Theriogenology xxx (2015) 1–7
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Some quantitative indicators of postovulatory aging and its effect on larval and juvenile development of Atlantic salmon (Salmo salar) Maren Mommens a, Arne Storset a, Igor Babiak b, * a b
Aqua Gen AS, Trondheim, Norway Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 October 2014 Received in revised form 25 February 2015 Accepted 5 March 2015
Modern out-of-season egg production in Atlantic salmon (Salmo salar) increases the risk of postovulatory aging (POA) of oocytes. Postovulatory aging is known to influence oocyte quality in salmonids, but reliable tests for POA are lacking in Atlantic salmon egg production. To address this problem, we have collected oocytes from the same 20 Atlantic salmon females sequentially in approximately 1-week intervals, from the start of ovulation until 28 days postovulation (dpo), to determine the effect of natural retention of matured oocytes in body coelomic cavity on further performance of embryos and juveniles produced from those oocytes. Also, we investigated oocyte water hardening and several coelomic fluid parameters as potential quantitative indicators of POA. Oocyte quality decreased significantly from 22 dpo onward, as inferred from decrease in fertilization success and survival of embryos, alevins, and juveniles and increase in alevin and juvenile deformity rates. The occurrence of head deformities was significantly related to postovulatory age of oocytes. Coelomic fluid pH decreased significantly at 28 dpo and correlated positively with fertilization rates (r ¼ 0.45), normal eyed embryo rates (r ¼ 0.67), and alevin relative survival rates (r ¼ 0.63) and negatively correlated with total alevin deformity rates (r ¼ 0.59). Oocyte weight gain at 60 minutes decreased significantly at 28 dpo and correlated negatively with total alevin deformities and the occurrence of cranial nodules (r ¼ 0.99). Generally, quality of ovulated oocytes remained stable for the first 2 weeks after ovulation. Later on, POA negatively influenced Atlantic salmon embryo, alevin, and juvenile performance. For the first time, we show a long-term effect of POA on salmonid juvenile performance. Standardized pH measurements of coelomic fluid could potentially improve embryo and juvenile production by identifying low-quality oocytes at an early stage during the production. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Aquaculture Atlantic salmon Embryonic development Gamete quality Postovulatory aging
1. Introduction The Atlantic salmon (Salmo salar) is one of the most important aquaculture species in the world. The global harvest of farmed Atlantic salmon in 2013 was 1.84 million metric tons with a value of 4.9 billion V (Salmon World
* Corresponding author. Tel.: þ47 75517922; fax: þ47 75517457. E-mail address: [email protected] (I. Babiak). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.03.001
2014, Kontali Analyse AS: http://kontali.no). The Atlantic salmon is an anadromous and iteroparous salmonid, although approximately 11% of spawners survive and breed in another year [1]. Held in captivity, Atlantic salmon do not spawn spontaneously and oocytes have to be expelled from the abdominal cavity manually by stripping. An experienced fish farmer can, to a great degree, decide the optimal timing for stripping by observing and palpating the belly of the brood fish. However, this has become more difficult in modern out-of-season egg production involving
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photoperiod and temperature manipulations to a brood stock. No research-based knowledge is available about the optimal timing for stripping of Atlantic salmon in relation to ovulation. The width of the spawning window is temperature dependent, with higher and lower temperatures curtailing and extending the period of optimum ripeness, respectively. Late stripping may induce postovulatory aging (POA) of oocytes retained in the abdominal cavity of a salmonid female after the ovulation. In rainbow trout (Oncorhynchus mykiss), POA leads to morphologic and biochemical changes in an oocyte with hardly noticeable morphologic changes in its appearance [2–4]. A reduction in oocyte quality is manifested in the form of decreased fertilization and further survival success accompanied by increase in morphologic abnormalities and spontaneous occurrence of triploidy [4–7]. Timing of POA depends on the fish species. In goldfish (Carassius auratus), fertilization and hatching rates are dramatically reduced within a few hours after ovulation [8]. In turbot (Scophthalmus maximus), no survival at hatching was recorded after fertilization of oocytes retained in the lumen for 24 hours postovulation [9]. In contrast, salmonids can hold matured oocytes in their body cavity for 1 to 3 weeks, depending on the holding temperature, without a significant decrease in developmental competence [6,10]. However, after the prolonged retention time in the abdominal cavity, oocyte quality can be highly variable among females [6]. Quality of oocytes can be evaluated on the basis of physiochemical properties of oocytes and ovarian or coelomic fluid [2–4,10–12]. In rainbow trout, positive correlations have been found between the weight of the hardened eggs, oocyte lipid and protein concentrations, and egg viability [2,3]. Decrease in coelomic fluid pH and increase in the level of proteins and fatty acids, as well as the activity of aspartate aminotransferase and acid phosphatase in the coelomic fluid, have been found significantly related to POA [3,6,11]. Despite the economic importance of Atlantic salmon production, the possible effect of POA on oocyte quality and further larval and juvenile performance has not been systematically quantified yet. Also, simple and reliable tests of oocyte quality in Atlantic salmon are lacking. In this study, we report how POA influences embryonic, alevin, and juvenile survival and performance in Atlantic salmon being held at the industrial facility of a commercial salmon egg producer. In addition, coelomic fluid parameters and egg water hardening were evaluated as possible predictors of oocyte quality during the production. 2. Materials and methods 2.1. Fish husbandry and gametes collection AquaGen’s brood stock production follows the Code of Good Practice for Farm Animal Breeding and Reproduction Organizations (Code-EFABAR, http://www.effab.org/ CODEEFABAR.aspx). The experiment in this study compiled with the guidelines set by the National Animal Research Authority (Forsøksdyrutvalget, Norway). Fish used in this study were 3-year-old elite breeders from AquaGen’s breeding brood stock population. On May 23, 2011, fish were moved from sea cages to 50 m3 tanks (14.9 1.2 C, pH
6.8 0.4, 104.4 5.0% O2, 10.8 4.9 mg/L CO2) at AquaGen AS facilities in Kyrksæterøra, Norway (63170 2600 N 09 050 2000 E). One month before expected stripping, water temperature was reduced to 7.39 0.3 C (pH 6.8 0.4, 105.1 5.7% O2, 8.9 2.9 mg/L CO2). Females were closely monitored for ovulation readiness once a week. One week before the start of experiment, the exact experimental group (n ¼ 20, mean weight standard deviation: 11.8 0.6 kg) was selected to secure similar timing of the start of ovulation predicted for the next week. Before stripping, females were anesthetized with Finquel vet. (0.0004 g/L; ScanVacc, Norway). All 20 females were hand stripped approximately once a week for 4 consecutive weeks. At each stripping, only a portion of oocytes (n ¼ 1000) was collected. Sampling dates were as follows: October 28, 2011 (Day 0), November 4, (Day 7 postovulation, dpo), November 11 (14 dpo), November 19 (22 dpo), and November 25 (28 dpo). On Day 0, fish were individually marked with unique ID tags (#601201; Sokymat Identification, Cynthiana, KY, USA). Two females died after the first stripping and were replaced with two new females from the same brood stock, which were stripped for eggs at the following dates: November 4, 2011 (Day 0), November 11 (7 dpo), November 19 (14 dpo), November 25 (22 dpo), and December 2 (28 dpo). Autopsy of the dead females did not reveal the cause of death. To reduce the paternal effect on embryonic performance, cryopreserved sperm from a single male was used throughout the whole study to fertilize batches of eggs. Sperm samples were collected on October 4, 2011, from a single male (3 years old, 13.5 kg). Sperm motility was greater than 90%. Sperm was cryopreserved in 12.5-mL SquarePacks with a sperm density of 5 109/mL (Cryogenetics, Hamar, Norway), following the routine protocol for Atlantic salmon applied in AquaGen production. 