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Sturgeon.su

Chapter 12
Influence of Temperature on the Sterlet
(Acipenser ruthenus L.) Ovarian Follicles State
B.F. Goncharov, M.N. Skoblina, O.B. Trubnikova, and M.S. Chebanov
Abstract We studied the effects of changes in the temperature of water in which the
sterlet (Acipenser ruthenus L.) females were kept on morphometrical and physiologi-
cal characteristics of the ovarian follicles: size of oocytes, their polarization index,
and their capacity to mature and ovulate in vitro in the presence of progesterone or
purified preparation of gonadotropic hormone of the sturgeon pituitary, as well as
‘spontaneously' in the culture medium. When the temperature was lowered from
13°C, the optimal for spawning, to 6°C, the morphometrical parameters remained
unchanged, but the indices of the physiological state underwent changes. After the
temperature was gradually elevated to the initial value, some indices returned fully or
partially to the initial state, while others continued to change in the same direction.
Key words Sturgeons, oocytes, maturation, ovulation, in vitro, temperature
Temperature is the main factor that regulates the seasonal character of reproduction in sturgeon females. Rich experience of the national sturgeon aquaculture based on utilization of spawners caught in nature made it possible to establish some patterns of the influence of this factor on the state of gonads and artificial reproduction. For example, it was shown that keeping females even for a short time (several days) at the spawning temperature may lead to the irreversible loss of the capacity to respond to hormonal stimulation in the production of high-quality eggs. It was proposed long ago (Kazanskii, 1963) to change the temperature regime for spawn-ers to preserve their reproduction capacity. The temperature could be lowered by B.F. Goncharov, M.N. Skoblina, and O.B. TrubnikovaKol'tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia M.S. ChebanovSouth Branch Federal Centre of Selections and Genetics for Aquaculture, Krasnodar, Russia R. Carmona et al. (eds.) Biology, Conservation and Sustainable Development of Sturgeons, 205 Springer Science + Business Media B.V. 2009 B.F. Goncharov et al.
3–4°C below the threshold of spawning temperature and maintained until a given moment and then gradually elevated to the optimal one. Under these conditions, the females could be used in the second cycle of artificial reproduction. This method was successfully used at sturgeon hatcheries.
With the advent of keeping sturgeons for a long term or full-cycle rearing, the problem of controlling seasonal reproduction became even more acute. It was shown that, given a certain temperature regime, mature gametes could be obtained in practically any season of the year (Chebanov et al., 2002). Nevertheless, the results of artificial reproduction are often far from ideal and sometimes even disap-pointing. The matter is that fish populations may be heterogeneous, especially fish reared in captivity, in which asynchronous gametogenesis may be rather pro-nounced. In this respect, the problem of the criteria for the selection of females suitable for reproduction remains urgent.
A method was proposed (Goncharov, 1993) for selection, based on the determi- nation of the time of in vitro maturation of 50% of oocytes in the presence of pro-gesterone (T50). For the stellate sturgeon (Acipenser stellatus Pal.) females caught in nature, this method allowed identification and removal from artificial reproduc-tion of a group of fishes that were not capable of producing mature high-quality eggs in this state and under standard conditions of hormonal stimulation. Later (Goncharov et al., 1999), a similar study was made on Siberian sturgeon (Acipenser baerii Brandt) females reared in captivity. The seasonal changes in some morpho-metrical and physiological characteristics of follicles were monitored and their prognostic value was determined. It was seen that all the characteristics studied underwent changes during the observation period from December until April, and the oocytes ovulated in vitro under the influence of hormonal preparations had the best prognostic value. The application of these criteria allowed us to increase the efficiency of artificial reproduction but none of them could ensure unmistakable sorting of females by the quality of gametes that they could produce after hormonal stimulation. Hence, it was necessary to continue the studies of factors affecting the physiological state of follicles. The seasonal changes in the reaction of follicles to hormones in vitro suggest their ontogenetic character, but the information we obtained led us to hypothesize that the competence of maturation and ovulation of oocytes is maintained by homeostatic mechanisms. This implies not only unidirec-tional changes of these characteristics, but also the possibility of their transient return to the preceding state. Here, we study the influence of a gradual decrease in the temperature at which sterlet females are reared in captivity and an increase in the initial value on some morphometrical and physiological characteristics of fol-licles, finding evidence in favor of the above hypothesis.
12.2 Materials and Methods
Studies were carried out at the Krasnodar (Adygean) Sturgeon Hatchery on sterlet females reared in ponds at ambient seasonal temperatures at the South Branch Federal Centre of Selection and Genetics for Aquaculture.
12 Influence of Temperature on the Sterlet (Acipenser ruthenus L.) The females were transferred from a pond, where the temperature reached 13°C, into large concrete basins with regulated temperature in a closed room. The temperature was gradually lowered to 6°C within 5 days, maintained at this level for another 5 days, and then gradually elevated to the initial level within 6 days.
