Full Text - American Society of Animal Science

The impact of a transgene for ovine growth hormone on the
performance of two breeds of sheep1
N. R. Adams*2, J. R. Briegel*, and K. A. Ward†
*CSIRO Livestock Industries, PO Bag 5, Wembley W.A. 6913, Australia and
†CSIRO Livestock Industries, Prospect N.S.W. 2148, Australia
ABSTRACT: The effect of a transgene encoding ovine
growth hormone and regulated by a metallothionein
promoter was examined in progeny of 69 Merino ewes
and 49 Poll Dorset ewes that were inseminated by rams
heterozygous for the gene construct. The presence of
the transgene had no effect on the progeny from one of
the three rams used, as evinced by a normal concentration and secretion pattern of growth hormone and normal growth rate and fatness. In progeny from the other
two rams that bore an actively transcribed and translated copy of the transgene, the mean concentration of
growth hormone in the plasma was twice that of controls, but the pulsatility of secretion was lost. These
animals grew faster (P < 0.001) and were leaner (P <
0.001), but had a greater parasite fecal egg count (P <
0.001). The impact of the transgene differed between
breeds with greater wool growth rate (P < 0.01) and live
weight increase (P = 0.06) in Merino progeny compared
with Poll Dorset cross. At 18 mo of age, the depth of
the eyemuscle was decreased (P < 0.001), particularly
in female sheep (P < 0.01). The results indicate that
the production effects of genetic manipulation may depend on the age, the breed, and the sex of the animal.
Furthermore, the transgene may fail to be expressed
in some progeny so that its activity cannot be detected,
even though the sheep bear the DNA construct.
Key Words: Genetic Engineering, Obesity, Sheep, Somatotropin, Wool Production
2002 American Society of Animal Science. All rights reserved.
Introduction
Growth hormone affects many of the important
characteristics of animal production, including growth
rate, fatness, and lactation. Injections of growth hormone are used to increase the efficiency of milk production in dairy cattle in the United States, and to
improve production efficiency of swine in Australia.
Accordingly, enhanced growth hormone expression is
an obvious target for genetic manipulation in domestic animals.
Early gene constructs inserted into sheep produced
10- to 20-fold elevations of growth hormone in the
plasma (Rexroad et al., 1988; Murray et al., 1989).
These sheep also had reduced growth rate and feed
efficiency and a shortened life span. More recently, a
modified gene construct (MTSGH10) has been de-
1
We thank B. W. Brown for supplying semen from the transgenic
rams, and for helpful advice, and Y.-P. Du for carrying out the Southern analysis. Antiserum and standards for the growth hormone RIA
were kindly provided by A. F. Parlow, National Hormone & Pituitary
Program of the NIDDKD.
2
Correspondence: phone +61 8 9333 6687; fax: +61 8 9383 7688;
E-mail: [email protected].
Received November 18, 2001.
Accepted May 13, 2002.
J. Anim. Sci. 2002. 80:2325–2333
scribed that resulted in lesser increases in plasma
growth hormone concentrations and improved rates
of gain in live weight (Ward and Brown, 1998). The
present study examines sheep produced using semen
from three of the transgenic rams described by Ward
and Brown (1998) to inseminate ewes of the Merino
and Poll Dorset breeds. The concentration of growth
hormone was measured in plasma of the progeny, and
their health and productivity monitored under field
conditions up to the age of 2 yr. The experiment aimed
to determine the impact of genetic background on the
effect of the transgene on traits of commercial interest,
such as growth rate, fatness, and wool production.
Materials and Methods
Experimental Animals
Semen was collected from three medium wool Merino rams from the fourth generation of transgenic
sheep described by Ward and Brown (1998). All three
of these rams descended from a single foundation sire.
Artificial insemination was carried out on 69 Merino
ewes and 49 Poll Dorset ewes on January 25, 1999.
Fifty-four ewes were inseminated to Ram #46 and 54
to Ram #8, and ten ewes were inseminated to Ram #5.
2325
2326
Adams et al.
Each lamb was identified with its mother at birth,
and a piece of the tail removed at lamb marking 3 wk
later was frozen for Southern blot analysis of its DNA.
To detect the presence of the transgene, DNA was
extracted by conventional proteinase K digestion and
chloroform/phenol extraction, digested with the restriction enzyme BamHI and subjected to Southern
blot analysis using as probe a 32P-labeled cDNA encoding the entire ovine GH coding sequence (Ward and
Brown, 1998).
