Comparative biochemical composition of wild and farm origin grayling (Thymallus thymallus) eggs

Abstract

In this study, the biochemical composition of wild and farm origin grayling eggs was compared, first, to evaluate the composition of fatty acids and protein content in grayling eggs for the first time, and second, to discover differences in biochemical composition between wild and farm origin eggs which is affected by broodstock feed. Farm origin grayling females were fed commercial trout starter feed, while wild origin females were feeding on food present in their natural environment. Fatty acid composition, protein content and colour were determined. Based on a principle component analysis (PCA) of the biochemical profile, farm and wild origin grayling eggs were clearly separated. Wild origin grayling eggs contained higher amount of SFA, but lower MUFA and PUFA than farm origin eggs. A considerable difference between wild and farm origin grayling eggs was observed also in essential fatty acids: C22:6 n-3 (DHA), C20:5 n-3 (EPA) and C20:4 n-6 (ARA), resulting in higher EPA/ARA and DHA/EPA ratio in farm origin eggs compared to wild origin eggs. Wild origin grayling eggs were characterised by higher protein content and were more intensely red-coloured than farm origin eggs, which were almost colourless. It could be concluded that the biochemical composition of farm origin eggs significantly differs from wild origin grayling eggs, which might have an influence on the embryonic development and hatching success of farm origin grayling. Therefore, the diet that has the greatest effect on egg composition needs to be optimised to meet the needs of the grayling broodstock and to improve the breeding efficiency of farm raised graylings.

HIGHLIGHTS

• Biochemical composition of grayling eggs differs significantly between the two lifestyles (wild and farm origin).

• Differences in the proportion of essential fatty acids: DHA, EPA and ARA.

• Suboptimal EPA/ARA and DHA/EPA ratios in farm origin eggs.

Keywords:

• Salmonidae
• fatty acid
• reproduction
• egg quality
• conservation

Introduction

Successful reproduction, production of larval fish and their subsequent growth, development and health are basic requirement for prosperous fish breeding, both for breeding for human consumption and for reintroduction and conservation intentions (Jeffery et al. 2006; Fraser 2008; Comizzoli and Holt 2019). This is dependent on the quality of eggs and relies on optimal breeding stock management practices, including knowledge of the nutritional and environmental needs of individual fish species. Egg quality is determined by nutrients delivered from the female, therefore, it is of paramount importance that broodstock nutrition is optimised (Izquierdo et al. 2001). However, for some species, including grayling, Thymallus thymallus, the knowledge on nutrition needs and importance of broodstock nutrition for reproductive performance and egg and larval quality, is very limited, which might result in low reproductive performance of farm raised grayling (Szmyt et al. 2013; Szmyt et al. 2015).

Yolk sac of fish eggs contains all the nutrients and other components required for normal embryonic and early larval development. Energetic reserves of the yolk sac are particularly important in Salmonids, as their development is dependent on yolk sac reserves not only through embryo stages but also for many days after hatching. During these stages, maternally derived egg components are the only source of energy and nutrients for metabolism, development, and growth (Wiegand 1996; Sargent et al. 2002). Recent studies have demonstrated that the composition of these endogenous nutritional resources depends, among other intrinsic and extrinsic factors (Izquierdo et al. 2001; Bobe and Labbé 2010), on parental diet and lifestyle (e.g. wild or hatchery) and this could affect egg quality, hatching rate, survival of embryos and their proper development in multiple fish species (Watanabe et al. 1984; Bromage and Roberts 1995; Johnson et al. 2017). These maternally derived nutrients consist of proteins, carbohydrates, lipids, vitamins, minerals, ions (Brooks et al. 1997; Reading et al. 2018), maternal RNA transcripts and microRNAs (Cheung et al. 2018). Biochemical composition of fish egg is therefore one of the major criteria for determining egg quality and can have decisive impact on hatching success and early life history of many fish species, including Salmonids.

Lipids are together with proteins a major component of egg yolk and are a source of energy and structural components of cell membranes, micronutrients and signalling molecules for developing embryos and larvae. The crude lipid content of freshwater fish eggs ranges from 2.5% to 10% of egg wet weight (Henderson and Tocher 1987; Czesny and Dabrowski 1998). Salmonids belong to the high lipid content group with lipid levels around 10%. Fatty acids (FA) are integral building blocks of lipids and are recognised as important molecules regulating several processes of early life history (Henderson and Tocher 1987; Czesny and Dabrowski 1998; Glatz 2011; de Carvalho and Caramujo 2018). Namely, the FA, especially the essential fatty acids (EFA), particularly the polyunsaturated FA (PUFA), are important structural components, serve as nutrient reserves, are precursors of eicosanoids and have been shown to affect fecundity, egg quality, hatching success and larval malformations in numerous fish species, including Salmonids (Bell et al. 1986; Tocher et al. 2002). It is not only the amounts of FAs but, even more importantly, the optimum ratio between them in fish eggs that influences early embryonic development and survival (Wiegand 1996; Sargent et al. 1999; Tocher et al. 2002). In particular, docosahexaenoic acid (C22:6 n-3, DHA) has important structural and functional roles in neural membranes, while eicosapentaenoic acid (C20:5 n-3, EPA), and arachidonic acid (C20:4 n-6, ARA) have unique roles in controlling and regulating cellular metabolism through production of bioactive eicosanoids (Schmitz and Ecker 2008; Glatz 2011; de Carvalho and Caramujo 2018). All three LC-PUFAs are linked to abnormalities in development, decreased hatching success, and low offspring survival in numerous teleost fishes when present in subnormal levels or ratios (Sargent 1995; Wiegand 1996; Czesny and Dabrowski 1998; Tocher 2010).