Studies were carried out on two groups (experiment and control) of 10 females in each. In the experimental group, the follicles were sampled at the beginning of the experiment (0 days), within 10 days, and at the end of the experiment (16 days). In the control group, which remained under the same temperature regime, the state of the follicles was estimated only at the end of the experiment (control to stress related to the sampling of follicles).
Ovarian follicles were taken using a special metallic probe and placed in a medium with hormonal preparations within 1 h. The follicles were repeatedly washed by Ringer solution for poikilotherms modified for sturgeons (RMS) (Goncharov, 1978) containing sodium bicarbonate at an increased (1.5 g/L) concen-tration. The follicles were incubated in the same medium in 55 mm plastic Petri dishes, 36 ± 3 follicles in 7.5 mL per dish, at 16 ± 1°C.
Oocyte maturation and ovulation were estimated with 38–42τ (τ is a dimen- sionless unit proposed by T.A. Dettlaff for measuring the development duration; see Dettlaff et al., 1993). Ovulation was estimated by counting the follicle envelopes separated from the oocytes. Maturation was estimated according to the germinal vesicle breakdown (GVBD). For this purpose, the oocytes were fixed by boiling and cut through with a safety blade under a dissection microscope.
Progesterone was introduced directly in Petri dishes as concentrated alcohol solution to a final concentration of 1 µg/mL (alcohol concentration 0.1%). Gonadotropic hormone of stellate sturgeon (aciGTH) was purified as described elsewhere (Burzawa-Gerard et al., 1975), with slight modifications. The lyophi-lized aciGTH powder was dissolved to a concentration of 2 µg/mL in the culture medium directly before the experiment.
The following characteristics of the follicles were determined in the course of the experiment: oocyte diameter, oocyte polarization index (OPI), percentage of oocytes capable of maturation and ovulation in vitro under the influence of proges-terone, aciGTH, or culture medium, and duration (in the number of τ ) of matura- tion of 50% oocytes in vitro in the presence of progesterone (T50).
Thirty follicles were fixed immediately after their isolation from the female and envelopes were removed with sharpened forceps. The diameter (mean of two meas-urements: between the poles and perpendicular one) and shortest distance between the GV surface and oocyte membrane were measured using ocular micrometer to a precision of 0.03 mm. The ratio of the distance from GV to the oocyte surface to the mean diameter (expressed in %) was designated as OPI.
In order to determine T50, maturation was monitored within 12τ after the beginning of incubation by fixing 12 to 15 oocytes with an interval of 1τ . From the moment of the appearance of the first mature oocytes, double numbers of oocytes were fixed with the same interval until all or almost all oocytes matured. T50 was determined using the graphic method after transformation of percentages into probits (Finney, 1971).
B.F. Goncharov et al.
To estimate the differences between the mean quantitative indices, the criteria for randomization for paired samples (for studying the changes in the follicles of females of the experimental group) and independent samples (for comparison of follicles of females of the experimental and control groups) were used (MegaStat software for Excel).
12.3 Results
The mean values of the morphometrical and physiological characteristics of the follicles and data for individual females are given in the Table 12.1 and Fig. 12.1, respectively.
At the beginning of the experiment, practically 100% of oocytes of the experimen- tal females matured in the presence of progesterone or aciGTH. This was only partly due to the effect of the hormones, since in different females 17 to 97% of oocytes matured in a hormone-free medium. The percentage of oocytes ovulated in the pres-ence of these hormonal preparations was much lower and progesterone was a more effective inducer. Ovulation was practically absent in the hormone-free medium.
Within 10 days of the beginning of the experiment, some parameters of the fol- licles underwent changes. The mean T50 value increased by approximately 6τ . The mean percentage of ovulated oocytes decreased approximately twice in the experiments with progesterone and somewhat more with aciGTH. The mean percentage of oocyte maturation in the hormone-free medium decreased as expected. On the contrary, the lowered temperature did not notably affect the percentage of oocyte maturation in the presence of progesterone or aciGTH, nor did it alter the mean morphometrical indices, such as oocyte diameter and OPI.
After the temperature rose to the initial level, some indices were fully or partly restored. The mean values of T50 and percentage of oocyte ovulation in the pres-ence of aciGTH practically returned to the initial level, while that in the presence of progesterone was only partially restored. On the contrary, the percentage of oocyte maturation in the hormone-free medium continued to decrease. The mor-phometrical parameters and percentage of oocyte maturation in the presence of hormones remained at the same level.
Comparison of the state of follicles of the experimental and control females at the end of the experiment did not reveal statistically significant differences for any of the morphometrical or physiological characteristics studied.
