Cryobiology 69 (2014) 451–456
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Hatching, survival and deformities of piracanjuba (Brycon orbignyanus) embryos subjected to different cooling protocols q Maria do Carmo Faria Paes a,⇑, Regiane Cristina da Silva a, Nivaldo Ferreira do Nascimento a, Fernanda Nogueira Valentin a, José Augusto Senhorini b, Laura Satiko Okada Nakaghi a a b
Aquaculture Centre of Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane, s/n., 14884-900 Jaboticabal, São Paulo, Brazil National Centre for Research and Continental Fish Conservation, CEPTA/ICMBio, Rodovia SP-201, Km 6,5, Mailbox 64, 13.630-970 Pirassununga, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 28 May 2013 Accepted 6 October 2014 Available online 14 October 2014 Keywords: Cryobiology Fish Reproduction Conservation Anomalies
a b s t r a c t Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me2SO); at different temperatures (0.0 ± 2.0 °C and 8.0 ± 2.0 °C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10 h and the temperature decreased from 8.0 ± 2.0 °C to 0.0 ± 2.0 °C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Piracanjuba (Brycon orbignyanus) is a tropical fish highly appreciated for its flavor, meat quality and the aggressiveness displayed in sport fishing [20]. However, the species has been endangered by the damming of rivers for the construction of hydroelectric plants, preventing their migration; the destruction of riparian vegetation, reducing natural food availability; pollution and overfishing [20,16,6]. This critical decrease in the B. orbignyanus population is daunting, as the loss of genetic diversity in the population increases the species vulnerability [15]. Thus, cryopreservation
q Statement of funding: This work was supported by the agency ‘‘Foundation for Research Support of the State of São Paulo/Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, Brazil’’, protocol number 2009/12340-5 and also by the agency ‘‘National Council for Scientific and Technological Development/ Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil’’, protocol 3315241710512807. ⇑ Corresponding author. Fax: +55 16 3209 2656. E-mail addresses: [email protected] (M.d.C.F. Paes), regianesilva_bio@ yahoo.com.br (R.C. da Silva), [email protected] (N.F. do Nascimento), [email protected] (F.N. Valentin), [email protected] (J.A. Senhorini), [email protected] (L.S.O. Nakaghi).
http://dx.doi.org/10.1016/j.cryobiol.2014.10.003 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
may be a valuable tool in achieving gamete and embryo availability all year round [18]. Cryopreservation of fish semen is well understood and satisfactory results have been achieved, including in piracanjuba [11]. However, the development of cryopreservation techniques for teleostean embryos is urgent, as aquaculture is still largely dependent on wild fish populations or on the maintenance of natural stocks in captivity, both of which are constantly affected by natural disasters, reproductive failures and diseases [14]. Knowledge of successful cooling protocols can aid storage and transportation of embryos, optimize reproduction, and allow genetic material to be stored in small spaces to increase genetic variability for restocking rivers [4]. Furthermore, it serves as a basis for cryopreservation experiments as greater understanding of embryo behavior under various cooling conditions, and of toxicity of cryoprotectants to embryo development and survival may lead to the development of successful cryopreservation protocols for fish embryos. Thus, due to the lack of information on B. orbignyanus embryo cryopreservation, the aim of this study was to evaluate the efficiency of various cooling protocols using different cryoprotectants, temperatures and refrigeration times; as well as to perform morphological analysis of eggs and larvae to detect any anomalies
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caused by the use of these protocols. Greater knowledge of cooling processes and cryopreservation of embryos could improve reproduction, restocking and the preservation techniques currently used for this species.
Materials and methods B. orbignyanus embryos were collected, during their reproductive period, from Duke Energy International Aquaculture and Hydrology Station, Salto Grande-SP, Brazil and from CEPTA/ICMBio – IBAMA, Pirassununga-SP, Brazil. The experiment lasted 9 weeks and each week was considered a repetition. Breeding adults received food containing 40% crude protein, 3 times a day, the temperature was kept between 27 and 29 °C and natural light was available. Sixty females and one hundred and twenty males were selected when secondary sexual characteristics, such as a red soft abdomen in females and a rough anal fin in males, became apparent. Both males and females were kept in 500 L ponds and induced to breed with commercial crude carp pituitary extract. Females received 0.5 mg carp pituitary extract/kg of live weight (LW) followed by a second dose of 5 mg carp pituitary extract/kg (LW) 12 h later. Males received a single dose containing 1 mg carp pituitary extract/kg (LW) concomitant to the females’ second dose. Spawning occurred around 6 h after the last induction, at 26.65 ± 2.5 °C. Embryos were collected and transferred to 7 L conical incubators with continuous water outflow, at a concentration of 6000 eggs per liter of water. One hundred healthy embryos, at the stage of blastopore closure, were selected for each treatment and repetition. Embryonic health was confirmed by the blue coloration of the eggs and the transparency of the chorion. Embryos from the positive control group were counted and transferred directly to the incubators without undergoing any cooling process. The protocol used was adapted from that employed by Fornari et al. [4] for the tropical species Rhinelepis aspera. Seven different combinations of cryoprotectants were tested. Sucrose at 0.85 M was used as extracellular cryoprotectant in all protocols while the following solutions were used as intracellular cryoprotectants, in three different concentrations: methanol – 2.8 M (9 mL), 1.4 M (4.5 mL) and 0.93 M (3 mL); ethylene glycol – 1.45 M (9 mL), 0.75 M (4.5 mL) and 0.48 M (3 mL) and dimethyl sulfoxide (Me2SO) – 1.15 M (9 mL), 0.57 M (4.5 mL) and 0.38 M (3 mL), as detailed in Table 1. The average water temperature and the dissolved-oxygen levels in the experimental incubators were 26.65 ± 2.5 °C and 6.36 ± 1.9 mg/L, respectively. To test cryoprotectant toxicity, 30 embryos were placed in each of the cryoprotectant solutions for 1 h, at room temperature, before being rinsed with water to remove excess cryoprotectant and incu-
Table 1 Different cryoprotectant solutions tested in the cooling of piracanjuba (B. orbignyanus) embryos. Treatments
1 2 3 4 5 6 7