The lambs were born in June and weaned at 3 mo
of age in September. After weaning, animals were
weighed every 2 wk throughout their lives. Sheep were
shorn at 6 mo of age and again at 18 mo. Animals
were maintained at pasture under conditions typical
of the Mediterranean-type climate of south Western
Australia. This climate sustains annual pastures with
large seasonal fluctuations in pasture quality and
quantity. The green pasture senesced and died at the
end of spring (November), but abundant dry feed was
available until the end of January (midsummer), when
supplementary feeding of approx 150 g lupin seed/d
began. The animals remained on dry pasture until
short green pasture became available following rainfall in autumn (early May), but this pasture was too
short to maintain live weight. Limited supplementation with lupin seed was continued until the beginning
of spring (early August, 2000), when the animals were
13 mo of age.
The CSIRO Floreat Park Animal Ethics Committee
monitored the welfare of animal involved in these
studies, and the genetically manipulated animals
were managed and contained as required by the Australian Government Genetic Manipulation Advisory
Committee, supervised by the CSIRO Floreat Park
Institutional Biosafety Committee.
Sample Collection and Analysis
Fat depth and eyemuscle depth at the C site, 45 mm
from the midline at the 12th rib were measured with
ultrasound by an accredited Lambplan operator (Lambplan, Univ. New England, Armidale, NSW, Australia) at 5 mo and 18 mo of age. Body condition was
scored as described by Russel et al. (1969) in November
and December of 1999, and January, May, June, July,
September, and October of 2000. Following shearing
in February 2000, dyebands were applied in April,
May, August, and removed in December (Wheeler et
al., 1977). Staples from each sheep were cut along each
dyeband, and average daily clean wool growth rates
were calculated for each period.
Single blood samples were collected from all animals
at 3, 5, 14, and 17 mo of age, and the plasma concentrations of growth hormone measured. In addition, the
pattern of pulsatile secretion of growth hormone was
measured in an animal house study at 8 mo of age in
two groups of 24 (total 48) castrate male lambs that
either carried the inserted DNA (iDNA) or did not.
Half of each group was sired by Ram #8 and half by
Ram #46, and the groups were balanced to include
equal numbers of Merino and Poll Dorset cross sheep.
One nontransgenic Merino was later found to have a
retained testis and was removed from the analysis.
Animals were fed to maintenance, as calculated by
Grazfeed (Freer et al., 1997), on a ration of hammermilled oaten hay with 20% lupin seed and 2.5% mineral supplement (Siromin, Compass Farm Feeds Pty.
Ltd., Mt. Compass, SA, Australia). Sheep were fitted
with jugular catheters, and blood samples collected
every 20 min for 10 h on d 32 upon introduction into
the animal house, after which they were returned to
the field.
Fecal egg counts (FEC) were carried out by the
Western Australian Department of Agriculture to determine the concentration of nematode eggs in feces.
Samples were collected from different sheep on each of
three occasions; 38 sheep in March 2000, 23 in August
2000, and 36 in November 2000. All sheep were
drenched in March 2000 and April 2001 with a macrocyclic lactone anthelmintic.
Plasma concentrations of ovine growth hormone
were measured as described by Adams et al. (1996).
The interassay CV for four pools averaging 1.29, 4.02,
8.69, and 11.95 ␮g/L were 6.1%, 3.4%, 4.3%, and 3.5%,
respectively, and the corresponding intra-assay CV
were 2.0%, 2.0%, 1.0%, and 2.0%. The minimum detectable concentration was 0.49 ␮g/L, and 50% maximal displacement on the standard curve occurred at
5.8 ␮g/L. The proportion of tracer bound in the absence
of unlabelled growth hormone (B0) was 40% and 4.5%
of the label bound nonspecifically (NSB). Pulses of
growth hormone were analyzed by the Munro Pulsar
algorithm, as described previously )Adams et al.,
1996). The G parameters (number of standard deviations by which a peak of one, two, three, four, or five
points must exceed the mean to be accepted) were set
at 4.0, 2.4, 1.7, 1.2, and 0.9, respectively.
