônia, Brazil, were used during the breeding season (December to February) in 2013. With this research, 16 males (5.4 ± 0.4 kg) and 16 females (7.9 ± 0.5 kg) were used.
With the goal of increasing the volume of semen collected, males were captured, and immediately carp pituitary extract 1 mg/kg was administered in the dorsal region of the animal. The extract was diluted in 0.5 mL of sterile saline (0.9% NaCl). Fish were kept in two handling tanks (no more than four animals in each) with a water column of 70 cm, with steady stream of water flow through the tanks. After 6.5 h at room temperature, the fish were cloth dried, and the urogenital orifice cleaned with a paper towel. Semen was collected in a 15 mL conical tube (one for each animal) through abdominal massage and the volume collected was recorded. Simultaneous extrusion of feces and/or urine was avoided so as to not activate sperm or contaminate samples (Billard et al., 1995). Prior to cryopreservation, sperm motility and latency time of each semen sample was evaluated by using an optical microscopy slide under a cover slip with the same trained technician assessing all samples. Semen was discarded when sperm activation occurred due to contamination with urine, feces or water. When there was no contamination activation, sperm motility was assessed by placing 1 L semen with 99 L distilled water (25 ◦ C) under the coverslip and an optical phase contrast microscope was used for sperm assessments with a magnification of 200× (BX Olympus 41, 400×). Sperm of all males had a motility value of greater than 80% in 10Ys after activation with distilled water. To evaluate the latency time, the time from activation to complete stoppage of progressive sperm movement was measured (in seconds). The sperm concentration was determined using a Neubauer chamber with semen being diluted 1:2000 in 4% formalin. Cells (after settling for 5 min) were counted under an optical phase contrast microscope (Olympus BX41, 400×). 2.2. Cryopreservation of semen The base solution was the diluent Beltsville Thawing Solution (BTS) comprising 37 g of glucose, 6 g of sodium citrate dihydrate, 1.25 g sodium bicarbonate, 1.25 g of ethylene diamine and 0.9 g of potassium chloride in 1YL of distilled water (Pursel and Johnson, 1975). All reagents used in this experiment were obtained from Sigma Chemical Company (St. Louis, MO, USA). Semen was diluted from 16 males and homogenized in BTS at 5%, 10%, 15%, and 20% DMSO with the final concentration being 800 million cells per mL. All treatments (nY= 12) were packaged into 0.25 mL straws and frozen using different freezing methods: Dry Shipper, Box Conditioner – slow freezing and Vitrification Methods. With the Dry Shipper Method, the straws were placed in a cylinder canister with liquid nitrogen vapor (N2 L), dry shipper type (Taylor-Wharton, CP 300 dry shipper model), at −170 ◦ C for 12 h, and were then transferred to a conventional cylinder containing N2 L (MVE, CP-34 model) for storage at −196 ◦ C. On the day preceding sperm processing for the Dry Shipper Method, the cylinder was filled with N2 L, to allow for overnight for stabilization before the sperm were placed in the cylinder. Before starting the process, excess N2 L was drained (Streit Junior et al., 2006). With the Box Conditioner Method, the N2 L was in
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the vapor form and the straws being held at room temperature were placed on a grid of polystyrene at a distance of 5 cm from the N2 L in a styrofoam box designed for this type of semen storage that was 32.5 cm in height that contained 15 cm of N2 L therein. Semen was pre-frozen for 10 min in the N2 L steam and, after this period, the straws were dipped in N2 L. Soon after this processing, the straws were placed in canister and stored in N2 L (−196 ◦ C). With the Vitrification Method, after being filled and sealed, the straws were plunged directly into N2 L (−196 ◦ C) (Isachenko et al., 2004, 2005; Merino et al., 2012).