Fish egg composition is species-specific and is at the same time a good indicator of broodstock diet (Lazzarotto et al. 2015; Cardona et al. 2022). Thus, the FA composition of broodstock diet can affect the female reproductive performance and change the composition of FAs in the resulting eggs, which influences the egg quality (Czesny and Dabrowski 1998; Pickova et al. 1999). Multiple studies on a number of species, e.g. common sole (Solea solea; (Lund et al. 2008)), Atlantic cod (Gadus morhua; (Lanes et al. 2012)), spotted rose snapper (Lutjanus guttatus; (Chacón Guzmán et al. 2020)) and European eel (Anguilla anguilla (Kottmann et al. 2020)) reported that biochemical composition of diet differs significantly between farm and wild origin broodstock and this is reflected in the composition of eggs, mainly FA profile (Lund et al. 2008; Lanes et al. 2012; Chacón Guzmán et al. 2020). In addition, fertilisation and hatching rates in multiple fish species are reported to be significantly higher for eggs from wild origin broodstock (hereafter referred as wild origin eggs) compared to eggs from farmed broodstock (hereafter referred to farm origin eggs) (Lund et al. 2008; Lahnsteiner and Kletzl 2012; Lanes et al. 2012) Species specific diet of farm raised broodstock is therefore of paramount importance and is believed to be best designed on biochemical composition of wild origin fish eggs (Tocher and Sargent 1984; Sargent et al. 2002; Szmyt et al. 2015).

One such species, for which broodstock diet needs to be improved, in order to successfully approach repopulation programs based on stocking of hatchery-reared juvenile fish, is European grayling, Thymallus thymallus (Turek et al. 2013). European grayling, distributed across parts of the Northern hemisphere, belongs to the Salmonidae family, subfamily Thymallinae. It inhabits cool rivers and streams with strong flows and clean well-oxygenated water but also occurs in lakes. Adult grayling is predominately benthic feeder consuming a wide variety of prey items (Ibbotson et al. 2001). Grayling achieves sexual maturity between the ages of two and three years (Northcote 1995; Cove et al. 2018). Spawning takes place in spring, from late March to early June (Parkinson et al. 1999) with a spawning season lasting between 2 and 24 days (Kratt and Smith 1977). European grayling is a highly fecund fish that can produce from 6400 to as many 31000 eggs per kg of body weight, with egg size ranging from 2.5 mm to 3.5 mm (Janković 1964; Northcote 1995; Kodela et al. 2023). Females release their eggs several times within two days, usually with the same male and at the same spawning ground.

Rearing grayling in captivity is challenging mainly due to high mortality during embryonic development (Lahnsteiner and Kletzl 2012; Szmyt et al. 2013; Szmyt et al. 2015). High mortality is believed to be surpassed when all phases of rearing, including the nutrition of the broodstock, which would be based on biochemical composition of wild origin grayling eggs, were optimised (Turek et al. 2013). Nowadays grayling broodstock is typically fed trout grower feed (Szmyt et al. 2015; Szmyt et al. 2021) that does not fulfil requirements of broodstock fish, or trout broodstock feed grounded to smaller particles (Kodela et al. 2023), none of them being optimised for grayling.

To the best of our knowledge, biochemical composition of grayling eggs has not been described yet, and differences in the composition of wild and farm origin eggs, which might be related to high embryo mortality rate in farmed grayling, are not known. The aim of the present study is to determine and compare the biochemical composition of eggs collected from wild origin grayling females on spawning ground and eggs collected from farm raised broodstock grayling females. Specifically, egg FA composition, protein content and colour were compared. In addition, biochemical composition of wild origin grayling eggs could set the basis for further development of proper grayling broodstock diet.

Materials and methods

No permit for animal experimentation from an ethical committee is required for experiments reported on in the manuscript, eggs were collected during standard grayling stripping protocols followed in hatchery and on field.

Grayling female origin

Eggs from five wild origin and 11 farm origin grayling females were included in the analyses. All females were 3+ years old. Farm origin eggs were collected at the fish farm Soča (Kobarid, Slovenia), and wild origin eggs were obtained at the grayling spawning ground at the mill race, which receives water from Kamniška Bistrica river (Slovenia, 46,148863°; 14,595641°). Grayling at the fish farm Soča origins from Unica river, that is together with Kamniška Bistrica river a part of Sava river system, harbouring very homogenous grayling population of one phylogenetic lineage and without detected population substructures (Sušnik et al. 2004). Wild and farm origin grayling included in this study therefore belong to the same genetic group.

