Furthermore, in some diallel crosses with clonal lines, high heterosis effects were found. Whether a breeding strategy including clone crosses is relevant for practical applications
depends on the performance of the clone crosses as well as the cost-effectiveness of development and maintenance of the clonal lines.
Until now, experiments to produce highly inbred lines of farm animals such as pig and poultry were not very successful. The relatively low increase in the inbreeding level
per generation and high losses of lines due to inbreeding depression made the practical Ž
. application of this strategy too expensive Ameli, 1989; Glodek, 1992 .
In fish, external fertilization and embryonic development allow the development of homozygous clones in only two generations by use of induced gynogenetic or androge-
Ž netic reproduction Thorgaard, 1986; Horstgen-Schwark, 1991; Horvath and Orban,
¨ ´
´
. 1995 . The high fecundity of fish increases the probability of development of homozy-
gous genotypes free from expression of harmful genes and, therefore, viable and reproductively competent.
Ž Although clones have been developed within eight fish species Brachydanio rerio:
Streisinger et al., 1981; Oryzias latipes: Naruse et al., 1985; Cyprinus carpio: Komen et al., 1991; Plectoglossus altiÕelis: Han et al., 1991; Paralichtys oliÕaceus: Hara et al.,
1993; Oncorhynchus rhodurus: Kobayashi et al., 1994; Onchorhynchus mykiss: Young .
et al., 1995; Oreochromis niloticus: Muller-Belecke and Horstgen-Schwark, 1995 , only
¨ ¨
few data of their reproductive and growth performance in comparison to normal heterozygous controls have been published. The aim of the present investigation is to
supply performance data during the development, maintenance and reproduction of O. niloticus clones for subsequent evaluation of clone crosses.
2. Materials and methods
2.1. DeÕelopment and reproduction of clones The production of clone mothers by mitotic gynogenesis and the verification of their
homozygous status has been described in detail by Muller-Belecke and Horstgen-Schwark
¨ ¨
Ž .
1995 . These mitotic gynogenetic females, derived from the ‘‘Lake Manzala’’ strain Ž
. Egypt have been used to develop homozygous clones by meiotic gynogenetic repro-
duction. For this purpose, groups of mitotic gynogenetic females have been transferred to glass aquaria when they reached an age of 9 month. When preparation of spawning
Ž .
was observed, eggs of ripe females were stripped into 0.2 l saline solution 0.9 NaCl . Ž
. Sperm from male fish delivering high quality sperm high density and motility and
showing characteristic allele configurations at genetic markers was stripped and mixed in saline solution 1:5. For the inactivation of the paternal genome, this mixture was
Ž .
y2
irradiated in a layer of 2 mm with UV-light 254 nm with an intensity of 58 mJ cm .
Ž .
The sperm was stirred 120 rpm throughout the process of irradiation. Five minutes Ž
. after activation of eggs with genetically inactivated sperm and water 288C , eggs were
transferred into a 418C water bath for 4.5 min to induce the retention of the 2nd polar Ž
. body Puckhaber and Horstgen-Schwark, 1996 . The first generation clonal females thus
¨
developed, were reproduced when they were 9 months old by a further step of meiotic gynogenesis to produce second generation clones.
2.2. Proof of the clonal status Successful gynogenetic reproduction was confirmed by the alloenzyme marker
system adenosine deaminase analysing blood samples of all potentially clonal adults Ž
. Ž
. G 50 g as described by Muller-Belecke and Horstgen-Schwark 1995 . Due to the
¨ ¨
recombination rate of 100, which was observed in O. niloticus in this marker system, it allows the proper differentiation between meiotic and mitotic gynogenetic origin
Ž .
Hussain et al., 1994 . Fish were assumed to be homozygous clones if the absence of characteristic allele
configurations of the sperm donor in their electropherograms proved them to be Ž
gynogenetic offspring, resulting from mitotic gynogenetic females first generation
. Ž
. clones or clonal females second generation clones . A verification of the clonal status
at the DNA level was conducted by multilocus DNA fingerprinting and random Ž
. Ž
. amplified polymorphic DNA RAPD as described by Jenneckens et al. 1999 .
2.3. Control groups Control groups were produced simultaneously with gynogenetic groups, but control
groups differed with regard to their genetic make-up depending on what kind of comparison they were used for.