2.2. Embryo production, larval and juvenile rearing, and egg quality measures Electric conductivity (EC) and pH were measured in coelomic fluid (HI 9321; Hanna Instruments, MI, USA), and 1 mL of coelomic fluid was frozen at 40 C for osmolality measurements (Fiske One-Ten Osmometer; Fiske Associates, MA, USA). Oocyte water hardening was estimated according to Lahnsteiner et al. [12]. For embryo production, oocytes from each batch were fertilized with 3 mL of cryopreserved sperm (diluted in Cryogenetic’s extender, Cryogenetics). Fertilized eggs were incubated in duplicates in raceways (8 0.4 C, pH 6.8 0.3, 105.4 3.0% O2, 8.8 2.9 mg/L CO2). Fertilization success (%) was estimated at 1 day after fertilization (two–eight-cell stage), and normal eyed embryos were estimated at 320 day-degree. Dead embryos were counted and removed. From hatching to yolk sac resorption (920 day-degree), dead alevins were collected to estimate relative survival (%). At yolk sac resorption, alevin deformities (%) were quantified. Deformities were categorized into spine deformities (scoliosis, lordosis), yolk sac deformities (yolk sac strangulation, edema), head deformities (cyclops, gaping), and Siamese. To start feeding, groups of alevins from each of the 5 dpo groups were pooled in duplicate tanks (0.5 m3), kept at 12 0.3 C (pH 6.5 0.2, 108.2 3.3% O2, 10.5 4.6 mg/L
M. Mommens et al. / Theriogenology xxx (2015) 1–7
CO2), and fed EWOA Start (EWOS, Bergen, Norway) until 5g body weight (mean), and the fry were fed EWOS Yngel diet afterward. After 180 days of feeding, weight (g), relative survival (%), and deformities of juveniles (%) were quantified. Deformities were categorized into spine deformities (lordosis, scoliosis, and shortened spine) and head deformities (shortened opercula, microstomia, prognathia, or missing eye). 2.3. Statistical analysis Statistical analyses of quantitative parameters and performance were conducted using SAS 9.3 software (SAS Institute Inc., Cary, NC, USA). Analysis of variance was used to test the effect of dpo, and unbalanced repeatedmeasures ANOVA was used to test the effect of female on coelomic fluid pH, EC, osmolality, oocyte dry weight, oocyte water hardening, fertilization success, and embryonic, alevin, and juvenile survival and deformities [13]. A post hoc least squares means test was performed to determine significance of differences between time points. Percentage data were arc sin square root transformed. Correlations were estimated using Pearson’s correlation coefficient (r). 3. Results 3.1. Coelomic fluid parameters Coelomic fluid pH was decreasing gradually in the course of the experiment, from 8.4 0.1 at Day 0 to 8.0 0.1 at 28 dpo (F(4, 93) ¼ 30.7, P < 0.0001, Fig. 1A). Electric conductivity remained stable in the course of the experiment (Fig. 1B). Coelomic fluid osmolality increased from 272.2 23.5 mOsm/kg at Day 0 to 299.8 15.3 mOsm/kg at 28 dpo (F(4, 91) ¼ 5.2, P < 0.001, Fig. 1C). No significant effect of females on coelomic fluid pH, EC, and osmolality was found. 3.2. Oocyte water hardening Oocyte dry weight stayed constant in 0 to 28 dpo oocytes (Fig. 1D). After 15 minutes of water hardening, oocyte weight gain decreased significantly in 22 dpo oocytes (F(4, 96) ¼ 7.0, P < 0.0001, Fig. 1E). No significant differences in oocyte weight gain were observed after 30 minutes (Fig. 1F). In 28 dpo oocytes, the differences were significant after 60 minutes (F(4, 96) ¼ 14.3, P < 0.0001, Fig. 1G). Between 15 and 30 minutes of water hardening, no significant differences in oocyte weight gain were observed (Fig. 1H). Between 30 and 60 minutes of water hardening, oocyte weight gain decreased in 28 dpo oocytes (F(4, 96) ¼ 6.6, P < 0.0001, Fig. 1I). No significant differences in oocyte water hardening were found between females. 3.3. Fertilization, embryonic survival, and alevin performance The effect of oocytes’ age on the fertilization rate was significant (F(4, 74) ¼ 7.5, P < 0.0001, Fig. 2A). Fertilization rates ranged from 92 5% at Day 0 to 66 25% at 22 dpo oocytes; however, the difference between 0 and 28 dpo was not significant (Fig. 2A). The rate of normally developing
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embryos at eyed embryo stage was decreasing gradually from 84 5% to 5.3 17% (F(4, 96) ¼ 87.3, P < 0.0001, Fig. 2B). Relative survival of alevins was decreasing from 96 2.3% to 27 34% in the course of the study (F(4, 95) ¼ 31.4, P < 0.0001, Fig. 2C). From 22 dpo onward, the decrease in both parameters was significant as compared to Day 0 (Fig. 2B, C). Occurrence of alevin deformities increased significantly at the end of the study, from 0.2 0.5% at 0 dpo to 6.4 7.1% at 28 dpo (F(4, 95) ¼ 12.5, P < 0.0001, Fig. 2D). The most common form of alevin deformity was yolk sac edema (56 6%, Supplementary Table 1). Variation (coefficient of variation, %) in fertilization success, normal eyed embryo, and relative alevin survival rates was increasing systematically in time with maximum rates at 28 dpo (Fig. 3A–C). 3.4. Juvenile performance Juvenile relative survival ranged from 87 7% at Day 0 to 93 3% at 7 dpo group, and it stayed constant in juveniles produced from 14 to 28 dpo oocytes (F(4, 4520) ¼ 166.6, P < 0.0001, Fig. 4A). Weight of juveniles ranged from 23 8 g (28 dpo group) to 30 10 g (22 dpo group), and fish from 14 to 22 dpo groups were significantly heavier than fish from the other groups (F(4, 4520) ¼ 96.2, P < 0.0001, Fig. 4B). Age of oocytes (dpo) did not influence the amount of total deformities in juveniles (Fig. 4C), but the type of deformity (F(4, 221) ¼ 79.0, P < 0.0001). Shortened opercula were the most frequent type of deformity (82%, Supplementary Table 2) followed by other head deformities, such as cranial nodules, microstomia, and prognathia. Deformities of the spinal column were manifested as lordosis or shortened spinal column anterior and posterior (mean: 2%, 1%, and 4%, respectively; Supplementary Table 2). Juveniles with shortened opercula were present in 0 to 22 dpo groups, whereas they were absent in the 28 dpo group (F(4, 4018) ¼ 3.8, P < 0.01). Juveniles suffering from cranial nodules were only present in the 28 dpo group (F(4,4018) ¼ 34.03, P < 0.0001, Fig. 4D). Microstomia was present in juveniles produced from 22 dpo and 28 dpo oocytes (F(4, 4018) ¼ 12.1, P < 0.0001, Fig. 4E), whereas prognathia was observed only in the 22 dpo group (Fig. 4F). 3.5. Correlations Coelomic fluid pH correlated significantly and positively with fertilization (r ¼ 0.45, P < 0.0001), normal eyed embryos (r ¼ 0.67, P < 0.0001), and alevin relative survival rates (r ¼ 0.63, P < 0.0001) and negatively correlated with total alevin deformities (r ¼ 0.59, P < 0.0001) throughout the study. Coelomic fluid osmolality correlated highly and positively with the occurrence of cranial nodes (r ¼ 0.87, P < 0.001). Oocyte weight gain at 15, 30, and 60 minutes after the start of water hardening correlated positively with fertilization (r ¼ 0.49, P < 0.0001; r ¼ 0.49, P < 0.0001; and r ¼ 0.39, P < 0.001), normal eyed embryos (r ¼ 0.49, P < 0.0001; r ¼ 0.39, P < 0.001; and r ¼ 0.48, P < 0.0001), and alevin survival rates (r ¼ 0.45, P < 0.0001; r ¼ 0.37, P < 0.001; and r ¼ 0.49, P < 0.0001) throughout the study. Oocyte weight gain at 60 minutes correlated negatively with total alevin deformities (r ¼ 0.56, P < 0.0001).
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Fig. 1. Atlantic salmon coelomic fluid parameters (A, pH; B, electric conductivity; C, osmolality) and oocyte weight gain (D, total dry weight; E-I, change in dry weight within 15 minutes, 30 minutes, 60 minutes, 15-30 minutes, and 30-60 minutes from fertilization, respectively) during water hardening in relation to postovulatory aging. Mean (standard deviation) values are given for oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters differ significantly from each other at P < 0.0001.