Diets

Farm origin graylings females were fed commercial trout starter feed containing 43–52% proteins, 22–24% crude fat, 6.2–7.7% ash and 0.8–3.7% of crude fibre (BioMar). Feeding was performed manually 2 - 3 times a day with feed quantity determined according to the manufacturer’s recommendations. Fish were fed this diet for seven months prior to spawning.

The analysis of FA composition of the commercial feed showed that the feed contained the following essential and metabolically important long-chain polyunsaturated fatty acids (g/100 g of feed): 3.25 g linoleic FA (C18:2 n-6), 1.17 g linolenic FA (C18:3 n-3), 0.06 g ARA, 0.70 g EPA and 1.16 g DHA. One hundred grams of commercial feed contained 3.63 g of saturated fatty acids (SFA), 11.10 g of monounsaturated fatty acids (MUFA), and 6.90 g PUFA, of which 3.45 g were n-6 PUFA and 3.45 g were n-3 PUFA. The ratio between n-3 and n-6 PUFA was 1:1, while EPA to ARA ratio was 12:1 and DHA to EPA ratio was 1.7:1.

Wild origin females were feeding on food present in their natural environment (aquatic and terrestrial insects, fish eggs and small fish).

Stripping and egg collection

Fish eggs were obtained using standard procedures for salmonids at the beginning of the spawning season (beginning of April 2022). Before handling, all fish were anaesthetized using anaesthetic Tricaine (100 mg/L; Pharmaq, Norway) and the total length (TL, cm) of fish was measured. The eggs were stripped by abdominal massage. Approximately 5 g of eggs from each female were collected in 5-ml plastic tube, frozen in dry ice and transported to the laboratory, where they were kept at −80° C till biochemical analyses. The diameter of 10 eggs from each female was measured.

Egg biochemical composition

Samples were homogenised using laboratory homogeniser (Grindomix, Retsch, Germany). Liquid nitrogen was used to facilitate homogenisation and to prevent lipid oxidation of the samples. All analyses were performed in two replicates.

Total nitrogen in egg samples was determined using the Kjeldahl method (AOAC 2000) (AOAC Official method 984.13: Protein (Crude) in animal feed and pet food. Copper catalyst method). To obtain crude protein level, factor 6.25 was used.

The FA composition of the samples was determined using gas chromatographic separation of fatty acid methyl esters (FAME). FAME was prepared using in situ transesterification, developed by Park and Goins (1994). Internal standard (C19:0) was added to the samples prior to in situ transesterification procedure for quantification purpose. A gas chromatography system 6890 (Agilent Technologies, Santa Clara, California, USA) equipped with an automatic liquid sampler 7683 series and flame ionisation detector was used. Separation of FAME was obtained using capillary column DB-FATWAX UI (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, California, USA). Helium was used as the carrier gas. FAME were identified based on their retention times. The instrument was calibrated using the GLC 85, GLC 423, GLC 411 and GLC 68a FAME standards (Nu-Check Prep, Elysian, Minnesota, USA). Chromatographic data were analysed using the ChemStation A.08.03 software (Agilent Technologies, Santa Clara, California, USA). Results are presented as proportion (mass) of total FA and in mg per 100 g of wet sample mass.

Egg colour

Egg colour parameters were measured in triplicate using chroma metre (Minolta CR 300, Osaka, Japan) with an 8-mm aperture, illuminate C and a 0° viewing angle. The standard white plate was used for calibration. The colour was expressed according to the CIE system, measuring three axes, one representing lightness (L*) and two colours (a* and b*). In the three-dimensional model, the chromatic a* axis extends from green (-a*) to red (+a*), and the chromatic b* axis extends from blue (-b*) to yellow (+b*). The lightness dimension (L*), ranges from 0 (pure black) to 100 (diffuse white). The mean value of the three replicates was used for further statistical analyses.

Preliminary fertilisation and hatching rate estimation

Fish eggs from one wild origin and one farm origin grayling female were obtained as described above. Stripped eggs in a dry plastic bowl were mixed with sperm from two males (wild origin sperm was used for fertilisation of wild origin eggs, and farm origin sperm for farm origin eggs). Sperm was activated using approximately 5 mL of extender (per 1000 mL water: 2.42 g Tris, 3.76 g glycine, 5.5 g NaCl). After 2 h of hardening, approximately 100 of fertilised egg were placed on a petri dish and incubated at 8 °C till hatching. The water in a petri dish was changed every day using a pipette. All white (dead) eggs from the petri dish were manually discarded each day.

The fertilisation rate was calculated as the number of fertilised eggs after two hours of hardening/number of all eggs × 100 (Pertiwi et al. 2018).