bated as normal. All treatments resulted in 100% hatching and no visible deformity. This test was repeated three times. The 100 healthy embryos were placed in sealed plastic tubes and seven groups received cryoprotectant solutions while the negative control group only received distilled water and water from the incubator. Embryos from all treatment groups, with the exception of the positive control group (embryos in water incubation and normal temperature conditions), were cooled to 20.0 °C for 10 min, followed by 10 more minutes at 10.0 °C. Subsequently, embryos were transferred to two temperature-controlled refrigerators, at 0.0 ± 2.0 °C and 8.0 ± 2.0 °C, for 6, 10, 24, 72 and 168 h. The sealed tubes were then transferred to a water bath containing water from the incubator at room temperature and acclimatized for 15 min. The embryos were removed from the tubes, washed with incubator water and randomly transferred to experimental incubators at room temperature. Three rates were determined: hatching, larvae that had ruptured the chorion; survival, larvae that displayed heartbeat, whether the chorion was ruptured or not; and deformity, larvae that presented any kind of abnormality. Survival and deformity rates were determined 6 h after hatching had ended (the 6 h that follow hatching are critical for the larval mortality in this species). Thus, although abnormal larvae were observed since hatching, for comparison purposes, we considered the deformity and survival rates together. Morphological analysis Embryos that did not survive were collected and fixed as soon as they were spotted in the incubators. The remaining embryos were collected 6 h after the end of hatching. Eggs and larvae were fixed in paraformaldehyde 2.5% + glutaraldehyde 2.5% solution for 24 h, washed in sodium cacodylate buffer, analyzed and microphotographed using a stereomicroscope (Leica DMZ 180) attached to a digital camera (Leica MZ8). For light microscopy, samples were dehydrated, processed and embedded into Historesin (Leica HistoresinÒ embedding kit). Two micrometer sections were mounted onto slides and stained with Hematoxylin–Floxine. Slide analysis and photography were performed using a photomicroscope (Leica DM2500) attached to a digital camera (DFC 280) and Leica Application Suite (LAS) software. For transmission electron microscopy, samples were fixed in 2% osmium tetroxide solution for 2 h, dehydrated, soaked in araldite and acetone and embedded in araldite. Sections (0,5 lm) were cut using a glass blade and stained with 1% Toluidine Blue in saturated boric acid. The best samples were further cut into ultrafine sections (60 nm), stained with uranyl acetate and lead citrate, analyzed and microphotographed using Jeol-JEM 100CX II transmission electron microscope. The statistical analysis was performed using Proc GLM and Proc CORR-SAS software (SAS Institute, Cary, NC, EUA) and the means compared by Turkey Test at 5%. Results
Cryoprotectants Sucrose (%)
Methanol (%)
Ethylene glycol (%)
Me2SO (%)
Distilled water (%)
17.1 17.1 17.1 17.1 17.1 17.1 17.1
9 – – 4.5 4.5 – 3
– 9 – 4.5 – 4.5 3
– – 9 – 4.5 4.5 3
73.9 73.9 73.9 73.9 73.9 73.9 73.9
⁄ Positive control did not use distilled water (just water of incubator) and had 2 types of negative controls: with distilled water and with water of incubator.
Piracanjuba is very sensitive to handling and the stress induced during artificial reproduction in captivity affects the quality of the spawn. The mean embryo hatching and survival rates of the positive control was 73.11 ± 11.5% and 52.44 ± 20.81%, respectively, which could be considered a very low rate for subsequent experiments. However, embryo cryopreservation in this case is justified by the high prolixity of the species, which can release over 200,000 oocytes per spawn. No embryos survived after 24, 72 and 168 h of refrigeration, therefore, these refrigeration times were tested only once.
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Maria do Carmo Faria Paes et al. / Cryobiology 69 (2014) 451–456 Table 2 Means and standard deviations for hatching rate considering the interaction of different combinations of cryoprotectants, temperature and storage time. Temp/time
Cryoprotectants – Mean hatching (%) 1
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h * ⁄⁄
2 Bb
5.22 ± 8.94 0.22 ± 0.66Bc 5.00 ± 6.81Bb 0.66 ± 2.00Bc
3
5.66 ± 13.08 0.0 ± 0.0Bc 0.0 ± 0.0Cc 0.0 ± 0.0Bc
Bb
4 Cb
20.62 ± 29.85 3.7 ± 8.45Cc 0.11 ± 0.33Cd 1.0 ± 3.0Bd
5 Bb
11.22 ± 17.73 2.22 ± 6.66Cc 4.33 ± 4.82Bc 0.0 ± 0.0Bd
6 Cb
21.4 ± 19.10 9.87 ± 10.20Dc 11.88 ± 18.10Dc 0.0 ± 0.0Bd
8*
7 Bb
BCb
9.0 ± 22.35 3.5 ± 6.23Cc 1.11 ± 2.97Cc 0.0 ± 0.0Bd
73.11 ± 11.5Aa 73.11 ± 11.5Aa 73.11 ± 11.5Aa 73.11 ± 11.5Aa
15.66 ± 22.07 1.0 ± 1.58BCc 2.77 ± 7.96BCc 1.77 ± 0.0Bc
Positive control group in normal conditions of incubator. Different uppercase letters in the same line differ from each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
It was observed that temperature, as well as refrigeration time, affected survival and hatching rates. When the temperature decreased and the refrigeration time increased, both survival and hatching rates declined significantly. When all variables were taken into account, treatments 3, 5 and 7 showed the best results at 8 ± 2 °C and after 6 h of refrigeration, with survival rates of up to 32.75 ± 35.0, 33.10 ± 23.23 and 24.88 ± 24.32, respectively. In general, deformity rates were proportional to survival and hatching rates. But comparing the best results of survival and hatching (treatments 3, 5 and 7), treatment 5 (sucrose + methanol + Me2SO) had the lowest rate of deformity, then the solution of, at the temperature 8.0 ± 2.0 °C in refrigeration time of 6 h can be considered the most recommended for the species (Tables 2–4). It is important to point out that, although considered dead, some embryos did not die immediately upon their removal from the refrigerator but continued to develop for 1 to 2 h during incubation. Other embryos, although alive (heartbeat present), were unable to hatch due to tail malformations, which did not allow for the movement necessary to break the chorion. Therefore, survival rate values were higher than the hatching rates.
Eggs Live eggs from the positive control group varied from several shades of blue to dark brown, had a round shape and an intact and transparent chorion (Fig. 1A). The blastoderm cells and the yolk syncytial layer were well defined with a visible nucleus (Fig. 1B and C) and the yolk granules were clearly outlined (Fig. 1D). It was observed that in the positive control group, blue colored embryos showed greater survival rates than their brown colored counterparts. All embryos subjected to cooling that did not survive had an intact but opaque chorion when visualized under a magnifying glass (Fig. 1E). The most common abnormalities observed on histology were: disorganized blastoderm cells (Fig. 1F), extravasated yolk sac with blastoderm cells and syncytial layer completely damaged and attached to the yolk sac (Fig. 1G), absence of the membrane that surrounds the yolk granules and yolk granules of irregular shape (Fig. 1H).
