Effect of dietary lipid source on semen fatty acids profile and sperm motility after
cryopreservation in rainbow trout (Oncorhynchus mykiss).
Efeito de diversas fontes alimentares lipídicas sobre o perfil ácido do sêmem de
truta arco íris (Oncorhynchus mykis) e sobre a mobilidade espermática depois do
descongelamento
Laura Gasco,* Ivo Zoccarato,* Carola Lussiana, * Hilton Amaral Júnior **
Abstract
In order to evaluate the effects of different lipid sources on sperm motility of
cryopreserved semen of rainbow trout (Oncorhynchus mykiss) 28 broodstock males 3+
were kept in four tanks and fed with: 1) a commercial diet as control; 2) a diet containing
cod liver oil; 3) a diet containing peanut oil; 4) a diet containing soybean oil. Fish were
stripped for milt collection after 150 days of feeding. The semen was analysed for fatty
acids composition, and fresh and post thawing sperm motility was evaluated. Vegetable
oils induced an increase of C 18:0 and C 18:1ω9 content in semen in comparison with
the commercial diet. An increase in C 18:2ω6 and C 18:3ω3 were induced by the diets
containing peanut and soybean oil, respectively. Milt from fish fed the diet containing
cod liver oil showed a high content in oleic and C 20:5ω3 acid, whereas C 22:6ω3 was
______________________________________________________________________
* Universita Degli Studi di Torino – Torino/Italia
** EPAGRI CP 20 – 88340.000 Camboriu SC/Brasil [email protected]
** CNPq. Brasilia DF/Brasil
significantly lower in sperm from fish fed the diet containing peanut oil. Fresh motility
was about 90%, while post-thawing motility reached 51,2%, 52,8%, 49,6% and 48,9% in
fish fed the commercial diet and those containing cod liver oil, peanut oil and soybean
oil, respectively. The results confirm that feeding may modify the semen composition but
does not affect significantly the post thawing motility of sperm.
Resumo
Com a intenção de avaliar o efeito de diferentes fontes lipídicas sobre a mobilidade
espermática do sêmen de truta arco-íris criopreservado, 28 reprodutores machos foram
mantidos em quatro tanques e alimentados no seguinte esquema: 1) dieta comercial
como controle; 2) dieta contendo óleo de fígado de bacalhau; 3) dieta contendo óleo de
amendoim; 4) dieta contendo óleo de soja. Após 150 dias se procedeu a coleta do
sêmen, sobre o qual se determinou o perfil dos ácidos graxos. Também foi avaliada a
mobilidade a fresco e depois do descongelamento. Os óleos vegetais induziram um
aumento no sêmen do nível de C 18:0 e C 18:1ω9 em comparação a dieta comercial. A
dieta contendo óleo de amendoim induziu ao um incremento de C 18:2ω6, enquanto o
conteúdo de C 18:3ω3 cresceu com a dieta contendo óleo de soja. O sêmen obtido dos
peixes alimentados com óleo de fígado de bacalhau mostrou um alto conteúdo de ácido
oléico e em C 20:5ω3, enquanto C 22:6ω3 resultou significativamente mais baixo no
sêmen obtido dos peixes alimentados com óleo de amendoim com respeito aos outros
3 tratamentos. No sêmen fresco, a mobilidade foi de cerca de 90%, enquanto que
depois do descongelamento o resultado foi de 51,2% nos peixes alimentados com a
dieta comercial. No tratamento com óleo de fígado de bacalhau a mobilidade foi de
52,8%, 49,8% com óleo de amendoim e 48,9% com óleo de soja. Os resultados obtidos
confirmam a possibilidade de influenciarem a composição do sêmen através da
alimentação,
todavia
a
mobilidade
pós-descongelamento
não
resultou
significativamente modificada.
Introduction
Many factors play a major role in regulating the gametes quality and thereby the
reproductive process. Hitherto in fish farming, owing to the close relationship with eggs
and larval quality, more attention has been paid to the female rather than the male. Less
importance has been given to the male’s role that finishes after spermiogenesis and milt
collection. It is known that feeding practice affects the nutritional condition and thus the
reproductive physiology. Previous studies have focused upon temperate species
particularly salmonids (Scott, 1962; Springate et al., 1985; Rowe and Thorpe, 1990;
Thorpe et al., 1990; Bromage et al., 1992).