Oocyte weight gain at 30 and 60 minutes correlated highly and negatively with cranial nodes occurrence in juveniles (r ¼ 0.96, P < 0.0001 and r ¼ 0.99, P < 0.0001). 4. Discussion A decrease in coelomic fluid pH has previously been found related with POA in rainbow trout, turbot, lake trout (Salmo trutta lacustris), Caspian brown trout (Salmo trutta caspius), channel catfish (Ictalurus punctatus) blue catfish (Ictalurus furcatus) hybrids, and Atlantic halibut (Hippoglossus hippoglossus) [3,9,14–17]. In the present study, Atlantic salmon coelomic fluid pH decreased with increasing dpo and correlated with oocyte quality at the embryo and alevin stage (Fig. 1A). Similarly, oocyte weight gain during water hardening decreased in time and correlated with oocyte quality at the embryo and alevin stage (Fig. 1E, G, I). Coelomic fluid pH correlated with oocyte weight gain; it suggests that pH could directly affect oocyte membrane potential during activation and hence oocyte water balance during water hardening. Low pH in oocytes has been shown to slow down the velocity of calcium ion
wave at the cortical reaction during oocyte activation in medaka (Oryzias latipes), reducing water hardening potential [18]. In rainbow trout, only partial extrusion of cortical vesicles in over-mature oocytes was observed along with a decrease in oocyte weight gain during the water hardening [3]. Because fertilization and activation of oocytes in the present study were performed after removal of the coelomic fluid, any potential effect of coelomic fluid pH on oocyte activation and quality must take place while the oocytes are stored in the abdominal cavity. In the present study, coelomic fluid osmolality increased, whereas oocyte dry weight remained the same in the course of the experiment (Fig. 1C, D). This is in contrast to previous findings in rainbow trout, where coelomic fluid osmolality decreased and oocyte dry weight increased along with dpo [3,6]. The release of osmolytes from degrading oocytes could hypothetically increase coelomic fluid osmolality; however, a leakage from intact, though over-mature oocytes, could not be confirmed previously in rainbow trout [3]. Also in the present study, no significant correlation between osmolality and oocyte dry weight was found. Also, no broken oocytes were observed at stripping in the course of this study that
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Fig. 2. Atlantic salmon embryonic and alevin performance in relation to postovulatory aging (A, fertilization; B, normal embryos at eyed stage; C, survival of alevins; D, deformities in alevins). Mean (standard deviation) values are given for oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters differ significantly from each other at P < 0.0001.
could explain an increase in osmolality. Therefore, the increase in coelomic fluid osmolality may indicate dehydration of the coelomic fluid due to either secretory changes in the postovulatory ovaries or water absorption in the abdominal cavity during the postovulatory storage; unfortunately, volumetric proportions between oocytes and
coelomic fluid could not be estimated during this study. In turbot, increased levels of proteins and Kþ were found in ovarian fluid with low pH [16]. In the present study, pH and osmolality correlated negatively (r ¼ 0.61, P < 0.001) which indicated a similar relation. As a quantitative measure, osmolality correlated significantly only with oocyte
Fig. 3. Coefficient of variation (%) for Atlantic salmon embryo and alevin survival and performance in relation to postovulatory aging. (A) Fertilization; (B) embryos at eyed stage; (C) survival of alevins from hatching; (D) deformities in alevins. dpo, days postovulation.
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Fig. 4. Juvenile performance in relation to postovulatory aging. (A) Survival from start feeding; (B) weight; (C) total deformities; (D) cranial nodules; (E) microstomia; (F) prognathia. Mean (standard deviation) values are given for juveniles produced from oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters are significantly different at P < 0.0001.
weight gain after 30 to 60 minutes of water hardening (r ¼ 0.25, P < 0.05) and not with fertilization, normal eyed embryos, and alevin survival rates. This points at pH as a more sensible indicator of oocyte quality in embryonic and alevin stages, compared to osmolality. Oocyte quality started to deteriorate from 22 dpo onward, as found previously in rainbow trout [2–4,6,12]. Variation in fertilization, normal eyed embryos, and relative alevin survival rates among females was increasing progressively with dpo, indicating individual differences in POA effect. Clearly homogenous survival parameters (eyed embryo and alevin survival rates) were obtained only in the 0 and 7 dpo groups (Fig. 3B, C). It indicates that the probability of POA effect in commercial production of eggs in Atlantic salmon has to be taken into consideration from the second week after ovulation onward, although the quantitative effects of POA become significant from the fourth week after ovulation onward (Fig. 2B, C). The effect of POA on juvenile performance has not been studied in salmonids before. Shortened operculum was the most frequent deformity type observed in juveniles; however, it was rather an effect of stocking density (r ¼ 0.94, P < 0.0001). Excluding juveniles with shortened opercula from the analysis did not significantly influence the effect of dpo on total deformities. Skull deformities increased significantly in juveniles produced from 22 and 28 dpo oocytes (Fig. 4D–F). Skeletal deformities in larval and juvenile fish, such as skull deformities, have been linked to a poorly understood relationship between nutrition, environment, and genetic factors during commercial farming [22]. In the present study, cranial nodules were found in juveniles produced from 28 dpo oocytes (Fig. 4D). They arise from the center of the frontal plates and are dorsal to
the optic lobes. Histologically, a fenestra in the front plate underlying the nodule has been observed [20,21]. Microstomia, observed in juveniles produced from 22 and 28 dpo oocytes, is characterized by a displacement of the lower end of the hyoid arch, both downward and backward, through a gap in the mouth floor (Fig. 4E). Prognathia was observed in juveniles produced from 22 dpo oocytes (Fig. 4F). Prognathia is characterized as a shortening of the frontal and maxilla bones of the skull, resulting in undershot maxilla [19]. Here, we describe for the first time, to the best of our knowledge, the maternal effect (POA) on skeletal and specifically skull deformities in a juvenile fish. 4.1. Conclusions Oocyte quality in Atlantic salmon is stably high in a time window of 2 weeks after ovulation. Postovulatory aging significantly decreases fertilization rates, embryo, alevin, and juvenile survival rates, as well as induces the occurrence of deformities in alevins and juveniles. Low coelomic fluid pH is a potential indicator for POA in Atlantic salmon. Standardized pH measurements of Atlantic coelomic fluid could potentially improve juvenile production by identifying low-quality oocytes. Acknowledgments The authors would like to thank Trude Reinholdens at Aqua Gen AS for help during sampling and the production staff at Aqua Gen AS for maintaining eggs, alevins, and juveniles during the experiment. This work was funded by the MABIT program (Norway; #AF0057).
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Appendix A. Supplementary Data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. theriogenology.2015.03.001. References [1] Flemming IA. Pattern and variability in the breeding system of Atlantic salmon (Salmo salar), with comparisons to other salmonids. Can J Fish Aquat Sci 1998;55:59–76. [2] Craik JCA, Harvey SM. Egg quality in rainbow trout: the relation between egg viability, selected aspects of egg composition, and time of stripping. Aquaculture 1984;40:115–34. [3] Lahnsteiner F. Morphological, physiological and biochemical parameters characterizing the over-ripening of rainbow trout eggs. Fish Physiol Biochem 2000;23:107–18. [4] Aegerter S, Jalabert B, Bobe J. Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing. Mol Reprod Dev 2005;385: 377–85. [5] Bromage N, Jones J, Randall C, Thrush M, Davies B, Springate J, et al. Broodstock management, fecundity, egg quality and the timing of egg production in the rainbow trout (Oncorhynchus mykiss). Aquaculture 1992;100:141–66. [6] Aegerter S, Jalabert B. Effects of post-ovulatory oocyte ageing and temperature on egg quality and on the occurrence of triploid fry in rainbow trout, Oncorhynchus mykiss. Aquaculture 2004;231:59–71. [7] Bonnet E, Fostier A, Bobe J. Characterization of rainbow trout egg quality: a case study using four different breeding protocols, with emphasis on the incidence of embryonic malformations. Theriogenology 2007;67:786–94. [8] Formacion MJ, Venkatesh B, Tan CH, Lam TJ. Overripening of ovulated eggs in goldfish, Carassius auratus: II. Possible involvement of postovulatory follicles and steroids. Fish Physiol Biochem 1995;14: 237–46.