Larvae Morphological analysis of deformities Newly hatched healthy larvae had a free and long tail, a well developed and slightly pigmented vesicle and an overall length of 1.63 ± 0.49 mm (Fig. 2A). The vast majority of abnormal larvae were smaller (average length 1.30 ± 0.37 mm) than the healthy larvae, however, a small
All treatment groups had eggs and/or larvae with some degree of abnormality. This was also observed in a very small percentage of the positive control group, when the water temperature of the incubator reached 30.0 °C during the seventh repetition.
Table 3 Means and standard deviations for survival rate considering the interaction of different combinations of cryoprotectants, temperature and storage time. Temp/estoc
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h ⁄
Crioprotetor – Mean survival (%) 1
2
3
4
5
6
7
8
14.22 ± 12.65Bb 0.55 ± 1.13Bc 15.88 ± 16.62Bb 2.55 ± 4.9Bc
9.0 ± 15.57Bb 0.0 ± 0.0Bc 0.0 ± 0.0Cc 0.0 ± 0.0Cc
32.75 ± 35.0Cb 10.30 ± 17.44CDc 6.88 ± 19.92Dc 3.22 ± 9.66Bc
19.66 ± 24.7Bb 5.55 ± 16.66Cc 8.44 ± 8.35Dc 0.44 ± 1.33Cd
33.10 ± 23.23Cb 20.37 ± 15.24Dc 26.44 ± 30.38Ec 2.55 ± 5.19Bd
13.9 ± 22.42Bb 6.37 ± 9.76Cc 2.44 ± 4.12CDc 0.0 ± 0.0Cd
24.88 ± 24.32BCb 6.33 ± 7.15Cc 6.22 ± 14.70Dc 2.0 ± 5.33Bc
52.44 ± 20.81Aa 52.44 ± 20.81Aa 52.44 ± 20.81Aa 52.44 ± 20.81Aa
Positive control group in normal conditions of incubator. Different uppercase letters in the same line differ from each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
⁄⁄
Table 4 Means and standard deviations for deformity rate considering the interaction of different combinations of cryoprotectants, temperature and storage time. Temp/estoc
Crioprotetor – Mean deformity (%) 1
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h
10.88 ± 9.37Aa 0.55 ± 1.13Ab 14.00 ± 14.95Aa 2.11 ± 3.65Ab
2 5.33 ± 7.43 * NS * NS * NS
3 Ba
4 Ca
26.5 ± 25.74 8.7 ± 15.11Bb 6.88 ± 19.92Cb 3.22 ± 9.66Ab
5 Aa
13.88 ± 17.76 1.88 ± 5.66Ab 3.33 ± 4.87Cb 5.11 ± 11.53Ab
6 Aa
15.8 ± 15.92 19.62 ± 12.61Ca 20.11 ± 24.59Aa 4.0 ± 7.15Ab
7 Aa
9.3 ± 15.95 4.62 ± 7.15Bb 3.66 ± 6.08Cb * NS
19.66 ± 18.24ACa 5.0 ± 6.32Bb 5.77 ± 13.04Cb 2.0 ± 4.97Ab
*
NS: No survival (so, no calculation of deformity rate). The positive control group did not show deformity rate, and the negative control groups did not show survival. each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
⁄⁄
⁄⁄⁄
Different uppercase letters in the same line differ from
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Fig. 1. Steromicrography, electronmicrography and photomicrography of piracanjuba (Brycon orbignyanus) eggs: A, B, C and D – normal development with blastoderm and syncytial layer cells present, round yolk granules and intact chorion (positive control); E, F, G and H – eggs subjected to cooling with irregular blastoderm, extravasated yolk sac and irregular yolk granules (Y – yolk sac; b – blastoderm; c – chorion; slc – syncytial layer cells) Staining: B and F – Toluidine Blue; C and F – Hematoxylin–Floxine.
Fig. 2. Steromicrography of the external morphology of piracanjuba (Brycon orbignyanus) larvae: (A) normal development (positive control); (B) malformation of the tail which curved upwards; (C) malformation of the tail which curved downwards; (D) malformation of the yolk sac, head and tail; (E) microcephaly and malformation of the tail and yolk sac; (F) malformation of tail and yolk sac; (G) malformation of the yolk sac, head and tail; (H) underdeveloped larva with abnormal tail and head (Y – yolk sac).
percentage was found to be bigger (average length 2.10 ± 0.87 mm). The main abnormalities observed under stereomicroscopy were: lordosis, scoliosis and kyphosis; short or sinuous tail due to poor tail development, which hindered hatching as the larvae
were unable to move adequately and break the chorion; yolk sac retraction; micro and macrocephaly (Fig. 2B–H). Histology analysis showed that healthy larvae had an intact yolk sac, normally developed prosencephalon (Fig. 3A) and otic vesicle (Fig. 3B) and an organized notochord with large vacuoles.
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Fig. 3. Photomicrography of (Brycon orbignyanus) larvae: A and B – normal development (positive control); C, D, E and F – larvae subjected to cooling with abnormal optic vesicle and prosencephalon (P – prosencephalon; VO – optic vesicle). Staining: A, C and E – Toluidine Blue; B, D and F – Hematoxylin–Floxine.
However, it was observed that although abnormal larvae from all treatment groups presented an intact yolk sac membrane with apparent normal content, the optic vesicle was either underdeveloped, poorly formed or misplaced, and the prosencephalon was abnormal (Fig. 3 C–F). It was noted that in some samples the cells from the cranial region presented ruptured plasma membranes, even though the nuclear membrane and organelles appeared to be intact. A great number of vacuoles and mitochondria were observed in the cells located between the embryo and the yolk sac and the presence of an unknown substance of colloidal aspect between the yolk granules and inter and intracellularly throughout the larval body.