Hatching rate was calculated as the number of hatched larvae to total incubated eggs (Bilguven 2014; Pertiwi et al. 2018). The presence of abnormally developed larvae was also registered.

Statistical analysis

Egg composition is described using summary statistics for FA content (in mg/g of fresh matter and % of total identified FA), FA ratios and protein content; the data are presented as mean ± standard deviation (SD) of wild origin and farm origin grayling egg samples. To determine significant differences between two groups, the results were compared using an independent samples t-test. Differences between mean values were regarded as significant when p < 0.05.

The principal component analysis (PCA) was used to visualise relationship between eggs from individual females based on FA composition, FA ratios and protein content, and to determine the egg components contributing most to egg distribution based on PCA. In other words, PCA was used to determine whether FA profile and protein content could be used to separate wild origin and farm origin grayling eggs. Statistical analyses were performed using SPSS for Windows statistical software package.

Results

The average total length of wild origin females was 29.7 ± 2.2 cm and the egg diameter 2.7 ± 0.3 mm. The average total length of farm origin females was 25.6 ± 0.2 cm and the egg diameter 2.6 ± 0.2 mm. The difference in fish egg diameter between the two grayling groups was not statistically significant.

PCA of grayling eggs based on all biochemical parameters analysed (see Table 1.) is presented in Figure 1. The first two principal components (PC) accounted for 85% of the total variance observed. PC1 accounted for 78% of the total variance separating two non-overlapping groups of samples, corresponding to wild and farm origin eggs (Figure 1A). PC1 was positively related to higher docosapentaenoic acid (C22:5 n-3, n-3 DPA), EPA, SFA, ARA and protein level, characteristic for wild origin grayling eggs. On the other hand, PC1 was negatively related to higher levels of MUFA, PUFA and DHA, characteristic for farm origin eggs (Figure 1B). PC2, accounting for 7% of total variance and separating grayling eggs within both groups, was positively related to higher n-3/n-6 PUFA ratio, protein and C14:0 level; and negatively related to higher C18:2n-6 and n-6 PUFA level.

Figure 1. Principal component analysis (PCA). (A) PCA of wild (N = 5) and farm origin grayling eggs (N = 11) based on fatty acid (FA) content (% of total), FA ratios and protein content (see Table 1). (B) Projections of loading densities for the first two principal components of FA content (% of total), FA ratios and protein content (same as in Table 1), affecting distribution of egg samples in (a).

Table 1. Sum of fatty acid (FA) and protein content in mg/100 g of grayling egg wet weight, fatty acid profile in wild and farm origin grayling eggs in mg/100 g of fresh matter and in % of individual FA of total identified. Composition of FA groups and ratios in wild and farm origin grayling eggs are also presented. Statistical significance of difference between the means of the two groups is indicated with P value.