The efficiency of artificial reproduction of sturgeons is determined by a number of factors. The obtaining of mature eggs capable of normal development after insemi-nation depends on two main factors. First, the follicles of the older generation should 12 Influence of Temperature on the Sterlet (Acipenser ruthenus L.) < 0.05.
p Morphometric characteristics wering and subsequent ele ferences between the mean v in the presence ofo arian follicles incubated alues. Identical letters within the column designate the absence of dif Physiological characteristics of o Changes in morphometrical and physiological characteristics of sterlet follicles after lo able 12.1
eeping of the females experiment (days) Numerals designate the mean v B.F. Goncharov et al.
Fig. 12.1 Changes in physiological characteristics of the sterlet ovarian follicles (a, T50; b, per-
centage of oocyte maturation in hormone-free medium; c, percentage of oocyte ovulation in the
presence of progesterone; d, percentage of oocyte ovulation in the presence of aciGTH) after
lowering and subsequent raising of the temperature of keeping females. Abscissa, time after the
beginning of experiment, days; ordinate, number of τ (a), GVBD, % (b), ovulated oocytes, %
(c and d). Identical lines and marks represent the data for the same female not be damaged and should be capable of responding to the hormonal preparations through oocyte maturation and ovulation. Second, an adequate hormonal treatment should be given to the females. As shown by the experience of artificial breeding of sturgeon fishes, the first factor is of paramount importance. In the beginning of the era of sturgeon aquaculture in the USSR, there was no problem concerning the 12 Influence of Temperature on the Sterlet (Acipenser ruthenus L.) production of high-quality gametes from the broodfish, as the fish used for reproduc-tion were caught in nature during anadromous migration, when the state of their gonads was close to spawning. Under these conditions, a single injection of pituitary suspension led to the desirable result in the vast majority of cases. However, as the ecological situation in the seas and rivers worsened, the efficiency of artificial repro-duction was reduced and the problem of selection of broodfish became very acute. The numbers of sturgeon individuals were then catastrophically decreased, forcing many hatcheries to resort to repeated utilization and, recently, to utilization of brood-fish reared in captivity. When the fishes are held or reared in captivity, the conditions are different from the natural ones: in the feed and often in the temperature regime. This may lead to disturbances of oogenesis and/or its desynchronization. Hence, the problem of selection of females for reproduction becomes especially acute.
It is known that as the oocytes grow and the reproduction season approaches, certain changes take place in the oocytes and, specifically, GV is displaced to the animal pole. This parameter can be rapidly and relatively easily estimated. It was proposed long ago (Trusov, 1964; Kazanskii et al., 1978) to use it for selecting the females suitable for reproduction, but its efficiency turned out to be far from always effective. Later another approach was proposed on the basis of estimating the physi-ological state of follicles according to their reaction to hormonal preparations in vitro (Goncharov, 1993). Among the indices studied, the most suitable were the duration of in vitro oocyte maturation in the presence of progesterone (T50) and, next, the percentage of in vitro oocyte ovulation in the presence of gonadotropic preparations (Goncharov et al., 1999). A correlation was found between T50 and the quality of eggs. It proved to be highly significant, but weak. The use of this criterion can increase the efficiency of artificial reproduction on a large scale, but cannot guarantee results for individual females. When studying the in vitro reaction of the Siberian sturgeon follicles to hormones, it was found that the pattern of seasonal changes in physiological parameters correlated quite well with seasonal temperature changes. Hence, it was confirmed that as the spawning season approached, the sen-sitivity of the gonads to hormones increased, the reaction to these hormones became more complete, and it occurred at a higher rate. All these data suggest unidirectional-ity of the processes in question. However, data accumulated suggesting their changes in the reverse direction as well. It was also reported (Dettlaff and Davydova, 1974, 1979) that the sensitivity of follicles to gonadotropic hormones in vitro could be lost after their sharp short-term cooling in vivo and in vitro and could be restored after injection of triiodothyronine to females, while such treatment did not affect the reac-tion of oocytes to progesterone. However, two conditions were not fulfilled in this work, which are essential for correct comparison of the results of different experi-ments on the induction of in vitro oocyte maturation by gonadotropic preparations. First, strict identity of the culture medium is necessary, but it could not be ensured because the medium pH was brought to a certain value by sodium bicarbonate. The data on the important role of the sodium bicarbonate concentration for the sensitivity of follicles to gonadotropins were obtained later (Goncharov, 1978). Second, the use of the hormonal preparation of standard activity is necessary.