Discussion The post-cooling survival rates found in this study can be considered satisfactory when taking into account that the piracanjuba is extremely sensitive to reproductive management. It is common for the female breeder to die after hormonal induction and spawning [7] and this reproductive stress is reflected on spawn quality [21]. In addition to the species sensitivity, other factors may influence larval survival rates, such as water quality during incubation and genetic inheritance from the female breeder [17]. Therefore, to minimize genetically inherited problems, the eggs used in this study were pooled from several spawns from different breeders and the embryos checked to ensure their viability prior to
treatment. The temperature and dissolved-oxygen levels of the incubator water were constantly monitored and kept within the range considered acceptable for the farming of piracanjuba [16]. Different cryoprotectant agents (CPAs) protocols have been tested for fish embryos. The use of cryoprotectants is essential when subjecting embryos to temperatures below 0 °C [4]. Zhang et al. [23] and Zhang et al. [22] tested the effects of different CPAs on the embryo survival rates of zebrafish (Danio rerio) and medaka (Oryzias latipes) at low temperatures and the results showed that the inclusion of methanol significantly increased the survival rates of zebrafish. On the other hand, when the concentration of CPA increased, the survival rates of medaka embryos decreased. In a different study, Zhang et al. [24] reported that, when incubated for 30 min at room temperature, the maximum non-toxic concentrations tolerated by Danio rerio embryos at different stages of development were: 2 M for methanol, Me2SO and ethylene glycol, 1 M for glycerol and 0.5 M for sucrose. Piracanjuba embryos under the experimental conditions of this study showed good tolerance to the concentrations and types of cryoprotectants used, demonstrating that cryoprotectant toxicity varies for different species. Cold sensitivity is directly related to the stage of embryonic development [17,10]. It has been reported that the highest survival rates of Piaractus mesopotamicus embryos subjected to cooling at four different stages of development were during the stage of blastopore closure [9]. Thus, this stage of embryonic development was chosen for this study based on the good results reported for cooling studies in tropical species such as pacu (P. mesopotamicus) and black catfish (R. aspera) [9,19,13,5,4].
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The temperature and refrigeration period which embryos can tolerate without compromising their survival should also be taken into consideration. A study in black catfish (R. aspera) using cryoprotectant solutions containing methanol and sucrose for 6 h at 8.0 °C reported a survival rate of approximately 50% [4]. Furthermore, other studies have shown that the survival rates of pacu (P. mesopotamicus) were 69%, 85% and 40.5%, when refrigerated for 6 h at 8.0 °C in various concentrations of methanol + sucrose cryoprotectant solution [9,4,13]. In this study, when the refrigeration time increased from 6 to 10 h and the temperature decreased from 8.0 ± 2.0 to 0.0 ± 2.0 °C, the mean hatching and survival rates of piracanjuba decreased to almost half, while the survival rate was zero after 24, 72 and 168 h of refrigeration. This result is in agreement with the study by Ahamad et al. [1], which reports that the survival rate of the common carp (Cyprinus carpio) decreases as the refrigeration time increases. Among the several factors that can cause embryo malformation, Leme dos Santos and Azoubel [8] reported that the water temperature of the incubator directly influences the embryonic development of fish. Low temperatures slow development, while high temperatures accelerate development and may cause malformations. This was also observed in B. orbignyanus as malformed larvae were present not only in the cooling treatments but also in the positive control group, where temperature in the incubators reached 30.0 °C. Fornari et al. [3] has also reported larvae malformation in pacu subjected to cooling, however, the nature of the abnormalities was not specified. This is an important factor to be recorded, as many studies report high survival rates but fail to consider that even though alive, malformed larvae will unlikely develop into adults. Neves et al. [12] points out that damage to the yolk granule membrane can be caused by various factors such as insufficient penetration of cryoprotectants, insufficient suspension time, cryoprotectant solution toxicity and low temperatures. Dobrinsky [2] notes that the formation of intracellular ice crystals may damage the cytoplasmic membrane and denature intracellular functions and organelles. Neves et al. [13] reported that pacu embryos subjected to freezing and defrosting appeared white and with a ruptured chorion when analyzed under light microscopy. Furthermore, it was observed that the syncytial yolk layer was of varied shape, size and thickness and often located atypically bellow the yolk sac or enveloping separate pieces of the sac, similarly to the damages observed in piracanjuba embryos. Thus, it can be concluded that cooling can cause damages as severe as freezing. As a final remark, the data and results discussed in this study provide significant information that can help optimize the reproduction of piracanjuba and emphasize the need for new protocols to reduce the damage caused by low temperatures. Acknowledgments The authors would like to thank FAPESP (2009/12340-5) and CNPq (3315241710512807) for their financial support, Prof. Dr. João Ademir de Oliveira for the statistical analysis and Duke Energy International and CEPTA/ICMBio – IBAMA for providing the infrastructure and the fish. References [1] M.M. Ahammad, D. Bhattacharyya, B.B. Jana, Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and –2 °C in different concentrations of methanol and sucrose, Theriogenology 60 (2003) 1409–1422. [2] J.R. Dobrinsky, Cellular approach to cryopreservation of embryos, Theriogenology 45 (1996) 17–26.