	Wild origin eggs	Farm origin eggs	Significance	Wild origin eggs	Farm origin eggs	Significance
	g/100 g of fresh material
FA sum	6.18 ± 0.36	6.67 ± 1.41
Proteins	22.48 ± 0.93	19.47 ± 2.11	p < 0.01
Fatty acid	mg/100g of fresh matter			% of total FAs
C14:0	104.5 ± 11.37	105.14 ± 15.12		1.72 ± 0.12	1.54 ± 0.08	p < 0.001
C14:1 n-5	1.67 ± 0.27	0.56 ± 0.08	p < 0.01	0.03 ± 0.01	0.01 ± 0.00	p < 0.001
C16:0	991.25 ± 67.84	874.29 ± 124.25		16.36 ± 0.26	12.91 ± 0.31	p < 0.001
sum C16:1*	311.61 ± 39.60	185.27 ± 25.28	p < 0.001	5.14 ± 0.54	2.72 ± 0.09	p < 0.001
C18:0	201.43 ± 17.07	146.13 ± 21.90	p < 0.001	3.33 ± 0.26	2.18 ± 0.12	p < 0.001
sum C18:1*	1487.98 ± 109.94	1894.80 ± 224.31	p < 0.01	24.55 ± 0.76	27.75 ± 0.90	p < 0.001
C18:2 n-6	597.19 ± 55.67	824.65 ± 98.78	p < 0.001	9.89 ± 1.20	12.05 ± 0.70	p < 0.001
C18:3 n-6	27.47 ± 6.97	23.13 ± 4.81		0.46 ± 0.12	0.35 ± 0.08	p < 0.05
C18:3 n-3	177.71 ± 39.22	245.90 ± 34.88	p < 0.01	2.92 ± 0.54	3.58 ± 0.17
C18:4 n-3	53.58 ± 7.54	73.65 ± 10.69	p < 0.01	0.88 ± 0.11	1.09 ± 0.14	p < 0.01
C20:0	1.99 ± 1.83	6.16 ± 1.09	p < 0.01	0.03 ± 0.03	0.09 ± 0.01	p < 0.05
C20:1 n-9	40.09 ± 3.77	98.72 ± 16.39	p < 0.001	0.67 ± 0.10	1.46 ± 0.12	p < 0.001
C20:2 n-6	67.80 ± 4.97	57.22 ± 9.24	p < 0.05	1.12 ± 0.11	0.84 ± 0.06	p < 0.001
C20:3 n-6	37.96 ± 10.76	25.02 ± 3.77		0.63 ± 0.21	0.38 ± 0.05
C20:4 n-6 (ARA)	176.48 ± 5.02	55.33 ± 8.78	p < 0.001	2.92 ± 0.18	0.83 ± 0.04	p < 0.001
C20:3 n-3	18.95 ± 4.45	15.67 ± 2.71		0.31 ± 0.06	0.23 ± 0.02	p < 0.01
C20:4 n-3	22.16 ± 3.24	40.07 ± 6.46	p < 0.001	0.37 ± 0.04	0.58 ± 0.05	p < 0.001
C20:5 n-3 (EPA)	486.47 ± 62.08	307.60 ± 47.44	p < 0.001	8.01 ± 0.70	4.56 ± 0.18	p < 0.001
C22:1 n-9c	0.00 ± 0.00	6.57 ± 1.34	p < 0.001	0.00 ± 0.00	0.10 ± 0.01	p < 0.001
C22:2 n-6	3.23 ± 0.88	4.54 ± 1.16	p < 0.05	0.05 ± 0.01	0.06 ± 0.01
C22:4 n-6	10.51 ± 0.72	2.34 ± 0.61	p < 0.001	0.17 ± 0.02	0.03 ± 0.01	p < 0.001
C22:5 n-6 (n-6 DPA)	49.61 ± 3.60	22.89 ± 3.19	p < 0.001	0.82 ± 0.07	0.34 ± 0.02	p < 0.001
C22:5 n-3 (n-3 DPA)	161.69 ± 23.38	79.44 ± 12.02	p < 0.01	2.66 ± 0.26	1.17 ± 0.07	p < 0.001
C22:6 n-3 (DHA)	918.85 ± 56.58	1629.81 ± 250.99	p < 0.001	15.18 ± 0.61	24.22 ± 1.24	p < 0.001
SFA	1372.80 ± 90.40	1166.48 ± 164.56	p < 0.05	22.66 ± 0.37	17.22 ± 0.37	p < 0.001
MUFA	1854.96 ± 135.06	2207.21 ± 262.75	p < 0.05	30.61 ± 1.00	32.35 ± 0.84	p < 0.001
PUFA	2819.21 ± 150.01	3412.07 ± 451.29	p < 0.05	46.56 ± 1.18	50.39 ± 0.71	p < 0.001
n-6 PUFA	942.78 ± 69.12	991.99 ± 117.53		15.62 ± 1.66	14.53 ± 0.67
n-3 PUFA	1839.41 ± 160.77	2392.15 ± 350.39	p < 0.01	30.33 ± 1.24	35.44 ± 1.26	p < 0.001
LC PUFA	1788.80 ± 101.97	2155.95 ± 326.76	p < 0.05	29.54 ± 0.73	32.01 ± 1.46	p < 0.01
LC n-6 PUFA	345.60 ± 14.77	167.34 ± 23.09	p < 0.001	5.73 ± 0.51	2.48 ± 0.08	p < 0.001
LC n-3 PUFA	1608.12 ± 135.56	2072.59 ± 316.22	p < 0.01	26.52 ± 1.12	30.77 ± 1.44	p < 0.001

*sum of positional and geometric isomers of FA.

ARA: Arachidonic acid, EPA: Eicosapentaenoic acid, DPA: Docosapentaenoic acid, DHA: Docosahexaenoic acid, SFA: Saturated fatty acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids.

Statistically significant difference was found in protein content (p < 0.01) between wild and farm origin grayling eggs; higher protein content was characteristic for wild origin grayling eggs (Figure 2). No difference between the egg groups was observed in sum of FA.

Figure 2. Sum of fatty acids (FA) and protein content in the wild and farm origin grayling eggs. Statistically significant difference between the means of two groups is denoted by asterisks (** - p < 0.01).

The amount of FA (in mg/100 g of fresh matter and % of total identified FAs), as well as sums of SFA, MUFA, PUFA, n-3 PUFA, n-6 PUFA, and n-6/n-3 PUFA ratios and amount of proteins in wild and farm origin grayling eggs are given in Table 1. Significant influence of lifestyle (wild vs farm) was observed for almost all tested FA (in % of total FA) except for C18:3 n-3, C20:3 n-6, C22:2 n-6 and n-6 PUFA. The most abundant FA in eggs of wild and farm origin are sum C18:1, C16:0, DHA and C18:2 n-6. The most abundant FA groups are PUFA, followed by MUFA and SFA in both egg groups (Figure 3). Wild origin grayling eggs contain a higher amount of SFA, but lower MUFA and PUFA than farm origin eggs with a highly significant difference. The content of almost all individual SFA and MUFA was higher in wild origin eggs. The lower content of MUFA compared to farm origin eggs is attributed to higher sum C18:1 (the most abundant FA group within MUFA) and C20:1 n-9 in farm origin eggs, while C19:1 n-9 was detected only in wild, and C22:1 n-11 and C22:1 n-9 only in farm origin eggs. A significantly higher total content of PUFA in farm origin eggs is mainly due to higher content of DHA, C18:2 n-6, C18:3 n-3 and C20:1 n-9 compared to wild origin eggs.