2.3. Thawing and assessments of semen quality To assess the fertilization rate, 16 females, one for each male was used. Female egg release was induced using carp pituitary extract, applying 5 mg/kg of body weight, diluted in 2 mL of sterile saline (0.9% NaCl) with administration occurring by use of a 5 mL syringe and injection in the dorsal lateral region. After 9 h, the eggs were extruded by abdominal massage and were collected in 1000 mL beaker. Of the eggs, 16 aliquots of 2 g were reserved. To obtain the rate of fertilization, semen samples were thawed in a water bath at 45 ◦ C for 5Ys (Streit Junior et al., 2006). Thawed samples of 12 treatments per male (three freezing methods × four DMSO concentrations), were added in aliquots of eggs for fertilization. For each aliquot of eggs (1800 eggs), 150 L (120 million sperm) were used from the thawed sample of each treatment. Two aliquots in which eggs were fertilized with fresh semen were used for control samples, collected from two other males at the time of preliminary methodology assessments. With fresh semen samples, the samples were evaluated using the same number of sperm cells (120 million) similar to treatments with frozen semen. Aliquots with fresh semen were used to fertilize the first and last aliquot of eggs, the difference in fertilization rate between these two aliquots and fertilization of other eggs from the same female, never exceeded 6%. Rates of fertilization with fresh semen served as control values to ensure the quality of the eggs, and the rate of fertilized eggs in all repetitions were above 70%. In implementing the fertilization tests for both samples of thawed semen, as well as for the rates of fertilization with fresh semen (eggs quality control), sperm cells were placed in contact with the eggs in a 50 mL beaker. After, the material was gently homogenized for about 5Ys, activation of semen was performed by the addition of 2 mL of distilled water at 29 ◦ C by gently homogenizing again (5Ys) with a resting time of 2 min. At the end of this period, 20 mL of distilled water at 29 ◦ C were added to hydrate the fertilized eggs. After these eggs were incubated under gentle steady upward 150 mL/min flow rates of water (29 ◦ C), in incubators (conical) of 2YL, the experimental fertilization rates were evaluated after 8 h of incubation. Evaluations occurred for the total number of dividing eggs compared with the total number of eggs evaluated. Hatching rate was calculated based on the number of embryos hatched compared with the total number of fertilized eggs (1800).
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To perform the seminal analysis, samples were transported to the Laboratory of Animal Reproduction, Faculty of Veterinary Medicine of the Federal University of Pelotas, REPROPEL (31◦ 48 8.62 S and 52◦ 24 44.97 ). At this stage of the study, two straws of each treatment from each male were thawed (45 ◦ C/5Ys), and each sample was resuspended in 400 L BTS (1:3, v/v) at a temperature of 22 ◦ C in a 1.5 mL conical tube to minimize the toxicity of cryoprotectant after thawing. The seminal analyses conducted after thawing were sperm: motility, latency time, mitochondrial function, DNA integrity, membrane integrity and cell viability. Mitochondrial functionality was evaluated by fluorescent staining Rhodamine 123, placing 10 L of thawed sample and 40 L of the working solution of rhodamine 123 (13 M), and incubation occurring at 20 ◦ C for 10 min (He and Woods, 2004). For the integrity of DNA, 45 L thawed semen was diluted in 50 L TNE (0.01 M Tris–HCl, 0.15 M NaCl, 0.001 M EDTA, pH 7.2), and after 30 s 200 L 1× Triton solution was added, then 50 L of Acridine Orange (2 mg per mL in deionized H2 O) was added, waiting 5 min to perform the assessment, not exceeding 1 min exposure of the blade fitted to fluorescence during evaluation (Bencharif et al., 2010). The membrane integrity of spermatozoa was assessed using the fluorescent probes carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) by adding 40 L of working solution to 10 L of thawed sample and incubating at 20 ◦ C for 10 min (Harrison and Vickers, 1990). The evaluations of the mitochondria functionality, DNA integrity and membrane integrity were performed using an epifluorescence microscope (Olympus BX 51, America, Inc., São Paulo, Brazil) with the samples being held in 1 L of solution with sperm on slides under a cover slip. There were evaluations of 200Ycells per sample. Rates were expressed as the percentage of intact/functional cells divided by the total number of evaluated cells. Cell viability was performed using histochemical eosin nigrosin dyes (Maria et al., 2012). There was 10 L of diluted semen in 1 mL of staining solution (1.6 g of eosin Y and 6 g of nigrosin in BTS) that were homogenized with the smear being applied after 2 min. Sperms were observed after drying the slides in brightfield (Olympus BX41) with an oil immersion objective (100×). There were 200Ycells evaluated per sample. 2.4. Statistical analysis For analysis of normality, the Shapiro–Wilk test was performed. Then analysis of variance was performed with subsequent comparison between averages through the Tukey test. Concentrations of DMSO and different freezing methods were considered independent variables, and the variables of sperm motility, latency time, mitochondria functionality, DNA integrity, membrane integrity, cell viability, fertilization rate as well as egg hatching rates were considered dependent variables. The model of analysis of variance considered the combinations between the concentrations of DMSO and freezing method, totalling 12 treatments. All data were expressed as an average ± standard deviation (SD). All analyses were performed using Statistix 9.0 (2008) software.