In particular, the effect of feeding rate on fecundity and egg dimension was evaluated by
Bromage et al. (1992). Several reports anticipate the possibility of improving the quality
of eggs and larvae of different fish species such as trout (Corraze et al., 1993), gilthead
sea bream (Rodriguez et al., 1998; Almansa et al., 1999), tilapia (Coward and Bromage,
1999) and in freshwater prawn as well (Samuel et al., 1999). About sperm, Ciereszko et
al. (1996) observed that vitamin C diet supplementation improves the quality of rainbow
trout semen, while diet composition was found to affect sea bass (Bell et al., 1996) and
rainbow trout (Labbé et al.,1993; 1995; McNiven et al., 1999) semen. An issue of great
interest in aquaculture is the cryopreservation of semen, a procedure that assumes a
primary role in artificial insemination and management of broodstock (Maisse et al.,
1998; Lahnsteiner et. al., 1998). In salmonids, semen cryopreservation shows
considerable points of uncertainty because of individual variation in potency of different
males and even of a single male during the breeding season (Babiak et. al., 1998).
Moreover, since the tolerance to freezing stress seems to be linked to the lipid
composition of the sperm membrane, it could be very interesting to verify the possibility
of modifying the semen composition. Labbé et. al. (1995) observed that the quality of
fatty acids ingested affects the fatty acids profile of the sperm membrane without
modifying its fluidity. In rainbow trout, spermatozoa characterised by a low
cholesterol/phospholipids molar membrane ratio display the best aptitude to
cryopreservation (Labbé and Maisse et. al., 1998). McNiven et.al. (1999) observed that
a diet characterised by a low unsaturated/saturated
fatty acids ratio improves the
tolerance of semen to freezing damage. Nevertheless, very few information is available
about the influence of feeding practice on semen cryopreservation (Labbé et. al., 1995;
Maisse, 1996).
The aim of this investigation was to evaluate the effects of diets differing by their lipid
composition (high in ω3 or ω6 fatty acids) on whole semen fatty acids profile and sperm
motility after cryopreservation.
Materials and methods
In all, 28 three years old rainbow trout males (mean length 46,62 ± 5,03 cm and weight
1327,75 ± 375,45g.) were kept in four 2500 l circular outdoor fibreglass tanks supplied
by an open system of ground water (130C, flow rate 12l/min., 7,6 mg/l-1 dissolved O2).
During the experimental period, from the 10th of July to the 8th of December 1998, the
males were held under natural photoperiod regime and fed four diets, at 1% of body
weight, as follows: 1) a commercial diet as control; 2) a diet with cod liver oil; 3) a diet
with peanut oil; 4) a diet with soybean oil. The ingredients, proximate composition and
fatty acids profile of the diets are reported in tab.1 and 2 (AOAC, 1990). Fish were
individually tagged by implanted electromagnetic tags (Fish Eagle, Lechlade, England).
At the end of the trial, after 150 days of feeding, males were anaesthetised in
benzocaine (Sedgwick, 1990) and hand stripped; semen was individually collected into
10 ml vials and stored on ice. Ejaculates contaminated with faeces were discarded.
Each batch of individual semen was divided in two sub-samples. The first was used,
after centrifugation (500 g. for 20 min.), for the fatty acids determination. Lipids from total
spermatozoa were extracted, according to Folch et al. (1957), by homogenising the
sample in chloroform-methanol-water. A biphasic system was obtained, and the lower
phase, containing the lipids, was evapored under vacuum with a rotary evaporator.
Fatty acid methyl esters (FAMEs) were prepared by methylation of total lipids, and a
rapid transesterification technique using boron trifluoride methanol (Metcalfe an
Schimitz, 1961). The analysis of fatty acid methyl esters was carried out by gas
chromatography (Perkin Elmer 8700 GC) using a fused silica column (Supelcowax TM
10-Supelco), 30 µm x 0,53 mm i.d. and 1,0µm film thickness and hydrogen as carrier
gas (25 cm/min.)
Tab. 1 Ingredients and proximate composition (%) of diets (means ± s.e.).
Diets
Fish meal
Soybean meal
Gelatinised starch
Cod liver oil
Peanut oil
Soybean oil
Binder
Premix
Antioxidant (ppm)
Dry matter
Crude protein (w/w)
Ether extract (w/w)
Ash (w/w)
Gross energy MJ/kg
Commercial
Cod liver oil
43
30
18,4
7,1
Peanut oil
43
30
18,4
Soybean oil
43
30
18,4
7,1
91,56
42,30±0,24
10,59±0,03
7,31±0,02
19,05
1
0,5
100
93,16
42,14±0,13
11,23±0,51
8,07±0,04
21,63
1
0,5
100
93,00
42,21±0,11
11,78±0,02
7,83±0,02
20,22
7,1
1
0,5
100
92,68
43,57±0,07
12,10±0,04
7,68±0,08
19,58
The splitter injector temperature was 2600C, the FID was 2900C. The oven temperature
was programmed as follows: 1900C for 4 min; from 1900C to 2400C at 50C/min; the final
temperature was held for 15 min. After a 0,30µl injection, the separated components
were identified by reference to standards.