Figure 3. Proportion (% of total) of the EFA and FA groups (DHA, EPA, ARA, SFA, MUFA, PUFA) in the wild and farm origin grayling eggs. Statistically significant difference between the means of two groups is denoted by asterisks (*** - p < 0.001). DHA: Docosahexaenoic acid, EPA: Eicosapentaenoic acid, ARA: Arachidonic acid, SFA: Saturated fatty acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids.

The highest difference in the FA content between wild and farm origin grayling eggs was observed for DHA, EPA and ARA, resulting in significantly different EPA/ARA and DHA/EPA ratios between wild and farm origin eggs (Figure 4); both ratios were higher in farm origin eggs.

Figure 4. Fatty acid ratios in wild and farm origin grayling eggs. Statistically significant difference between the means of two groups is denoted by asterisks (*** - p < 0.001). EPA: Eicosapentaenoic acid, ARA: Arachidonic acid, DHA: Docosahexaenoic acid, PUFA: Polyunsaturated fatty acids.

All three parameters of colorimetric values were significantly different between wild and farm origin eggs. Wild origin eggs were darker and had more intense red colour than farm origin eggs, which were lighter and almost colourless (Table 2, for egg colour see also Figure 5).

Figure 5. Embryo and larval development. (A) Normal developing wild origin grayling larvae before hatching (A, B, C) and after hatching (C, D). Wild origin eggs are intensively yellow/orange coloured. (B) Deformities noticed in farm origin grayling larvae before hatching (A, B, C) and after hatching (D, E, F). Spine malformations, such as coiled tail (A, B, C) or bended spine (D, E) and jaw malformations (E, F) are shown. Farm origin eggs are pale, light yellow coloured.

Table 2. Colorimetric values L*, a* and b* measured for wild and farm origin grayling eggs. Average ± SD of all three parameters, and significance of comparison between both groups of eggs is presented.

	Wild origin eggs	Farm origin eggs	Significance
L* (lightness)	50 ± 4.9	62.4 ± 4.3	p < 0.001
a* (redness)	14.7 ± 5.1	−0.8 ± 1.2	p < 0.01
b* (yellowness)	42.7 ± 6.5	22.7 ± 8.4	p < 0.001

Fertilisation rate and hatching rate of wild origin grayling eggs were evidently higher compared to farm origin eggs (98% and 68% in wild origin compared to 58% and 40% in farm origin eggs, respectively). Numerous deformities (spine and cranio-facial malformations) were noticed in hatched larvae from farm origin eggs (Figure 5A), while no deformities were noticed in wild origin larvae (Figure 5B). Some malformations were severe and a number of such larvae have not survived till swim-up stage, which further reduced the survival rate in farm origin larvae.

Discussion

The present study is to the best of our knowledge the first report on biochemical composition of grayling eggs, providing a detailed analysis of differences between farm and wild origin grayling eggs, which further contributes to observed variation in egg quality and in early life-history traits in grayling. Namely, FA compositions, protein level and colour of eggs differed significantly between farm and wild origin grayling eggs resulting in two clearly separated and non-overlapping egg groups on PCA graph (Figure 1).

The principal function of egg proteins is the provision of nutrients for the developing embryo, protection of the egg from microbial attack and transport of nutrients into the developing embryo (Stevens 1996). Egg proteins are also important for fertilisation success and normal development of embryonic tissues and organs (Stevens 1996; Sun and Zhang 2015). In different salmonid species the fish eggs contain between 17% and 27% of crude protein (Mol and Turan 2008; Bekhit et al. 2009; Kowalska-Góralska et al. 2020). Protein content in grayling eggs, being around 20%, therefore, agrees with the results of the studies listed above. Significantly lower protein content was found in farm origin grayling eggs compared to wild origin eggs. Interestingly, eggs of Oncorhynchus kisutch fed with addition of algal oil compared to the eggs of fish in the fish oil treatment group had higher DHA levels but also lower protein content, with no effect on embryo survival and growth (Johnson et al. 2011). Besides the amount, the type of proteins and amino acid composition are the most important in this context but were not analysed in this study. Further analyses are thus necessary if proteins should be accounted for in grayling egg quality and specific broodstock feed.