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[9] McEvoy LA. Ovulatory rhythms and over-ripening of eggs in cultivated turbot, Scophthalmus maximus L. J Fish Biol 1984;24:437–48. [10] Bahrekazemi M, Matinfar A, Soltani M, Abtahi B, Pusti I, Mohagheghi A. The relation between egg viability, selected aspects of egg and ovarian fluid composition and time of stripping in endangered caspian brown trout. J Fish Aquat Sci 2009;4:306–15. [11] Rime H, Guitton N, Pineau C, Bonnet E, Bobe J, Jalabert B. Postovulatory ageing and egg quality: a proteomic analysis of rainbow trout coelomic fluid. Reprod Biol Endocr 2004;2:26–36. [12] Lahnsteiner F, Patzner RA. Rainbow trout egg quality determination by the relative weight increase during hardening: a practical standardization. J Appl Ichthyol 2002;18:24–6. [13] Zar JH. Biostatistical analysis. Fourth edition. Upper Saddle River: Pearson higher education; 1999. [14] Lahnsteiner F, Weismann T, Patzner RA. Physiological and biochemical parameters for egg quality determination in lake trout, Salmo trutta lacustris. Fish Physiol Biochem 1999;20:375–88. [15] Chatakondi NG, Torrans EL. The influence of ovarian fluid pH of stripped unfertilized channel catfish, Ictalurus punctatus, eggs on the hatching success of channel catfish \ x blue catfish, Ictalurus furcatus_, hybrid catfish eggs. J World Aquacult Soc 2012;43:585–93. [16] Fauvel C, Omnès M, Suquet M, Normant Y. Reliable assessment of overripening in turbot (Scophthalmus maximus) by a simple pH measurement. Aquaculture 1993;117:107–13. [17] Skaalsvik TH, Bolla SL, Thornqvist P-O, Babiak I. Quantitative characteristics of Atlantic halibut (Hippoglossus hippoglossus L.) egg quality throughout the reproductive season. Theriogenology 2015; 83:38–47. [18] Gilkey JC. Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes. J Cell Biol 1983;97:669–78. [19] Bruno DW, Poppe TT. A colour atlas of salmonid diseases. London: Academic Press, Harcourt Brace & Company; 1996. [20] Kent ML, Wellings SR, Yasutake WT, Elston RA. Cranial nodules associated with cranial fenestrae in juvenile Atlantic salmon, Salmo salar L. J Fish Dis 1987;10:419–21. [21] Bruno DW. Cranial nodules in farmed Atlantic salmon, Salmo salar L., fry. J Appl Ichtyol 1997;13:47–8. [22] Lall SP, Lewis-McCrea LM. Role of nutrients in skeletal metabolism and pathology in fishdan overview. Aquaculture 2007;267:3–19.
Parameter
Days after ovulation 0
7
n
Mean SD
20 20 20
8.4 0.1 3.3 0.4 272.2 23.5
14
CV (%)
n
Mean SD
1.2 12.1 4.8
19 19 19
7
22
CV (%)
n
Mean SD
8.4 0.1 3.3 0.4 278.8 21.0
1.2 12.1 7.5
18 19 18
8.3 0.1 3.6 0.5 275.6 20.5
20
117 9
7
19
117 9
28
CV (%)
n
Mean SD
1.4 13.9 7.4
18 18 18
8.3 0.1 3.4 0.3 286.5 17.4
8
18
119 8
25.2 17.2 11.6 25.7 116.6
16 16 18 18 18
8.0 14.3 17.5 6.3 3.2
n
Mean SD
1.2 8.8 6.1
18 16 16
8.0 0.1 3.2 0.7 299.8 15.3
1.3 21.9 5.1
7
18
123 11
9.0
24.5 27.4 18.3 28.6 93.8
16 16 18 18 18
6.6 8.9 8.9 2.3 1.4
6.8 10.2 4.1 2.0 4.0
104.1 114.6 46.1 86.9 282.4
76.1 35.9 5.3 16.7
47.2 188.3
126.8 111.0 282.1 285.4 285.4 70.9 92.6 0 0
CV (%)
20
118 8
20 20 19 20 19
11.7 16.2 17.4 4.5 1.2
1.9 1.6 2.7 2.0 1.2
16.7 9.9 15.7 44.4 100.0
20 20 20 20 20
11.4 15.4 17.5 4.0 2.1
1.7 2.2 1.4 1.8 1.9
14.9 14.3 8.9 46.6 90.5
19 19 19 19 19
10.3 15.7 18.1 5.4 2.4
20 20
92.2 5.2 84.4 5.4
5.6 6.4
20 20
86.0 6.5 78.2 7.6
8.8 9.3
19 19
83.9 8.6 59.0 27.9
10.3 47.6
19 19
66.1 25.0 14.6 20.3
37.8 80.9
18 18
18 18 18 18 18 18 18 18 18
96.1 0.2 4.8 2.5 5.0 55.6 32.5 13.4 5.5
2.4 250.0 457.2 448.0 448.0 83.8 143.7 247.8 258.2
20 20 20 20 20 20 20 20 20
95.1 0.4 19.7 12.0 5.1 74.3 33.3 31.3 1.0
2.3 150.0 150.8 220.0 249.0 45.5 100.6 128.1 450.0
19 19 19 19 19 19 19 19 19
93.9 0.3 7.5 4.0 0.0 38.2 29.6 17.3 7.0
24.6 200.0 312.0 245.0 0 121.5 124.3 159.6 330.0
13 12 11 13 13 12 13 13 11
39.9 2.2 20.5 13.2 7.7 45.8 44.8 0.6 0.0
96.7 177.3 167.3 231.1 359.7 102.6 104.1 366.6 0
8 8 8 8 8 8 8 8 8
2.3 0.5 21.8 11.2 22.4 46.6 46.7 33.2 14.2
2.2 0.6 29.7 26.4 12.7 33.8 33.5 40.1 4.5
Abbreviations: CV, coefficient of variation; EC, electric conductivity; SD, standard deviation.
2.6 2.7 2.1 1.4 2.8
23.1 0.6 23.4 9.8 0.0 46.4 36.8 27.6 23.1
2.0 3.9 3.2 1.8 3.0
38.6 3.9 34.3 30.5 27.7 47.0 46.6 2.2 0.0
26.9 6.4 8.4 4.1 4.1 66.6 54.2 0.0 0.0
34.1 7.1 23.7 11.7 11.7 47.2 50.2 0.0 0.0
CV (%)
M. Mommens et al. / Theriogenology xxx (2015) 1–7
Coelomic fluid pH EC (mS/m) Osmolality (mOsm/kg) Water hardening Dry weight (mg/egg) Weight increase (%) 0–15 min 0–30 min 0–60 min 15–30 min 30–60 min Embryo Fertilization (%) Eyed egg (%) Alevin Relative survival (%) Total deformities (%) Total spine (%) Scoliosis (%) Lordosis (%) Total yolk (%) Yolk sac edema (%) Yolk sac abscission (%) Twins (%)
7.e1
Supplementary Table 1 Atlantic salmon coelomic fluid parameters, oocyte weight gain during water hardening, and embryonic and juvenile performance in relation to postovulatory aging.
M. Mommens et al. / Theriogenology xxx (2015) 1–7
7.e2
Supplementary Table 2 Juvenile performance in relation to postovulatory aging. Parameter
Relative survival (%) Weight (g) Total deformities Total deformities without short operculum Types of deformities Short operculum Lordosis Short spinal column posterior Short spinal column anterior Cranial nodules Microstomia Prognathia
Days after ovulation 0
7
14
22
28
n ¼ 1105
n ¼ 1174
n ¼ 1086
n ¼ 817
n ¼ 338
Mean SD
CV (%)
83.1 26.7 5.2 0.4
6.5 7.5 0.4 0.0
7.8 28.1 7.7 0.0
96.5 2.0 0.0 1.5 0.0 0.0 0.0
0.7 2.8 0.0 2.1 0.0 0.0 0.0
0.7 140.0 0.0 140.0 0.0 0.0 0.0
Mean SD
CV (%)
Mean SD
CV (%)
Mean SD
CV (%)
Mean SD
CV (%)
89.1 27.7 6.0 0.4
4.0 7.7 1.2 1.4
4.5 27.6 20.0 350.0
92.3 31.9 4.9 0.1
0.6 7.9 1.1 0.7
0.6 24.7 22.4 700.0
92.8 30.1 5.7 2.1
4.2 9.5 4.4 2.1
4.5 31.7 77.2 100.0
88.9 22.7 7.4 7.4
5.1 8.3 0.4 0.7
5.8 36.4 5.4 9.5
94.5 1.3 1.7 2.6 0.0 0.0 0.0
3.1 1.8 2.4 3.6 0.0 0.0 0.0
3.3 138.5 141.2 138.5 0.0 0.0 0.0
98.4 0.0 0.0 1.6 0.0 0.0 0.0
2.3 0.0 0.0 2.3 0.0 0.0 0.0
2.3 0.0 0.0 143.8 0.0 0.0 0.0
54.8 0.0 9.5 0.0 0.0 19.0 4.8
16.8 0.0 6.7 0.0 0.0 13.5 6.7
30.7 0.0 70.5 0.0 0.0 71.1 139.6
0.0 6.3 6.3 0.0 46.4 27.7 0.0
0.0a 8.8 8.8 0.0 5.0 21.5 0.0
0.0 139.7 139.7 0.0 11.0 77.6 0.0
Rates of deformities (%, mean SD) are given for total number of juveniles (n) from each days postovulation group. Abbreviations: CV, coefficient of variation; SD, standard deviation.