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Table 1 Rates of fertilization and hatching (average ± standard deviation) obtained with Tambaqui (Colossoma macropomum) fresh and thawed semen after different cryopreservation techniques. Cryopreservation technique
Concentration DMSO (%)
Fertilization rate
5 10 15 20 5 10 15 20 5 10 15 20
84.9 55.5 51.9 41.4 32.8 43.0 37.8 21.6 5.4 7.0 2.9 0.0 0.0
Fresh semen Box conditioner
Dry shipper
Vitrification
± ± ± ± ± ± ± ± ± ± ± ± ±
6.5a 13.0b 13.0bc 12.6de 8.4e 11.1cd 10.8de 16.9f 2.9g 6.3g 3.3g 0.0g 0.0g
Hatching rate 75.2 44.1 40.6 31.2 21.4 29.1 27.5 11.1 2.1 0.4 0.6 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ± ±
11.6a 16.3b 14.9bc 10.9cd 13.6de 12.5d 13.2d 12.8ef 2.6fg 0.7g 0.9fg 0.0g 0.0g
The variable of plasma membrane integrity when the Box Conditioner Method was used with 5% DMSO was superior as compared with values for this variable when other treatments were used, independent of DMSO concentrations (P < 0.05). The DNA integrity with use of the Box Conditioner Method at all DSMO concentrations, and with use of the Vitrification and Dry Shipper Methods with 5% and 10% DMSO was similar (P > 0.05) and greater than with use of the Vitrification Method when 20% DMSO was used (P < 0.05). With the use of increased concentrations of DMSO, the DNA integrity was reduced. The mitochondria functionality was similar when the Box Conditioner Method was used with 5%, 10% and 15% DMSO and Dry Shipper Method was used with 10% and 15% of cryoprotectant (P > 0.05). Use of the Vitrification Method with 5% and 10% DMSO had the smallest mitochondrial function (P < 0.05) compared with the other groups (Table 3).
DMSO, dimethyl sulfoxide; a–g: averages ± SEM with different superscripts in the same column are different (P < 0.001).
4. Discussion 3. Results The average volume of fresh semen collected (n = 16), motility and latency time were, respectively, 4.5 ± 0.3 mL, 95.7 ± 2.0% and 122.6 ± 5.0Ys. Fertilization and hatching rates using the Box Conditioner Method with BTS added and using 5% and 10% DMSO were similar (P > 0.05). With use of a concentration of 5% DMSO, these rates were greater than for the other treatments tested (P < 0.05). With use of the Vitrification Method, rates were less for fertilization and hatching (Table 1). Sperm motility, viability and latency time were influenced by the freezing method and concentration of DMSO used (Table 2). Sperm motility and the latency time after cryopreservation when the Box Conditioner Method was used at the 5% DMSO concentration were greater than when other concentrations of cryoprotectants were used with the same methods of freezing as well as when all concentrations of DMSO were used in samples cryopreserved using the Dry Shipper and Vitrification Methods (P < 0.05). The viability rate, however, was greatest when the Dry Shipper Method was used with 20% DMSO and this rate was similar to that when the Box Conditioner Method was used with 15% DMSO (P > 0.05).
The present study is the first to simultaneously evaluate the Vitrification, Box Conditioner and Dry Shipper Methods for freezing of semen of fish. The Dry Shipper Method is the most widely used method in semen storage for species of migratory Brazilian fish (Viveiros and Godinho, 2009). Maria et al. (2012) found that the Dry Shipper Method was superior to the automated system of semen freezing (TK 4000® Machine, Uberaba, MG, Brazil) in maintaining the mobility and membrane integrity of frozen sperm of Tambaqui. In the present study, it was demonstrated by assessing different variables that the Box Conditioner Method of freezing in nitrogen vapor at a 5 cm spacing from the N2 L when the quality of sperm cells were compared to those of sperm cells where freezing occurred with the Dry Shipper and Vitrification Methods. Evaluation of alternative methods for fish sperm freezing is important because of the inherent shortfalls of each method, such as the cost of using a bottled dry-shipper, the great expense of N2 L with use of the Box Conditioner Method. It is also important to ascertain the most efficient method for preservation of the quality of the C. macropomum sperm cells. The use of the Vitrification Method for semen cryopreservation of C. macropomum was not satisfactory based on
Table 2 Motility and sperm viability and latency time (average ± standard deviation) after use of different techniques of cryopreservation of Tambaqui (Colossoma macropomum) semen with different DMSO concentrations. Cryopreservation technique
Box conditioner
Dry shipper
Vitrification
DMSO concentration (%)
Motility (%)
5 10 15 20 5 10 15 20 5 10 15 20
51.2 35.0 26.1 3.1 19.4 15.0 1.3 0.6 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ±
17.3a 21.4b 9.0c 5.3d 8.2c 4.6c 3.5d 1.2d 0.0d 0.0d 0.0d 0.0d
Latency time (s) 67.4 37.1 28.0 4.1 25.4 22.7 2.4 1.4 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ±
13.6a 16.9b 5.9c 4.0d 8.9c 7.6c 3.7d 3.9d 0.0d 0.0d 0.0d 0.0d
Sperm viability 56.9 57.0 59.0 54.3 48.5 52.4 57.7 70.9 52.3 49.9 41.6 43.7
± ± ± ± ± ± ± ± ± ± ± ±
16.7bc 15.5bc 20.2ab 23.6bcd 9.7bcd 4.9bcd 10.8ab 12.5a 8.0bcd 6.5bcd 7.7d 16.3cd
DMSO, dimethyl sulfoxide; a–c: average ± SEM with different superscripts in the same column are different (P < 0.001).