Statistical Analysis
Values for single point measurements of plasma
growth hormone at each age were transformed to logarithms to homogenize the variances, and back transformed means are presented. These data were analyzed by analysis of variance (ANOVA) using Systat
10 (SPSS Inc., Chicago, IL) with a model that included
sire, presence of iDNA, and breed. Significant sire ×
iDNA interactions were detected at each age. Further
analysis indicated a similar interaction between sire
and iDNA for the pulsatile secretion of growth hormone; effectively, the presence of iDNA did not affect
either the mean level nor pulsatile secretion of growth
hormone in the progeny of one of the rams (Ram #8).
This sire × iDNA interaction obscured analyses of
the way in which the active transgene interacted with
factors such as breed, sex, or age. Accordingly,
throughout this paper the description ‘transgenic’ is
2327
An inserted GH gene in two sheep breeds
Table 1. Numbers of lambs born to Merino or Poll Dorset mothers from rams
transgenic for growth hormone and the lambs’ mean birth weight
Merino
Item
Male (N)
Female (N)
Total
Birth wt (kg)
Poll Dorset
Controla
Transgenic
Controla
Transgenic
33
22
55
4.14
13
8
21
4.33
22
20
42
3.78
8
5
13
3.36
a
Includes lambs without iDNA and lambs in which iDNA was not expressed.
restricted to the progeny of Ram #5 and Ram #46 that
were shown by Southern analysis to bear the inserted
gene construct. All these animals had elevated concentrations of growth hormone in their plasma. The other
sheep which had normal concentrations of growth hormone were classified as nontransgenic, regardless of
whether they had iDNA or not. The term iDNA is used
to classify all animals in which transgene DNA could
be detected by Southern analysis, regardless of
whether it affected growth hormone concentrations.
Using these classifications, data were analyzed by
ANOVA or repeated measures ANOVA for live weight
and condition score. Fixed effects included breed, sex,
and transgene status. Results presented are the least
squares means and standard error means from
these analyses.
Results
Birth, Growth, and Early Development
A total of 63 lambs with iDNA and 68 control lambs
were born. However, 29 of the iDNA lambs and 34 of
the lambs without iDNA were born to Ram #8, so only
34 lambs that expressed the transgene were born. The
distribution of the progeny at weaning among breeds
and sex is indicated in Table 1. There was no effect
of active or inactive transgene on lamb birthweight or
lamb survival.
The Merino ewes had a longer gestation period
(153.0 vs 150.1 d for Poll Dorset ewes, P < 0.001). Male
lambs were heavier than female lambs (4.75 vs 4.17
± 0.14 kg, P < 0.001), but there was no effect of
transgene on lamb birth weight (Table 1).
Activity of the Inserted DNA from Different Sires
Plasma samples from each animal showed that the
concentration of growth hormone was not elevated by
iDNA in progeny of Ram #8, but was in progeny from
the other two rams (Table 2). As a result, an interaction (P < 0.001) was observed between sire and iDNA
for concentration of growth hormone (Table 2) at each
of the four times measured, but there was no significant effect of breed at any time. The average value of
growth hormone changed with time, reaching a maximum at 14 mo, but the interaction between sire ×
iDNA remained constant over time (P = 0.46), so the
overall effect was well represented by the mean across
times (Table 2).
The interaction between sire and iDNA was further
investigated by measuring pulsatile concentrations of
growth hormone in the plasma of progeny from two of
the rams (#8 and #46). The results summarized in
Table 3 again indicate an interaction between sire and
iDNA status. Transgenic progeny of Ram #46 differed
from the other groups, having fewer and smaller
pulses and a tendency to higher mean concentration
of growth hormone. Breed effects were not statistically
significant for any component of growth hormone secretion.
These patterns are displayed in Figure 1, indicating
the secretion of growth hormone was pulsatile in progeny of Ram #8, even though they carried iDNA. In
contrast, secretion of growth hormone in transgenic
progeny from Ram #46 was constant over the sampling
period. Figure 1 indicates that amplitude of some of
the pulses tended to be higher in progeny from Ram
#8 bearing iDNA, but this effect was not statistically
significant (comparison with non-iDNA progeny from
Ram 8, P = 0.25).
The differences in secretion of growth hormone were
reflected in body characteristics. By 6 mo of age, there
was a significant interaction (P = 0.04) between sire
and iDNA for fat thickness measured by ultrasound,
the body condition score differed among sires (P =
0.05), and the live weight showed a similar tendency
to differ among sires (P = 0.08). As shown in Figure
2, these effects occurred because the progeny of Ram
#8 with the iDNA construct were no different from
controls.