Ž . 1 During the development of first generation clones normal outbred progeny of the
Ž .
Lake Manzala population 72 batches from 42 females and six males was included as control to compare survival rates of gynogenetically reproduced progeny of mitotic
gynogenetic females up to the first feeding stage with survival rates of this outbred Ž
. progeny Table 1 . Incubation of approximately 200 eggs per batch and first feeding of
the hatched larvae was conducted, following the same procedure as gynogenetically reproduced eggs of mitotic gynogenetic females.
Ž . Ž
. 2 For the evaluation of egg quality of first generation clonal females Table 2
approximately 200 eggs of each batch from these clonal females undergoing gyno-
Table 1 Survival rates of meiotic gynogenetic O. niloticus offspring of six mitotic gynogenetic clone mothers in
comparison to offspring of normal outbred females Mother
Mother Mother
Mother Mother
Mother Normal
Ž .
Clone I Clone II
Clone III Clone IV
Clone V Clone VI
females ns 42
a a
a a
a a
Number n
2812 1836
1306 3474
958 3691
13334 of eggs
U U
U U
U U
Developing 19.8
31.7 15.9
6.30 15.5
6.48 69.4
embryos
U U
U U
U U
First 5.26
16.2 8.50
2.13 5.22
1.46 43.9
feeding fry Adult fish
n 73
171 42
14 23
20 data not recorded
Ž .
generation 1 2.59
9.31 3.22
0.40 2.40
0.54
a
Undergoing meiotic gynogenesis
U
Ž .
Significantly different from normal heterozygous offspring P F 0.05 .
Table 2 Reproductive traits of first generation clonal O. niloticus females from six different clonal lines
Clone I Clone II
Clone III Clone IV
Clone V Clone VI
No. of egg batches collected n
21 32
4 7
3 8
egg batches with first feeding fry 66.7
40.6 25.0
28.6 66.7
25.0 Ž
. No. of untreated eggs controls
n 4012
3465 317
1746 343
2121 developing embryos
42.4 19.0
3.15 15.6
14.3 14.3
a
min.–max. 0.0–90.8
0.0–83.2 0.0–58.8
0.0–59.8 1.8–27.5
0.0–72.1 first feeding fry
19.2 7.68
1.26 7.90
2.92 7.40
min.–max. 0.0–52.3
0.0–45.1 0.0–23.5
0.0–34.1 0.0–6.4
0.0–60.6 Ž
. No. of shock-treated eggs clones
n 9733
6021 3015
6365 507
4833 developing embryos
20.4 12.6
5.00 20.0
8.88 4.95
min.–max. 0.0–49.2
0.0–77.9 0.0–46.9
0.0–53.4 0.6–15.1
0.0–30.9 first feeding fry
4.88 2.21
0.99 4.67
0.39 2.09
min.–max. 0.0–20.5
0.0–13.0 0.0–9.4
0.0–11.4 0.0–0.6
0.0–16.3 adult fish
n 159
75 28
158 2
72 Ž
. generation 2
1.63 1.25
0.93 2.48
0.39 1.49
a
Minimum and maximum values among different egg batches.
genetic reproduction were fertilised with untreated sperm of high quality and raised without any special treatment to serve as controls.
Ž . 3 To provide all-female heterozygous control groups for growth comparisons with
Ž .
the all-female homozygous clonal groups Table 3 , normal outbred females of the same age as clonal females were mated with mitotic gynogenetic males that produced
Ž all-female offspring in earlier experiments
Muller-Belecke and Horstgen-Schwark,
¨ ¨
. 1995 .
2.4. Performance testing Reproduction and growth experiments were carried out in the warm water recircula-
tion system of the Institut fur Tierzucht und Haustiergenetik under constant environmen-
¨
Ž .
tal conditions water quality parameters, photoperiod the year round. Spawners were consecutively incorporated into reproduction experiments when they reached an age of 9
months. As not all clones have been developed at the same time, growth experiments were performed with first generation and second generation clonal groups ensuring that
spawners had a similar age when producing the progenies.