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Some quantitative indicators of postovulatory aging and its effect on larval and juvenile development of Atlantic salmon (Salmo salar) Maren Mommens a, Arne Storset a, Igor Babiak b, * a b
Aqua Gen AS, Trondheim, Norway Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 October 2014 Received in revised form 25 February 2015 Accepted 5 March 2015
Modern out-of-season egg production in Atlantic salmon (Salmo salar) increases the risk of postovulatory aging (POA) of oocytes. Postovulatory aging is known to influence oocyte quality in salmonids, but reliable tests for POA are lacking in Atlantic salmon egg production. To address this problem, we have collected oocytes from the same 20 Atlantic salmon females sequentially in approximately 1-week intervals, from the start of ovulation until 28 days postovulation (dpo), to determine the effect of natural retention of matured oocytes in body coelomic cavity on further performance of embryos and juveniles produced from those oocytes. Also, we investigated oocyte water hardening and several coelomic fluid parameters as potential quantitative indicators of POA. Oocyte quality decreased significantly from 22 dpo onward, as inferred from decrease in fertilization success and survival of embryos, alevins, and juveniles and increase in alevin and juvenile deformity rates. The occurrence of head deformities was significantly related to postovulatory age of oocytes. Coelomic fluid pH decreased significantly at 28 dpo and correlated positively with fertilization rates (r ¼ 0.45), normal eyed embryo rates (r ¼ 0.67), and alevin relative survival rates (r ¼ 0.63) and negatively correlated with total alevin deformity rates (r ¼ 0.59). Oocyte weight gain at 60 minutes decreased significantly at 28 dpo and correlated negatively with total alevin deformities and the occurrence of cranial nodules (r ¼ 0.99). Generally, quality of ovulated oocytes remained stable for the first 2 weeks after ovulation. Later on, POA negatively influenced Atlantic salmon embryo, alevin, and juvenile performance. For the first time, we show a long-term effect of POA on salmonid juvenile performance. Standardized pH measurements of coelomic fluid could potentially improve embryo and juvenile production by identifying low-quality oocytes at an early stage during the production. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Aquaculture Atlantic salmon Embryonic development Gamete quality Postovulatory aging
1. Introduction The Atlantic salmon (Salmo salar) is one of the most important aquaculture species in the world. The global harvest of farmed Atlantic salmon in 2013 was 1.84 million metric tons with a value of 4.9 billion V (Salmon World
* Corresponding author. Tel.: þ47 75517922; fax: þ47 75517457. E-mail address: [email protected] (I. Babiak). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.03.001
2014, Kontali Analyse AS: http://kontali.no). The Atlantic salmon is an anadromous and iteroparous salmonid, although approximately 11% of spawners survive and breed in another year [1]. Held in captivity, Atlantic salmon do not spawn spontaneously and oocytes have to be expelled from the abdominal cavity manually by stripping. An experienced fish farmer can, to a great degree, decide the optimal timing for stripping by observing and palpating the belly of the brood fish. However, this has become more difficult in modern out-of-season egg production involving
2
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photoperiod and temperature manipulations to a brood stock. No research-based knowledge is available about the optimal timing for stripping of Atlantic salmon in relation to ovulation. The width of the spawning window is temperature dependent, with higher and lower temperatures curtailing and extending the period of optimum ripeness, respectively. Late stripping may induce postovulatory aging (POA) of oocytes retained in the abdominal cavity of a salmonid female after the ovulation. In rainbow trout (Oncorhynchus mykiss), POA leads to morphologic and biochemical changes in an oocyte with hardly noticeable morphologic changes in its appearance [2–4]. A reduction in oocyte quality is manifested in the form of decreased fertilization and further survival success accompanied by increase in morphologic abnormalities and spontaneous occurrence of triploidy [4–7]. Timing of POA depends on the fish species. In goldfish (Carassius auratus), fertilization and hatching rates are dramatically reduced within a few hours after ovulation [8]. In turbot (Scophthalmus maximus), no survival at hatching was recorded after fertilization of oocytes retained in the lumen for 24 hours postovulation [9]. In contrast, salmonids can hold matured oocytes in their body cavity for 1 to 3 weeks, depending on the holding temperature, without a significant decrease in developmental competence [6,10]. However, after the prolonged retention time in the abdominal cavity, oocyte quality can be highly variable among females [6]. Quality of oocytes can be evaluated on the basis of physiochemical properties of oocytes and ovarian or coelomic fluid [2–4,10–12]. In rainbow trout, positive correlations have been found between the weight of the hardened eggs, oocyte lipid and protein concentrations, and egg viability [2,3]. Decrease in coelomic fluid pH and increase in the level of proteins and fatty acids, as well as the activity of aspartate aminotransferase and acid phosphatase in the coelomic fluid, have been found significantly related to POA [3,6,11]. Despite the economic importance of Atlantic salmon production, the possible effect of POA on oocyte quality and further larval and juvenile performance has not been systematically quantified yet. Also, simple and reliable tests of oocyte quality in Atlantic salmon are lacking. In this study, we report how POA influences embryonic, alevin, and juvenile survival and performance in Atlantic salmon being held at the industrial facility of a commercial salmon egg producer. In addition, coelomic fluid parameters and egg water hardening were evaluated as possible predictors of oocyte quality during the production. 2. Materials and methods 2.1. Fish husbandry and gametes collection AquaGen’s brood stock production follows the Code of Good Practice for Farm Animal Breeding and Reproduction Organizations (Code-EFABAR, http://www.effab.org/ CODEEFABAR.aspx). The experiment in this study compiled with the guidelines set by the National Animal Research Authority (Forsøksdyrutvalget, Norway). Fish used in this study were 3-year-old elite breeders from AquaGen’s breeding brood stock population. On May 23, 2011, fish were moved from sea cages to 50 m3 tanks (14.9 1.2 C, pH
6.8 0.4, 104.4 5.0% O2, 10.8 4.9 mg/L CO2) at AquaGen AS facilities in Kyrksæterøra, Norway (63170 2600 N 09 050 2000 E). One month before expected stripping, water temperature was reduced to 7.39 0.3 C (pH 6.8 0.4, 105.1 5.7% O2, 8.9 2.9 mg/L CO2). Females were closely monitored for ovulation readiness once a week. One week before the start of experiment, the exact experimental group (n ¼ 20, mean weight standard deviation: 11.8 0.6 kg) was selected to secure similar timing of the start of ovulation predicted for the next week. Before stripping, females were anesthetized with Finquel vet. (0.0004 g/L; ScanVacc, Norway). All 20 females were hand stripped approximately once a week for 4 consecutive weeks. At each stripping, only a portion of oocytes (n ¼ 1000) was collected. Sampling dates were as follows: October 28, 2011 (Day 0), November 4, (Day 7 postovulation, dpo), November 11 (14 dpo), November 19 (22 dpo), and November 25 (28 dpo). On Day 0, fish were individually marked with unique ID tags (#601201; Sokymat Identification, Cynthiana, KY, USA). Two females died after the first stripping and were replaced with two new females from the same brood stock, which were stripped for eggs at the following dates: November 4, 2011 (Day 0), November 11 (7 dpo), November 19 (14 dpo), November 25 (22 dpo), and December 2 (28 dpo). Autopsy of the dead females did not reveal the cause of death. To reduce the paternal effect on embryonic performance, cryopreserved sperm from a single male was used throughout the whole study to fertilize batches of eggs. Sperm samples were collected on October 4, 2011, from a single male (3 years old, 13.5 kg). Sperm motility was greater than 90%. Sperm was cryopreserved in 12.5-mL SquarePacks with a sperm density of 5 109/mL (Cryogenetics, Hamar, Norway), following the routine protocol for Atlantic salmon applied in AquaGen production. 