Live Weights
Repeated measures ANOVA of all live weight data
indicated that transgenesis was the major factor affecting live weight (P < 0.001). Although birth weights
were similar, the transgenic lambs were slightly heavier by weaning and became progressively heavier than
the controls as the experiment progressed (Figure 3).
Live weight change over time was substantially affected by the quality and quantity of the pasture available. Nevertheless, the difference in live weight in-
2328
Adams et al.
Table 2. Back-transformed means (and SE range) of the concentration of growth
hormone in single plasma samples taken at different ages and the mean value
for each control (Cont) and transgenic (iDNA) animal across time
Ram 5
Item
Age (mo)
3
5
14
17
Mean
Ram 8
Ram 46
Cont (4)a
iDNA (4)
Cont (34)
iDNA (28)
Cont (25)
iDNA (24)
2.0
(1.5–2.7)
1.4
(1.0–1.8)
3.5
(2.3–5.4)
1.4
(1.0–1.9)
2.1
(1.6–2.9)
4.3
(3.1–6.1)
4.7
(3.4–6.5)
7.7
(4.7–12.6)
5.6
(3.8–8.1)
5.2
(3.8–7.0)
2.2
(2.0–2.5)
1.2
(1.1–1.4)
3.3
(2.8–3.8)
1.6
(1.4–1.8)
2.1
(1.9–2.4)
1.7
(1.5–1.9)
1.3
(1.2–1.5)
4.0
(3.4–4.8)
1.6
(1.4–1.8)
2.3
(2.1–2.6)
1.8
(1.6–2.1)
1.6
(1.4–1.8)
3.8
(3.1–4.5)
1.9
(1.6–2.2)
2.6
(2.3–3.0)
3.3
(2.9–3.8)
4.0
(3.5–4.5)
8.7
(7.3–10.5)
7.0
(6.1–8.0)
6.2
(5.8–7.4)
a
Number of progeny.
transgenesis for eyemuscle depth (P < 0.01); eye muscle depth was suppressed less by transgenesis in castrate males (22.8 vs 26.3 mm) than in females (20.5
vs 27.6 mm). Similarly, at this age fatness was suppressed less by transgenesis in castrate males (4.17
vs 5.05 mm) than in females (3.31 vs 6.49 mm; interaction P = 0.001).
Repeated measures ANOVA indicated that body
condition score was higher in the Poll Dorset cross
sheep than the Merinos (2.31 ± 0.04 vs 2.08 ± 0.03; P
< 0.001). Presence of an active transgene reduced body
condition score from 2.38 ± 0.02 to 2.01 ± 0.04 (P <
0.001), and there was a significant interaction between transgene and sex (P < 0.001), with the
transgene suppressing condition score more in females (2.47 vs 1.99) than in males (2.28 vs 2.03).
creased regardless of whether sheep were gaining or
holding live weight.
Poll Dorset cross sheep were heavier (P < 0.05), and
there was an interaction between breed and transgenesis (P = 0.06), with transgenesis having a greater
effect in the Merinos than in the Poll Dorset cross. As
a means of illustrating these differences, the average
of all live weights collected across the whole study
was calculated for each sheep. The mean control and
transgenic live weights for Poll Dorset cross were 57.0
± 0.9 vs 61.1 ± 1.9 kg and for Merino were 51.2 ± 0.8
vs 62.9 ± 1.4 kg.
Body Composition and Condition Score
Compared with Merinos, Poll Dorset cross sheep
were fatter and had a greater eye muscle depth (P <
0.01; Table 4). Transgenic sheep (i.e., those sired by
Rams #5 or #46) were leaner at all ages (P < 0.01). At
18 mo of age, the depth of eyemuscle was reduced by
transgenesis (P < 0.001). The reduction in eyemuscle
depth caused by the transgene was greater in the Poll
Dorset cross sheep than in Merinos (Table 4; P =
0.001).