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Injuries in pacu embryos (Piaractus mesopotamicus) after freezing and thawing, Zygote (2012). Available at: http://dx.doi.org/10.1017/ S096719941200024X, (accessed January 2013). [14] A. Ninhaus-Silveira, Preservação dos gametas de peixes e suas aplicações [Preservation of fish gametes and their applications], Revista Colombiana de Ciências Pecuárias 20 (2004) 516–517. [15] R.S. Panarari-Antunes, A.J. Prioli, S.M.A.P. Prioli, A.S. Galdino, H.F. Julio Junior, L.M. Prioli, Genetic Variability of Brycon orbignyanus (Valenciennes,1850) (Characiformes: Characidae) in cultivated and natural populations of the upper paraná river, and implications for the conservation of the species, Braz. Arch. Biol. Technol. Int. J. 54 (2011) 839–848. Available at: http://dx.doi.org/ 10.1590/S1516-89132011000400025, (accessed January 2013). [16] D. Reynalte-Tataje, E. Zaniboni-Filho, J.R. Esquivel, Embryonic and larvae development of piracanjuba, Brycon orbignyanus Valenciennes, 1849 (Pisces, Characidae), Acta Sci. Biol. Sci. 26 (2004) 67–71. [17] E. Saillant, B. Chatain, A. Fostier, C. Przybyla, C. Fauvel, Parental influence on early development in the European sea bass, J. Fish Biol. 8 (2001) 1585–1600. [18] G. Sansone, I.A. Nascimento, M.B.N.L. Leite, M.M.S. Araújo, S.A. Pereira, A.M. Mariani, Toxic effects of cryoprotectants on oyster gametes and embryos: a preliminary step towards establishing cryopreservation protocols, Biociências 13 (2005) 11–18. [19] D.P. Streit Jr, M. Digmayer, R.P. Ribeiro, R.N. Sirol, G.V. Moraes, J.M. Galo, Embriões de pacu submetidos a diferentes protocolos de resfriamento [Pacu embryos under differents cooling protocols], Pesquisa Agropecuária Brasileira 42 (2007) 1199–1202. [20] M.M. Vaz, V.C. Torquato, N.D.C. Barbosa, Guia ilustrado de peixes da Bacia do Rio Grande. [Illustrated guide of fish of the Rio Grande Basin]. CEMIG/CETEC, Belo Horizonte, 2000, p. 144. [21] E. Zaniboni-Filho, A.P.O. Nuñer, Fisiologia da reprodução e propagação artificial dos peixes, in: Tópicos especiais em piscicultura de água doce tropical intensiva [Physiology of reproduction and artificial propagation of fish, in: Special topics in freshwater tropical fish intensive], TecArt, São Paulo, 2004, pp. 45–73. [22] Q.J. Zhang, G.B. Zhou, Y.P. Wang, X.W. Fu, S.E. Zhu, Cryoprotectants protect medaka (Oryzias latipes) embryos from chilling injury, CryoLetters 33 (2) (2012) 107–116. [23] T. Zhang, X.H. Liu, D. Rawnson, Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos, Theriogenology 59 (7) (2003) 1545–1556. [24] T. Zhang, D.M. Rawson, G.J. Morris, Cryopreservation of pre-hatch embryos of zebrafish (Brachydanio rerio) embryos, Aquat. Living Resour. 6 (1993) 145–153.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Hatching, survival and deformities of piracanjuba (Brycon orbignyanus) embryos subjected to different cooling protocols q Maria do Carmo Faria Paes a,⇑, Regiane Cristina da Silva a, Nivaldo Ferreira do Nascimento a, Fernanda Nogueira Valentin a, José Augusto Senhorini b, Laura Satiko Okada Nakaghi a a b
Aquaculture Centre of Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane, s/n., 14884-900 Jaboticabal, São Paulo, Brazil National Centre for Research and Continental Fish Conservation, CEPTA/ICMBio, Rodovia SP-201, Km 6,5, Mailbox 64, 13.630-970 Pirassununga, São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 28 May 2013 Accepted 6 October 2014 Available online 14 October 2014 Keywords: Cryobiology Fish Reproduction Conservation Anomalies
a b s t r a c t Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me2SO); at different temperatures (0.0 ± 2.0 °C and 8.0 ± 2.0 °C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10 h and the temperature decreased from 8.0 ± 2.0 °C to 0.0 ± 2.0 °C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Piracanjuba (Brycon orbignyanus) is a tropical fish highly appreciated for its flavor, meat quality and the aggressiveness displayed in sport fishing [20]. However, the species has been endangered by the damming of rivers for the construction of hydroelectric plants, preventing their migration; the destruction of riparian vegetation, reducing natural food availability; pollution and overfishing [20,16,6]. This critical decrease in the B. orbignyanus population is daunting, as the loss of genetic diversity in the population increases the species vulnerability [15]. Thus, cryopreservation
q Statement of funding: This work was supported by the agency ‘‘Foundation for Research Support of the State of São Paulo/Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, Brazil’’, protocol number 2009/12340-5 and also by the agency ‘‘National Council for Scientific and Technological Development/ Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Brazil’’, protocol 3315241710512807. ⇑ Corresponding author. Fax: +55 16 3209 2656. E-mail addresses: [email protected] (M.d.C.F. Paes), regianesilva_bio@ yahoo.com.br (R.C. da Silva), [email protected] (N.F. do Nascimento), [email protected] (F.N. Valentin), [email protected] (J.A. Senhorini), [email protected] (L.S.O. Nakaghi).
http://dx.doi.org/10.1016/j.cryobiol.2014.10.003 0011-2240/Ó 2014 Elsevier Inc. All rights reserved.
may be a valuable tool in achieving gamete and embryo availability all year round [18]. Cryopreservation of fish semen is well understood and satisfactory results have been achieved, including in piracanjuba [11]. However, the development of cryopreservation techniques for teleostean embryos is urgent, as aquaculture is still largely dependent on wild fish populations or on the maintenance of natural stocks in captivity, both of which are constantly affected by natural disasters, reproductive failures and diseases [14]. Knowledge of successful cooling protocols can aid storage and transportation of embryos, optimize reproduction, and allow genetic material to be stored in small spaces to increase genetic variability for restocking rivers [4]. Furthermore, it serves as a basis for cryopreservation experiments as greater understanding of embryo behavior under various cooling conditions, and of toxicity of cryoprotectants to embryo development and survival may lead to the development of successful cryopreservation protocols for fish embryos. Thus, due to the lack of information on B. orbignyanus embryo cryopreservation, the aim of this study was to evaluate the efficiency of various cooling protocols using different cryoprotectants, temperatures and refrigeration times; as well as to perform morphological analysis of eggs and larvae to detect any anomalies
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caused by the use of these protocols. Greater knowledge of cooling processes and cryopreservation of embryos could improve reproduction, restocking and the preservation techniques currently used for this species.