The present work was undertaken to test whether the follicles may return to a less advanced state and, if so, whether this return is an initial sign of pathological B.F. Goncharov et al.
changes or, under certain conditions, progression to the prespawning state may be restored. We chose temperature as a natural factor, essential for sturgeons and, pos-sibly, capable of altering the physiological state of follicles, and we switched the direction of its changes to the opposite with reference to the seasonal changes. It is believed that the range of spawning temperatures for the sterlet lies within the limits of 9 to 20°C (cf. Dettlaff et al., 1993). Hence, our experimental females were at the temperature conditions favourable for spawning and we might have expected that their gametes were ready for maturation. Actually, a study of the initial state showed that this was the case. In all the females, practically 100% of oocytes were capable of responding to both progesterone and aciGTH by maturation, the level of in vitro ovulation in the presence of hormones was sufficiently high, while T50 and OPI were rather low. The work on artificial reproduction of the sterlet at the hatch-ery was under way at that time, which also suggested that the experimental females were ready for reproduction.
After the decrease in temperature, OPI remained unchanged, and some physiologi- cal parameters, such as percentage of oocyte maturation in the presence of progester-one and aciGTH, also remained unchanged and others were displaced to a less advanced state. The mean T50 increased from 13.7 to 19.6τ and in 6 females out of 10 it exceeded 18τ . It was shown for the stellate sturgeon (Goncharov, 1993), Siberian sturgeon (Goncharov et al., 1999), and sterlet (B.F. Goncharov et al., unpub-lished data) that the females with T50≥18τ are not capable of producing high-quality mature gametes. The transformation of the data on T50 for the Siberian sturgeon became possible after the dependence of τ on temperature was studied (Gisbert and Williot, 2002). The percentages of oocyte ovulation in the presence of progesterone and aciGTH and in the hormone-free medium were also predictably decreased.
To determine whether these changes in the state of follicles are a normal reaction to the lowered temperature or whether they reflect the initial stages of pathological change, we gradually elevated the temperature so that, by the end of the experiment, the females were again under the initial temperature conditions. Analysis of the follicles sampled at this time showed that the characteristics that were invariable at the first stage of experiment (temperature lowering) again remained unchanged. These characteristics include the percentage of oocyte maturation in the presence of progesterone and aciGTH, as well as oocyte diameter and OPI. The mean T50 returned almost to the initial value. It was noted that, despite different initial levels of this parameter in different females, the pattern of changes proved to be similar: increase and then decrease to the initial level. The mean percentage of oocyte ovu-lation in the presence of progesterone increased, but did not reach the initial level. In this case, however, the pattern of changes at the level of individual females was not entirely similar, especially during the second stage of experiment. If at the low-ered temperature, this parameter decreased in all females, the reaction to tempera-ture elevation was more variable: from return to the initial level to the absence of changes or further slight decrease. Similar heterogeneity was also observed in the case of oocyte ovulation in the presence of aciGTH. At a certain state, the oocytes placed in RMS are capable of maturing in the absence of hormonal preparations. This phenomenon is called ‘spontaneous maturation' and has been described for some fishes and amphibians. Spontaneous maturation is increasingly expressed as 12 Influence of Temperature on the Sterlet (Acipenser ruthenus L.) the spawning season approaches. Goncharov et al. (1999) demonstrated that this characteristic also bears some prognostic significance for the selection of females. In our study, this characteristic diminished in practically all the females both during temperature lowering and the subsequent raising to the initial level.
The aim of this work was to study the influence of temperature changes on the state of follicles, but we know that the parameters in question may be affected also by stress related to manipulation with the broodfish (Goncharov and Polupan, 1997). To identify the changes that may be related to stress influences, we subjected a group of 10 females to the same temperature regime, but the state of their follicles was assessed only at the end of the experiment and, hence, they were not subjected to stress influ-ences connected with double sampling of follicles from the females. Although there were no statistically significant differences for any of the characteristics studied, T50 in the control group was somewhat higher and OPI somewhat lower. It cannot be ruled out that in a larger sample, the differences in T50 could be significant. This suggestion is based on the fact that the procedure of follicle extraction may lead to a decreased T50. In all likelihood, OPI is a less labile parameter. In many of our experi-ments on different sturgeon species we found no correlation between these parameters and T50 had the best prognostic significance, while OPI had the lowest.
The results suggest that the characteristics of the follicles studied combine the features of the ontogenetic process, as there is, undoubtedly, a vector of their changes over the course of oogenesis. However, the maintenance of their state at any given moment appears to be determined by a homeostatic mechanism permit-ting differently directed deviations under the influence of different factors. Both vitellogenesis and terminal stages of oogenesis, oocyte maturation and ovulation, are regulated by gonadotropic hormones. It can be proposed that the observed return of the follicles to an earlier ontogenetic state is related to a decreased level of gonadotropins and/or sensitivity of the follicle to them, but, as was shown in the present study, this deviation may be fully or partly reversible.
Unfortunately, the circumstances did not allow us to test the capacities of all these females to reproduction but, according to the evidence of fish culturists, who worked with these females after our departure, these temperature influences did not lead to significant losses, and high-quality gametes were obtained from at least some of these females.
Acknowledgements This study was partially supported by the Russian Foundation for Basic
Research, project no. 05-04-48433.
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