The testing procedure started with the incubation of eggs from clonal and control
3
Ž .
groups for 9 days in 35 cm hatching jars at 288C Habitzky-Biester, 1987 . The number Ž
. Ž
. of eggs 1st day of life , the percentage of developing embryos 2nd day of life and the
Ž .
percentage of first feeding fry 9th day of life were recorded for each group. From the 9th to the 24th day of life, the fry were kept at a density of 50 fish per l in 2 l plastic
Ž .
aquaria, and fed with a high protein diet 47 crude protein . At the 24th day of life, the fish designated for growth performance testing were transferred into 80-l glass aquaria.
Depending on the number of available fish per egg batch the 80 l tanks were stocked with a maximum of 80 individuals. Between their 24th and 136th day of life fish were
counted every two weeks. All groups were fed three times a day ad libitum with trout
Ž .
Ž diet 43 crude protein up to the 80th day of life, and with carp diet 35 crude
Table 3 Mean body weights and coefficients of variation of body weight from all homozygous clonal groups and
corresponding all-female heterozygous control groups kept among three different density classes
a
Density class Group
No. of tanks No. of fish
Mean body CV
F-test weight in g
1–20 fishrtank control
8 86
60.7 45.9
Clone I 6
81 55.4
21.7 Clone II
3 37
45.7 15.9
Clone III –
– Clone IV
– –
Clone V –
– Clone VI
3 21
65.5 30.3
U b
all clones 12
139 55.4 n.s.
22.6 21–50 fishrtank
control 11
385 49.6
31.1 Clone I
5 140
43.2 18.2
Clone II 4
155 41.7
25.3 Clone III
2 90
68.3 21.4
Clone IV 1
29 62.5
21.0 Clone V
1 39
22.2 19.7
Clone VI –
–
U
all clones 13
453 48.0 n.s.
22.5 51–80 fishrtank
control 3
199 40.3
29.2 Clone I
1 54
29.7 28.3
Clone II 1
52 40.3
20.1 Clone III
– –
Clone IV 1
54 30.7
33.3 Clone V
– –
Clone VI 1
73 31.5
16.2
U
all clones 4
233 33.0 n.s.
24.2 all density classes
control 22
670 50.2
34.1
U
all clones 29
825 45.5 n.s.
23.1
a
Coefficient of variation.
b
Not significantly different from control.
U
Ž .
Significantly different from control P F 0.05 .
. protein from the 81st to 136th day of life. At the 136th day of life the fish were
individually tagged and weighed. After finishing this growth performance testing, fish were communally reared in 700-l tanks. When the fish reached an individual body
weight above 50 g, blood samples for genetic marker studies were taken.
2.5. Data analysis To test differences in the number of surviving fish between clones and controls for
their significance, Chi square-tests were applied. No significant differences could be detected between the growth performance of
isogenic groups of the generations 1 and 2, which allowed to use the data of both generations commonly for further analysis. To obtain information about the influence of
stocking density on growth during the standard performance testing, groups were assigned to three density classes, depending on the number of fish per tank at the 136th
day of life. Represented by the number of tanks from clonal groups or control groups
within density classes, between three and 13 replicates were available for the analysis of growth performance data. Differences in mean body weights between clones and
controls kept among the three different density classes were evaluated by ANOVA, Ž
. applying The Generalised Linear Models GLM procedure on the following statistical
model:
U U
Y s m q G q C q G C
q T G C
q e
Ž .
Ž .
i j i j
i jk l i
j k
i jk l
where Y s value observed in the ijklth fish; m s overall mean, G s effect of the ith
i jk l i
Ž genetic category i s 1, 2; 1 s homozygous isogenic groups; 2 s all-female heterozy-
. Ž
gous control groups ; C s effect of the jth density class j s 1, 2, 3; 1 s one to 20 fish
j
. Ž .
per tank; 2 s 21 to 50 fish per tank; 3 s 51 to 80 fish per tank ; GC s effect of the
i j
wŽ . x
interaction between the ith genetic category and the jth density class; T GC
s
k i j
random effect of the k th tank within the ith genetic category and the jth density class; and e
s error term.
i jk l
To prove the effect of the genetic category for its significance the G -term was tested
i
wŽ . x
against the T GC
-term in an F-test.
k i j
Differences in variances of body weight observed between clonal- and control groups were analysed using F-tests from pooled within tank variances.
3. Results