The total lipid fraction of eggs from a range of freshwater fish contains high levels of LC-PUFA and they are predominantly rich in n-3 PUFA (Henderson and Tocher 1987). Levels of PUFA in salmonid fish eggs range from 25 to 60% of total FA (Blanchet et al. 2005; Haring et al. 2016; Yesilayer and Türk 2018; Appert 2022). With 46% PUFA of total FA in wild origin grayling eggs and 50% in farm origin eggs, the values in grayling are in the upper range of total PUFA level compared to other salmonids. Interestingly, farm origin grayling eggs also contained higher levels of n-3 PUFA than wild origin eggs. The higher PUFA content in farm origin grayling eggs is mainly due to almost twice the amount of DHA, the most abundant PUFA in grayling eggs, compared to wild origin eggs, while wild origin eggs are characterised by higher content of EPA and ARA compared to farm origin eggs. These three EFA were reported to be linked to abnormalities in development, decreased hatching success, low offspring survival and early mortality syndrome (EMS) in numerous teleost fishes when present in suboptimal levels or ratios between them (Sargent 1995; Wiegand 1996; Czesny and Dabrowski 1998; Torniainen et al. 2017; Appert 2022). Similar content of EFA as in grayling was reported for Coregonus muksun (Lyutikov 2022) when wild and farm origin eggs were compared. On the other hand, wild origin salmon (Salmo salar) eggs had lower EPA, higher ARA and almost equal DHA compared to the farm origin eggs (Pickova et al. 1999), while only minor differences in these three FA were observed between wild and farm origin eggs of Scottish salmon (Migaud et al. 2013). Despite significant differences in FA intake of different salmon populations, the egg values of DHA in salmon were approximately the same (Pickova et al. 1999; Migaud et al. 2013b), pointing to the fact that accumulation of FAs in eggs is not only dependent on maternal diet but is also species-specific. In addition, selection pressure is believed to maintain certain levels of DHA in eggs within a species-specific range (Ashton et al. 1993; Wiegand 1996; Pickova et al. 1999; Migaud et al. 2013). Farm origin grayling (this study), Coregonus (Lyutikov 2022), spotted rose snapper (Lutjanus guttatus) (Chacón Guzmán et al. 2020) and also one population of Atlantic salmon (Blanchet et al. 2005) on the other hand, deposited much more DHA to the eggs than their wild counterparts, contradicting this theory or its generalisation.

It has been shown that the amount of FA itself is not the critical point affecting egg quality, but it is the ratio between EFA that has the major impact (Sargent 1995; Pickova et al. 1999). The ratios between EPA/ARA and DHA/EPA found in this study indicate suboptimal relationship of these three FA in farm origin grayling eggs. Fish eggs should contain enough DHA and EPA for normal embryonic development (Bell et al. 1998), with the DHA/EPA ratio in the wild fish egg commonly being 2:1 (Sargent et al. 1995; Sargent et al. 1999; Torniainen et al. 2017). The DHA/EPA ratio in wild origin grayling eggs was close to this value (1.91), but very high ratio (5.31) was observed in farm origin eggs potentially indicating low-quality eggs, that might result in abnormal embryo growth and survival. Namely, a high DHA/EPA ratio was associated with an increased risk for EMS (Czesny and Dabrowski 1998; Torniainen et al. 2017).

Also, the ARA (n-6 PUFA) is considered as an EFA and its higher content an indicator of higher egg viability (Czesny and Dabrowski 1998; Pickova et al. 1999). ARA is the preferred FA as precursor for eicosanoids (Czesny and Dabrowski 1998; Sargent et al. 2002). Eicosanoid production and many other physiological functions, including reproduction, are further influenced by the cellular ratio of EPA/ARA (Pickova et al. 1999; Tocher et al. 2002). Wild origin grayling eggs are characterised with more than three-times higher amount of ARA (2.92%) compared to farm origin eggs (0.83%). In addition, the EPA/ARA ratio was significantly lower in wild origin eggs (2.76) compared to farm origin ones (5.52). Interestingly, the same trend of higher ARA content and lower EPA/ARA ratio was noticed in multiple fish species when wild origin eggs were compared to farm origin eggs; e.g. in salmon (Pickova et al. 1999), Arctic char (Salvelinus alpinus) (Pickova et al. 2007), rainbow trout (O. mykiss) (Blanchet et al. 2005), Atlantic cod (Gadus morhua) (Johnston and Leggett 2002) and others.

High levels of PUFA in fish eggs can increase the risk for lipid oxidation (Awada et al. 2012), therefore, a highly efficient antioxidant protection system is essential for proper physiological functions. Carotenoids, mainly astaxanthin, are deposited in the eggs of numerous fish species during egg maturation and are potential antioxidants (Torrissen et al. 1989; Watanabe and Miki 1993), thus, their presence enhance the egg quality. Wild origin grayling eggs were characterised by higher total pigment content, they were intensely red coloured compared to farm origin eggs, which were lighter and almost colourless (Figure 5). Low concentrations of astaxanthin may cause oxidative stress and could trigger lipid peroxidation (Pettersson and Lignell 1999). Due to the lower content of astaxanthin and higher levels of PUFA in farm origin eggs, the possibility of lipid oxidation in these eggs is even higher. Furthermore, low carotenoid concentration in eggs (loss of egg colour) has been shown to be correlated with lower fertility, embryo survival and disease resistance in salmonids (Pettersson and Lignell 1999; Janhunen et al. 2011), again pointing to reduced quality of farm origin grayling eggs. Almost all the carotenoids in salmonid fish are provisioned from females’ diet, therefore, carotenoids in the broodstock diet directly affect their accumulation in eggs, where they might play an important role in proper embryo development (Tyndale et al. 2008).