These measurements were not affected by sex, but
at 18 mo there was an interaction between sex and
Wool
All wool characteristics differed significantly between breeds but were not affected by sex. At 6 mo,
the only significant effect of transgenesis was a decrease in wool yield (Table 5). At 18 mo, transgenic
sheep again had a lower yield, especially in the Poll
Dorset cross sheep (interaction P < 0.05). The clean
fleece weight (CFW) was increased by 12% in
Table 3. Characteristics of secretion of growth hormone by progeny of two different
rams heterozygous for an inserted growth hormone gene (iDNA). Progeny were
determined by Southern analysis as either not carrying (no iDNA) or carrying (iDNA)
the inserted gene
No iDNA
Item
Ram Number:
N
Mean (ng/mL)
Pulses/10 h
Amplitude (ng)
Pulse area (ng2)
iDNA
Significance
8
46
8
46
SEM
iDNA
Ram
iDNA × R
11
3.3
3.8
4.7
151
12
3.7
4.2
6.0
172
12
3.7
3.7
6.4
241
12
6.1
1.3
1.2
44
1.3
0.5
2.0
57
0.07
0.000
0.60
0.05
0.08
0.001
0.02
0.91
0.21
0.000
0.004
0.002
An inserted GH gene in two sheep breeds
Figure 1. Patterns of secretion of growth hormone over
10 h from individual wethers that (a) did not carry inserted DNA, (b) carried inserted DNA from Ram 46 or
(c) carried inserted DNA from Ram 8. Each symbol within
a panel indicates an individual animal.
transgenic Merino sheep, but decreased 11% in Poll
Dorset cross sheep (interaction P < 0.01). Fiber diameter tended to be greater in transgenic sheep of both
breeds (P = 0.05). Transgenic sheep had a greater coefficient of variation in fiber diameter (CVfd).
Dyebands indicated that the pattern of wool growth
responded in a similar way in transgenic and control
sheep to the changes in pasture availability and quality (data not shown).
Animal Health
The transgenic sheep had higher average fecal worm
egg count at all ages sampled (9, 13, and 16 mo) when
compared with controls (means 441 ± 52 vs 153 ± 32,
2329
Figure 2. Mean (and SEM bars) at 6 mo of age for (a)
condition score, (b) depth of subcutaneous fat, and (c) live
weight for control lambs (solid bars) or lambs carrying
inserted DNA (open bars) from three different heterozygous sires.
P < 0.001). There was no significant effect of breed, sex,
or time of sampling and no significant interactions.
Retained testis was found at weaning in four of the
21 male transgenics and one of the 55 control males
(P < 0.05). The testes failed to descend over the next
6 mo, and the animals were removed from the experiment. A further transgenic animal had the external
genitalia of a ewe, but the vaginal canal extended for
only 100 mm, and on slaughter the animal was found
to have small male gonads in the external inguinal
canal.
Four of the transgenics and six controls died in the
field (P = 0.28) in the 22 mo following weaning. Most
causes of death were undiagnosed, but cases in which
2330
Adams et al.
of growth hormone was only twice that of controls
(Table 2). Furthermore, the transgene prevented
pulses of endogenous growth hormone, resulting in
chronic, nonpulsatile secretion of growth hormone,
which probably has a diminished biological effectiveness (Veldhuis et al., 1995). Nevertheless, the small
increase was sufficient to produce significant effects
on growth rate, leanness, and wool production.
The failure of progeny of Ram #8 to express the
transgene was unexpected. Transgenes may become
“silenced,” so that although present, they are not expressed (Parker et al., 1998), but this has not been
reported commonly in transgenic domestic livestock.
A broad range of potential mechanisms for silencing
was described by Marx (2000), and silencing may be
stimulated by a high number of copies of the inserted
gene in each cell (Garrick et al., 1998). This is unlikely
in our case, however, since the copy number of progeny
from Ram #8 appeared, on the basis of band intensity
on Southern blot autoradiographs, to be either similar
or possibly lower than that of the animals in which
the gene was active. Furthermore, copy number does
not affect growth hormone secretion in transgenic
mice (Bartke et al., 1994; Cecim et al., 1991); or in
normal sheep (Gootwine et al., 1998).
Phenotypic effects of the additional growth hormone
varied with breed, sex, and age. Compared with Merino sheep, the Poll Dorset cross transgenic sheep had
a lesser increase in live weight, a greater decrease in
muscle depth, and a depression in fleece weight rather
than an increase. These differences became more
marked as the animals matured (Tables 4 and 5).