Materials and methods B. orbignyanus embryos were collected, during their reproductive period, from Duke Energy International Aquaculture and Hydrology Station, Salto Grande-SP, Brazil and from CEPTA/ICMBio – IBAMA, Pirassununga-SP, Brazil. The experiment lasted 9 weeks and each week was considered a repetition. Breeding adults received food containing 40% crude protein, 3 times a day, the temperature was kept between 27 and 29 °C and natural light was available. Sixty females and one hundred and twenty males were selected when secondary sexual characteristics, such as a red soft abdomen in females and a rough anal fin in males, became apparent. Both males and females were kept in 500 L ponds and induced to breed with commercial crude carp pituitary extract. Females received 0.5 mg carp pituitary extract/kg of live weight (LW) followed by a second dose of 5 mg carp pituitary extract/kg (LW) 12 h later. Males received a single dose containing 1 mg carp pituitary extract/kg (LW) concomitant to the females’ second dose. Spawning occurred around 6 h after the last induction, at 26.65 ± 2.5 °C. Embryos were collected and transferred to 7 L conical incubators with continuous water outflow, at a concentration of 6000 eggs per liter of water. One hundred healthy embryos, at the stage of blastopore closure, were selected for each treatment and repetition. Embryonic health was confirmed by the blue coloration of the eggs and the transparency of the chorion. Embryos from the positive control group were counted and transferred directly to the incubators without undergoing any cooling process. The protocol used was adapted from that employed by Fornari et al. [4] for the tropical species Rhinelepis aspera. Seven different combinations of cryoprotectants were tested. Sucrose at 0.85 M was used as extracellular cryoprotectant in all protocols while the following solutions were used as intracellular cryoprotectants, in three different concentrations: methanol – 2.8 M (9 mL), 1.4 M (4.5 mL) and 0.93 M (3 mL); ethylene glycol – 1.45 M (9 mL), 0.75 M (4.5 mL) and 0.48 M (3 mL) and dimethyl sulfoxide (Me2SO) – 1.15 M (9 mL), 0.57 M (4.5 mL) and 0.38 M (3 mL), as detailed in Table 1. The average water temperature and the dissolved-oxygen levels in the experimental incubators were 26.65 ± 2.5 °C and 6.36 ± 1.9 mg/L, respectively. To test cryoprotectant toxicity, 30 embryos were placed in each of the cryoprotectant solutions for 1 h, at room temperature, before being rinsed with water to remove excess cryoprotectant and incu-
Table 1 Different cryoprotectant solutions tested in the cooling of piracanjuba (B. orbignyanus) embryos. Treatments
1 2 3 4 5 6 7
bated as normal. All treatments resulted in 100% hatching and no visible deformity. This test was repeated three times. The 100 healthy embryos were placed in sealed plastic tubes and seven groups received cryoprotectant solutions while the negative control group only received distilled water and water from the incubator. Embryos from all treatment groups, with the exception of the positive control group (embryos in water incubation and normal temperature conditions), were cooled to 20.0 °C for 10 min, followed by 10 more minutes at 10.0 °C. Subsequently, embryos were transferred to two temperature-controlled refrigerators, at 0.0 ± 2.0 °C and 8.0 ± 2.0 °C, for 6, 10, 24, 72 and 168 h. The sealed tubes were then transferred to a water bath containing water from the incubator at room temperature and acclimatized for 15 min. The embryos were removed from the tubes, washed with incubator water and randomly transferred to experimental incubators at room temperature. Three rates were determined: hatching, larvae that had ruptured the chorion; survival, larvae that displayed heartbeat, whether the chorion was ruptured or not; and deformity, larvae that presented any kind of abnormality. Survival and deformity rates were determined 6 h after hatching had ended (the 6 h that follow hatching are critical for the larval mortality in this species). Thus, although abnormal larvae were observed since hatching, for comparison purposes, we considered the deformity and survival rates together. Morphological analysis Embryos that did not survive were collected and fixed as soon as they were spotted in the incubators. The remaining embryos were collected 6 h after the end of hatching. Eggs and larvae were fixed in paraformaldehyde 2.5% + glutaraldehyde 2.5% solution for 24 h, washed in sodium cacodylate buffer, analyzed and microphotographed using a stereomicroscope (Leica DMZ 180) attached to a digital camera (Leica MZ8). For light microscopy, samples were dehydrated, processed and embedded into Historesin (Leica HistoresinÒ embedding kit). Two micrometer sections were mounted onto slides and stained with Hematoxylin–Floxine. Slide analysis and photography were performed using a photomicroscope (Leica DM2500) attached to a digital camera (DFC 280) and Leica Application Suite (LAS) software. For transmission electron microscopy, samples were fixed in 2% osmium tetroxide solution for 2 h, dehydrated, soaked in araldite and acetone and embedded in araldite. Sections (0,5 lm) were cut using a glass blade and stained with 1% Toluidine Blue in saturated boric acid. The best samples were further cut into ultrafine sections (60 nm), stained with uranyl acetate and lead citrate, analyzed and microphotographed using Jeol-JEM 100CX II transmission electron microscope. The statistical analysis was performed using Proc GLM and Proc CORR-SAS software (SAS Institute, Cary, NC, EUA) and the means compared by Turkey Test at 5%. Results
Cryoprotectants Sucrose (%)
Methanol (%)
Ethylene glycol (%)
Me2SO (%)
Distilled water (%)
17.1 17.1 17.1 17.1 17.1 17.1 17.1
9 – – 4.5 4.5 – 3
– 9 – 4.5 – 4.5 3
– – 9 – 4.5 4.5 3
73.9 73.9 73.9 73.9 73.9 73.9 73.9
⁄ Positive control did not use distilled water (just water of incubator) and had 2 types of negative controls: with distilled water and with water of incubator.
Piracanjuba is very sensitive to handling and the stress induced during artificial reproduction in captivity affects the quality of the spawn. The mean embryo hatching and survival rates of the positive control was 73.11 ± 11.5% and 52.44 ± 20.81%, respectively, which could be considered a very low rate for subsequent experiments. However, embryo cryopreservation in this case is justified by the high prolixity of the species, which can release over 200,000 oocytes per spawn. No embryos survived after 24, 72 and 168 h of refrigeration, therefore, these refrigeration times were tested only once.
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Cryoprotectants – Mean hatching (%) 1
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h * ⁄⁄
2 Bb
5.22 ± 8.94 0.22 ± 0.66Bc 5.00 ± 6.81Bb 0.66 ± 2.00Bc
3
5.66 ± 13.08 0.0 ± 0.0Bc 0.0 ± 0.0Cc 0.0 ± 0.0Bc
Bb
4 Cb
20.62 ± 29.85 3.7 ± 8.45Cc 0.11 ± 0.33Cd 1.0 ± 3.0Bd
5 Bb
11.22 ± 17.73 2.22 ± 6.66Cc 4.33 ± 4.82Bc 0.0 ± 0.0Bd
6 Cb
21.4 ± 19.10 9.87 ± 10.20Dc 11.88 ± 18.10Dc 0.0 ± 0.0Bd
8*
7 Bb
BCb
9.0 ± 22.35 3.5 ± 6.23Cc 1.11 ± 2.97Cc 0.0 ± 0.0Bd
73.11 ± 11.5Aa 73.11 ± 11.5Aa 73.11 ± 11.5Aa 73.11 ± 11.5Aa
15.66 ± 22.07 1.0 ± 1.58BCc 2.77 ± 7.96BCc 1.77 ± 0.0Bc
Positive control group in normal conditions of incubator. Different uppercase letters in the same line differ from each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
It was observed that temperature, as well as refrigeration time, affected survival and hatching rates. When the temperature decreased and the refrigeration time increased, both survival and hatching rates declined significantly. When all variables were taken into account, treatments 3, 5 and 7 showed the best results at 8 ± 2 °C and after 6 h of refrigeration, with survival rates of up to 32.75 ± 35.0, 33.10 ± 23.23 and 24.88 ± 24.32, respectively. In general, deformity rates were proportional to survival and hatching rates. But comparing the best results of survival and hatching (treatments 3, 5 and 7), treatment 5 (sucrose + methanol + Me2SO) had the lowest rate of deformity, then the solution of, at the temperature 8.0 ± 2.0 °C in refrigeration time of 6 h can be considered the most recommended for the species (Tables 2–4). It is important to point out that, although considered dead, some embryos did not die immediately upon their removal from the refrigerator but continued to develop for 1 to 2 h during incubation. Other embryos, although alive (heartbeat present), were unable to hatch due to tail malformations, which did not allow for the movement necessary to break the chorion. Therefore, survival rate values were higher than the hatching rates.