The observed differences in grayling egg FA composition could be linked to the observed low embryo survival rate in farm raised grayling (Kodela et al. 2023), which is confirmed by pilot results of laboratory hatching rate estimation for eggs derived from one grayling female of wild and one of farm origin being 68% for wild origin eggs and 40% for farm origin eggs. Furthermore, many hatched larvae from farm origin eggs were deformed (spine and jaw deformations, see Figure 5) and would probably not survive during the early larval developmental stages, while no deformities were noticed in wild origin larvae. These preliminary data further support the importance of egg biochemical composition for proper embryo development in grayling.

It has been reported previously that wild origin fish produce better quality eggs compared to farmed fish (Brooks et al. 1997; Lund et al. 2008); one of the important factors influencing this is parental diet (Lazzarotto et al. 2015). Wild fish feed on natural food containing all essential nutrients, while farm raised fish are fed by commercial feed that is optimised only for some commercially important fish species, such as Oncorhynchus sp. and Salmo sp. among salmonids. Specific diet that meets the need of the species and its different life stages, is reflected in better performance of these fish, including reproduction, i.e. in Chinook salmon (O. tshawytscha) no significant difference in biochemical composition of wild or farm origin eggs was observed (Haring et al. 2016), which suggests that parental diet, affecting the composition and therefore the quality of Chinook salmon eggs in captivity are similar to those in the wild. On the contrary, specific feed has not been developed for grayling, which is therefore fed with commercially available starter or grower feed for S. salar or O. mykiss. As we observed in this study, such feed does not fulfil the requirements of grayling breeding stock to produce high-quality eggs. Therefore, there is a demand for optimising diet for farm raised broodstock grayling and this is believed to be best designed on biochemical composition of wild origin grayling eggs (Tocher and Sargent 1984; Szmyt et al. 2015).

Presented results thus set the basis for further development of more suitable broodstock diet for grayling, which should consider the FA composition of their natural diets and the possible limitations in the ability of grayling for de novo synthesis of LC-PUFA from their precursors (linoleic (LA) and linolenic (ALA)). Our study indicates that n-3 LC-PUFA (EPA and DHA) are efficiently absorbed from the diet, and/or de novo synthesised from precursors (ALA), and incorporated into eggs’ lipids in grayling, as their proportion in farm derived eggs is higher than in the diet. Opposite was observed for n-6 LC-PUFA, mainly ARA, as the levels and proportion of LA were higher in farm derived grayling eggs, but ARA proportions and levels were lower, which would indicate limited ability of grayling to elongate and desaturate LA to ARA, especially when n-3 PUFA, which are competitive substrates for the enzymes in biosynthetic pathway, predominate. Further studies are needed to develop the grayling-specific broodstock diet which should consider changes in FA composition (lower amount of DHA and higher amount of EPA and ARA) of the broodstock feed in ratios that would mimic as much as possible the ratios among these LC-PUFA in wild origin grayling eggs. Elucidation of grayling biosynthetic pathway together with its possible limitations for LC-PUFA (ARA, EPA and DHA) is also needed, to produce high-quality farm derived eggs for the conservation of threatened grayling population.

Conclusion

This study is the first to compare the biochemical composition of eggs from wild and farm origin grayling females, showing statistically highly significant differences in protein and fatty acid content. Differences in the content of LC-PUFA (DHA, EPA and ARA) and their ratios are of particular importance, because they were previously related to egg quality in other fish species. The results of this study indicate inadequate grayling broodstock nutrition resulting in low quality of farm origin eggs. Basic reproduction parameters including fertilisation, hatching and survival rate, and the presence of deformities at embryo and larval stage are of the most importance in rearing for conservation purposes and should be optimised by fully meet the nutritional requirements of spawning individuals reared in hatchery.

Acknowledgements

The authors thank technical assistant Ivana Štular from Head of nutrition for her assistance with laboratory analyses, Erik Sivec from Soča fish farm (Kobarid, Fisheries Research Institute of Slovenia) for his help with grayling rearing and gamete collection and colleagues from Fisheries Research Institute of Slovenia, fishing clubs Bistrica and Bled for organizing field sampling. The authors acknowledge the financial support of the Slovenian Research Agency (Research core funding No. P4–0220) and Ministry of Education, Science and Sport of the Republic of Slovenia.

Disclosure statement

The authors report there are no competing interests to declare

Data availability statement

The data presented in this study are available on request from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by the Slovenian Research Agency (Research core funding No. P4–0220) and Ministry of Education, Science and Sport of the Republic of Slovenia.