2.2. Embryo production, larval and juvenile rearing, and egg quality measures Electric conductivity (EC) and pH were measured in coelomic fluid (HI 9321; Hanna Instruments, MI, USA), and 1 mL of coelomic fluid was frozen at 40 C for osmolality measurements (Fiske One-Ten Osmometer; Fiske Associates, MA, USA). Oocyte water hardening was estimated according to Lahnsteiner et al. [12]. For embryo production, oocytes from each batch were fertilized with 3 mL of cryopreserved sperm (diluted in Cryogenetic’s extender, Cryogenetics). Fertilized eggs were incubated in duplicates in raceways (8 0.4 C, pH 6.8 0.3, 105.4 3.0% O2, 8.8 2.9 mg/L CO2). Fertilization success (%) was estimated at 1 day after fertilization (two–eight-cell stage), and normal eyed embryos were estimated at 320 day-degree. Dead embryos were counted and removed. From hatching to yolk sac resorption (920 day-degree), dead alevins were collected to estimate relative survival (%). At yolk sac resorption, alevin deformities (%) were quantified. Deformities were categorized into spine deformities (scoliosis, lordosis), yolk sac deformities (yolk sac strangulation, edema), head deformities (cyclops, gaping), and Siamese. To start feeding, groups of alevins from each of the 5 dpo groups were pooled in duplicate tanks (0.5 m3), kept at 12 0.3 C (pH 6.5 0.2, 108.2 3.3% O2, 10.5 4.6 mg/L
M. Mommens et al. / Theriogenology xxx (2015) 1–7
CO2), and fed EWOA Start (EWOS, Bergen, Norway) until 5g body weight (mean), and the fry were fed EWOS Yngel diet afterward. After 180 days of feeding, weight (g), relative survival (%), and deformities of juveniles (%) were quantified. Deformities were categorized into spine deformities (lordosis, scoliosis, and shortened spine) and head deformities (shortened opercula, microstomia, prognathia, or missing eye). 2.3. Statistical analysis Statistical analyses of quantitative parameters and performance were conducted using SAS 9.3 software (SAS Institute Inc., Cary, NC, USA). Analysis of variance was used to test the effect of dpo, and unbalanced repeatedmeasures ANOVA was used to test the effect of female on coelomic fluid pH, EC, osmolality, oocyte dry weight, oocyte water hardening, fertilization success, and embryonic, alevin, and juvenile survival and deformities [13]. A post hoc least squares means test was performed to determine significance of differences between time points. Percentage data were arc sin square root transformed. Correlations were estimated using Pearson’s correlation coefficient (r). 3. Results 3.1. Coelomic fluid parameters Coelomic fluid pH was decreasing gradually in the course of the experiment, from 8.4 0.1 at Day 0 to 8.0 0.1 at 28 dpo (F(4, 93) ¼ 30.7, P < 0.0001, Fig. 1A). Electric conductivity remained stable in the course of the experiment (Fig. 1B). Coelomic fluid osmolality increased from 272.2 23.5 mOsm/kg at Day 0 to 299.8 15.3 mOsm/kg at 28 dpo (F(4, 91) ¼ 5.2, P < 0.001, Fig. 1C). No significant effect of females on coelomic fluid pH, EC, and osmolality was found. 3.2. Oocyte water hardening Oocyte dry weight stayed constant in 0 to 28 dpo oocytes (Fig. 1D). After 15 minutes of water hardening, oocyte weight gain decreased significantly in 22 dpo oocytes (F(4, 96) ¼ 7.0, P < 0.0001, Fig. 1E). No significant differences in oocyte weight gain were observed after 30 minutes (Fig. 1F). In 28 dpo oocytes, the differences were significant after 60 minutes (F(4, 96) ¼ 14.3, P < 0.0001, Fig. 1G). Between 15 and 30 minutes of water hardening, no significant differences in oocyte weight gain were observed (Fig. 1H). Between 30 and 60 minutes of water hardening, oocyte weight gain decreased in 28 dpo oocytes (F(4, 96) ¼ 6.6, P < 0.0001, Fig. 1I). No significant differences in oocyte water hardening were found between females. 3.3. Fertilization, embryonic survival, and alevin performance The effect of oocytes’ age on the fertilization rate was significant (F(4, 74) ¼ 7.5, P < 0.0001, Fig. 2A). Fertilization rates ranged from 92 5% at Day 0 to 66 25% at 22 dpo oocytes; however, the difference between 0 and 28 dpo was not significant (Fig. 2A). The rate of normally developing
3
embryos at eyed embryo stage was decreasing gradually from 84 5% to 5.3 17% (F(4, 96) ¼ 87.3, P < 0.0001, Fig. 2B). Relative survival of alevins was decreasing from 96 2.3% to 27 34% in the course of the study (F(4, 95) ¼ 31.4, P < 0.0001, Fig. 2C). From 22 dpo onward, the decrease in both parameters was significant as compared to Day 0 (Fig. 2B, C). Occurrence of alevin deformities increased significantly at the end of the study, from 0.2 0.5% at 0 dpo to 6.4 7.1% at 28 dpo (F(4, 95) ¼ 12.5, P < 0.0001, Fig. 2D). The most common form of alevin deformity was yolk sac edema (56 6%, Supplementary Table 1). Variation (coefficient of variation, %) in fertilization success, normal eyed embryo, and relative alevin survival rates was increasing systematically in time with maximum rates at 28 dpo (Fig. 3A–C). 3.4. Juvenile performance Juvenile relative survival ranged from 87 7% at Day 0 to 93 3% at 7 dpo group, and it stayed constant in juveniles produced from 14 to 28 dpo oocytes (F(4, 4520) ¼ 166.6, P < 0.0001, Fig. 4A). Weight of juveniles ranged from 23 8 g (28 dpo group) to 30 10 g (22 dpo group), and fish from 14 to 22 dpo groups were significantly heavier than fish from the other groups (F(4, 4520) ¼ 96.2, P < 0.0001, Fig. 4B). Age of oocytes (dpo) did not influence the amount of total deformities in juveniles (Fig. 4C), but the type of deformity (F(4, 221) ¼ 79.0, P < 0.0001). Shortened opercula were the most frequent type of deformity (82%, Supplementary Table 2) followed by other head deformities, such as cranial nodules, microstomia, and prognathia. Deformities of the spinal column were manifested as lordosis or shortened spinal column anterior and posterior (mean: 2%, 1%, and 4%, respectively; Supplementary Table 2). Juveniles with shortened opercula were present in 0 to 22 dpo groups, whereas they were absent in the 28 dpo group (F(4, 4018) ¼ 3.8, P < 0.01). Juveniles suffering from cranial nodules were only present in the 28 dpo group (F(4,4018) ¼ 34.03, P < 0.0001, Fig. 4D). Microstomia was present in juveniles produced from 22 dpo and 28 dpo oocytes (F(4, 4018) ¼ 12.1, P < 0.0001, Fig. 4E), whereas prognathia was observed only in the 22 dpo group (Fig. 4F). 3.5. Correlations Coelomic fluid pH correlated significantly and positively with fertilization (r ¼ 0.45, P < 0.0001), normal eyed embryos (r ¼ 0.67, P < 0.0001), and alevin relative survival rates (r ¼ 0.63, P < 0.0001) and negatively correlated with total alevin deformities (r ¼ 0.59, P < 0.0001) throughout the study. Coelomic fluid osmolality correlated highly and positively with the occurrence of cranial nodes (r ¼ 0.87, P < 0.001). Oocyte weight gain at 15, 30, and 60 minutes after the start of water hardening correlated positively with fertilization (r ¼ 0.49, P < 0.0001; r ¼ 0.49, P < 0.0001; and r ¼ 0.39, P < 0.001), normal eyed embryos (r ¼ 0.49, P < 0.0001; r ¼ 0.39, P < 0.001; and r ¼ 0.48, P < 0.0001), and alevin survival rates (r ¼ 0.45, P < 0.0001; r ¼ 0.37, P < 0.001; and r ¼ 0.49, P < 0.0001) throughout the study. Oocyte weight gain at 60 minutes correlated negatively with total alevin deformities (r ¼ 0.56, P < 0.0001).
4
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Fig. 1. Atlantic salmon coelomic fluid parameters (A, pH; B, electric conductivity; C, osmolality) and oocyte weight gain (D, total dry weight; E-I, change in dry weight within 15 minutes, 30 minutes, 60 minutes, 15-30 minutes, and 30-60 minutes from fertilization, respectively) during water hardening in relation to postovulatory aging. Mean (standard deviation) values are given for oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters differ significantly from each other at P < 0.0001.