Eggs Live eggs from the positive control group varied from several shades of blue to dark brown, had a round shape and an intact and transparent chorion (Fig. 1A). The blastoderm cells and the yolk syncytial layer were well defined with a visible nucleus (Fig. 1B and C) and the yolk granules were clearly outlined (Fig. 1D). It was observed that in the positive control group, blue colored embryos showed greater survival rates than their brown colored counterparts. All embryos subjected to cooling that did not survive had an intact but opaque chorion when visualized under a magnifying glass (Fig. 1E). The most common abnormalities observed on histology were: disorganized blastoderm cells (Fig. 1F), extravasated yolk sac with blastoderm cells and syncytial layer completely damaged and attached to the yolk sac (Fig. 1G), absence of the membrane that surrounds the yolk granules and yolk granules of irregular shape (Fig. 1H).
Larvae Morphological analysis of deformities Newly hatched healthy larvae had a free and long tail, a well developed and slightly pigmented vesicle and an overall length of 1.63 ± 0.49 mm (Fig. 2A). The vast majority of abnormal larvae were smaller (average length 1.30 ± 0.37 mm) than the healthy larvae, however, a small
All treatment groups had eggs and/or larvae with some degree of abnormality. This was also observed in a very small percentage of the positive control group, when the water temperature of the incubator reached 30.0 °C during the seventh repetition.
Table 3 Means and standard deviations for survival rate considering the interaction of different combinations of cryoprotectants, temperature and storage time. Temp/estoc
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h ⁄
Crioprotetor – Mean survival (%) 1
2
3
4
5
6
7
8
14.22 ± 12.65Bb 0.55 ± 1.13Bc 15.88 ± 16.62Bb 2.55 ± 4.9Bc
9.0 ± 15.57Bb 0.0 ± 0.0Bc 0.0 ± 0.0Cc 0.0 ± 0.0Cc
32.75 ± 35.0Cb 10.30 ± 17.44CDc 6.88 ± 19.92Dc 3.22 ± 9.66Bc
19.66 ± 24.7Bb 5.55 ± 16.66Cc 8.44 ± 8.35Dc 0.44 ± 1.33Cd
33.10 ± 23.23Cb 20.37 ± 15.24Dc 26.44 ± 30.38Ec 2.55 ± 5.19Bd
13.9 ± 22.42Bb 6.37 ± 9.76Cc 2.44 ± 4.12CDc 0.0 ± 0.0Cd
24.88 ± 24.32BCb 6.33 ± 7.15Cc 6.22 ± 14.70Dc 2.0 ± 5.33Bc
52.44 ± 20.81Aa 52.44 ± 20.81Aa 52.44 ± 20.81Aa 52.44 ± 20.81Aa
Positive control group in normal conditions of incubator. Different uppercase letters in the same line differ from each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
⁄⁄
Table 4 Means and standard deviations for deformity rate considering the interaction of different combinations of cryoprotectants, temperature and storage time. Temp/estoc
Crioprotetor – Mean deformity (%) 1
8 ± 2 °C/6 h 8 ± 2 °C/10 h 0 ± 2 °C/6 h 0 ± 2 °C/10 h
10.88 ± 9.37Aa 0.55 ± 1.13Ab 14.00 ± 14.95Aa 2.11 ± 3.65Ab
2 5.33 ± 7.43 * NS * NS * NS
3 Ba
4 Ca
26.5 ± 25.74 8.7 ± 15.11Bb 6.88 ± 19.92Cb 3.22 ± 9.66Ab
5 Aa
13.88 ± 17.76 1.88 ± 5.66Ab 3.33 ± 4.87Cb 5.11 ± 11.53Ab
6 Aa
15.8 ± 15.92 19.62 ± 12.61Ca 20.11 ± 24.59Aa 4.0 ± 7.15Ab
7 Aa
9.3 ± 15.95 4.62 ± 7.15Bb 3.66 ± 6.08Cb * NS
19.66 ± 18.24ACa 5.0 ± 6.32Bb 5.77 ± 13.04Cb 2.0 ± 4.97Ab
*
NS: No survival (so, no calculation of deformity rate). The positive control group did not show deformity rate, and the negative control groups did not show survival. each other. Different lowercase letters within the same column differ from each other by Tukey (P < 0.05).
⁄⁄
⁄⁄⁄
Different uppercase letters in the same line differ from
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Fig. 1. Steromicrography, electronmicrography and photomicrography of piracanjuba (Brycon orbignyanus) eggs: A, B, C and D – normal development with blastoderm and syncytial layer cells present, round yolk granules and intact chorion (positive control); E, F, G and H – eggs subjected to cooling with irregular blastoderm, extravasated yolk sac and irregular yolk granules (Y – yolk sac; b – blastoderm; c – chorion; slc – syncytial layer cells) Staining: B and F – Toluidine Blue; C and F – Hematoxylin–Floxine.
Fig. 2. Steromicrography of the external morphology of piracanjuba (Brycon orbignyanus) larvae: (A) normal development (positive control); (B) malformation of the tail which curved upwards; (C) malformation of the tail which curved downwards; (D) malformation of the yolk sac, head and tail; (E) microcephaly and malformation of the tail and yolk sac; (F) malformation of tail and yolk sac; (G) malformation of the yolk sac, head and tail; (H) underdeveloped larva with abnormal tail and head (Y – yolk sac).
percentage was found to be bigger (average length 2.10 ± 0.87 mm). The main abnormalities observed under stereomicroscopy were: lordosis, scoliosis and kyphosis; short or sinuous tail due to poor tail development, which hindered hatching as the larvae
were unable to move adequately and break the chorion; yolk sac retraction; micro and macrocephaly (Fig. 2B–H). Histology analysis showed that healthy larvae had an intact yolk sac, normally developed prosencephalon (Fig. 3A) and otic vesicle (Fig. 3B) and an organized notochord with large vacuoles.