Tab. 2 major fatty acids (% of total FAMEs) of diets
Diets
C 14:0
C 16:0
C 16:1
C 18:0
C 18:1 ω9
C 18:2 ω6
C 18:3 ω3
C 18:4 ω3
C 20:1
C 20:4 ω6
C 20:4 ω3
C 20:5 ω3
C 22:0
C 22:1 ω11
C 22:4 ω6
C 22:5 ω6
C 22:5 ω3
C 22:6 ω3
C 23:0
Total ω3
Total ω6
ω3/ω6
Commercial
5,13
23,77
6,31
4,38
22,65
15,38
0,83
1,82
3,67
5,58
5,31
3,62
11,85
15,38
0,77
Cod liver oil
7,10
16,08
5,39
2,51
11,55
5,23
1,35
2,47
8,29
0,43
0,60
8,42
14,71
0,15
0,13
0,73
9,39
0,39
23,08
6,31
3,65
Peanut oil
2,45
15,54
2,68
3,59
36,86
22,43
1,05
0,96
2,22
0,14
2,66
1,19
1,85
0,06
0,57
2,91
0,12
8,44
22,59
0,37
Soybean oil
4,73
16,98
4,27
3,67
19,52
29,17
3,27
1,25
2,93
0,25
4,18
3,84
0,19
3,40
0,16
12,78
29,30
0,43
The second sub sample was utilised for the evaluation of spermatozoa motility at fresh
and after cryopreservation. For cryopreservation fish semen was directly mixed (1:4)
with 1,5 M DMSO+0,6 M sucrose and frozen according to Conget et al. (1996), in plastic
straws (250 µl) using a liquid nitrogen programmable freezing system (planer Biomed
Cryo 10). After 10 days, the semen was thawed by a 10 s immersion in water at 370C.
The motility and the percentage of motile spermatozoa were estimated by “Frame
analysis” on five straws per fish, by recording the spermatozoa activation with a
microscope coupled to a VHS video camera (Rana et al.,1990; Leveroni Calvi et al.,
1993). For each straw, four random fields were recorded for later motility evaluation. In
order to check motility, semen was activated (1:400) with M532 medium (0,55 NaCl,
0,38% Glycine, 0,24% Tris HCl, pH 8,8) (Billard, 1992). Whole semen composition and
motility data obtained were evaluated by Anova. Significant differences among treatment
means were revealed by New-man-Keus test (P≤0,01) (SPSS, 1999).
Results and Discussion
Experimental diets showed a good acceptance and no mortality was recorded until the
end of spawning season and, in spite of the high variability, the mean weight of
broodstock increased of 294±173 g after stripping. As shown in table 3 the lipid
composition of the diets affected significantly (P≤0,01), the profile of sperm fatty acids.
The vegetable oils (diets 3 and 4) induced an increase of C 18:0 content in sperm in
comparison with both the commercial diet and the diet containing cod liver oil. The
content of C 18:1ω9 differed significantly reaching the maximal level in broodstock fed
peanut oil diet. A similar trend was also found for the content of C 18:2ω6 that increased
in sperm obtained from fish fed diet 3, whereas the use of soybean oil increased the
content of C 18:3ω3. Milt from fish fed diet containing cod liver oil showed a high content
in oleic and C 20:5ω3 acid, while the sperm of fish fed the diet containing peanut oil
contained a significantly lower amount of C 22:6ω3. Anyway, the semen levels of C
20:5ω3 and C 22:6ω3 were higher than those found in diets. Ours results confirm that
the lipid composition of diets affects the fatty acids profile of trout sperm as observed by
Watanabe et al. (1984), Leray and Pelletier (1985) and Pustowka (1998), but whole milt
fatty acids profile did not reflect the trend of fatty acids of the diet.
The goal of this research was to evaluate the effects of dietary lipids on post-thawing
sperm motility and from this point of view no significant differences were observed. As
reported in figure 1 the highest percentage of motile sperm was obtained with cod liver
oil (mean ±s.e.) (58,2±0,96) bud did not differ significantly from the others treatments.
The lack of significant differences in post=thawing motility between treatments is in
agreement with the results of Labbé et al. (1995).
Tab3 – Percentage of major fatty acids in sperm (% of total FAMEs) (n=7; mean±s.e.)