Oocyte weight gain at 30 and 60 minutes correlated highly and negatively with cranial nodes occurrence in juveniles (r ¼ 0.96, P < 0.0001 and r ¼ 0.99, P < 0.0001). 4. Discussion A decrease in coelomic fluid pH has previously been found related with POA in rainbow trout, turbot, lake trout (Salmo trutta lacustris), Caspian brown trout (Salmo trutta caspius), channel catfish (Ictalurus punctatus) blue catfish (Ictalurus furcatus) hybrids, and Atlantic halibut (Hippoglossus hippoglossus) [3,9,14–17]. In the present study, Atlantic salmon coelomic fluid pH decreased with increasing dpo and correlated with oocyte quality at the embryo and alevin stage (Fig. 1A). Similarly, oocyte weight gain during water hardening decreased in time and correlated with oocyte quality at the embryo and alevin stage (Fig. 1E, G, I). Coelomic fluid pH correlated with oocyte weight gain; it suggests that pH could directly affect oocyte membrane potential during activation and hence oocyte water balance during water hardening. Low pH in oocytes has been shown to slow down the velocity of calcium ion
wave at the cortical reaction during oocyte activation in medaka (Oryzias latipes), reducing water hardening potential [18]. In rainbow trout, only partial extrusion of cortical vesicles in over-mature oocytes was observed along with a decrease in oocyte weight gain during the water hardening [3]. Because fertilization and activation of oocytes in the present study were performed after removal of the coelomic fluid, any potential effect of coelomic fluid pH on oocyte activation and quality must take place while the oocytes are stored in the abdominal cavity. In the present study, coelomic fluid osmolality increased, whereas oocyte dry weight remained the same in the course of the experiment (Fig. 1C, D). This is in contrast to previous findings in rainbow trout, where coelomic fluid osmolality decreased and oocyte dry weight increased along with dpo [3,6]. The release of osmolytes from degrading oocytes could hypothetically increase coelomic fluid osmolality; however, a leakage from intact, though over-mature oocytes, could not be confirmed previously in rainbow trout [3]. Also in the present study, no significant correlation between osmolality and oocyte dry weight was found. Also, no broken oocytes were observed at stripping in the course of this study that
M. Mommens et al. / Theriogenology xxx (2015) 1–7
5
Fig. 2. Atlantic salmon embryonic and alevin performance in relation to postovulatory aging (A, fertilization; B, normal embryos at eyed stage; C, survival of alevins; D, deformities in alevins). Mean (standard deviation) values are given for oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters differ significantly from each other at P < 0.0001.
could explain an increase in osmolality. Therefore, the increase in coelomic fluid osmolality may indicate dehydration of the coelomic fluid due to either secretory changes in the postovulatory ovaries or water absorption in the abdominal cavity during the postovulatory storage; unfortunately, volumetric proportions between oocytes and
coelomic fluid could not be estimated during this study. In turbot, increased levels of proteins and Kþ were found in ovarian fluid with low pH [16]. In the present study, pH and osmolality correlated negatively (r ¼ 0.61, P < 0.001) which indicated a similar relation. As a quantitative measure, osmolality correlated significantly only with oocyte
Fig. 3. Coefficient of variation (%) for Atlantic salmon embryo and alevin survival and performance in relation to postovulatory aging. (A) Fertilization; (B) embryos at eyed stage; (C) survival of alevins from hatching; (D) deformities in alevins. dpo, days postovulation.
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Fig. 4. Juvenile performance in relation to postovulatory aging. (A) Survival from start feeding; (B) weight; (C) total deformities; (D) cranial nodules; (E) microstomia; (F) prognathia. Mean (standard deviation) values are given for juveniles produced from oocyte groups collected at five different days postovulation (dpo; 0, 7, 14, 22, and 28). Values marked with different letters are significantly different at P < 0.0001.
weight gain after 30 to 60 minutes of water hardening (r ¼ 0.25, P < 0.05) and not with fertilization, normal eyed embryos, and alevin survival rates. This points at pH as a more sensible indicator of oocyte quality in embryonic and alevin stages, compared to osmolality. Oocyte quality started to deteriorate from 22 dpo onward, as found previously in rainbow trout [2–4,6,12]. Variation in fertilization, normal eyed embryos, and relative alevin survival rates among females was increasing progressively with dpo, indicating individual differences in POA effect. Clearly homogenous survival parameters (eyed embryo and alevin survival rates) were obtained only in the 0 and 7 dpo groups (Fig. 3B, C). It indicates that the probability of POA effect in commercial production of eggs in Atlantic salmon has to be taken into consideration from the second week after ovulation onward, although the quantitative effects of POA become significant from the fourth week after ovulation onward (Fig. 2B, C). The effect of POA on juvenile performance has not been studied in salmonids before. Shortened operculum was the most frequent deformity type observed in juveniles; however, it was rather an effect of stocking density (r ¼ 0.94, P < 0.0001). Excluding juveniles with shortened opercula from the analysis did not significantly influence the effect of dpo on total deformities. Skull deformities increased significantly in juveniles produced from 22 and 28 dpo oocytes (Fig. 4D–F). Skeletal deformities in larval and juvenile fish, such as skull deformities, have been linked to a poorly understood relationship between nutrition, environment, and genetic factors during commercial farming [22]. In the present study, cranial nodules were found in juveniles produced from 28 dpo oocytes (Fig. 4D). They arise from the center of the frontal plates and are dorsal to
the optic lobes. Histologically, a fenestra in the front plate underlying the nodule has been observed [20,21]. Microstomia, observed in juveniles produced from 22 and 28 dpo oocytes, is characterized by a displacement of the lower end of the hyoid arch, both downward and backward, through a gap in the mouth floor (Fig. 4E). Prognathia was observed in juveniles produced from 22 dpo oocytes (Fig. 4F). Prognathia is characterized as a shortening of the frontal and maxilla bones of the skull, resulting in undershot maxilla [19]. Here, we describe for the first time, to the best of our knowledge, the maternal effect (POA) on skeletal and specifically skull deformities in a juvenile fish. 4.1. Conclusions Oocyte quality in Atlantic salmon is stably high in a time window of 2 weeks after ovulation. Postovulatory aging significantly decreases fertilization rates, embryo, alevin, and juvenile survival rates, as well as induces the occurrence of deformities in alevins and juveniles. Low coelomic fluid pH is a potential indicator for POA in Atlantic salmon. Standardized pH measurements of Atlantic coelomic fluid could potentially improve juvenile production by identifying low-quality oocytes. Acknowledgments The authors would like to thank Trude Reinholdens at Aqua Gen AS for help during sampling and the production staff at Aqua Gen AS for maintaining eggs, alevins, and juveniles during the experiment. This work was funded by the MABIT program (Norway; #AF0057).
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Appendix A. Supplementary Data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. theriogenology.2015.03.001. References [1] Flemming IA. Pattern and variability in the breeding system of Atlantic salmon (Salmo salar), with comparisons to other salmonids. Can J Fish Aquat Sci 1998;55:59–76. [2] Craik JCA, Harvey SM. Egg quality in rainbow trout: the relation between egg viability, selected aspects of egg composition, and time of stripping. Aquaculture 1984;40:115–34. [3] Lahnsteiner F. Morphological, physiological and biochemical parameters characterizing the over-ripening of rainbow trout eggs. Fish Physiol Biochem 2000;23:107–18. [4] Aegerter S, Jalabert B, Bobe J. Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing. Mol Reprod Dev 2005;385: 377–85. [5] Bromage N, Jones J, Randall C, Thrush M, Davies B, Springate J, et al. Broodstock management, fecundity, egg quality and the timing of egg production in the rainbow trout (Oncorhynchus mykiss). Aquaculture 1992;100:141–66. [6] Aegerter S, Jalabert B. Effects of post-ovulatory oocyte ageing and temperature on egg quality and on the occurrence of triploid fry in rainbow trout, Oncorhynchus mykiss. Aquaculture 2004;231:59–71. [7] Bonnet E, Fostier A, Bobe J. Characterization of rainbow trout egg quality: a case study using four different breeding protocols, with emphasis on the incidence of embryonic malformations. Theriogenology 2007;67:786–94. [8] Formacion MJ, Venkatesh B, Tan CH, Lam TJ. Overripening of ovulated eggs in goldfish, Carassius auratus: II. Possible involvement of postovulatory follicles and steroids. Fish Physiol Biochem 1995;14: 237–46.