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Fig. 3. Photomicrography of (Brycon orbignyanus) larvae: A and B – normal development (positive control); C, D, E and F – larvae subjected to cooling with abnormal optic vesicle and prosencephalon (P – prosencephalon; VO – optic vesicle). Staining: A, C and E – Toluidine Blue; B, D and F – Hematoxylin–Floxine.
However, it was observed that although abnormal larvae from all treatment groups presented an intact yolk sac membrane with apparent normal content, the optic vesicle was either underdeveloped, poorly formed or misplaced, and the prosencephalon was abnormal (Fig. 3 C–F). It was noted that in some samples the cells from the cranial region presented ruptured plasma membranes, even though the nuclear membrane and organelles appeared to be intact. A great number of vacuoles and mitochondria were observed in the cells located between the embryo and the yolk sac and the presence of an unknown substance of colloidal aspect between the yolk granules and inter and intracellularly throughout the larval body.
Discussion The post-cooling survival rates found in this study can be considered satisfactory when taking into account that the piracanjuba is extremely sensitive to reproductive management. It is common for the female breeder to die after hormonal induction and spawning [7] and this reproductive stress is reflected on spawn quality [21]. In addition to the species sensitivity, other factors may influence larval survival rates, such as water quality during incubation and genetic inheritance from the female breeder [17]. Therefore, to minimize genetically inherited problems, the eggs used in this study were pooled from several spawns from different breeders and the embryos checked to ensure their viability prior to
treatment. The temperature and dissolved-oxygen levels of the incubator water were constantly monitored and kept within the range considered acceptable for the farming of piracanjuba [16]. Different cryoprotectant agents (CPAs) protocols have been tested for fish embryos. The use of cryoprotectants is essential when subjecting embryos to temperatures below 0 °C [4]. Zhang et al. [23] and Zhang et al. [22] tested the effects of different CPAs on the embryo survival rates of zebrafish (Danio rerio) and medaka (Oryzias latipes) at low temperatures and the results showed that the inclusion of methanol significantly increased the survival rates of zebrafish. On the other hand, when the concentration of CPA increased, the survival rates of medaka embryos decreased. In a different study, Zhang et al. [24] reported that, when incubated for 30 min at room temperature, the maximum non-toxic concentrations tolerated by Danio rerio embryos at different stages of development were: 2 M for methanol, Me2SO and ethylene glycol, 1 M for glycerol and 0.5 M for sucrose. Piracanjuba embryos under the experimental conditions of this study showed good tolerance to the concentrations and types of cryoprotectants used, demonstrating that cryoprotectant toxicity varies for different species. Cold sensitivity is directly related to the stage of embryonic development [17,10]. It has been reported that the highest survival rates of Piaractus mesopotamicus embryos subjected to cooling at four different stages of development were during the stage of blastopore closure [9]. Thus, this stage of embryonic development was chosen for this study based on the good results reported for cooling studies in tropical species such as pacu (P. mesopotamicus) and black catfish (R. aspera) [9,19,13,5,4].
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The temperature and refrigeration period which embryos can tolerate without compromising their survival should also be taken into consideration. A study in black catfish (R. aspera) using cryoprotectant solutions containing methanol and sucrose for 6 h at 8.0 °C reported a survival rate of approximately 50% [4]. Furthermore, other studies have shown that the survival rates of pacu (P. mesopotamicus) were 69%, 85% and 40.5%, when refrigerated for 6 h at 8.0 °C in various concentrations of methanol + sucrose cryoprotectant solution [9,4,13]. In this study, when the refrigeration time increased from 6 to 10 h and the temperature decreased from 8.0 ± 2.0 to 0.0 ± 2.0 °C, the mean hatching and survival rates of piracanjuba decreased to almost half, while the survival rate was zero after 24, 72 and 168 h of refrigeration. This result is in agreement with the study by Ahamad et al. [1], which reports that the survival rate of the common carp (Cyprinus carpio) decreases as the refrigeration time increases. Among the several factors that can cause embryo malformation, Leme dos Santos and Azoubel [8] reported that the water temperature of the incubator directly influences the embryonic development of fish. Low temperatures slow development, while high temperatures accelerate development and may cause malformations. This was also observed in B. orbignyanus as malformed larvae were present not only in the cooling treatments but also in the positive control group, where temperature in the incubators reached 30.0 °C. Fornari et al. [3] has also reported larvae malformation in pacu subjected to cooling, however, the nature of the abnormalities was not specified. This is an important factor to be recorded, as many studies report high survival rates but fail to consider that even though alive, malformed larvae will unlikely develop into adults. Neves et al. [12] points out that damage to the yolk granule membrane can be caused by various factors such as insufficient penetration of cryoprotectants, insufficient suspension time, cryoprotectant solution toxicity and low temperatures. Dobrinsky [2] notes that the formation of intracellular ice crystals may damage the cytoplasmic membrane and denature intracellular functions and organelles. Neves et al. [13] reported that pacu embryos subjected to freezing and defrosting appeared white and with a ruptured chorion when analyzed under light microscopy. Furthermore, it was observed that the syncytial yolk layer was of varied shape, size and thickness and often located atypically bellow the yolk sac or enveloping separate pieces of the sac, similarly to the damages observed in piracanjuba embryos. Thus, it can be concluded that cooling can cause damages as severe as freezing. As a final remark, the data and results discussed in this study provide significant information that can help optimize the reproduction of piracanjuba and emphasize the need for new protocols to reduce the damage caused by low temperatures. Acknowledgments The authors would like to thank FAPESP (2009/12340-5) and CNPq (3315241710512807) for their financial support, Prof. Dr. João Ademir de Oliveira for the statistical analysis and Duke Energy International and CEPTA/ICMBio – IBAMA for providing the infrastructure and the fish. References [1] M.M. Ahammad, D. Bhattacharyya, B.B. Jana, Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and –2 °C in different concentrations of methanol and sucrose, Theriogenology 60 (2003) 1409–1422. [2] J.R. Dobrinsky, Cellular approach to cryopreservation of embryos, Theriogenology 45 (1996) 17–26.
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