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* CSIRO Livestock Industries, PO Bag 5, Wembley W.A. 6913, Australia and
and
CSIRO Livestock Industries, Prospect N.S.W. 2148, Australia
2 Correspondence:
phone +61 8 9333 6687; fax: +61 8 9383 7688; E-mail:
n.adams{at}ccmar.csiro.au.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Genetic Engineering • Obesity • Sheep • Somatotropin • Wool Production
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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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 described 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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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 hammer-milled 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 µg/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 µg/L, and 50% maximal displacement on the standard curve occurred at 5.8 µg/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 x 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 x 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 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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. There was no effect of active or inactive transgene on lamb birthweight or lamb survival.
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).
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 x iDNA remained constant over time (P = 0.46), so the overall effect was well represented by the mean across times (Table 2).
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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.
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, 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).
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, these effects occurred because the progeny of Ram #8 with the iDNA construct were no different from controls.
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). Live weight change over time was substantially affected by the quality and quantity of the pasture available. Nevertheless, the difference in live weight increased regardless of whether sheep were gaining or holding live weight.
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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).
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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).
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 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).
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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, 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 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 insulin-resistant diabetes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). 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 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 secondary 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).
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication November 18, 2001. Accepted for publication May 13, 2002.
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Literature Cited |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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This article has been cited by other articles:
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  N. R. Adams and J. R. Briegel Multiple effects of an additional growth hormone gene in adult sheep J Anim Sci, August 1, 2005; 83(8): 1868 - 1874. [Abstract] [Full Text] [PDF]
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