Plasma concentrations of growth hormone were similar in both breeds, so this interaction probably reflects
differences in the responsiveness of the breeds to the
altered growth hormone secretion. Although interactions between the transgene and heterosis effects cannot be discounted, it is more likely that the result
depended on breed sensitivity, because breeds of sheep
differ in their responsiveness to injected growth hormone (Sinnett-Smith et al., 1989). Furthermore, the
magnitude of the response to a growth hormone
transgene was determined by the genetic background
Figure 3. Mean and SEM bars of live weight in control
(solid symbols) and transgenic (open symbols) sheep run
at pasture in a typical Mediterranean climate. Arrows
indicate loss of fleece weight associated with shearing.
a diagnosis could be reached included enterotoxemia
and snake bite. There were no visible abnormalities
in the transgenic sheep at 12 mo of age, but by 16 mo
of age it was necessary to trim horn-growth in the feet
of 62% of transgenics and 42% of controls (P = 0.08).
Over the next 6 mo, horn overgrowth became more
severe in the transgenic sheep and monthly foot-trimming became necessary. Three transgenic sheep commenced to lose body condition at 20 mo of age, were
euthanased at 24 mo, following a diagnosis of insulinresistant diabetes.
Discussion
Impact of Transgenesis
The MTSGH10 construct has overcome problems of
excessive secretion of growth hormone observed with
previous constructs (Rexroad et al., 1988; Murray et
al., 1989). The mean increase in plasma concentration
Table 4. Ultrasound measurements of subcutaneous fat depth and eyemuscle depth in
control (Cont) and transgenic (Trans) sheep born to Merino or Poll Dorset mothers
Merino
Item
Poll Dorset
Significance
Cont
Trans
Cont
Trans
SEM
Trans
Breed
T×B
5 mo of age
Number
Ca Fat depth (mm)
Eyemuscle depth (mm)
54
1.95
19.7
17
1.62
18.8
40
2.61
22.6
12
1.94
21.9
0.14
0.5
0.003
0.19
0.003
0.000
0.30
0.89
18 mo of age
Number
Ca Fat depth (mm)
Eyemuscle depth (mm)
51
4.9
25.2
17
3.3
21.8
38
6.7
28.7
9
4.2
21.5
0.3
0.5
0.000
0.000
0.000
0.008
0.18
0.001
a
The C site is 45 mm from the middle of the back at the level of the 12th rib.
2331
An inserted GH gene in two sheep breeds
Table 5. Fleece characteristics of transgenic (Trans) and control (Cont) sheep
from two ewe breeds
Merino
Item
Poll Dorset
Significance
Cont
Trans
Cont
Trans
SEM
Trans
Breed
T×B
6 mo
CFW (kg)
FD (␮m)
Yield
CVfd (%)
1.47
20.3
71.9
21.4
1.51
20.3
70.7
22.6
1.20
23.6
75.3
22.3
1.17
24.0
71.6
23.9
0.06
0.3
0.9
0.5
0.99
0.52
0.02
0.07
0.000
0.000
0.04
0.003
0.56
0.53
0.21
0.30
18 mo
CFW (kg)
FD (␮m)
Yield
CVfd (%)
2.83
21.1
66.6
21.97
3.17
21.8
61.0
22.27
2.02
25.4
63.4
22.37
1.82
26.2
52.6
24.54
0.08
0.3
0.9
0.4
0.45
0.05
0.000
0.03
0.000
0.000
0.000
0.02
0.007
0.89
0.03
0.10
in mice (Siewerdt et al., 2000) and trout (Devlin et
al., 2001).
There was also an interaction between the effects of
transgenesis and sex on fatness and eyemuscle depth,
with the impact being greater in females than in castrate males. Sex hormones modulate the response to
growth hormone treatment in humans (Span et al.,
2000) and to growth hormone transgenesis in mice
(Kaps et al., 1999; Wanke et al., 1999). Therefore, the
results of the current study are consistent with reports
in other species. However, the extent to which the
phenotype of the transgenic animal depended on characteristics such as breed, sex, and age indicates that
even tighter control of the level of expression of a
transgene for production efficiency may be required,
depending on the specific nature of the commercial
target.
Effect of Growth Hormone
The most obvious effects of the additional growth
hormone secretion in the transgenic sheep were reduced fatness and increased live weight. In ruminants, growth hormone has its primary effect on energy metabolism, where it reduces lipogenesis by reducing the capacity of adipose tissue to respond to
insulin (Dunshea et al., 1995). Energy expenditure is
greater, due in part to increased activity of the Na+
pump and to greater turnover of glucose and protein
(O’Sullivan et al., 1994). The effects on protein accretion are more difficult to predict. Growth hormone
increases both protein synthesis and degradation (Lobley, 1998), and the increased energy expenditure may
increase feed intake (Reklewska, 1974). Effects on
wool growth rate and on muscle depth depend on the
balance among these processes.