Diets
Commercial
Cod liver oil
Peanut oil
Soybean oil
C 18:0
2:41±0,05 C
3,67±0,18 C
7,45±0,39 A
5,46±0,30 B
C 18:1ω9
9,11±0,61 D
27,12±2,56 B
35,43±2,23 A
17,03±2,20 C
C 18:2ω6
2,13±0,06 C
0,91±0,09 C
8,08±0,51 A
3,45±0,37 B
C 18:3ω3
1,04±0,07 B
1,33±0,003 B
0,94±0,09 B
10,35±0,80 A
C 20:5ω3
11,08±0,47 C
20,99±2,07 A
13,29±1,38 BC
15,73±1,34 B
C 22:6ω3
14,13±0,40 A
15,82±1,73 A
8,67±1,39 B
17,38±1,80 A
A,B,C,D: (P≤0,01)
The latter observed a modification in fatty acids profile but no changes in fluidity in
purified membrane of spermatozoa obtained from trout fed diets containing corn oil or
cod liver oil. Very little information is available about the effects of diets on tolerance of
semen to cryopreservation.
Moreover, Labbé et al. (1993) found that the fertilising
ability of cryopreserved trout semen was affected by rearing water temperature, but did
not differ significantly whether the animals were fed a diet containing corn oil or cod liver
oil. Although our attention was focused to the C18 fatty acids family and we have
analysed the whole trout semen, the level of mayor fatty acids showed a trend similar to
that observed by Labbé et al. (1995) for the semen fatty acids profile in the lipid class of
phosphatidycholine.
In whole trout semen, in accordance with Pustowka (1998), we have found a relatively
high percentage of EPA and DHA. The increased level of PUFA was likely to be due to
in vivo synthesis by the elongation of linoleic and linolenic acids. EPA plays an important
and not well-defined role in prostaglandin formation in fish (bell et al., 1986), probably as
an inhibitory regulator of testosterone production as suggested by Wade et al. (1994).
We have not explored this aspects, however the significant differences in EPA and DHA
content, among the semen obtained from fish fed different diets, did not modify the
tolerance to cryopreservation of rainbow trout sperm. The ω3/ω6 ratio in our diets, that
ranged from 3,65 to 0,37 (see Tab. 2), did not affect significantly sperm motility, even if
the best motility was observed in semen from fish fed the diet characterised by highest
ω3/ω6 ratio.
This result, indirectly, supports the observations of Baynes and Scott (1987) that
improved sperm post-thawing motility by increasing the ω3/ω6 ratio in the diet from 3,33
to 6,25. The supplementation of diets with vegetable oil allowed us to reduce the ω3/ω6
ratio below 0,5, but this did not affect sperm cryopreservation tolerance. No effects on
post-thawing fertilising ability was observed by Labbé et al. (1993) between diets
containing corn oil or cod liver oil, characterised by a ω3/ω6 ratio below 1. |On the
contrary, Pustowka (1998) obtained a significant decrease of cryopreservation-induced
damage in rainbow trout spermatozoa membranes by feeding the broodstock with a diet
containing tallow, high in saturated fatty acids, with a ω3/ω6 ratio of 0,3.
It is important to remember That the attempts to improve membrane fluidity by changing
the degree of unsaturation, could induce a degeneration of the testis tissue due to a
deficiency of vitamin E or zinc, as observed in the rat (Chanmugam et al., 1991).
Besides, Bell et al. (1991) observed that a decreased ω3/ω6 ratio can induce changes in
fatty acids metabolism with a consequent increase of stress susceptibility and cardiac
lesions in salmonid fish. From this point of view it is know that an increase of PUFA in
bio membranes can induce a high risk of membrane per oxidation and fragility, as
observed by Cowry et al. (1984). As to the post thawing motility, the results obtained
ranged between 48,9% (soybean oil treatment) and 52,8% (cod liver oil treatment).
These values, within the limit of the VCR evaluation system, are in agreement with those
recorded by Conget et al. (1996) using the same cryopreservation method. A similar
percentage of motility was recorded, using the same system, by Rana (1990) in
cryopreserved sperm of tilapia.
In the present trial we did not perform egg-fertilisation tests, however in accordance to
Stoss (1983), Ciereszko and Dabrowski (1994) and Lahnsteiner et al. (1998), we can
consider that sperm motility provides a useful parameter to estimate the viability of
cryopreserved spermatozoa.
Our results confirm the possibility to modify the semen fatty acids profile, but the
cryopreservation tolerance, is not apparently improved. On the contrary, recent results
by Pustowka (1998) and McNiven et al. (1999) suggest that lipid sources high in
saturated fatty acids reduce the freezing damage of spermatozoa. This is in contrast
with the idea to improve the spermatozoa membrane fluidity by increasing the dietary
levels of unsaturated fatty acids, but is very interesting and opens new perspectives.
Another interesting topic, requiring more investigations, is the evaluation of the aptitude
of each male to produce a semen characterized by a high tolerance to cryopreservation.
In conclusion, fish nutrition represents an interesting tool to after positively the viability of
cryopreserved semen. However, more investigations are needed in order to improve this
experimental technology and spread it in the farming system.
Acknowledgements
Research supported by MURST (ex 40% “Miglioramento dell’efficienza riproduttiva nelle
zoocolture”.
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