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[9] McEvoy LA. Ovulatory rhythms and over-ripening of eggs in cultivated turbot, Scophthalmus maximus L. J Fish Biol 1984;24:437–48. [10] Bahrekazemi M, Matinfar A, Soltani M, Abtahi B, Pusti I, Mohagheghi A. The relation between egg viability, selected aspects of egg and ovarian fluid composition and time of stripping in endangered caspian brown trout. J Fish Aquat Sci 2009;4:306–15. [11] Rime H, Guitton N, Pineau C, Bonnet E, Bobe J, Jalabert B. Postovulatory ageing and egg quality: a proteomic analysis of rainbow trout coelomic fluid. Reprod Biol Endocr 2004;2:26–36. [12] Lahnsteiner F, Patzner RA. Rainbow trout egg quality determination by the relative weight increase during hardening: a practical standardization. J Appl Ichthyol 2002;18:24–6. [13] Zar JH. Biostatistical analysis. Fourth edition. Upper Saddle River: Pearson higher education; 1999. [14] Lahnsteiner F, Weismann T, Patzner RA. Physiological and biochemical parameters for egg quality determination in lake trout, Salmo trutta lacustris. Fish Physiol Biochem 1999;20:375–88. [15] Chatakondi NG, Torrans EL. The influence of ovarian fluid pH of stripped unfertilized channel catfish, Ictalurus punctatus, eggs on the hatching success of channel catfish \ x blue catfish, Ictalurus furcatus_, hybrid catfish eggs. J World Aquacult Soc 2012;43:585–93. [16] Fauvel C, Omnès M, Suquet M, Normant Y. Reliable assessment of overripening in turbot (Scophthalmus maximus) by a simple pH measurement. Aquaculture 1993;117:107–13. [17] Skaalsvik TH, Bolla SL, Thornqvist P-O, Babiak I. Quantitative characteristics of Atlantic halibut (Hippoglossus hippoglossus L.) egg quality throughout the reproductive season. Theriogenology 2015; 83:38–47. [18] Gilkey JC. Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes. J Cell Biol 1983;97:669–78. [19] Bruno DW, Poppe TT. A colour atlas of salmonid diseases. London: Academic Press, Harcourt Brace & Company; 1996. [20] Kent ML, Wellings SR, Yasutake WT, Elston RA. Cranial nodules associated with cranial fenestrae in juvenile Atlantic salmon, Salmo salar L. J Fish Dis 1987;10:419–21. [21] Bruno DW. Cranial nodules in farmed Atlantic salmon, Salmo salar L., fry. J Appl Ichtyol 1997;13:47–8. [22] Lall SP, Lewis-McCrea LM. Role of nutrients in skeletal metabolism and pathology in fishdan overview. Aquaculture 2007;267:3–19.
Parameter
Days after ovulation 0
7
n
Mean SD
20 20 20
8.4 0.1 3.3 0.4 272.2 23.5
14
CV (%)
n
Mean SD
1.2 12.1 4.8
19 19 19
7
22
CV (%)
n
Mean SD
8.4 0.1 3.3 0.4 278.8 21.0
1.2 12.1 7.5
18 19 18
8.3 0.1 3.6 0.5 275.6 20.5
20
117 9
7
19
117 9
28
CV (%)
n
Mean SD
1.4 13.9 7.4
18 18 18
8.3 0.1 3.4 0.3 286.5 17.4
8
18
119 8
25.2 17.2 11.6 25.7 116.6
16 16 18 18 18
8.0 14.3 17.5 6.3 3.2
n
Mean SD
1.2 8.8 6.1
18 16 16
8.0 0.1 3.2 0.7 299.8 15.3
1.3 21.9 5.1
7
18
123 11
9.0
24.5 27.4 18.3 28.6 93.8
16 16 18 18 18
6.6 8.9 8.9 2.3 1.4
6.8 10.2 4.1 2.0 4.0
104.1 114.6 46.1 86.9 282.4
76.1 35.9 5.3 16.7
47.2 188.3
126.8 111.0 282.1 285.4 285.4 70.9 92.6 0 0
CV (%)
20
118 8
20 20 19 20 19
11.7 16.2 17.4 4.5 1.2
1.9 1.6 2.7 2.0 1.2
16.7 9.9 15.7 44.4 100.0
20 20 20 20 20
11.4 15.4 17.5 4.0 2.1
1.7 2.2 1.4 1.8 1.9
14.9 14.3 8.9 46.6 90.5
19 19 19 19 19
10.3 15.7 18.1 5.4 2.4
20 20
92.2 5.2 84.4 5.4
5.6 6.4
20 20
86.0 6.5 78.2 7.6
8.8 9.3
19 19
83.9 8.6 59.0 27.9
10.3 47.6
19 19
66.1 25.0 14.6 20.3
37.8 80.9
18 18
18 18 18 18 18 18 18 18 18
96.1 0.2 4.8 2.5 5.0 55.6 32.5 13.4 5.5
2.4 250.0 457.2 448.0 448.0 83.8 143.7 247.8 258.2
20 20 20 20 20 20 20 20 20
95.1 0.4 19.7 12.0 5.1 74.3 33.3 31.3 1.0
2.3 150.0 150.8 220.0 249.0 45.5 100.6 128.1 450.0
19 19 19 19 19 19 19 19 19
93.9 0.3 7.5 4.0 0.0 38.2 29.6 17.3 7.0
24.6 200.0 312.0 245.0 0 121.5 124.3 159.6 330.0
13 12 11 13 13 12 13 13 11
39.9 2.2 20.5 13.2 7.7 45.8 44.8 0.6 0.0
96.7 177.3 167.3 231.1 359.7 102.6 104.1 366.6 0
8 8 8 8 8 8 8 8 8
2.3 0.5 21.8 11.2 22.4 46.6 46.7 33.2 14.2
2.2 0.6 29.7 26.4 12.7 33.8 33.5 40.1 4.5
Abbreviations: CV, coefficient of variation; EC, electric conductivity; SD, standard deviation.
2.6 2.7 2.1 1.4 2.8
23.1 0.6 23.4 9.8 0.0 46.4 36.8 27.6 23.1
2.0 3.9 3.2 1.8 3.0
38.6 3.9 34.3 30.5 27.7 47.0 46.6 2.2 0.0
26.9 6.4 8.4 4.1 4.1 66.6 54.2 0.0 0.0
34.1 7.1 23.7 11.7 11.7 47.2 50.2 0.0 0.0
CV (%)
M. Mommens et al. / Theriogenology xxx (2015) 1–7
Coelomic fluid pH EC (mS/m) Osmolality (mOsm/kg) Water hardening Dry weight (mg/egg) Weight increase (%) 0–15 min 0–30 min 0–60 min 15–30 min 30–60 min Embryo Fertilization (%) Eyed egg (%) Alevin Relative survival (%) Total deformities (%) Total spine (%) Scoliosis (%) Lordosis (%) Total yolk (%) Yolk sac edema (%) Yolk sac abscission (%) Twins (%)
7.e1
Supplementary Table 1 Atlantic salmon coelomic fluid parameters, oocyte weight gain during water hardening, and embryonic and juvenile performance in relation to postovulatory aging.
M. Mommens et al. / Theriogenology xxx (2015) 1–7
7.e2
Supplementary Table 2 Juvenile performance in relation to postovulatory aging. Parameter
Relative survival (%) Weight (g) Total deformities Total deformities without short operculum Types of deformities Short operculum Lordosis Short spinal column posterior Short spinal column anterior Cranial nodules Microstomia Prognathia
Days after ovulation 0
7
14
22
28
n ¼ 1105
n ¼ 1174
n ¼ 1086
n ¼ 817
n ¼ 338
Mean SD
CV (%)
83.1 26.7 5.2 0.4
6.5 7.5 0.4 0.0
7.8 28.1 7.7 0.0
96.5 2.0 0.0 1.5 0.0 0.0 0.0
0.7 2.8 0.0 2.1 0.0 0.0 0.0
0.7 140.0 0.0 140.0 0.0 0.0 0.0
Mean SD
CV (%)
Mean SD
CV (%)
Mean SD
CV (%)
Mean SD
CV (%)
89.1 27.7 6.0 0.4
4.0 7.7 1.2 1.4
4.5 27.6 20.0 350.0
92.3 31.9 4.9 0.1
0.6 7.9 1.1 0.7
0.6 24.7 22.4 700.0
92.8 30.1 5.7 2.1
4.2 9.5 4.4 2.1
4.5 31.7 77.2 100.0
88.9 22.7 7.4 7.4
5.1 8.3 0.4 0.7
5.8 36.4 5.4 9.5
94.5 1.3 1.7 2.6 0.0 0.0 0.0
3.1 1.8 2.4 3.6 0.0 0.0 0.0
3.3 138.5 141.2 138.5 0.0 0.0 0.0
98.4 0.0 0.0 1.6 0.0 0.0 0.0
2.3 0.0 0.0 2.3 0.0 0.0 0.0
2.3 0.0 0.0 143.8 0.0 0.0 0.0
54.8 0.0 9.5 0.0 0.0 19.0 4.8
16.8 0.0 6.7 0.0 0.0 13.5 6.7
30.7 0.0 70.5 0.0 0.0 71.1 139.6
0.0 6.3 6.3 0.0 46.4 27.7 0.0
0.0a 8.8 8.8 0.0 5.0 21.5 0.0
0.0 139.7 139.7 0.0 11.0 77.6 0.0
Rates of deformities (%, mean SD) are given for total number of juveniles (n) from each days postovulation group. Abbreviations: CV, coefficient of variation; SD, standard deviation.