Transgenic sheep weighed more, but the eyemuscle
depth was not increased and indeed was decreased in
the 2nd yr. The increased live weight appeared to be
due to bigger skeletons and viscera, as observed in
sheep treated with growth hormone (Johnsson et al.,
1985). Anabolic hormones may reduce fleece weight
by diverting amino acids from wool to muscle (Nash
et al., 1994), but muscle depth was also decreased in
Poll Dorset cross sheep in the 2nd yr, so it is likely
that overall protein synthesis in the body was reduced.
Fleece weight is closely related to total protein synthesis in skin (Adams et al., 2000), supporting the interpretation that overall rates of protein synthesis were
reduced. This was probably due to amino acids being
diverted to gluconeogenesis, as the energy status of
the sheep declined.
It was anticipated that effects of transgenesis would
be observed only while animals were receiving abundant pasture. Previous work showed that the performance of sheep with reduced endogenous growth hormone following immunization against GHRH was not
affected on poor quality pastures, but live weight gain
and wool growth were suppressed on good pasture
(Adams et al., 1996). Furthermore, lambs treated with
growth hormone increased their nitrogen retention
only if protein supply was adequate (MacRae et al.,
1991). However, the difference in live weight between
transgenic and control sheep continued to increase
even while the sheep were losing weight (Figure 3).
Therefore, the current study indicates that growth
hormone may also have a significant role even when
nutritional conditions are limiting.
Treatment with growth hormone has been reported
to increase (Johnsson et al., 1985) or decrease (Wynn
et al., 1988) fleece weight. The variation in response
has been attributed to ability to increase their feed
intake (Reklewska, 1974), or to repartitioning of nutrients among wool and other tissues (Wynn et al., 1988).
In the present study, fleece weight was increased in
Merinos and decreased in Poll Dorset cross sheep (Table 5), indicating that genotype rather than nutrient
supply was the primary determinant of the wool
growth response to growth hormone.
The decrease in wool yield was probably due to stimulation of the sebaceous gland secretion by growth
hormone (Deplewski and Rosenfield, 1999), which
would have a greater effect in the Poll Dorset cross
sheep because they have a lower proportion of second-
2332
Adams et al.
ary wool follicles that lack a sebaceous gland. The
reason for the increase in variability of fiber diameter
(CVfd) is unknown, but a similar increase was seen
by Piper et al. (2001). The similar changes in wool
growth rate through the year, indicated by the dyeband results, suggests that the enhanced variability
was not due to greater variation in diameter along
the fiber, and so must be due to greater variation in
diameter between fibers.
Houdijk et al. (2001) suggested that a deficiency in
metabolizable protein may reduce immunity to intestinal parasites and result in a higher FEC. However,
the increase in FEC, also observed by Bell et al. (2001),
is unlikely to have resulted from a reduction in amino
acids available to mount an immune response, because
changes in partitioning to fleece weight or live weight
appear relatively minor, particularly early in the
study when differences in FEC were observed. Additional growth hormone may have had a direct effect
on the immune system, because an increased FEC was
observed at 9 mo of age, at a time when few other
effects were observed. Increased hoof growth and
splaying of the digits was not observed until 16 mo of
age. It is not clear whether the excessive hoof growth
was due to stimulation by growth hormone or the
changed shape of the feet. Foot problems are recognized in dairy cows injected with growth hormone
(Collier et al., 2001) and appear to be the counterpart
of bone growth in the digits of humans suffering acromegaly due to excessive growth hormone stimulation
(Giustina et al., 2000).
Implications
Sheep expressing a transgene for ovine growth hormone grew to a marketable weight faster than controls
and were leaner. However, growth hormone secretion
was unaffected by the presence on the inserted DNA
in the progeny of one of the rams used, indicating
that the transgene was not expressed in some sheep.
Furthermore, the effect of the growth hormone
transgene on specific production characteristics depended on the breed, age, and sex of the animal. For
example, wool growth was increased in the 2nd yr in
Merinos, but reduced in Poll Dorset cross sheep. Thus,
the optimal level of expression of a transgene may
depend on the commercial target that is desired. Animal health problems were negligible in the 1st yr,
except for a greater number of nematode eggs in the
feces, but further attention needs to be paid to potential animal health issues that may arise later in life.
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