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Table 3 Integrity of plasma membrane. DNA integrity and mitochondrial functionality (average ± standard deviation) after use of different techniques of cryopreservation of Tambaqui (Colossoma macropomum) semen with different concentrations of DMSO. Cryopreservation technique
Box conditioner
Dry Shipper
Vitrification
DMSO concentration (%) 5 10 15 20 5 10 15 20 5 10 15 20
Integrity of plasma membrane 65.9 56.5 26.1 10.4 47.9 45.4 30.0 26.9 10.9 9.9 5.7 4.1
± ± ± ± ± ± ± ± ± ± ± ±
a
21.7 28.2ab 9.0c 18.0d 17.3b 4.2b 13.0c 13.4c 10.8d 9.8d 5.3d 4.6d
DNA integrity 98.0 99.0 97.1 96.4 98.4 98.7 95.5 94.7 97.5 96.6 95.2 91.7
± ± ± ± ± ± ± ± ± ± ± ±
ab
13.1 8.9a 17.4abcd 18.9abcd 12.4a 15.8a 5.5bcd 16.2d 18.5abc 8.0abcd 13.3cd 8.8e
Mitochondrial functionality 90.7 76.6 82.9 59.7 73.1 86.7 75.1 72.0 26.0 40.4 44.4 48.0
± ± ± ± ± ± ± ± ± ± ± ±
7.6a 8.6ab 10.6ab 21.2cd 13.0bc 5.9ab 9.2abc 21.6bc 21.0f 21.0f 22.8de 18.8de
DMSO, dimethylsulfoxide; a–g: averages ± SEM with different superscripts in the same column differ (P < 0.001).
the results from the present study. The values for variables evaluated after the freezing and thawing processes were generally less than when the other methods were used, indicating the Vitrification Method is not an acceptable method for sperm storage with this species. If the Vitrification Method is used, the use of cryoprotectants at concentrations other than those used in the present study should be considered. Semen cryopreservation with use of the Vitrification Method, ultra-fast freezing, is an unconventional process for the C. macropomum cell type, being mainly used for freezing of embryos and tissues where it is important that the formation of intra- and extra-cellular ice crystals is prevented (Vajta and Kuwayama, 2006). Results using the Vitrification Method for rabbit sperm storage were not promising with poor or no survival of rabbit sperm (Rosato and Iaffaldano, 2013) and use of this method had a negative impact on sperm variables in humans (Khalili et al., 2014). The detrimental impacts when using this method occurs due to high-speed freezing, the lack of appropriate dehydrating and thus a lethal formation of ice crystals (Yang and Tiersch, 2009). Fertilization and hatching rates were greater with the use of fresh semen (P < 0.05) in the present study, which was expected, because the sperm cells did not suffer the stress of freezing and thawing. Among the freezing techniques, the use of the Box Conditioner Method with 5% DMSO achieved the most desirable fertilization and hatching rates (P < 0.05), similar to with the use of the Box Conditioner Method with 10% cryoprotectant (P > 0.05). These are, therefore, effective techniques for obtaining desirable fertilization rates after freezing sperm cells from C. macropomum. With semen freezing of Lopardus pardalis, the sperm quality did not differ (P > 0.05) when the Dry Shipper and Box Conditioner Methods were used with these methods being superior to freezing semen with dry ice (Stoops et al., 2007). The equipment used with the Dry Shipper Method for semen freezing is practical and safe for the cryopreservation process, and the equipment is convenient and safe for transporting with leakage problems being minimized. This equipment has already been used for cryopreservation of semen of some species of fish (Viveiros and Godinho, 2009), and has been used for the freezing of genetic material of
the C. macropomum (Varela Junior et al., 2012; Maria et al., 2012). However, the equipment needed for the Dry Shipper Method has a significant acquisition cost, so alternatives that allow the cryopreservation of semen with similar or superior results for sperm quality being achieved to that obtained with Dry Shipper Method will allow for a wider dissemination of the biotechnique with increase in the use of frozen semen in Brazilian fish farming. Sperm motility and latency time after cryopreservation using the Box Conditioner Method at the 5% DMSO concentration were superior compared to values for these variables when other techniques and concentrations of cryoprotectants were used (P < 0.05). Even though values for both variables were different when the Box Conditioner Method was used at the 5% and 10% DMSO concentrations, the values for these variables when both concentrations were used were superior to these values when than other groups were evaluated and compared with the values when the Box Conditioner Method was used (P < 0.05). The results obtained with the use of Box Conditioner Method at a distance of 5 cm from the N2 L in the present study are promising, indicating the possibility of its use in breeding systems, enabling for a reduction of cost at the time of freezing for the preservation of genetic material. When the Box Conditioner Method was used, sperm membrane integrity with use of 5% and 10% DMSO was similar (P > 0.05). However, results with use of the lesser DMSO concentration were superior to when other techniques with different DMSO concentrations were used (P < 0.05). Results with use of Dry Shipper Method for sperm storage in the present study with 5% and 10% of cryoprotectant were similar to the results with the Box Conditioner Method with 10% DMSO (P > 0.05), and greater than the results for the other groups (P < 0.05), except for when the Box Conditioner method was used with 5% DMSO. The use of the Vitrification Method resulted in injuries in the sperm plasma membrane, yielding a maximum average of 10.9 ± 10.8 intact cells when the smallest concentration of DMSO was used, similar to the value when 20% cryoprotectant was used with the Box Conditioner Method indicating there may be a concentration–toxicity relationship with cryoprotectant with practically all groups in the present study.
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The DNA integrity was apparently unaffected by freezing method or by DMSO, with over 90% of cells with intact DNA in all groups. It has been previously reported that alterations in the DNA integrity occurred that were cryopreservation-induced, whereas others have reported no influence of sperm freezing conditions on this variable (Duru et al., 2001; Waterhouse et al., 2010; Zribi et al., 2010; Isachenko et al., 2004). In the present study, there was only a detrimental impact on DNA integrity when the Vitrification Method was used with 20% DMSO (P < 0.05) but even under these conditions there were 90% of cells with intact DNA. The inconsistent results between the present and some previous research may result from different freezing processes, methods for preparation of semen before cryopreservation and in the DNA integrity tests utilized in the studies (Zribi et al., 2010). Semen cryopreservation processes can lead to two types of damage to mitochondria, affecting sperm motility: direct damage to the genetic material or its membrane, and indirect damage caused by the fragmentation of nuclear DNA (Kurland and Andersson, 2000). The sperm mitochondria functionality was less when the Vitrification Method was used in the present study with the sperm motility after cryopreservation being zero, reinforcing the importance of the integrity of this organelle for sperm movement. A possible explanation for the damage to mitochondria in the present study was the direct damage to the sperm cell membrane because the nuclear DNA was not significantly affected. Thus, the results of the present study provide evidence that under the experimental conditions imposed, the freezing with the Box Conditioner Method using 5% or 10% DMSO was effective and superior to other freezing techniques, demonstrating that the use of a thermal ice chest for cryopreservation of C. macropomum semen is a fiscally inexpensive and viable alternative in global fish farming enterprises with this species. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was sponsored by FAPERGS (ARD 2012 process n◦ 141212-5). The authors thank the staff of Piscicultura Boa Esperanc¸a (Pimenta Bueno, RO, Brazil) for their invaluable contributions to this study and to CAPES for the financial support to the second author. C.D. Corcini is a research fellow from the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) process n◦ 306356/2014-7. References Bencharif, D., Amirat, L., Pascal, O., Anton, M., Schmitt, E., Desherces, S., Delhomme, G., Langlois, M.-L., Barrière, P., Larrat, M., Tainturier, D., 2010. The advantages of combining low-density lipoproteins with glutamine for cryopreservation of canine semen. Reprod. Domest. Anim. 45, 189–200. Billard, R., Cosson, J., Crim, L.W., 1995. Broodstock management and seed quality – general considerations. In: Bromage, N., Roberts, R.J. (Eds.),
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